Fibroblast growth factor 21 (FGF21) is a potent regulator of glucose and lipid metabolism and is currently being pursued as a therapeutic agent for insulin resistance and type 2 diabetes. However, the cellular mechanisms by which FGF21 modifies insulin action in vivo are unclear. To address this question, we assessed insulin action in regular chow- and high-fat diet (HFD)-fed wild-type mice chronically infused with FGF21 or vehicle. Here, we show that FGF21 administration results in improvements in both hepatic and peripheral insulin sensitivity in both regular chow- and HFD-fed mice. This improvement in insulin responsiveness in FGF21-treated HFD-fed mice was associated with decreased hepatocellular and myocellular diacylglycerol content and reduced protein kinase Cε activation in liver and protein kinase Cθ in skeletal muscle. In contrast, there were no effects of FGF21 on liver or muscle ceramide content. These effects may be attributed, in part, to increased energy expenditure in the liver and white adipose tissue. Taken together, these data provide a mechanism by which FGF21 protects mice from lipid-induced liver and muscle insulin resistance and support its development as a novel therapy for the treatment of nonalcoholic fatty liver disease, insulin resistance, and type 2 diabetes.

Comparative gene identification 58 (CGI-58) is a lipid droplet-associated protein that promotes the hydrolysis of triglyceride by activating adipose triglyceride lipase. Loss-of-function mutations in CGI-58 in humans lead to Chanarin-Dorfman syndrome, a condition in which triglyceride accumulates in various tissues, including the skin, liver, muscle, and intestines. Therefore, without adequate CGI-58 expression, lipids are stored rather than used for fuel, signaling intermediates, and membrane biosynthesis. CGI-58 knockdown in mice using antisense oligonucleotide (ASO) treatment also leads to severe hepatic steatosis as well as increased hepatocellular diacylglycerol (DAG) content, a well-documented trigger of insulin resistance. Surprisingly, CGI-58 knockdown mice remain insulin-sensitive, seemingly dissociating DAG from the development of insulin resistance. Therefore, we sought to determine the mechanism responsible for this paradox. Hyperinsulinemic-euglycemic clamp studies reveal that the maintenance of insulin sensitivity with CGI-58 ASO treatment could entirely be attributed to protection from lipid-induced hepatic insulin resistance, despite the apparent lipotoxic conditions. Analysis of the cellular compartmentation of DAG revealed that DAG increased in the membrane fraction of high fat-fed mice, leading to PKCe activation and hepatic insulin resistance. However, DAG increased in lipid droplets or lipid-associated endoplasmic reticulum rather than the membrane of CGI-58 ASO-treated mice, and thus prevented PKCe translocation to the plasma membrane and induction of insulin resistance. Taken together, these results explain the disassociation of hepatic steatosis and DAG accumulation from hepatic insulin resistance in CGI-58 ASO-treated mice, and highlight the importance of intracellular compartmentation of DAG in causing lipotoxicity and hepatic insulin resistance.nonalcoholic fatty liver disease | type 2 diabetes N onalcoholic fatty liver disease (NAFLD) is now the most common chronic liver disease in the United States and is strongly associated with hepatic insulin resistance and type 2 diabetes (1, 2). Although NAFLD is characterized by excessively high triglycerides in the liver, the triglycerides do not appear to be detrimental to hepatic insulin sensitivity (3, 4). Rather, other lipid moieties, such as diacylglycerols (DAG) and ceramides, have been implicated as the molecular triggers of insulin resistance (5-7). The mechanism whereby these lipids cause insulin resistance are diverse: DAGs cause insulin resistance through activation of PKCe in liver, leading to the inhibition of insulin-receptor kinase activity (8, 9), and PKCθ in skeletal muscle, leading to insulinreceptor substrate-1 serine phosphorylation on sites that interfere with insulin action (10-12). Ceramides have been proposed to inhibit AKT2 activation by either activating PP2A, which dephosphorylates and deactivates AKT2, or activating PKCζ, which phosphorylates AKT on an inhibitory residue and prevents its translocation to the plasma ...

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.

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...

Background: Endoplasmic reticulum (ER) stress has been implicated in causing hepatic insulin resistance.Results: Fructose-fed XBP1 knock-out mice were protected from hepatic insulin resistance despite increased hepatic ER stress and JNK activation.Conclusion: ER stress and hepatic JNK activation can be disassociated from hepatic insulin resistance.Significance: Hepatic ER stress is not a direct causal factor in hepatic insulin resistance.