Suppression Of Glucose Production Research Articles

Dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, ameliorates type 2 diabetes via reduced gluconeogenesis

AimsPyruvate dehydrogenase (PDH) catalyzes the decarboxylation of pyruvate to acetyl-CoA, which plays a key role in linking cytosolic glycolysis to mitochondria metabolism. PDH is physiologically inactivated by pyruvate dehydrogenase kinases (PDKs). Thus, activation of PDH via inhibiting PDK may lead to metabolic benefits. In the present study, we investigated the antidiabetic effect of PDK inhibition using dichloroacetate (DCA), a PDK inhibitor. Main methodsWe evaluated the effect of single dose of DCA on plasma metabolic parameters in normal rats. Next, we investigated the antidiabetic effect of DCA in diabetic ob/ob mice. In addition, we performed in vitro assays to understand the effect and mechanism of action of DCA on gluconeogenesis in mouse myoblast cell line C2C12 and rat hepatoma cell line FaO. Key findingsIn normal rats, a single dose of DCA decreased the plasma level of pyruvate, the product of glycolysis, and the plasma glucose level only in the fasting state. Meanwhile, a single dose of DCA lowered the plasma glucose level, and a three-week treatment decreased the fructosamine level in diabetic ob/ob mice. In vitro experiments demonstrated concentration-dependent suppression of lactate production in C2C12 myotubes. In addition, DCA suppressed glucose production from pyruvate and lactate in FaO hepatoma cells. Thus, DCA-mediated restricted supply of gluconeogenic substrates from the muscle to liver, and direct suppression of hepatic gluconeogenesis might have contributed to its glucose-lowering effect in the current models. SignificancePDK inhibitor may be considered as a potential antidiabetic agent harboring inhibitory effect on gluconeogenesis.

Comparative analysis of oral and intraperitoneal glucose tolerance tests in mice

ObjectiveThe glucose tolerance test (GTT) is widely used in preclinical research to investigate glucose metabolism, but there is no standardised way to administer glucose. The aim of this study was to directly compare the effect of the route of glucose administration on glucose and insulin kinetics during a GTT in mice. MethodsA GTT was performed in lean male and female mice and obese male mice and glucose was administered via the oral or intraperitoneal (I.P.) route. Samples were collected frequently during the GTT to provide a full time-course of the insulin and glucose excursions. In another cohort of lean male mice, plasma concentrations of insulin, c-peptide, and incretin hormones were measured at early time points after glucose administration. A stable-isotope labelled GTT (SiGTT) was then performed to delineate the contribution of exogenous and endogenous glucose to glycemia during the GTT, comparing both methods of glucose administration. Finally, we present a method to easily measure insulin from small volumes of blood during a GTT by directly assaying whole-blood insulin using ELISA and show a good concordance between whole-blood and plasma insulin measurements. ResultsWe report that I.P. glucose administration results in an elevated blood glucose excursion and a largely absent elevation in blood insulin and plasma incretin hormones when compared to oral administration. Utilising stable-isotope labelled glucose, we demonstrate that the difference in glucose excursion between the two routes of administration is mainly due to the lack of suppression of glucose production in I.P. injected mice. Additionally, rates of exogenous glucose appearance into circulation were different between lean and obese mice after I.P., but not after oral glucose administration. ConclusionReflecting on these data, we suggest that careful consideration be given to the route of glucose administration when planning a GTT procedure in mice and that in most circumstances the oral route of glucose administration should be preferred over the I.P. route to avoid possible artifacts originating from a non-physiological route.

Circulating metabolite homeostasis achieved through mass action.

Homeostasis maintains serum metabolites within physiological ranges. For glucose, this requires insulin, which suppresses glucose production while accelerating its consumption. For other circulating metabolites, a comparable master regulator has yet to be discovered. Here we show that, in mice, many circulating metabolites are cleared via the tricarboxylic acid cycle (TCA) cycle in linear proportionality to their circulating concentration. Abundant circulating metabolites (essential amino acids, serine, alanine, citrate, 3-hydroxybutyrate) were administered intravenously in perturbative amounts and their fluxes were measured using isotope labelling. The increased circulating concentrations induced by the perturbative infusions hardly altered production fluxes while linearly enhancing consumption fluxes and TCA contributions. The same mass action relationship between concentration and consumption flux largely held across feeding, fasting and high- and low-protein diets, with amino acid homeostasis during fasting further supported by enhanced endogenous protein catabolism. Thus, despite the copious regulatory machinery in mammals, circulating metabolite homeostasis is achieved substantially through mass action-driven oxidation.

Chronic Dietary Branched-Chain Amino Acids Induced Lipid Oxidation and Blunted Insulin-Stimulated Glucose Production in Mice With NAFLD

Abstract Objectives Elevated circulating branched-chain amino acids (BCAAs) during insulin resistance are strong predictors of type 2 diabetes mellitus onset. Defects in BCAA degradation are evident in several tissues during insulin resistance and non-alcoholic fatty liver disease (NAFLD). Furthermore, alterations in BCAA metabolism are associated with changes in several aspects lipid metabolism, including lipogenesis, ketogenesis and mitochondrial TCA cycle activity. Considering the crosstalk between BCAAs and lipid metabolism, we hypothesized that chronic supplementation of BCAAs will modulate hepatic insulin resistance and mitochondrial lipid oxidation during NAFLD. Methods Mice (C57BL/6N) were reared on either a low-fat (LF; 10% fat kcal), high-fat (HF; 60% fat kcal or high-fat diet supplemented with BCAA (HFBA; 150% BCAA) for 24 weeks. Metabolic profiling was conducted under fed or overnight fasted (14–16 hrs) conditions. A subset of overnight fasted mice from the HF and HFBA groups were subjected to hyperinsulinemic euglycemic clamps, following implantation of jugular vein catheters. Results Feeding HF and HFBA diets resulted in NAFLD. Circulating BCAAs were higher in ‘fed’ mice consuming HFBA diet (e.g., Valine, µM ± SEM; 311 ± 38 in HF, 432 ± 34 in HFBA, P ≤ 0.05). Overnight fasting significantly reduced BCAA levels in all groups, but the fasting levels of BCAAs remained similar between groups. Fed-to-fasted fold changes in blood glucose, serum insulin and c-peptide were higher in HFBA mice (P ≤ 0.05). Insulin stimulated suppression of glucose production (% ± SEM; HF = 38 ± 11, HFBA = 16 ± 16) was blunted in HFBA mice. Furthermore, fed-to-fasted expression of hepatic genes involved in lipid oxidation, including LCAD, MCAD, PPARa and CPT1a were significantly higher (P ≤ 0.05) in the HFBA mice. Conclusions In summary, chronic BCAA supplementation induced hepatic lipid oxidation gene expression, without any apparent improvements in insulin sensitivity. In conclusion, while the induction of lipid oxidation by BCAAs could explain certain beneficial effects associated with their supplementation, the longer-term impact of the BCAAs on insulin sensitivity need to be further explored. Funding Sources National Institutes of Health (NIH) grant RO1-DK-112865

921-P: Do Glucose and Lipid Metabolism Differ by Adiposity in Adolescents with Type 1 Diabetes (T1D)? Preliminary Results of a 2-Step Clamp Study

Puberty is a known stage of insulin resistance in lean adolescents with or without T1D. Moreover, 25% of adolescents with T1D in clinical practice are overweight, which may further contribute to insulin resistance. The present study examines whether higher body fat aggravates insulin resistance in the liver, muscle, and adipose tissue in pubertal adolescents with T1D by performing a 2-step, 8 and 80 mU/m2/min hyperinsulinemic euglycemic clamp. Body Composition was measured by DEXA, abdominal fat by MRI and hepatic fat by MRI-proton density fat fraction (PDFF). Endogenous glucose production and glycerol turnover were traced with [6,6-2H2] glucose and [2H5]-glycerol (Figure). Participants (mean age 14.4±1.2 years, A1c 7.6±0.5%) with lower (22.9) and higher (36.7) mean % body fat had mean weights of 61.6 and 68.1 kg, BMI% 68.3 and 84.8, and PDFF 1.4 and 1.7%, respectively. Endogenous glucose production was partially suppressed across the body fat spectrum during the low dose insulin infusion. In contrast, those with higher body fat tended to have diminished suppression of glucose production during the high dose insulin infusion. Rates of lipolysis appear to be unaffected. Hence our preliminary findings suggest that on the spectrum of increasing body fat in pubertal adolescents with T1D, we start to see metabolic inflexibility in youth with a moderate degree of adiposity. Disclosure M. A. Van name: None. S. Siebel: None. E. M. Tichy: None. S. Caprio: None. W. V. Tamborlane: Consultant; Self; Medtronic, Sanofi, Other Relationship; Self; Tolerion, Inc. N. Santoro: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (1K23DK115894-01A1)

ABCB10 exports mitochondrial biliverdin, driving metabolic maladaptation in obesity.

Although the role of hydrophilic antioxidants in the development of hepatic insulin resistance and nonalcoholic fatty liver disease has been well studied, the role of lipophilic antioxidants remains poorly characterized. A known lipophilic hydrogen peroxide scavenger is bilirubin, which can be oxidized to biliverdin and then reduced back to bilirubin by cytosolic biliverdin reductase. Oxidation of bilirubin to biliverdin inside mitochondria must be followed by the export of biliverdin to the cytosol, where biliverdin is reduced back to bilirubin. Thus, the putative mitochondrial exporter of biliverdin is expected to be a major determinant of bilirubin regeneration and intracellular hydrogen peroxide scavenging. Here, we identified ABCB10 as a mitochondrial biliverdin exporter. ABCB10 reconstituted into liposomes transported biliverdin, and ABCB10 deletion caused accumulation of biliverdin inside mitochondria. Obesity with insulin resistance up-regulated hepatic ABCB10 expression in mice and elevated cytosolic and mitochondrial bilirubin content in an ABCB10-dependent manner. Revealing a maladaptive role of ABCB10-driven bilirubin synthesis, hepatic ABCB10 deletion protected diet-induced obese mice from steatosis and hyperglycemia, improving insulin-mediated suppression of glucose production and decreasing lipogenic SREBP-1c expression. Protection was concurrent with enhanced mitochondrial function and increased inactivation of PTP1B, a phosphatase disrupting insulin signaling and elevating SREBP-1c expression. Restoration of cellular bilirubin content in ABCB10 KO hepatocytes reversed the improvements in mitochondrial function and PTP1B inactivation, demonstrating that bilirubin was the maladaptive effector linked to ABCB10 function. Thus, we identified a fundamental transport process that amplifies intracellular bilirubin redox actions, which can exacerbate insulin resistance and steatosis in obesity.

Pyrvinium Treatment Confers Hepatic Metabolic Benefits via β-Catenin Downregulation and AMPK Activation.

Genetic evidence has indicated that β-catenin plays a vital role in glucose and lipid metabolism. Here, we investigated whether pyrvinium, an anthelmintic agent previously reported as a down-regulator of cellular β-catenin levels, conferred any metabolic advantages in treatment of metabolic disorders. Glucose production and lipid accumulation were analyzed to assess metabolic response to pyrvinium in hepatocytes. The expression of key proteins and genes were assessed by immunoblotting and RT-PCR. The in vivo efficacy of pyrvinium against metabolic disorders was evaluated in the mice fed with a high fat diet (HFD). We found that pyrvinium inhibited glucose production and reduced lipogenesis by decreasing the expression of key genes in hepatocytes, which were partially elicited by the downregulation of β-catenin through AXIN stabilization. Interestingly, the AMPK pathway also played a role in the action of pyrvinium, dependent on AXIN stabilization but independent of β-catenin downregulation. In HFD-fed mice, pyrvinium treatment led to improvement in glucose tolerance, fatty liver disorder, and serum cholesterol levels along with a reduced body weight gain. Our results show that small molecule stabilization of AXIN using pyrvinium may lead to improved glucose and lipid metabolism, via β-catenin downregulation and AMPK activation.

Olanzapine-induced insulin resistance may occur via attenuation of central KATP channel-activation

Antipsychotic use is associated with an increased risk of type 2 diabetes. Recent work suggests antipsychotics can induce insulin resistance immediately and independently of weight gain, and that this may occur via the central nervous system (CNS). We have previously shown that the highly effective and widely prescribed antipsychotic, olanzapine inhibits CNS insulin-mediated suppression of hepatic glucose production, but the mechanisms remain unknown. The ATP-sensitive potassium (KATP) channel is a key metabolic sensor downstream of hypothalamic insulin signalling, involved in the maintenance of glucose homeostasis. Thus, the possibility arises that olanzapine inhibits central KATP channel activation to disrupt glucose metabolism. We replicate that intracerebroventricular (ICV) administration of the KATP channel activator, diazoxide, suppresses hepatic glucose production and additionally demonstrate stimulation of peripheral glucose utilization. We report that olanzapine inhibits the effects of central KATP channel activation resulting in perturbation of whole body insulin sensitivity, specifically via inhibition of glucose utilization, while leaving central KATP channel-mediated suppression of glucose production intact. Perturbation of KATP channel action in the CNS could represent a novel mechanism of antipsychotic-induced diabetes.

Hepatic Insulin Resistance Is Not Pathway Selective in Humans With Nonalcoholic Fatty Liver Disease.

Both glucose and triglyceride production are increased in type 2 diabetes and nonalcoholic fatty liver disease (NAFLD). For decades, the leading hypothesis to explain these paradoxical observations has been selective hepatic insulin resistance wherein insulin drives de novo lipogenesis (DNL) while failing to suppress glucose production. Here, we aimed to test this hypothesis in humans. We recruited obese subjects who met criteria for bariatric surgery with (n = 16) or without (n = 15) NAFLD and assessed 1) insulin-mediated regulation of hepatic and peripheral glucose metabolism using hyperinsulinemic-euglycemic clamps with [6,6-2H2]glucose, 2) fasting and carbohydrate-driven hepatic DNL using deuterated water (2H2O), and 3) hepatocellular insulin signaling in liver biopsy samples collected during bariatric surgery. Compared with subjects without NAFLD, those with NAFLD demonstrated impaired insulin-mediated suppression of glucose production and attenuated-not increased-glucose-stimulated/high-insulin lipogenesis. Fructose-stimulated/low-insulin lipogenesis was intact. Hepatocellular insulin signaling, assessed for the first time in humans, exhibited a proximal block in insulin-resistant subjects: Signaling was attenuated from the level of the insulin receptor through both glucose and lipogenesis pathways. The carbohydrate-regulated lipogenic transcription factor ChREBP was increased in subjects with NAFLD. Acute increases in lipogenesis in humans with NAFLD are not explained by altered molecular regulation of lipogenesis through a paradoxical increase in lipogenic insulin action; rather, increases in lipogenic substrate availability may be the key.

Hypothalamic BMP9 suppresses glucose production by central PI3K/Akt/mTOR pathway.

Bone morphogenetic proteins (BMPs) are secreted ligands that belong to the transforming growth factor-β (TGF-β) superfamily. BMP7 has been reported to play a role in reversing obesity and regulating appetite in the hypothalamus. Whether BMP9 plays a central role in regulating glucose metabolism and insulin sensitivity remains unclear. Here, we investigated the impact of central BMP9 signaling and possible route of transmission. We performed intracerebroventricular (ICV) surgery and injected adenovirus expressing BMP9 (Ad-BMP9) into the cerebral ventricle of mice. Metabolic analysis, hyperinsulinemic-euglycemic clamp test, and analysis of phosphatidylinositol 3,4,5-trisphosphate (PIP3) formation were then performed. Real-time PCR and Western blotting were performed to detect gene expression and potential pathways involved. We found that hypothalamic BMP9 expression was downregulated in obese and insulin-resistant mice. Overexpression of BMP9 in the mediobasal hypothalamus reduced food intake, body weight, and blood glucose level, and elevated the energy expenditure in high-fat diet (HFD)-fed mice. Importantly, central treatment with BMP9 improved hepatic insulin resistance (IR) and inhibited hepatic glucose production in HFD-fed mice. ICV BMP9-induced increase in hepatic insulin sensitivity and related metabolic effects were blocked by ICV injection of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) signaling. In addition, ICV BMP9 promoted the ability of insulin to activate the insulin receptor/phosphoinositide 3-kinase (PI3K)/Akt pathway in the hypothalamus. Thus, this study provides insights into the potential mechanism by which central BMP9 ameliorates hepatic glucose metabolism and IR via activating the mTOR/PI3K/Akt pathway in the hypothalamus.

The importance of the AMPK gamma 1 subunit in metformin suppression of liver glucose production

Metformin has been used to treat patients with type 2 diabetes for over 60 years, however, its mechanism of action is still not completely understood. Our previous reports showed that high-fat-diet (HFD)-fed mice with liver-specific knockout of both AMPK catalytic α1 and α2 subunits exhibited significantly higher fasting blood glucose levels and produced more glucose than floxed AMPK catalytic α1 and α2 mice after long-term metformin treatment, and that metformin promotes the formation of the functional AMPK αβγ heterotrimeric complex. We tested the importance of each regulatory γ subunit isoform to metformin action in this current study. We found that depletion of γ1, but not γ2 or γ3, drastically reduced metformin activation of AMPK. HFD-fed mice with depletion of the γ1 subunit are resistant to metformin suppression of liver glucose production. Furthermore, we determined the role of each regulatory cystathionine-β-synthase (CBS) domain in the γ1 subunit in metformin action and found that deletion of either CBS1 or CBS4 negated metformin’s effect on AMPKα phosphorylation at T172 and suppression of glucose production in hepatocytes. Our data indicate that the γ1 subunit is required for metformin’s control of glucose metabolism in hepatocytes. Furthermore, in humans and animal models, metformin treatment leads to the loss of body weight, we found that the decrease in body weight gain in mice treated with metformin is not directly attributable to increased energy expenditure.

Механізми дії метформіну за діабету та пов’язаних із діабетом патологій

Метформін (МФ) — основний пероральний препарат, який використовується в клініці для лікування пацієнтів із цукровим діабетом 2-го типу (ЦД2) понад 60 років. Предіабет — метаболічний стан, що характеризується резистентністю до інсуліну та первинною або вторинною дисфункцією β-клітин, що збільшує ризик ЦД2. Отримані дані підтверджують роль МФ у запобіганні ЦД2 в людей із предіабетом. Його ефективність для затримки або запобігання виникненню діабету доведено у великих, добре обгрунтованих і спланованих ран- домізованих дослідженнях. За діабету МФ знижує гіперглікемію, пригнічуючи глюконеогенез у печінці через активацію шляху LKB/AMPK, а також підвищує чутливість до інсуліну — ефект, що підсилює опосередковану інсуліном супресію продукції глюкози в печінці й утилізацію глюкози в скелетних м’язах. Дію МФ пов’язано з руйнуванням мітохондріального комплексу I і зменшенням продукції АТР, посиленням автофагії та іншими механізмами. Отримані дані надійно свідчать про зв’язок ЦД із порушеннями когнітивних здібностей людини. За діабету відбуваються структурні та функціональні зміни в нервовій системі та її кровоносних судинах. ЦД прискорює розвиток хвороби Альцгеймера, стимулюючи утворення сенильних бляшок і нейрофібрилярних клубочків. Хворі на діабет, які приймають МФ, меншою мірою схильні до когнітивних розладів. МФ запобігає апоптозу в первинних нейронах, ефективно посилює споживання глюкози в інсулінорезистентних клітинах. Нейропротекторні ефекти МФ, мабуть, пов’язано з його цукрознижуючими, антиоксидантними й антиапоптотичними властивостями. Відомо, що хворі на ЦД характеризуються підвищеним ризиком канцерогенезу, а в разі захворювання на рак їхня здатність до виживання є нижчою, ніж у недіабетичних пацієнтів. Антидіабетична терапія гальмує клітинний ріст, проліферацію та метаболізм і, як наслідок, впливає на потенціал онкогенезу. Гіперінсулінемія та гіперглікемія є добре встановленими чинниками ризику канцерогенезу, тому зниження їх рівня є важливим моментом у запобіганні злоякісній трансформації клітин. МФ має протипухлиннівластивості та може пригнічувати онкогенез за допомогою системних і клітинних механізмів. Цей препарат справляє пряму, спрямовану на ракові клітини, та непряму дію, впливаючи на системну інсулінемію та глікемію.

1883-P: Metformin Increases Protein Phosphatase 2A Activity in Primary Skeletal Muscle Cells Derived from Lean Healthy Insulin-Sensitive Human Participants

Skeletal muscle insulin resistance is one of the primary contributors of type 2 diabetes (T2D). Metformin is the first-line drug for the treatment of T2D. Main known effects of metformin include inhibition of glucose production in liver and attenuation of insulin resistance in skeletal muscle. However, molecular mechanism of metformin’s action in skeletal muscle is not well understood. Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, plays a pivotal role in cellular processes (e.g., signal transduction, apoptosis), and acts through dephosphorylating key signaling molecules such as AKT, AMPK, etc. However, whether or not PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells remains to be elucidated. In the present study, hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells (shown to retain metabolic characteristics of donors) were cultured from these muscle biopsies from 8 lean insulin sensitive participants (4 Female and 4 male; age: 21.4±0.8 yrs; BMI: 22.1±0.7 kg/m2; 2-hour OGTT: 96.5± 6.3 mg/dl; HbA1c: 5.3±0.1 %; fasting plasma glucose: 87.5±1.4 mg/dl; M-value; 10.9±0.8 mg/kgBW/min). The cells were expanded, differentiated into myotubes, and treated with 50µM metformin (the physiologic concentration) for 24 hours before harvesting. PP2A activity was measured by a phosphatase activity assay (Millipore). The results indicated that metformin increased the activity of PP2A in the myotubes derived from all 8 participants, and the average fold increase is 1.59±0.13 (p<0.05). These results provided the first evidence to suggest that metformin promotes activation of PP2A in human skeletal muscle cells derived from lean insulin sensitive participants, and may help to understand metformin’s action in skeletal muscle in humans. Disclosure A.H. Mestareehi: None. B. Seyoum: None. Z. Msallaty: None. A.P. Mallisho: None. X. Zhang: None. A. Kowluru: None. Z. Yi: None. Funding American Diabetes Association (1-13-TS-27 to Z.Y.); National Institutes of Health (R01DK107666, R01DK081750 to Z.Y.), (T32HL120822 to A.H.M.); DOTS Scholars (to A.H.M., Z.Y.)

1333-P: The Adipokine Fatty Acid Binding Protein 4 Promotes Glucose Production in Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) is characterized by maternal hyperglycemia and insulin resistance that develops during pregnancy. Altered secretion of adipokines that modulate insulin action during pregnancy has been implicated in GDM pathogenesis. FABP4 is an adipokine with an important role in promoting insulin resistance in various animal models. In humans, FABP4 was suggested to regulate systemic metabolism. Although elevated levels of circulating FABP4 were demonstrated in GDM, its differential contribution to GDM pathophysiology is unclear. In this study, we aimed to determine the tissue source of elevated circulating FABP4 levels and to assess FABP4 differential contribution in promoting hepatic glucose production in GDM. We included 71 pregnant women defined as normal glucose tolerant (NGT) and 31 diagnosed with GDM. Maternal level of FABP4 before delivery was measured using ELISA. FABP4 circulating levels were increased in GDM compared to NGT (14.1 IQR: 11.5-21.4 ng/ml vs.10.3 IQR: 6.6-12.4 ng/ml, respectively, p=0.0014), with a rapid post-partum decline in FABP4 levels observed in both groups. Using biopsies of placenta, sub-cutaneous (sWAT) and visceral (vWAT) adipose tissues, we observed that FABP4 is secreted from these tissues during pregnancy. FABP4 secretion from vWAT of GDM women was ? 2 fold higher than that of NGT women. Depletion of FABP4 from vWAT conditioned media of NGT or GDM women using monoclonal antibodies, significantly suppressed glucose production in primary hepatocytes by ? 20% and ? 36%, respectively (p<0.05). Taken together, our data highlight the importance of FABP4 in the pathophysiology of enhanced hepatic glucose production in GDM. Disclosure R. Madah: None. I. Ron Ronen: None. M. Rathaus: None. R. Zemet: None. S. Amazaki Tovi: None. A. Tirosh: Advisory Panel; Self; Abbott, AstraZeneca, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi-Aventis. Consultant; Self; DreaMed Diabetes. Research Support; Self; Medtronic. Funding Israeli Diabetes Association

367-OR: High-Fat and Fructose Feeding Severely Impairs Hepatic Glucose Effectiveness

Diets high in fat and fructose (HFFD) disrupt glucose metabolism. The aim of this study was to determine if HFFD impairs hepatic glucose effectiveness (i.e., the ability of glucose per se to stimulate net hepatic glucose uptake [NHGU] and/or to suppress glucose production [Ra]). In overnight fasted dogs fed either a chow diet or HFFD for 1 month, somatostatin, 3-3H-glucose, and basal hepatic portal vein infusions of insulin and glucagon were given. After a basal sampling period, these infusions continued for 4h while glucose was infused to create a 3-fold rise in plasma glucose. Since hepatic glucose uptake is also stimulated by a negative arterial to portal vein glucose gradient, such as occurs with glucose ingestion, glucose was either all infused into a peripheral vein (Chow-PeG, n=6; HFFD-PeG, n=6), or a portion was also given into the portal vein (4 mg/kg/min; Chow-PoG, n=5; HFFD-PoG, n=5). There were no differences in plasma insulin, glucagon, arterial glucose, or hepatic glucose load between groups. In response to hyperglycemia, the liver switched from making (basal period) to storing (last h) glucose in the chow fed groups (1.1±0.1 to -2.0±0.5* mg/kg/min in Chow-PeG, and 1.3±0.1 to -3.0±0.9* in Chow-PoG; a negative rate indicates uptake). Conversely, HFFD markedly reduced liver glucose effectiveness. In fact, with HFFD the liver did not take up glucose in response to hyperglycemia whether the portal signal was absent (1.4±0.1 to 1.0±0.3 in HFFD-PeG) or present (1.6±0.3 to 0.0±0.3* in HFFD-PoG). Thus, in the Chow vs. HFFD groups, the impact of hyperglycemia on overall net hepatic glucose balance was 3.1±0.5 vs. 0.4±0.4* (13% of normal) in the absence of portal glucose delivery and 4.3±0.9 vs. 1.6±0.5* (37% of normal) in its presence. Hyperglycemia suppressed Ra by 75±12 vs. 22±6%* (last h) in Chow vs. HFFD-PeG, respectively, and by 81±14 vs. 34±7%* in Chow vs. HFFD-PoG. In summary, HFFD induced a severe impairment in hepatic glucose effectiveness but did not affect the liver’s response to the portal glucose signal. (*P<0.05). Disclosure D.S. Edgerton: None. G. Kraft: None. B. Farmer: None. M. Scott: None. M.S. Smith: None. A.D. Cherrington: Advisory Panel; Self; Biocon, Fractyl Laboratories, Inc., Metavention, Sekkei Bio, Sensulin, LLC., vTv Therapeutics, Zafgen, Inc. Consultant; Self; Thetis Pharmaceuticals LLC. Research Support; Self; Diasome Pharmaceuticals, Inc. Other Relationship; Self; Abvance Therapeutics, Novo Nordisk Inc. Funding National Institutes of Health (R01DK18243)

120-LB: Small Molecule Activator of Pyruvate Kinase Regulates In Vivo Glucose Homeostasis

Background: Nonalcoholic fatty liver disease (NAFLD) is a major factor in the pathogenesis of type-2 diabetes (T2D). Liver pyruvate kinase (PK) inactivating mutations are associated with diabetes in human subjects and PK deficiency associated with severe liver dysfunction and fat accumulation. Aim: To determine whether small molecule activators of PK (PKa) could improve liver metabolism in rats fed a high fat diet. Methods: Sprague Dawley regular chow (RC) or high fat diet (HFD) rats were either treated acutely (IV bolus 11mg/kg/min then primed continuous 0.06 mg/kg/min) or chronically (50 mg/kg/day x 3 weeks). Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp studies. Results: PKa dose dependently suppressed glucose production from primary rat hepatocytes. Acute PKa infusion suppressed endogenous glucose production (EGP) by 20% in RC rats. Chronic PKa exposure did not affect body weight in HFD-fed rats. Three-week PKa treatment did not change activity, food intake, whole-body energy expenditure, or body weight in HFD-fed mice. Nevertheless, PKa improved whole-body insulin sensitivity compared with control HFD-fed rats (glucose infusion rate: control 11.8±1.4 vs. PKa 15.3±0.7 mg/(kg-min), P<0.05). Differences in whole-body insulin sensitivity were accounted for by a 15% reduction in EGP and increased insulin-stimulated peripheral glucose uptake (21.2±0.6 vs. 26.4±1.7 mg/(kg-min), P<0.05). Insulin-stimulated hepatic AKT phosphorylation was also significantly improved. Consistent with improving of peripheral insulin sensitivity, liver and muscle triglyceride content (liver: 27.2±2.6 vs. 16.9±1.5 mg/g, P<0.05. muscle: 9.8 ±1.4 vs. 6.7±1.0 mg/g, P<0.05) were improved in PKa-treated rats. In conclusion, activation of pyruvate kinase in rodents protects against HFD-induced insulin resistance. By reducing EGP and insulin resistance, activation of PK may be an exciting potential therapeutic target for T2D. Disclosure A. Abulizi: None. R.L. Cardone: None. S. Siebel: None. C. Kung: Employee; Self; Agios Pharmaceuticals. Stock/Shareholder; Self; Agios Pharmaceuticals. R. Kibbey: None. Funding American Diabetes Association (1-16-IBS-158 to R.K.); National Institutes of Health (R01DK108283-01A1)

Berberine Improves Glucose and Lipid Metabolism in HepG2 Cells Through AMPKα1 Activation.

AimThis study is designed to investigate whether or not AMP-activated protein kinase α1 (AMPKα1) is required for natural product berberine (BBR) to improve glucose and lipid metabolism in HepG2 cells.MethodsAMPKα1 knocked-out (KO, AMPKα1-/-) cells were obtained by co-transfection of the CRISPR/Cas9 KO and HDR (homology-directed repair) plasmid into HepG2 cells, as well as subsequent screen with puromycin. The expression levels of target proteins or mRNAs were determined by western blot or real-time RT-PCR, respectively. Cellular AMPK activity, glucose consumption, lactate release, glucose production, and lipid accumulation were determined by kits.ResultsThe results showed that the AMPKα1 gene was successfully KO in HepG2 cells. In AMPKα1-/- cells, the protein expression of AMPKα1 and phosphorylated-AMPKα1 (p-AMPKα1) disappeared, the level of total AMPKα declined to about 45–50% of wild type (p < 0.01), while p-AMPKα level and AMPK activity were reduced to less than 10% of wild type (p < 0.001). BBR increased p-AMPKα1, p-AMPKα, AMPK activity, and stimulated glucose consumption, lactate release, inhibited glucose production in wild type HepG2 cells (p < 0.05 or p < 0.01). BBR also reduced intracellular lipid accumulation and suppressed the expression of lipogenic genes in oleic acid (OA) treated wild type HepG2 cells (p < 0.05 or p < 0.01). In AMPKα1-/- HepG2 cells, the stimulating effects of BBR on p-AMPKα1, p-AMPKα, AMPK activity, and its improving effects on glucose and lipid metabolism were completely abolished.ConclusionOur study proves that AMPKα1 plays a critical role for BBR to improve glucose and lipid metabolism in HepG2 cells. Our results will provide new information to further understand the molecular mechanisms of BBR.

Ceramides, crucial actors in the development of insulin resistance and type 2 diabetes

In healthy subjects, the balance between glucose production and its usage is precisely controlled. When circulating glucose reaches a critical threshold, pancreatic β-cells secrete insulin, which has two major actions: lowering circulating glucose concentrations by facilitating its uptake mainly in skeletal muscles and the liver, and inhibiting glucose production. Triglycerides are the main source of fatty acids to meet the energy needs of oxidative tissues and any excess is stored in adipocytes. Thus, adipose tissue acts as a trap for excess fatty acids released from plasma triglycerides. When the buffering action of adipose tissue to store fatty acids is impaired, they accumulate in other tissues where they are metabolized in several lipid species, including sphingolipid derivatives such as ceramides. Numerous studies have shown that ceramides are among the most active lipid second messengers to inhibit insulin signalling. This review describes the major role played by ceramides in the development of insulin resistance in peripheral tissues.

Inhibition of Calcineurin and Glycogen Synthase Kinase-3β by Ricinoleic Acid Derived from Castor Oil.

Ricinoleic acid (RA) is the main fatty acid component of castor oil and was found to inhibit Ca2+ -signal transduction pathway-mediated cell cycle regulation in a yeast-based drug screening assay. RA is expected to have antidiabetic, antiallergy, and/or anticancer properties but its target molecule is unknown. To identify a novel pharmacological effect of RA, we investigated its target molecule in the Ca2+ -signal transduction pathway. RA inhibition of calcineurin (CN) was examined in a yeast-based CN inhibitor screening assay using the rsp5A401E mutant and in a phosphatase assay using recombinant human CN. RA showed growth-restoration activity at 5 μg/spot in the CN inhibitor screening assay with the rsp5A401E yeast strain. Furthermore, it directly inhibited CN without immunophilins at Ki = 33.7 μM in a substrate-competitive manner. The effects of RA on CN in mammalian cells were further evaluated by measuring β-hexosaminidase (β-HEX) release in RBL-2H3 cells. RA at 50 μM suppressed the release of β-HEX from RBL-2H3 cells. Moreover, this compound was found to inhibit glycogen synthase kinase-3β (GSK-3β), as determined by a kinase assay using recombinant human GSK-3β. RA inhibited GSK-3β at Ki = 1.43 μM in a peptide substrate-competitive manner. The inhibition of GSK-3β by this molecule was further assessed in mammalian cells by measuring the inhibition of glucose production in H4IIE rat hepatoma cells. RA at 25 μM suppressed glucose production in these cells. These findings indicate that RA and/or castor oil could be a useful functional fatty acid to treat allergy or type 2 diabetes.

Purβ promotes hepatic glucose production by increasing Adcy6 transcription

ObjectiveEnhanced glucagon signaling and hepatic glucose production (HGP) can account for hyperglycemia in patients with obesity and type 2 diabetes. However, the detailed molecular mechanisms underlying the enhanced HGP in these patients are not fully understood. Here, we identify Purβ as a positive regulator of HGP and study its molecular mechanisms in the regulation of HGP both in vivo and in vitro. MethodsAdenovirus-mediated knockdown or overexpression of Purβ was performed in either primary hepatocytes or the livers of db/db mice. Glucose metabolism, insulin sensitivity, and HGP were determined by glucose, insulin, and lactate tolerance tests, respectively. Purβ/ADCY6 protein levels, glucagon signaling (p-CREB/CREB), and insulin signaling (p-Akt/Akt) were measured by immunoblotting. Gene expression was measured by RNA-seq and real-time quantitative polymerase chain reaction. Luciferase reporter and chromatin immunoprecipitation assays were used to study the interaction between Purβ and the Adcy6 promoter. ResultsPurβ was abnormally elevated in obese mice and was also increased under fasting conditions or via the glucagon signaling pathway, which promoted HGP by increasing Adcy6 expression. Liver-specific knockdown of Purβ in db/db mice significantly ameliorated hyperglycemia and glucose intolerance by suppressing the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway. Consistent with this observation, the knockdown of Purβ also inhibited glucose production in isolated primary hepatocytes by inhibiting the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway, whereas the overexpression of Purβ promoted glucose production by activating this signaling pathway. Mechanistically, Purβ directly binds to the promoter of the Adcy6 gene and thereby promotes its transcription. ConclusionsTaken together, these results illustrate a new model in which Purβ functions to regulate the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway to help maintain glucose homeostasis.