Glucose Output Research Articles

Discovery of potent and orally bioavailable indazole-based glucagon receptor antagonists for the treatment of type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is characterized by chronically elevated plasma glucose levels. The inhibition of glucagon-induced hepatic glucose output via antagonism of the glucagon receptor (GCGR) using a small-molecule antagonist is a promising mechanism for improving glycemic control in the diabetic state. The present work discloses the discovery of indazole-based β-alanine derivatives as potent GCGR antagonists through an efficient enantioselective synthesis and structure-activity relationship (SAR) exploration and optimization. Compounds within this class exhibited excellent pharmacokinetic properties in multiple preclinical species. In an acute dog glucagon challenge test, compound 13K significantly inhibited glucagon-mediated blood glucose increase when dosed orally at 10 mg/kg.

Female mice are protected against acute olanzapine-induced hyperglycemia

Olanzapine is a second-generation antipsychotic (SGA) used frequently in the treatment of schizophrenia and a growing list of off-label conditions. Though effective in reducing psychoses, acute olanzapine treatment causes rapid increases in blood glucose that are believed to be mediated by increases in liver glucose output, skeletal muscle insulin resistance, and beta cell dysfunction. Further, the acute lipidemic response to olanzapine has been largely unexplored. While females have been reported to be more susceptible to olanzapine-induced weight gain, there is little known about the impact of sex on the acute response to SGAs. The purpose of this study was to determine if the acute effects of SGAs on glucose and lipid metabolism display a sexually dimorphic response in C57BL/6 J mice and examine potential mechanisms mediating this effect. Age matched male and female C57BL/6 J mice were treated with olanzapine (5 mg/ kg, IP) or vehicle control and blood glucose was measured at baseline, 15, 30, 60, 90, and 120 min post-treatment and tissues and serum harvested. These experiments were repeated, and mice underwent an insulin (0.5 IU/kg) or pyruvate tolerance test (2 g/kg) following 60 min of olanzapine treatment. Females were protected against olanzapine-induced increases in blood glucose and pyruvate intolerance compared to male mice, and this occurred despite the development of severe insulin resistance. In male mice olanzapine increased the glucagon:insulin ratio whereas in females this ratio was reduced. When challenged with exogenous glucagon (1 mg/kg IP), females were less responsive than males. Male and female mice displayed similar increases in whole body fatty acid oxidation, serum fatty acids and liver triglyceride accumulation. Our findings provide evidence that while there are no apparent sex differences in the lipid metabolism response to olanzapine, that females are protected from acute olanzapine-induced excursions in blood glucose. This is likely due in part to reductions in the glucagon:insulin ratio and glucagon responsiveness which could impact olanzapine induced increases in liver glucose production.

Lipid nanoparticles as vehicles for oral delivery of insulin and insulin analogs: preliminary ex vivo and in vivo studies.

Subcutaneous administration of insulin in patients suffering from diabetes is associated with the distress of daily injections. Among alternative administration routes, the oral route seems to be the most advantageous for long-term administration, also because the peptide undergoes a hepatic first-pass effect, contributing to the inhibition of the hepatic glucose output. Unfortunately, insulin oral administration has so far been hampered by degradation by gastrointestinal enzymes and poor intestinal absorption. Loading in lipid nanoparticles should allow to overcome these limitations. Entrapment of peptides into such nanoparticles is not easy, because of their high molecular weight, hydrophilicity and thermo-sensitivity. In this study, this objective was achieved by employing fatty acid coacervation method: solid lipid nanoparticles and newly engineered nanostructured lipid carriers were formulated. Insulin and insulin analog-glargine insulin-were entrapped in the lipid matrix through hydrophobic ion pairing. Bioactivity of lipid entrapped peptides was demonstrated through a suitable in vivo experiment. Ex vivo and in vivo studies were carried out by employing fluorescently labelled peptides. Gut tied up experiments showed the superiority of glargine insulin-loaded nanostructured lipid carriers, which demonstrated significantly higher permeation (till 30% dose/mL) compared to free peptide. Approximately 6% absolute bioavailability in the bloodstream was estimated for the same formulation through in vivo pharmacokinetic studies in rats. Consequently, a discrete blood glucose responsivity was noted in healthy animals. Given the optimized ex vivo and in vivo intestinal uptake of glargine insulin from nanostructured lipid carriers, further studies will be carried out on healthy and diabetic rat models in order to establish a glargine insulin dose-glucose response relation.

First High-Resolution Crystal Structures of the Glucocorticoid Receptor Ligand-Binding Domain–Peroxisome Proliferator-Activated γ Coactivator 1-α Complex with Endogenous and Synthetic Glucocorticoids

Both synthetic and endogenous glucocorticoids are important pharmaceutic drugs known to bind to the ligand-binding domain (LBD) of glucocorticoid receptor (GR), a member of the nuclear receptor (NR) superfamily. Ligand binding induces conformational changes within GR, resulting in subsequent DNA binding and differential coregulator recruitment, ultimately activating or repressing target gene expression. One of the most crucial coregulators is peroxisome proliferator-activated γ coactivator 1-α (PGC1α), which acts to regulate energy metabolism by directly interacting with GR to modulate gene expression. However, the mechanisms through which PGC1α senses GR conformation to drive transcription are not completely known. Here, an ancestral variant of the GR (AncGR2) LBD was used as a tool to produce stable protein for biochemical and structural studies. PGC1α is found to interact more tightly and form a more stable complex with AncGR2 LBD than nuclear receptor coactivator 2. We report the first high-resolution X-ray crystal structures of AncGR2 LBD in complex with PGC1α and dexamethasone (DEX) or hydrocortisone (HCY). Structural analyses reveal how distinct steroid drugs bind to GR with different affinities by unique hydrogen bonds and hydrophobic interactions. Important charge clamps are formed between the activation function 2 and PGC1α to mediate their specific interactions. These interactions lead to a high level of protection from hydrogen-deuterium exchange at the coregulator interaction site and strong intramolecular allosteric communication to ligand binding site. This is the first structure detailing the GR-PGC1α interaction providing a foundation for future design of specific therapeutic agents targeting these critical metabolic regulators. SIGNIFICANCE STATEMENT: High-resolution structures of AncGR2 LBD bound to DEX and HCY in complex with PGC1α reveal the molecular mechanism of PGC1α binding to AncGR2 LBD as well as the distinct affinities between DEX and HCY binding. Identifying the structural mechanisms that drive drug affinity is of pharmacologic interest to the glucocorticoid receptor field as an avenue to guide future drug design targeting GR-PGC1α signaling, which plays a crucial role in controlling hepatic glucose output.

A dual mixture of persistent organic pollutants modifies carbohydrate metabolism in the human hepatic cell line HepaRG

Individuals as well as entire ecosystems are exposed to mixtures of Persistent Organic Pollutants (POPs). Previously, we showed, by a non-targeted approach, that the expression of several genes involved in carbohydrate metabolism was almost completely inhibited in the human hepatic cell line HepaRG following exposure to a mixture of the organochlorine insecticide alpha-endosulfan and 2,3,7,8 tetrachlorodibenzo-p-dioxin. In this European HEALS project, which studies the effects of the exposome on human health, we used a Physiologically Based BioKinetic model to compare the concentrations previously used in vitro with in vivo exposures for humans. We investigated the effects of these POPs on the levels of proteins, on glycogen content, glucose production and the oxidation of glucose into CO2 and correlated them to the expression of genes involved in carbohydrate metabolism as measured by RT-qPCR. Exposure to individual POPs and the mixture decreased the expression of the proteins investigated as well as glucose output (up to 82%), glucose oxidation (up to 29%) and glycogen content (up to 48%). siRNAs that specifically inhibit the expression of several xenobiotic receptors were used to assess receptor involvement in the effects of the POPs. In the HepaRG model, we demonstrate that the effects are mediated by the aryl hydrocarbon receptor and the estrogen receptor alpha, but not the pregnane X receptor or the constitutive androstane receptor. These results provide evidence that exposure to combinations of POPs, acting through different signaling pathways, may affect, more profoundly than single pollutants alone, metabolic pathways such as carbohydrate/energy metabolism and play a potential role in pollutant associated metabolic disorders.

Non-peptidyl small molecule, adenosine, 5\u2032-Se-methyl-5\u2032-seleno-, 2\u2032,3\u2032-diacetate, activates insulin receptor and attenuates hyperglycemia in type 2 diabetic Leprdb/db mice

The pathophysiology of type 2 diabetes mellitus (T2D) is characterized by reduced or absent insulin receptor (INSR) responsiveness to its ligand, elevated hepatic glucose output and impaired glucose uptake in peripheral tissues, particularly skeletal muscle. Treatments to reduce hyperglycemia and reestablish normal insulin signaling are much sought after. Any agent which could be orally administered to restore INSR function, in an insulin-independent manner, would have major implications for the management of this global disease. We have discovered a non-peptidyl small molecule, adenosine, 5′-Se-methyl-5′-seleno-, 2′,3′-diacetate [referred to as non-peptidyl compound #43 (NPC43)], which restores INSR signaling in the complete absence of insulin. Initial screening of numerous compounds in human HepG2 liver cells revealed that NPC43 significantly inhibited glucose production. The compound was potently anti-hyperglycemic and anti-hyperinsulinemic in vivo, in insulin-resistant T2D Leprdb/db mice, following either acute or chronic treatment by oral gavage and intraperitoneal injection, respectively. The compound acted at the level of INSR and activated it in both liver and skeletal muscle of Leprdb/db mice. In cell culture, the compound activated INSR in both liver and skeletal muscle cells; furthermore, it cooperated with insulin to depress glucose-6-phosphatase catalytic subunit (G6pc) expression and stimulate glucose uptake, respectively. Our results indicated that the compound directly interacted with INSRα, triggering appropriate phosphorylation and activation of the receptor and its downstream targets. Unlike insulin, NPC43 did not activate insulin-like growth factor 1 receptor in either liver or skeletal muscle. We believe this compound represents a potential oral and/or injectable insulin replacement therapy for diabetes and diseases associated with insulin resistance.

Anabolic and Pro-metabolic Functions of CREB-CRTC in Skeletal Muscle: Advantages and Obstacles for Type 2 Diabetes and Cancer Cachexia.

cAMP is one of the earliest described mediators of hormone action in response to physiologic stress that allows acute stress responses and adaptation in every tissue. The classic role of cAMP signaling in metabolic tissues is to regulate nutrient partitioning. In response to acute stress, such as epinephrine released during strenuous exercise or fasting, intramuscular cAMP liberates glucose from glycogen and fatty acids from triglycerides. In the long-term, activation of Gs-coupled GPCRs stimulates muscle growth (hypertrophy) and metabolic adaptation through multiple pathways that culminate in a net increase of protein synthesis, mitochondrial biogenesis, and improved metabolic efficiency. This review focuses on regulation, function, and transcriptional targets of CREB (cAMP response element binding protein) and CRTCs (CREB regulated transcriptional coactivators) in skeletal muscle and the potential for targeting this pathway to sustain muscle mass and metabolic function in type 2 diabetes and cancer. Although the muscle-autonomous roles of these proteins might render them excellent targets for both conditions, pharmacologic targeting must be approached with caution. Gain of CREB-CRTC function is associated with excess liver glucose output in type 2 diabetes, and growing evidence implicates CREB-CRTC activation in proliferation and invasion of different types of cancer cells. We conclude that deeper investigation to identify skeletal muscle specific regulatory mechanisms that govern CREB-CRTC transcriptional activity is needed to safely take advantage of their potent effects to invigorate skeletal muscle to potentially improve health in people with type 2 diabetes and cancer.

High-content analysis of constitutive androstane receptor (CAR) translocation identifies mosapride citrate as a CAR agonist that represses gluconeogenesis

The constitutive androstane receptor (CAR) plays an important role in hepatic drug metabolism and detoxification but has recently been projected as a potential drug target for metabolic disorders due to its repression of lipogenesis and gluconeogenesis. Thus, identification of physiologically-relevant CAR modulators has garnered significant interest. Here, we adapted the previously characterized human CAR (hCAR) nuclear translocation assay in human primary hepatocytes (HPH) to a high-content format and screened an FDA-approved drug library containing 978 compounds. Comparison of hCAR nuclear translocation results with the Tox21 hCAR luciferase reporter assay database in 643 shared compounds revealed significant overlap between these two assays, with approximately half of hCAR agonists also mediating nuclear translocation. Further validation of these compounds in HPH and/or using published data from literature demonstrated that hCAR translocation exhibits a higher correlation with the induction of hCAR target genes, such as CYP2B6, than the luciferase assay. In addition, some CAR antagonists which repress CYP2B6 mRNA expression in HPH, such as sorafenib, rimonabant, and CINPA1, were found to translocate hCAR to the nucleus of HPH. Notably, both the translocation assay and the luciferase assay identified mosapride citrate (MOS), a gastroprokinetic agent that is known to reduce fasting blood glucose levels in humans, as a novel hCAR activator. Further studies with MOS in HPH uncovered that MOS can repress the expression of gluconeogenic genes and decrease glucose output from hepatocytes, providing a previously unidentified liver-specific mechanism by which MOS modulates blood glucose levels.

The Modulatory Action of Vitamin D on the Renin-Angiotensin System and the Determination of Hepatic Insulin Resistance.

Vitamin D deficiency or hypovitaminosis D is associated with increased risks of insulin resistance, type 2 diabetes mellitus (T2DM) and its related non-alcoholic fatty liver disease (NAFLD). Meanwhile, inappropriate over-activation of the renin–angiotensin system (RAS) in the liver leads to the hepatic dysfunction and increased risk of T2DM, such as abnormalities in lipid and glucose metabolism. Our previous findings have shown that calcitriol, an active metabolite of vitamin D, reduces hepatic triglyceride accumulation and glucose output in diabetic db/db mice and human hepatocellular cell HepG2 cells under insulin-resistant conditions. Notwithstanding the existence of this evidence, the protective action of vitamin D in the modulation of overexpressed RAS-induced metabolic abnormalities in the liver under insulin resistance remains to be elusive and investigated. Herein, we have reported the potential interaction between vitamin D and RAS; and its beneficial effects on the expression and function of the RAS components in HepG2 cells and primary hepatocytes under insulin-resistance states. Our study findings suggest that hormonal vitamin D (calcitriol) has modulatory action on the inappropriate upregulation of the hepatic RAS under insulin-resistant conditions. If confirmed, vitamin D supplementation might provide a nutraceutical potential as a cost-effective approach for the management of hepatic metabolic dysfunction as observed in T2DM and related NAFLD.

A Short Review on Anti-diabetics for Uncontrolled Type 2 Diabetes Mellitus

Introduction: This article reviews the treatments available for patients with T2DM, with an emphasis on agents introduced within the last decade. Materials and Methods: This review is based on a search of Medline, the Cochrane Database of Systemic Reviews, and citation lists of relevant publications. Subject heading and key words used include type 2 diabetes mellitus, prevalence, current diagnosis, and current treatment. Only articles in English were included.
 Materials & Methods: Screening and diagnosis is still based on World Health Organization (WHO) and American Diabetes Association (ADA) criteria which include both clinical and laboratory parameters. No cure has yet been found for the disease; however, treatment modalities include lifestyle modifications, treatment of obesity, oral hypoglycemic agents, and insulin sensitizers is still the recommended first line medication. Other effective medications include non-sulfonylurea secretagogues, thiazolidinediones, alpha glucosidase inhibitors, and insulin. Recent research into the pathophysiology of type 2 DM has led to the introduction of new medications like glucagon-like peptide 1 analogoues: dipeptidyl peptidase-IV inhibitors, inhibitors of the sodium-glucose cotransporter 2 and 11ß-hydroxysteroid dehydrogenase 1, insulin-releasing glucokinase activators and pancreatic-G-protein-coupled fatty-acid-receptor agonists, glucagon-receptor antagonists, metabolic inhibitors of hepatic glucose output and quick-release bromocriptine.
 Discussion: Screening and diagnosis is still based on World Health Organization (WHO) and American Diabetes Association (ADA) criteria which include both clinical and laboratory parameters. No cure has yet been found for the disease; however, treatment modalities include lifestyle modifications, treatment of obesity, oral hypoglycemic agents, and insulin sensitizers is still the recommended first line medication. Other effective medications include non-sulfonylurea secretagogues, thiazolidinediones, alpha glucosidase inhibitors, and insulin. Recent research into the pathophysiology of type 2 DM has led to the introduction of new medications like glucagon-like peptide 1 analogoues: dipeptidyl peptidase-IV inhibitors, inhibitors of the sodium-glucose cotransporter 2 and 11ß-hydroxysteroid dehydrogenase 1, insulin-releasing glucokinase activators and pancreatic-G-protein-coupled fatty-acid-receptor agonists, glucagon-receptor antagonists, metabolic inhibitors of hepatic glucose output and quick-release bromocriptine.
 Conclusion: Metformin remains the first choice of treatment for most patients. Other alternative or second-line treatment options should be individualized depending on the characteristics of each patient.
 Medicine Today 2019 Vol.31(2): 120-127

Metabolic Effects of Metformin in Humans.

Both insulin deficiency and insulin resistance due to glucagon secretion cause fasting and postprandial hyperglycemia in patients with diabetes. Metformin enhances insulin sensitivity, being used to prevent and treat diabetes, although its mechanism of action remains elusive. Patients with diabetes fail to store glucose as hepatic glycogen via the direct pathway (glycogen synthesis from dietary glucose during the post-prandial period) and via the indirect pathway (glycogen synthesis from "de novo" synthesized glucose) owing to insulin deficiency and glucagoninduced insulin resistance. Depletion of the hepatic glycogen deposit activates gluconeogenesis to replenish the storage via the indirect pathway. Unlike healthy subjects, patients with diabetes experience glycogen cycling due to enhanced gluconeogenesis and failure to store glucose as glycogen. These defects raise hepatic glucose output causing both fasting and post-prandial hyperglycemia. Metformin reduces post-prandial plasma glucose, suggesting that the drug facilitates glucose storage as hepatic glycogen after meals. Replenishment of glycogen store attenuates the accelerated rate of gluconeogenesis and reduces both glycogen cycling and hepatic glucose output. Metformin also reduces fasting hyperglycemia due to declining hepatic glucose production. In addition, metformin reduces plasma insulin concentration in subjects with impaired glucose tolerance and diabetes and decreases the amount of insulin required for metabolic control in patients with diabetes, reflecting improvement of insulin activity. Accordingly, metformin preserves β-cell function in patients with type 2 diabetes. Several mechanisms have been proposed to explain the metabolic effects of metformin, but evidence is not conclusive and the molecular basis of metformin action remains unknown.

Unconventional Secretion of Adipocyte Fatty Acid Binding Protein 4 Is Mediated By Autophagic Proteins in a Sirtuin-1-Dependent Manner.

Fatty acid binding protein 4 (FABP4) is a leaderless lipid carrier protein primarily expressed by adipocytes and macrophages that not only functions intracellularly but is also secreted. The secretion is mediated via unconventional mechanism(s), and in a variety of species, metabolic dysfunction is correlated with elevated circulating FABP4 levels. In diabetic animals, neutralizing antibodies targeting serum FABP4 increase insulin sensitivity and attenuate hepatic glucose output, suggesting the functional importance of circulating FABP4. Using animal and cell-based models, we show that FABP4 is secreted from white, but not brown, adipose tissue in response to lipolytic stimulation in a sirtuin-1 (SIRT1)-dependent manner via a mechanism that requires some, but not all, autophagic components. Silencing of early autophagic genes such as Ulk1/2, Fip200, or Beclin-1 or chemical inhibition of ULK1/2 or VPS34 attenuated secretion, while Atg5 knockdown potentiated FABP4 release. Genetic knockout of Sirt1 diminished secretion, and serum FABP4 levels were undetectable in Sirt1 knockout mice. In addition, blocking SIRT1 by EX527 attenuated secretion while activating SIRT1 by resveratrol-potentiated secretion. These studies suggest that FABP4 secretion from adipocytes is regulated by SIRT1 and requires early autophagic components.

1845-P: Divergent Effects on T2D of Alleles Associated with Increased Liver Fat Reflect Differential Impact on Hepatic Glucose and Lipid Output: An IMI DIRECT Study

Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) often coincide but the physiological relationship is unclear. Important clues are provided by human genetics: variants associated with NAFLD-risk have divergent effects on T2D. At TM6SF2 the NAFLD-risk allele (rs58542926:T) predisposes to T2D whilst at GCKR (rs1260326:T), it protects. We hypothesize that these divergent effects result from the impact of NAFLD-raising alleles on hepatic lipid output (HLO) and hepatic glucose output (HGO) respectively. To test this hypothesis, we applied structural equation modeling (SEM) to data from the IMI-DIRECT study, including 1350 European participants, 923 nondiabetic (C1) and 427 with recently-diagnosed T2D (C2). Insulin sensitivity (IS) was modeled from frequently sampled OGTT (C1) and mixed-meal tolerance tests (C2), and liver fat (LF) measured by MRI. SEM fit was assessed by comparing model χ2s with comparable null models: variables randomized to nodes in identical SEM definition giving comparable degrees of freedom and structure, 10,000 iterations. The hypothesized model provided a better fit than null models (C1: χ2=242, P=0.005; C2: χ2=63, P=0.01). Decreasing HLO (via TM6SF2) was associated with an increase in LF (C1: ß=0.28 [SE 0.09], P=0.001; C2: ß=0.28 [0.12], P=0.02) and fasting plasma glucose (FG): these were mediated by LF and IS (C1: ß=0.09 [0.03], P=0.001; C2: ß=0.07 [0.03], P=0.027). Decreasing HGO (via GCKR) was not associated with LF in either cohort but was associated with a direct (non-mediated) decrease in FG (C1 only: ß=-0.08 [0.04], P=0.04). The results support a model whereby increased genetic risk of NAFLD, arising from defects in two processes (HLO and HGO) results in opposing glycemic effects. This has important consequences for therapeutic strategies for combatting NAFLD: approaches to reduce NAFLD through targeting of HLO (as opposed to HGO) may also reduce risk of hyperglycaemia and T2D. Disclosure R.W. Koivula: None. P.W. Franks: Board Member; Self; Zoe Ltd. Research Support; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Lilly Diabetes, Novo Nordisk A/S, Novo Nordisk Foundation, Sanofi, Servier. T.H. Hansen: None. M. Laakso: None. E. Pearson: None. F. Rutters: None. F. Karpe: None. J.W. Tomlinson: Advisory Panel; Self; Novo Nordisk Inc., Pfizer Inc., Poxel SA. A. Mahajan: None. M. McCarthy: Advisory Panel; Self; European Association for the Study of Diabetes, Pfizer Inc. Consultant; Self; Eli Lilly and Company, Merck & Co., Inc. Consultant; Spouse/Partner; Merck & Co., Inc. Research Support; Self; AbbVie Inc., Boehringer Ingelheim International GmbH. Research Support; Spouse/Partner; Diabetes UK. Research Support; Self; Janssen Pharmaceuticals, Inc., Merck & Co., Inc., National Institutes of Health. Research Support; Spouse/Partner; National Institutes of Health. Research Support; Self; Novo Nordisk A/S. Research Support; Spouse/Partner; Novo Nordisk A/S. Research Support; Self; Novo Nordisk Foundation, Roche Pharma, Sanofi-Aventis, Servier, Takeda Pharmaceutical Company Limited. Funding Novo Nordisk Foundation (NNF18OC0031650); Innovative Medicines Initiative Joint Undertaking (115317)

1056-P: Characterization of Multi-timescale Biological Rhythms in Individuals with Type 1 Diabetes

Background: Longitudinal biological rhythms in glucose and insulin outputs have not been mapped in individuals with T1D. Physiologically based, within-individual predictions of ultradian (UR), circadian (CR) or longer timescale changes in glucose and insulin are not currently possible, in part due to a lack of understanding of population heterogeneity. This is the first longitudinal analysis of biological rhythms in T1D, compared to non-T1D individuals. Methods: Frequency decomposition using the continuous morlet wavelet transformation were created to assess change in rhythmic composition of normalized blood glucose data from 5 non-T1D individuals, and anonymized, retrospective CGM data from 19 T1D individuals using a DIY closed loop APS in the OpenAPS Data Commons. Results: T1D individuals showed a wide range of rhythmic power at the circadian and long-period ultradian timescales compared to individuals without T1D. There is not much UR power lower than 6-hour periodicity, likely due to insulin DIA effects. Conclusion: Such analysis of biological rhythms should be further studied across a broader T1D population, including both APS and non-users, and knowledge of the rhythms could be used for improving personalization and prediction capabilities of APS. Disclosure D.M. Lewis: Consultant; Self; Diabeloop SA, Roche Diabetes Care. A.D. Grant: None.

280-LB: Role of A1 and A3 Adenosine Receptors in Whole Body Glucose Metabolism

A1 and A3 adenosine receptors (ARs) belong to the group of GPCRs that primarily couple to Gi/o protein and mediate adenosine’s biological actions. ARs are widely expressed in the body and regulate various metabolic functions. We generated mice lacking two ARs, Adora1-/-; Adora3-/- (double knockout [A1A3DKO]) to enable investigation of the role of Gi-coupled ARs on glucose metabolism. The Adora1-/- (A1KO), Adora3-/- (A3KO) and WT mice generated as littermates were used as controls. A1A3DKO mice showed no difference in body weight, glucose tolerance, insulin sensitivity, fed blood glucose and plasma insulin levels on regular chow (RC). Fasting blood glucose levels were reduced in A1KO and A1A3DKO mice compared to WT mice. No difference in body weight was observed between A1A3DKO and control groups on high fat diet (HFD). A1KO mice showed improved glucose tolerance compared to A3KO mice without any difference in insulin sensitivity. No difference in plasma insulin levels was observed between the groups. Fasting blood glucose levels were reduced in A1A3DKO mice and A1KO mice compared to WT and A3KO mice, respectively. We investigated the contribution of liver to the observed decrease in fasting blood glucose levels. Pyruvate tolerance test revealed decreased glucose output from the liver in A1KO and A1A3DKO mice. Expression levels of hepatic genes involved in gluconeogenesis, lipid uptake, lipogenesis and inflammation were reduced in A1KO and A1A3DKO mice. Activation of A1AR by specific agonist (MRS7469) increased the phosphorylation of c-Jun N-terminal Kinases (JNK). Increased hepatic glucose production because of Gi signaling-mediated JNK activation is well documented. Taken together, our data support the concept that A1AR antagonists may prove valuable in enhancing whole-body glucose homeostasis by improving hepatic metabolism and could be beneficial in diabetes treatment. Disclosure S. Jain: None. D.K. Tosh: None. M. Reitman: None. K.A. Jacobson: None.

293-LB: The Suppression of Xanthine Oxidoreductase (XOR) Improves Skeletal Muscle Insulin Resistance

Hyperuricemia is closely associated with insulin resistance. Xanthine oxidoreductase (XOR), a key enzyme known to catalyze purines to uric acid, is expressed in ubiquitous tissues. In addition, XOR expression is upregulated in obesity. Therefore, we speculated that XOR may exacerbate insulin resistance. But, the effects of XOR on insulin sensitivity have not been fully elucidated. Febuxostat is a selective XOR inhibitor. In this study we investigated the effect of suppression of XOR using febuxostat on insulin sensitivity in vivo and in vitro. Male Wistar rats were fed with 60% high fat diet containing with either febuxostat or not, for 4 weeks. Euglycemic-hyperinsulinemic clamp studies were performed after an 8-h fast. Differentiated C2C12 cells were incubated with 0 or 10 μM febuxostat. For Xor silencing experiments, we used the Xor RNAi adenovirus. The glucose infusion rate and insulin stimulated-glucose disposal rate were significantly increased by 12% (P<0.05) and 17% (P<0.05), respectively, in febuxostat fed rats. But the clamp hepatic glucose output was no significant change between two groups. Consistent with the clamp data, the insulin-stimulated phosphorylation of Akt and AMPK were significantly increased by 90% (P<0.05) and 53% (P<0.05), respectively, in skeletal muscle of febuxostat fed rats. In differentiated C2C12 cells, febuxostat treatment significantly increased the insulin-stimulated phosphorylation of Akt and AMPK by 65% (P<0.05) and 43% (P<0.05), respectively. Furthermore, knockdown of XOR with XOR RNAi adenovirus significantly increased the insulin-stimulated phosphorylation of Akt by 60% (P<0.05). These results suggest that the suppression of XOR in skeletal muscle improves the skeletal muscle insulin resistance. In conclusion, our findings demonstrated that suppression of XOR had beneficial effects on skeletal muscle insulin sensitivity in an insulin resistant state. The upregulation of XOR in skeletal muscle might cause the insulin resistance. Disclosure C.T. Moriya: None. H. Satoh: None. S. Kakehi: None. H. Watada: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Kissei Pharmaceutical Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Pfizer Inc., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited. Speaker's Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Sanofi, Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. Other Relationship; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Kowa Pharmaceutical Europe Co. Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Sanwa Chemical Industry Co. Ltd., Takeda Pharmaceutical Company Limited.

371-P: Hypoglycemia in the Brain Is Required to Augment Counterregulatory Responses to Hypoglycemia when Liver Glycogen Levels Are Enhanced

In a previous study, we showed that elevated hepatic glycogen levels can generate a signal in the liver during insulin (INS)-induced hypoglycemia that is relayed to the brain, resulting in enhanced counterregulatory (CR) hormone secretion and net hepatic glucose output (NHGO). The purpose of this study was to determine whether hypoglycemia in the brain is required for this augmentation to occur. Fasted (18 hour) dogs were studied and each experiment began with a 4h glycogen deposition period in which each animal received somatostatin and basal intraportal infusions of both INS and glucagon (GGN). Plasma glucose (PG) was doubled by an intravenous (IV) glucose infusion and liver glycogen content was enhanced in all animals (76 ± 6 and 68 ± 4 mg/g in Brain EU and HYPO, respectively) by an intraportal infusion of fructose. A 2h control period followed, during which hyperglycemia and basal replacement of hormones were continued but fructose infusion was stopped. In the final 2h hypoglycemic period, somatostatin and GGN infusions were stopped and the plasma INS level was increased 16-fold by an IV infusion of INS. During the final period, whole body PG was clamped at 52 ± 1 mg/dL in one group (HYPO; n=6). In a second group, PG in the head was clamped at 92 ± 3 mg/dL by infusing glucose into the carotid and vertebral arteries (Brain EU; n=8) while the glucose level in the rest of the body remained at 51 ± 1 mg/dL. During the final hour of the hypoglycemic period, NHGO was lower in Brain EU compared to HYPO (0.8 ± 0.2 vs. 2.2 ± 0.4 mg/kg/minute, p<0.01). This decrease in NHGO was associated with a decrease in CR hormone secretion, as GGN (27 ± 6 vs. 53 ± 8 pg/mL, p<0.01) and epinephrine (451 ± 103 vs. 1106 ± 143 pg/mL, p<0.01) were lower when the brain was euglycemic (Brain EU vs. HYPO, respectively). These data suggest that the augmented CR responses that occur during INS-induced hypoglycemia when liver glycogen content is enhanced, require hypoglycemia in the brain. Disclosure S.O. Warner: None. M.S. Smith: None. B. Farmer: None. J.J. Winnick: None. Funding National Institutes of Health

1904-P: The Hepatocyte Integrin ß1 Subunit Links ECM-Integrin Signaling to Insulin Resistance in Obese Mice

Insulin resistance (IR) is a primary contributor to obesity related pathologies including type 2 diabetes. Hepatic IR manifests as a loss of insulin’s suppressive action on hepatic glucose output, and correlates with extracellular matrix (ECM) expansion. Integrin receptors are the primary input for ECM derived signals to the intracellular environment. Given the correlation of ECM deposition with hepatic IR our goal is to define contributions of integrin signaling to this pathologic process. Integrin receptors are heterodimeric membrane receptors with an α and β subunit. Hepatocytes are involved in hepatic insulin responsiveness and express four integrin subunits. Utilizing mice with hepatocyte specific knockout of integrin α5 (hepα5-KO) or β1 (hepβ1-KO) we aimed to determine contributions of these integrin subunits to IR. A high-fat (HF) fed model of diet induced obesity (DIO) caused weight gain in hepα5-KO, hepβ1-KO, and respective control littermates. Using hyperinsulinemic-euglycemic clamps in the conscious catheterized mouse we demonstrate that hepα5-KO mice on a standard diet (chow) have normal insulin sensitivity and develop IR during DIO similar to controls. While insulin sensitivity of chow fed hepβ1-KO is no different from controls, DIO hepβ1-KO mice are protected from IR. Significant increases in glucose disappearance (Rd) and suppression of endogenous glucose appearance (EndoRa) during the clamp drive preservation of insulin sensitivity in these mice. Interestingly the primary hepatocyte knockout of the β1 subunit has effects on extrahepatic tissue as DIO hepβ1-KO mice also demonstrate increased glucose metabolic indexes (Rg) in several tissues, likely contributing to enhanced Rd. Overall, these findings demonstrate the direct link between IR and ECM-integrin signaling through integrin β1. This further establishes the involvement of ECM-integrin signaling during metabolic disease and the potential for this pathway as a novel therapeutic avenue. Disclosure E. Trefts: None. D.A. Roby: None. A. Pozzi: None. R. Zent: None. D. Wasserman: None. Funding National Institutes of Health (DK007563), (DK112553 to E.T.), (DK069221, DK083187 to R.Z.), (CA162433, DK095761 to A.P.), (DK050277, DK054902 to D.W.)

1879-P: The Liver Mitochondrial Oxidative Phosphorylation Increases in Diabetic Mice

Mitochondria play critical role in energy metabolism. However, the alteration of mitochondrial function in diabetes is still controversial. To address this issue, we measured the liver and muscle mitochondrial function in animal models characterizing different stages of type 1 and 2 diabetes. Streptozotocin-induced diabetic mice, db/db mice and high-fat diet-induced obese mice were used in this study. Both in early stage (3 weeks after STZ injection) and late stage (3 months after STZ injection) of type 1 diabetic mice, liver mitochondrial oxidative phosphorylation (OXPHOS) increased significantly, among which the complex IV-dependent oxygen consumption rate (OCR) was the most prominent, indicating that complex IV may play an important role in the pathophysiology of type 1 diabetes. The liver mitochondrial complex I and II-dependent OXPHOS increased greatly in prediabetes and early stage (12-week-old db/db mice) of type 2 diabetes. At the late stage (42-week-old db/db mice) of type 2 diabetes, the OXPHOS declined to almost normal, and alteration of complex I-dependent OCR was the most significant. In contrast, no change of mitochondrial OXPHOS was observed in the muscle of all disease models. Besides, no alteration of ATP, ADP and AMP contents in the tissues was observed. Our in vitro experiments showed that among specific chemical inhibitors to complex I, II, III or IV in mitochondrial respiratory chain, rotenone (complex I inhibitor) and myxothiazol (complex III inhibitor) enhanced glucose consumption and lactate release. But only rotenone decreased glucose output in primary hepatocytes in the absence of cytotoxicity. In summary, the mitochondrial OXPHOS of liver rather than muscle increases in diabetes. And inhibition of complex I may ameliorate hyperglycemia by reducing hepatic glucose production and inducing glycolysis. Our results provide evidences for using complex I inhibitors (e.g., metformin and berberine) as oral antidiabetic agents. Disclosure M. Alimujiang: None. M. Yu: None. J. Yin: None. Funding National Natural Science Foundation of China (81670790, 31171128)

Novel aspects of the role of the liver in carbohydrate metabolism

Malfunction of the liver is a central factor in metabolic disease. Glucose production by liver is complex and controlled via indirect mechanisms; insulin regulates adipose tissue lipolysis, and free fatty acids in turn regulate liver glucose output. This latter concept is confirmed by studies in L-Akt-Foxo1 knockout mice. The adipocyte is a likely locus of hepatic insulin resistance. Also, kidneys play a role in regulating glucose production; denervated kidneys abrogate the effect of fat feeding to cause insulin resistance. Glucose itself is an important regulator of liver metabolism (“glucose effectiveness”); after entering liver, glucose is phosphorylated and can be exported as lactate. Using the dynamic glucose/lactate relationship, we have been able to estimate glucose effectiveness in intact animals and human subjects. Families have been identified with a glucokinase regulatory protein defect; modeling demonstrates elevated glucokinase activity. Insulin clearance by liver is highly variable among normal individuals, and is under environmental control: high fat diet reduces clearance by 30%. Liver insulin clearance is significantly lower in African American (AA) adults and children compared to European American participants, accounting for fasting hyperinsulinemia in AA. We hypothesize that reduced hepatic insulin clearance causes peripheral insulin resistance and increased Type 2 diabetes in AA.