Glucose Production In Hepatocytes Research Articles

Pioglitazone inhibits mitochondrial pyruvate metabolism and glucose production in hepatocytes.

Pioglitazone is used globally for the treatment of type 2 diabetes mellitus (T2DM) and is one of the most effective therapies for improving glucose homeostasis and insulin resistance in T2DM patients. However, its mechanism of action in the tissues and pathways that regulate glucose metabolism are incompletely defined. Here we investigated the direct effects of pioglitazone on hepatocellular pyruvate metabolism and the dependency of these observations on the purported regulators of mitochondrial pyruvate transport, MPC1 and MPC2. In cultured H4IIE hepatocytes, pioglitazone inhibited [2-14 C]-pyruvate oxidation and pyruvate-driven oxygen consumption and, in mitochondria isolated from both hepatocytes and human skeletal muscle, pioglitazone selectively and dose-dependently inhibited pyruvate-driven ATP synthesis. Pioglitazone also suppressed hepatocellular glucose production (HGP), without influencing the mRNA expression of key HGP regulatory genes. Targeted siRNA silencing of MPC1 and 2 caused a modest inhibition of pyruvate oxidation and pyruvate-driven ATP synthesis, but did not alter pyruvate-driven HGP and, importantly, it did not influence the actions of pioglitazone on either pathway. In summary, these findings outline a novel mode of action of pioglitazone relevant to the pathogenesis of T2DM and suggest that targeting pyruvate metabolism may lead to the development of effective new T2DM therapies.

MicroRNA-206 prevents hepatosteatosis and hyperglycemia by facilitating insulin signaling and impairing lipogenesis

Background & AimsThe paradox of selective hepatic insulin resistance, wherein the insulin-resistant liver fails to suppress glucose production but continues to produce lipids, has been central to the pathophysiology of hepatosteatosis and hyperglycemia. Our study was designed to investigate the mechanism(s) by which microRNA-206 alleviates the pathogenesis of hepatosteatosis and hyperglycemia.MethodsDietary obese mice induced by a high fat diet were used to study the role of microRNA-206 in the pathogenesis of hepatosteatosis and hyperglycemia. A mini-circle vector was used to deliver microRNA-206 into the livers of mice.ResultsLipid accumulation impaired biogenesis of microRNA-206 in fatty livers of dietary obese mice and human hepatocytes (p <0.01). Delivery of microRNA-206 into the livers of dietary obese mice resulted in the strong therapeutic effects on hepatosteatosis and hyperglycemia. Mechanistically, miR-206 interacted with the 3′ untranslated region of PTPN1 (protein tyrosine phosphatase, non-receptor type 1) and induced its degradation. By inhibiting PTPN1 expression, microRNA-206 facilitated insulin signaling by promoting phosphorylation of INSR (insulin receptor) and impaired hepatic lipogenesis by inhibiting Srebp1c transcription. By simultaneously modulating lipogenesis and insulin signaling, microRNA-206 reduced lipid (p = 0.006) and glucose (p = 0.018) production in human hepatocytes and livers of dietary obese mice (p <0.001 and p <0.01 respectively). Re-introduction of Ptpn1 into livers offset the inhibitory effects of microRNA-206, indicating that PTPN1 mediates the inhibitory effects of microRNA-206 on both hepatosteatosis and hyperglycemia.ConclusionsMicroRNA-206 is a potent inhibitor of lipid and glucose production by simultaneously facilitating insulin signaling and impairing hepatic lipogenesis. Our findings potentially provide a novel therapeutic agent for both hepatosteatosis and hyperglycemia.

Pathways of Acetyl-CoA Metabolism Involved in the Reversal of Palmitate-Induced Glucose Production by Metformin and Salicylate.

The pathways through which fatty acids induce insulin resistance have been the subject of much research. We hypothesise that by focussing on the reversal of insulin resistance, novel insights can be made regarding the mechanisms by which insulin resistance can be overcome. Using global gene and lipid expression profiling, we aimed to identify biological pathways altered during the prevention of palmitate-induced glucose production in hepatocytes using metformin and sodium salicylate. FAO hepatoma cells were treated with palmitate (0.075 mM, 48 h) with or without metformin (0.25 mM) and sodium salicylate (2 mM) in the final 24 h of palmitate treatment, and effects on glucose production were determined. RNA microarray measurements followed by gene set enrichment analysis were performed to investigate pathway regulation. Lipidomic analysis and measurement of secreted bile acids and cholesterol were also performed. Reversal of palmitate-induced glucose production by metformin and sodium salicylate was characterised by co-ordinated down-regulated expression of pathways regulating acetyl-CoA to cholesterol and bile acid biosynthesis. All 20 enzymes that regulate the conversion of acetyl-CoA to cholesterol were reduced following metformin and sodium salicylate. Selected findings were confirmed using primary mouse hepatocytes. Although total intracellular levels of diacylglycerol, triacylglycerol and cholesterol esters increased with palmitate, these were not, however, further altered by metformin and sodium salicylate. 6 individual diacylglycerol, triacylglycerol and cholesterol ester species containing 18:0 and 18:1 side-chains were reduced by metformin and sodium salicylate. These results implicate acetyl-CoA metabolism and C18 lipid species as modulators of hepatic glucose production that could be targeted to improve glucose homeostasis.

Activation of the cAMP-PKA pathway Antagonizes Metformin Suppression of Hepatic Glucose Production

Metformin is the most commonly prescribed oral anti-diabetic agent worldwide. Surprisingly, about 35% of diabetic patients either lack or have a delayed response to metformin treatment, and many patients become less responsive to metformin over time. It remains unknown how metformin resistance or insensitivity occurs. Recently, we found that therapeutic metformin concentrations suppressed glucose production in primary hepatocytes through AMPK; activation of the cAMP-PKA pathway negatively regulates AMPK activity by phosphorylating AMPKα subunit at Ser-485, which in turn reduces AMPK activity. In this study, we find that metformin failed to suppress glucose production in primary hepatocytes with constitutively activated PKA and did not improve hyperglycemia in mice with hyperglucagonemia. Expression of the AMPKα1(S485A) mutant, which is unable to be phosphorylated by PKA, increased both AMPKα activation and the suppression of glucose production in primary hepatocytes treated with metformin. Intriguingly, salicylate/aspirin prevents the phosphorylation of AMPKα at Ser-485, blocks cAMP-PKA negative regulation of AMPK, and improves metformin resistance. We propose that aspirin/salicylate may augment metformin's hepatic action to suppress glucose production.

The Methionine Transamination Pathway Controls Hepatic Glucose Metabolism through Regulation of the GCN5 Acetyltransferase and the PGC-1α Transcriptional Coactivator

Methionine is an essential sulfur amino acid that is engaged in key cellular functions such as protein synthesis and is a precursor for critical metabolites involved in maintaining cellular homeostasis. In mammals, in response to nutrient conditions, the liver plays a significant role in regulating methionine concentrations by altering its flux through the transmethylation, transsulfuration, and transamination metabolic pathways. A comprehensive understanding of how hepatic methionine metabolism intersects with other regulatory nutrient signaling and transcriptional events is, however, lacking. Here, we show that methionine and derived-sulfur metabolites in the transamination pathway activate the GCN5 acetyltransferase promoting acetylation of the transcriptional coactivator PGC-1α to control hepatic gluconeogenesis. Methionine was the only essential amino acid that rapidly induced PGC-1α acetylation through activating the GCN5 acetyltransferase. Experiments employing metabolic pathway intermediates revealed that methionine transamination, and not the transmethylation or transsulfuration pathways, contributed to methionine-induced PGC-1α acetylation. Moreover, aminooxyacetic acid, a transaminase inhibitor, was able to potently suppress PGC-1α acetylation stimulated by methionine, which was accompanied by predicted alterations in PGC-1α-mediated gluconeogenic gene expression and glucose production in primary murine hepatocytes. Methionine administration in mice likewise induced hepatic PGC-1α acetylation, suppressed the gluconeogenic gene program, and lowered glycemia, indicating that a similar phenomenon occurs in vivo These results highlight a communication between methionine metabolism and PGC-1α-mediated hepatic gluconeogenesis, suggesting that influencing methionine metabolic flux has the potential to be therapeutically exploited for diabetes treatment.

Investigation of salicylate hepatic responses in comparison with chemical analogues of the drug

Anti-hyperglycaemic effects of the hydroxybenzoic acid salicylate might stem from effects of the drug on mitochondrial uncoupling, activation of AMP-activated protein kinase, and inhibition of NF-κB signalling. Here, we have gauged the contribution of these effects to control of hepatocyte glucose production, comparing salicylate with inactive hydroxybenzoic acid analogues of the drug. In rat H4IIE hepatoma cells, salicylate was the only drug tested that activated AMPK. Salicylate also reduced mTOR signalling, but this property was observed widely among the analogues. In a sub-panel of analogues, salicylate alone reduced promoter activity of the key gluconeogenic enzyme glucose 6-phosphatase and suppressed basal glucose production in mouse primary hepatocytes. Both salicylate and 2,6 dihydroxybenzoic acid suppressed TNFα-induced IκB degradation, and in genetic knockout experiments, we found that the effect of salicylate on IκB degradation was AMPK-independent. Previous data also identified AMPK-independent regulation of glucose but we found that direct inhibition of neither NF-κB nor mTOR signalling suppressed glucose production, suggesting that other factors besides these cell signalling pathways may need to be considered to account for this response to salicylate. We found, for example, that H4IIE cells were exquisitely sensitive to uncoupling with modest doses of salicylate, which occurred on a similar time course to another anti-hyperglycaemic uncoupling agent 2,4-dinitrophenol, while there was no discernible effect at all of two salicylate analogues which are not anti-hyperglycaemic. This finding supports much earlier literature suggesting that salicylates exert anti-hyperglycaemic effects at least in part through uncoupling.

A decrease in hepatic microRNA-9 expression impairs gluconeogenesis by targeting FOXO1 in obese mice.

MicroRNA-9 (miR-9) is involved in the regulation of pancreatic beta cell function. However, its role in gluconeogenesis is still unclear. Our objective was to investigate the role of miR-9 in hepatic glucose production (HGP). MiR-9 expression was measured in livers of high-fat diet (HFD) mice and ob/ob mice. The methylation status of the miR-9-3 promoter regions in hepatocytes was determined by the methylation-specific PCR procedure. The binding activity of DNA methyltransferase (DNMT)1, DNMT3a and DNMT3b on the miR-9-3 promoter was detected by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR assays. HGP was evaluated in vitro and in vivo. Glucose tolerance, insulin tolerance and pyruvate tolerance tests were also performed. Reduced miR-9 expression and hypermethylation of the miR-9-3 promoter were observed in the livers of obese mice. Further study showed that the binding of DNMT1, but not of DNMT3a and DNMT3b, to the miR-9-3 promoter was increased in hepatocytes from ob/ob mice. Knockdown of DNMT1 alleviated the decrease in hepatic miR-9 expression in vivo and in vitro. Overexpression of hepatic miR-9 improved insulin sensitivity in obese mice and inhibited HGP. In addition, deletion of hepatic miR-9 led to an increase in random and fasting blood glucose levels in lean mice. Importantly, silenced forkhead box O1 (FOXO1) expression reversed the gluconeogenesis and glucose production in hepatocytes induced by miR-9 deletion. Our observations suggest that the decrease in miR-9 expression contributes to an inappropriately activated gluconeogenesis in obese mice.

Pharmacological inhibition of S6K1 increases glucose metabolism and Akt signalling in vitro and in diet-induced obese mice.

The mammalian target of rapamycin complex 1 (mTORC1)/p70 ribosomal S6 kinase (S6K)1 pathway is overactivated in obesity, leading to inhibition of phosphoinositide 3-kinase (PI3K)/Akt signalling and insulin resistance. However, chronic mTORC1 inhibition by rapamycin impairs glucose homeostasis because of robust induction of liver gluconeogenesis. Here, we compared the effect of rapamycin with that of the selective S6K1 inhibitor, PF-4708671, on glucose metabolism in vitro and in vivo. We used L6 myocytes and FAO hepatocytes to explore the effect of PF-4708671 on the regulation of glucose uptake, glucose production and insulin signalling. We also treated high-fat (HF)-fed obese mice for 7days with PF-4708671 in comparison with rapamycin to assess glucose tolerance, insulin resistance and insulin signalling in vivo. Chronic rapamycin treatment induced insulin resistance and impaired glucose metabolism in hepatic and muscle cells. Conversely, chronic S6K1 inhibition with PF-4708671 reduced glucose production in hepatocytes and enhanced glucose uptake in myocytes. Whereas rapamycin treatment inhibited Akt phosphorylation, PF-4708671 increased Akt phosphorylation in both cell lines. These opposite effects of the mTORC1 and S6K1 inhibitors were also observed in vivo. Indeed, while rapamycin treatment induced glucose intolerance and failed to improve Akt phosphorylation in liver and muscle of HF-fed mice, PF-4708671 treatment improved glucose tolerance and increased Akt phosphorylation in metabolic tissues of these obese mice. Chronic S6K1 inhibition by PF-4708671 improves glucose homeostasis in obese mice through enhanced Akt activation in liver and muscle. Our results suggest that specific S6K1 blockade is a valid pharmacological approach to improve glucose disposal in obese diabetic individuals.

Interaction of ApoA-IV with NR4A1 and NR1D1 Represses G6Pase and PEPCK Transcription: Nuclear Receptor-Mediated Downregulation of Hepatic Gluconeogenesis in Mice and a Human Hepatocyte Cell Line.

We have previously shown that the nuclear receptor, NR1D1, is a cofactor in ApoA-IV-mediated downregulation of gluconeogenesis. Nuclear receptor, NR4A1, is involved in the transcriptional regulation of various genes involved in inflammation, apoptosis, and glucose metabolism. We investigated whether NR4A1 influences the effect of ApoA-IV on hepatic glucose metabolism. Our in situ proximity ligation assays and coimmunoprecipitation experiments indicated that ApoA-IV colocalized with NR4A1 in human liver (HepG2) and kidney (HEK-293) cell lines. The chromatin immunoprecipitation experiments and luciferase reporter assays indicated that the ApoA-IV and NR4A1 colocalized at the RORα response element of the human G6Pase promoter, reducing its transcriptional activity. Our RNA interference experiments showed that knocking down the expression of NR4A1 in primary mouse hepatocytes treated with ApoA-IV increased the expression of NR1D1, G6Pase, and PEPCK, and that knocking down NR1D1 expression increased the level of NR4A1. We also found that ApoA-IV induced the expression of endogenous NR4A1 in both cultured primary mouse hepatocytes and in the mouse liver, and decreased glucose production in primary mouse hepatocytes. Our findings showed that ApoA-IV colocalizes with NR4A1, which suppresses G6Pase and PEPCK gene expression at the transcriptional level, reducing hepatic glucose output and lowering blood glucose. The ApoA-IV-induced increase in NR4A1 expression in hepatocytes mediates further repression of gluconeogenesis. Our findings suggest that NR1D1 and NR4A1 serve similar or complementary functions in the ApoA-IV-mediated regulation of gluconeogenesis.

Short-Term Curcumin Gavage Sensitizes Insulin Signaling in Dexamethasone-Treated C57BL/6 Mice1–3

Background: Long-term dietary curcumin (>12 wk) improves metabolic homeostasis in obese mice by sensitizing insulin signaling and reducing hepatic gluconeogenesis. Whether these occur only secondary to its chronic anti-inflammatory and antioxidative functions is unknown.Objective: In this study, we assessed the insulin sensitization effect of short-term curcumin gavage in a rapid dexamethasone-induced insulin resistance mouse model, in which the chronic anti-inflammatory function is eliminated.Methods: Six-week-old male C57BL/6 mice received an intraperitoneal injection of dexamethasone (100 mg/kg body weight) or phosphate-buffered saline every day for 5 d, with or without simultaneous curcumin gavage (500 mg/kg body weight). On day 7, insulin tolerance tests were performed. After a booster dexamethasone injection and curcumin gavage on day 8, blood glucose and insulin concentrations were measured. Liver tissues were collected on day 10 for quantitative polymerase chain reaction and Western blotting to assess gluconeogenic gene expression, insulin signaling, and the expression of fibroblast growth factor 21 (FGF21). Primary hepatocytes from separate, untreated C57BL/6 mice were used for testing the in vitro effect of curcumin treatment.Results: Dexamethasone injection impaired insulin tolerance (P < 0.05) and elevated ambient plasma insulin concentrations by ∼2.7-fold (P < 0.01). Concomitant curcumin administration improved insulin sensitivity and reduced hepatic gluconeogenic gene expression. The insulin sensitization effect of curcumin was demonstrated by increased stimulation of S473 phosphorylation of protein kinase B (P < 0.01) in the dexamethasone-treated mouse liver, as well as the repression of glucose production in primary hepatocytes (P < 0.001). Finally, curcumin gavage increased FGF21 expression by 2.1-fold in the mouse liver (P < 0.05) and curcumin treatment increased FGF21 expression in primary hepatocytes.Conclusion: These observations suggest that the early beneficial effect of curcumin intervention in dexamethasone-treated mice is the sensitization of insulin signaling, involving the stimulation of FGF21 production, a known insulin sensitizer.

Treatment of Obese Insulin-Resistant Mice With an Allosteric MAPKAPK2/3 Inhibitor Lowers Blood Glucose and Improves Insulin Sensitivity.

The prevalence of obesity-induced type 2 diabetes (T2D) is increasing worldwide, and new treatment strategies are needed. We recently discovered that obesity activates a previously unknown pathway that promotes both excessive hepatic glucose production (HGP) and defective insulin signaling in hepatocytes, leading to exacerbation of hyperglycemia and insulin resistance in obesity. At the hub of this new pathway is a kinase cascade involving calcium/calmodulin-dependent protein kinase II (CaMKII), p38α mitogen-activated protein kinase (MAPK), and MAPKAPK2/3 (MK2/3). Genetic-based inhibition of these kinases improves metabolism in obese mice. Here, we report that treatment of obese insulin-resistant mice with an allosteric MK2/3 inhibitor, compound (cmpd) 28, ameliorates glucose homeostasis by suppressing excessive HGP and enhancing insulin signaling. The metabolic improvement seen with cmpd 28 is additive with the leading T2D drug, metformin, but it is not additive with dominant-negative MK2, suggesting an on-target mechanism of action. Allosteric MK2/3 inhibitors represent a potentially new approach to T2D that is highly mechanism based, has links to human T2D, and is predicted to avoid certain adverse effects seen with current T2D drugs.

Hepatic SirT1-Dependent Gain of Function of Stearoyl-CoA Desaturase-1 Conveys Dysmetabolic and Tumor Progression Functions.

Obesity is associated with higher incidence of cancer, but the predisposing mechanisms remain poorly understood. The NAD(+)-dependent deacetylase SirT1 orchestrates metabolism, cellular survival, and growth. However, there is no unifying mechanism to explain the metabolic and tumor-related effects of SirT1. In this work, we demonstrate that genetic ablation of the endogenous inhibitor of SirT1, Deleted-in-Breast-Cancer-1 (Dbc1), unexpectedly results in obesity and insulin resistance. Dbc1 deficiency promoted SirT1-dependent gain of function of stearoyl-coenzyme A desaturase 1 (Scd1), increasing plasma and tissue levels of unsaturated fatty acids. The metabolic abnormalities in Dbc1(-/-) mice were reversed by ablation of hepatic SirT1 or by inhibition of Scd1 activity. Furthermore, loss of Dbc1 impaired activation of the master tumor suppressor p53 and treatment with an Scd1 inhibitor extended survival of tumor-prone TP53(-/-) mice by decreasing tumor-related death. Together, our findings illustrate a shared mechanism of obesity and tumor progression mediated by hepatic SirT1 and resulting in the activation of a key lipid synthetic enzyme, with potential therapeutic implications.

Metformin Activates AMP-activated Protein Kinase by Promoting Formation of the αβγ Heterotrimeric Complex

Metformin is the most widely prescribed oral anti-diabetic agent. Recently, we have shown that low metformin concentrations found in the portal vein suppress glucose production in hepatocytes through activation of AMPK. Moreover, low concentrations of metformin were found to activate AMPK by increasing the phosphorylation of AMPKα at Thr-172. However, the mechanism underlying the increase in AMPKα phosphorylation at Thr-172 and activation by metformin remains unknown. In the current study, we find that low concentrations of metformin promote the formation of the AMPK αβγ complex, resulting in an increase in net phosphorylation of the AMPK α catalytic subunit at Thr-172 by augmenting phosphorylation by LKB1 and antagonizing dephosphorylation by PP2C.

The LKB1-salt-inducible kinase pathway functions as a key gluconeogenic suppressor in the liver.

LKB1 is a master kinase that regulates metabolism and growth through adenosine monophosphate-activated protein kinase (AMPK) and 12 other closely related kinases. Liver-specific ablation of LKB1 causes increased glucose production in hepatocytes in vitro and hyperglycaemia in fasting mice in vivo. Here we report that the salt-inducible kinases (SIK1, 2 and 3), members of the AMPK-related kinase family, play a key role as gluconeogenic suppressors downstream of LKB1 in the liver. The selective SIK inhibitor HG-9-91-01 promotes dephosphorylation of transcriptional co-activators CRTC2/3 resulting in enhanced gluconeogenic gene expression and glucose production in hepatocytes, an effect that is abolished when an HG-9-91-01-insensitive mutant SIK is introduced or LKB1 is ablated. Although SIK2 was proposed as a key regulator of insulin-mediated suppression of gluconeogenesis, we provide genetic evidence that liver-specific ablation of SIK2 alone has no effect on gluconeogenesis and insulin does not modulate SIK2 phosphorylation or activity. Collectively, we demonstrate that the LKB1–SIK pathway functions as a key gluconeogenic gatekeeper in the liver.

Low Concentrations of Metformin Suppress Glucose Production in Hepatocytes through AMP-activated Protein Kinase (AMPK)*♦

Metformin is a first-line antidiabetic agent taken by 150 million people across the world every year, yet its mechanism remains only partially understood and controversial. It was proposed that suppression of glucose production in hepatocytes by metformin is AMPK-independent; however, unachievably high concentrations of metformin were employed in these studies. In the current study, we find that metformin, via an AMP-activated protein kinase (AMPK)-dependent mechanism, suppresses glucose production and gluconeogenic gene expression in primary hepatocytes at concentrations found in the portal vein of animals (60-80 μM). Metformin also inhibits gluconeogenic gene expression in the liver of mice administered orally with metformin. Furthermore, the cAMP-PKA pathway negatively regulates AMPK activity through phosphorylation at Ser-485/497 on the α subunit, which in turn reduces net phosphorylation at Thr-172. Because diabetic patients often have hyperglucagonemia, AMPKα phosphorylation at Ser-485/497 is a therapeutic target to improve metformin efficacy.

Effects of eugenol on hepatic glucose production and AMPK signaling pathway in hepatocytes and C57BL/6J mice

Eugenol is a phenylpropanoid with many pharmacological activities, but its anti-hyperglycemic activity is not yet fully explored. For in vitro study, HepG2 cells and primary rat hepatocytes were used, and glucose production was induced by adding 100nM of glucagon in the presence of gluconeogenic substrates. In animal study, hyperglycemia was induced by high fat diet (HFD) in male C57BL/6J mice, and eugenol was orally administered at 20 or 40mg per kg (E20, E40) for 15weeks. Eugenol significantly inhibited glucagon-induced glucose production and phosphorylated AMPK in the HepG2 and primary rat hepatocytes, and these effects were reversed in the presence of compound C (an AMPK inhibitor) or STO-609 (a CAMKK inhibitor). In addition, the protein and gene expression levels of CREB, CRTC2·CREB complex, PGC-1α, PEPCK and G6Pase were all significantly suppressed. Moreover, inhibition of AMPK by over-expression of dominant negative AMPK prevented eugenol from suppressions of gluconeogenic gene expression and hepatic glucose production. In animal study, plasma glucose and insulin levels of the E40 group were decreased by 31% and 63%, respectively, when compared to those of HFD control. In pyruvate tolerance tests, pyruvate-induced glucose excursions were decreased, indicating that the anti-hyperglycemic activity of eugenol is primarily due to the suppression of hepatic gluconeogenesis. In summary, eugenol effectively ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis via modulating CAMKK-AMPK-CREB signaling pathway. Eugenol or eugenol-containing medicinal plants could represent a promising therapeutic agent to prevent type 2 diabetes.

Study of CRTC2 Pharmacology Using Antisense Oligonuceotides

The cAMP response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is a key component of the transcription complex regulating glucagon driven hepatic glucose production and previous evidence suggests that "inhibition" of CRTC2 improves glucose homeostasis in multiple rodent models of type 2 diabetes. Here we describe a process of identifying potential therapeutic antisense oligonucleotides (ASOs) directed against CRTC2. These ASOs were designed as locked nucleic acid (LNA) gapmers and a panel of approximately 400 sequences were first screened in vitro within both human and mouse liver cell lines. A group of active and selective compounds were then profiled in acute studies in mice to determine the level of CRTC2 mRNA reduction in liver as well as to obtain a preliminary indication of safety and tolerability. The compounds with the best activity and safety profiles were then evaluated in subchronic efficacy studies using the diet induced obese (DIO) mouse model of type 2 diabetes and primary human hepatocytes. Efficacy findings broadly confirmed the beneficial effect of reducing CRTC2 mRNA levels towards improving glucose control and other markers of metabolic function. Additionally, for the first time, translation to human cells has been established with demonstration of a reduction in glucagon-mediated glucose production in primary human hepatocytes and a potential clinical biomarker source identified to assess modulation of CRTC2 mRNA following ASO treatment. While the compounds identified herein did not demonstrate a therapeutic index sufficient for further development, this study should facilitate more efficient prosecution of compounds within an in vivo setting.

Adipocyte Lipid Chaperone aP2 Is a Secreted Adipokine Regulating Hepatic Glucose Production

Proper control of hepatic glucose production is central to whole-body glucose homeostasis, and its disruption plays a major role in diabetes. Here, we demonstrate that although established as an intracellular lipid chaperone, aP2 is in fact actively secreted from adipocytes to control liver glucose metabolism. Secretion of aP2 from adipocytes is regulated by fasting- and lipolysis-related signals, and circulating aP2 levels are markedly elevated in mouse and human obesity. Recombinant aP2 stimulates glucose production and gluconeogenic activity in primary hepatocytes in vitro and in lean mice in vivo. In contrast, neutralization of secreted aP2 reduces glucose production and corrects the diabetic phenotype of obese mice. Hyperinsulinemic-euglycemic and pancreatic clamp studies upon aP2 administration or neutralization demonstrated actions of aP2 in liver. We conclude that aP2 is an adipokine linking adipocytes to hepatic glucose production and that neutralizing secreted aP2 may represent an effective therapeutic strategy against diabetes.

Liver Sinusoidal Endothelial Cells Link Hyperinsulinemia to Hepatic Insulin Resistance

Insulin signaling in vascular endothelial cells (ECs) is critical to maintain endothelial function but also to mediate insulin action on peripheral glucose disposal. However, gene knockout studies have reached disparate conclusions. Thus, insulin receptor inactivation in ECs does not impair insulin action, whereas inactivation of Irs2 does. Previously, we have shown that endothelial ablation of the three Foxo genes protects mice from atherosclerosis. Interestingly, here we show that mice lacking FoxO isoforms in ECs develop hepatic insulin resistance through excessive generation of nitric oxide (NO) that impairs insulin action in hepatocytes via tyrosine nitration of insulin receptors. Coculture experiments demonstrate that NO produced in liver sinusoidal ECs impairs insulin’s ability to suppress glucose production in hepatocytes. The effects of liver sinusoidal ECs can be mimicked by NO donors and can be reversed by NO inhibitors in vivo and ex vivo. The findings are consistent with a model in which excessive, rather than reduced, insulin signaling in ECs predisposes to systemic insulin resistance, prompting a reevaluation of current approaches to insulin sensitization.

Serum visfatin in type 2 diabetes mellitus

Abstract Introduction Obesity is commonly associated with insulin resistance and hyperinsulinemia and is the most important risk factor for type 2 diabetes. Visfatin is an adipokine that exerts insulin-mimetic effects that stimulate muscle and adipocyte glucose transport and inhibit hepatocyte glucose production. Purpose The aim of this study was to assess levels of visfatin and its relationship to obesity and insulin resistance in type 2 diabetes mellitus. Patients and methods This study included 40 patients with type 2 diabetes as the patient group: 20 of them were obese (BMI ≥ 30) and 20 were not (BMI &lt; 25). Forty apparently healthy individuals matched for age and sex were included as the control group: 20 of them were obese (BMI ≥ 30) and the other 20 were not (BMI &lt; 25). All patients and controls underwent history taking, physical examination including determination of BMI, and the following laboratory investigations: determination of levels of fasting blood glucose, 2 h postprandial blood glucose, serum cholesterol, triglycerides, fasting insulin, and fasting visfatin; kidney and liver function tests and calculation of homeostasis model of assessment-insulin resistance (HOMA-IR) were also performed. Neither the patients nor controls suffered from any chronic disease other than diabetes. Results The results of this study revealed a highly significant increase in the fasting serum insulin level, HOMA-IR, and fasting serum visfatin level among diabetic patients (26.10 ± 6.04μIU/ml, 12.18 ± 5.24, 36.70 ± 6.86 ng/ml, respectively) when compared with controls (12.10 ± 3.45μIU/ml, 2.42 ± 0.79, 13.63 ± 3.98 ng/ml, respectively; P &lt; 0.01). Fasting insulin levels, HOMA-IR, and visfatin levels were significantly higher in obese diabetic patients (31.13 ± 4.34μIU/ml, 14.71 ± 6.22, 42.36 ± 4.11 ng/ml, respectively) than in obese controls (14.31±3.11mIU/ml, 2.89±0.77, 16.72 ± 3.16 ng/ml, respectively; P &lt; 0.01). Visfatin levels were higher in nonobese diabetic patients than in nonobese controls. Moreover, visfatin levels were higher in obese diabetic patients (31.04 ± 3.49 ng/ml) than in nonobese diabetic patients (10.54 ± 1.53 ng/ml; P &lt; 0.01). The present study revealed a highly significant positive correlation between levels of visfatin and fasting insulin in both obese and nonobese diabetic patients. Although there was a significant positive correlation between visfatin levels and HOMA-IR, there was no significant correlation between visfatin levels and BMI in obese diabetic patients. Conclusion Visfatin levels are increased in patients with type 2 diabetes regardless the degree of adiposity.