PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of diabetesSubscribeSearchDiabetes JournalAmerican Diabetes Association
 
Diabetes. 2009 December; 58(12): 2788–2796.
Published online 2009 September 9. doi:  10.2337/db09-0763
PMCID: PMC2780886

Insulin Resistance and Altered Systemic Glucose Metabolism in Mice Lacking Nur77

Abstract

OBJECTIVE

Nur77 is an orphan nuclear receptor with pleotropic functions. Previous studies have identified Nur77 as a transcriptional regulator of glucose utilization genes in skeletal muscle and gluconeogenesis in liver. However, the net functional impact of these pathways is unknown. To examine the consequence of Nur77 signaling for glucose metabolism in vivo, we challenged Nur77 null mice with high-fat feeding.

RESEARCH DESIGN AND METHODS

Wild-type and Nur77 null mice were fed a high-fat diet (60% calories from fat) for 3 months. We determined glucose tolerance, tissue-specific insulin sensitivity, oxygen consumption, muscle and liver lipid content, muscle insulin signaling, and expression of glucose and lipid metabolism genes.

RESULTS

Mice with genetic deletion of Nur77 exhibited increased susceptibility to diet-induced obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp studies revealed greater high-fat diet–induced insulin resistance in both skeletal muscle and liver of Nur77 null mice compared with controls. Loss of Nur77 expression in skeletal muscle impaired insulin signaling and markedly reduced GLUT4 protein expression. Muscles lacking Nur77 also exhibited increased triglyceride content and accumulation of multiple even-chained acylcarnitine species. In the liver, Nur77 deletion led to hepatic steatosis and enhanced expression of lipogenic genes, likely reflecting the lipogenic effect of hyperinsulinemia.

CONCLUSIONS

Collectively, these data demonstrate that loss of Nur77 influences systemic glucose metabolism and highlight the physiological contribution of muscle Nur77 to this regulatory pathway.

The NR4A family includes three highly homologous isotypes, NR4A1, NR4A2, and NR4A3, also known as Nur77, Nurr1, and Nor1, respectively (1). Although these receptors possess a putative ligand-binding domain, X-ray crystallography studies suggest that the ligand-binding pocket is occluded by bulky hydrophobic residues and is unable to accommodate ligands (2,3). Instead, NR4A activity is regulated primarily at the transcriptional level by stimuli that signal through the cAMP pathway as well as posttranslational modification. NR4A receptors have been implicated in a range of biological processes, including apoptosis, dopaminergic neuron development, and tumorigenesis (46).

Several members of the nuclear receptor superfamily function as downstream regulators of metabolic pathways in response to nutritional perturbations. In particular, the ligand-responsive nuclear receptors of the peroxisomal proliferator–activated receptors (PPARs) family, liver X receptors, and glucocorticoid receptor have been characterized as transcriptional coordinators of specific metabolic programs (710). In addition, ligand-independent orphan nuclear receptors, such as estrogen-related receptor-α, chicken ovalbumin upstream transcriptional factors, and retinoic acid receptor–related orphan receptors have also been implicated in metabolic regulation (1114). In recent years, the NR4A family of orphan nuclear receptors has also joined the cadre of nuclear receptors involved in metabolic regulation.

Work by our group and others have pointed to NR4A1 (Nur77) as a transcriptional regulator of glucose metabolism in liver and skeletal muscle (1,1517). Ectopic expression of Nur77 in vivo enhances hepatic glucose production and elevates plasma blood glucose (16). In skeletal muscle, Nur77 regulates the expression of a battery of glucose utilization genes, including GLUT4 and multiple genes involved in glycolysis (17). However, whether Nur77-mediated changes in the expression of muscle-glucose utilization genes are sufficient to modulate systemic glucose metabolism is unknown. Furthermore, the relative contribution of liver versus muscle Nur77 to maintaining systemic glucose homeostasis has not been explored previously.

Here, we show that mice lacking Nur77 develop hepatic steatosis and exacerbated insulin resistance in both skeletal muscle and liver when challenged with a high-fat diet (HFD). In addition to diminished expression of GLUT4 and glycolytic genes, Nur77 null mice demonstrated impaired muscle insulin signaling and increased intramuscular lipid content. Our findings highlight the importance of skeletal muscle Nur77 in the regulation of systemic glucose metabolism.

RESULTS

Nur77 null mice develop worsened glucose tolerance after high-fat feeding.

Prior work has shown that Nur77 regulates metabolic pathways that have opposing effects on peripheral glucose clearance. In the liver, Nur77 is a transcriptional modulator of gluconeogenesis (16). In skeletal muscle, Nur77 regulates the expression of a panel of genes that promote glucose utilization (17). To investigate the net functional outcome of Nur77 activity on systemic glucose metabolism, we performed metabolic analysis on Nur77 null mice. Global Nur77 knockout mice (4), devoid of Nur77 in both skeletal muscle and liver, exhibited no obvious difference in glucose tolerance when fed a standard rat diet (data not shown). To determine whether Nur77 affected the development of diet-induced obesity and diabetes, we challenged male control and Nur77 null mice with a HFD (60% calories from fat; Research Diets, New Brunswick, NJ) for a period of 3 months. As shown in Fig. 1A, Nur77 null mice exhibited slightly greater body weight at baseline and continued to gain more weight than wild-type controls over the course of the high-fat feeding, despite similar food intake (Fig. 1B). After 1 month of high-fat feeding, Nur77 null mice had worsened glucose tolerance compared with wild-type controls (Fig. 1C). In addition, Nur77 null mice displayed markedly higher fasting insulin levels (Fig. 1D), indicating that the glucose intolerance observed was attributable to insulin resistance rather than insulinopenia. This difference in fasting insulin level was reproduced in a separate cohort of high-fat–fed Nur77 null mice despite comparable body weight, adiposity, and adiponectin level (Fig. 1E, supplementary Table 1 and supplementary Fig. 1, available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0763/DC1), suggesting that the insulin resistance we observed was not entirely attributable to differences in adiposity.

FIG. 1.
Nur77 null mice develop diet-induced obesity and insulin resistance on a HFD. Body weight (A) and food intake (B) of wild-type (WT, ○) and Nur77 knockout (KO, ●) mice. C: Intraperitoneal glucose tolerance test (1 g/kg) performed after ...

Nur77 null mice have reduced oxygen consumption.

To determine the effect of Nur77 deletion on energy expenditure, we subjected high-fat–fed Nur77 knockout mice to indirect calorimetry. Mice were housed individually in the Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments, OH) and were fed ad libitum. As shown in Fig. 2A, Nur77 null mice showed reduced oxygen consumption (a measure of energy expenditure) during both light and dark cycles. This reduction in energy expenditure may contribute to the increased weight accretion in Nur77 null mice on a HFD, despite comparable ambulatory activities (Fig. 2B) and food intake. Although Nur77 regulates the expression of glycolytic genes in skeletal muscle, there was no significant effect of Nur77 deletion on substrate preference based on comparable respiratory exchange ratios between the genotypes, either during fasting or fed states (data not shown).

FIG. 2.
Indirect calorimetry of Nur77 null mice on HFD. Oxygen consumption (A) and ambulatory activity (B) of wild-type (WT) and Nur77 knockout (KO) mice after 3 months of high-fat feeding. Mice were fed ad libitum, male, n = 7–8. *P < 0.05.

High-fat feeding elicits insulin resistance in both skeletal muscle and liver of Nur77 null mice.

To determine tissue-specific contributions to the impaired insulin sensitivity of Nur77 null mice, we performed hyperinsulinemic-euglycemic clamps. In diet-fed animals, there was no difference in basal glucose turnover between wild-type and Nur77 null mice in the fasted state (Table 1 and Fig. 3B). Furthermore, Nur77 null and wild-type control mice had comparable insulin-stimulated glucose disposal rates (IS-GDR), reflecting similar skeletal muscle insulin sensitivity (Fig. 3A). In contrast, Nur77 null mice subjected to high-fat feeding exhibited marked reduction in IS-GDR, indicative of impaired skeletal muscle insulin sensitivity (Fig. 3C). Thus, these results highlight Nur77 as an important regulator of insulin sensitivity in skeletal muscle.

Table 1
Metabolic parameters
FIG. 3.
Muscle and liver insulin sensitivity of Nur77 null mice. A and B: IS-GDR and HGP of diet-fed wild-type (WT) and Nur77 knockout (KO) mice; male, n = 7–8. C and D: Basal state, white bars; postclamp, black bars. IS-GDR and HGP of mice on HFD for ...

Although muscle insulin sensitivity was unchanged in diet-fed Nur77 null mice, Nur77 deletion enhanced liver insulin sensitivity. As shown in Fig. 3B, insulin suppressed hepatic glucose production more effectively in diet-fed Nur77 knockout mice, reflecting enhanced hepatic insulin sensitivity. The increased hepatic insulin sensitivity is also supported by a higher glucose infusion rate required to maintain euglycemia during the clamp (Table 1). This result confirmed our previous finding that Nur77 regulates hepatic gluconeogenesis (16). Surprisingly, however, the protective effect of Nur77 on hepatic insulin sensitivity was not preserved during high-fat feeding, as Nur77 null mice showed a blunted suppression of hepatic glucose production (HGP) by insulin (Fig. 3D). When integrating both diet and genotype in two-way ANOVA, high-fat–fed Nur77 null livers actually exhibited worsened insulin sensitivity.

Impaired insulin receptor phosphorylation in Nur77 null mice.

We performed immunoblot analysis on quadriceps muscle from HFD fed mice to examine insulin-signaling pathways. Caveolin 3, a muscle-specific plasma membrane protein (18), was used as a loading control. As shown in Fig. 4A, GLUT4 protein level was markedly reduced in Nur77 null muscle, consistent with our previous finding that GLUT4 is a direct transcriptional target of Nur77 (17). We found that insulin receptor phosphorylation was reduced in Nur77 null muscle (Fig. 4A). Insulin receptor phosphorylation was unaffected in Nur77 null livers (supplementary Fig. 2). Both the reduced GLUT4 protein level and insulin receptor phosphorylation would be predicted to contribute to the observed impairment in muscle insulin sensitivity. Interestingly, Akt phosphorylation was not appreciably altered in Nur77 null mice. Although this result was unexpected, dissociation between insulin resistance and diminished Akt phosphorylation has been reported in several models of insulin resistance (1921). As well, the level of insulin-regulated aminopeptidase, a GLUT4 vesicle protein (22), was unchanged, thus confirming the specificity of Nur77 regarding genes of glucose utilization.

FIG. 4.
Effect of Nur77 on glycolysis and insulin signaling. A: Immunoblot of cell lysates prepared from high-fat–fed Nur77 null quadriceps muscle. B: Gene expression of wild-type (WT) and Nur77 knockout (KO) tibialis anterior muscle from high-fat–fed ...

Nur77 stimulates glycolysis in C2C12 muscle cells.

The observation that Nur77 null mice display impaired insulin-stimulated glucose disposal is concordant with our previous finding that Nur77 regulates the expression of glycolytic genes in skeletal muscle (17). Indeed, the expression of several glycolytic genes (enolase 3, 2,3-bisphosphoglycerate mutase, phosphoglycerate kinase 1) was reduced in tibialis anterior muscle of Nur77 null mice on a HFD (Fig. 4B). This defect would be expected to diminish insulin-stimulated glycolysis and glucose disposal. We found that the expression of lipin1 was reduced in Nur77 null muscle. Although the physiologic function of lipin1 in skeletal muscle remains unclear, our finding is consistent with the observation that muscle lipin1 expression is reduced in other models of insulin resistance (K. Reue, unpublished results).

At least in the Nur77 null model, regulation of glycolytic genes by Nur77 appears to be specific to skeletal muscle, as there was no appreciable loss of glycolytic gene expression in other metabolically active tissues such as brain and heart (supplementary Fig. 3), although we cannot exclude the potential compensatory actions of the remaining two NR4A receptors. In the liver, the expression of these enzymes was upregulated in high-fat–fed Nur77 null mice (supplementary Fig. 3). However, as most glycolytic enzymes also catalyze the reverse gluconeogenic reactions, the induction of these enzymes may reflect excessive gluconeogenesis observed in the high-fat–fed Nur77 knockout mice (Fig. 3D and supplementary Fig. 4). Interestingly, we also observed an increase in muscle Nur77 expression in response to HFD feeding (supplementary Fig. 5).

To test the hypothesis that Nur77-dependent regulation of glycolytic gene expression is sufficient to alter glycolytic activity, we expressed Nur77 in mouse C2C12 muscle cells with an adenoviral vector and measured in vivo glycolytic activity 3 days after infection. Glycolysis was measured by the Extracellular Flux Analyzer (Seahorse Bioscience, Chicopee, MA). Lactate accumulation during glycolysis lowers extracellular pH; extracellular acidification rates reflect glycolytic activity (23). Glycolytic activity can be further augmented by the addition of an uncoupling agent such as 2,4-dinitrophenol (DNP). As shown in Fig. 4C, Nur77-expressing cells exhibited enhanced glycolysis both at baseline and after DNP treatment. The specificity of DNP-stimulated glycolysis was confirmed by inhibiting glycolysis, using excess 2-deoxyglucose, which is phosphorylated by hexokinase but cannot be further metabolized in the glycolytic pathway. These results demonstrate that Nur77-driven changes in glycolytic gene expression increase cellular glycolytic function. Conversely, lactate and citrate content, two byproducts of glycolytic flux, was reduced in Nur77 null muscles (supplementary Fig. 6). These data support our hypothesis that impaired muscle insulin sensitivity in high-fat–fed Nur77 null mice is in part attributable to attenuated glycolysis.

Nur77 deletion increases intramuscular lipid content.

Increased intramuscular lipid content has been associated with development of skeletal muscle insulin resistance in human and rodent models (24,25). Recent evidence suggests that although intramuscular triglyceride content correlates with skeletal muscle insulin resistance, it is the proinflammatory lipid intermediates such as diacylglycerol (DAG) and ceramides that may contribute to lipid-induced insulin resistance (2629). We sought to determine whether impairment in muscle insulin sensitivity of high-fat–fed Nur77 null mice was associated with increased intramuscular lipid accumulation. Intramuscular triacylglycerol (TAG) and DAG content in tibialis anterior muscle of Nur77 null mice was increased, whereas ceramide level was unchanged (Fig. 5A). In addition, as shown in Fig. 5B, Nur77 null mice had increased accumulation of multiple even-chain acylcarnitine species, reflecting accumulation of the cognate acyl CoA species in muscle mitochondria. Possibly related to this finding, the expression of lipoprotein lipase was upregulated in Nur77 null mice (Fig. 5C), suggesting that fatty acid uptake was increased in Nur77 null muscle. The expression of PPARα and other mitochondrial fatty acid metabolism genes (including carnitine palmitoyltransferase 1b, long-chain acyl-CoA dehydrogenase, and medium-chain acyl-CoA dehydrogenase) was unchanged. Interestingly, the expression of peroxisomal bifunctional β-oxidation enzyme, 3-hydroxyacyl CoA dehydrogenase (Ehhadh), was reduced by 54% (P = 0.01) in Nur77 null muscle (Fig. 5C). The expression of peroxisomal acyl-CoA oxidase also tended to be lower in Nur77 null mice. The reduction in peroxisomal β-oxidation enzyme suggests that long-chain fatty acid oxidation (>C16) was impaired in Nur77 null mice. The expression of lipogenic enzymes was largely unchanged (supplementary Fig. 7). In addition, the expression of pyruvate dehydrogenase kinase, isozyme 4 (Pdk4), was reduced in Nur77 null muscle (Fig. 4A). Pdk4 inactivates the pyruvate dehydrogenase complex, limiting the flux of pyruvate through the Kreb cycle, effectively reducing carbohydrate oxidation and favoring lipid catabolism (30). The decrease in Pdk4 and the increase in lipoprotein lipase may represent attempts to compensate for reduced glucose uptake and glycolytic activity in Nur77 null muscle, as changes in expression of these genes might enhance glucose entry into the trichloroacetic acid cycle and fatty acid uptake into the cell to provide alternative sources of energy.

FIG. 5.
Intramuscular lipid content and expression of lipid metabolism genes in high-fat–fed Nur77 null mice. A: TAG, DAG, and ceramide concentrations from tibialis anterior muscle. B: Quantitation of fatty-acylcarnitine species in gastrocnemius muscle. ...

Hepatic steatosis in high-fat–fed Nur77 null mice.

In addition to increased intramuscular lipid content, high-fat–fed Nur77 null mice also developed hepatic steatosis (Fig. 6A). Quantitation of liver lipid content confirmed that liver from Nur77 null mice accumulated more triglyceride and cholesterol than wild-type mice (Fig. 6B). Not surprisingly, quantitative real-time PCR confirmed that the expression of lipogenic genes including sterol regulatory element binding protein (SREBP-1c), fatty acid synthase, and stearoyl-CoA desaturase 1 was increased ~twofold in Nur77 null livers. To investigate whether the hepatic steatosis observed in high-fat–fed Nur77 null mice could be linked to a direct effect of Nur77 on SREBP pathway, we overexpressed a dominant-negative form of Nur77 (Nur77-898) (16) using adenoviral vectors in HepG2 cells and primary murine hepatocytes. As shown in supplementary Fig. 8, Nur77 overexpression did not suppress SREBP-1c or stearoyl-CoA desaturase 1 expression. Likewise, Nur77 had no effect on lipogenic gene expression when the liver X receptor was activated by ligand GW3965 (1 μmol/l). To test the effect of Nur77 on SREBP-1c activity, we also performed transient transfection assays in HEK293T cells, using the rat fatty acid synthase promoter that contains the SREBP-1c binding site as the reporter. As shown in supplementary Fig. 8C, cotransfecting Nur77 had little effect on SREBP-1c activity with or without liver X receptors-α and retinoid X receptor-α. Collectively, our data suggest that the hepatic steatosis observed in high-fat–fed Nur77 null mice is not attributable to loss of Nur77 suppression of lipogenesis but rather the lipogenic effect of hyperinsulinemia.

FIG. 6.
Hepatic steatosis in high-fat–fed Nur77 null livers. A: Hematoxylin-eosin stain of wild-type (WT) and Nur77 knockout (KO) liver section. B: Liver lipid content. C: Lipogenic gene expression in liver. *P < 0.05, **P < 0.01. (A high-quality ...

DISCUSSION

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 (Fig. 7). 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 ...

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 (31,32). 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 (35,36). 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 (41,42). 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.

RESEARCH DESIGN AND METHODS

Animal husbandry.

Mice were fed ad libitum and maintained on a 12-h light-dark cycle and were age and sex matched for all experiments. Intraperitoneal glucose tolerance test was performed after a 6-h fast by intraperitoneal injection of 1 g/kg of dextrose. Animal studies were performed in accordance with University of California at Los Angeles animal research committees guidelines.

Tissue culture.

Care of C2C12 cells, HEK293T cells, and primary murine hepatocytes has been previously described (16,17). HepG2 cells (ATCC, Rockville, MD) were maintained in 10% FBS/DMEM and incubated with 5% CO2.

Plasma and tissue chemistry.

Measurement of fasting mouse insulin, free fatty acid, triglyceride concentrations, and liver lipids were as described previously (44,45). Insulin during clamp experiments was determined by Linco-Millipore RIA kit (St. Charles, MO). Adiponectin concentration was measured by ELISA kit from B-Bridge International (Mountain View, CA). Concentrations of adipokines listed in supplementary Table 1 were measured by the Mouse Serum Adipokine Panel–7-plex kit (Millipore, MA). Total cholesterol was determined by Cholesterol E kit (Wako, VA). Muscle lipids, including DAG and ceramide, were extracted from skeletal muscle and assessed as previously described (46). TAGs were saponified, and glycerol was assessed (Free Glycerol Reagent; Sigma-Aldrich, MO). Determination of fatty-acylcarnitine and organic acid concentrations was described previously (39).

Indirect calorimetry.

Mice were individually housed in the CLAMS and acclimated for 7 h in the cages. Data were collected for the next 48 h, spanning 2 day and 2 night periods. Effective mass coefficient was set at 0.75. Ambulatory activity level was measured as interruption of dual-axis infrared beams. Data were analyzed by Oxymax software (Columbus Instruments, OH).

Body composition.

Body composition was determined by dual-energy X-ray absorptiometry densitometry (PIXImus, Madison, WI).

Insulin sensitivity in vivo.

Hyperinsulinemic-euglycemic clamps using an insulin infusion of 12 mU · kg−1 · min−1 were performed as described previously (47). Differences in IS-GDR and HGP between treatment groups were detected using ANOVA, and significance was set a priori at P < 0.05.

Quantitative real-time PCR.

Total RNA preparation and quantitative real-time PCR were performed as described (17). Expression was normalized to 36B4 expression. Primer sequences are listed in supplementary Table 2 and as previously described (16,17,48).

In vitro glycolysis.

C2C12 myoblasts were plated in V7 plates (Seahorse Biosciences) 1 day before adenovirus infection. Adenovirus infection was performed as described (17). On the day of assay, media were refreshed with freshly made unbuffered Delbecco' modified Eagle's medium (plus glutamine 2 mmol/l, sodium pyruvate 1 mmol/l, glucose 25 mmol/l, NaCl 31.7 mmol/l, Phenol Red 15 mg/l, and pH 7.4); 2,4-dinitrophenol 1 mmol/l and 2-deoxyglucose 200 mmol/l were made in the same media. The XF24 Extracellular Flux Analyzer (Seahorse Bioscience) (49) was set up with measure:mix:wait cycles of 3:2:3 min. Protein quantitation was performed using the DC Protein Assay (BioRad, Richmond, CA).

Immunoblot.

Monoclonal anti-GLUT4 antibody 1F8 29 was made in the Pilch lab, and affinity purified anti–insulin-regulated aminopeptidase polyclonal antibody was made by 21st Century Biochemical (Hopkinton, MA). Phospho-insulin receptor antibody was purchased from Cell Signaling (#3021, Beverly, MA). Other antibodies used were described previously (16,17,50). Mouse quadriceps muscle and liver were isolated and flash frozen. Tissue lysate and immunoblots were prepared as described (17).

Histology.

Liver sections were fixed in 10% formalin and stained with hematoxylin and eosin by UCLA Translational Pathology Core Laboratory.

Statistics.

Statistical analysis was performed by Student t test unless otherwise noted.

Supplementary Material

Online-Only Appendix:

Acknowledgments

L.C.C. was supported by the National Institutes of Health Grants HD-00850 and DK-081683-01. P.F.P. was supported by the National Institutes of Health Grants DK-30425 and the American Diabetes Association. P.T. was supported by the National Institutes of Health Grant HL030568 and is an investigator of the Howard Hughes Medical Institute.

No potential conflicts of interest relevant to this article were reported.

This work was presented at the 2009 Deuel Conference on Lipids, La Casa Del Zorro, Borrego Springs, California, 3–6 March 2009.

We thank Drs. P. Cohen and J. Milbrandt for the Nur77 null mice and Drs. K.W. Park and C. Villanueva for helpful discussions.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

1. Maxwell MA, Cleasby ME, Harding A, Stark A, Cooney GJ, Muscat GE.: Nur77 regulates lipolysis in skeletal muscle cells. Evidence for cross-talk between the β-adrenergic and an orphan nuclear hormone receptor pathway. J Biol Chem 2005; 280: 12573– 12584. [PubMed]
2. Flaig R, Greschik H, Peluso-Iltis C, Moras D.: Structural basis for the cell-specific activities of the NGFI-B and the Nurr1 ligand-binding domain. J Biol Chem 2005; 280: 19250– 19258. [PubMed]
3. Wang Z, Benoit G, Liu J, Prasad S, Aarnisalo P, Liu X, Xu H, Walker NP, Perlmann T.: Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors. Nature 2003; 423: 555– 560. [PubMed]
4. Lee SL, Wesselschmidt RL, Linette GP, Kanagawa O, Russell JH, Milbrandt J.: Unimpaired thymic and peripheral T cell death in mice lacking the nuclear receptor NGFI-B (Nur77). Science 1995; 269: 532– 535. [PubMed]
5. Mullican SE, Zhang S, Konopleva M, Ruvolo V, Andreeff M, Milbrandt J, Conneely OM.: Abrogation of nuclear receptors Nr4a3 and Nr4a1 leads to development of acute myeloid leukemia. Nat Med 2007; 13: 730– 735. [PubMed]
6. Saucedo-Cardenas O, Quintana-Hau JD, Le WD, Smidt MP, Cox JJ, De Mayo F, Burbach JP, Conneely OM.: Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Natl Acad Sci U S A 1998; 95: 4013– 4018. [PubMed]
7. Sugden MC, Holness MJ.: Role of nuclear receptors in the modulation of insulin secretion in lipid-induced insulin resistance. Biochem Soc Trans 2008; 036: 891– 900. [PubMed]
8. Juurinen L, Kotronen A, Graner M, Yki-Jarvinen H.: Rosiglitazone reduces liver fat and insulin requirements and improves hepatic insulin sensitivity and glycemic control in patients with type 2 diabetes requiring high insulin doses. J Clin Endocrinol Metab 2008; 93: 118– 124. [PubMed]
9. Laffitte BA, Chao LC, Li J, Walczak R, Hummasti S, Joseph SB, Castrillo A, Wilpitz DC, Mangelsdorf DJ, Collins JL, Saez E, Tontonoz P.: Activation of liver X receptor improves glucose tolerance through coordinate regulation of glucose metabolism in liver and adipose tissue. Proc Natl Acad Sci U S A 2003; 100: 5419– 5424. [PubMed]
10. Tomlinson JW, Stewart PM.: Modulation of glucocorticoid action and the treatment of type-2 diabetes. Best Pract Res Clin Endocrinol Metab 2007; 21: 607– 619. [PubMed]
11. Achatz G, Holzl B, Speckmayer R, Hauser C, Sandhofer F, Paulweber B.: Functional domains of the human orphan receptor ARP-1/COUP-TFII involved in active repression and transrepression. Mol Cell Biol 1997; 17: 4914– 4932. [PMC free article] [PubMed]
12. Lau P, Fitzsimmons RL, Raichur S, Wang SC, Lechtken A, Muscat GE.: The orphan nuclear receptor, RORα, regulates gene expression that controls lipid metabolism: staggerer (SG/SG) mice are resistant to diet-induced obesity. J Biol Chem 2008; 283: 18411– 18421. [PubMed]
13. Villena JA, Kralli A.: ERR[α]: a metabolic function for the oldest orphan. Trends Endocrin Metab 2008; 19: 269– 276. [PMC free article] [PubMed]
14. Myers SA, Wang SCM, Muscat GEO.: The chicken ovalbumin upstream promoter-transcription factors modulate genes and pathways involved in skeletal muscle cell metabolism. J Biol Chem 2006; 281: 24149– 24160. [PubMed]
15. Pearen MA, Ryall JG, Maxwell MA, Ohkura N, Lynch GS, Muscat GE.: The orphan nuclear receptor, NOR-1, is a target of β-adrenergic signaling in skeletal muscle. Endocrinology 2006; 147: 5217– 5227. [PubMed]
16. Pei L, Waki H, Vaitheesvaran B, Wilpitz DC, Kurland IJ, Tontonoz P.: NR4A orphan nuclear receptors are transcriptional regulators of hepatic glucose metabolism. Nat Med 2006; 12: 1048– 1055. [PubMed]
17. Chao LC, Zhang Z, Pei L, Saito T, Tontonoz P, Pilch PF.: Nur77 coordinately regulates expression of genes linked to glucose metabolism in skeletal muscle. Mol Endocrinol 2007; 21: 2152– 2163. [PMC free article] [PubMed]
18. Parton RG, Simons K.: The multiple faces of caveolae. Nat Rev Mol Cell Biol 2007; 8: 185– 194. [PubMed]
19. Kruszynska YT, Worrall DS, Ofrecio J, Frias JP, Macaraeg G, Olefsky JM.: Fatty acid-induced insulin resistance: decreased muscle PI3K activation but unchanged Akt phosphorylation. J Clin Endocrinol Metab 2002; 87: 226– 234. [PubMed]
20. Kim YB, Zhu JS, Zierath JR, Shen HQ, Baron AD, Kahn BB.: Glucosamine infusion in rats rapidly impairs insulin stimulation of phosphoinositide 3-kinase but does not alter activation of Akt/protein kinase B in skeletal muscle. Diabetes 1999; 48: 310– 320. [PubMed]
21. Storgaard H, Jensen CB, Bjornholm M, Song XM, Madsbad S, Zierath JR, Vaag AA.: Dissociation between fat-induced in vivo insulin resistance and proximal insulin signaling in skeletal muscle in men at risk for type 2 diabetes. J Clin Endocrinol Metab 2004; 89: 1301– 1311. [PubMed]
22. Hosaka T, Brooks CC, Presman E, Kim SK, Zhang Z, Breen M, Gross DN, Sztul E, Pilch PF.: p115 Interacts with the GLUT4 vesicle protein, IRAP, and plays a critical role in insulin-stimulated GLUT4 translocation. Mol Biol Cell 2005; 16: 2882– 2890. [PMC free article] [PubMed]
23. Ferrick DA, Neilson A, Beeson C.: Advances in measuring cellular bioenergetics using extracellular flux. Drug Discov Today 2008; 13: 268– 274. [PubMed]
24. Sinha R, Dufour S, Petersen KF, LeBon V, Enoksson S, Ma Y-Z, Savoye M, Rothman DL, Shulman GI, Caprio S.: Assessment of skeletal muscle triglyceride content by 1H nuclear magnetic resonance spectroscopy in lean and obese adolescents: relationships to insulin sensitivity, total body fat, and central adiposity. Diabetes 2002; 51: 1022– 1027. [PubMed]
25. Manco M, Mingrone G, Greco AV, Capristo E, Gniuli D, De Gaetano A, Gasbarrini G.: Insulin resistance directly correlates with increased saturated fatty acids in skeletal muscle triglycerides. Metabolism 2000; 49: 220– 224. [PubMed]
26. Liu L, Zhang Y, Chen N, Shi X, Tsang B, Yu YH.: Upregulation of myocellular DGAT1 augments triglyceride synthesis in skeletal muscle and protects against fat-induced insulin resistance. J Clin Invest 2007; 117: 1679– 1689. [PMC free article] [PubMed]
27. Timmers S, Schrauwen P, de Vogel J.: Muscular diacylglycerol metabolism and insulin resistance. Physiol Behav 2008; 94: 242– 251. [PubMed]
28. Levin MC, Monetti M, Watt MJ, Sajan MP, Stevens RD, Bain JR, Newgard CB, Farese RV, Sr, Farese RV., Jr: Increased lipid accumulation and insulin resistance in transgenic mice expressing DGAT2 in glycolytic (type II) muscle. Am J Physiol Endocrinol Metab 2007; 293: E1772– E1781. [PubMed]
29. Turinsky J, O'Sullivan DM, Bayly BP.: 1,2-diacylglycerol and ceramide levels in insulin-resistant tissues of the rat in vivo. J Biol Chem 1990; 265: 16880– 16885. [PubMed]
30. Sugden MC, Holness MJ.: Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases. Arch Physiol Biochem 2006; 112: 139– 149. [PubMed]
31. Hickey MS, Carey JO, Azevedo JL, Houmard JA, Pories WJ, Israel RG, Dohm GL.: Skeletal muscle fiber composition is related to adiposity and in vitro glucose transport rate in humans. Am J Physiol 1995; 268: E453– E457. [PubMed]
32. Oberbach A, Bossenz Y, Lehmann S, Niebauer J, Adams V, Paschke R, Schon MR, Bluher M, Punkt K.: Altered fiber distribution and fiber-specific glycolytic and oxidative enzyme activity in skeletal muscle of patients with type 2 diabetes. Diabetes Care 2006; 29: 895– 900. [PubMed]
33. Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, Bayuga-Ocampo CR, Ham J, Kang H, Evans RM.: Regulation of muscle fiber type and running endurance by PPARΔ. PLoS Biol 2004; 2: e294. [PMC free article] [PubMed]
34. Izumiya Y, Hopkins T, Morris C, Sato K, Zeng L, Viereck J, Hamilton JA, Ouchi N, LeBrasseur NK, Walsh K.: Fast/glycolytic muscle fiber growth reduces fat mass and improves metabolic parameters in obese mice. Cell Metab 2008; 7: 159– 172. [PMC free article] [PubMed]
35. Morino K, Petersen KF, Shulman GI.: Molecular mechanisms of insulin resistance in humans and their potential links with mitochondrial dysfunction. Diabetes 2006; 55( Suppl. 2): S9– S15. [PMC free article] [PubMed]
36. Yu C, Chen Y, Cline GW, Zhang D, Zong H, Wang Y, Bergeron R, Kim JK, Cushman SW, Cooney GJ, Atcheson B, White MF, Kraegen EW, Shulman GI.: Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem 2002; 277: 50230– 50236. [PubMed]
37. Choi CS, Savage DB, Abu-Elheiga L, Liu ZX, Kim S, Kulkarni A, Distefano A, Hwang YJ, Reznick RM, Codella R, Zhang D, Cline GW, Wakil SJ, Shulman GI.: Continuous fat oxidation in acetyl-CoA carboxylase 2 knockout mice increases total energy expenditure, reduces fat mass, and improves insulin sensitivity. Proc Natl Acad Sci U S A 2007; 104: 16480– 16485. [PubMed]
38. Koves TR, Li P, An J, Akimoto T, Slentz D, Ilkayeva O, Dohm GL, Yan Z, Newgard CB, Muoio DM.: Peroxisome proliferator-activated receptor-{γ} co-activator 1{α}-mediated metabolic remodeling of skeletal myocytes mimics exercise training and reverses lipid-induced mitochondrial inefficiency. J Biol Chem 2005; 280: 33588– 33598. [PubMed]
39. Koves TR, Ussher JR, Noland RC, Slentz D, Mosedale M, Ilkayeva O, Bain J, Stevens R, Dyck JR, Newgard CB, Lopaschuk GD, Muoio DM.: Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance. Cell Metab 2008; 7: 45– 56. [PubMed]
40. Finck BN, Bernal-Mizrachi C, Han DH, Coleman T, Sambandam N, LaRiviere LL, Holloszy JO, Semenkovich CF, Kelly DP.: A potential link between muscle peroxisome proliferator- activated receptor-α signaling and obesity-related diabetes. Cell Metab 2005; 1: 133– 144. [PubMed]
41. Kim YB, Peroni OD, Aschenbach WG, Minokoshi Y, Kotani K, Zisman A, Kahn CR, Goodyear LJ, Kahn BB.: Muscle-specific deletion of the GLUT4 glucose transporter alters multiple regulatory steps in glycogen metabolism. Mol Cell Biol 2005; 25: 9713– 9723. [PMC free article] [PubMed]
42. Herman MA, Kahn BB.: Glucose transport and sensing in the maintenance of glucose homeostasis and metabolic harmony. J Clin Invest 2006; 116: 1767– 1775. [PMC free article] [PubMed]
43. Pols TW, Ottenhoff R, Vos M, Levels JH, Quax PH, Meijers JC, Pannekoek H, Groen AK, de Vries CJ.: Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity. Biochem Biophys Res Commun 2008; 366: 910– 916. [PubMed]
44. Bradley MN, Hong C, Chen M, Joseph SB, Wilpitz DC, Wang X, Lusis AJ, Collins A, Hseuh WA, Collins JL, Tangirala RK, Tontonoz P.: Ligand activation of LXR β reverses atherosclerosis and cellular cholesterol overload in mice lacking LXR α and apoE. J Clin Invest 2007; 117: 2337– 2346. [PMC free article] [PubMed]
45. Waki H, Park KW, Mitro N, Pei L, Damoiseaux R, Wilpitz DC, Reue K, Saez E, Tontonoz P.: The small molecule harmine is an antidiabetic cell-type-specific regulator of PPARγ expression. Cell Metab 2007; 5: 357– 370. [PubMed]
46. Watt MJ, Dzamko N, Thomas WG, Rose-John S, Ernst M, Carling D, Kemp BE, Febbraio MA, Steinberg GR.: CNTF reverses obesity-induced insulin resistance by activating skeletal muscle AMPK. Nat Med 2006; 12: 541– 548. [PubMed]
47. Hevener AL, Olefsky JM, Reichart D, Nguyen MT, Bandyopadyhay G, Leung HY, Watt MJ, Benner C, Febbraio MA, Nguyen AK, Folian B, Subramaniam S, Gonzalez FJ, Glass CK, Ricote M.: Macrophage PPARγ is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones. J Clin Invest 2007; 117: 1658– 1669. [PMC free article] [PubMed]
48. Yang J, Goldstein JL, Hammer RE, Moon YA, Brown MS, Horton JD.: Decreased lipid synthesis in livers of mice with disrupted Site-1 protease gene. Proc Natl Acad Sci U S A 2001; 98: 13607– 13612. [PubMed]
49. Wu M, Neilson A, Swift AL, Moran R, Tamagnine J, Parslow D, Armistead S, Lemire K, Orrell J, Teich J, Chomicz S, Ferrick DA.: Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am J Physiol Cell Physiol 2007; 292: C125– C136. [PubMed]
50. Liu L, Brown D, McKee M, Lebrasseur NK, Yang D, Albrecht KH, Ravid K, Pilch PF.: Deletion of Cavin/PTRF causes global loss of caveolae, dyslipidemia, and glucose intolerance. Cell Metab 2008; 8: 310– 317. [PMC free article] [PubMed]

Articles from Diabetes are provided here courtesy of American Diabetes Association