A Whole-Body Model for Glycogen Regulation Reveals a Critical Role for Substrate Cycling in Maintaining Blood Glucose Homeostasis

Timely, and sometimes rapid, metabolic adaptation to changes in food supply is critical for survival as an organism moves from the fasted to the fed state, and vice versa. These transitions necessitate major metabolic changes to maintain energy homeostasis as the source of blood glucose moves away from ingested carbohydrates, through hepatic glycogen stores, towards gluconeogenesis. The integration of hepatic glycogen regulation with extra-hepatic energetics is a key aspect of these adaptive mechanisms. Here we use computational modeling to explore hepatic glycogen regulation under fed and fasting conditions in the context of a whole-body model. The model was validated against previous experimental results concerning glycogen phosphorylase a (active) and glycogen synthase a dynamics. The model qualitatively reproduced physiological changes that occur during transition from the fed to the fasted state. Analysis of the model reveals a critical role for the inhibition of glycogen synthase phosphatase by glycogen phosphorylase a. This negative regulation leads to high levels of glycogen synthase activity during fasting conditions, which in turn increases substrate (futile) cycling, priming the system for a rapid response once an external source of glucose is restored. This work demonstrates that a mechanistic understanding of the design principles used by metabolic control circuits to maintain homeostasis can benefit from the incorporation of mathematical descriptions of these networks into “whole-body” contextual models that mimic in vivo conditions

Metabolic and hormonal responses to altered carbohydrate availability and its effect on fatigue development

Includes bibliographical references (leaves 200-220).The main aims of the series of studies comprising this thesis were to investigate the effect of altered endogenous carbohydrate (CHO) availability, achieved primarily by pre-exercise dietary manipulation and antecedent exercise exposure, on interindividual variability in metabolic and hormonal responses to dynamic, steady-state exercise. Further, this thesis examined the impact of altered blood glucose availability on fatigue development during prolonged exercise. In this regard, it was hypothesized that endogenous CHO availability and the associated metabolic sequelae would impact on effort perception during exercise and fatigue development

Metabolomic responses to acute exercise and AMPK-glycogen binding disruption in mice

Background: Exercise is widely accepted as a potent intervention to promote whole-body metabolic health and help prevent and/or treat metabolic diseases. Exercise represents a major challenge to energy homeostasis, both at the whole-body and cellular level. Numerous molecular metabolic responses to acute exercise are activated to preserve energy homeostasis. Central to maintaining cellular energy balance is the AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that senses cellular energy levels by competitively binding to adenosine mono-, di- and triphosphate (AMP, ADP and ATP, respectively). In response to energy stress, AMPK becomes activated and switches on energy-producing catabolic processes while simultaneously switching off energy-consuming anabolic processes. Through its regulatory β subunit, AMPK also binds glycogen – an important energy reserve primarily stored in liver and skeletal muscle. Although growing evidence from AMPK double knock-in (DKI) mice has highlighted physiological consequences of disrupting AMPK-glycogen binding in exercise and metabolic control, the underlying molecular pathways and mechanisms remain unclear. Metabolomics is the unbiased collection and study of small molecules (< 1500 daltons) involved in metabolic reactions to capture molecular snapshots of metabolic pathways, for example associated with given stimuli (e.g., exercise) or genotype. Therefore, metabolomic analysis of biofluids and tissues represents a promising approach to better understand the molecular metabolic responses to acute exercise and the physiological effects of disrupting AMPK-glycogen binding in vivo. Methods: Plasma, gastrocnemius muscle and liver samples were collected from age-matched male WT and DKI mice with disrupted AMPK-glycogen binding at rest and immediately following 30-min submaximal treadmill running. An untargeted mass spectrometry-based metabolomic approach was utilised to determine changes in plasma and/or tissue metabolites occurring in response to acute exercise and the disruption of AMPK-glycogen interactions in DKI mice. Complementary whole-body mouse phenotyping and real-time metabolic phenotyping assays using the Seahorse XFe24 Analyzer and Oroboros O2k high-resolution respirometer were performed to compare energy metabolism and substrate utilisation profiles in mouse embryonic fibroblast (MEF) cells and skeletal muscle from WT and DKI mice. Results/Discussion: Relative to WT mice, DKI mice had reduced maximal running speed, concomitant with increased total body mass and adiposity. In plasma, a total of 83 metabolites were identified/annotated, with 17 metabolites significantly different in exercised versus rested mice. These included amino acids, acylcarnitines and steroid hormones. Distinct plasma metabolite profiles were observed between the rest and exercise conditions and between WT and DKI mice at rest, while metabolite profiles of both genotypes converged following exercise. These differences in metabolite profiles were primarily explained by exercise-associated increases in acylcarnitines and steroid hormones as well as decreases in amino acids and derivatives following exercise. DKI mice showed greater decreases in plasma amino acid levels following exercise versus WT. In liver and skeletal muscle, 150 and 92 metabolites were identified/annotated, respectively. Similar to the plasma metabolite responses observed across genotypes and conditions, significant overall metabolite profile shifts were observed between WT and DKI mice at rest, as well as significant metabolite profile differences between the rested and exercised conditions. Differential muscle metabolite responses to acute exercise were also observed between genotypes. Markers of mitochondrial respiration in permeabilised gastrocnemius fibres were not affected by AMPK DKI mutation, although there were reduced total ATP rate and relative contribution of glycolysis in DKI versus WT MEF cells. Conclusion: The plasma metabolomic analyses performed in Study 1 represent the first study to map mouse plasma metabolomic changes following acute exercise in WT mice and the effects of disrupting AMPK-glycogen interactions using DKI mice. Untargeted metabolomics uncovered alterations in plasma, skeletal muscle and liver metabolite profiles between rested and exercised mice in both genotypes, and between genotypes at rest. This study has uncovered known and previously unreported plasma metabolite responses to acute exercise in WT mice, as well as greater decreases in amino acids following exercise in DKI plasma. These mouse tissue metabolomic datasets, combined with cell and tissue respirometry data complement previous whole-body, tissue and molecular characterisation of WT and DKI mice, revealing potential metabolic pathways and novel molecular biomarkers underlying exercise’s metabolic health benefits and the physiological effects of disrupting AMPK-glycogen binding in mice

Uncovering a sugar tolerance network : SIK3 and Cabut as downstream effectors of Mondo-Mlx

Simple carbohydrates constitute a big part of our everyday diet, as significant consumption increases have occurred in recent decades. This has coincided with a dramatic rise in people suffering from metabolic disorders, such as diabetes and obesity. However, the genetic factors ensuring a healthy response to sugar intake remain poorly understood. To keep blood glucose in a healthy range even upon overnight fasting or following a rich meal, animals need to be able to adapt quickly. In a healthy organism, high sugar intake leads to rapid conversion of excess sugars into stored glycogen and triacylglycerols, while starvation triggers glucose production through gluconeogenesis and glycogen breakdown. To cope with these fluctuating nutritional conditions organisms possess glucose-sensing mechanisms. Such pathways have a key role in monitoring changes in cellular and organismal nutrient status and readjusting animal physiology accordingly to maintain homeostasis. Intracellular sugar metabolites are sensed by the conserved Mondo family transcription factors (TFs)(MondoA and MondoB/ChREBP in mammals, Mondo in Drosophila), which act together with TF Mlx. Together, they control the expression of metabolic target genes by binding to the carbohydrate response elements present in their promoters. The known Mondo-Mlx targets include genes involved in carbohydrate metabolism and biosynthesis of fatty acids. Yet, the physiological output of these transcription factors has remained largely elusive. As these TFs are very well conserved in Drosophila, this model organism has been used in this thesis to understand the physiological role of Mondo-Mlx and their target genes in vivo. We show that Mondo-Mlx interact in the fly and are crucial for dietary sugar tolerance. Loss of either transcription factor leads to impaired growth and lethality upon high sugar diet. We further characterize the mlx mutant phenotype to reveal high circulating glucose, and increased glycogen levels. We uncover transcriptional repressor Cabut as a direct target of Mondo-Mlx. The Cabut promoter is directly bound by Mondo-Mlx in a sugar-dependent manner. Loss of Cabut similarly results in dietary sugar intolerance, pointing to a crucial function in carbohydrate metabolism. Mondo-Mlx through Cabut are essential for repressing the expression of pepck – the rate limiting gene in gluconeogenesis and glyceroneogenesis. A failure to regulate this step results in pepck over-expression, leading to several fold higher levels of circulating glycerol, and pupal lethality of mlx mutants. Moreover, we reveal that Cabut interconnects nutrient sensing with the circadian clock, and contributes to circadian gene expression oscillation. We also identify Salt inducible kinase 3 (SIK3) as a direct transcriptional target of Mondo-Mlx required for dietary sugar tolerance. Moreover, we show that the activity of the rate-limiting enzyme in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD), is increased following sugar feeding. The sugar-augmented increase in G6PD activity is achieved through SIK3-dependent activating G6PD phosphorylation. Collectively, Mondo-Mlx-mediated transcriptional upregulation, as well as SIK3-dependent phosphorylation promote G6PD enzyme activity in response to high high sugar diet. The increased G6PD activity is required for elevating NADPH/NADP+ ratio in order to reduce glutathione in high sugar conditions. We determine that upregulating G6PD activity through Mondo-Mlx and SIK3 is essential for redox balance maintenance, and dietary sugar tolerance. In sum, thesis demonstrates that maintaining redox balance, directing the flow of carbon backbones, and regulating the expression of metabolic circadian genes is essential for dietary sugar tolerance. We highlight the central role of Mondo-Mlx in orchestrating the necessary transcriptional response required for safe glucose utilization, and ultimately, for maintaining metabolic homeostasis.Yksinkertaiset hiilihydraatit, sokerit, ovat merkittävä osa päivittäistä ravintoamme ja sokerien kulutus on lisääntynyt huomattavasti viime vuosikymmeninä. Samanaikaisesti metabolisten sairauksien, kuten diabeteksen ja lihavuuden, esiintyvyys on lisääntynyt voimakkaasti. Ymmärrämme kuitenkin edelleen huonosti, miten yksilön geenit vaikuttavat ruuan sokerien aiheuttamiin terveysriskeihin. Eläimet pystyvät nopeasti sopeuttamaan elimistönsä aineenvaihdunnan muuttuviin ravitsemusolosuhteisiin. Näinollen esimerkiksi veren glukoosi pysyy tasapainossa aterian jälkeen. Tämä on mahdollista, koska elimistössä on ns. glukoosinaistintamekanismeja, jotka toimivat hormonaalisesti (esim. insuliini ja glukagoni) tai paikallisesti solujen sisällä. Nämä mekanismit reagoivat elimistön muuttuviin glukoosipitoisuuksiin ja muuttavat eläimen fysiologiaa niin, että aineenvaihdunnan tasapaino palautuu. Solun sisäisen glukoosinaistinnan kulmakiviä ovat Mondo-transkriptiotekijät ChREBP (MondoB) ja MondoA, jotka toimivat yhdessä Mlx transkriptiotekijän kanssa. ChREBP/MondoA-Mlx-kompleksi aktivoituu solunsisäisen glukoosin lisääntyessä ja se säätelee monia aineenvaihduntaan vaikuttavia geenejä. Monet aineenvaihdunnan säätelyyn vaikuttavat mekanismit ovat evoluutiossa hyvin säilyneitä, joten ne ovat samankaltaisia ihmisellä ja yksinkertaisemmilla eläimillä, kuten banaanikärpäsellä (Drosophila melanogaster). Tämä mahdollistaa aineenvaihdunnan säätelyn perusmekanismien tutkimisen käyttäen Drosophilaa tutkimusmallina. Myös Drosophilalla on solunsisäinen glukoosinaistintajärjestelmä, joka on hieman yksinkertaisempi kuin ihmisellä: Drosophilalla on yksi Mondo-transkriptiotekijä ja yksi Mlx. Niiden toimintaa ei ollut juurikaan aiemmin tutkittu. Tässä väitöstyössä osoitimme, että Drosophilan Mondo ja Mlx muodostavat yhdessä samanlaisen kompleksin kuin ihmisellä. Lisäksi havaitsimme, että toiminnallinen Mondo-Mlx kompleksi on välttämätön edellytys Drosophilan kyvylle säilyä hengissä sokeripitoisella ravinnolla. Lisäksi löysimme useita Mondo-Mlx:n säätelemiä tekijöitä, jotka osallistuvat solunsisäiseen glukoosinaistintaan. Yksi näistä on transkriptiotekijä Cabut, joka vaikuttaa solunsisäiseen glukoosiaineenvaihduntaan inhiboimalla gluko- ja glyseroneogeneesiä. Lisäksi Cabut toimii yhdistävänä linkkinä glukoosiaineenvaihdunnan ja vuorokausirytmin säätelyn välillä. Toinen löytämämme Mondo-Mlx:n kohdegeeni on SIK3, joka on aineenvaihduntaa säätelevä kinaasi. Me havaitsimme, että SIK3 säätelee solujen hapetus-pelkistys-tasapainoa, mikä puolestaan on keskeinen säätelykohde eläinten sokerinsietokyvyn kannalta. Yhteenvetona, tässä työssä on löydetty uusia säätelyverkostoja, jotka mahdollistavat eläimen fysiologisen adaptoitumisen sokeripitoiseen ravintoon. Koska väitöstyössä tutkitut säätelijät ovat evoluutiossa hyvin säilyneitä, saavuttamamme tieto saattaa olla tärkeää myös ihmisen aineenvaihduntasairauksien näkökulmasta

Lipoic acid prevents fructose-induced changes in liver carbohydrate metabolism: Role of oxidative stress

Fructose administration rapidly induces oxidative stress that triggers compensatory hepatic metabolic changes. We evaluated the effect of an antioxidant, R/S-α-lipoic acid on fructose-induced oxidative stress and carbohydrate metabolism changes. METHODS: Wistar rats were fed a standard commercial diet, the same diet plus 10% fructose in drinking water, or injected with R/S-α-lipoic acid (35mg/kg, i.p.) (control+L and fructose+L). Three weeks thereafter, blood samples were drawn to measure glucose, triglycerides, insulin, and the homeostasis model assessment-insulin resistance (HOMA-IR) and Matsuda indices. In the liver, we measured gene expression, protein content and activity of several enzymes, and metabolite concentration. RESULTS: Comparable body weight changes and calorie intake were recorded in all groups after the treatments. Fructose fed rats had hyperinsulinemia, hypertriglyceridemia, higher HOMA-IR and lower Matsuda indices compared to control animals. Fructose fed rats showed increased fructokinase gene expression, protein content and activity, glucokinase and glucose-6-phosphatase gene expression and activity, glycogen storage, glucose-6-phosphate dehydrogenase mRNA and enzyme activity, NAD(P)H oxidase subunits (gp91phox and p22phox) gene expression and protein concentration and phosphofructokinase-2 protein content than control rats. All these changes were prevented by R/S-α-lipoic acid co-administration. CONCLUSIONS: Fructose induces hepatic metabolic changes that presumably begin with increased fructose phosphorylation by fructokinase, followed by adaptive changes that attempt to switch the substrate flow from mitochondrial metabolism to energy storage. These changes can be effectively prevented by R/S-α-lipoic acid co-administration. GENERAL SIGNIFICANCE: Control of oxidative stress could be a useful strategy to prevent the transition from impaired glucose tolerance to type 2 diabetes.Fil: Castro, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico. Centro de Endocrinologia Experimental y Aplicada (i); ArgentinaFil: Massa, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico. Centro de Endocrinologia Experimental y Aplicada (i); ArgentinaFil: Gagliardino, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico. Centro de Endocrinologia Experimental y Aplicada (i); ArgentinaFil: Francini, Flavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico. Centro de Endocrinologia Experimental y Aplicada (i); Argentin

Cellular Links between Neuronal Activity and Energy Homeostasis

Neuronal activity, astrocytic responses to this activity, and energy homeostasis are linked together during baseline, conscious conditions, and short-term rapid activation (as occurs with sensory or motor function). Nervous system energy homeostasis also varies during long-term physiological conditions (i.e., development and aging) and with adaptation to pathological conditions, such as ischemia or low glucose. Neuronal activation requires increased metabolism (i.e., ATP generation) which leads initially to substrate depletion, induction of a variety of signals for enhanced astrocytic function, and increased local blood flow and substrate delivery. Energy generation (particularly in mitochondria) and use during ATP hydrolysis also lead to considerable heat generation. The local increases in blood flow noted following neuronal activation can both enhance local substrate delivery but also provides a heat sink to help cool the brain and removal of waste by-products. In this review we highlight the interactions between short-term neuronal activity and energy metabolism with an emphasis on signals and factors regulating astrocyte function and substrate supply

PERIPHERAL AND CENTRAL GLUCOSE FLUX IN TYPE I DIABETES

Diabetes is a complex metabolic disorder, of which high blood glucose concentration is the primary hallmark. Type I diabetes mellitus (T1DM) is characterized by the lack of insulin production, due to a poorly understood autoinflammatory cascade. In the words of historian Barnett “Diabetes may no longer be a death sentence, but for more and more people in the 21st century, it will become a life sentence”, making it the focal point of many research groups. It is estimated that around 20 million individuals worldwide live with T1DM. Effects of long-term chronically elevated blood glucose are not only seen in micro/macro-vascular diseases, retinopathy, peripheral neuropathy, and liver disease but also in the brain as people with T1DM show decreased mental speed and flexibility. Despite these clinical observations, the brain’s role in hyperglycemia remains to be elucidated and could be key to identifying potential insulin-independent interventions to alleviate these effects. In recent years, insulin-independent mechanisms involved in glucose homeostasis have been discovered, most notably the brain’s capacity to regulate blood glucose. The brainstem dorsal vagal complex (DVC) is the main neuronal center responsible for parasympathetic visceral regulation and has been identified as a microcircuit that is important for the regulation of blood glucose. The present work, utilizing a designer receptor exclusively activated by the designer drug (DREADDs) system to selectively activate GABA neurons within hindbrain circuitry in vivo, demonstrates hindbrain inhibitory microcircuitry as a key mediator of whole-body glucose levels, indicating the role of the parasympathetic nervous system. Neuronal function is affected by the brain metabolome, especially as glucose metabolism is highly heterogeneous among brain regions. To accurately capture physiological brain metabolome we developed a method utilizing a high-power focused microwave to euthanize animals, and fix and preserve metabolites. To understand how hyperglycemia modulates the central carbon metabolism of several brain regions (neocortex, hippocampus, and dorsal vagal complex) we employed gas chromatography-mass spectroscopy. Utilizing untargeted metabolomics we found that glucose concentration was significantly elevated across all regions but glycogen and glucose-6-phosphate remained unchanged with hyperglycemia only in DVC. Interestingly pyruvate and lactate were unchanged across all regions indicating that hyperglycemia does not affect anaerobic cellular respiration. Intermediates of the tricarboxylic acid (TCA) malate and fumarate are significantly decreased with hyperglycemia only in DVC, suggesting that hyperglycemia in DVC preferentially affects TCA cycle. Furthermore, we observed a significant decrease in glutamate across all regions while glutamine and GABA were unchanged, suggesting neurotransmitter regulation disturbance. Stable isotopic tracing of uniformly labeled 13C6 glucose was employed to assess carbon flux in different brain regions perturbed by T1DM to understand its effect on glycolysis, tricarboxylic acid cycle, and neurotransmitter synthesis. We found that hyperglycemia results in metabolic reprogramming with a significant decrease in glucose utilization and we demonstrated decrease in immunofluorescent labeling of GLUT2, a neuronal glucose transporter, as well as enzymes pyruvate dehydrogenase and pyruvate carboxylase, responsible for anaplerosis of TCA cycle intermediates, indicating glucose hypometabolism phenotype. This work is the first of its kind to demonstrate the effects of hyperglycemia not on the brain as a whole but rather on specific regions; neocortex, hippocampus and DVC. We shown heterogeneous effects of hyperglycemia on the central carbon metabolism pathways in the brain, where TCA cycle and neurotransmitter regulation are selectively affected in the DVC. Collectively, these data demonstrate that peripheral hyperglycemia results in glucose hypometabolism in the brain and will serve as a starting point in understanding the brain’s metabolic adaptations during hyperglycemia

Maintaining Glucose Homeostasis in Response to Aging and Stress: The Role of Pcif1, Bmi1, and Pdx1

A sufficient number of functioning beta cells is necessary for maintaining glucose homeostasis. Reduction of beta cell mass or function leads to diabetes. Investigation into the maintenance of both beta cell mass and function is important for the development of therapies to prevent and/or restore functional beta cells. Here, the networks surrounding three proteins in the beta cell, Pcif1, Bmi1, and Pdx1, were studied as they relate to beta cell function and number. The Polycomb protein, Bmi1, has been shown to influence beta cell replication via epigenetic repression of the Ink4a/Arf locus, resulting in suppression of p16 protein translation. The adapter protein, Pcif1, facilitates the ubiquitination of Bmi1 and influences beta cell replication, as Pcif1 heterogyzous mice have increased rates of beta cell proliferation. I hypothesized that Pcif1 regulates beta cell proliferation through a Bmi1-dependent mechanism. Analysis of Pcif1 heterozygous islets revealed that p16 protein levels were indistinguishable from controls, thus making a p16-dependent mechanism unlikely. Further investigation of Bmi1 targets may reveal another pathway by which Pcif1 and Bmi1 influence beta cell replication. The role of Bmi1 has not been well-described in adult animals. Analysis of Bmi1 heterozygous animals revealed increased insulin sensitivity, as compared to wildtype. This was found to be due to an enhancement of Akt phosphorylation, with the upstream insulin signaling pathway unaffected. Bmi1 also appears to play a role in the development of insulin resistance, as Bmi1 levels are high in insulin-resistant animals. I also began to explore the possibility that the action of Pcif1 on Bmi1 is responsible for the role Bmi1 plays in insulin signaling. The transcription factor, Pdx1, regulates numerous processes specific to the beta cell, including multiple pathways regulating translation. Pdx1 levels have been shown to affect the ability of beta cells to respond to ER stress. A global analysis of translational efficiencies using the TRAP methodology indicated that Pdx1 activity may result in repression of translation of some transcripts. Further analysis of these transcripts will help determine how Pdx1 regulates the translatome of the beta cell and, potentially, how Pdx1 influences the beta cell stress response

Mechanistic Role of ARNT/HIF-1β in the Regulation of Glucose-Stimulated Insulin Secretion

Loss of glucose-stimulated insulin secretion (GSIS) from the pancreatic beta-cells is one of the earliest detectable defects in the pathogenesis of type 2 diabetes. However, despite its relevance, the mechanisms that govern GSIS are still not completely understood. ARNT/HIF-1β is a member of the bHLH-PAS family of transcription factors, with a prominent role in the transcriptional regulation of enzymes required for the metabolism of xenobiotics as well as regulation of genes that are critical for cellular responses to hypoxia. Recent research has uncovered a previously unknown function for ARNT/HIF-1β in the pancreatic beta-cells, where the gene was found to be 90% down-regulated in human type 2 diabetic islets and loss of ARNT/HIF-1β protein leads to defective GSIS in pancreatic beta-cells of mice. The main focus of this thesis was to understand the mechanisms by which ARNT/HIF-1β maintains normal GSIS from pancreatic beta-cells and understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice. ARNT/HIF-1β was found to positively regulate GSIS in both INS-1 derived 832/13 cell line and mice islets. In the 832/13 cells, loss of ARNT/HIF-1β leads to a reduction in glycolysis without affecting the glucose oxidation and the ATP/ADP ratio suggesting that the regulation of GSIS takes place in a manner that is independent of the KATP channels. In order to further assess the mechanism of lowered GSIS in the absence of ARNT/HIF-1β in the 832/13 cells, a metabolite profiling was performed which revealed a significant reduction in the metabolite levels of glycolysis and the TCA cycle intermediates and glucose-induced fatty acid production, suggesting the involvement of ARNT/HIF-1β in regulating glucose-stimulated anaplerosis, which is believed to play a key role in the regulation of GSIS from the pancreatic beta-cells. The changes in metabolite levels in the absence of ARNT/HIF-1β were associated with corresponding changes in the gene expression pattern of key enzymes regulating glycolysis, the TCA cycle and fatty acid synthesis in beta-cells. In an attempt to understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice, a pancreatic beta-cell specific ARNT/HIF-1β knock out mouse (β-ARNT KO) was generated using the Cre-loxP technology. Functional characterization of islets from both male and female β-ARNT KO mice revealed a significant impairment in GSIS, which was attributed due to a small, but significant reduction in rise in intracellular calcium upon glucose stimulation. Further analysis revealed reduced secretory response to glucose in the presence of KCl and diazoxide indicating a defect in the amplifying pathway of GSIS in β-ARNT KO islets. Expression of pyruvate carboxylase (PC) was significantly reduced in β-ARNT KO islets suggesting possible impairments in anaplerosis and consistent with this, defect in GSIS in β-ARNT KO islets could be almost completely rescued by treatment with membrane permeable TCA intermediates. Surprisingly, both male and female β-ARNT KO mice have normal glucose homeostasis. In an attempt to assess how β-ARNT KO mice maintained normal blood glucose levels, indirect calorimetry was used to understand changes in whole-body energy expenditure. This investigation revealed that β-ARNT KO mice exhibited a small but significant increase in respiratory exchange ratio (RER), suggesting a preference in utilizing carbohydrates as a fuel source, possibly leading to improved glucose uptake from the blood stream. Response to exogenous insulin was completely normal in β-ARNT KO mice suggesting intact functioning of the skeletal muscles. To conclude, based on our in vitro data, we believe that ARNT/HIF-1β plays an indispensable role in maintaining normal beta-cell secretory function, however, results from β-ARNT KO mice indicates that these mice are protected from the adverse effects of hyperglycemia. Although loss of ARNT/HIF-1β alone is not sufficient for the genesis of type 2 diabetes, it creates a perfect storm in the pancreatic beta-cells that may eventually lead to an imbalance in the whole body glucose homeostasis. Our study provides significant information to the scientific community that engages in assessing the pharmacological potential of gene targets for the treatment of type 2 diabetes.1 yea