Dissertations / Theses on the topic 'Glucose metabolism'

The current theories about endothelial inflammation in the adipose tissue in metabolic syndrome point to hypoxia as one of its main causes. Nevertheless, our group has found that both 3T3L1 adipocytes, under normoxic conditions, and WAT, in vivo, consume large amount of glucose, with high production of lactate and glycerol irrespective of oxygen availability. This thesis is based on the hypothesis that adipocytes act essentially as glycolytic cells impervious to hypoxia, and their metabolism may help reduce the blood glucose levels. We assumed that hypoxia, could affect the cells of the stromal fraction, eliciting an inflammatory response. To this purpose, we have studied ex vivo the glycolytic and anaerobic capacity of adult adipocytes and stromal vascular cells of both sex and obtained from different WAT sites, developing the methodology needed for a quantitative comparative analysis of data obtained from the same cultured cells well. We found that adipocytes, despite being the cells present in WAT in lower numbers, occupied almost the whole volume of the tissue, a consequence of their huge size due to their inert fat vacuole. The overall “live cell" volume represented only about 1.5% of the tissue, thus showing a very high metabolic activity of WAT in relative terms. Adipocytes ex vivo incubated with glucose, also took large amounts of the sugar, irrespective of its concentration, releasing instead 3C metabolites, such as lactate and glycerol to the medium. Lactate was fully derived from glucose and was produced at a steady pace, irrespective the presence of oxygen, to form the ATP needed for cell functions. Glycerol efflux increased over time and its origin shifted from glycolitic to glycolytic-lipolytic: new-formed glycerol was incorporated into TAG, by esterification with acyl-CoA, derived from the same TAG lipolisis. The coexistence of these processes appears as a “futile cycle”, with the probable function further to waste excess energy. Lipogenesis was limited because the size of cells limits the access to oxidative mitochondrial pathways. The release of 3C metabolites seems to be a mechanism to lower glycemia, defend WAT against excess of substrate, and provide 3C fragments as more accessible substrates for other tissues. Mesenteric WAT adipocytes presented the highest metabolic activity, probably to help the hepatic handling of NEFA and reduce the flow of intestinal glucose to the liver. While in almost all WAT sites, excess mitochondrial pyruvate is returned to the cytoplasm to keep forming lactate; in female mesenteric adipocytes it is in part oxidized to acetyl-CoA to fuel lipogenesis. Stromal vascular cells also released lactate, even more than adipocytes per unit of tissue weight, but not glycerol nor NEFA. Red blood cells produced lactate, but its contribution was quantitatively minimal. Thus, stromal cells, acted in consonance with adipocytes, in all sites and sexes examined, wasting glucose in an anaerobic way, producing high amounts of 3C units. Thus, reinforcing the idea that WAT may be an active protagonist both in energy handling and in the body control of glycemia.
Las teorías actuales sobre la inflamación endotelial en el tejido adiposo en el síndrome metabólico apuntan a la hipoxia como una de sus causas. Nuestro grupo ha encontrado que adipocitos 3T3L1, bajo condiciones normóxicas, consumen grandes cantidades de glucosa, con elevada producción de lactato y glicerol. La hipótesis de esta tesis es que los adipocitos actuan como células glucolíticas y su metabolismo ayuda a reducir la glucemia, mientre la hipoxia podría afectar las células estromales. Hemos estudiado ex vivo la capacidad glucolítica de adipocitos adultos y células estromales de ambos sexos y de diferentes ubicaciones del TAB, desarrollando la metodología para un análisis cuantitativo. Hallamos que los adipocitos eran las células menos númerosas del tejido y la parte "viva" representaba solo el 1,5% del total, mostrando así el TAB una actividad metabólica muy elevada. Los adipocitos ex vivo captaban grandes cantidades de glucosa, independientemente de su concentración; liberando metabolitos de 3C. El lactato, liberado a ritmo constante y independientemente de la presencia de oxígeno, procedía totalmente de la glucosa. El eflujo de glicerol aumentó con el tiempo y su origen cambió de glucolítico a glucolítico-lipolítico. El glicerol neoformado se incorporaba a los TAG, reciclado acil-CoA de la misma lipolisis. Este"ciclo fútil", probablemente sirve para desperdiciar e energía, disminuir la glucemia, y proporcionar sustratos energéticos para otros tejidos. Los adipocitos del TAB presentaban la más alta actividad metabólica; probablemente para ayudar al manejo hepático de ácidos grasos y reducir el flujo de glucosa intestinal. Mientras que en todas las localizaciones, el exceso de piruvato mitocondrial regresaba al citoplasma y seguía formando lactato; en los adipocitos de las hembras de mesentérico, parte se oxidaba a acetil-CoA para sostener la lipogénesis. Las células estromales, por unidad de tejido, liberaban más lactato que los adipocitos, pero no glicerol ni ácidos grasos. Los eritrocitos produjeron mínimas cantidades de lactato.Así las células estromales, actúan en consonancia con los adipocitos, en todas las localizaciones estudiadas y en ambos sexos. Estos resultados refuerzan la idea de que el TAB puede ser un protagonista activo en el manejo de la energía y en el control de la glucemia.

Under normal conditions, non-transformed cells rely on glycolysis followed by oxidative phosphorylation to generate ATPs. When oxygen is scarce or when cells are actively proliferating, cellular ATPs come mainly from glycolysis. Pyruvate is converted into lactate to allow glycolysis to continue. Interestingly, cancer cells have adapted to favor lactate production even at normal oxygen tensions, exhibiting a metabolic shift known as the Warburg effect. However, the metabolic state of other cellular constituents within the tumor remains mostly unknown. Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells. They aid tumor growth and metastasis by providing growth factors, cytokine, ECM remodeling proteins and interacting with other tumor stromal cells. Here I show that the Warburg effect also operates in stromal fibroblasts of the tumor microenvironment. Using mass spectrometry, genetic mouse models, gene expression and methylation studies, I demonstrate that CAFs from human and mouse mammary tumors exhibit hyperactive glycolysis and a metabolic shift towards lactate production. Furthermore, this phenotype may be sustained through epigenetic modifications of endogenous hypoxia-inducible factor 1α, key regulatory enzymes fructose-bisphosphatase 1 and pyruvate kinase M2. Depletion of stromal fibroblasts or suppression of lactate production specifically in these cells alters the metabolic profile of not only the tumors but also the cancer cells and results in impeded tumor growth. These results collectively suggest that tumor growth is dependent on metabolic state and metabolic support of stromal fibroblasts, highlighting these cells as attractive therapeutic targets in controlling cancer progression.

Type II diabetes is one of the leading cause of morbidity in the U.S. and other parts of the world. Insulin resistance which precedes Type II diabetes is a complex state of the body where the body fails to respond to insulin. Its complexity lies in its multifactorial origin that is to say various environmental and polygenic components come into play. Here we try to dissect one of these components - `Alox8' in transgenic mice and try to see if it affects blood glucose homeostasis. Comparison of glucose tolerance and insulin sensitivity among sixteen mice comprising of six wild type, five heterozygous and five knockout mice with respect to Alox8 gene showed that wild type mice had relatively more glucose tolerance than knockout mice and this corresponded with relatively more insulin sensitiveness of wild type mice with respect to the knock out. However, these findings were not significant statistically at p=0.05. In search of any relevant biological significance, periodic acid schiff staining of the liver sections from these mice in three independent repeated experiments revealed that the knockout phenotype led to accumulation of glycogen deposits as compared to the wild type mice, an indication of insulin resistance. Taken together, our data suggests that these findings when extrapolated to human which carries ALOX15B instead of mice orthologue Alox8, could lead to a benefit of administration of lower doses of insulin in the wild type phenotype as compared to its polymorphic alleles carrying individuals.

La actividad sináptica regula una larga lista de procesos esenciales para el desarrollo y la supervivencia neuronal mediante cambios en los niveles de Ca2+ citoplasmático. Modula desde la morfología del árbol dendrítico neuronal hasta la plasticidad neuronal o los mecanismos de protección contra el daño oxidativo. La entrada de Ca2+ a través de los receptores sinápticos promueve diferentes cambios a nivel celular que van desde la remodelación del citoesqueleto a la estimulación de diferentes cascadas de señalización que finalmente activan diferentes factores de transcripción implicados en la regulación de toda una serie de programas de transcripción. Durante las primeras semanas tras el nacimiento tiene lugar el período, enmarcado dentro del desarrollo del sistema nervioso, en que se da una mayor tasa de crecimiento de dendritas y axones. El crecimiento neuronal debe ir acompañado de la síntesis de nuevos lípidos, necesarios para la generación de nuevas membranas. A pesar de que los mecanismos implicados en la regulación del crecimiento de dendritas y axones han sido ampliamente estudiados, se conoce muy poco con respecto a los cambios a nivel metabólico destinados a la síntesis de las biomoléculas necesarias para abastecer la generación de nuevas membranas. El principal objetivo de este trabajo ha sido estudiar el mecanismo por el que la actividad sináptica es capaz de regular el metabolismo neuronal para promover la síntesis de diferentes metabolitos precursores para la síntesis de los lípidos necesarios para el crecimiento neurítico. El principal modelo utilizado en este estudio ha sido los cultivos primarios de neuronas corticales de embriones de rata. La actividad sináptica se ha estimulado mediante un protocolo basado en la desinhibición de la red neuronal, que consiste en el tratamiento combinado con Bicuculina y 4-aminopiridina. Los resultados de esta tesis demuestran que la actividad sináptica estimula la captación y el metabolismo de la glucosa incrementando la transcripción del principal transportador de glucosa en neuronas (Glut3) y de diferentes enzimas implicadas en la glucólisis (HK2, PKM1/2 y PFKFB3). Todo el proceso está regulado por un mecanismo caracterizado por la activación de dos factores de transcripción: CREB y HIF-1α. La activación de CREB, uno de los principales factores de transcripción regulados por la actividad sináptica, promueve la expresión de Glut3 y de la enzima ubiquitina ligasa, Siah2. La actividad de esta última es necesaria para promover la estabilización y, en consecuencia, la activación de HIF-1α, que finalmente promueve la expresión de las enzimas implicadas en la glucólisis. La inhibición de la glucólisis o el bloqueo de la actividad de HIF-1α son suficientes para inhibir la estimulación del crecimiento neuronal por la actividad sináptica. De acuerdo con estos resultados en cultivos celulares, la inhibición de esta vía o la eliminación de este factor de transcripción durante las primeras semanas de desarrollo postnatal en roedores tiene como consecuencia una reducción de la complejidad del árbol dendrítico. Tal como se muestra en el presente trabajo, la actividad sináptica también podría estar implicada en la regulación de diferentes orgánulos como son los peroxisomas y las mitocondrias. En el primer caso se ha observado que la actividad sináptica promueve la síntesis de diferentes implicados tanto el desarrollo y el mantenimiento de los peroxisomas (Pex5, Pex11b y Pex13) como en el metabolismo lipídico peroxisomal (ABCD2 y ACOT8). Cuanto a las mitocondrias se ha comprobado que las neuronas estimuladas sinápticamente activas presentan un incremento en el transporte anterógrado mitocondrial a lo largo del axón que depende de la actividad de HIF-1α.
During the first weeks after birth occurs the period of the nervous system development in which takes place the highest dendritic and axonal growth ratio. Neuronal growth must be accompanied by the synthesis of new lipids, which are necessary for the formation of new membranes. Although the mechanisms involved in the regulation of dendrites and axons growth have been widely studied, there is very little known about the changes at the metabolic level involved in the synthesis of the biomolecules necessary to supply the formation of new membranes. The main goal of this doctoral thesis has been studying the mechanism by which synaptic activity, one of the most important inducers of neurite growth, regulates neuronal metabolism to promote the synthesis of different precursor metabolites involved in the synthesis of lipids required for neuritic growth. The results of this thesis show that synaptic activity stimulates glucose uptake and metabolism by increasing the transcription of the main glucose transporter in neurons, Glut3 and different enzymes involved in glycolysis. The whole process is regulated by a mechanism characterized by the activation of two transcription factors: CREB and HIF-1α. The activation of CREB, one of the main transcription factors regulated by synaptic activity, promotes the expression of Glut3 and the ubiquitin-protein ligase, Siah2. The activity of the latter is necessary to promote the stabilization and, consequently, the activation of HIF-1α, which finally promotes the expression of enzymes involved in glycolysis. Glycolysis inhibition or blocking of HIF-1α activity are sufficient to inhibit stimulation of neuronal growth by synaptic activity. A shown in this thesis, synaptic activity may also be involved in the regulation of peroxisomes and mitochondria. In the first case, it has been observed that synaptic activity promotes the synthesis of different agents involved in the development and maintenance of peroxisomes and in peroxisomal lipid metabolism. In regard to mitochondria, it has been proven that synaptically active neurons exhibit an increase in mitochondrial anterograde transport along the axon which requires HIF-1α activity.

Abstract Type 1 diabetes is considered to be a T cell-mediated autoimmune disease characterized by destruction of the pancreatic beta cells. Its prediction is currently based on diabetes-associated autoantibodies, giving a cumulative risk of 84% during 15 years of follow-up since seroconversion. Prediction of the timing of clinical onset has remained challenging, however. This thesis examines glucose metabolism in autoantibody-positive children with a high risk of developing type 1 diabetes. Out of a total of 14,876 children with an increased genetic risk followed up from birth in the Finnish DIPP study, 567 developed ≥2 autoantibodies during the follow-up and 255 of these (45%) were diagnosed with type 1 diabetes until the end of December 2011. The glucose parameters measured were HbA1c, OGTT and random plasma glucose with 3 to 12 months interval. Seven-day continuous glucose monitoring (CGM) was performed on an age and sex-matched cohort. We showed that rising HbA1c, impaired glucose tolerance in OGTT, random plasma glucose values of ≥7.8mmol/l and potentially CGM can predict type 1 diabetes with a median time to diagnosis of approximately one year. Our results suggest that especially HbA1c and random plasma glucose are cost-effective and improve the prediction of diabetes. These markers may be useful for monitoring the response to treatment in prevention studies
Tiivistelmä Tyypin 1 diabetesta pidetään T-soluvälitteisenä autoimmuunitautina, joka johtaa haiman beetasolujen tuhoutumiseen. Tyypin 1 diabeteksen ennustaminen perustuu tällä hetkellä diabetekseen assosioituviin vasta-aineisiin, jotka antavat 84% kumulatiivisen riskin 15 vuoden seurannassa. Taudin puhkeamisen ajankohdan ennustaminen on kuitenkin edelleen vaikeaa. Tämä väitöskirja käsittelee glukoosiaineenvaihduntaa vasta-ainepositiivisilla lapsilla, joilla on suurentunut riski sairastua tyypin 1 diabetekseen. Suomalaisessa DIPP-tutkimuksessa vasta-aineiden kehittymistä on seurattu yhteensä 14876 lapselta. Seurannan aikana 567 lasta kehitti ≥2 autovasta-ainetta ja näistä 255 (45%) sairastui tyypin 1 diabetekseen joulukuun loppuun 2011 mennessä. Glukoosiaineenvaihduntaa seurattiin tutkimalla HbA1c, OGTT ja satunnaisia verensokeriarvoja 3-12 kuukauden välein. Ikä ja sukupuolivakioidussa kohortissa tehtiin jatkuvan sokeripitoisuuden seuranta (CGM). Tutkimuksessamme nouseva HbA1c, heikentynyt sokerin sieto OGTT-kokeessa, satunnainen verensokeri ≥7.8 mmol/l ja mahdollisesti CGM ennustavat tyypin 1 diabeteksen puhkeamista. Tulostemme perusteella erityisesti kustannustehokkaat HbA1c ja satunnainen verensokeri parantavat diabeteksen ennustamista. Nämä parametrit saattavat olla hyödyllisiä myös preventiotutkimuksissa hoitovasteen seurannassa

It has been suggested that coffee drinking may confer a beneficial effect on health by reducing the risk of developing Type 2 Diabetes Mellitus (T2DM) and indeed there is much epidemiological evidence for a reduced incidence of T2DM in habitual coffee drinkers. However, many acute studies have reported a temporary worsening in postprandial glycaemia following caffeinated coffee (CC) consumption. Varied methodologies have been employed by these studies with many giving their participants large doses of coffee. In the acute studies conducted for this thesis, a single serving of CC increased the postprandial glycaemic response more than control (p=0.008), with no apparent dose-response effect. Furthermore, a single serving of decaffeinated coffee (DC) consumed in the morning, produced no effect on postprandial glycaemia, although a trend was observed for a reduction in the postprandial glucose peak (p=0.060) when DC was consumed at lunchtime. The majority of longer-term investigations have recruited habitual coffee drinkers who are likely to have already obtained any potential benefits of coffee consumption. The longer-term intervention reported in this thesis found no overall effects of twelve weeks of CC drinking on glucose and lipid metabolism in coffee-naïve individuals. However, differences were observed between fast and slow caffeine metabolisers when the analysis was split by phenotype. The fast caffeine metabolisers displayed a lower postprandial glucose response (p=0.019) and greater NEFA suppression (p=0.001) at baseline. Furthermore, significant interaction effects were observed between visit and phenotype for postprandial glucose (p=0.048) and NEFA (p=0.019), with the intervention producing an apparent increase in postprandial glycaemia in fast metabolisers and reduced NEFA suppression in slow metabolisers. In conclusion, no evidence was found for a beneficial effect of coffee drinking on glucose and lipid metabolism in the general population, however, individual differences in response to longer-term coffee drinking were observed which warrant further investigation.

Glucose is a key nutrient for T cells. Despite that T cell activation is impaired when they are deprived of glucose, it has also been shown that T effector responses can be elicited in vivo in glucose-poor environments, such as the intratumoral niche, which raises the question of how these cells can maintain their function in nutrient-restricted sites. In this work, we analyzed the ability of T effector cells to be activated by pro-inflammatory polarizing conditions under limiting glucose availability, using an in vitro model in which effector Th0 cells were restimulated to secondary effector T cells in glucose deficiency (0.3 mM). We found that secondary effector T cells could express characteristic Th1 and Th17 cytokines such as IL-17A and IFNγ when exposed to low glucose, but they lost expression of IL-22. Secondary effector T cells adapted to low glucose by reducing their rate of glucose consumption and expression of glycolysis genes, although they still kept using glucose as the main fuel for ATP production. In addition, we found that glucose limitation caused a mild, progressive impairment on mTORC1 activity in these cells that explained in part the downregulation of IL-22, an mTORC1-dependent cytokine. Our results also showed that secondary effector T cells that had experienced glucose stress acquired a nutrient-trained phenotype, and when they were later restimulated under glucose sufficiency they induced an altered cytokine expression pattern with exacerbated production of IL-22 and reduced IFNγ production. Finally, we observed that effector CD4+ T cells generated in different activation contexts in vivo exhibited different patterns of glucose-sensitive genes upon restimulation ex vivo, which suggested that the context in which T effector cells are induced might be a relevant determinant in shaping different response patterns to glucose limitation upon further stimulation. Altogether, our results uncover a previously unappreciated robustness of T cells to maintain effector function under nutrient-restricted conditions, also revealing that a prior history of nutrient stress can influence future effector T cell responses.
La glucosa és un nutrient essencial per les cèl·lules T. Malgrat que l’activació T es veu disminuïda per la manca de glucosa, s’ha vist que respostes T efectores tenen lloc in vivo en entorns amb nivells baixos de glucosa, com són els tumors. Això planteja la incògnita de saber com aquestes cèl·lules poden mantenir les seves funcions en ambients pobres de nutrients. En aquest treball hem analitzat la capacitat de les cèl·lules T efectores (Th0) de ser activades en condicions pro-inflamatòries i nivells baixos de glucosa (0.3 mM). Hem vist que les cèl·lules T efectores secundàries poden induir citocines característiques de respostes Th1 i Th17 com la IL-17A i l’IFNγ en condicions de nivells baixos de glucosa, però perden la capacitat d’expressar la IL-22. Aquestes cèl·lules s’adapten a un entorn baix de glucosa reduint-ne el consum i reduint l’expressió de gens de la glicòlisi, malgrat tot, la glucosa segueix sent la seva principal font d’energia (ATP). A més a més, hem observat que nivells limitats de glucosa provoquen una lleu però progressiva deficiència en l’activitat d’mTORC1, necessària per la producció de la IL-22 i que explicaria en part la disminució dels nivells d’aquesta citocina. Els nostres resultats també mostren que les cèl·lules T efectores secundàries que han experimentat un estrès de glucosa adquireixen un fenotip de memòria que fa que responguin de manera alterada (producció exagerada de IL-22) a un segon estímul en presència de nivells normals de glucosa. Finalment, hem observat que les cèl·lules T CD4 efectores activades in vivo expressen diferencialment gens sensibles a glucosa quan són re-estimulades ex vivo. Això suggereix que el context d’activació d’una cèl·lula T és important per determinar la resposta d’aquestes cèl·lules a posteriors estimulacions en situació de baixa glucosa. En resum, els nostres resultats mostren que els limfòcits T son capaços de mantenir un ventall de funcions efectores en situacions de restricció de nutrients, però que el haver passat per una etapa d’estrès de nutrients pot condicionar els seus perfils d’expressió gènica en respostes efectores futures.

Low back pain is a major social and economic dilemma in the United States. Despite its high impact, the origins of low back remain unclear. Nonetheless, degenerative changes to the intervertebral discs (IVD) of the spine have been implicated as a possible source leading to pain. Poor nutritional supply to the IVD is believed to play a primary role in the pathophysiology of disc degeneration. Since the disc is avascular, vital nutrients, such as glucose, must be supplied by surrounding blood vessels. However, the transport and metabolic properties of glucose in the IVD have not been fully delineated. This knowledge is necessary in order to elucidate the nutrition-related mechanisms of disc degeneration. Therefore, in this dissertation, experimental and theoretical methods are used to investigate the transport and metabolism of glucose in the intervertebral disc. Strain-dependent and anisotropic (i.e., direction-dependent) transport of glucose in human annulus fibrosus (AF) was investigated using custom apparatuses. Results indicate that diffusivity and partitioning of glucose in human AF decreases with increasing compressive strain. Furthermore, diffusivity of glucose is anisotropic, being lower in the radial direction than the axial or circumferential directions at all strain levels. Transport of glucose in human AF was also found to diminish with increasing disc degeneration. A new method was developed to measure the rate of glucose consumption by IVD cells; this method was then validated with porcine AF and nucleus pulposus (NP) cells at varying levels of oxygen tension. Results show a positive Pasteur effect, with the glucose consumption rate by AF and NP cells increasing at low levels of oxygen. Moreover, results indicate that the rate of consumption of glucose by NP cells is significantly higher than that by AF cells. A new, three-dimensional finite element model of the IVD was developed in order to theoretically predict nutrient distributions in the disc. This model incorporated anatomical disc geometry, nutrient transport coupled to cellular metabolism, and mechanical loading conditions. The model was used to investigate the effects of endplate calcification and in vivo loading conditions on glucose distributions in the disc. Both calcification and compressive loading resulted in diminished glucose concentrations in the tissue. The model was also used to analyze the effects of degeneration and compression on cell viability in IVD by incorporating viability criteria. Our model could predict cell death in degenerated tissue, and compressive loading augmented this effect. The model prediction can be used to supplement experimental results, and may also serve as a useful tool in developing new strategies for the treatment of disc degeneration. The findings of this dissertation greatly enhance the knowledge of glucose transport and metabolism in the intervertebral disc. Given that glucose is a critical nutrient for disc cell survival, this knowledge can provide important insight into nutritional pathways and mechanisms in the IVD, as well as related disc degeneration.

Cardiovascular disease is the leading cause of death globally. This thesis has critically evaluated aspects related to two cardiovascular risk factors, namely hyperlipidaemia and hyperglycaemia, in healthy volunteers. Free fatty acids produced by visceral fat and transported to the liver in the portal circulation are thought to be one mechanism by which obesity contributes to increased insulin resistance and therefore reduced glucose disposal and a propensity to hyperglycaemia. The first study assessed the bioavailability of eicosapentaenoic acid, a cardio-protective fatty acid, in different forms of fish oil and then by incorporation with spores or exines. Potential use of the latter as a carrier of fatty acids to the portal circulation to increase insulin resistance and reduced glucose disposal was also studied. As short term plasma glucose fluctuations have been linked to an increased production of oxygen radicals, and thereby the microvascular complications of diabetes, a controlled assessment of this has been investigated in the final study. The first study examined in a double blind design the short term bioavailability, in the form of24 hour area under the curve, of eicosapentaenoic acid obtained from 5 forms of fish oil. The second study was an open label study which tested the change in the bioavailability of eicosapentaenoic acid in the form of ethyl ester by their incorporation with spores or exines. The latter was used in the third open label study to test whether the exines are capable of delivering what is inside them directly to the portal circulation. This could directly deliver oleic acid to the portal circulation thereby simulating the portal influx of farty acid from the central adipose tissue. In the final study, urinary isoprostane, a marker of oxidative stress, linked to the microvascular complications of diabetes was measured in three short term glycaemic states using the hyperglycaemic clamps in a cohort of healthy volunteers. This study aim was to establish whether glycaemic variability is associated with significant change in oxidative stress over and above that already present due to hyperglycaemia.

Background/Aim: Hepatitis C virus infection is a known risk factor of impaired glucose metabolism and diabetes mellitus. Whether hepatitis B virus (HBV) infection is also associated with impaired glucose tolerance remains uncertain. The aim of the study was to conduct a systematic review on the association between HBV infection and impaired glucose metabolism Methods: Studies reporting the association between HBV infection and markers of impaired glucose metabolism were identified through keyword search in PubMed and Google Scholar. 10 studies (out of 320) were included in this systematic review. Results were included. Majority (n=7) of the included studies were conducted among the Asian populations. Of the 10 included studies, eight studies reported a significant association between HBV infection and impaired glucose metabolism, proxied by impaired glucose tolerance, impaired fasting glucose, diabetes mellitus, insulin resistance, and metabolic syndromes. The remaining two studies using diabetes mellitus and insulin resistance as outcome measures did not find a positive association with HBV infection. Conclusions: The association between HBV and impaired glucose metabolism is suggestive from the evidence compiled from included articles. However, whether the development of glucose intolerance or diabetes mellitus is linked to an infectious cause of HBV is still inconclusive. Further studies that could improve on the current understanding of the associations between HBV infection and impaired glucose metabolism are necessary.
published_or_final_version
Public Health
Master
Master of Public Health

The neonatal period in mammals is characterized by high growth rates and is dominated by skeletal muscle hypertrophy. Low birth weight (LBWT) neonates experience restricted growth and development of skeletal muscle, leading to metabolic perturbations later in life. The overall hypothesis of this dissertation was that in utero disturbances in glucose metabolism and increased energy requirements predisposes LBWT neonatal pigs to metabolic disturbances after birth. We sought to increase growth of skeletal muscle and improve glucose production through increasing dietary energy and to determine the changes in glucose catabolism and metabolic flexibility in different skeletal muscle fiber types in LBWT neonates. Piglets were considered normal birth weight (NBWT) and LBWT when birth weight was within 0.5 SD and below 2 SD of the litter average, respectively. Increasing dietary energy increased lean deposition in the longissimus dorsi (LD) in both NBWT and LBWT neonates. Although glucose rate of appearance was greater in LBWT compared to their NBWT sibling, glucose concentrations were reduced in LBWT compared to NBWT pigs, regardless of diet fed. Postprandial glucose concentrations were lower in LBWT compared to NBWT pigs, regardless of diet fed, although rate of appearance did not differ between them. This would suggest that glucose is being absorbed in the peripheral tissues to be utilized. However, expression of enzymes related to glycolysis were downregulated in both the soleus and LD of LBWT compared to NBWT neonatal pigs. In addition, expression of enzymes related to the catabolism of glucose in the serine biosynthetic pathway were decreased in both the soleus and LD muscles of LBWT compared to NBWT neonatal pigs. Expression of the pentose phosphate pathway was slightly increased in LBWT compared to NBWT siblings in both muscle types. Increased expression of pyruvate dehydrogenase 4 was exhibited in both the soleus and LD of LBWT pigs compared to NBWT siblings. This would indicate a switch in fuel utilization to more fatty acid oxidation. By contrast, CO2 production from the oxidation of palmitate was reduced in LBWT compared with NBWT pigs along with reduced oxidation of glucose and pyruvate. In conclusion, lipid supplementation increased growth at the expense of fat deposition in the liver of NBWT and LBWT pigs. However, supplementing with fat did not increase glucose production due to the contribution of glycerol remaining constant. Hypoglycemia cannot be attributed to greater catabolism in skeletal muscle due to decreased expression of glycolytic genes and the addition of fatty acids did not spare glucose oxidation in skeletal muscle of LBWT pigs.
PHD

Since steroid hormones have well characterised programming properties, excessive fetal exposure to glucocorticoids in early in life may produce lifelong effects. In order to examine the role of glucocorticoids in the programming of glucose metabolism, fetal glucocorticoid load was increased by administration of dexamethasone, a poor substrate for 11β-HSD, during the different trimesters of rat pregnancy. Dexamethasone given selectively in the last week of pregnancy reduced birth weight by 10%, and produced hyperglycaemia and hyperinsulinaemia in the adult offspring. In addition, rats exposed to dexamethasone during the last week of intrauterine life had permanent increases in hepatic expression of glucocorticoid receptor (GR) mRNA and phosphoenolpyruvate carboxykinase (PEPCK) mRNA (and activity). In contrast dexamethasone, when given in the first or second week of gestation, had no effect on offspring insulin/glucose responses or hepatic PEPCK and GR expression. Similarly, administration of dexamethasone to pups postnatally did not alter glucose control in the adult animals. These observations suggest that excessive glucocorticoid exposure late in pregnancy predisposes the offspring to glucose intolerance in adulthood. Programmed hepatic PEPCK overexpression, perhaps mediated by increased GR, may promote this process by increasing gluconeogenesis. In order to examine further the molecular basis for hepatic PEPCK overexpression, levels of liver enriched transcription factors known to co-operate with GR in stimulation of PEPCK gene transcription were analysed. Levels of hepatic nuclear factor (HNF)-1, HNF-4, and CCAAT/enhancer binding protein (C/EBP)α (but not C/EBPβ) did alter with prenatal dexamethasone treatment. However, unlike GR mRNA, expression of none of these factors correlated closely with changes in PEPCK level or hyperglycaemia. These studies have confirmed that in utero overexposure to glucocorticoids can programme hyperglycaemia which may be in part due to glucocorticoid mediated increase in hepatic gluconeogenesis, and provide support for the hypothesis that excessive fetal exposure to glucocorticoids underlies the link between low birth weight and disease in adulthood observed in human epidemiological studies.

Diabetes mellitus is not only a very serious disease, causing discomfort and pain to millions across the world, but with the aging of the population and the prevalence of a sedentary lifestyle, it is assuming the proportion of a real epidemic, becoming a public health and social emergency. In answer to this call, research on diabetes has been intensely carried out in the past decades and the knowledge and understanding of its etiology have been significantly improved. However, investigation is still ongoing, many important questions are still unanswered, and the causes eliciting the pathogenesis and progression of the diseases are not yet fully understood. During all these years of research, sophisticated tools have been developed to study the glucose-insulin metabolic system in vivo, and cope with the inaccessibility to direct measurement of some of the key phenomena underlying the glycemic control. Such tools, as complex test protocols and model-based approaches used to interpret the experimental data, have proven powerful weapons of investigation, but, considering the epidemic proportion of the disease, there is great demand for approaches that are less invasive, less expensive and therefore more suitable for large clinical studies. Mathematical and statistical techniques that collectively go under the name of “population approaches” have already been developed and are largely employed for pharmacokinetics and pharmacodynamics studies in drug development. However, in spite of their interesting potential, they have not found significant application yet in the context of metabolism research. Thus, investigation is required to probe the feasibility and relevance of such approaches in the study of diabetes. The research presented here addresses these issues, and is aimed at applying these sophisticated techniques to the modeling of glucose metabolism, first assessing the applicability of these approaches and tailoring them to the problem under investigation, then employing them to the analysis of data from population studies. First, a simulated but physiologically plausible dataset is created based on previous real data and employed as a benchmark to assess the applicability of population approaches to the Intra Venous Glucose Tolerance Test (IVGTT) minimal model of glucose disappearance. Various population algorithms have been proposed in the literature, therefore a thorough comparison of the available methodologies is performed, and a sparse data situation is replicated to test the robustness of these methods in such cases. The results select the First-Order Conditional Estimation as method of choice and show its robustness to poor sampling. Then, a larger real dataset is employed and analyzed with the same techniques, this time assessing the quality of the results with a Monte Carlo sampling approach to profile the likelihood function. Then the population model is optimized, to provide a base model for the following covariate analysis. In fact, at the time of the experiments, demographic data about the subjects has been collected, and the purpose of the covariate analysis is to determine whether some of these variables are significantly correlated with the model parameters and can be successfully used to explain part of the differences among the subjects. After a first exploratory regression analysis, different models are tested, integrating the most significant covariates directly as predictors into the model. In agreement with previous findings in literature, basal insulinemia, age and visceral abdominal fat are shown to be good predictors of insulin sensitivity and their introduction in the model is able to account for about a third of the between-subject variability of the values of this parameter. The use of covariates enhances the explanatory power of the model and opens the way for devising new lighter experimental protocols. One of the main benefits of the population approaches, in fact, consists in their ability to borrow information across the population and use it to improve the individual parameter estimates. As a result, the experimental protocols can be less demanding, both in invasiveness and economic cost, allowing in this way a broader use in large clinical studies. The results presented here, in fact, show that population approaches are very robust and able to cope with sparse data situations. In addition, the use of covariates in the model enhances even further the power of such techniques and makes them very appealing approaches to the study of glucose-insulin metabolism. In addition, a population approach is proposed to solve the problem of the estimation of the Disposition Index (DI) of glucose tolerance in a population. Since both insulin sensitivity and beta-cell response must be taken into account to assess the actual efficiency of the glucose disposal system, the DI was proposed to condense the information conveyed by both these parameters in a single value. Traditionally, approaches based on a geometrical fit are used to determine the value of DI in a population of subjects characterized by the same degree of glucose tolerance. However, all these methods rely on the assumption that all the subjects in the population share exactly the same value of DI and are therefore not able to account for the population variability, which is inevitably inherent to biological data. In this work, a NonLinear Mixed-Effects Approach is proposed to analyze the distribution of the insulin sensitivity and beta-cell response indices across a population, and then obtaining the information on the DI from the population features thus estimates. Comparisons on simulated datasets between the newly proposed method and its competitors prove that a proper model of the variability structure is essential to avoid severe bias in the estimates.
Il diabete mellito è non solo una patologia molto seria, che causa disagi e sofferenze a milioni di persone nel mondo, ma, anche a causa dell’affermarsi di uno stile di vita sedentario e dell’invecchiamento della popolazione, negli ultimi decenni ha raggiunto proporzioni epidemiche, diventando una vera e propria emergenza sanitaria e sociale. Per fronteggiare questo problema, molte risorse sono state dedicate all’attività di ricerca scientifica, che ha permesso una più profonda conoscenza dell’eziologia del diabete. Tuttavia, il diabete è a tutt’oggi ancora inguaribile e molte questioni rimangono aperte, fra cui la completa comprensione dei fattori che causano e fanno progredire la malattia. Anni di ricerca hanno permesso di sviluppare molti sofisticati strumenti per studiare il sistema metabolico glucosio-insulina in vivo e poter così fronteggiare il problema dell’inaccessibilità diretta di alcuni dei fenomeni chiave che controllano la glicemia. Tali strumenti, fra cui protocolli di studio e approcci basati su modello usati per interpretare i dati sperimentali, si sono rivelati armi molto potenti nelle mani dei ricercatori, ma le proporzioni epidemiche della malattia e il parziale cambiamento delle strategie e obiettivi della ricerca hanno sollevato l’esigenza di poter disporre di metodologie meno invasive, più economiche, e quindi più adatte ad essere applicate ad estesi studi clinici. Alcuni strumenti matematici e statistici che sono collettivamente conosciuti con il nome di “approcci di popolazione” sono già stati sviluppati e vengono largamente impiegati in studi di farmacocinetica e farmacodinamica, per lo sviluppo di farmaci. Tali approcci si prefiggono come obiettivo primario di stimare la distribuzione dei parametri di un modello all’interno di una popolazione e pertanto si avvalgono, per la stima individuale, delle informazioni disponibili sull’intero gruppo di soggetti. Sono particolarmente adatti a situazioni in cui il campionamento intensivo in un singolo soggetto non è possibile, e quando l’interesse del ricercatore è focalizzato sulla variabilità inter-individuale. Tuttavia, nonostante le loro interessanti potenzialità, gli approcci di popolazione non sono ancora apprezzati all’interno dell’ambiente di ricerca sulle malattie metaboliche, e la loro applicazione in tali studi è stata molto limitata. Pertanto è necessaria dell’attività di ricerca per saggiare l’effettiva fattibilità e rilevanza dell’utilizzo di tali approcci nello studio del diabete. La ricerca qui presentata risponde a queste esigenze, proponendosi come obiettivo l’applicazione di queste sofisticate tecniche ai modelli di metabolismo del glucosio, prima testandone la fattibilità e adattandole al problema in esame, e poi impiegandole nell’analisi di dati raccolti in studi di popolazione. Poiché in letteratura sono stati proposti molti diversi algoritmi, come primo passo, un dataset simulato è stato utilizzato per effettuare un confronto delle metodologie quando applicate al modello minimo del glucosio per il Test di Tolleranza IntraVenosa al Glucosio (IVGTT). First-Order Conditional Estimation (FOCE) si è rivelato come l’algoritmo più soddisfacente, in quanto ha fornito i risultati più accurati e robusti in caso di scarsità o rumorosità dei campioni. Successivamente, per validare i risultati trovati su dati reali, l’analisi è stata ripetuta su un dataset più esteso, relativo a 204 soggetti sani testati con IVGTT. Per poter saggiare la bontà delle soluzioni fornite dai vari algoritmi, è stato impiegato un sistema di stima della likelihood function basato su campionamento Monte Carlo. Questa analisi, non solo ha permesso di confermare la scelta di FOCE come metodo preferenziale, ma si è anche rivelata come un potente strumento per valutare la precisione delle stime dei parametri di popolazione. Successivamente, è stato messo a punto e ottimizzato un modello di popolazione, conservando nella matrice di covarianza solo i termini di correlazione fra i parametri SI-P2 e SG-VOL. Questo modello è servito come base per la successiva integrazione di covariate nel modello. Al momento dell’esecuzione degli esperimenti, infatti, sono stati raccolti alcuni dati sui pazienti, fra cui altezza, peso, sesso, età, glicemia e insulinemia basali, informazioni sul grasso corporeo. È stata effettuata una analisi per determinare quali fra queste variabili potessero essere usate per spiegare parte della variabilità nei valori dei parametri del modello minimo fra i diversi soggetti. Il risultato è un modello che integra queste informazioni direttamente nelle sue equazioni, mentre i coefficienti di regressione per ognuno dei predittori diventano veri e propri parametri del modello e il loro valore viene ottimizzato insieme agli altri parametri di popolazione. L’analisi effettuata ha trovato come buoni predittori per SI e P2 l’ età, l’insulinemia basale e il grasso addominale, che in ambo i parametri riescono a spiegare una buona fetta della variabilità inter-individuale. Sia l’impiego di metodologie di popolazione, sia l’introduzione delle covariate nel modello, permettono di aumentarne il potere predittivo, e sono in grado di usare informazioni indipendenti dai soli dati sperimentali. Questo permette di mettere a punto dei protocolli di studio meno invasivi, meno costosi, e pertanto più adatti ad un impiego su larga scala: ulteriore ricerca potrebbe avere come obiettivo l’ottimizzazione di una sampling schedule ridotta, che si avvantaggi dell’utilizzo degli approcci di popolazione. Ad ogni modo, il dataset utilizzato in questa analisi comprende solo soggetti sani, ed è quindi caratterizzato da una quantità limitata di variabilità di popolazione. Pertanto, sarebbe necessario ripetere l’analisi su altri dataset, per poter confermare questi risultati, in particolare sulle covariate. Inoltre, in una sezione successiva, un metodo di popolazione è stato applicato anche ad un altro problema diverso, la stima del Disposition Index (DI) del glucosio. Questo è un indice calcolato combinando sensitività e responsività all’insulina, che serve per testare l’effettiva efficacia del sistema di controllo della glicemia. Ci sono due versioni proposte per la formula, una semplificata, che consiste semplicemente nel prodotto (da cui il nome di Legge Iperbolica), e una con un parametro aggiuntivo ad esponente della sensitività all’insulina. Per poter calcolare il DI medio in una popolazione, e per poter saggiare quale delle due formule sia effettivamente più adatta, in letteratura si trovano alcuni approcci basati su un fit geometrico. Tuttavia, alcune approssimazioni sono utilizzate per semplificare il fit, e sono molte le questioni metodologiche spesso sottovalutate. Pertanto viene presentato qui un nuovo metodo Total Least Squares (TLS) che affronta il problema senza l’impiego di approssimazioni. Grazie ad alcune simulazioni, si è effettuato un paragone fra i vari metodi disponibili, e il nuovo algoritmo è risultato migliore rispetto ai predecessori. Tuttavia, tutti gli algoritmi basati su fit si fondano sull’ipotesi che i soggetti appartenenti alla popolazione abbiano lo stesso valore di DI, e l’unica fonte di incertezza nei dati sia dovuta alla stima degli indici di secrezione e sensitività. Questa ipotesi sembra una forte semplificazione e, in effetti, l’analisi di un dataset reale sembra confermare la presenza di variabilità di popolazione nei valori del DI. Ulteriori simulazioni hanno confermato che tutti metodi basati su fit, TLS compreso, falliscono quando la variabilità di popolazione è presente. Pertanto, è stato ideato un altro metodo basato su approcci di popolazione e, in particolare, su NonLinear Mixed-Effects Models (NLMEM), che è in grado di separare la variabilità nei dati, poiché fondato su ipotesi meno restrittive. Tale algoritmo stima i parametri della distribuzione di probabilità congiunta degli indici di secrezione e sensitività, e poi estrae le informazioni sul DI dalla matrice di covarianza. NLMEM si è rivelato equivalente a TLS quando non c’è variabilità di popolazione, ma di gran lunga più affidabile quando le ipotesi per il fit geometrico non sono rispettate, pertanto si è deciso di utilizzarlo sul dataset reale per testare la validità della legge iperbolica. Anche se una validazione su altri dataset è auspicabile per validare i risultati qui presentati, il modello con il parametro aggiuntivo sembra spiegare i dati in maniera più soddisfacente, e il valore del parametro sembra dipendere dalla coppia di parametri usata per la definizione del DI, più che dalla popolazione in esame (anziani piuttosto che giovani). Inoltre, nello studio qui proposto, il punto di partenza sono stati i valori degli indici di secrezione già calcolati, insieme con la loro precisione, grazie ad un metodo tradizionale; un approccio ancora più potente consisterebbe nell’utilizzare un modello di popolazione per stimare contemporaneamente sia gli indici di secrezione che sensitività, sia i parametri della loro distribuzione di popolazione, da cui ricavare le informazioni sul DI. Riassumendo, in questo lavoro si sono messi in luce i vantaggi dell’applicazione di approcci di popolazione nello studio nel diabete. Le potenzialità sono molte, dal miglioramento delle stime dei parametri individuali grazie all’uso dei prior di popolazione o di covariate e la relativa possibilità di mettere a punto protocolli di studio più leggeri, fino all’analisi di situazioni in cui la struttura gerarchica della variabilità è un aspetto cruciale.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.
This study investigates whether an 8-week lifestyle-based, smartphone-delivered intervention targeting reduction in sedentary behavior (i.e., sitting) significantly reduces objectively measured time spent sitting and improves fasting glucose and insulin. The incidence of type II diabetes has continued to increase in the United States and increases in sedentary behavior along with reductions in physical activity throughout the day have contributed to the increase of disease. There were 31 participants in the study and they started with a 3-week run-in period where a basic self- monitoring component was installed on their smartphone. After this run-in period, participants were randomly assigned to one of the eight experimental conditions. All participants received a basic self-monitoring with feedback component where they self-reported sleep, sedentary, and more active behaviors. Sitting time was measured with the activPAL3c, which is a device that they wore 24/7. Study visits occurred at week 0 (immediately after the 3-week run-in period), week 4, and week 8. Fasting glucose and insulin were measured at each of these visits. Participants logged approximately 60% of their sleep, sedentary, and exercise behaviors, which took 3– 4 min/day to complete. The impact of the intervention was not significant, such that decreases in sedentary time in those assigned to the sedentary component did not significantly differ from those not assigned to the sedentary component at 8 weeks (beta (SE) = -1.19 (.32), p>0.05); however, the effect size was moderate (Cohen’s d = 0.29). There was no significant impact on fasting glucose or insulin.

The purpose of this investigation was to determine the relationship between calcium and glucose uptake following muscle contraction with the use of the calcium channel blocker dantrolene. In previous studies an exercise model has been used to investigate the role of calcium during post-exercise glucose uptake. This study utilized electrical stimulation. It has been shown that exercise-induced glucose uptake is calciummediated, but to date no one has shown that glucose transport induced by electrical stimulation is calcium-mediated. Twenty four male Sprague Dawley rats weighing 140 g were sacrificed and their epitrochlearis muscles were removed. Four treatment groups were established: control, muscle incubated in glucose (4mM); insulin, muscles incubated in glucose (4mM) and insulin (1000uU/ml); electrical stimulation, at 50 Hz for two five minute intervals separated by one minute rest periods; insulin (1000uU/ml) and electrical stimulation at 50 Hz for two five minute intervals separated by one minute intervals. Each group consisted of contain 8-10 muscle preparations. Glucose uptake was measured through the use of a double label of radioactive mannitol and 3-O-methylglucose and analyzed using liquid scintillation. This project followed a randomized group design. Treatments were measured with a one way ANOVA.
School of Physical Education

Thesis advisor: Thomas C. Chiles
B-lymphocytes respond to environmental cues for their survival, growth, and differentiation through receptor-mediated signaling pathways. Naïve Blymphocytes must acquire and metabolize external glucose in order to support the bioenergetics associated with maintaining cell volume, ion gradients, and basal macromolecular synthesis. The up-regulation of glycolytic enzyme expression and activity via engaged B-cell receptor mediated-events was glucose-dependent. This suggests an essential role for glucose energy metabolism in the promotion of B cell growth, survival, and proliferation in response to extracellular stimuli. In addition, the activity of ATP-citrate lyase (ACL) was determined to be crucial for ex vivo splenic B cell differentiation to antibody-producing cells wherein B cells undergo endomembrane synthesis and expansion. This investigation employed knockout murine models as well as chemical inhibitors to determine the signaling components and enzymes responsible for glucose utilization and incorporation into membrane lipids. These results point to a critical role for phosphatidylinositol 3- kinase (PI3K) in orchestrating cellular glucose energy metabolism and glucosedependent de novo lipogenesis for B lymphocyte responses
Thesis (PhD) — Boston College, 2012
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Biology
Ph.D.
Regulation of Glucose metabolism is known to play an important role in pathogenesis of many diseases. Primarily because deregulation of this metabolic pathway can lead to either apoptosis or extended life span of the cells involved. Viruses are parasitic in nature, they utilize the host cellular pathways to support their own progeny; hence it is expected that viruses would regulate the central glucose metabolism of infected host cells. Human immunodeficiency virus type 1 (HIV-1) causes acquired immune deficiency syndrome, and it uniquely infects both activated CD4+ T cells and terminally differentiated macrophages during the course of HIV-1 pathogenesis. While HIV-1 infection of CD4+ T cells induces G2 arrest and cell death within 2-3 days, HIV-1 infection of macrophages results in longer survival of infected cells and low constitutive viral production, generating viral reservoirs. Our studies show that HIV-1 infection lead to significant changes in the glycolytic pathway of infected cells by altering the enzymatic activity and protein expression of various glycolytic components. The data suggests that the two HIV-1 target cell types exhibit very different metabolic outcomes. During viral replication in monocyte/macrophage lineage cells we observe increase in glycolytic protein expression and the same proteins show no modulation in T-cell lines post viral replication. Similar differential regulation is observed in case of enzymatic activity of glycolytic enzymes as well. We also conducted proteomic studies in collaboration with the proteomics core. HIV-1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Vpr is known to cause cell cycle block in infected cell and bring about cell death. However, macrophages are resistant to cell death and are viral reservoir, even Vpr over expression does not cause apoptosis in these cell types. The goal of the study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. We observed that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. We then focused on infected monocyte macrophages to identify if glycolytic components such as HK and G6PD were regulated by HIV-1 infection/replication. We report that Hexokinase-1 (HK-1) enzyme expression increases post infection of PBMCs where as the enzymatic activity of HK decreases. Similar effect is seen with HIV-1 replication in latently infected monocyte cell lines U1. The G6PD enzyme activity and expression both increases in infected PBMCs and in U1 cells post induction of viral replication with PMA. We also found that HK-1 translocate to the mitochondria of U1 cells post induction of HIV-1. It is known that the product of HK activity, Glucose 6-phosphate (G6P) releases HKI from the outer leaflet of mitochondria. Hence we conclude that the viral infection decreases HK activity to have less G6P produced in cell and increases G6PD enzyme activity ensuring the remaining G6P is quickly used up, supporting the adherence of outer mitochondrial membrane bound HK1. This sequence of cellular events ensures longer survival of infected cells supporting the viral progeny to propagate in the cell. We further show that suppressing the Pentose phosphate pathway (PPP) by blocking G6PD activity is not only detrimental to the survival of the infected cells it also suppresses viral replication and promoter level transactivation of the viral LTR. Next we sought to identify if glycolytic enzyme PKM2, that is also known to play a nonmetabolic dual role as a protein kinase regulating gene transcription has any effect on the transcription of HIV-LTR. Our study demonstrates upregulation of pyruvate kinase isoform M2 (PKM2) expression in whole cell extracts and nuclear extracts of HIV-1JRFL infected PBMCs and during reactivation of HIV-1 in chronically infected U1 cells. We then focused on understanding the potential role of PKM2 on HIV-1 LTR transactivation. Our studies demonstrate that over expression of PKM2 leads to transactivation of the HIV-1 LTR reporter construct. Using various deletions constructs of HIV-1 LTR, we mapped the region spanning between -120 bp to -80 bp to be essential for PKM2 mediated transactivation. This region contains the NFKB DNA binding site and mutation of NFKB binding site attenuated PKM2 mediated transactivation of HIV-LTR. Chromatin immune-precipitation (ChIP) analysis confirmed interaction of PKM2 with HIV-1 LTR. Our studies suggest that PKM2 is a transcriptional co-activator of HIV-1 LTR. Hence it opens up another possible target to curb HIV-1 replication at transcriptional level. This study sheds light on the regulation of glycolytic pathway of host cells by HIV-1 infection and its consequences for the virus, opening up new avenues to target viral replication and identify glycolytic markers of HIV-1 pathogenesis.
Temple University--Theses

Type 2diabetes is an adult-onset condition in which the body is not able to tightly regulate the amount of glucose in the blood and it results from a combination of causes including genetic, behavioral and environmental factors. Krüppel-like factor 3 (KLF3) is a zinc finger transcription factor encoded by the klf3 gene. It has three Krüppel-like zinc fingers that bind to DNA. KLF3 inhibits adipogenesis in vitro through its ability to inhibit an adipogenic master regulator, C/EBPα expression (Sue et al., 2008). klf3-/- mice have fewer and smaller fat cells in adipose tissue. Reasons for different phenotype of the klf3-/- mice than the in vitro results could be prevention of expansion of adipocyte progenitors by premature expression of C/EBPα, likelihood of indirect processes influencing fat pad size in vivo (Sue et al., 2008) and less food intake of klf3-/- mice. klf3-/- mice have increased fasting blood glucose level (Unpublished data, Bell-Anderson Lab). This variation in fasting blood glucose level suggests a role for KLF3 in glucose metabolism. Therefore, this study was designed to investigate the mechanism underlying high fasting blood glucose levels observed in klf3-/- mice. Mice were sacrificed at 13 weeks of age at 5pm in a naturally fasted state and 9pm in a fed state. Blood was collected and blood glucose and plasma glucagon levels were measured. Liver and red quadriceps muscles were collected in order to measure glycogen and glucose-6-phosphatase (G6Pase) activity. Body weight of klf3-/- mice was significantly lighter than that of klf3+/+ mice (p=0.003). We observed that klf3-/- mice had significantly higher fasting blood glucose level than klf3+/+ mice (9.8mmol/L ± 0.89 vs 8.7mmol/L ± 0.71 respectively) (p<0.05). klf3+/+ mice had significantly higher liver glycogen level than KLF3 deficient mice in the fed state (233.47µmol/g ± 54.48 vs 189.68µmol/g ± 38.33 respectively) (p=0.027). There was no significant difference in muscle glycogen levels in klf3-/- and klf3+/+ mice in both fed and fasted states. klf3-/- mice had significantly higher G6Pase activity compared to klf3+/+ mice in the fed state (p=0.008). In the fasted state and fed state, klf3-/- mice had more or less similar G6Pase activities which were respectively 14.1 and 14.9 µmol/g/10 minutes. There was no significant difference in plasma glucagon levels of klf3-/- mice and klf3+/+ mice in the fasted state. Insulin levels unchanged in KLF3 knockout and wild type mice in fed state but KLF3 knockout mice had significantly higher insulin level in fasted state (185.6µU/mL ± 57.5 vs 112.4µU/mL ± 35.7) (p=0.004). Food intake data showed that KLF3 knockout mice eat more food in the afternoon in which we considered as a fasted state. Therefore, the higher fasting blood glucose levels observed in klf3-/- mice is not a result of changes in glycogen levels, G6Pase activity, plasma glucagon and could be due to higher food intake and increased insulin level . Results showed that KLF3 could potentially have an impact on glucose metabolism in the fed and fasted state but further analysis on other glucose metabolic pathways like gluconeogenesis should be performed to confirm the underlying mechanisms for increased fasting blood glucose level of klf3-/- mice.

Cell metabolism disorder is considered as both direct and indirect consequence of oncogenic mutations for tumoigenesis. Autophagy is a metabolic stress response and a mechanism of natural cellular degradation. It is believed that autophagy, as well as metabolism, is a crucial process for the adaptation of cancer cells to changes in nutrient availability. Understanding the relationship between metabolism and autophagy and targeting on the key steps are regarded as a promising strategy to treat cancer. Halofuginone (HF), a semisynthetic quinazolinone alkaloid originally derived from the plant Dichroa febrifuga Lour., has gained attention for its potential therapeutic effects in a variety of cancers. We hypothesize that HF may inhibit cancer cell proliferation by inducing changes in glucose metabolism and modulating related autophagy. A series of studies, from in vitro to in vivo, were designed to approve this concept in colorectal cancer (CRC);Firstly, we found that HF inhibited human CRC cell proliferation and induced the generation of reactive oxygen species (ROS) and apoptosis. As expected, a reduced level of NADPH was also observed, at least in part due to inactivation of glucose-6-phosphate dehydrogenase (G6PD) in pentose phosphate pathway (PPP) upon HF treatment. Given these findings, we further investigated metabolic regulation of HF through Akt/mTORC1-mediated aerobic glycolysis and found that HF downregulated the Akt/mTORC1 signaling pathway. Moreover, metabolomics found slower rates in both glycolytic flux and glucose-derived tricarboxylic acid cycle flux. Meanwhile, both glucose transporter GLUT1 and hexokinase-2 in glycolysis were suppressed in CRC cells by HF. These findings support our notion that HF regulates the Akt/mTORC1 signaling pathway to dampen glucose uptake and glycolysis in CRC cells. Furthermore, HF retarded tumor growth in nude mice inoculated with HCT116 cells and reduced the viability of primary cells from the tissues of CRC patients. This finding further confirmed our hypothesis that HF inhibits CRC cell growth through metabolic regulation of Akt/mTORC1. Because mTORC1 can inhibit autophagy through phosphorylation and inactivation of the initiating kinase ULK1 in cancer cells, we further studied the HF effects on CRC in different nutritional conditions. The results showed that HF in nutrient-rich conditions could reduce SQSTM1/p62 through mTORC1-mediated phosphorylation at Ser757 of ULK1. More interestingly, HF elevated SQSTM1 protein level in low nutrient condition through AMPK-mediated phosphorylation at Ser317/777 of ULK1. It showed that HF could regulate nutrient-sensing mTORC1-ULK1 or AMPK-ULK1 to dually modulate autophagy in CRC cells. Further study by using a variety of methods, including mRFP-GFP-LC3 puncta formation, transmission electron microscope (TEM) analysis and monodansylcadaverine (MDC) staining, found that HF could induce autophagosome formation and inhibit autophagosome membrane elongation, depending on nutrient-sensing pathways. Furthermore, we found HF pronouncedly enhanced expression level of Atg7 under nutrient-rich conditions while it decreased Atg7 in CRC cells under nutrient-poor conditions. These two findings imply that Atg7 is required in dual regulation of autophagic flux depending on nutrient conditions. This conclusion was then validated by comparing with autophagy-related proteins in Atg7 knockout (KO) MEFs and Wild-type (WT) MEFs upon HF treatment. Importantly, through analysis of metabolome and metabolic enzymes, we found that HF inhibited glycolysis under nutrient-rich conditions while it inhibited gluconeogenesis under nutrient-poor conditions in an Atg7-dependent manner. In subsequent studies, we found that caloric restriction (CR) in a xenograft mouse model, which mimics low nutrition in vitro, enhanced the anticancer activity of HF. Further analysis of the expression of TQTSM1 and LC3 in tumor tissues demonstrated that HF is an autophagic inducer in xenograft-bearing nude mice with ad libitum feeding, whereas it is an autophagic inhibitor when using CR.;In summary, this study explains how HF controls CRC cell growth through its influences on glucose metabolism and autophagy regulation. HF not only dually regulates autophagy in vitro and in vivo to inhibit cancer cell growth and proliferation through nutrient-sensing pathways under different conditions, but also modulates glycolysis/gluconeogenesis through an autophagic pathway. These results suggest that HF could turn out to be a potent therapeutic drug for treating CRC.

This thesis investigates the ability of rainbow trout to modulate hepatic glucose production (Ra) and disposal (Rd). My goals were to determine: (1) if resting trout can modulate fluxes to cope with exogenous glucose; (2) how fluxes change during graded swimming; (3) how exogenous glucose affects swimming kinetics; and (4) if exogenous glucose affects cost of transport or performance. Results show that resting trout suppress Ra completely and stimulate Rd from 10.6 to 27.6 μmol kg-1 min-1. During swimming, fluxes increase from 15.6 to 21.9 μmol kg-1 min-1, but only at speeds >2.4 BL s-1. When given glucose, trout suppress Ra from 16.4 to 4.1 μmol kg-1 min-1 and stimulate Rd from 16.4 to 40.1 μmol kg-1 min-1. Glucose lowers metabolic rate but does not affect critical swimming speed. Therefore, this research shows that rainbow trout have a much better capacity for glucoregulation than generally suggested by current literature.

L'hépatite C chronique est une des étiologies principales du carcinome hépatocellulaire. En revanche, les mécanismes de tumorigenèse sont peu connus. Récemment, plusieurs modifications du métabolisme du glucose ont été décrites dans les cellules infectées par le HCV. Celles-ci évoquent les reprogrammations métaboliques mises en place dans les cellules cancéreuses. L'effet Warburg, ou glycolyse aérobie, est une des caractéristiques principales des cellules tumorales. Ce phénomène permet d'assurer une production énergétique ainsi qu'un stock en précurseurs de macromolécules suffisants pour permettre la prolifération. Par ailleurs, en complément de l'utilisation de glucose, les cellules tumorales deviennent dépendantes de la métabolisation de la glutamine pour alimenter leur métabolisme énergétique et les différentes voies de biosynthèse. Mes travaux de thèse ont porté sur l'étude des changements métaboliques caractéristiques des cellules cancéreuses, et plus précisément sur le métabolisme de la glutamine, dans les cellules infectées par le HCV. Dans le modèle de culture cellulaire HCVcc, une activation de l'utilisation de la glutamine par le virus a pu être mise en évidence. L'infection par HCV entraine une augmentation du facteur de transcription MYC, de plusieurs transporteurs de glutamine ainsi que de la glutaminase, l'enzyme limitante de la glutaminolyse. De façon intéressante, ces changements semblent survenir également chez les personnes chroniquement infectés par le virus, comme le suggère l'analyse des biopsies de patients. Ces altérations métaboliques pourraient participer à la mise en place d'un environnement favorable au développement tumoral
Chronic infection with hepatitis C virus (HCV) is one of the main etiologies of hepatocellular carcinoma (HCC). However, mechanisms of HCV-related tumorigenesis are ill-defined. Recent literature data suggest that HCV infection may reprogram glucose metabolism in a cancerlike fashion. The Warburg effect, or aerobic glycolysis, is a hallmark of cancer. Activation of this pathway allows tumor cells to sustain high rates of energy production and provide sufficient biosynthetic precursors for proliferation. Likewise, the induction of similar metabolic alterations may favor HCV multiplication through the rapid production of nucleotides, amino acids and lipids. To complement aerobic glycolysis, tumor cells become frequently dependent on glutamine. The partial oxidation of glutamine through the glutaminolytic pathway can fuel their energy metabolism and several anabolic pathways. However, the role of glutamine metabolism in HCV life cycle has not been documented so far. I focused my PhD research project on the characterization of metabolic alterations triggered by HCV. In particular, I evaluated the occurrence of distinctive features of tumor cell metabolism in HCVinfected cells, with a specific attention on glutamine utilization. In the HCVcc cell culture model, I report the induction of a metabolic reprogramming towards higher rates of glutaminolysis upon HCV infection. HCV-induced transcriptional activation of MYC, along with several glutamine transporters and glutaminase, is likely to be responsible for this metabolic shift. Interestingly, increases in transcript levels of these factors in liver biopsies of patients with chronic hepatitis C suggest that this metabolic reprogramming may be relevant in vivo. Moreover, these metabolic changes may expose new drug targets against HCV as suggested by the inhibition of the virus replication upon suppression of glutaminolysis via different strategies. Altogether, these findings uncover a potential link between chronic hepatitis C and HCC through the installation of a favorable metabolic environment for tumor development

Obese individuals present with an increased inflammatory tone as compared to healthy, normal-weight individuals, which is associated with insulin resistance. One factor hypothesized to contribute to increased inflammation in obese and diabetic states is elevated blood endotoxin levels, also known as metabolic endotoxemia. In healthy rodents (non-obese and insulin sensitive), there is evidence that blood endotoxin levels fluctuate over the course of the day with elevations in the post-prandial state that return to baseline levels in the post-absorptive state. High-fat feeding in these animals altered these fluctuations causing endotoxin levels to remain high throughout the day. The effects of alterations in endotoxin levels on glucose metabolism are not understood. The goal of this study was to determine the effects of short-term and long-term increases in endotoxin of a low magnitude on insulin signaling in a human primary cell line as well as the effects of short-term endotoxin treatments on glucose homeostasis in a C57/Bl6 mouse model. First, we tested the hypothesis in cell culture that short-term low-dose endotoxin treatments would enhance insulin-signaling and glycogen synthesis while long-term treatments would have inhibitory effects. Under our second hypothesis, we examined whether short-term low-dose treatments of endotoxin would contribute to improvements in glucose tolerance in a mouse model. In contrast to our first hypothesis, short-term endotoxin treatments did not improve insulin signaling or glycogen synthesis although long-term treatments did contribute to decreases in glycogen synthesis. Interestingly, short-term endotoxin treatments resulted in significant improvements in glucose clearance in the mouse model; this is believed to be partly attributed to LPS inhibiting gluconeogenesis. Future studies are necessary to understand the mechanisms responsible for altered glucose metabolism in response to low magnitude changes in LPS levels.
Ph. D.