To determine the net effect of tissue-specific Nur77 regulation of glucose metabolism, we studied the effect of high-fat feeding on Nur77 null mice. We found that Nur77 null mice developed exacerbated skeletal muscle insulin resistance after high-fat feeding. Skeletal muscle insulin resistance in Nur77 null mice may be explained by defects in multiple steps of the glucose utilization pathway (). As Nur77 is a transcriptional regulator of GLUT4 and multiple glycolytic enzymes, reduction in the abundance of these glucose uptake and glycolytic genes would be predicted to diminish insulin-stimulated glucose disposal. The reduced glucose utilization may contribute to a compensatory increase in lipid uptake in skeletal muscle. In addition, we found that insulin receptor phosphorylation was reduced in Nur77 null skeletal muscle, which may in part be attributed to increased intramuscular lipid accumulation. In the liver, Nur77 deletion was unable to protect high-fat–fed mice from hepatic insulin resistance. Rather, Nur77 null livers became more steatotic than wild-type livers because of increased lipogenesis, likely secondary to hyperinsulinemia. These findings highlight skeletal muscle Nur77 as a physiologic regulator of systemic glucose metabolism.
FIG. 7. Proposed mechanism of insulin resistance in Nur77 null mice. Nur77 deletion leads to reduced abundance of GLUT4 as well as diminished glycolysis in skeletal muscle. Reduced glucose utilization leads to compensatory increase in fatty acid uptake, which (more ...)
The metabolic benefit of oxidative metabolism in skeletal muscle is well established. Studies in patients with diabetes suggest that insulin resistance correlates with decreased oxidative enzyme activity in skeletal muscle (31
). Muscle-specific overexpression of PPARδ results in not only increased abundance of slow-twitch oxidative fibers but also protects mice from diet-induced obesity and diabetes (33
). On the other hand, the metabolic impact of glycolytic activity in skeletal muscle is less clear. Recent evidence suggests that selective fast-twitch/glycolytic muscle fiber growth also protects mice from diet-induced obesity and diabetes (34
). We have previously shown that Nur77-mediated regulation of glycolytic genes occurred selectively in fast-twitch/glycolytic, not slow-twitch/oxidative, fibers (17
). Our current finding that Nur77 deletion exacerbates diet-induced insulin resistance further supports the notion that enhancing glycolytic activity in skeletal muscle is metabolically advantageous.
Lipotoxicity has been implicated as a contributor to insulin resistance in both skeletal muscle and liver, although the relationship between increased lipid accumulation and tissue insulin responsiveness is not always direct. Increased muscle lipid content, particularly long-chain acyl-CoAs, DAGs, and ceramides, has been implicated in activation of various kinases that phosphorylate insulin receptor substrate on serine residues and thereby impairs insulin signaling (35
). An implication of these studies is that decreased fatty acid oxidation contributes to insulin resistance and impaired glucose metabolism. In support of this idea, mice with global acetyl-CoA carboxylase 2 deletion have diminished malonyl-CoA levels, increased CPT-1 activity, and are protected from diet-induced obesity and insulin resistance (37
). In contrast, Koves et al. (38
) showed that chronic exposure of muscle to elevated lipids induced β-oxidation of fatty acids without concurrent upregulation of downstream metabolic pathways such as the trichloroacetic acid cycle and electron transport chain. This results in incomplete metabolism of fatty acids in the β-oxidation pathway and exacerbation of insulin resistance (39
). Additional evidence supporting this argument includes the diabetic phenotype of muscle-specific PPARα transgenic mouse, which is reversed by pharmacologic inhibition of CPT-1 (40
). In HFD-challenged Nur77 null mice, we showed that loss of Nur77 led to decreased glycolytic flux with reduced lactate and citrate levels, upregulation of lipoprotein lipase mRNA, downregulation of PDK4 and peroxisomal bifunctional β-oxidation enzyme, as well as increased intramuscular TAG and DAG. This constellation of findings is most consistent with a model in which decreased glucose metabolism leads to compensatory responses, including an attempt to increase glucose oxidation via PDK4 dowregulation, increased fatty acid uptake into skeletal muscle, diminished peroxisomal β-oxidation of long-chain fatty acids, and subsequent accumulation of TAG and DAG, which, if any of these events contributed to impaired insulin signaling in Nur77 null mice, remains to be investigated.
Given that Nur77 deletion enhanced hepatic insulin sensitivity in diet-fed mice, it was somewhat surprising that Nur77 null mice challenged with a HFD actually developed exacerbated hepatic insulin sensitivity. This finding suggests that Nur77 deletion is insufficient to overcome the metabolic stress of lipid oversupply. It is conceivable that in response to muscle insulin resistance, nutritional or humoral factors could alter insulin action in other tissues, as secondary phenotypes were shown in muscle-specific GLUT4 knockout mice (41
). Alternatively, if the hepatic steatosis observed in Nur77 null mice is a primary result of hepatic Nur77 deficiency, lipotoxicity may contribute to hepatic insulin resistance. Based on findings by Pols et al. (43
) that Nur77 diminishes SREBP-1c activity, we investigated whether Nur77 directly suppresses hepatic lipogenesis. However, we were unable to demonstrate reduction of lipogenic gene expression when Nur77 was ectopically expressed in HepG2 cells and primary murine hepatocytes, nor when SREBP1c activity was measured in HEK293T cells, suggesting that the hepatic steatosis we observed may be secondary to the lipogenic effect of hyperinsulinemia.
One limitation of our analysis of the global Nur77 knockout mouse is the potential for additional metabolic effects of Nur77 in tissues not studied. We have determined that loss of hepatic Nur77 does not protect mice from diet-induced obesity and diabetes. However, the metabolic effect of Nur77 deletion on other tissues has not been explored. Our finding that Nur77 null mice have increased susceptibility for skeletal muscle insulin resistance is nevertheless consistent with the biologic pathways Nur77 regulates in muscle (17
) and illustrates specifically the importance of muscle Nur77 in the regulation of whole-body glucose metabolism. The generation of tissue-specific Nur77 transgenic and knockout mouse models will be necessary to delineate the physiologic roles of Nur77 in metabolism.