Mice overexpressing acylCoA:diacylglycerol (DAG) acyltransferase 2 in the liver (Liv-DGAT2) have been shown to have normal hepatic insulin responsiveness despite severe hepatic steatosis and increased hepatic triglyceride, diacylglycerol, and ceramide content, demonstrating a dissociation between hepatic steatosis and hepatic insulin resistance. This led us to reevaluate the role of DAG in causing hepatic insulin resistance in this mouse model of severe hepatic steatosis. Using hyperinsulinemic-euglycemic clamps, we studied insulin action in Liv-DGAT2 mice and their wild-type (WT) littermate controls. Here, we show that Liv-DGAT2 mice manifest severe hepatic insulin resistance as reflected by decreased suppression of endogenous glucose production (0.8 ± 41.8 vs. 87.7 ± 34.3% in WT mice, P < 0.01) during the clamps. Hepatic insulin resistance could be attributed to an almost 12-fold increase in hepatic DAG content (P < 0.01) resulting in a 3.6-fold increase in protein kinase Cε (PKCε) activation (P < 0.01) and a subsequent 52% decrease in insulin-stimulated insulin receptor substrate 2 (IRS-2) tyrosine phosphorylation (P < 0.05), as well as a 64% decrease in fold increase pAkt/Akt ratio from basal conditions (P < 0.01). In contrast, hepatic insulin resistance in these mice was not associated with increased endoplasmic reticulum (ER) stress or inflammation. Importantly, hepatic insulin resistance in Liv-DGAT2 mice was independent of differences in body composition, energy expenditure, or food intake. In conclusion, these findings strengthen the link between hepatic steatosis and hepatic insulin resistance and support the hypothesis that DAG-induced PKCε activation plays a major role in nonalcoholic fatty liver disease (NAFLD)-associated hepatic insulin resistance.protein kinase Cε | ceramides, insulin receptor kinase | type 2 diabetes H epatic insulin resistance associated with nonalcoholic fatty liver disease (NAFLD) is a major risk factor for the development of type 2 diabetes (1-6). However, whether there is a causal link between NAFLD and hepatic insulin resistance remains controversial (7,8). Although intracellular triglyceride is unlikely to be the direct mediator of insulin resistance (3, 4), other related intracellular lipid intermediates such as diacylglycerol (3, 4, 9-12) and ceramide (13) have been postulated to block insulin signaling at the level of the insulin receptor kinase and Akt2, respectively, leading to insulin resistance. Indeed, diacylglycerol (DAG) has been shown to activate protein kinase Cε (PKCε) in the liver, which in turn binds to the insulin receptor and inhibits its tyrosine kinase activity (14).However, a recent paper reported that mice overexpressing acylCoA:diacylglycerol acyltransferase 2 (Liv-DGAT2) in the liver were not insulin resistant despite severe hepatic steatosis associated with increased liver DAG content (15). This study was of critical importance because it dissociated hepatic DAG content from hepatic insulin resistance and implied that hepatic DAG was not a causal fa...

Estrogen replacement therapy reduces the incidence of type 2 diabetes in postmenopausal women; however, the mechanism is unknown. Therefore, the aim of this study was to evaluate the metabolic effects of estrogen replacement therapy in an experimental model of menopause. At 8 weeks of age, female mice were ovariectomized (OVX) or sham (SHAM) operated, and OVX mice were treated with vehicle (OVX) or estradiol (E2) (OVX+E2). After 4 weeks of high-fat diet feeding, OVX mice had increased body weight and fat mass compared with SHAM and OVX+E2 mice. OVX mice displayed reduced whole-body energy expenditure, as well as impaired glucose tolerance and whole-body insulin resistance. Differences in whole-body insulin sensitivity in OVX compared with SHAM mice were accounted for by impaired muscle insulin sensitivity, whereas both hepatic and muscle insulin sensitivity were impaired in OVX compared with OVX+E2 mice. Muscle diacylglycerol (DAG), content in OVX mice was increased relative to SHAM and OVX+E2 mice. In contrast, E2 treatment prevented the increase in hepatic DAG content observed in both SHAM and OVX mice. Increases in tissue DAG content were associated with increased protein kinase Cε activation in liver of SHAM and OVX mice compared with OVX+E2 and protein kinase Cθ activation in skeletal muscle of OVX mice compared with SHAM and OVX+E2. Taken together, these data demonstrate that E2 plays a pivotal role in the regulation of whole-body energy homeostasis, increasing O(2) consumption and energy expenditure in OVX mice, and in turn preventing diet-induced ectopic lipid (DAG) deposition and hepatic and muscle insulin resistance.

Non-alcoholic fatty liver disease (NAFLD) and insulin resistance have recently been found to be associated with increased plasma concentrations of apolipoprotein CIII (APOC3) in humans carrying single nucleotide polymorphisms within the insulin response element of the APOC3 gene. To examine whether increased expression of APOC3 would predispose mice to NAFLD and hepatic insulin resistance, human APOC3 overexpressing (ApoC3Tg) mice were metabolically phenotyped following either a regular chow or high-fat diet (HFD). After HFD feeding, ApoC3Tg mice had increased hepatic triglyceride accumulation, which was associated with cellular ballooning and inflammatory changes. ApoC3Tg mice also manifested severe hepatic insulin resistance assessed by a hyperinsulinemic-euglycemic clamp, which could mostly be attributed to increased hepatic diacylglycerol content, PKCε activation and decreased insulin-stimulated Akt2 activity. Increased hepatic triglyceride content in the HFD fed ApoC3Tg mice could be attributed to a ~70% increase in hepatic triglyceride uptake and ~50% reduction hepatic triglyceride secretion. In conclusion these data demonstrate that increase plasma APOC3 concentrations predispose mice to diet-induced NAFLD and hepatic insulin resistance.

ApoA5 has a critical role in the regulation of plasma TG concentrations. In order to determine whether ApoA5 also impacts ectopic lipid deposition in liver and skeletal muscle, as well as tissue insulin sensitivity, we treated mice with an antisense oligonucleotide (ASO) to decrease hepatic expression of ApoA5. ASO treatment reduced ApoA5 protein expression in liver by 60–70%. ApoA5 ASO-treated mice displayed approximately 3-fold higher plasma TG concentrations, which were associated with decreased plasma TG clearance. Furthermore, ApoA5 ASO-treated mice fed a high-fat diet (HFD) exhibited reduced liver and skeletal muscle TG uptake and reduced liver and muscle TG and diacylglycerol (DAG) content. HFD-fed ApoA5 ASO-treated mice were protected from HFD-induced insulin resistance, as assessed by hyperinsulinemic-euglycemic clamps. This protection could be attributed to increases in both hepatic and peripheral insulin responsiveness associated with decreased DAG activation of protein kinase C (PKC)-ε and PKCθ in liver and muscle, respectively, and increased insulin-stimulated AKT2 pho­sphory­lation in these tissues. In summary, these studies demonstrate a novel role for ApoA5 as a modulator of susceptibility to diet-induced liver and muscle insulin resistance through regulation of ectopic lipid accumulation in liver and skeletal muscle.

Abstract. The pharmacological effects of rivoglitazone, a novel thiazolidinedione-derivative peroxisome proliferator-activated receptor (PPAR)-γ agonist, were characterized in vitro and in vivo. Rivoglitazone activated human PPARγ more potently compared with rosiglitazone and pioglitazone and had little effect on PPARα and PPARδ activity in luciferase reporter assays. In Zucker diabetic fatty (ZDF) rats, 14-day administration of rivoglitazone decreased the plasma glucose and triglyceride (TG) levels in a dose-dependent manner. The glucose-lowering effect of rivoglitazone was much more potent than those of pioglitazone (ED 50 : 0.19 vs. 34 mg/kg) and rosiglitazone (ED 50 : 0.20 vs. 28 mg/kg). In addition, rivoglitazone showed potent antidiabetic effects in diabetic db/db mice. In Zucker fatty rats, rivoglitazone at a dose of 0.1 mg/kg clearly ameliorated insulin resistance and lowered plasma TG levels by accelerating the clearance of plasma TG. Gene expression analysis in the liver and heart of ZDF rats treated with rivoglitazone for 14 days suggested that rivoglitazone may reduce hepatic glucose production and modulate the balance of the cardiac glucose/fatty acid metabolism in diabetic animals. In summary, we showed that rivoglitazone is a potent and selective PPARγ agonist and has a potent glucoselowering effect via improvement of the insulin resistance in diabetic animal models.

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Aims/hypothesis Aerobic exercise increases muscle glucose and improves insulin action through numerous pathways, including activation of Ca2+/calmodulin-dependent protein kinases (CAMKs) and peroxisome proliferator γ coactivator 1α (PGC-1α). While overexpression of PGC-1α increases muscle mitochondrial content and oxidative type I fibres, it does not improve insulin action. Activation of CAMK4 also increases the content of type I muscle fibres, PGC-1α level and mitochondrial content. However, it remains unknown whether CAMK4 activation improves insulin action on glucose metabolism in vivo. Methods The effects of CAMK4 activation on skeletal muscle insulin action were quantified using transgenic mice with a truncated and constitutively active form of CAMK4 (CAMK4*) in skeletal muscle. Tissue-specific insulin sensitivity was assessed in vivo using a hyperinsulinaemic–euglycaemic clamp and isotopic measurements of glucose metabolism. Results The rate of insulin-stimulated whole-body glucose uptake was increased by ~25% in CAMK4* mice, which was largely attributed to an increase of ~60% in the glucose uptake in the quadriceps, the largest hindlimb muscle. These mice had improvements in insulin signalling, as reflected by increased phosphorylation of Akt and its substrates and an increase in the level of GLUT4 protein. In addition, there were extramuscular effects: CAMK4* mice had improved hepatic and adipose insulin action. These pleiotropic effects were associated with increased levels of PGC-1α-related myokines in CAMK4* skeletal muscle. Conclusions/interpretation Activation of CAMK4 enhances mitochondrial biogenesis in skeletal muscle while also coordinating improvements in whole-body insulin-mediated glucose utilisation.