Dissertations / Theses on the topic 'Obesity – Animal models'

Theses (Ph. D.)--Marshall University, 2006.
Title from document title page. Includes abstract. Document formatted into pages: contains xiii, 138 p. including illustrations. Bibliography: p. 109-121.

Obesity has been described as a global health crisis due to the rapid increases seen worldwide (Whitlock et al., 2005; Harris et al., 2009; Yetter, 2009). The consequences of obesity are far-reaching and include the physiological and psychological implications for obese individuals, as well as the financial impact it has on both the individual and national health care. Children, especially those of minority ethnic background and lower socioeconomic status, are at increased risk for developing obesity (Yetter, 2009; Veldhuis et al., 2009). Intervention programs targeting underlying causes of childhood obesity have been developed, but little consistent success has been achieved (Summerbell et al., 2005; Sherry, 2005). One factor that could be influencing the lack of success is the stigmatization that can be felt by children taking part in intervention programs. Furthermore, many programs have targeted behavior change without determining underlying attitudes about behaviors. It is critical that effective obesity intervention programs be developed for children at high risk of developing obesity. This study used indirect messaging to address health issues related to overweight and obesity in children. An education program about animal health was presented to two groups of eight and nine year old children. The program included a combination of classroom instruction and practical application both in the classroom and at the Palm Beach Zoo with real animals. The children's attitude, knowledge, and practice of healthy behavior was measured before and after exposure to the program to evaluate its effect. It was hypothesized that learning about what being healthy entails for animals will have positive implications for the children's own health. It was found that children who participated in this study were already knowledgeable about healthy behaviors and also had overall positive attitudes towards health. However, they did not have high levels of health behavior practices. Participation in the program did not significantly improve the knowledge, attitudes, or practice of health behavior in the children. Zoos should consider designing program that specifically target increasing the practice of health behaviors in children.

Obesity is one of the leading causes of world-wide life-threatening diseases, such as type 2 diabetes mellitus, atherosclerosis and cardiovascular diseases. So far, there is no effective, promising and safe remedy. Development of cost-effective anti-obesity therapies and promising biomarkers for obesity-related diseases have become a demanding task. Lipocalins, such as LCN13, were recently identified as potential drug target because of its beneficial effects on glucose and fat metabolism in mouse. LCN14 is a putative lipocalin that share high degree of homology with LCN13. In this study, it is experimentally proved that LCN14 is a secretory protein that is mainly expressed in white adipose tissues. It is also demonstrated that serum LCN14 level was significantly increased in mice with HFD treatment, and it was significantly reduced in diet-induced obese and diabetic (db/db) mice. In addition, the degree of suppression of circulating LCN14 was greater than that of LCN13 in diet-induced obese and db/db mice. Therefore, serum LCN14 level could be a promising marker for risk prediction of obesity and its complications. Further investigation is urgently needed to unveil the important roles of LCN14 in metabolism.
published_or_final_version
Medicine
Master
Master of Medical Sciences

Background and objectives: A lot of studies proved that adipocyte fatty acid binding protein (A-FABP), an adipokine mainly expressed in adipocytes and macrophages, is the key link between obesity and inflammation which is suggested to be a therapeutic target for obesity-related diseases. Loss-of-function study was employed by using A-FABP knockout (KO) mice generated by our group to investigate role of A-FABP in high fat high cholesterol (HFHC) diet-induced obesity. Key findings: 1. Our study confirmed that HFHC diet-induced A-FABP KO mice have a significantly increased body weight when compared to the wild-type (WT) control mice. 2. Higher adiposity was the major reason for the A-FABP KO mice to be heavier than the WT controls under HFHC diet induction. 3. The marked increase of the weight of subcutaneous fat and peri-renal fat contributed to the higher adiposity of the HFHC-diet induced A-FABP KO mice when compared to the WT controls. 4. The HFHC-diet induced A-FABP KO mice significantly consumed less oxygen and produced less carbon dioxide suggesting the reduced energy expenditure but had higher weekly energy intake when compared with the WT controls, leading to higher adiposity. 5. The A-FABP KO mice were protected against HFHC diet induced glucose intolerance, insulin resistance, hyperglycemia and hyperinsulinemia when compared with the WT controls. There was also a better insulin secretion in response to glucose stimulation in A-FABP KO mice under prolonged HFHC diet induction when compared with the WT controls. 6. The A-FABP KO mice were protected against the development of hypercholesterolemia and hypertriglycemia when compared the WT controls under HFHC diet induction. However, there was no significant difference in the fasting serum free fatty acids (FFA) level among A-FABP WT and KO mice fed with standard chow (STC) or HFHC diet. 7. A-FABP KO mice were protected against isolated systolic hypertension (ISH) under HFHC diet induction. 8. The A-FABP KO mice were protected against HFHC diet-induced liver injury as indicated by a lower serum ALT level suggesting a better liver function when compared with the WT controls. 9. Under HFHC diet induction, M1 macrophage polarization was dominant in fat tissues of A-FABP WT mice but M2 macrophage polarization was dominant in fat tissues of A-FABP KO mice, suggesting an improved inflammatory status in the adipose tissue of the A-FABP KO mice when compared with the WT controls. This may also be the reason for why HFHC diet-induced A-FABP KO mice have an increased body weight but are metabolically healthier compared to their WT controls. Conclusions: A-FABP KO mice had a significant higher body weight and higher adiposity due to the reduced energy expenditure and increased weekly food intake as indicated in the metabolic cage study and the reason for metabolic healthier is due to the alleviated HFHC diet induced M1 macrophage polarization in various adipose tissues suggesting an improved inflammatory status in A-FABP KO mice comparing to the WT controls.
published_or_final_version
Medicine
Master
Master of Philosophy

Thesis (MScMedSc)--University of Stellenbosch, 2011.
ENGLISH ABSTRACT: INTRODUCTION: Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase that was first discovered as a regulator of glycogen synthase thus playing a role in glycogen synthesis (Embi et al. 1980). GSK-3 has also been shown to down regulate the expression of SERCA-2a (a calcium ATPase pump) thus playing a role in myocardial contractility (Michael et al. 2004). However, SERCA-2a activity is regulated by phospholamban (PLM) and sarcolipin (SLN) (Asahi et al. 2003). GSK-3 is constitutively active in cells and can be acutely inactivated by insulin through phosphorylation by PKB/Akt. However, GSK-3 is known to phosphorylate and inhibit IRS-1 protein, thus disrupting insulin signaling (Eldar-Finkelman et al. 1996). In addition, abnormally high activities of GSK-3 protein has been implicated in several pathological disorders which include type 2 diabetes, neuron degenerative and affective disorders (Eldar-Finkelman et al 2009). This led to the development of new generations of inhibitors with specific clinical implications to treat these diseases (Martinez 2008). GSK-3 inhibition has been shown to improve insulin and blood glucose levels and to be cardioprotective during ischemia/reperfusion (Nikoulina et al. 2002; Kumar et al. 2007). AIMS: To determine whether myocardial GSK-3 protein and its substrate proteins are dysregulated in obesity and insulin resistance, and whether a specific GSK-3 inhibitor can prevent or reverse the cardiovascular pathology found in obese and insulin resistant animals. OBJECTIVES: To correlate the alterations in expression and activation of GSK-3 protein in a well characterised rat model of obesity coupled to insulin resistance with: i) myocardial contractile dysfunction and an inability of hearts to withstand ischemia/reperfusion, ii) the activation and expression of phospholamban and SERCA-2a in the sarcoplasmic reticulum, iii) the activation of intermediates (IRS-1, IRS-2 and PKB/Akt) that lie upstream in the activation pathway of GSK-3 and iv) to determine the effects of inhibition of GSK-3 on the abovementioned parameters. METHODS: Age and weight matched male Wistar rats (controls and diet induced obese (DIO) animals) were used in the present study. Controls were fed normal rat chow, while DIOs were fed a rat chow diet supplemented with sucrose and condensed milk, for 8 or 16 weeks. Half of each group of animals were treated with the GSK-3 inhibitor for 4 weeks (from 12 to 16 weeks). After the feeding and treatment period, animals were weighed, sacrificed, hearts removed and freeze clamped immediately or perfused with Krebs-Henseleit buffer and subjected to low flow ischemia (25 min) followed by 30 min reperfusion. Biometric (body weight, intraperitoneal fat, ventricular weight and tibia length) and biochemical (fasting blood glucose and insulin levels) parameters were determined. Expression of GSK-3, PKB/Akt, IRS-1, IRS-2, SERCA-2a and Phospholamban were determined by Western blotting. Ca2+ ATPase activity was determined spectrophotometrically. RESULTS: At both 8 and 16 weeks DIO animals were significantly bigger than control animals and this was associated with increased intraperitoneal fat in DIOs. In DIO animals: IRS-1 was downregulated at 8 weeks and both IRS-1 and IRS-2 as well as PKB/Akt at 16 weeks. There was an increased tendency of GSK-3 expression at both 8 and 16 weeks in DIO animals while SERCA-2a was severely downregulated from 8 weeks onwards and associated with lower Ca2+-ATPase activity. PLM expression was upregulated but its phosphorylation was attenuated. At 16 weeks, baseline heart rate (225 vs 275 in control, P<0.0001, n=6) and rate pressure product (21000 vs 30000 in control, P=0.019, n=6) were significantly lower in hearts from DIO animals. Functional recovery was unchanged but the time to ischemic contracture development was increased (11.6±0.4 control vs 16.2±0.5 min DIO, P<0.01, n=6). Treatment had no effect on total GSK-3 expression. However, GSK-3 phosphorylation was significantly increased in treated controls, while there was no significant difference in DIO animals. However, there was a tendency for an increased GSK-3 phosphorylation in treated DIO animals. GSK-3 inhibitor, improved hypertrophy in DIO animals, while it led to its development in control animals. GSK-3 inhibitor improved IRS-2 expression in both control and DIO animals while it had no effect on IRS-1 and SERCA-2a expression and activity. However, GSK-3 inhibition increased PKB/Akt and phospholamban phosphorylation in DIO animals. CONCLUSION: These findings show that high calorie diet as well as imbalance between energy intake and expenditure lead to the development of obesity and insulin resistance in male Wistar rats. We showed that GSK-3 and its substrate proteins are dysregulated in obesity and insulin resistance. The reduced SERCA-2a expression at baseline may have a negative impact on cardiac function. By treating the animals with GSK-3 inhibitor, we showed that GSK-3 protein may not be responsible for changes seen at baseline. The decreased IRS-1 and SERCA-2a expression may have been caused by a different mechanism other than the actions of GSK-3. However, according to this study, GSK-3 may play a role in regulation of IRS-2 expression but not in IRS-1. Increased PKB/Akt phosphorylation may contribute to the GSK-3 inhibition. In addition, GSK-3 inhibition may reverse cardiac hypertrophy in DIO animals, thus acting as a negative regulator of hypertrophy.
AFRIKAANSE OPSOMMING: Inleiding: Glikogeen sintase kinase-3 (GSK-3), 'n serien/threonien proteïen kinase, is oorspronklik ontdek as 'n rolspeler in glikogeen sintese, aangesien dit 'n reguleerder van glikogeen sintase is (Embi et al.1980). Intussen is dit ook bevind dat GSK-3 die uitdrukking van SERCA-2a ('n kalsium ATPase pomp) kan afreguleer en dus sodoende 'n rol speel in miokardiale kontraktiliteit (Michael et al. 2004). Die aktiwiteit van SERCA-2a kan egter ook gereguleer word deur fosfolamban (PLM) en sarkolipin (Asahi et al. 2003). GSK-3 is deurgaans aktief, maar kan tydelik geïnaktiveer word onder kondisies van insulien stimulasie deur PKB/Akt gemedieerde fosforilering. Aan die ander kant is dit bekend dat GSK-3 die IRS-1 proteïen kan fosforileer om dus sodoende insulien sein-transduksie af te reguleer (Eldar-Finkelman et al. 1996). Daarmee saam is abnormaal hoë vlakke van GSK-3 aktiwiteit geassosieer met verskeie patologiese versteurings, insluitend tipe 2 diabetes, neuron degeneratiewe en affektiewe versteurings (Eldar-Finkelman et al. 2009). Daar is dus nuwe generasies GSK-3 inhibitore ontwikkel met die kliniese potensiaal om hierdie patologieë te behandel (Martinez 2008). Dit is al bevind dat GSK-3 inhibisie geassosieer kan word met beide die normalisering van plasma insulien- en glukose vlakke, asook kardiobeskerming in die konteks van iskemie/herperfusie (Nikoulina et al. 2002; Kumar et al. 2007). Doelwitte: Om te bepaal of GSK-3 proteïen en sy substraat proteïene gedisreguleer is onder kondisies van obesiteit en insulien weerstandigheid, asook om vas te stel of 'n spesifieke GSK-3 inhibitor die kardiovaskulêre patologie wat gevind word in obese en insulien weerstandige diere kan verhoed of omkeer. Mikpunte: Om veranderinge in uitdrukking en aktiwiteit van GSK-3 proteïen in 'n goed gekarakteriseerde rotmodel van obesiteit, gekoppel aan insulien weerstandigheid, te korreleer met die volgende: i) miokardiale kontraktiele disfunksie en onvermoë om kardiale iskemie/herperfusie besering te weerstaan, ii) aktivering en uitdrukking van PLM en SERCA-2a in die sarkoplasmiese retikulum, iii) die aktivering van intermediêres wat proksimaal geleë is in die insulienseintransduksiepad van GSK-3 (IRS-1, IRS-2 en PKB/Akt) en iv) om die effek van behandeling met 'n spesifieke inhibitor van GSK-3 op die bogenoemde punte te bepaal. Metodes: Ouderdoms- en gewigsgepaarde manlike Wistar rotte (kontrole en dieet geïnduseerde obees (DIO) diere) is in die studie gebruik. Kontrole diere was normale rotkos gevoer, terwyl die DIO diere op 'n dieet van rotkos aangevul met sukrose en kondensmelk geplaas is vir 'n periode van 8 of 16 weke. Helfte van die diere van elke groep is behandel met die GSK-3 inhibitor vir 4 weke (vanaf week 12 tot 16). Na afloop van die voer- en behandelingsperiode is die diere geweeg, doodgemaak en die harte verwyder om dan of onmiddelik gevriesklamp te word, of retrograad geperfuseer te word met Krebs-Hensleit buffer. Ex vivo geperfuseerde harte is dan blootgestel aan 25 minute lae vloei iskemie gevolg deur 30 minute herperfusie. Biometriese (liggaamsgewig, intraperitoneale vet, ventrikulêre gewig en tibia lengte) en biochemiese (vastende bloedglukose en -insulien vlakke) parameters is telkens bepaal. Western klad tegnieke is gebruik om die uitdrukking en fosforilering van GSK-3, PKB/Akt, IRS-1, IRS-2, SERCA-2a en PLM te bepaal. Ca2+-ATPase aktiwiteit is spektrofotometries bepaal. Resultate: Na beide 8 en 16 weke was die DIO diere beduidend swaarder as die kontrole diere. Hierdie gewigstoename was geassosieer met meer intraperitoneale vet in die DIO diere. Verder, in die DIO diere was IRS-1 afgereguleer na 8 weke, terwyl beide IRS-1 en IRS-2 asook PKB/Akt afgereguleer was na 16 weke. GSK-3 uitdrukking het 'n neiging getoon om toe te neem na beide 8 en 16 weke in die DIO diere, terwyl SERCA-2a beduidend afgereguleer was reeds vanaf 8 weke, geassosieer met laer Ca2+-ATPase aktiwiteit. PLM uitdrukking het toegeneem en die fosforilering daarvan was verlaag. Op 16 weke was die basale harttempo (225 vs 275 in die kontrole groep, P<0.0001, n=6) en tempo druk produk (21000 vs 30000 in die kontrole groep, P=0.019, n=6) betekenisvol laer in die DIO diere. Funksionele herstel het onveranderd gebly, alhoewel die tyd tot iskemiese kontraktuur toegeneem het in die DIO groep (kontrole: 11.6±0.4 min vs DIO: 16.2±0.5 min, P<0.01, n=6). Toediening van die inhibitor het geen effek op totale GSK-3 uitdrukking gehad nie. Fosforilering van GSK-3 was egter wel beduidend verhoog in die behandelde kontrole diere, terwyl daar geen verskille in die DIO groep was nie. Die fosforilering van GSK-3 het wel geneig na 'n toename in die behandelde DIO diere. Die GSK-3 inhibitor het kontrasterende effekte op hipertrofie gehad: dit het dit omgekeer in die DIO groep, maar veroorsaak in die kontrole diere. Daarmee saam het die inhibitor die uitdrukking van IRS-2 in beide DIO en kontrole diere gestimuleer, maar geen effek op IRS-1 en SERCA-2a uitdrukking en aktiwiteit gehad nie. GSK-3 inhibisie het wel PKB/Akt en PLM fosforilering in die DIO diere verhoog. Gevolgtrekking: Hierdie bevindinge toon dat 'n hoë kalorie dieet, tesame met 'n wanbalans tussen energie inname en verbruiking, lei tot die ontwikkeling van obesiteit en insulien weerstand in manlike Wistar rotte. Die studie het ook getoon dat GSK-3 en sy substraat proteïene wel gedisreguleer is in obesiteit en insulien weerstandigheid. Die verlaagde basale uitdrukking van SERCA-2a mag dalk 'n negatiewe impak hê op kardiale funksie. Behandeling van die diere met 'n GSK-3 inhibitor het getoon dat GSK-3 moontlik nie verantwoordelik is vir die basislyn veranderinge nie. Die afname in IRS-1 en SERCA-2a uitdrukking kan moontlik toegeskryf word aan ander meganismes buiten die effekte van GSK-3. Hierdie studie toon wel dat GSK-3 moontlik 'n rol speel in die regulering van die uitdrukking van IRS-2, maar nie IRS-1 nie. Verhoogde PKB/Akt fosforilering mag dalk bydra tot die inhibisie van GSK-3. Daarmee saam blyk dit dat GSK-3 inhibisie hipertrofie kan omkeer in DIO diere, om dan sodoende op te tree as 'n negatiewe reguleerder van hipertrofie, maar in normale kontrole diere, hipertrofie in die hand werk.
South African Medical Research Council
University of Stellenbosch, Dept. of medical Physiology

The global number of obese people has reached pandemic proportions. High caloric diets and reduced physical exercise are to blame for this growing epidemic. Obesity has a very complex association with several other metabolic disorders, such as insulin resistance (IR), diabetes mellitus type 2 (DMT2) and cardiovascular disease. This puts a huge burden on health care systems world wide and claims many lives. Sutherlandia frutescens is a traditionally used herb, which is known to have anti-diabetic properties. However, the direct mode of action of S. frutescens still remains to be elucidated. The aim of this study was to investigate the developmental stages of high fat diet (HFD)-induced IR, to illuminate the pathogenesis of IR with a focal point on modifications in the lipid metabolism. Furthermore, the effects of S. frutescens as a treatment or prevention drug for IR and associated metabolic changes were examined. Two sets of experiments were conducted on male Wistar rats. In the first experiment rats, one week post weaning received a low fat diet (LFD), high fat diet (HFD) or HFD supplemented with S. frutescens (50mg/kg BW/d). Rats were sacrificed at week 0, 1, 2, 4, 8 and 12 in the feeding regime. In a second experiment rats were fed with a LFD or a HFD for 12 weeks and treated thereafter with S. frutescens (50mg/kg BW/d), metformin (13mg/kg BW/d) or water (control) for 28 days. Rats in the second experiment were sacrificed at week 12 to confirm IR while concurrently run rats were sacrificed after 28 days of treatment. For all the experiments rats were anaesthetized, blood was removed and rats were dissected. Plasma samples were analyzed for insulin, glucose, blood lipid parameters and cytokines. Liver, muscle and adipose tissue were analyzed for glucose uptake, total lipid content, lipid profile and fatty acid profile. It was shown that the intake of HFD caused IR and hyperinsulinaemia. The developmental stages in experiment one confirmed that an increase in plasma free fatty acids preceeded the onset of IR. Plasma and tissue lipid parameters (free fatty acid-, triglyceride- and cholesterol concentrations) showed pathological modifications in the HFD group. An ectopic accumulation of fat was observed in muscle and liver, as well as a change in membrane fatty acid profile. The results for circulating cytokines were somewhat inconclusive. Rats supplemented with S. frutescens did not develop HFD-induced IR (study one) or IR was reversed (study two). S. frutescens treatment also resulted in positive changes in plasma and tissue lipid parameters. In summary, an animal model for HFD-induced IR was established and the detrimental effect of elevated plasma FFA on glucose and lipid metabolism was observed. A novel discovery suggests that the anti-diabetic mode of action of S. frutescens is through modulation of lipid metabolism. It was also established that S. frutescens has the potential to prevent IR in vivo.

The rapid increase of obesity has encouraged the study of the aetiology of eating disorders. Besides genetic, social and metabolic factors, obesity is caused by over-eating and has high rate of relapse to abnormal food-taking habits, which is mainly provoked by food-associated cues. High-palatable food intake shares similarities with the consumption of addictive drugs since it engages brain reward systems and produces comparable behavioural adaptations. However, the mechanism of the transition from normal food consumption to pathological over-eating remains obscure. Animal models are crucial to study the underlying mechanisms and are also excellent tools to facilitate the research of new pharmacological targets and to evaluate the benefit/risk ratio of potential novel treatments. We have focussed our research in the study of novel potential targets for eating disorders using new animal models developed in our laboratory. Firstly, we investigated the effects of the chronic blockade of the endocannabinoid system as anti-obesity treatment in a novel model of diet-induced obese rats and the role of the endocannabinoid system on cognitive processes and metabolism in mice using new behavioural devices. Secondly, we validated new sophisticated behavioural models of relapse and frustration in mice to investigate these key elements in the development of overeating.
L’augment de l’obesitat promou l’estudi de l’etiologia dels trastorns alimentaris. A més dels factors genètics, socials i metabòlics, l’obesitat es deu a la ingesta excessiva i hi ha una elevada recaiguda a hàbits alimentaris anormals per factors ambientals associats al menjar. El consum de menjar d’alta pal•latabilitat s’assembla al de substàncies addictives donat que implica els sistemes cerebrals de recompensa produint adaptacions comportamentals comparables. Tanmateix, el mecanisme de transició d’un consum normal de menjar a un consum patològic excessiu es coneix poc. Els models animals són molt importants per conèixer els mecanismes d’aquests processos patològics i eines excel•lents per trobar noves dianes farmacològiques, així com per avaluar el risc/benefici de possibles nous tractaments. Hem centrat el nostre treball en l’estudi de noves dianes per als trastorns alimentaris emprant nous models animals posats a punt en el nostre laboratori. Primer, hem estudiat els efectes del bloqueig crònic del sistema endocannabinoide com un tractament anti-obesitat en un nou model d’obesitat induïda en rates i la implicació del sistema endocannabinoide en els processos cognitius i metabòlics en ratolins emprant nous sistemes comportamentals. Segon, hem validat nous models de recaiguda i frustració en ratolins per estudiar aquests elements claus en el desenvolupament de l’obesitat.

Obesity has been attributed in a major risk factor for developing and accelerating disease progression in osteoarthritis. To date, there is a lack of clinically proven therapies to halt osteoarthritis, the developments of such therapies are therefore a national as well as an international research priority. This research provides a new overview of the involvement of synovitis in promoting the destruction of synovial joints in obesity-induced osteoarthritis and might therefore by used as a therapeutic strategy for the development of disease-modifying anti-osteoarthritis drugs.

Thesis (MScMedSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Obesity has reached epidemic proportions worldwide and is currently a serious health problem. It is associated with metabolic abnormalities, oxidative stress, hypertension, insulin resistance and an increased disposition for the development of cardiovascular disease. Elucidation of the pathophysiological mechanisms underlying obesity and its relationship with metabolic and cardiovascular diseases is essential for prevention and management of these disorders. Melatonin, the pineal gland hormone, is a powerful antioxidant and has been shown to protect the myocardium against ischaemia/reperfusion (I/R) injury. Long- as well as shortterm melatonin treatment also reversed several of the harmful effects of obesity in an animal model of hyperphagia-induced obesity (DIO). However, its effects on myocardial I/R injury and intracellular signalling in obesity induced by a high fat diet (HFD) are still unknown. Aims of study: (i) To evaluate the ability of a high fat diet (HFD) to induce obesity in rats. Apart from evaluating its effects on the biometric parameters and resistance to ischaemia/reperfusion injury (as indicated by infarct size in regional ischaemia and functional recovery after global ischaemia), special attention will be given on the interplay between adiponectin, AMPK, leptin, and FFA in this model. (ii) To evaluate the effect of daily oral administration of melatonin to rats on the HFD as well as their littermate controls, on the parameters listed above as well as on the development of obesity. In this study melatonin will be administered from the onset of the feeding of the high fat diet. Methods: Male Wistar rats were divided into 4 groups: (i) control rats (receiving normal rat chow) (C); (ii) control rats receiving melatonin (CM); (iii) obese rats (receiving HFD) (HFD); (iv) obese rats receiving melatonin (HM). Animals were kept on the diet for 16 weeks and melatonin treatment (10mg/kg/day, added to the drinking water) started at the onset of the feeding. Following feeding and treatment, the animals were grouped into fasted/ non-fasted of which biometric parameters were recorded and blood collected at the time of sacrifice for metabolic and biochemical assays. Hearts were perfused in the working mode for evaluation of myocardial function and infarct size determination after exposure to 35min regional ischaemia/60min reperfusion. For study of intracellular signaling, hearts were perfused in the working mode, subjected to 20min global ischaemia/10min reperfusion and freeze-clamped for Western blotting. Plasma leptin, adiponectin, free fatty acid, triglycerides, total cholesterol, phospholipids, conjugated dienes and thiobarbituric reactive substances (TBARS) levels were determined. Several kinases were investigated including, the RISK (reperfusion injury salvage kinase) (PKB/Akt and ERK p44/42) and SAFE (survivor activating factor enhancement) (STAT-3) pathways, AMPK and JNK under baseline conditions or following 10 min reperfusion. In addition, expression of UCP-3 and PGC1-α was determined. Results: Significant increases in body weight, visceral fat, blood glucose, insulin, HOMA index and leptin and a reduction in adiponectin levels were observed in the fasted high fat diet (HFD) group when compared with controls (C). Significant increases in free fatty acid and triglyceride levels were also noted the HFD group while other serum lipid parameters, including TBARS, remained unchanged. No differences in functional recovery during reperfusion or infarct size after exposure to 35 min regional ischaemia, as well as functional recovery during reperfusion after 20 min global ischaemia were observed between the control and HFD groups. Baseline and 10 min reperfusion data were similar for the RISK and SAFE pathway kinases for the control vs HFD groups. The HFD also had no effect on the expression and phosphorylation of myocardial AMPK and JNK, as well as on the expression of UCP-3 and PGC1-α, when compared to the controls. Treatment with melatonin significantly reduced body weight, visceral fat, blood glucose, HOMA index and serum leptin levels in HFD treated groups, while having no effect on the lipid profile. Although melatonin significantly reduced infarct size in both control [% of area at risk: 20.59 ± 2.29 (CM) vs 38.08 ± 2.77 (C)] and high-fat diet groups [% of area at risk: 11.43 ± 2.94 (HM) vs 38.06 ± 3.59 (H)], it was without effect on myocardial functional recovery during reperfusion. Melatonin had no effect on the intracellular signaling pathways studied. Conclusions: The HFD proved to be a useful model of diet-induced obesity with a more pronounced impact on biometric and metabolic changes compared to the DIO model. Long-term melatonin treatment successfully prevented the development of metabolic abnormalities associated with the high fat diet and obesity as well as significantly reduced myocardial infarct size. The mechanisms involved in melatonin-induced cardioprotection in obesity have not been fully elucidated in this study and require further investigation. However, the anti-obesogenic and cardioprotective properties of melatonin were very significant indeed and support the suggestion of this hormone as a potential tool in the treatment of obesity and associated cardiovascular complications.
AFRIKAANSE OPSOMMING: Inleiding: Vetsug (obesiteit) het wêreldwyd epidemiese afmetings aangeneem en word tans as ‘n ‘n ernstige gesondheidsprobleem beskou. Vetsug word geassosieer met metaboliese afwykings, oksidatiewe stres, hipertensie, insulienweerstandigheid en is‘n belangrike risikofaktor vir die ontwikkeling van kardiovaskulêre siekte. Ten spyte hiervan, het onlangse studies ‘n gunstige effek van vetsug op die uitkomste van miokardiale infarksie in pasiënte gerapporteer, die sg obesiteitsparadoks. Kennis van die patofisiologiese meganismes onderliggend aan vetsug en die ontstaan van metaboliese afwykinge en hartsiekte is noodsaaklik vir die voorkoming en behandeling van hierdie toestande. Melatonien, die hormoon afgeskei deur die pineaalklier, is ‘n kragtige antioksidant en vry radikaal opruimer. Dit is voorheen aangetoon dat dit die hart teen iskemie/herperfusie (I/H) besering kan beskerm en sommige van die skadelike gevolge van vetsug in diermodelle kan omkeer. Die effek van melatonien op miokardiale I/H besering en intrasellulêre seintransduksie prosesse in vetsug geïduseer deur ‘n hoë vet dieet is egter nog onbekend. Doelstellings: (i) Die ontwikkeling en karakterisering van ‘n nuwe model van vetsug en insulienweerstandigheid geïnduseer deur 'n hoë vet dieet (HVD) en die evaluering van die effek daarvan op miokardiale I/H besering en die gepaardgaande intrasellulêre seintransduksieprosesse; (ii) Bepaling van die effek van daaglikse toediening van melatonien aan rotte op die HVD sowel as aan kontroles op ‘n standard dieet, op die ontwikkeling van dieet-geïnduseerde metaboliese veranderinge, miokardiale infarktgrootte en funksionele herstel na koronêre arterie afbinding, sowel as intrasellulêre seintransduksie. Metodiek: Vier groepe van manlike Wistar rotte is bestudeer: (i) kontrole rotte (op‘n standaard dieet) (K); (ii) kontrole rotte op ‘n standard dieet plus melatonien (KM); (iii) dieetrotte (op‘n HVD); (iv) HVD rotte wat melatonien ontvang (HM). Die HVD en melatonien (10mg/kg/dag in die drinkwater) is vir 16 weke toegedien. Na die periode van behandeling, is die diere in vastende en nie-vastende groepe verdeel, die biometriese parameters genoteer en bloedmonsters vir metaboliese en biochemiese bepalings versamel, tydens verwydering van die harte. Harte is geperfuseer volgens die werkhartmodel vir bepaling van miokardiale funksie en infarktgrootte na blootstelling aan 35min streeksiskemie. Vir evaluering van intrasellulêre seintransduksie, is geperfuseerde werkende rotharte blootgestel aan 15min globale iskemie/10 min herperfusie en gevriesklamp vir latere analises volgens die Western kladtegniek.hart. Serum leptien, adiponektien, vryvetsure, trigliseried, totale cholesterol, fosfolipiede, gekonjugeerde diene en tiobarbituursuur reaktiewe stowwe (TBARS) is bepaal. Met gebruik van Western kladtegniek, is die aktivering en/of uitdrukking van die RISK (PKB/ Akt en ERK p44/42) en SAFE (STAT-3) seintransduksiepaaie, AMPK, JNK, UCP-3 en PGC1-α, onder basislyn toestande of na 10 min herperfusie bestudeer. Resultate:‘n Beduidende toename in liggaamsgewig, visserale vet, die HOMA indeks, insulien en leptien vlakke is in die HVD groep waargeneem vergeleke met die kontrole (K) rotte. Adiponektien vlakke was laer in die HVD groep. Die HVD groep is ook gekenmerk deur ‘n beduidende styging in serum vryvetsuur en trigliseried vlakke, terwyl die ander lipied parameters, insluitende die TBARS vlakke, onveranderd was. Infarktgrootte en funksionele herstel tydens herperfusie na blootstelling aan 35 min streeksiskemie, asook funksionele herstel tydens herperfusie na 20 min globale iskemie het nie verskil tussen harte van die kontrole en HVD rotte nie. Aktivering van PKB/Akt, ERK p44/p42, STAT3, AMPK en JNK by basislyn en na 10 min herperfusie was soortgelyk in die kontrole en HFD groepe. Die HVD het ook geen effek op die uitdrukking van UCP-3 en PGC1-α in vergelyking met die kontrole gehad nie. Behandeling met melatonien het die liggaamsgewig, visserale vet, bloedglukose, HOMA indeks en serum leptien vlakke in die HVD groepe statisties beduidend verlaag, terwyl dit geen invloed op die lipiedprofiel gehad het nie. Melatonien behandeling het die miokardiale infarktgrootte beduidend en tot dieselfde mate verminder in beide kontrole [20.59 ± 2.29 (KM) vs 38.08 ± 2.77% (K)] en HVD groepe [11.43 ± 2.94 (HM) vs 38.06 ± 3.59% (HVD)]. Geen verskille is egter tussen die funksionele herstel gedurende herperfusie van die behandelde en onbehandelde kontrole en HVD groepe waargeneem nie. Melatonien het ook geen uitwerking op die intrasellulêre seintransduksiepaaie gehad nie. Gevolgtrekkings: Die resultate het getoon dat die HFD 'n goeie model van dieetgeïnduseerde vetsug en insulien weerstandigheid ontlok, met 'n meer uitgesproke impak op biometriese en metaboliese veranderinge as die voorheen gebruikte hoë-sukrose dieet. Langtermyn melatonien- behandeling het die ontwikkeling van metaboliese abnormaliteite geassosieer met die HVD, voorkom, asook miokardiale infarktgrootte na koronêre afbinding beduidend verminder. Die meganismes betrokke in melatonien-geïnduseerde miokardiale beskerming moet egter in meer detail ondersoek word. Die resultate verkry steun die voorstel dat melatonientoediening voordelig sal wees in die behandeling van vetsug en sy kardiovaskulêre komplikasies.

RESUMO A acção hipoglicemiante da insulina é máxima no estado pós-prandial e depende da substância hepática sensibilizadora da insulina (HISS). Esta dissertação visa o estudo do mecanismo de acção da insulina no estado pós-prandial e em particular da via dependente da HISS, em modelos animais fisiológicos e patológicos (obesidade e diabetes mellitus tipo 2). Avaliaram-se diferentes tipos de refeição quanto ao seu efeito potenciador da acção da insulina, em ratos Sprague-Dawley (modelo fisiológico). A administração intragástrica de glícidos não afecta a acção da insulina, mas a refeição mista (lípidos, glícidos e proteínas), promove a sensibilização para a acção da insulina, através de um processo que parece ser iniciado no intestino e envolve a activação da via da HISS. Nos estudos de obesidade, o primeiro modelo utilizado foi o rato alimentado com dieta hiperlipídica (HFD), no qual se observou uma insulinorresistência pós-prandial devida quase exclusivamente à perda de acção da HISS, que se correlaciona com a adiposidade (corporal e abdominal) e parece ser devida à diminuição da sua síntese. O segundo modelo de obesidade usado foi o rato Zucker obeso (OZR), modelo genético que apresenta uma diminuição idêntica de ambas as componentes de acção da insulina (dependente e independente da HISS). A alteração na via da HISS parece localizar-se a jusante da sua síntese, sugerindo que um ou vários pontos comuns entre as vias de sinalização intracelular da HISS e da insulina per se estão alterados, resultando num diminuto aporte de glucose. No OZR, a acção da HISS não se altera com a idade, apresentando-se baixa também às 52 semanas de idade. Em ratos não obesos (LZR), a acção da HISS diminui entre as 9 e 52 semanas, sendo acompanhada por um decréscimo menos acentuado, embora significativo, da acção da insulina per se. A diminuição da acção da HISS com a idade parece ser a principal causa de insulinorresistência pós-prandial em LZR velhos, não se agravando no OZR. No modelo de diabetes tipo 2 estudado, o rato Zucker diabético (ZDF), também ambas as componentes de acção da insulina estavam diminuídas. No entanto, a alimentação com ração Purina, ligeiramente mais energética e lipídica do que a ração standard, agrava a disfunção da via da HISS nestes animais, sugerindo que a sensibilidade à insulina em ratos ZDF é muito susceptível a factores nutricionais. A via da HISS é essencial para potenciar a acção da insulina do estado de jejum para o pós-prandial e a sua disfunção é em grande medida responsável pela insulinorresistência observada nos modelos animais de obesidade e diabetes estudados. xix SUMMARY Hypoglycemic insulin action is maximal in the postprandial state and depends on the hepatic insulin sensitizing substance (HISS). The present thesis focus on the postprandial insulin action and, in particular, on the HISS-dependent pathway, both in physiological and pathological (obesity and type 2 diabetes mellitus) animal models. Different meals were tested in Sprague-Dawley rats (physiological model) for their capacity to potentiate insulin action. It was observed that intragastric administration of either glucose or sucrose does not affect insulin sensitivity, unlike the mixed meal, composed of lipids carbohydrates and proteins, which significantly potentiated insulin action through a process that seems to be initiated at the intestine and involves activation of the HISS pathway. For the obesity studies, the first of the two obesity models used was the high fat-fed rat (HFD), in which the postprandial insulin resistance was almost exclusively caused by the decrease of HISS action, probably due to the impairment of HISS synthesis. This impairment correlates with both corporal and abdominal adiposity. The second obesity model used was the obese Zucker rat (OZR), a genetic model, which presented a similar impairment of both components of insulin action (HISSdependent and –independent). The modification in HISS pathway in OZR seems to be located downstream from HISS synthesis, that is, at its site of action – the skeletal muscle -, suggesting that one or several points common to both HISS and insulin per se signaling cascades are defective, resulting in a decreased glucose uptake. In OZR, HISS action does not decrease with age and is also low at 52 weeks of age. In non-obese rats (LZR), HISS action decreases from 9 to 52 weeks and it is accompanied by a lower, although significant, impairment of insulin action per se. HISS action impairment with aging seems to be the major cause of insulin resistance in old LZR, whereas insulin resistance is not aggravated in aging OZR. In the type 2 diabetes model, the diabetic Zucker rat (ZDF), both components of insulin action were also equally impaired. However, feeding the animals with Purina rat chow, which is slightly more caloric and more lipidic, induces additional HISS deterioration when compared with the standard lab diet, suggesting that insulin sensitivity in ZDF is very susceptible to nutritional factors. In conclusion, HISS pathway is essential to potentiate insulin action from the fasted to the fed state and its dysfunction is highly responsible for the insulin resistance observed in the obesity and diabetes animal models studied.

Background and objectives: Nonalcoholic steatohepatitis (NASH), which is characterized by concurrent existence of hepatic steatosis and predominantly lobular necroinflammation, represents the more advanced stage in the spectrum of nonalcoholic fatty liver disease (NAFLD). NASH exhibits dramatically increased risk of progression to end-stage liver diseases than simple steatosis. Therefore, the progression of hepatic steatosis to steatohepatitis is the crucial step in the development of obesity-related NASH. Toll like receptor 4 (TLR4), a master regulator of innate immunity, is the principal receptor for endotoxin, which is a central mediator of liver inflammation associated with both alcoholic and nonalcoholic liver disease. However, due to a lack of suitable animal models which fully recapitulate the natural history of obesity-induced NASH, the precise pathophysiological function of TLR4 signaling in the development of this disease remains poorly understood. The objective of this study is to investigate the role of TLR4 in mediating inflammatory responses in obesity-induced NASH using both in vivo and ex vivo approaches, and to unveil cellular and molecular mechanisms responsible for TLR4 actions. Key findings: 1. To address the role of TLR4 in the pathogenesis of NASH, we crossed ApoEdeficient mice (ApoE-/-) with TLR4 mutant mice (TLR4-/-) to generate ApoE-/- /TLR4 wild type mice (ApoE-/-/TLR4-WT) and ApoE-/-/TLR4-/- mice. Noticeably, when fed with high fat high cholesterol (HFHC) diet, ApoE-/-/TLR4-WT mice developed the typical pathology of NASH (hepatic steatosis, lobular inflammation, and hepatocyte ballooning) in the context of obesity and metabolic syndrome, suggesting HFHC-fed ApoE-/- mice as a suitable animal model for NASH. 2. TLR4 inactivation protected ApoE-/- mice against HFHC diet-induced liver injury, as indicated by a significant improvement in liver histology, a a marked reduction in serum ALT activity, a dramatic repression of inflammatory infiltrates, as well as an obvious decrease in hepatic production of pro-inflammatory cytokines. 3. In ApoE-/-/TLR4-WT mice, TLR4 expression was selectively elevated in Kupffer cells in response to HFHC diet feeding. 4. The activation of XBP1, a transcription factor involved in endoplasmic reticulum stress, was markedly elevated in liver of ApoE-/-/TLR4-WT mice fed with HFHC diet, whereas this change was abrogated in HFHC diet-fed ApoE-/-/TLR4-/- mice. 5. In rat primary Kupffer cells, treatment with anti-oxidants blocked endotoxininduced activation of XBP1 and NF-κB, leading to decreased cytokine production. In addition, siRNA-mediated knockdown of XBP1 inhibited NF-κB activation and cytokine production resulted from the treatment with the TLR4 agonist LPS. 6. In ApoE-/-/TLR4-WT mice, adenovirus-mediated expression of dominant negative XBP1 had no obvious effect on HFHC diet-induced hepatic steatosis and ROS production, but markedly decreased lobular inflammation, NF-κB activation, cytokine production in the liver and significantly reduced serum levels of ALT. Conclusions: These findings support the role of TLR4 in Kupffer cells as a key player in mediating the progression of simple steatosis to NASH, by inducing ROS-dependent activation of XBP1. In light of the obligatory role of XBP1 in TLR4-induced liver inflammation and injury, therapeutic interventions that inhibit TLR4/XBP1 activation may represent a promising strategy for treatment of NASH.
published_or_final_version
Medicine
Doctoral
Doctor of Philosophy

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009.
Insulin stimulates the production of nitric oxide (NO) in endothelial cells and cardiac myocytes by a signalling pathway that involves the insulin receptor substrate (IRS)-1, phosphatidylinositol-3-kinase and protein kinase B (PKB/Akt). Physiological concentrations of NO play an important part in maintaining normal vascular function. It has been suggested that nitric oxide synthase (NOS) activity and NO production are chronically impaired in diabetes mellitus by an unknown mechanism. The reninangiotensin system and subsequent production of angiotensin II (Ang II) are elevated in obesity and diabetes while antagonism of the AT1 receptor with Losartan has beneficial effects in patients with insulin resistance and type II diabetes. Aims: We therefore aimed to investigate (i) the effect of Ang II on myocardial insulin signalling with regards to key proteins (IRS-1, PKB/Akt, eNOS and p38 MAPK) in correlation with NO production, (ii) the effect of Losartan on these parameters. Methods: Hyperphagia-induced obese, insulin resistant rats (DIO=diet supplemented with sucrose and condensed milk) were compared to age-matched controls. Half the animals were treated with 10mg/kg Losartan per day for 1 week. Isolated hearts were perfused with or without 0.03 μIU/mL insulin for 15 min. Blood glucose, bodyweight, intraperitoneal fat and plasma insulin and Ang II were recorded. Proteins of interest and their phosphorylation were determined by Western blotting. NO production was flow cytometrically analyzed. ANOVA followed by the Bonferroni correction was used with a p< 0.05 considered significant. Results: DIO animals had significant elevated bodyweight, blood glucose, plasma insulin and Ang II levels. Our data showed that the hearts from the DIO animals are insulin resistant, ultimately reflected by the attenuated activation of the key proteins (IRS-1, PKB/Akt and eNOS) involved in insulin signalling as well as NO production. AT1 receptor antagonism improved NO production in isolated adult ventricular myocytes from DIO animals while concurrently enhancing expression of eNOS, PKB/Akt and p38 MAPK. In contrast, NO production as well as expression of eNOS and PKB/Akt was attenuated in control animals after Losartan treatment. Conclusion: These results suggested that Ang II via AT1 or AT2 receptors, modulates protein expression of both PKB/Akt and eNOS. This encouraged us to investigate the involvement of AT2 receptors in the observed changes. To investigate this we needed to establish a culture of neonatal rat cardiac myocytes treated with raised fatty acids and Ang II. If similar changes were induced as observed in the hearts of DIO animals, the involvement of the AT1 and AT2 receptors could be investigated using specific antagonists against these receptors. Primary cultured ventricular myocytes were isolated from 1-3 day old Wistar rat pups. They were cultured for 48 hours before the addition of palmitate and oleate at a concentration of 0.25 mM each and were treated with or without the fatty acids for a period of 4 days. After 18 hours of serum starvation, cells were stimulated with or without 10 nM insulin for 15 minutes. The effect of fatty acid treatment on cell viability and glucose uptake were assessed by trypan blue and propidium iodide staining and 2-deoxy-D-3[H] glucose uptake respectively. Protein levels and phosphorylation of key proteins (PKB/Akt, PTEN and p38 MAPK) in insulin signalling was determined by Western blotting. 0.25 mM Fatty acids did not result in the loss of cell viability. Contrary to expectation, fatty acid treatment led to enhanced basal glucose uptake but lower Glut 1 protein expression. Basal protein expression of PPARα was, however, upregulated as was the expression of the phosphatase, PTEN. The latter could explain the lower PKB/Akt phosphorylation also documented. From these results we conclude that neonatal cardiac myocytes, cultured in the presence of elevated fatty acids, did not respond in a similar manner as the intact hearts of our animals and further modifications of the system might be needed before it can be utilized as initially planned.

Durante a vida intrauterina, o desenvolvimento do embrião e do feto é suscetível a mudanças ambientais que podem alterar o fenótipo do indivíduo na vida pós-natal. Eventos que ocorrem durante períodos críticos de rápida divisão celular, nos quais são formados os diversos órgãos e tecidos, podem alterar a estrutura e função de sistemas orgânicos gerando consequências precoces (baixo peso ao nascimento) e tardias (doenças na vida adulta). Os protocolos experimentais da maior parte dos estudos sobre a programação fetal reduz o tamanho da ninhada logo após o nascimento. Essa abordagem dificulta a interpretação e reprodutibilidade dos resultados observados. O objetivo deste estudo foi determinar se a pressão sanguínea, o metabolismo de carboidratos e gasto energético em proles adultas é influenciado pelo tamanho da ninhada. Ratas Wistar foram alimentadas com ração padrão ad libitum e foram acasaladas com ratos machos com 12 semanas de idade. Após o nascimento, a prole foi dividida em três grupos: tamanho da ninhada sem redução (Gc), proles reduzido a oito filhotes (G8) e proles reduzidos a quatro filhotes (G4). Ao fim de 12 semanas de idade, o peso corporal, pressão arterial, consumo de ração, glicemia, insulina, colesterol e triacilgliceróis, massa de tecido adiposo marrom, índice de adiposidade, massa renal e cardíaca foram determinados. O peso corporal, índice de adiposidade, glicemia, nível de insulina e índice HOMA foram maiores em machos e fêmeas no grupo G4 do que nos grupos G8 e Gc. No entanto, o consumo de ração foi menor no grupo G4. A pressão arterial foi maior no grupo Gc em machos e fêmeas. Em resumo, a redução do tamanho da ninhada está relacionada com a obesidade, resistência à insulina e possíveis alterações no gasto energético na prole adulta
During intrauterine life, the developing fetus is susceptible to environmental changes that can alter the phenotype of the individual in postnatal life. This phenomenon is called fetal programming. Events that occur during critical periods of rapid cell division may alter the structure and function of organ systems, resulting in consequences both early (low birth weight) and late (diseases in adulthood) in life. The experimental protocols of most of the studies on fetal programming involve a reduction in litter size soon after birth. This approach hampers the interpretation and reproducibility of the observed results. The purpose of this investigation was to determine if blood pressure, carbohydrate metabolism and energy expenditure in adult offspring are influenced by litter size. Female Wistar rats were fed standard rat chow ad libitum and were mated with male rats at 12 weeks of age. After birth, the offspring were divided into three groups: unchanged litter size (GU), culled to eight neonates (G8) and culled to four neonates (G4). At 12 weeks of age, the body weight; blood pressure; food intake; glucose, insulin, cholesterol and triacylglycerol levels; brown adipose tissue mass; adiposity index; renal mass; and cardiac mass were determined. The body weight, adiposity index, glucose level, insulin level and HOMA index were higher in males and females in the G4 group than in the G8 and GU groups. However, food consumption was lower in G4 males. The blood pressure was higher in the GU group. In summary, a small litter size is related to obesity, possible alterations in energy expenditure and insulin resistance in adult offspring

Thesis (MScMedSc)--University of Stellenbosch, 2011.
ENGLISH ABSTRACT: Introduction: A westernized diet rich in saturated fats and sugars, together with a sedentary lifestyle, has contributed to the dramatic increase in obesity during the last decade (Zimmett et al, 2001; Wild et al, 2004). Obesity is associated with dyslipidemia and insulin resistance which are major risk factors for the development of type 2 diabetes (T2D) (Zimmet et al, 2001, Kahn et al, 2006; Schröder et al, 2007). High-fat feeding in rodents induces symptoms similar to the human metabolic syndrome without progression to T2D (Woods et al, 2002; Weir and Bonner-Weir, 2007). The addition of fructose to a high-fat diet exacerbates the insulin resistance and leads to impaired pancreatic function of insulin secretion and glucose intolerance (Basciano et al, 2005; Stanhope et al, 2009). Aims: The aim of this study was to establish the effect of a high-fat and sucrose/fructose diet on glucose metabolism, the development of insulin resistance and β-cell dynamics. Methods: Weanling Wistar rats were randomized into two study groups; study one over an experimental period for three months and study two for twelve months. Each study consisted of a control group that received standard rat chow and water; and two experimental groups receiving either a high-fat diet and water (HFD) or a café diet consisting of HFD with the addition of 15% sucrose/fructose (CFD). Fasting glucose and insulin concentrations, intravenous glucose tolerance test (IVGTT), glucose stimulated insulin secretion rates and 2-deoxy-[3H]-D-glucose uptake in muscle, liver and fat were measured. The pancreata were harvested for immunohistochemical labeling of β-cells (insulin), α-cells (glucagon), GLUT2 (glucose transport) and MIB5 (proliferation). Samples of the pancreata were also collected for electron microscopy. Results and discussion: Feeding Wistar rats a CFD induced obesity, insulin resistance and glucose intolerance. By twelve months the rats had an impaired glucose response with increased IVGTT peak values, area under the curve (AUC) values and glucose clearance rates. Concomitantly, the glucose stimulated insulin secretion rate (GS-ISR) was attenuated. Stimulated glucose disposal as measured by 2-deoxy-[3H]-D-glucose uptake was reduced in muscle and adipose tissue at three months. By twelve months, due to the age of the rats, stimulated glucose uptake declined compared to three months with no difference between groups. After three months the diets had no observable effect on the islets using light microscopy. However, by twelve months morphological changes were observed in both the HFD and CFD groups. In the HFD group large hypertrophied irregular islets with fibrous changes were observed. In the CFD group these morphological changes were more prominent with fibrous segregation and disruption of the normal endocrine arrangement. In addition, the presence of inflammatory cells within the affected islets is consistent with T2D. Conclusion: High-fat diet fed to Wistar rats induced obesity, abdominal adiposity and insulin resistance. The addition of sucrose/fructose to a high-fat diet exacerbated the insulin resistance and resulted in glucose intolerance and mild hyperglycemia. Morphological changes in the large islets were observed which are consistent with the development of T2D.
AFRIKAANSE OPSOMMING: Inleiding: ‘n Verwesterde dieët, ryk aan versadigde vette en suikers tesame met 'n passiewe lewenstyl, het bygedra tot die dramatiese verhoging in vetsug gedurende die laaste dekade (Zimmett et al, 2001; Wild et al, 2004). Vetsug word met dislipidemie en insulienweerstandigheid geassosieer wat hoof risikofaktore is vir die ontwikkeling van tipe 2 diabetes (T2D) (Zimmet et al, 2001; Kahn et al, 2006; Schröder et al, 2007). Hoë-vet voeding in knaagdiere induseer simptome soortgelyk aan menslike metaboliese sindroom sonder die ontwikkeling van T2D (Woods et al, 2002; Weir and Bonner-Weir, 2007). Die byvoeging van fruktose tot 'n hoë-vet dieët vererger insulienweerstandigheid en lei tot verswakte pankreas funksie, insuliensekresie en glukoseintoleransie (Basciano et al, 2005; Stanhope et al, 2009). Doelwitte: Die doelwitte van die studie was om die effek van hoë-vet en sukrose/fruktose voeding op glukosemetabolisme, die ontwikkeling van insulienweerstandigheid en β-sel dinamika te bepaal. Metodes: Gespeende Wistar rotte was in twee groepe gerandomiseer; studie een oor ʼn tydperk van drie maande en studie twee oor ʼn tydperk van twaalf maande onderskeidelik. Elke studie het 'n kontrole groep met standaard rot kos en water (control); en twee experimentele diëte wat of ʼn hoë-vet dieët en water (HFD) of 'n kafeedieët groep wat die HFD met die byvoeging van 15% sukrose/fruktose in hul drink water (CFD) ontvang. Fastende glukose en insulien, binneaarse glukose toleransie toets (IVGTT), glukose gestimuleerde insulien sekresie tempo en 2-deoxi-[3H]-D-glukose opname in spier, lewer en vet is gebruik om die effek van die dieët op glukosemetabolisme te bepaal. Die pankreata is uitgehaal vir immunohistochemiese identifisering van β-selle (insulien), α-selle (glukagoon), GLUT2 (glukose transport) en MIB5 (proliferasie). Monsters van die pankreata was ook vir elektronmikroskopie versamel. Resultate en bespreking: Voeding van ʼn CFD aan Wistar rotte induseer vetsug, insulienweerstandigheid en glukose-intoleransie Teen twaalf maande toon die rotte 'n verswakte respons tot glukose met verhoogde IVGTT piekwaardes, AUC waardes en glukose opruimingswaardes. Terselfdetyd is die glukose gestimuleerde insuliensekresie tempo (GS-ISR) ook verswak. Gestimuleerde glukose opruiming, soos deur 2-deoxi-[3H]-D-glukose opname bepaal, was verlaag in spier en vetweefsel teen drie maande. Teen twaalf maande, weens die ouderdom van die rotte, is die gestimuleerde glukose opname verlaag in vergelyking met drie maande sonder 'n verskil tussen groepe. Na drie maande kon geen sigbare morfologiese verskille met ligmikroskopie tussen die diëte waargeneem word nie. Teen twaalf maande is morfologiese verskille waargeneem in beide die HFD en die CFD groepe. In die HFD groep is groot hipertrofiese onreëlmatige eilande met fibrotiese verandering waargeneem. In die CFD groep was die morfologiese verandering meer gevorder met fibrotiese onderverdeling en ontwrigting van die normale endokriene rangskikking. Die teenwoordigheid van inflammatoriese selle in die geaffekteerde eilande is verenigbaar met T2D. Afleiding: Die voer van 'n hoë-vet dieët aan Wistar rotte veroorsaak vetsug, abdominale adipositeit en insulienweerstandigheid. Die byvoeging van sukrose/ fruktose tot die hoë-vet dieët vererger die insulienweerstandigheid en veroorsaak glukoseintoleransie en matige hiperglukemie. Morfologiese veranderings in die groter eilande was verenigbaar met T2D.

A síndrome metabólica (SM) é uma condição clínica que agrupa uma variedade de morbidades, como hiperglicemia, pressão arterial elevada, dislipidemia aterogênica e obesidade (particularmente na região abdominal). Nessa conjuntura, os principais tecidos-alvo da ação da insulina sofrem alterações metabólicas que aumentam o risco de ocorrência de doenças cardiovasculares e diabetes tipo 2. As alterações teciduais observadas são caracterizadas por infiltrados de células do sistema imune, especialmente macrófagos. Citocinas pró-inflamatórias, como TNF-α, são liberadas e alcançam a corrente sanguínea, promovendo nesses indivíduos um estado de inflamação crônica e sistêmica. O tecido adiposo intra-abdominal parece ser de particular importância no estabelecimento desse quadro inflamatório, e estratégias direcionadas no sentido de modular os processos inflamatórios nesse tecido podem atenuar as consequências da SM. Os reconhecidos benefícios da terapia com luz de baixa potência em condições inflamatórias nos permitem supor que essa poderia ser uma proposta terapêutica para a SM. Sendo esse o nosso foco de estudo, camundongos adultos, machos, das linhagens C57BL/6 e BALB/c receberam dieta hiperlipídica durante 8 semanas para indução do quadro de SM. Os animais foram então irradiados sobre a superfície abdominal no decorrer de 21 dias, usando um LED (λ = 850 nm, 6 sessões, 300 s por sessão, potência = 60 mW, fluência = 6 J/cm², taxa de fluência = 19 mW/cm²). Antes e durante o tratamento, amostras de sague foram coletadas para quantificação de glicose, colesterol total e triglicérides plasmáticos. Considerando os parâmetros de irradiação adotados, a terapia com luz de baixa potência não se mostrou efetiva para alterar massa corporal, glicemia, colesterol total e triglicérides de camundongos alimentados com dieta hiperlipídica.
Metabolic syndrome comprises a constellation of morbidities such as insulin resistance, hyperinsulinemia, atherogenic dyslipidemia, dysglycemia and obesity (especially abdominal). Metabolic alterations are observed in major insulin target organs, increasing the risk of cardiovascular diseases, type-2 diabetes and therefore mortality. Tissue alterations are characterized by immune cells infiltrates (especially activated macrophages). Released inflammatory mediators such as TNF-α induce chronic inflammation in subjects with metabolic syndrome, since inflammatory pathways are activated in the neighboring cells. The intra-abdominal adipose tissue appears to be of particular importance in the onset of the inflammatory state, and strategies contributing to modulate the inflammatory process within this adipose tissue can mitigate the metabolic syndrome consequences. Considering the low level light therapy (LLLT) recognized benefits in inflammatory conditions, we hypothesized this therapeutic approach could promote positive effects in modulating the inflammatory state of metabolic syndrome. That being the scope of this study, male C57BL/6 AND BALB/c mice were submitted to a high-fat/high-fructose diet among 8 weeks to induce metabolic syndrome. Animals were then irradiated on the abdominal region during 21 days using an 850 nm LED (6 sessions, 300 seconds per session, 60 mW output power, ~6 J/cm² fluence, ~19 mW/cm² fluence rate). Before and during treatment, blood was sampled either from the retro-orbital plexus or from tail puncture for glucose, total cholesterol and triglycerides analysis. Our results indicate no alterations on these metabolic parameters after LLLT.

Colon cancer and obesity are two significant and related pathological states with multiple etiological factors. In this dissertation, it was hypothesized that tumor growth is accelerated in the altered state of obesity due to their resistance towards tumor necrosis factor-alpha (TNF-alpha) mediated cytotoxicity. Physiologically elevated TNF-alpha in an obese state induces increased nuclear transcription factor-kB (NF-kB) activity, known to transcribe genes crucial to cell survival. Insulin resistance, oxidative stress, and a pro-inflammatory environment are few of the biological consequences of TNF-alpha and NF-kB pathway activation, and further contribute to disease progression.

Three major studies were conducted to investigate phenotypical changes in obesity associated tumors. Firstly, characteristics of the TNF-alpha resistant phenotype were preliminarily assessed by evaluating the effects of exogenous TNF-alpha treatment to HT-29 cells. Elevated levels of NF-kB in response to exogenous TNF-alpha gave an indication that this pathway is critical for cell survival. Furthermore, upregulation of TNF-alpha receptor 2 (TNFR2) suggested another strategy by which the cells were utilizing exogenous TNF-alpha for a survival advantage. Inhibition of NF-kB via St. John?s Wort treatment demonstrated that HT-29 cells may be sensitized towards TNF-alpha mediated cytotoxicity.

Zucker obese (Zk-Ob), Zucker lean (Zk-Ln), and Sprague Dawley (SD) animal models were used to assess tumor phenotype in vivo. Remarkable physiological differences between genotypes were observed. Zk-Ob rats had significantly higher body and organ weights as well as plasma TNF- alpha, insulin, leptin, and oxidative markers than Zk-Ln and SD animals. Tumor incidence and multiplicity were also notably higher in Zk-Ob rats. Protein analyses demonstrated increased levels of TNF-alpha, TNFR2, NF-kB, IkB kinase beta (IKKbeta), insulin receptor (IR), insulin like growth factor-I-receptor (IGF-IR), and mitogen activated protein kinase (MAPK) in Zk-Ob tumors than Zk-Ln counterparts. In all groups, tumors generally had higher protein expression than surrounding, normal appearing colonic mucosa. It is well known that these molecules are involved in signaling pathways that influence and co-operate with each other in rendering growth autonomy to tumor tissue.

A higher number of lesions in the distal than proximal colon in Zk-Ob rats was observed, supporting the emerging concept that genotype/physiological state of the host affects development and distribution of tumors. Thus, a third study was conducted to explore differences between distal and proximal tumor phenotype. Results demonstrated that expression of TNFR2, NF-kB, IR, IGF-IR, and MAPK p44 were significantly higher in distal than proximal tumors. This observation suggested that development of tumors in different regions of the colon varied under the same physiological conditions. Moreover, phenotype of distal tumors appeared to be upregulating survival pathways in comparison to proximal lesions, possibly explaining the higher tumor incidence in the distal colon.

Research documented in this thesis supported the hypothesis that the physiological status of the host intricately affects tumor phenotype. In particular, the TNF-alpha resistant phenotype was most prominent in Zk-Ob tumors, and appeared to be associated with upregulation of multiple signaling pathways cooperating towards tumorigenesis.

Diante da ausência de dados prévios, foi pretendido estabelecer uma correlação entre as possíveis modificações metabólicas e moleculares na resistência insulínica e inflamação em modelos animais de obesidade, induzida pela dieta, recebendo a suplementação de ômega 3/ALA. Além disto, pretendeu-se obter dados que permitam uma melhor elucidação dos mecanismos envolvidos na resistência insulínica neste modelo e um possível efeito preventivo da administração de ALA sobre este processo, podendo, desta forma, auxiliar no desenvolvimento de novas terapias. O objetivo do estudo foi demonstrar que a suplementação ALA reduz a resistência à insulina e a inflamação em modelo animal de obesidade. Foram divididos 40 camundongos machos (C57/BL6) em 4 grupos: controle (C), controle + ômega 3/ALA (CW), obesos (O) e obesas + ômega 3/ALA (OW). Por um período de oito semanas, os grupos O e OW receberam uma dieta hiperlipídica com 60% de lipídeos, enquanto o C e CW receberam ração padrão. Depois, os grupos CW e OW receberam suplementação de 10% de ômega 3/ALA liofilizado, extraído de semente de linhaça, diariamente, por mais 8 semanas. Observando os resultados, todos os grupos de animais tiveram o mesmo ganho de peso, assim como consumo alimentar, eficiência enérgica e eficiência alimentar. O uso do ALA diminuiu o peso da gordura subcutânea no grupo OW comparado ao O e manteve os valores semelhantes entre os outros grupos, no entanto, as comparações do peso do fígado, gordura epididimal, pâncreas e músculo gastrocnemio, foram semelhantes entre todos os grupos. Observou-se uma diminuição na resistência insulínica nos animais OW comparado ao O pelo teste de IPGTT, sendo a área sob a curva de glicose similar entre C e CW. Além disso, os níveis totais de gordura no fígado foram significativamente menores no CW e OW, em comparação com C e O, estes resultados são reforçados pela análise dos tecidos em avaliação histopatológica. Os níveis séricos de glicemia e insulina, ao final do estudo, mostraram uma redução importante em OW comparado a CW, porém, não foi observado diferenças entre os animais obesos suplementados ou não com ômega-3/ALA. Entretanto, quanto à avaliação de resistência insulínica pelo cálculo do HOMA IR, este mostrou-se menor comprando-se OW com O. Os níveis séricos de colesterol total e HDL foram maiores no grupo CW comparados ao C, sendo que os níveis de colesterol total foram menores, também, no grupo OW comparados ao O. No entanto, os valores de triglicérides séricos foram semelhantes entre todos os grupos, assim como valores de triglicérides hepáticos. Colesterol total hepático teve um aumento significativo entre OW e O. As dosagens séricas de IL1, IL6 e MCP1 mostraram uma redução importante em O comparadas com OW. Já quanto a IL17 e TNF, ambas foram equivalentes, nas comparações entre diferentes grupos. A avaliação da ativação do estresse do retículo endoplasmático mostrou que a proteína BIP teve um aumento importante tanto na comparação entre C e CW e também entre O e OW. A também chaperona HSP70 mostrou aumento significativo em ambas as comparações entre grupos, tanto em C e CW quanto em O e OW. GRP94 e IRE1 tiveram resultados semelhantes, sem diferenças entre os grupos, assim como a DAPK1. CHOP teve diminuição importante comparando-se C e CW, e O e OW. Em contrapartida, XBP1 teve diminuição importante na comparação entre os grupos C e CW. Por fim, a suplementação de ômega 3/ALA mostrou ser eficaz na prevenção de esteatose hepática, redução de resistência insulínica, diminuição do processo inflamatório, e redução da ativação do estresse do retículo endoplasmático em tecido hepático.
Given the absence of previous data, it is intended to establish a possible correlation between metabolic and molecular changes in insulin resistance and inflammation in animals receiving supplementation of -linoleic acid (ALA) found in flaxseeds oil. Furthermore, we intend to obtain data for an elucidation of the mechanisms involved in insulin resistance and preventive effect of administration of ALA on this process and may thus aid the development of new therapies. The aim of study was prove the ALA supplementation reduces insulin resistance and inflammation in an obesity animal model. 40 male mice (C57/BL6) were divided into 4 groups: control (C), control + omega 3/ALA (CW), obese (O) + omega 3/ALA and obese (OW). For a period of eight weeks, the groups O and OW received a high-fat diet with 60% fat, while the C and CW received regular chow. Then, the CW and OW groups were supplemented with 10% omega 3/ALA lyophilized, extracted from flaxseed daily, for another 8 weeks. Analyzing the results, all groups of animals had the same weight gain, and food consumption, food efficiency and energetic efficiency. The use of ALA decreased subcutaneous fat weight in group OW compared to O and remained similar values between the other groups, however, comparisons of the size of liver tissue, epididymal fat, pancreas and gastrocnemius muscle were similar among all groups. There was a decrease in insulin resistance in animals OW compared to O by GTT test, with an area under the curve of glucose similar between C and CW. Moreover, the total liver fat levels were significantly lower in CW and OW compared to C and O, these results are reinforced by histopathological tissues analysis. Serum glucose and insulin levels, at the end of the study, showed a significant reduction in OW compared to CW, however, no differences were observed between the obese animals supplemented or not with omega 3/ALA. Meanwhile, about the valuation of insulin resistance calculating by HOMA IR, this was lower in OW compared with O. Serum total cholesterol and HDL levels were higher in CW compared to the group C, and total cholesterol levels were also lower in the OW group compared to O. However, the serum levels of triglyceride were similar among all groups as well as hepatic triglycerides values. Hepatic total cholesterol increased significantly between OW and O. Serum levels of IL1, IL6 and MCP1 showed a significant reduction in O compared to OW. As for IL17 and TNF were similar for both comparisons between different groups. The evaluation of endoplasmic reticulum stress activation showed the BIP protein had a significant increase in the comparison between CW and C and also between OW and O. The also chaperone HSP70 showed a significant increase in both comparisons between groups, both in C and CW as for O and OW. GRP94 and IRE1 had similar results, with no differences between groups, as well as DAPK1. CHOP had significant decrease comparing CW and C, and O and OW. In contrast, XBP1 had significant reduction in the comparison between groups C and CW. Finally, omega 3/ALA supplementation showed to be effective in preventing hepatic esteatose, the reduction of insulin resistance, inflammation decrease, and reduction of activation of endoplasmic reticulum stress in liver tissue.

A obesidade é uma doença que vem aumentando de maneira assustadora em todo o mundo e está implicada em várias condições patológicas, como resistência à insulina, diabetes tipo 2, dislipidemia, esteatose hepática, hipertensão e risco de doenças cardiovasculares. Evidências demonstram que a obesidade é um estado de estresse oxidativo crônico que está associado às alterações metabólicas e fisiológicas presentes no organismo humano que incidem no aparecimento das patologias citadas. Para combater o excesso de espécies reativas geradas, os organismos possuem um complexo sistema de defesa que inclui mecanismos enzimáticos e não enzimáticos de desintoxicação. O presente trabalho utilizou camundongos SWISS alimentados com dieta hiperlipídica, modelo experimental de obesidade, tratados com CAPE, composto isolado da própolis descrito como tendo várias atividades biológicas, nas doses de 13 e 30 mg/kg de peso vivo e dois tempos de tratamento, 15 e 22 dias. Neste trabalho, as respostas das enzimas superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GSH-Px) e glutationa redutase (GR) foram analisadas nos tecidos hepático e adiposo. Além disso, outros parâmetros importantes foram determinados, como a quantificação da peroxidação lipídica, o conteúdo de peróxido de hidrogênio (H2O2), a atividade da enzima hepática -GT e o controle do ganho de peso, peso relativo do fígado e da gordura (%). Os resultados mostraram que o CAPE não interferiu no ganho de peso dos animais. A dieta hiperlipídica e o tratamento com o CAPE não levaram a alterações significativas nos níveis séricos da enzima hepática, -GT. Os resultados obtidos na resposta ao estresse oxidativo estão de acordo com os encontrados na literatura que têm o CAPE como um potente antioxidante, sendo capaz de melhorar a atividade de algumas enzimas antioxidantes nos tecidos estudados. No tecido hepático, pôde-se observar que a dose de 13 mg/kg de peso vivo foi mais eficiente no combate ao estresse oxidativo induzido pela obesidade, combatendo a produção de peróxido de hidrogênio e, conseqüente, peroxidação lipídica. Já no tecido adiposo, a dose de 30 mg/kg de peso vivo melhorou a atividade da GSH-Px, mas não afetou de forma significativa a atividade das outras enzimas avaliadas. Com isso, podese sugerir que o CAPE na dose de 13 mg/kg de peso vivo por 15 dias e a dose de 30 mg/kg de peso vivo por 22 dias melhora a resposta ao estresse oxidativo induzido pela obesidade nos tecidos hepático e adiposo, respectivamente.
Obesity is a disease that is increasing in a drastically way around the world and is implicated in many pathological conditions, such as insulin resistance, type 2 diabetes, dyslipidemia, fatty liver disease, hypertension and cardiovascular risk. Evidence has shown that obesity is a chronic oxidative stress state that is associated with metabolic and physiological changes present in human organism that emerge pathologies mentioned. To prevent excessive reactive oxygen species (ROS) generated, the organisms have a complex system of defense mechanisms including enzymatic and non-enzymatic detoxification. The present study utilized SWISS mice fed with high fat (HF) diet, an experimental model of obesity, treated with CAPE, an isolated compound from propolis that present many biological activities, at doses of 13 and 30 mg / kg body weight and two treatment times, 15 and 22 days. In this study, the responses of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were analyzed in liver and adipose tissues. In addition, other important parameters were performed, such as lipid peroxidation, hydrogen peroxide (H2O2) content, liver enzyme activity ?-GT and the weight gain, relative weight of liver and adipose tissue (%). The results showed that CAPE did not interfere with the daily weight gain of animals. The high fat diet and treatment with CAPE did not lead to significant changes in serum liver enzyme ?-GT. The results obtained in response to oxidative stress are in agreement with the literature that have CAPE as a potent antioxidant, being able to improve the activity of some antioxidant enzymes in the tissues studied. In liver, it was observed that the dose of 13 mg / kg body weight was more effective preventing oxidative stress induced by obesity, the production of hydrogen peroxide and, consequently, lipid peroxidation. In adipose tissue, the dose of 30 mg / kg body weight improved the activity of GSH-Px, but did not affect significantly the activity of other enzymes evaluated. Thus, we can suggest that CAPE at a dose of 13 mg / kg body weight for 15 days and 30 mg / kg body weight for 22 days improves the response to oxidative stress induced by obesity in the liver and adipose tissue, respectively

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FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Introdução: O tratamento da hipertensão arterial (HA) em indivíduos com síndrome metabólica (SM) é um desafio, uma vez que terapias não medicamentosas são de difícil implementação e o tratamento farmacológico ideal não está totalmente estabelecido.Objetivo: O presente trabalho objetivou avaliar o bloqueio do sistema renina angiotensina aldosterona (SRAA) na pressão arterial (PA), na função e na morfologia renais em modelo experimental de SM, induzida por dieta hiperlipídica.Material e métodos: Ratos Wistar receberam ração hiperlipídica a partir da quarta semana de vida, por 20 semanas. Os grupos tratados receberam Losartana (10mg/kg/dia), Espironolactona (40mg/Kg/dia) ou quercetina (10mg/kg/dia), por gavagem, a partir da oitava semana de vida. Avaliou-se semanalmente o peso corporal e a PA de cauda por pletismografia. Ao final do experimento, foram realizados testes orais de tolerância à glicose, perfil lipídico, clearance de creatinina, medida direta da PA e análise morfométrica de área e volume glomerular.Resultados: A administração de dieta hiperlipídica se associou ao desenvolvimento de SM, caracterizada por acúmulo central de gordura, hipertensão arterial, hiperglicemia e hipertrigliceridemia. Nesse modelo, não foram observadas alterações da histomorfometria glomerular. O bloqueio do receptor AT1 da angiotensina II preveniu o desenvolvimento da HA. O bloqueio mineralocorticóide não apresentou eficácia anti-hipertensiva, porém se associou à redução da gordura abdominal. A quercetina, por sua vez, não alterou de forma significante o perfil nutricional, metabólico e pressórico dos animais. Conclusão: Ratos Wistar alimentados com dieta hiperlipídica desenvolveram Síndrome Metabólica, porém não foram observadas alterações morfológicas e funcionais renais.
Introduction: The treatment of arterial hypertension (AH) in patients with metabolic syndrome (MS) is a challenge, since non drug therapies are difficult to implement and optimal pharmacological treatment is not fully established. Objectives: The present study had as objective to evaluate the blockade of the rennin angiotensin aldosterone system (RAAS) in blood pressure (BP) in renal function and morphology in an experimental model of MS induced by high fat diet. Material e methods: Wistar rats were fed on high fat diet from the fourth week of life, for 20 weeks. The groups received Losartan (10mg/Kg/day), Spironolactone (40mg/Kg/day) or Quercetin (10mg/Kg/day) from the eighth week of life. We weekly evaluated the body weight and BP by tail plethysmography. At the end of the experiment oral glucose tolerance, lipid profile, creatinine clearance tests, and the direct measurement of BP were performed. A morphometric kidney analysis was performed. Results: The administration of high-fat diet was associated with the development of MS, characterized by central fat accumulation, hypertension, hyperglycemia and hypertriglyceridemia. In this model there were no changes in renal histomorphometry. The blockade of angiotensin II receptor AT1 prevented the development of hypertension. The mineralocorticoid blockage did not have antihypertensive efficacy but was associated with reduction of abdominal fat. Quercetin, in turn, does not significantly alter animals nutritional, pressure and metabolic profile. Conclusion: Wistar rats fed high fat diet developed metabolic syndrome but no changes were observed morphological and functional kidney.

A descoberta de aminopeptidase regulada por insulina (IRAP), a qual hidrolisa peptídeos como ocitocina e vasopressina e é receptora de angiotensina IV, destaca a importância do estudo do envolvimento de peptidases na função endócrina do adipócito. Estudos recentes detectaram alterações das atividades de aminopeptidase neutra e de dipeptidil peptidase IV (DPPIV) no plasma e no hipotálamo e hipocampo na obesidade induzida por glutamato monossódico (MSG). A presente tese propôs (i) a existência de atividades aminopeptidásicas ácida (APA), básica (APB), neutra insensível (APM) e sensível à puromicina (PSA), metionil (MetAP) e DPPIV, além da já conhecida (leucil (LAP)/cistil (CAP)/IRAP), nas frações de membrana plasmática (FM) e de alta (HDM) e baixa (LDM) densidade microssomal de adipócitos isolados do depósito de gordura retroperitoneal; (ii) que suas atividades catalíticas, expressões gênicas e tráfego subcelular seriam diferenciados entre obesos induzidos por MSG, submetidos ou não à privação alimentar e em animais controle sadios, submetidos ou não a privação alimentar; (iii) e influenciadas por insulina, angiotensina II, angiotensina IV e vasopressina. A existência de todas essas aminopeptidases no adipócito foi demonstrada, sendo a APM a que apresenta maior Vmax e maior eficiência catalítica e a APA a maior afinidade. Os animais obesos apresentaram aumento do diâmetro médio dos adipócitos e aumento do lipócrito, caracterizando hipertrofia adipocítica, enquanto os controles privados de alimento tiveram um aumento no número de adipócitos e aumento no lipócrito, caracterizando hiperplasia adipocítica. Pela primeira vez, um perfil variado de atividades aminopeptidásicas distribuídas em diferentes compartimentos subcelulares do adipócito é evidenciado juntamente com a demonstração de diferenças no padrão de distribuição destas atividades entre os ratos obesos e sadios, privados ou não de alimento. Dentre as novas aminopeptidases detectadas no adipócito não há nenhuma com a característica clássica de IRAP. No geral, as alterações das atividades catalíticas nas diferentes situações sob estudo mostram o envolvimento dessas novas aminopeptidases na regulação endócrina do balanço energético (provavelmente via ação hidrolítica sobre angiotensina II e vasopressina) sob modulação por angiotensina IV (APB, APM, DPPIV, LAP/IRAP, MetAP e PSA em animais controle sadios e APB em animais controle privados de alimento), angiotensina II (APB e PSA nos animais obesos) e vasopressina (APB nos animais obesos privados de alimento; e influenciadas em seu tráfego subcelular por angiotensina II (CAP, DPPIV e LAP/IRAP) e angiotensina IV (LAP/IRAP). O tráfego subcelular da conhecida atividade CAP/IRAP mostrou-se suscetível à insulina e vasopressina
The discovery of insulin-regulated aminopeptidase (IRAP), which hydrolyzes peptides such as oxytocin and vasopressin and is an angiotensin IV receptor, highlights the importance of the involvement of peptidases in the endocrine function of adipocyte. Recent studies have detected changes in aminopeptidase activities of neutral aminopeptidase and dipeptidyl peptidase IV (DPPIV) in the plasma and in the hypothalamus and hippocampus of rats with obesity induced by monosodium glutamate (MSG). The present thesis proposes (i) the existence of acid (APA), basic (APB), neutral insensitive (APM) and puromycin sensitive (PSA) aminopeptidases, methionyl aminopeptidase (MetAP) and dipeptidyl peptidase IV (DPPIV), besides the well-known cystyl (CAP) / leucine aminopeptidase (LAP) / IRAP) in plasma membrane (MF) and in high (HDM) and low (LDM) density microsomes of isolated adipocytes from retroperitoneal fat pad; (ii) that their catalytic activities, gene expression and subcellular trafficking were different among MSG-induced obese and healthy control rats (food deprived or not); (iii) and influenced by insulin, angiotensin II and IV, and vasopressin. The existence of all these adypocite aminopeptidases was demonstrated. APM has a highest Vmax and catalytic efficiency, while APA has a highest substrate affinity. The obese animals have increased mean diameter of adipocytes and lipocrit, characterizing hypertrophy, while the food deprived rats had an increased number of adipocytes and lipocrit, characterizing hyperplasia. For the first time, a profile of varied aminopeptidases activities distributed in different subcellular compartments of adipocyte is evidenced together with the demonstration of differences in the distribution pattern of these activities among the obese and healthy rats, food deprived or not. Among these novel aminopeptidases detected in adipocytes there is no one with the classical characteristic of IRAP. In general, changes on catalytic activities in these different situations show the involvement of these novel aminopeptidases in the endocrine regulation of energy balance (probably via hydrolytic action on angiotensin II and vasopressin) under modulation by angiotensin IV (APB, APM, DPPIV, LAP/IRAP, MetAP and PSA in healthy animals and APB in food deprived healthy animals), by angiotensin II (APB and PSA in obese) and by vasopressin (APB in food deprived obese animais); and under influence of by angiotensin II (CAP, DPPIV, LAP/IRAP) and angiotensin IV (LAP/IRAP) on their subcellular trafficking. Subcellular trafficking of the well-known CAP/IRAP was susceptible to insulin and vasopressin

O aumento nos casos de obesidade em todo o mundo tem gerado grande preocupação e estimulado pesquisas na prevenção e tratamento dessa condição patológica. A combinação de diabetes tipo 2 ou resistência insulínica com obesidade agrava o potencial evolutivo da enfermidade. Mesmo pacientes submetidos com sucesso à cirurgia bariátrica ou metabólica, podem não se curar do diabetes, pois a melhora das taxas circulantes de glicose e insulina nem sempre corresponde à recuperação da massa beta pancreática. Até o momento, não há consenso sobre como medir a massa de células beta in vivo. As ferramentas disponíveis padecem de baixa sensibilidade e especificidade, muitas vezes revelando-se também complexas e dispendiosas. Incretinas radiomarcadas ,como os análogos do peptídeo glucagon-like-peptide-1 / GLP-1, têm-se revelado promissoras para avaliação de células beta pancreáticas, em diabetes e insulinoma. O objetivo do presente trabalho foi o desenvolvimento de dois conjugados de análogo de incretina GLP-1, marcados com tecnécio-99m, a fim de propor um método não invasivo de imagem, para monitoração da massa de células beta pancreáticas, em organismos afetados por obesidade. O estudo foi conduzido em diferentes modelos animais, incluindo obesidade induzida por dieta hiperlipídica, estado pós-obesidade em que o distúrbio inicialmente gerado foi parcialmente corrigido, e como controle, diabetes induzido com aloxana. Nos resultados, os radiotraçadores alcançaram um rendimento radioquímico superior a 97%. O melhor radiomarcador, dentre os dois análogos ensaiados, foi o 99mTc-HYNIC-βAla-Exendin-4. Animais com obesidade induzida por dieta revelaram captação reduzida nas células beta pancreáticas. A restrição dietética (estado pós-obesidade) não se seguiu de recuperação completa, embora notável melhora de glicemia haja sido observada. Estudos futuros são indicados em modelos de obesidade, diabetes tipo 2 e tratamento dietético, incluindo cirurgia bariátrica e metabólica.
Increased prevalence of obesity worldwide, has become a vast concern, stimulating investigations focusing prevention and therapy of this condition. The association of type 2 diabetes or insulin resistance aggravates the prognosis of obesity. Even patients successfully submitted to bariatric or metabolic surgery, may not be cured of diabetes, as improvement of circulating values of glucose and insulin not necessarily reflects recovery of pancreatic beta cell mass. There is no consensus about how to estimate beta cell mass in vivo. Available tools suffer from low sensitivity and specificity, often being as well cumbersome and expensive. Radiolabeled incretins, such as glucagon-like-peptide 1 (GLP-1) analogs, seem to be promising options for the measurement of beta cell mass in diabetes and insulinoma. The objective of this study was the development of two conjugates of GLP-1 analog, radiolabeled with 99m Technetium, as a noninvasive imaging method for the estimation of pancreatic beta cell mass, in the presence of obesity. Animal models were selected, including hyperlipidic diet-induced obesity, diet restricted obesity, and as controls, alloxan diabetes. Results indicated that both radiotracers achieved over 97% radiochemical yield. The most successful product was 99mTc-HYNIC-βAla-Exendin-4. Low beta cell mass uptake occurred in diet-induced obesity. Diet-restricted obesity, with substantial shedding of excess body weight, was followed by remarkable decrease of fasting blood glucose, however beta cell mass uptake was only mildly improved. Future studies are recommended in obesity, type 2 diabetes, and dieting, including bariatric and metabolic operations.

Abstract Atherosclerosis is the major cause of cardiovascular diseases and the leading cause of death globally. Atherosclerosis is a complex, chronic disease characterized by lipid accumulation and inflammation within the intima layer of vessel wall. Novel biomarkers and therapeutics are still being sought to provide both better diagnosis and treatment. Ghrelin represents an attractive target for studies into atherosclerosis. Ghrelin is a gastric peptide hormone, which has multiple functions, including regulation of appetite and energy metabolism. Emerging evidence suggests that it may also have a role in the cardiovascular and immune systems. The aim of the present study was to explore the role of ghrelin in atherosclerosis. The specific aims were 1) to investigate the association between the plasma ghrelin level and early atherosclerosis as determined by carotid artery intima media thickness (IMT) in a large (n = 1024) cross-sectional population-based study of middle-aged subjects, 2) to measure the associations between plasma ghrelin levels and already established risk factors of atherosclerosis in human subjects, 3) to assess the effects of ghrelin on atherogenesis in vitro by analyzing monocyte adhesion to endothelial cells, oxidized low density lipoprotein (LDL) binding and acetylated LDL uptake using macrophages, and 4) to study the influence of ghrelin on atherosclerosis using ghrelin vaccination in a mouse model of atherosclerosis. Plasma total ghrelin levels were positively associated with carotid IMT in male subjects. Association studies demonstrated plasma ghrelin levels to be negatively associated with total and LDL cholesterol, and triglyceride concentrations as well as with body mass index (BMI), and positively assocated with high density lipoprotein (HDL) cholesterol concentration in postmenopausal women and in a population-based study. In addition, estrogen increased plasma acylated ghrelin levels in postmenopausal women. Cell culture studies demonstrated that ghrelin could increase the binding of oxidized LDL and monocytes to endothelial cells. Interestingly, when endothelial cells were stimulated with tumor necrosis factor α (TNFα), then ghrelin prevented monocyte adhesion. The study with LDL receptor knockout mice, revealed that ghrelin vaccination could increase plasma ghrelin levels but had no effects on the development of atherosclerosis. However, the plasma MCP-1 level decreased in mice immunized with ghrelin vaccine. In conclusion, these studies suggest that ghrelin has modulatory functions in the vascular system and atherogenesis though the effect may not be as dominant as that of the known traditional risk factors. Whether this effect of ghrelin is positive or negative in atherogenesis will be clarified in further studies
Tiivistelmä Sydän- ja verisuonitaudit ovat suurin kuolinsyy niin Suomessa kuin useimmissa länsimaissakin. Näiden sairauksien taustalla on yleensä valtimonkovettumatauti eli ateroskleroosi, joka voi kliinisesti ilmentyä mm. sepelvaltimotautina, aivoveritulppana ja laskimotautina. Ateroskleroosissa tulehdussoluja ja kolesterolia kertyy verisuonen seinämään muodostaen ahtauman eli ateroomaplakin valtimoon. Valtimonkovettumataudin riskitekijäitä tunnetaan jo hyvin, mutta uusia tautia ennustavia merkkiaineita sekä hoitomuotoja tarvitaan yhä. Greliini on mahalaukusta eritettävä peptidihormoni, joka osallistuu elimistössä mm. ruokahalun, energiametabolian, tulehdustekijöiden sekä sydän- ja verenkiertoelimistön toiminnan säätelyyn. Tämän työn tavoitteena oli tutkia greliinin yhteyttä ihmisen valtimonkovettumatautiin. Tutkimus toteutettiin käyttämällä kahta eri potilasaineistoa, soluviljelykokeita sekä valtimonkovettumataudin hiirimallia. Laajassa väestöpohjaisessa potilasaineistossa tutkittiin veren greliinipitoisuuden yhteyttä kaulavaltimon seinämän paksuuteen, jota pidetään valtimonkovettumista kuvaavana tekijänä. Veren greliinipitoisuuden yhteyttä valtimonkovettumataudin tunnettuihin riskitekijöihin tutkittiin myös laajassa potilasaineistossa sekä vaihdevuosi-ikäisillä naisilla, joille annettiin estrogeenikorvaushoitoa. Solukokeilla selvitettiin greliinin vaikutusta tärkeisiin valtimonkovettumataudin syntyvaiheisiin käyttäen monosyytti-, endoteelisolu- sekä makrofaagi-soluviljelmiä. Greliinin vaikutusta ateroskleroosiin in vivo selvitettiin rokottamalla LDL-reseptoripuutteiset hiiret greliini-rokotteella. Tutkimuksessa havaittiin yhteys veren korkean greliinipitoisuuden ja kaulavaltimon seinämän paksuuden välillä miehillä laajassa potilasaineistossa (n = 1024). Tulosta tukivat soluilla tehdyt kokeet, joissa greliini lisäsi hapettuneen LDL:n sitoutumista makrofaageihin sekä monosyyttien tarttumista endoteelisolujen pinnalle. Greliinin vaikutukset monosyyttien tarttumiseen endoteelisolujen pinnalle olivat päinvastaiset silloin, kun endoteelisolut käsiteltiin tulehdusta stimuloivalla tekijällä. Matalat veren greliinipitoisuudet olivat myös yhteydessä korkeisiin LDL-kolesteroli- ja triglyseriditasoihin sekä painoindeksiin ja matalaan HDL-kolesterolitasoon potilasaineistoissa. Estrogeeni nosti veren greliinipitoisuutta vaihdevuosi-ikäisillä naisilla. Greliinirokote ei vaikuttanut ateroskleroosin kehittymiseen hiirimallissa. Tutkimustulosten perusteella greliinillä näyttäisi osallistuvan valtimonkovettumataudin kehitykseen, vaikkakin sen vaikutus on pienempi kuin aiemmin tunnetuilla taudin riskitekijöillä

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A obesidade atinge proporções epidêmicas em países industrializados e está relacionada a uma série de doenças metabólicas e circulatórias. Nesse contexto, a atividade física, tratamento não farmacológico da obesidade, acessível a diversas populações e está relacionada com a redução do risco cardiovasvascular mesmo. O objetivo deste trabalho foi avaliar, após mudança ou não da dieta, associação ou não a um programa de treinamento aeróbico (PTA) durante 8 semanas, a possível reversibilidade dos danos causados por uma dieta hiperlipídica por 12 semanas. Para tal, 120 hamsters machos da espécie Mesocricetus auratus, com massa corporal de 60 g, foram distribuídos em quatro grupos, cada um subdividido em três subgrupos, com dez animais para diferentes análises. Os grupos Obeso Controle (OBC) e Obeso Exercitado (OBEX) receberam a ração hiperlipídica por 20 semanas, com adição do PTA ao grupo OBEX nas últimas 8 semanas. Os Obeso Ração Padrão (OBRP) e Obeso Ração Padrão/Exercício (OBRP/EX) tiveram a ração modificada para comercial padrão e adição do PTA ao grupo OBRP/EX após as 12 semanas iniciais. Para as análises microcirculatórias, a bolsa da bochecha foi usada para determinação do número máximo de extravasamentos induzidos por 30 min de isquemia seguida de reperfusão e da reatividade microvascular após a aplicação tópica de acetilcolina e nitroprussiato de sódio. No sangue coletado foi avaliado o perfil lipídico, glicemias quinzenais e leptina. As expressões de eNOS e iNOS foram determinadas na aorta por imunoblotting e a composição corporal avaliada nos tecidos adiposos visceral, urogenital e retroperitoneal, retirados no dia do experimento. Os resultados foram analisados com os métodos o teste estatístico de análise de variância (One Way ANOVA - Teste de Kruskal-Wallis), seguido pelo pós-teste de Dunn. Resultados mostram que a modificação dietética, associada ou não ao PTA, reduziu significativamente a massa corporal (p<0,0001), comprimento naso-anal (p=0,0011) e tecido adiposo (visceral [p<0,0001], urogenital [p=0.0004] e retroperitoneal [p= 0,0083]). Nas análises sanguíneas não foram encontradas diferenças com relação ao perfil lipídico e glicemia, já na leptina houve uma redução significativa (p=0,0039). A análise da reatividade microvascular mostrou melhora significativa na vasodilatação endotélio-dependente nos grupos submetidos à modificação dietética associada ou não ao PTA. Nas medidas de permeabilidade a macromoléculas houve redução significativa no número de extravasamentos nos grupos submetidos à modificação dietética associada ou não ao PTA, (5 min [p= 0,0207] e 10 min [p= 0,0057]). Houve um aumento na expressão de eNOS nos grupos submetidos à modificação dietética associada ou não ao PTA, em comparação ao grupo OBC (p=0,0352). Os resultados mostraram que a modificação dietética, associada ao protocolo de treinamento aeróbico melhora a vasodilatação endotélio-dependente, aumenta a expressão da óxido nítrico sintase endotelial e reduz o número de extravasamentos induzidos por isquemia e reperfusão, mesmo sem melhoras nos marcadores bioquímicos tradicionais como glicemia e perfil lipídico.
Obesity is a growing public health problem reaching epidemic proportions in industrialized countries and an independent risk to metabolic and circulatory diseases. Physical activity is seen as a non-pharmacological treatment to obesity and its co-morbities, accessible to the population. Physical benefits are not necessarily related to adiposity loss but on improvement of vascular hemodynamic. The objective of this work was to evaluate if changes on diet associated or not to an aerobic training program (ATP) for 8 weeks could counteract damages on obese hamsters, elicited by high fat chow given during 12 weeks. One hundred and twenty male hamsters (Mesocricetus auratus) with body mass around 60g were distributed into four groups, subdivided into three subgroups with ten animals each for different analysis. Obese Control (OBC) and Obese + Exercise (OBEX) groups received high fat chow for 20 weeks, being the last one combined to AET. Groups Obese + Standard Chow (OBSC) and Obese + Standard Chow/Exercise (OBSC/EX) received high fat chow for 12 weeks, returned to the standard chow, being the last one combined to AET. The cheek pouch preparation was used to evaluate the maximum number of extravasations induced by 30 min ischemia followed by reperfusion and microvascular reactivity in response to topical applications of acetylcholine and sodium nitroprusside. Blood was collected for lipid profile, biweekly glycemia and leptin. On the day of the experiment, the aorta was excised for immunoblotting of eNOS and iNOS and the body composition was determined by collection of different adipose tissues. For statistical analysis Kruskal Wallis ANOVA for transversal analysis, followed by the Dunn post test. Results have shown that dietary modification associated or not to AET significantly reduced body mass (p<0.0001), naso-anal length (p=0.0011) and the amount of adipose tissues (visceral [p<0.0001], urogenital [p=0.0004] and retroperitoneal [p=0.0083]) in treated groups. Blood analysis did not show any differences between groups in relation to lipid profile and glycemia. Leptin had a significant reduction in all treated groups (p=0.0039). Microcirculatory analysis showed that groups subjected to dietary modification associated or not to AET had better endothelium-dependent responses after topical application of acetylcholine, in all concentrations (10-8M [p=0.0001], 10-6M [p<0.0001] and 10-4M [p<0.0001]). For ischemia/reperfusion, animals subjected to AET associated or not to dietary modification had significantly lower number of leaks (5 min [p=0.0207] and 10 min [p=0.0057]). Immunoblotting assays of eNOS showed linear improvement after AET associated to dietary modification in comparison to obese control animals (p=0.0352). Results have shown that the dietary modification and its association to an aerobic exercise training, could detect substantial improvement on the endothelial response and reduction in the number of leaks on ischemia/reperfusion. These modifications occurred without any significant changes on biochemical markers.

Orientador: Mario Jose Abdalla Saad
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A resistência seletiva à insulina através da via IRS/PI3-K/Akt/eNOS associada à ativação normal ou exacerbada da via de crescimento MAPK tem sido proposta como um possível elo entre situações de resistência à insulina e doença cardiovascular. Inicialmente demonstramos que animais com resistência à insulina e doença cardiovascular (o rato espontaneamente hipertenso, SHR) apresentam menor ativação da via IRS/PI3-K/Akt/eNOS e hiperativação/hiperexpressão da via da MAPK na aorta torácica, enquanto a ativação normal da via IRS/PI3-K/Akt/eNOS pode proteger o animal obeso, resistente à insulina e que não apresenta doença cardiovascular. Posteriormente, outras vias estimulatórias do crescimento celular, como a via JAK/STAT, foram estudadas no vaso de outro modelo animal de resistência à insulina e doença cardiovascular - o rato com obesidade induzida por dieta. Este modelo demonstrou que a redução da ativação da via PI3-K/Akt/eNOS ocorre em paralelo à hiperativação das vias da MAPK e JAK/STAT, e isso pode desempenhar função importante da patogênese da doença cardiovascular neste estado patológico. Em outro estudo foi demonstrado pela primeira vez que a acetilcolina pode ativar a eNOS de maneira cálcio-independente, através da via IRS/PI3-K/Akt utilizando para isso uma tirosina quinase intracelular, a JAK2. Em ratos com obesidade induzida por dieta, resistentes à insulina e com disfunção endotelial, foi demonstrado que há resistência na via da PI3-K/Akt/eNOS tanto em resposta à insulina quanto à acetilcolina, em decorrência de maior fosforilação inibitória do IRS-1 e da redução dos níveis teciduais da eNOS. Assim, o desequilíbrio entre a ativação reduzida da via IRS/PI3-K/Akt/eNOS e a maior ativação das vias de crescimento (MAPK e JAK/STAT) pode contribuir para o desenvolvimento de doença cardiovascular em estados de resistência à insulina
Abstract: The actions of acetylcholine on endothelium are mainly mediated through muscarinic receptors, which are members of the G protein-coupled receptor family. In the present study we show that acetylcholine induces rapid tyrosine phosphorylation and activation of Janus kinase 2 (JAK2) in rat aorta. Upon JAK2 activation, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) is detected. In addition, acetylcholine induces JAK2/IRS-1 and IRS-1/phosphatidylinositol (PI) 3-kinase associations, downstream activation of Akt/protein kinase B, endothelial cell-nitric oxide synthase (eNOS), and extracellular signal-regulated kinase (ERK1/2). The pharmacological blockade of JAK2 or PI 3-kinase reduced acetylcholine-stimulated eNOS phosphorylation, NOS activity and aorta relaxation. These data indicate a new signal transduction pathway for IRS-1/PI 3- kinase/Akt/eNOS activation and ERK1/2 by means of JAK2 tyrosine phosphorylation stimulated by acetylcholine in vessels. Moreover, we demonstrate that, in aorta of obese rats (high-fat diet), there is an impairment in insulin- and acetylcholinestimulated IRS-1/PI 3-kinase pathway, leading to reduced activation with lower protein levels of eNOS associated with a hyperactivated ERK/MAP kinase pathway. These results suggest that in aorta of obese rats, there is not only insulin resistance, but also acetylcholine resistance, probably mediated by a common signaling pathway that controls the activity and the protein levels of eNOS
Doutorado
Medicina Experimental
Doutor em Fisiopatologia Medica

L'objectif de ce travail est de comparer la liaison et les effets biologiques de l'hormone de croissance (gh) 1) dans les adipocytes du tissu inguinal de jeunes rats zucker obeses fa/fa et minces fa/fa; 2) dans les preadipocytes de rats zucker en culture primaire au cours de la differenciation adipocytaire. 1. Les etudes de liaison de la gh et les courbes dose-reponse pour le transport et le metabolisme du glucose sont realisees avec des adipocytes soit fraichement isoles (f) soit preincubes 3 h (p)

Obesity can lead to a range of health problems including type 2 diabetes (T2DM), cardiovascular disease and non-alcoholic fatty liver disease (NAFLD), and causes an estimated 2.8 million deaths annually (2016). It is a growing epidemic affecting over 600 million people worldwide (in 2014), with 26.8% of the adult population in England alone being obese, an increase of 10% in the last decade, and 62.9% overweight or obese. This trend is predicted to continue, and is attributed to an increasingly sedentary lifestyle, coupled with a high calorie “western diet”, which is estimated to cost >£25billion/year in the UK (2015), which is predicted to rise to £49.9 billion by 2050. It is clear that both sex and genetics affect the extent to which individuals exposed to a high fat diet develop adiposity and its associated morbidities, although the mechanisms underlying these differences are not well understood. Here we explore this aetiology, focusing on poly(A)-binding protein 4 (PABP4), an RNA-binding protein in which polymorphisms associated with altered cholesterol levels and cardiovascular disease risk were identified in human GWAS studies. To this end, I take advantage of an unpublished Pabp4 knock-out mouse, maintained on either normal (ND) or high fat diet (HFD), to explore the role of PABP4 in determining the response to high fat diet. PABP4 is a poorly characterised member of the PABP family, which are multifunctional central regulators of global and mRNA-specific translation, and stability. In cell lines, PABP4 is predominantly cytoplasmic, consistent with such functions. However, analogously to PABP1, the prototypical PABP family member, PABP4 is relocalised to stress granules or the nucleus in response to specific cellular stresses and/or viral infections, suggesting a role in altering gene expression programs in responses to changing cellular conditions. Whilst the expression pattern of PABP4 within tissues has not been previously characterised, western blotting of adult mouse tissues revealed that PABP4 is highly expressed in tissues relevant to obesity, T2DM and NAFLD, such as adipose, pancreas, liver and muscle, consistent with the idea that it may play a role in regulating gene expression programs in response to HFD. Immunohistochemistry of tissue sections provided additional insight, revealing a distinct cellular distribution of PABP4 in some tissues, when compared to the well characterised PABP1. Birth weight and post-birth growth can affect adult metabolism. In particular, low birth weight and catch-up growth, characterised by preferentially putting down adipose over lean mass, increases the risk of metabolic conditions in adulthood, such as obesity, T2DM and cardiovascular disease. Therefore, Pabp4-/- and wildtype mice were weighed at birth and daily until weaning. Interestingly this revealed that Pabp4-/- mice have a reduced weight at birth that is exacerbated to weaning (21days (P21)) (5.7% and 18.3% reductions respectively). This analysis also uncovered a reduced survival to weaning, with both male and female Pabp4-/- mice being present at sub-Mendelian ratios by P21 (p=0.0056). Whilst most death occurred neonatally, Pabp4-/- mice showed an increased rate of attrition until weaning, preceded in some cases by an arrest of weight gain. Weight gain was also tracked from 4 weeks to 12 weeks of age on normal diet showing that Pabp4-/- mice had reduced weight into adulthood (12% reduction at 12wks). Analysis of weight gain by sex uncovered a sexually dimorphic effect of Pabp4-deficiency, with female, but not male, Pabp4-/- mice remaining reduced in weight compared to wildtype after 8 weeks on ND (13.4% reduction in female weight). Body composition analysis showed that fat mass was equivalent to wildtype at 12 weeks of age in both sexes but that female Pabp4-/- mice had a 14.3% reduction in lean mass. Neither the catch-up growth in males nor the reduced lean mass in females was sufficient to result in a change in glucose homeostasis. As the risk of developing metabolic disorders in adult life is a consequence of both genetic and environmental factors, such as diet, Pabp4-/- were placed on a HFD at 4 weeks of age for 8 weeks. HFD models the ‘western’ diet, and has been shown to induce obesity, insulin resistance and glucose intolerance in wildtype mice. Whereas Pabp4-/- mice were only distinguishable from wild-type in terms of female lean mass on normal diet, pronounced sexually dimorphic differences were observed in HFD fed mice. Male Pabp4-/- mice appeared to be partially protected from the negative effects of an 8 week HFD regimen, with a 44% decrease in adipose mass gain compared to wildtype despite equal lean mass. Pabp4-/- male mice also had significantly reduced ectopic lipid stores, with an 81% decrease in hepatic triglyceride concentration compared to wildtype, meaning that NAFLD has not developed. Furthermore, Pabp4- /- male mice did not develop hyperinsulinemia on HFD and retained insulin sensitisation (assessed via glucose tolerance test (GTT) and insulin tolerance test (ITT)), although they displayed wildtype-like elevated plasma glucose concentrations (compared to ND). Western blotting had detected high PABP4 levels in the pancreas, indicating a possible pancreatic origin of these alterations. However, immunofluorescence revealed that PABP4 was confined to the exocrine portion of the pancreas, and was undetectable in the insulin producing pancreatic beta cells, suggesting this phenotype may not be beta cell in origin. This is consistent with the fact that the Pabp4-/- male mice retained an appropriate glucose-induced burst of insulin secretion, and therefore insulin production appears unimpaired. Thus, the primary defect may reside in the exocrine pancreas, which aids digestion, or in other key metabolism related tissues (e.g. muscle, liver, adipose and brain), or a combination thereof. In HFD fed wildtype mice, insulin resistance is caused by increased adiposity and ectopic lipid depots, which blunt insulin stimulated signalling cascades, meaning that the normal responses to insulin (e.g. cellular up take of glucose in muscle and arrested glucose production in liver, to decrease plasma glucose concentrations), are impaired. Therefore, the absence of insulin resistance in HFD fed Pabp4-/- male mice may be a consequence of the reduced increase in adipose mass and ectopic lipid deposits detected in these mice, and their consequent lack of inhibition on insulin signalling pathways. The reduced adiposity was not a result of reduced food intake or dietary fat absorption as male Pabp4-/- mice did not eat less nor exhibit apparent steatorrhea (fatty stools). These results highlight that the Pabp4-/- male mice appear to have an alteration in energy use/storage, and the investigation of this will form the basis of future work. When fed HFD, female Pabp4-/- mice revealed a divergent phenotype to that of wildtype female mice and Pabp4-/- male mice. HFD fed Pabp4-/- female mice showed no difference to HFD-fed wildtype mice in terms of weight, but still exhibited the reduction in lean mass seen on ND, but now with a 22.8% increase in volume of adipose tissue. Together, this means that HFD fed Pabp4-/- females have a higher body fat percentage (32.6% compared to 25.9 % for wildtype females). In contrast to the males, there was no difference in terms of hepatic triglycerides in HFD fed Pabp4-/- female mice and they showed greater hyperglycaemia than wildtype (GTT), although like males they retained insulin sensitisation (ITT). These potentially conflicting results in terms of insulin sensitivity and plasma glucose concentrations may result from the alterations in body composition, which can confound results when lean mass is altered and total body weight is used for calculating doses for GTT/ITT. Interestingly, adiponectin, an adipokine normally found in inverse proportion to adipose mass, was increased in plasma from HFD fed Pabp4-/- female mice (21% increase from HFD fed wildtype mice). Whilst surprising given the increase fat mass of Pabp4-/- females, the insulin sensitising properties of adiponectin may help to explain the retained insulin sensitivity detected in the female Pabp4-/- mice.
The finding that HFD revealed metabolic differences in the Pabp4-/- mice lead to the question of whether Pabp4-/- mice have issues adapting to other situations which require modulation of energy storage and glucose homeostasis. One such event is pregnancy, when maternal regulation of insulin resistance is tightly modulated throughout gestation. We therefore characterised the maternal Pabp4-/- environment in late pregnancy (E18.5), when insulin sensitivity decreases to 40-60% lower than pre-pregnancy which results reduced maternal glucose uptake, freeing the glucose up for the rapidly developing foetus. Pregnant Pabp4-/- mice had elevated plasma insulin concentration post fasting (63.7% increase), however glucose homeostasis was wildtype-like, both in terms of plasma glucose and insulin concentrations, throughout a GTT. However, plasma glucose and insulin concentrations in E18.5 Pabp4-/- foetuses were significantly decreased (9% and 44.3% respectively). Pabp4-/- foetuses also had reduced foetal and placental weight/length parameters. This establishes that the differences in weight observed at birth were present by late gestation and secondly, that the reductions in both foetal glucose and insulin concentrations which may contribute to or underlie the reduced growth. It also suggests that the differences seen in adulthood on HFD may be a consequence of metabolic differences present during pregnancy. Taken together, these data support the hypothesis that PABP4 plays a key role in the regulation of mRNAs which are important in growth, post-natal survival and metabolic adaption to high fat diet.

Introdução: Obesidade é uma questão de saúde pública em muitos países, inclusive no Brasil. O excesso de peso atinge 1 bilhão de adultos no mundo e, no Brasil, já é um problema maior que a desnutrição. O glutamato monossódico (GMS) é um agente flavorizante utilizado em níveis crescentes nos alimentos industrializados. O GMS administrado em ratos ocasiona obesidade e diminuição do hormônio do crescimento (GH). Objetivo: Avaliar o efeito do GMS nos fetos de ratas prenhas através da comparação do peso, comprimento nasal-anal (CNA) e índice de Lee (IL) ao nascimento e com 21 dias de vida. Métodos: Utilizamos ratas prenhas da linhagem Wistar divididas em três grupos: grupo controle (GC), G10 e G20. Estes, respectivamente, foram alimentados com ração contendo 0, 10 e 20% de GMS desde o período de acasalamento até o final da amamentação. Resultados: O peso e o CNA não foram diferentes entre os grupos ao nascimento. O grupo G20, ao nascimento, teve IL menor que o grupo GC (p < 0,05) e, aos 21 dias de vida, apresentou peso e CNA menores que o grupo G10, o qual foi menor que o GC (p < 0,01). O grupo G20, aos 21 dias de vida, teve IL semelhante aos outros dois grupos. O percentual de ganho de peso do nascimento ao 21º dia de vida foi menor no G20 em relação aos outros dois grupos (p < 0,01). O grupo G20 teve percentual de aumento de CNA do nascimento ao 21º dia de vida menor que o grupo G10, e este menor que o grupo GC (p < 0,01). Conclusões: O GMS nas concentrações de 10 e 20% na ração de ratas prenhas Wistar apresentou uma relação dose-dependente nas variáveis peso e CNA. Houve diminuição no padrão de ganho de peso e de aumento de CNA do nascimento ao 21º dia de vida com uso de GMS. O IL na prole do grupo G20 aumentou em relação ao do grupo GC após 3 semanas de acompanhamento.
Objective: Determine the effects of the MSG (monosodium glutamate) in the offspring of pregnant rats through the comparison of the weight, NAL (nasal-anal length) and IL (Index of Lee) at birth and with 21 days of life. Research Methods & Procedures: We have investigated pregnant Wistar rats and their offspring and divided them into 3 groups: GC, G10 and G20. Each of the groups received 0%, 10% and 20% of MSG, respectively from coupling until the end of the weaning period. Results: Neither weight nor NAL were different among the groups at birth. The group G20 at birth had an IL lower than the group GC (p<0,05) and with 21 days of life presented weight and NAL lower than the groups G10 and this lower than the GC (p<0,01). Otherwise the G20 at 21 days of life had the IL similar to the other two groups. The weight profit percentage from birth to the 21st day of life was lower in the G20 regarding the other two groups (p<0,01). The G20 had a NAL increase percentage from birth to the 21st day of life lower than the G10 and this lower than the GC (p<0,01). Conclusions: MSG presented a dose-dependent relation in the variables weight and NAL. It caused a decrease in the growth pattern as well as in the weight gain pattern until the 21st day of life. The IL of the group 20% had an increased in relation to the control group after 3 weeks of follow up.

A prevalência da obesidade tem aumentado em todo mundo, afetando também mulheres em idade reprodutiva. Estudos têm demonstrado que o acúmulo excessivo de tecido adiposo resulta em prejuízos à reprodução feminina. O mecanismo pelo qual a obesidade diminui a fertilidade não está totalmente estabelecido. Atualmente, são propostas a condição inflamatória e a indução de estresse oxidativo como potenciais mecanismos de ação para as patologias relacionadas à obesidade. Adicionalmente, a angiotensina II (Ang II) é mais um fator que tem sido relacionado com alterações associadas à obesidade, e os efeitos deste peptídeo incluem ações pró-inflamatórias e pró-oxidativas. O presente estudo analizou o efeito da obesidade na ovulação e no metabolismo oxidativo ovariano, e a participação da Ang II como um possível modulador da formação de espécies reativas de oxigênio em ovários de ratas obesas e seus efeitos na ovulação. Foram utilizadas ratas submetidas a uma dieta hipercalórica composta por alimentos palatáveis, conhecida como dieta de cafeteria, a partir do desmame até a idade adulta, por um período de 17 semanas. Os animais foram divididos em grupos: CTL (controle), CTL LOS (controle + losartan), CAF (cafeteria) e CAF LOS (cafeteria + losartan). Avaliamos o consumo de alimentos e líquidos, o peso corporal, o peso da gordura abdominal e retroperitonial, a concentração de insulina plasmática, o número de oócitos, as atividades das enzimas antioxidantes (superóxido dismutase e catalase), concentração de peróxido de hidrogênio (H2O2) e parâmetros de dano oxidativo (lipoperoxidação e oxidação de proteínas). As fêmeas obesas do grupo CAF apresentaram maior consumo de energia e reduzido consumo de água e ração padrão, hiperinsulinemia, aumento das gorduras abdominal e retroperitonial, e aumento de peso corporal. A obesidade não reduziu significativamente a ovulação, mas aumentou a atividade das enzimas antioxidantes e a concentração de H2O2 no ovário. A administração do losartan, em ratas alimentadas com a dieta de cafeteria, reduziu a ingestão energética total, a concentração plasmática de insulina e o ganho de gordura abdominal, mas não evitou o desenvolvimento da obesidade. O losartan inibiu a atividade da enzima antioxidante superóxido dismutase no ovário das ratas obesas do grupo CAF, porém não alterou a atividade da enzima antioxidante catalase. Os resultados deste estudo demonstram que a obesidade resulta em alterações no metabolismo e sugerem a participação da Ang II na formação de espécies reativas de oxigênio no ovário.
The prevalence of obesity has increased worldwide, also affecting women of reproductive age. Studies have shown that excessive accumulation of adipose tissue results in impaired female reproduction. The mechanism by which obesity reduces fertility is not fully established. Currently, proposals are the inflammatory condition and the induction of oxidative stress as a potential mechanism of action for diseases related to obesity. Additionally, angiotensin II (Ang II) is another factor that has been related to changes associated with obesity, and the effects of this peptide include pro-inflammatory and pro-oxidative actions. The present study considers the effect of obesity on ovulation and ovarian oxidative metabolism, and the participation of Ang II as a possible modulator of the formation of reactive oxygen species in ovaries of obese rats and their effects on ovulation. Female rats fed a diet consisting of high calorie foods palatable, known as cafeteria diet from weaning to adulthood, for a period of 17 weeks. The animals were divided into groups: Control (CTL) CTL LOS (control + losartan), CAF (cafeteria) and CAF LOS (losartan + cafeteria). We evaluate the consumption of food and fluids, body weight, abdominal and retroperitoneal fat weight, the plasma insulin concentration, the number of oocytes, the activities of antioxidant enzymes (superoxide dismutase and catalase), concentration of hydrogen peroxide (H2O2 ) and parameters of oxidative damage (lipid peroxidation and protein oxidation). The females of the CAF group had higher energy consumption and reduced consumption of water and standard chow, hyperinsulinemia, increased abdominal and retroperitoneal fat, and weight gain. Obesity not significantly reduced ovulation, but increased the activity of antioxidant enzymes and the concentration of H2O2 in the ovary. The administration of losartan in rats fed the cafeteria diet, reduced total energy intake, plasma insulin concentration and abdominal fat gain, but did not prevent the development of obesity. Losartan inhibited the activity of the antioxidant enzyme superoxide dismutase in the ovary of rats CAF group, but did not alter the activity of the catalase antioxidant enzyme. The results of this study demonstrate that the cafeteria diet results in changes in metabolism and suggests the involvement of Ang II in the formation of ROS in the ovary.

La stéatose hépatique affecte 20 à 40% de la population et progresse de façon constante. Il s’agit d’une pathologie chronique fortement associée au syndrome métabolique. Sa pathogenèse est mal comprise. Une altération du métabolisme des lipides dans le foie entraînant une accumulation intra-hépatique de lipides est probablement la cause majeure de la stéatose hépatique. A ce jour, il n’existe pas de traitement spécifique de la stéatose hépatique. La protéine kinase activée par l’AMP (AMPK) est un régulateur clé du métabolisme énergétique. Notamment, l’AMPK contrôle le métabolisme des lipides en inhibant la synthèse des acides gras et du cholestérol, et en stimulant l'oxydation des acides gras. Plusieurs études ont montré l’existence d’une association entre l’accumulation intracellulaire de lipides et une perte d’activité de l’AMPK dans le foie. Ces observations suggèrent que l’AMPK pourrait être un facteur impliqué dans la physiopathologie de la stéatose hépatique. Pour étudier cette hypothèse, nous avons généré un nouveau modèle de souris knockout dépourvu des sous-unités catalytiques α1 et α2 de l’AMPK spécifiquement dans le foie. Nous avons analysé les conséquences de cette délétion sur le métabolisme lipidique dans différentes situations nutritionnelles. La délétion de l’AMPK dans le foie ne modifie pas le contenu hépatique en triglycérides et en cholestérol au cours d’un jeûne ou après une réalimentation riche en glucides. Egalement, l’expression des gènes de la lipogenèse n’est pas modifiée dans le foie de ces animaux. De plus, l’oxydation des acides gras n’est pas altérée même après un jeûne de 24h. Etonnamment, l’absence de l’AMPK dans le foie n’amplifie pas la stéatose hépatique, ni l’hyperglycémie ou l’intolérance au glucose lorsque les souris sont nourries avec un régime riche en lipides. Cependant, l’activation de l’AMPK in vivo avec l'activateur direct, A-769662, normalise la stéatose hépatique chez des souris lipodystrophiques aP2-SREBP-1c et chez des souris obèses nourries avec un régime riche en lipides. Cet effet est dépendant de l’AMPK car il est totalement perdu chez des souris dépourvues d’AMPK dans le foie. Dans des hépatocytes de souris en culture primaire, l’activation de l'AMPK par un activateur direct (A-769662) ou par des activateurs indirects (metformine et AICAR) réduit le flux lipogénique et augmente l’oxydation des acides gras. Ces effets sont totalement abolis dans des hépatocytes AMPK KO, démontrant l’action spécifique de l'AMPK sur le métabolisme lipidique en réponse à ces composés. Ces résultats obtenus chez la souris sont extrapolables à l'homme puisque nous avons montré que l'activation de l'AMPK dans des hépatocytes humains en culture primaire inhibe de manière efficace la synthèse des acides gras et du cholestérol. En conclusion, nos résultats démontrent que l’inactivation de l’AMPK dans le foie n’est pas un facteur déclenchant ou aggravant dans la physiopathologie de la stéatose hépatique. En revanche, l’activation pharmacologique de l’AMPK améliore efficacement la stéatose hépatique. Ainsi, l’AMPK est une cible potentielle pour le développement d'activateurs dans le but de traiter la stéatose hépatique chez l’homme
Fatty liver disease affects between 20-40% of the population. This pathology is usually associated with metabolic disease. Its pathogenesis is poorly understood. Altered lipids metabolism in the liver resulting on hepatic fat accumulation is probably due to fatty liver. There is no specific treatment for fatty liver disease. AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism. In particular, AMPK regulates lipid metabolism by inhibiting fatty acids and cholesterol synthesis, and stimulating fatty acids oxidation. Several studies have shown an association between intracellular lipid accumulation and loss of AMPK activity in the liver. These observations suggest that AMPK may be a factor involved in the pathogenesis of hepatic steatosis. To investigate this hypothesis, we generated a new model of knockout mice lacking the catalytic subunits of AMPK α1 and α2 specifically in the liver. We analyzed the consequences of this deletion on lipid metabolism in different nutritional conditions. Deletion of AMPK in the liver does not affect hepatic triglyceride and cholesterol content in fasted or in refed conditions with a high carbohydrate diet. Also, lipogenic genes expression is not altered in the liver of these animals. Moreover, the oxidation of fatty acids is not impaired after 24 hour of fasting. Surprisingly, lacking AMPK specifically in the liver does not aggraving fatty liver, hyperglycemia, or impaired glucose tolerance when the mice are on high fat diet condition. However, the activation of AMPK in vivo with a direct activator, A-769662, normalizes hepatic steatosis in lipodystrophyc aP2-SREBP-1c mice and in obese mice placed on high-fat diet. This effect is AMPK dependent because it is completely abolished in mice lacking AMPK specifically in liver. In primary mice hepatocytes, AMPK activation by a direct activator (A-769662) or by indirect activators (metformin and AICAR) reduces lipogenesis rates and increases fatty acids oxidation rates. These effects were completely abolished in hepatocytes lacking AMPK, showing the specific action of AMPK on lipid metabolism in response to these compounds. These results obtained in mice can be extrapolated to humans. Indeed, we have shown that AMPK activation in primary humain hepatocytes inhibits effectively fatty acid and cholesterol synthesis rates. In conclusion, our results showed that inactivation of AMPK in the liver is not a triggering or an aggraving factor in the pathogenesis of hepatic steatosis. Nevertheless, AMPK re-activation has a therapeutic benefit for the treatment of fatty liver disease. Thus, AMPK is a potential target to treat fatty liver disease in human

Des études récentes ont montré que le microbiote participe à l’homéostasie intestinale en contribuant au développement morphologique, à l’éducation du système immunitaire, aux mécanismes de défense de l’hôte et à la régulation du métabolisme. Une dysbiose de ce microbiote ainsi qu’une réduction de la diversité bactérienne a été observé dans diverses pathologies chroniques telles que les maladies inflammatoires chroniques (MICI) et l’obésité. Le microbiote constitue donc une cible thérapeutique de choix dans la prise en charge de ces maladies chroniques. Les probiotiques, microorganismes bénéfiques pour l’hôte représentent une alternative intéressante, mais dont les critères de sélection nécessitent d’être améliorés.Dans une première étude, nous avons pu mettre en évidence les propriétés bénéfiques d’un mélange de deux probiotiques comprenant un bifide et un lactobacille dans un modèle murin d’obésité résultant d’une alimentation riche en graisses (Alard et al, 2016). Ce mélange probiotique a réduit significativement la prise de poids, amélioré les paramètres inflammatoires et métaboliques dont l’insulino-résistance, et augmenté l’expression intestinale des récepteurs aux acides gras à chaine courte (AGCC). Il a également favorisé dans un système d’intestin artificiel la production de butyrate et propionate ; principaux AGCCs. Les effets protecteurs ont été associés à l’amélioration de la dysbiose du microbiote, notamment la restauration de l’abondance d’Akkermansia muciniphila.L’objectif principal de cette thèse a été ensuite de sélectionner au sein d’une collection de 23 souches bactériennes provenant de la société PiLèJe, une ou plusieurs souche(s) probiotique(s) possédant des propriétés protectrices contre les MICI et l’obésité. Les propriétés immuno-modulatrices des souches ainsi que leur capacité à renforcer la barrière intestinale ont été étudiées in vitro à l’aide cellules mononuclées sanguines humaines, puis dans un modèle in vitro de perméabilité membranaire, induite par la sensibilisation d’une monocouche de cellules Caco-2 par de l’eau oxygénée. Six souches ont été sélectionnées, cinq souches induisant de forts niveaux de la cytokine anti-inflammatoire IL-10 et capables de restaurer la barrière intestinale et une souche capable de renforcer fortement cette barrière. Ces souches ont été ensuite évaluées en modèles in vivo de colite chronique et aigüe induite par du TNBS (2,4,6 trinitrobenzene sulfonic acid). De façon intéressante les souches protégeant en colite aigüe ne protègent pas aussi efficacement en colite chronique et inversement.Nous avons poursuivi la sélection de souches ou mélanges de souches dans le contexte de l’obésité et des maladies métaboliques associées. Nous avons utilisé les mêmes critères que précédemment (capacités anti-inflammatoires et à restaurer la barrière intestinale) complétés par l’étude de la capacité des souches à limiter l’accumulation des lipides dans un modèle in vitro de différenciation adipocytaire basé sur l’utilisation de la lignée 3T3-L1 et à induire la sécrétion de peptides entéro-endocrines impliqués notamment dans la satiété par l’utilisation de la lignée murine de cellules entéro-endocrine STC-1. Trois mélanges de souches et une souche seule ont été sélectionnées et évaluées dans un modèle murin d’obésité induite par un régime hyperlipidique à 45% de gras. Nous avons pu mettre en évidence des capacités positives d’un mélange de deux souches et d’une souche seule à réduire la prise de poids, ainsi que l’inflammation dans le tissu adipeux.Ces résultats indiquent que des criblages in vitro basés sur l’étude des propriétés immunomodulatrices, des capacités à restaurer la barrière, à diminuer l’accumulation des lipides et à induire des peptides de satiété, permettent une pré-sélection de souches ou mélanges de souches ayant un effet protecteur et démontrent à nouveau que les capacités bénéfiques des probiotiques sont souche-dépendantes et spécifiques des modèles ciblés
Recent studies have reported that the microbiota is involved in intestinal homeostasis by contributing to the morphological development, the education of the immune system, the mechanisms of defense, and to metabolic regulation. Dysbiosis of this microbiota as well as reduction in bacterial diversity has been observed in various chronic pathologies such as chronic inflammatory diseases (IBD) and obesity. The microbiota thus constitutes a therapeutic target of choice in the management of these chronic diseases. Probiotics, which are beneficial microorganisms for the host represent therefore an interesting alternative, however their selection criteria need to be improved.In a first study, we were able to highlight the beneficial properties of a mixture of two probiotics comprising a bifidobacteria and a lactobacilli, in a murine model of high fat diet (HFD)-induced obesity (Alard et al., 2016). This probiotic mixture significantly reduced weight gain, improved inflammatory and metabolic parameters including insulin resistance, and increased intestinal expression of receptors involved in short-chain fatty acid (AGCC) recognition. It also promoted in an artificial intestinal system the production of butyrate and propionate, the two main AGCCs. The protective effects were associated with the improvement of microbiota dysbiosis, in particular the restoration of the abundance of Akkermansia muciniphila.The main objective of this thesis was then to select within a collection of 23 bacterial strains provided by PiLèJe, one or more probiotic strain (s) possessing protective properties against IBD and obesity. Immunomodulatory properties of the strains and their ability to strengthen the intestinal barrier were studied in vitro using human mononuclear blood cells and an in vitro model of epithelial permeability induced by the sensitization of a Caco-2 cells monolayer with hydrogen peroxide. Six strains were selected, five strains inducing high levels of the anti-inflammatory cytokine IL-10 and capable of restoring the intestinal barrier and a strain capable of strongly reinforcing this barrier. These strains were then evaluated in in vivo models of TNBS (2,4,6 trinitrobenzene sulfonic acid)-induced chronic and acute colitis. Interestingly, strains able to rescue mice from acute colitis did not protect as efficiently in chronic colitis and vice versa.The selection of strains or mixtures was then pursued in the context of obesity and associated metabolic diseases. We used the same criteria as previously (anti-inflammatory capacities and to restore the intestinal barrier) in addition with the capacity of the strains to limit the accumulation of lipids in an in vitro model of adipocyte differentiation based on the use of the 3T3-L1 cell line and to induce the secretion of entero-endocrine peptides, notably involved in satiety, by the use of the murine STC-1 entero-endocrine cell line. Three mixtures and one single strain were selected and evaluated in a mouse model of obesity induced by 45% HFD diet. We demonstrated the positive capacities of a mixture composed of two strains and the single strain to reduce weight gain, as well as adipose tissue inflammation.These results indicate that in vitro screenings based on the immunomodulatory properties, the capacity to restore the gut barrier, to decrease lipid accumulation and to induce gut peptides allow pre-selection of strains or mixtures exhibiting protective effects and demonstrate that the beneficial capacities of probiotics are strain-dependent and specific to the targeted models

Le projet de cette thèse porte sur le développement de dépôts lipidiques ectopique et leur modulation suite à des intervenions thérapeutiques par imagerie résonance magnétique.Dans notre première étude, nous avons établi l’ordre chronologique d’apparition de graisses ectopiques et d’anomalies cardiaques dans un modèle de souris soumises à un régime riche en graisse et en sucre. Un traitement de courte durée à l’exendine-4 permet une amélioration de tous les paramètres altérés. Dans la deuxième étude, nous avons évalué l’impact d’un traitement de l’obésité sur les dépôts ectopique de graisse cardiaque (TAE et stéatose), hépatique et pancréatique à deux temps (6 mois et 32 mois) après chirurgie bariatrique. Nous avons montré que ce traitement chirurgical permet une réduction de tous ces dépôts, avec une cinétique différente. Enfin, dans la troisième étude, nous nous sommes intéressés à l’effet du poids de naissance sur le développement de tissu adipeux épicardique. Cette étude nous a permis de mettre en évidence qu’il existe une accumulation plus importante de TAE à l’âge adulte lorsque le poids de naissance est augmenté ; et que les paramètres poids de naissance et croissance entre 2 et 12 ans, jouent un rôle important dans la mise en place de ce dépôts de graisse ectopique. En somme, ces résultats permettent une avancée dans la compréhension du développement des dépôts de graisses et de leur modulation
The project of this thesis mainly focuses on ectopic lipid deposition development and their flexibility following therapeutic intervention. In our first study, we set out chronological order of ectopic fat onset and cardiac abnormalities in a high fat high sucrose mice model. Short duration exendin-4 treatment reverses every altered parameter. In the second study, we assessed treatment of obesity effect on cardiac ectopic fat deposition (EAT and steatosis), as well as hepatic and pancreatic fat at two different time points (6 months and 32 months) after bariatric surgery. We show significant reduction of every ectopic fat deposition, however in different kinetic. Finally, in a third study, we investigate birth weight effect on epicardial adipose tissue development. This study demonstrate important EAT accumulation in adulthood when birth weight is increased. Furthermore, birth weight and catch up growth in childhood between 2 and 12 years parameters impact significantly the development of epicardial fat.In summary, these results provide better understanding of ectopic fat deposition development and modulation

碩士
長庚大學
生物醫學研究所
101
Resveratrol (RSV, trans-3, 4’, 5-trihydroxystilbene), a phenolic compound, has been reported to have anti-oxidation, anti-inflammation, anti-platelet aggregation, anti-cancer, protecting against cardiac vascular disease, and promotion longevity. Recently, several researches pointed out that RSV might attenuate high-fat-diet (HFD) induced obesity; however, its underlying mechanisms are not fully elucidated. In the present study, the HFD-fed mice and 3T3-L1 preadipocytes were employed to further explore the anti-obese mechanisms of RSV. The 10-week-old male C57BL/6C mice were fed with HFD and treated HFD mice with or without RSV (1, 3, 10, or 30 mg/kg) for 10 weeks simultaneously. In RSV-treated groups, the body weight gain, adipose tissues weight and adipocytes size were reduced significantly compared to HFD fed mice, which demonstrated anti-obesity effect of RSV. In respect of in vitro study, RSV (10-100 μM) treatment significantly decreased cell viability and increased apoptosis in both 3T3-L1 preadipocytes and differentiated adipocytes. In contrast, RSV (100 μM) slightly reduced cell viability in matured adipocytes. RSV (1-10 μM) suppressed preadipocytes differentiation by decreasing PPARγ and perilipin protein expressions. RSV (0.1-100 μM) also inhibited TNF-induced lipolysis in mature adipocyte. Taking together, these results indicate that RSV exerted anti-obese effects may through inhibition of preadipocyte differentiation. Moreover, the anti-lipolysis of RSV may contribute to maintain the lipids storage function in matured adipocyte which might have potential to prevent ectopic lipid accumulation in other tissue organs.

博士
國立臺灣海洋大學
食品科學系
104
Gelidium amansii (GA) is an edible red algae that is distributed mainly in northeastern Taiwan. GA is widely used in food and chemical fields. The metabolic syndrome has become one of the major public-health challenges worldwide and in Taiwan. Hyperglycaemia, dyslipidaemia, hypertension and obesity were main risk factors for metabolic syndrome. In this study, we investigated the effect of GA and Gelidium amansii hot-water extract (GHE) on the metabolic syndrome by the animal models such as type 2 diabetes mellitus, hyperlipidemia and obesity models, respectively. Experiment (1): Diabetes is the fifth among the top ten leading causes of death in Taiwan. In Taiwan, an estimate shows that about ten thousand people died with diabetes every year. Type 2 diabetes accounts for 90-95% of patients with diabetes. After streptozotocin-nicotinamide induced rats can develop symptoms similar to human with type 2 diabetes. The first study was designed to investigate the effects of GA on plasma glucose, lipids and adipocytokines in rats with streptozotocin-nicotinamide-induced diabetes. Streptozotocin-nicotinamide induced diabetic rats were fed with 5% GA for 11 weeks, significantly reduced the fasting blood glucose level and improved the insulin resistance index HOMA-IR (homeostatic model assessment- insulin resistance). GA feeding significantly reduced the total cholesterol and triglycerides both in the plasma and liver. Furthermore, feeding of 5% GA can also significantly increase the excretion of fecal lipids and reduced liver weight, triglycerides and total cholesterol. Diabetic rats fed with GA could induce the rate of lipolysis and thus lower adipose tissue weight and thus reducing the inflammatory factors such as TNFα (tumor necrosis factor alpha), IL-6 (interleukin-6) and PAI-1 (plasminogen activator inhibitor type 1) concentrations. The results suggest that GA feeding can regulate plasma glucose and lipid levels and prevent adipose tissue accumulation in rats with diabetes. Experiment (2): Type 2 diabetes often combined with hyperlipidemia. The first part of study results show that feeding GA improved hyperlipidemia in diabetic rats. Therefore, the purpose of second part study was to investigate the effects of Gelidium amansii hot-water extracts (GHE) on lipid metabolism in hyperlipidmia hamsters. GHE is rich in water-soluble fiber (68.6%). Hyperlipidmia hamsters fed 1.5% GHE had lower triglycerides and total cholesterol concentrations both in plasma and liver after 6 weeks of feeding period. In addition, GHE feeding significantly decreased body weight, liver weight, and adipose tissue (perirenal and paraepididymal) weight. GHE supplementation increased the phosphorylation of AMPK (AMP-activated protein kinase) and reduced SREBP1 (sterol regulatory element binding protein 1) and SREBP2 protein expression, and thus reduced lipid synthesis enzyme activity such as FAS (fattyacid synthase) and ACC (acetyl-CoA carboxylase) in the liver. In addition, intaking of GHE can also significantly increase the excretion of total cholesterol, triglyceride and cholic acid. These results suggest that GHE feeding ameliorate lipid meatabolism in hyperlipidemia hamsters. Experiment (3): In the type 2 diabetes and hyperlipidemia animal model, feeding GA or GHE can improve plasma and liver lipid metabolism and have reduced the white adipose tissue weight. Therefore, the purpose of third study was to investigate the mechanism of GHE on plasma, liver lipids, adipocytokines and adipose tissues in high-fat diet induced obese hamsters. Obese hamster supplementation with 3% GHE extract for 9 weeks had decreased concentrations of plasma and liver total cholesterol, triglyceride and weight of the adipose tissue (paraepididymal and perirenal). Furthermore, feeding of GHE can also significantly increase the excretion of fecal total cholesterol and triglyceride. In addition, supplementation of GHE significantly reduced plasma levels of TNFα and IL-6. However, GHE supplementation had no effect on plasma concentrations of GLP-1 and food intake. GHE supplementation increased the phosphorylation of AMPK protein expression and increased hepatic levels of PPARα (peroxisome proliferator activated receptor alpha) and UCP 2 (uncoupling protein). Moreover, GHE feeding had higher HSL (hormone sensitive lipase) and lower LPL (lipoprotein lipase) enzyme activities in both paraepididymal and perirenal adipose tissues thus had lower triglyceride content of adipose tissues. In addition, hamsters feeding with GHE had lower body weight and adipose tissue than guar gum (GG), which due to the higher fecal total cholesterol excretion and adipose tissue HSL activity. Results from the present study show that GA and GHE can reduce the risk factors of metabolic syndrome.

碩士
中山醫學大學
營養學系碩士班
106
Obesity has become a known globalized issue. Accompanied with many negative effects on the human body, it is also a major contributing factor for many diseases. In recent years, scientists are constantly searching for alternative to prevent or reduce obesity. Mei-Gin is a concentrated extract of plum fruit which is proven to reduce blood triglyceride and blood sugar. However, a known issue of the functional components contained in the plum fruit could be destroyed during production at the long heated procedure. This experiment will be using combinations of Mei-Gin with different proportions of plum fruit extract, black garlic extract and hsian-tsao extract to recombine a new Mei-Gin formula and to investigate the anti-obesity effect of the Mei-Gin Formula. The experiment is divided into three parts: (1) Assess the anti-obesity potential of Mei-Gin formulas by using the in vitro cell model, and will select the most effective samples among the seven tested Mei-Gin formulas. (2) Evaluate the anti-obesity potential of Mei-Gin formula with the in vivo animal model. By using 6-week-old male Wistar rats as experimental animals, giving high-fat diet to induce obesity and are treated orally with different doses of Mei-Gin Formula-3 and -7 (100 and 300 mg/kg rat) to find most effective formula and it will be involved in the third part of the experiment. (3) Evaluate the anti-obesity effect of Mei-Gin Formula-7 with 6-week-old high-fat diet-induced obese male Wistar rats. The obese rats are treated orally with different doses of Mei-Gin Formula-7 (50, 100, and 300 mg/kg rat) to investigate the anti-obesity effects of Mei-Gin Formula-7. The results have shown that (1) 3T3-L1 cell which treated with Mei-Gin Formula-3 and -7 have significantly decreased in triglyceride and glycerol-3-phosphate dehydrogenase activity. Mei-Gin Formula-3 and 7 are the most effective samples among 7 kinds of Mei-Gin formulas. (2) In animal model, the results indicated that Mei-Gin Formula-7 has better anti-obesity potential compare to Mei-Gin Formula-3. Mei-Gin Formula-7 (100 and 300 mg/kg rat) has significantly reduced the final body weight, liver weight and total body fat of the high-fat diet-induced obese rats. (3) The results have shown that Mei-Gin Formula-7 has significantly reduced the final body weight, liver weight, total body fat, hepatic total lipid and increased the excretion of fecal total lipid. In gene expressions, treated with Mei-Gin Formula-7 upregulated the gene expressions of Sirt1, ATGL, FOXO1, HSL, ACO, CPT-1, PGC-1 beta, adiponectin, and UCP1, whereas it downregulated the gene expression of FAS, FATP-1, perilipin in the perirenal adipose tissue of high-fat diet-induced obese rats. In addition, Mei-Gin Formula-7 also upregulated the gene expression of Sirt1, ATGL, FOXO1, HSL, ACO, CPT-1, PGC-1 beta, PPAR-alpha, and AMPK in the liver of high-fat diet-induced obese rats. Moreover, Mei-Gin Formula-7 could upregulate the gene expression of C/EBPβ, PPAR-gamma, PRDM16, UCP-1, BMP7, PGC-1 alpha, AMPK, and Ebf2, but downregulated ACC gene expression in the brown adipose tissue of high-fat diet-induced obese rats. The results demonstrated the anti-obesity effect of Mei-Gin Formula-7, therefore Mei-Gin Formula-7 could be developed as an anti-obesity potential nutraceutical product.

Peptide YY (PYY) is a gut-derived hormone that is renowned for its effects on satiety. Reduced satiety in obese people has been attributed to low fasting and postprandial PYY levels. However, it has not been determined whether low PYY levels are the cause or the outcome of obesity. Moreover, the long-term role of PYY in regulating energy balance is unclear. Results presented in this thesis, using PYY-deficient mice (PYY-/-) and PYY transgenic mice (PYYtg) highlight that PYY indeed has an important role in regulating energy balance and glucose homeostasis in vivo. PYY knockout mice became obese with ageing or high-fat feeding linked to a hyperinsulinemic phenotype associated with hypersecretion of insulin from isolated pancreatic islets. These findings suggested that PYY deficiency may be a predisposing factor for the development of obesity and type 2 diabetes. On the other hand, PYYtg mice exhibited decreased adiposity and increased metabolism under high-fat feeding. Furthermore, PYYtg/ob mice had improved glucose tolerance and decreased adiposity. These latter studies suggested that high circulating PYY levels may protect against the development of obesity and type 2 diabetes. Interestingly, both animal models support PYY as an important regulator of the somatotropic axis. These preliminary findings prompted investigations in understanding whether low PYY levels may be a predisposing factor for the development of obesity and type 2 diabetes in human subjects. In a population of healthy human subjects that had a predisposition to the development of type 2 diabetes and obesity, fasting PYY levels were lower than in normal subjects. Moreover, low fasting PYY levels strongly correlated with decreased insulin sensitivity and high levels of fasting insulin. Collectively, these findings suggest that low circulating levels of PYY could contribute to increased adiposity, insulin resistance and type 2 diabetes. Therefore determination of PYY levels may be a method of detecting whether people are predisposed to becoming obese and insulin resistant. This work also suggests that treatments that enhance circulating PYY levels may be protective in the development of obesity and type 2 diabetes.

The foz/foz mouse is a murine model of Alstr{u00F6}m syndrome, a monogenetic disorder characterised in humans by childhood obesity, hearing loss, blindness, hyperinsulinaemia, early-onset type 2 diabetes and liver disease. In 2006, research from the host laboratory reported that foz/foz mice inherit a spontaneous mutation (foz) , an 11 base pair deletion in exon 8 of the Alms1 gene, and develop a similar phenotype to patients suffering from Alstr{u00F6}m syndrome. Thus, foz/foz mice are obese and exhibit high circulating insulin and leptin levels as well as fatty liver disease and metabolic syndrome. The purpose of the studies presented in this thesis was to further characterise the pathogenesis of obesity in foz/foz mice, by studying the role of Alms1 and hypothalamic appetite-regulating neuropeptide expression during the development of obesity. ALMS1 has been shown to localise at the base of primary cilia in what is termed the basal body or centrosome. Primary cilia are ubiquitously expressed hair-like organelles. Therefore, the first approach was to study primary cilia in the hypothalamus as well as hypothalamic Alms1 gene expression and Alms1 localisation in foz/foz and wildtype (WT) mice from birth until the obese phenotype is evident. At birth, foz/foz mice showed similar number of ciliated hypothalamic neurons to WT mice. However, from weaning and onwards the number of cilia was significantly decreased in foz/foz mice. In addition, while cilia were present in primary neuronal cultures from foz/foz and WT mice, Alms1 was only detected in WT neurons, appearing as two perinuclear dots at the base of cilia. After weaning, serum leptin levels become greatly elevated in foz/foz compared to WT mice. Leptin decreases appetite by acting in the hypothalamus and inducing or inhibiting the release of appetite-regulating neuropeptides. A detailed study of key hypothalamic neuropeptides demonstrated no differences in their gene and/or protein expression or localisation between foz/foz and WT mice. This failure of elevated leptin levels to decrease appetite and body weight is defined as leptin resistance. Further studies were therefore performed on hypothalamic leptin receptor (Ob-R) expression and signalling pathways to characterise leptin resistance in foz/foz mice. These results demonstrated induction of two proteins, SOCS3 and PTP1B, which negatively regulate Ob-R signalling and have been implicated in leptin resistance. Taken together, the data presented in this thesis strongly support the proposal that foz/foz mice develop leptin resistance, which correlates molecularly with over-expression of at least two negative feedback regulators of Ob-R. In addition, the post-natal reduction in ciliated hypothalamic neurons in foz/foz mice, in combination with the lack of Alms1 detection are consistent with the proposal that primary cilia stability/maintenance could be impaired as a consequence of the Alms1 mutation. In conclusion, foz/foz mice provide new opportunities for studying the role of Alms1 and neuronal cilia in appetite regulation, particularly with respect to the onset of leptin resistance. A better understanding of Alms1, cilial stability and behavioural responses that underlie obesity could provide clues to novel therapeutic approaches to combat more common forms of obesity.

Lam Tsz Yan.
Thesis submitted in: October 2007.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references.
Abstracts in English and Chinese.
Abstract (English) --- p.i
論文摘要 --- p.iv
Acknowledgements --- p.vi
Publications --- p.vii
Abbreviations --- p.ix
Contents --- p.x
Chapter 1. --- General Introduction --- p.1
Chapter 1.1. --- Obesity --- p.1
Chapter 1.1.1. --- Overview --- p.1
Chapter 1.1.2. --- Pathophysiology --- p.1
Chapter 1.1.3. --- Central obesity --- p.3
Chapter 1.2. --- Diabetes --- p.7
Chapter 1.2.1. --- Overview --- p.7
Chapter 1.2.2. --- Pathophysiology --- p.8
Chapter 1.3. --- Lipolysis --- p.9
Chapter 1.3.1. --- Proteins participating in triglyceride lipolysis --- p.10
Chapter 1.3.1.1. --- Hormone-sensitive lipase (HSL) --- p.10
Chapter 1.3.1.2. --- Adipose triglyceride lipase (ATGL) --- p.10
Chapter 1.3.1.3. --- Perilipins --- p.11
Chapter 1.3.2. --- Abnormal regulation of lipolysis in obesity --- p.11
Chapter 1.3.3. --- Disturbed lipolysis in insulin resistance --- p.13
Chapter 1.4. --- Pharmacotherapy --- p.13
Chapter 1.4.1. --- Obesity --- p.13
Chapter 1.4.1.1. --- Orlistat --- p.13
Chapter 1.4.1.2. --- Sibutramine --- p.14
Chapter 1.4.1.3. --- Others --- p.15
Chapter 1.4.2. --- Diabetes --- p.15
Chapter 1.4.2.1. --- Modulation of the β-cells functions --- p.15
Chapter 1.4.2.2. --- Control of glucose output --- p.16
Chapter 1.4.2.3. --- Modulation of carbohydrate absorption --- p.16
Chapter 1.4.2.4. --- Thiazolidinediones (TZDs) --- p.16
Chapter 1.5. --- Animal models used in type 2 diabetes and obesity research --- p.17
Chapter 1.6. --- Aim of study --- p.18
Chapter 2. --- β-Adrenoceptors (β-ARs) --- p.21
Chapter 2.1. --- Introduction --- p.21
Chapter 2.1.1. --- Hormonal control of lipolysis --- p.21
Chapter 2.1.1.1. --- Catecholamines --- p.21
Chapter 2.1.1.2. --- Insulin --- p.23
Chapter 2.1.2. --- Folic acid (folate) --- p.23
Chapter 2.1.2.1. --- Physiological roles of folate --- p.23
Chapter 2.1.2.2. --- Folate deficiency and its consequences --- p.24
Chapter 2.1.2.3. --- Hyperhomocysteinemia --- p.24
Chapter 2.1.2.4. --- Pleiotropic effects of folate --- p.25
Chapter 2.1.2.5. --- Role of folate in type 2 diabetes and obesity --- p.26
Chapter 2.1.3. --- Lingzhi --- p.28
Chapter 2.1.3.1. --- Triterpenoids --- p.29
Chapter 2.1.3.2. --- Polysaccharides --- p.30
Chapter 2.2. --- Materials and methods --- p.32
Chapter 2.2.1. --- Materials --- p.32
Chapter 2.2.1.1. --- Composition of physiological salt solution --- p.32
Chapter 2.2.1.2. --- Materials used in lipolysis experiment --- p.32
Chapter 2.2.1.3. --- Materials used in reverse transcription polymerase chain reaction (RT-PCR) --- p.34
Chapter 2.2.1.4. --- Materials used in Western blotting --- p.34
Chapter 2.2.2. --- Methods --- p.36
Chapter 2.2.2.1. --- Lipolysis experiment --- p.36
Chapter 2.2.2.1.1. --- Animals --- p.36
Chapter 2.2.2.1.2. --- Drug administration --- p.36
Chapter 2.2.2.1.3. --- Isolation of adipocytes --- p.37
Chapter 2.2.2.1.4. --- Lipolysis measurement --- p.37
Chapter 2.2.2.1.5. --- Data analysis --- p.38
Chapter 2.2.2.2. --- RT-PCR --- p.38
Chapter 2.2.2.2.1. --- Tissue preparation --- p.39
Chapter 2.2.2.2.2. --- RNA extraction --- p.39
Chapter 2.2.2.2.3. --- Reverse transcription (RT) --- p.40
Chapter 2.2.2.2.4. --- Polymerase chain reaction (PCR) --- p.40
Chapter 2.2.2.2.5. --- Agarose gel electrophoresis --- p.41
Chapter 2.2.2.2.6. --- Data representation and analysis --- p.41
Chapter 2.2.2.3. --- Western blotting --- p.42
Chapter 2.2.2.3.1. --- Tissue preparation --- p.42
Chapter 2.2.2.3.2. --- Protein extraction --- p.42
Chapter 2.2.2.3.3. --- Western blotting --- p.42
Chapter 2.2.2.3.4. --- Data representation and analysis --- p.43
Chapter 2.3. --- Results --- p.43
Chapter 2.3.1. --- Studies on the β-adrenoceptor-mediated lipolytic response in +m/+db and +db/+db mice --- p.43
Chapter 2.3.1.1. --- Effect of β2-adrenoceptor agonist on lipolysis --- p.43
Chapter 2.3.1.2. --- Effect of β3-adrenoceptor agonists and their antagonists on lipolysis --- p.44
Chapter 2.3.1.3. --- Effect of non-selective β-adrenoceptor agonists and their antagonists on lipolysis --- p.45
Chapter 2.3.1.4. --- Effect of modulators of intracellular cyclic nucleotide monophosphate on lipolysis --- p.46
Chapter 2.3.1.5. --- Effect of exogenously delivered nitric oxide on lipolysis --- p.47
Chapter 2.3.1.6. --- Gene expression of β-adrenoceptors in white adipose tissue --- p.47
Chapter 2.3.1.7. --- Protein expression of β-adrenoceptors in white adipose tissue --- p.47
Chapter 2.3.2. --- Effect of folic acid treatment on lipolysis --- p.48
Chapter 2.3.2.1. --- Determination of body weight --- p.48
Chapter 2.3.2.2. --- Effect of β2-adrenoceptor agonist on lipolysis --- p.48
Chapter 2.3.2.3. --- Effect of β-adrenoceptor agonists on lipolysis --- p.49
Chapter 2.3.2.4. --- Effect of non-selective β-adrenoceptor agonist on lipolysis --- p.50
Chapter 2.3.2.5. --- Effect of modulators of intracellular cyclic nucleotide monophosphate on lipolysis --- p.51
Chapter 2.3.2.6. --- Effect of exogenously delivered nitric oxide on lipolysis --- p.52
Chapter 2.3.2.7. --- Gene expression of β-adrenoceptors in white adipose tissue --- p.52
Chapter 2.3.2.8. --- Protein expression of β-adrenoceptors in white adipose tissue --- p.53
Chapter 2.3.3. --- Effect of Lingzhi (water-extract) treatment on lipolysis --- p.54
Chapter 2.3.3.1. --- Determination of body weight --- p.54
Chapter 2.3.3.2. --- Lipolytic effect of forskolin --- p.54
Chapter 3. --- Peroxisome Proliferator-Activated Receptor-y (PPAR-γ) --- p.91
Chapter 3.1. --- Introduction --- p.91
Chapter 3.1.1. --- Peroxisome proliferator-activated receptors --- p.91
Chapter 3.1.1.1. --- Peroxisome proliferator-activated receptor-γ --- p.91
Chapter 3.1.1.1.1. --- "PPAR-γ in obesity, lipid metabolism and type 2 diabetes" --- p.91
Chapter 3.1.1.1.2. --- PPAR-γ in inflammation and atherosclerosis --- p.92
Chapter 3.1.1.2. --- PPAR-γ and thiazolidinediones --- p.93
Chapter 3.2. --- Materials and method --- p.95
Chapter 3.2.1. --- Materials --- p.95
Chapter 3.2.1.1. --- Composition of physiological salt solution --- p.95
Chapter 3.2.1.2. --- Materials used in lipolysis experiment --- p.95
Chapter 3.2.1.3. --- Materials used in RT-PCR --- p.95
Chapter 3.2.1.4. --- Materials used in Western blotting --- p.95
Chapter 3.2.2. --- Methods --- p.96
Chapter 3.2.2.1. --- Lipolysis experiment --- p.96
Chapter 3.2.2.2. --- RT-PCR --- p.96
Chapter 3.2.2.3. --- Western blotting --- p.97
Chapter 3.3. --- Results --- p.97
Chapter 3.3.1. --- Effect of PPAR-γ agonists on lipolysis --- p.97
Chapter 3.3.2. --- Gene expression of PPAR-γ in white adipose tissue --- p.97
Chapter 3.3.3. --- Protein expression of PPAR-γ in white adipose tissue --- p.97
Chapter 4. --- 3-Hydoxy-3-MethylgIutaryl Coenzyme A (HMG-CoA) Reductase --- p.106
Chapter 4.1. --- Introduction --- p.106
Chapter 4.1.1. --- Cholesterol metabolism and cardiovascular diseases --- p.106
Chapter 4.1.2. --- Statins --- p.106
Chapter 4.1.2.1. --- Modes of action --- p.107
Chapter 4.1.2.2. --- Therapeutic efficacy of statins --- p.108
Chapter 4.1.2.2.1. --- Diabetes --- p.108
Chapter 4.1.2.2.2. --- Coronary artery disease --- p.109
Chapter 4.1.3. --- Distribution and expression of HMG-CoA reductase --- p.109
Chapter 4.2. --- Materials and method --- p.110
Chapter 4.2.1. --- Materials --- p.110
Chapter 4.2.1.1. --- Composition of physiological salt solution --- p.110
Chapter 4.2.1.2. --- Materials used in lipolysis experiment --- p.110
Chapter 4.2.1.3. --- Materials used in RT-PCR --- p.110
Chapter 4.2.1.4. --- Materials used in Western blotting --- p.110
Chapter 4.2.2. --- Methods --- p.110
Chapter 4.2.2.1. --- Lipolysis experiment --- p.110
Chapter 4.2.2.2. --- RT-PCR --- p.111
Chapter 4.2.2.3. --- Western blotting --- p.111
Chapter 4.3. --- Results --- p.112
Chapter 4.3.1. --- Effect of statins on lipolysis --- p.112
Chapter 4.3.2. --- Gene expression of HMG-CoA reductase in various internal organs --- p.112
Chapter 4.3.3. --- Protein expression of HMG-CoA reductase in various internal organs --- p.113
Chapter 5. --- Discussion --- p.122
Chapter 5.1. --- β-adrenoceptor-mediated lipolysis --- p.122
Chapter 5.2. --- Studies on peroxisome proliferator-activated receptor-γ --- p.140
Chapter 5.3. --- Studies on HMG-CoA reductase --- p.142
Chapter 5.4. --- Further studies --- p.147
Chapter 5.5. --- Conclusions --- p.148
Chapter 6. --- References --- p.152