Pathogenesis Of Hepatic Insulin Resistance Research Articles

Hepatocyte NLRP3 interacts with PKCε to drive hepatic insulin resistance and steatosis

Hepatic insulin resistance (IR), as a downstream sequela of nonalcoholic fatty liver disease (NAFLD), is strongly associated with liver steatosis. Despite numerous mechanism advancements, the molecular underpinnings and pathogenesis of hepatic IR, especially regarding the pattern recognition receptors in hepatocytes, remain elusive. Here, we identified hepatocyte NLRP3 as a direct and previously-unresolved driver of hepatic IR to promote steatosis response. Under the model of NAFLD, we identified hepatocyte NLRP3 as a crucial inducer of hepatic IR by undertaking multilayer transcriptomic searches and further confirmed that its expression was increased in the liver tissues from NAFLD patients and mouse models (high-fat diet (HFD), leptin-receptor-deficient (db/db) mice), and in palmitic acid (PA)-induced hepatocytes. Loss- or gain-of-function of hepatocyte-specific NLRP3 in HFD-induced mice ameliorated or exacerbated hepatic IR and steatosis, respectively. Mechanistically, NLRP3 directly bound to and promoted protein kinase C epsilon (PKCε) activation to impair insulin signaling and increase liver steatosis, while inhibition of PKCε activation dampened the beneficial effects seen in HFD-induced NLRP3-deficient mice. Moreover, we performed screening and discovered that the transcription factor Yin Yang 1 (YY1) positively controlled NLRP3 expression. In translational potential, adeno-associated virus serotype 8 (AAV8)-mediated NLRP3 knockdown in the liver alleviated hepatic IR and steatosis in db/db mice, and pharmacological inhibition of NLRP3 markedly alleviated diet-induced metabolic disorders. This finding reveals a previously-unexpected regulatory axis from YY1 to PKCε via NLRP3 induction for metabolic diseases and establishes the YY1-NLRP3-PKCε axis as a potential therapeutic target for NAFLD.

MiR-183-5p Induced by Saturated Fatty Acids Hinders Insulin Signaling by Downregulating IRS-1 in Hepatocytes.

Excessive saturated fatty acids (SFA) uptake is known to be a primary cause of obesity, a widely acknowledged risk factor of insulin resistance and type 2 diabetes. Although specific microRNAs (miRNAs) targeting insulin signaling intermediates are dysregulated by SFA, their effects on insulin signaling and sensitivity are largely unknown. Here, we investigated the role of SFA-induced miR-183-5p in the regulation of proximal insulin signaling molecules and the development of hepatic insulin resistance. HepG2 hepatocytes treated with palmitate and the livers of high-fat diet (HFD)-fed mice exhibited impaired insulin signaling resulting from dramatic reductions in the protein expressions of insulin receptor (INSR) and insulin receptor substrate-1 (IRS-1). Differential expression analysis showed the level of miR-183-5p, which tentatively targets the 3′UTR of IRS-1, was significantly elevated in palmitate-treated HepG2 hepatocytes and the livers of HFD-fed mice. Dual-luciferase analysis showed miR-183-5p bound directly to the 3′UTR of IRS-1 and reduced IRS-1 expression at the post-transcriptional stage. Moreover, transfection of HepG2 hepatocytes with miR-183-5p mimic significantly inhibited IRS-1 expression and hindered insulin signaling, consequently inhibiting insulin-stimulated glycogen synthesis. Collectively, this study reveals a novel mechanism whereby miR-183-5p induction by SFA impairs insulin signaling and suggests miR-183-5p plays a crucial role in the pathogenesis of hepatic insulin resistance in the background of obesity.

Role of Chitinase 3-Like 1 Protein in the Pathogenesis of Hepatic Insulin Resistance in Nonalcoholic Fatty Liver Disease.

A recently discovered human glycoprotein, chitinase 3-like 1 (Chi3L1), may play a role in inflammation, tissue remodeling, and visceral fat accumulation. We hypothesize that Chi3L1 gene expression is important in the development of hepatic insulin resistance characterized by the generation of pAKT, pGSK, and pERK in wild type and Chi3L1 knockout (KO) murine liver following insulin stimulation. The Chi3L1 gene and protein expression was evaluated by Real Time PCR and ELISA; lipid accumulation in hepatocytes was also assessed. To alter Chi3L1 function, three different anti-Chi3L1 monoclonal antibodies (mAbs) were administered in vivo and effects on the insulin signaling cascade and hepatic lipid deposition were determined. Transmission of the hepatic insulin signal was substantially improved following KO of the CHi3L1 gene and there was reduced lipid deposition produced by a HFD. The HFD-fed mice exhibited increased Chi3L1 expression in the liver and there was impaired insulin signal transduction. All three anti-Chi3L1 mAbs partially restored hepatic insulin sensitivity which was associated with reduced lipid accumulation in hepatocytes as well. A KO of the Chi3L1 gene reduced lipid accumulation and improved insulin signaling. Therefore, Chi3L1 gene upregulation may be an important factor in the generation of NAFLD/NASH phenotype.

Protectin DX ameliorates palmitate‐induced hepatic insulin resistance through AMPK/SIRT1‐mediated modulation of fetuin‐A and SeP expression

The role as well as the molecular mechanisms of protectin DX (PDX) in the prevention of hepatic insulin resistance, a hallmark of type 2 diabetes, remains unknown. Therefore, the present study was designed to explore the direct impact of PDX on insulin resistance and to investigate the expression of fetuin-A and selenoprotein P (SeP), hepatokines that are involved in insulin signalling, in hepatocytes. Human serum levels of PDX as well as fetuin-A and SeP were determined by high-performance liquid chromatography (HPLC). Human primary hepatocytes were treated with palmitate and PDX. NF-κB phosphorylation as well as expression of insulin signalling associated genes and hepatokines were determined by Western blotting analysis. FOXO1 binding levels were measured by quantitative real-time PCR. Selected genes from candidate pathways were evaluated by small interfering (si) RNA-mediated gene suppression. Serum PDX levels were significantly (P<0.05) downregulated, whereas serum fetuin-A and SeP levels were increased (P<0.05) in obese subjects compared with healthy subjects. In in vitro experiments, PDX treatment increased AMP-activated protein kinase (AMPK) phosphorylation and SIRT1 expression and attenuated palmitate-induced fetuin-A and SeP expression and insulin resistance in hepatocytes. AMPK or SIRT1 siRNA mitigated the suppressive effects of PDX on palmitate-induced fetuin-A through NF-κB and SeP expression linked to FOXO1 and insulin resistance. Recombinant fetuin-A and SeP reversed the suppressive effects of fetuin-A and SeP expression on palmitate-mediated impairment of insulin signalling. The current finding provides novel insight into the underlying mechanism linking hepatokines to the pathogenesis of hepatic insulin resistance.

19-OR: Controlled-Release Mitochondrial Protonophore (CRMP) Reverses Hypertriglyceridemia and Hepatic Steatosis in Dysmetabolic Nonhuman Primates

NAFLD is estimated to affect up to one third of the general population and is a major predisposing factor in the pathogenesis of hepatic insulin resistance, NASH, and T2D. While weight loss is highly effective at reversing NAFLD, insulin resistance and T2D, it is difficult to sustain and new therapies are required. Our lab has recently developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides by promoting a subtle sustained increase in hepatic mitochondrial inefficiency. While we have previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, and hepatic inflammation/fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess the safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of CRMP treatment on hepatic mitochondrial oxidation and the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in a spontaneous nonhuman primate model of NAFLD and the metabolic syndrome. Using Positional Isotopomer NMR Tracer Analysis (PINTA), we demonstrate that acute CRMP treatment (single dose of 5 mg/kg) significantly increases rates of hepatic mitochondrial fat oxidation by 40% (P&amp;lt;0.05). Importantly, CRMP treatment (5 mg/kg/day x 6 weeks) reduced hepatic and plasma triglycerides by 20% (both P=0.05) independently of changes in body weight, food intake, body temperature or adverse reactions. Furthermore, administration of CRMP significantly reduced endogenous glucose production by 18% (P&amp;lt;0.05), which could be attributed to a ~20% reduction in hepatic acetyl-CoA content (as assessed by whole-body βOHB turnover) and pyruvate carboxylase flux. Conclusion: These studies provide important proof-of-concept data to support the development of novel liver-targeted mitochondrial uncouplers for the treatment of NAFLD/NASH and T2D in humans. Disclosure L. Goedeke: None. V. Montalvo Romeral: None. G. Butrico: None. M. Kahn: None. S. Dufour: None. X. Zhang: None. G. Cline: None. K. Petersen: Advisory Panel; Spouse/Partner; Aegerion Pharmaceuticals, AstraZeneca, AstraZeneca, Janssen Research &amp; Development, Merck &amp; Co., Inc., Novo Nordisk A/S. Consultant; Spouse/Partner; Gilead Sciences, Inc. Consultant; Self; Merck &amp; Co., Inc. Consultant; Spouse/Partner; Nimbus Discoverey. Consultant; Self; Novo Nordisk A/S. K. Chng: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research &amp; Development, Merck &amp; Co., Inc. Advisory Panel; Spouse/Partner; Merck &amp; Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health

High-throughput sequencing of small RNAs and analysis of differentially expressed microRNAs associated with high-fat diet-induced hepatic insulin resistance in mice

BackgroundHepatic insulin resistance (IR) plays a crucial role in the development of many metabolic diseases, such as type 2 diabetes. MicroRNAs (miRNAs) are involved in the pathogenesis of IR and related diseases; however, studies of miRNAs in hepatic IR are limited.MethodIn this study, we adopted a high-throughput sequencing approach to construct small RNA libraries in the livers of normal mice and high-fat diet-induced hepatic IR mice.ResultsThrough analysis of data, 107 known and 56 novel miRNAs were identified as differentially expressed miRNAs between the two groups. Additionally, bioinformatics methods were used to predict targets of the differentially expressed miRNAs and to explore the potential downstream Gene Ontology categories and Kyoto Encyclopedia of Genes and Genomes pathways. Meanwhile, some differentially expressed miRNAs (miR-34a-5p, miR-149-5p, miR-335-3p, miR-10b-5p, miR-1a-3p, miR-411-5p, and miR-592-5p) were validated by quantitative-time PCR, and their potential target genes related to IR or glycolipid metabolism were also predicted and presented in this study.ConclusionTaken together, our results defined miRNA expression signature that may lead to hepatic IR in mice, and the findings provided a foundation for future studies to further explore the effects and underlying mechanisms of the miRNAs and their target genes in the pathogenesis of hepatic IR and related diseases.

Prolactin regulatory element-binding (PREB) protein regulates hepatic glucose homeostasis

Prolactin regulatory element-binding (PREB) protein is a transcription factor that regulates prolactin (PRL) gene expression. PRL, also known as luteotropic hormone or luteotropin, is well known for its role in producing milk. However, the role of PREB, in terms of hepatic glucose metabolism, is not well elucidated. Here, we observed expression of Preb in the mouse liver, in connection with glucose homeostasis. Morevoer, Preb was downregulated in db/db, ob/ob and high-fat diet-induced obese (DIO) mice, concurrent with upregulation of the liver genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase-1 (Pck). Administration of adenovirus-Preb (Ad-Preb) to db/db, ob/ob, and DIO mice diminished glucose, insulin, and pyruvate tolerance, which analogously, were impaired in normal (C57BL/6) mice knocked down for Preb, via infection with Ad-shPreb (anti-Preb RNA), indicating Preb to be a negative regulator of liver gluconeogenic genes. We further demonstrate that Preb negatively influences gluconeogenic gene expression, by directly binding to their promoters at a prolactin core-binding element (PCBE).A better understanding of Preb gene expression, during the pathogenesis of hepatic insulin resistance, could ultimately provide new avenues for therapies for metabolic syndrome, obesity, and type-2 diabetes mellitus, disorders whose worldwide incidences are increasing drastically.

PKM2 aggravates palmitate-induced insulin resistance in HepG2 cells via STAT3 pathway

Studies have identified that PKM2 is related to the development of glucose intolerance and insulin resistance in rodents and humans. However, the underlying mechanism remains largely unknown. In the present study, we found that PKM2 expression was significantly elevated in insulin-resistant hepatic tissues and hepatocytes, implicating an association between PKM2 expression and hepatic insulin resistance (IR). In vitro study revealed that overexpression of PKM2 impaired the insulin signaling pathway by decreasing the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3β (GSK3β). Furthermore, PKM2 overexpression enhanced the effects of PA on the lipid accumulation, the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) and hepatic glucose uptake. Intriguingly, PA-induced insulin resistance was suppressed following by the ablation of PKM2 in HepG2 cells. We also found that STAT3 was significantly activated by PKM2 overexpression. Moreover, we identified that PKM2 could interact directly with STAT3. Taken together, these studies demonstrate that PKM2 may promote hepatic IR via STAT3 pathway and would provide a new insight into dissecting the molecular pathogenesis of hepatic insulin resistance.

Expression of resistin in the liver of patients with non-alcoholic fatty liver disease.

Adipokines are cytokines that presumably connect the pathologies of metabolic syndrome. One of the adipokines is resistin, the role of which in insulin resistance, obesity, and non-alcoholic fatty liver disease (NAFLD) needs to be determined. Liver biopsy specimens were obtained intraoperatively from 214 obese patients. Histological assessment was based on NAFLD activity score according to Kleiner. Statistical analysis involved semi-quantitive immunohistochemistry assessment of resistin staining and: NAFLD status in obese patients compared with a non-obese control group, selected clinical data (age, sex, body mass index - BMI), selected biochemical data, comorbidities (hypertension, type 2 diabetes mellitus, dyslipidaemia), and metformin treatment in patients with type 2 diabetes mellitus. Resistin expression was observed in the histiocytes of inflammatory infiltrate, Kupffer cells, and histiocytes surrounding the hepatocytes with steatosis. There was a positive correlation between the total expression of resistin and: (1) NAFLD advancement (NAFLD Activity Score- NAS), (2) AST, ALT, BMI, glucose, insulin, Homeostasis Model Assessment (HOMA), LDH, GGT, triglycerides (TG), and glycated haemoglobin (HbA1c). Resistin expression was more intense in patients with type 2 diabetes mellitus and dyslipidaemia and less intense in the control group. Resistin probably plays a role in the pathogenesis of hepatic insulin resistance and aggravates pathologic changes in the liver of patients with NAFLD.

Induction of miR-96 by Dietary Saturated Fatty Acids Exacerbates Hepatic Insulin Resistance through the Suppression of INSR and IRS-1.

Obesity is defined as the excessive accumulation of body fat that ultimately leads to chronic metabolic diseases. Diets rich in saturated fatty acids (SFA) exacerbate obesity and hepatic steatosis, which increase the risk of hepatic insulin resistance and type 2 diabetes (T2DM). Although microRNAs (miRNAs) play an important role in a range of biological processes, the implications of SFA-induced miRNAs in metabolic dysregulation, particularly in the pathogenesis of hepatic insulin resistance, are not well understood. This study investigated the implications of miR-96, which is induced strongly by SFA, in the development of hepatic insulin resistance. The liver of HFD mice and the palmitate-treated hepatocytes exhibited an impairment of insulin signaling due to the significant decrease in INSR and IRS-1 expression. According to expression profiling and qRT-PCR analysis of the miRNAs, the expression level of miR-96 was higher in hepatocytes treated with palmitate. Moreover, miR-96 was also upregulated in the liver of HFD mice. Interestingly, miR-96 targeted the 3’UTRs of INSR and IRS-1 directly, and repressed the expression of INSR and IRS-1 at the post-transcriptional level. Accordingly, the overexpression of miR-96 was found to cause a significant decrease in INSR and IRS-1 expression, thereby leading to an impairment of insulin signaling and glycogen synthesis in hepatocytes. These results reveal a novel mechanism whereby miR-96 promotes the pathogenesis of hepatic insulin resistance resulted from SFA or obesity.

MiR-696 plays a role in hepatic gluconeogenesis in ob/ob mice by targeting PGC-1α.

MicroRNAs(miRNAs or miRs) are known to play a vital role in type2 diabetes, and peroxisome proliferator-activated receptor-γ(PPARγ) coactivator-1α(PGC-1α) is involved in the pathogenesis of hepatic insulin resistance. However, the correlation, if any, between PGC-1α and miRNAs in the disease has not yet been determined. Thus, in the present study, we aimed to examine the correlation between PGC-1α and miRNAs in diabetes. For this purpose, we used primary hepatocytes isolated from C57BL/6 mice and ob/ob mice. First, we found an inverse correlation between miR‑696 and PGC-1α protein levels invivo. Second, invitro evidence demonstrated that PGC-1α expression was significantly decreased by infection with pre-miR‑696-LV, whereas infection with anti-miR‑696-LV increased the PGC-1α protein levels. Third, a luciferase reporter assay confirmed that miR‑696 directly recognizes a specific location within the 3'-untranslated region of PGC-1α transcripts. Furthermore, the biological consequences of miR‑696 targeting PGC-1α were determined by measuring the expression levels of the characteristic hepatic gluconeogenic enzyme, PEPCK, which is regulated by PGC-1α in the liver via the coactivation of transcription factors. Taken together, our findings demonstrate that miR‑696 plays an important role in the development of hepatic gluconeogenesis and insulin resistance through the inhibition of PGC-1α translation in the liver.

Obesity-induced miR-15b is linked causally to the development of insulin resistance through the repression of the insulin receptor in hepatocytes.

Obesity increases intracellular lipid accumulation in key tissues or organs, which often leads to metabolic dysfunction and insulin resistance. Diets rich in saturated fatty acid (SFA) exacerbate obesity and hepatic steatosis, which accentuate the risk of insulin resistance and type 2 diabetes (T2DM). Although microRNAs (miRNAs) play a critical role in the regulation of gene expression, the implication of obesity-induced miRNAs in metabolic disorders particularly in the development of insulin resistance is largely unknown. Here, we investigated the implication of miR-15b, which is induced by SFA palmitate or obesity, in hepatic insulin resistance. Diet-induced obesity (DIO) in mice developed hyperglycemia and insulin resistance, accompanying with a reduction of insulin receptor (INSR) expression. Palmitate impaired insulin signaling as well as a decrease of INSR in hepatocytes. The expression of miR-15b was upregulated by DIO or palmitate in hepatocytes. Furthermore, the overexpression of miR-15b suppressed the protein expression of INSR through targeting INSR 3' untranslated region directly, resulting in an impairment of the insulin signaling and glycogen synthesis in hepatocytes. These results unveil a novel mechanism whereby miR-15b is linked causally to the pathogenesis of hepatic insulin resistance in SFA-induced obesity.

PCBP2 regulates hepatic insulin sensitivity via HIF-1α and STAT3 pathway in HepG2 cells

Elevated free fatty acids (FFAs) are fundamental to the pathogenesis of hepatic insulin resistance. However, the molecular mechanisms of insulin resistance remain not completely understood. Transcriptional dysregulation, post-transcriptional modifications and protein degradation contribute to the pathogenesis of insulin resistance. Poly(C) binding proteins (PCBPs) are RNA-binding proteins that are involved in post-transcriptional control pathways. However, there are little studies about the roles of PCBPs in insulin resistance. PCBP2 is the member of the RNA-binding proteins and is thought to participate in regulating hypoxia inducible factor-1 (HIF-1α) and signal transducers and activators of transcription (STAT) pathway which are involved in regulating insulin signaling pathway. Here, we investigated the influence of PCBP2 on hepatic insulin resistance. We showed that the protein and mRNA levels of PCBP2 were down-regulated under insulin-resistant conditions. In addition, we showed that over-expression of PCBP2 ameliorates palmitate (PA)-induced insulin resistance, which was indicated by elevated phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3β (GSK3β). We also found that over-expression of PCBP2 inhibits HIF1α and STAT3 pathway. Furthermore, glucose uptake was found to display a similar tendency with the phosphorylation of Akt. The expressions of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), two key gluconeogenic enzymes, were down-regulated following Over-expression of PCBP2. Accordingly, PA-induced intracellular lipid accumulation was suppressed in over-expression of PCBP2 HepG2 cells. In addition, we found that over-expression of PCBP2 inhibits HIF1α and STAT3 pathway. Our results demonstrate that PCBP2 was involved in hepatic insulin sensitivity might via HIF-1α and STAT3 pathway in HepG2 cells.

Association of Liver Enzymes and Computed Tomography Markers of Liver Steatosis with Familial Longevity

ObjectiveFamilial longevity is marked by enhanced peripheral but not hepatic insulin sensitivity. The liver has a critical role in the pathogenesis of hepatic insulin resistance. Therefore we hypothesized that the extent of liver steatosis would be similar between offspring of long-lived siblings and control subjects. To test our hypothesis, we investigated the extent of liver steatosis in non-diabetic offspring of long-lived siblings and age-matched controls by measuring liver enzymes in plasma and liver fat by computed tomography (CT).Research Design and Methods:We measured nonfasting alanine transaminase (ALT), aspartate aminotransferase (AST), and Υ-glutamyl transferase (GGT) in 1625 subjects (736 men, mean age 59.1 years) from the Leiden Longevity Study, comprising offspring of long-lived siblings and partners thereof. In a random subgroup, fasting serum samples (n = 230) were evaluated and CT was performed (n = 268) for assessment of liver-spleen (L/S) ratio and the prevalence of moderate-to-severe non-alcoholic fatty liver disease (NAFLD). Linear mixed model analysis was performed adjusting for age, gender, body mass index, smoking, use of alcohol and hepatotoxic medication, and correlation of sibling relationship.ResultsOffspring of long-lived siblings had higher nonfasting ALT levels as compared to control subjects (24.3 mmol/L versus 23.2 mmol/L, p = 0.03), while AST and GGT levels were similar between the two groups. All fasting liver enzyme levels were similar between the two groups. CT L/S ratio and prevalence of moderate-to-severe NAFLD was similar between groups (1.12 vs 1.14, p = 0.25 and 8% versus 8%, p = 0.91, respectively).ConclusionsExcept for nonfasting levels of ALT, which were slightly higher in the offspring of long-lived siblings compared to controls, no differences were found between groups in the extent of liver steatosis, as assessed with liver biochemical tests and CT. Thus, our data indicate that the extent of liver steatosis is similar between offspring of long-lived siblings and control subjects.

Treating fatty liver and insulin resistance

Treating fatty liver and insulin resistance

Role of patatin-like phospholipase domain-containing 3 on lipid-induced hepatic steatosis and insulin resistance in rats

Genome-wide array studies have associated the patatin-like phospholipase domain-containing 3 (PNPLA3) gene polymorphisms with hepatic steatosis. However, it is unclear whether PNPLA3 functions as a lipase or a lipogenic enzyme and whether PNPLA3 is involved in the pathogenesis of hepatic insulin resistance. To address these questions we treated high-fat-fed rats with specific antisense oligonucleotides to decrease hepatic and adipose pnpla3 expression. Reducing pnpla3 expression prevented hepatic steatosis, which could be attributed to decreased fatty acid esterification measured by the incorporation of [U-13C]-palmitate into hepatic triglyceride. While the precursors for phosphatidic acid (PA) (long-chain fatty acyl-CoAs and lysophosphatidic acid [LPA]) were not decreased, we did observe an ∼20% reduction in the hepatic PA content, ∼35% reduction in the PA/LPA ratio, and ∼60%-70% reduction in transacylation activity at the level of acyl-CoA:1-acylglycerol-sn-3-phosphate acyltransferase. These changes were associated with an ∼50% reduction in hepatic diacylglycerol (DAG) content, an ∼80% reduction in hepatic protein kinase Cε activation, and increased hepatic insulin sensitivity, as reflected by a 2-fold greater suppression of endogenous glucose production during the hyperinsulinemic-euglycemic clamp. Finally, in humans, hepatic PNPLA3 messenger RNA (mRNA) expression was strongly correlated with hepatic triglyceride and DAG content, supporting a potential lipogenic role of PNPLA3 in humans. Conclusion: PNPLA3 may function primarily in a lipogenic capacity and inhibition of PNPLA3 may be a novel therapeutic approach for treatment of nonalcoholic fatty liver disease-associated hepatic insulin resistance. ((Hepatology 2013;57:1763-1772))

Dissociation of Inositol-requiring Enzyme (IRE1α)-mediated c-Jun N-terminal Kinase Activation from Hepatic Insulin Resistance in Conditional X-box-binding Protein-1 (XBP1) Knock-out Mice

Hepatic insulin resistance has been attributed to both increased endoplasmic reticulum (ER) stress and accumulation of intracellular lipids, specifically diacylglycerol (DAG). The ER stress response protein, X-box-binding protein-1 (XBP1), was recently shown to regulate hepatic lipogenesis, suggesting that hepatic insulin resistance in models of ER stress may result from defective lipid storage, as opposed to ER-specific stress signals. Studies were designed to dissociate liver lipid accumulation and activation of ER stress signaling pathways, which would allow us to delineate the individual contributions of ER stress and hepatic lipid content to the pathogenesis of hepatic insulin resistance. Conditional XBP1 knock-out (XBP1Δ) and control mice were fed fructose chow for 1 week. Determinants of whole-body energy balance, weight, and composition were determined. Hepatic lipids including triglyceride, DAGs, and ceramide were measured, alongside markers of ER stress. Whole-body and tissue-specific insulin sensitivity were determined by hyperinsulinemic-euglycemic clamp studies. Hepatic ER stress signaling was increased in fructose chow-fed XBP1Δ mice as reflected by increased phosphorylated eIF2α, HSPA5 mRNA, and a 2-fold increase in hepatic JNK activity. Despite JNK activation, XBP1Δ displayed increased hepatic insulin sensitivity during hyperinsulinemic-euglycemic clamp studies, which was associated with increased insulin-stimulated IRS2 tyrosine phosphorylation, reduced hepatic DAG content, and reduced PKCε activity. These studies demonstrate that ER stress and IRE1α-mediated JNK activation can be disassociated from hepatic insulin resistance and support the hypothesis that hepatic insulin resistance in models of ER stress may be secondary to ER stress modulation of hepatic lipogenesis.

Diabetes and Nonalcoholic Fatty Liver Disease

Diabetes and Nonalcoholic Fatty Liver Disease

Whole body overexpression of PGC-1α has opposite effects on hepatic and muscle insulin sensitivity

Type 2 diabetes is characterized by fasting hyperglycemia, secondary to hepatic insulin resistance and increased glucose production. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) is a transcriptional coactivator that is thought to control adaptive responses to physiological stimuli. In liver, PGC-1alpha expression is induced by fasting, and this effect promotes gluconeogenesis. To examine whether PGC-1alpha is involved in the pathogenesis of hepatic insulin resistance, we generated transgenic (TG) mice with whole body overexpression of human PGC-1alpha and evaluated glucose homeostasis with a euglycemic-hyperinsulinemic clamp. PGC-1alpha was moderately (approximately 2-fold) overexpressed in liver, skeletal muscle, brain, and heart of TG mice. In liver, PGC-1alpha overexpression resulted in increased expression of hepatocyte nuclear factor-4alpha and the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. PGC-1alpha overexpression caused hepatic insulin resistance, manifested by higher glucose production and diminished insulin suppression of gluconeogenesis. Paradoxically, PGC-1alpha overexpression improved muscle insulin sensitivity, as evidenced by elevated insulin-stimulated Akt phosphorylation and peripheral glucose disposal. Content of myoglobin and troponin I slow protein was increased in muscle of TG mice, indicating fiber-type switching. PGC-1alpha overexpression also led to lower reactive oxygen species production by mitochondria and reduced IKK/IkappaB signaling in muscle. Feeding a high-fat diet to TG mice eliminated the increased muscle insulin sensitivity. The dichotomous effect of PGC-1alpha overexpression in liver and muscle suggests that PGC-1alpha is a fuel gauge that couples energy demands (muscle) with the corresponding fuel supply (liver). Thus, under conditions of physiological stress (i.e., prolonged fast and exercise training), increased hepatic glucose production may help sustain glucose utilization in peripheral tissues.

Inhibition of protein kinase Cepsilon prevents hepatic insulin resistance in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance and type 2 diabetes mellitus, but the molecular signals linking hepatic fat accumulation to hepatic insulin resistance are unknown. Three days of high-fat feeding in rats results specifically in hepatic steatosis and hepatic insulin resistance. In this setting, PKCepsilon, but not other isoforms of PKC, is activated. To determine whether PKCepsilon plays a causal role in the pathogenesis of hepatic insulin resistance, we treated rats with an antisense oligonucleotide against PKCepsilon and subjected them to 3 days of high-fat feeding. Knocking down PKCepsilon expression protects rats from fat-induced hepatic insulin resistance and reverses fat-induced defects in hepatic insulin signaling. Furthermore, we show that PKCepsilon associates with the insulin receptor in vivo and impairs insulin receptor kinase activity both in vivo and in vitro. These data support the hypothesis that PKCepsilon plays a critical role in mediating fat-induced hepatic insulin resistance and represents a novel therapeutic target for type 2 diabetes.