Overexpression of the orphan receptor Nur77 alters glucose metabolism in rat muscle cells and rat muscle in vivo
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- Kanzleiter, T., Preston, E., Wilks, D. et al. Diabetologia (2010) 53: 1174. doi:10.1007/s00125-010-1703-2
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A hallmark feature of the metabolic syndrome is abnormal glucose metabolism which can be improved by exercise. Recently the orphan nuclear receptor subfamily 4, group A, member 1 (NUR77) was found to be induced by exercise in muscle and was linked to transcriptional control of genes involved in lipid and glucose metabolism. Here we investigated if overexpression of Nur77 (also known as Nr4a1) in skeletal muscle has functional consequences for lipid and/or glucose metabolism.
L6 rat skeletal muscle myotubes were infected with a Nur77-coding adenovirus and lipid and glucose oxidation was measured. Nur77 was also overexpressed in skeletal muscle of chow- and fat-fed rats and the effects on glucose and lipid metabolism evaluated.
Nur77 overexpression had no effect on lipid oxidation in L6 cells or rat muscle, but did increase glucose oxidation and glycogen synthesis in L6 cells. In chow- and high-fat-fed rats, Nur77 overexpression by electrotransfer significantly increased basal glucose uptake and glycogen synthesis, but no increase in insulin-stimulated glucose metabolism was observed. Nur77 electrotransfer was associated with increased production of GLUT4 and glycogenin and increased hexokinase and phosphofructokinase activity. Interestingly, Nur77 expression in muscle biopsies from obese men was significantly lower than in those from lean men and was closely correlated with body-fat content and insulin sensitivity.
Our data provide compelling evidence that NUR77 is a functional regulator of glucose metabolism in skeletal muscle in vivo. Importantly, the diminished content in muscle of obese insulin-resistant men suggests that it might be a potential therapeutic target for the treatment of dysregulated glucose metabolism.
KeywordsElectrotransferGlucose metabolismInsulin resistanceNur77ObesitySkeletal muscle
Extensor digitorum longus
Green fluorescent protein
Nuclear receptor subfamily 4, group A
Nuclear receptor subfamily 4, group A, member 1
In the last two decades there has been a dramatic increase in the prevalence of obesity, such that it now represents one of the most significant health concerns worldwide . Obesity can give rise to a multitude of pathological conditions commonly referred to as the metabolic syndrome. One possible outcome of this rapid increase in obesity-related diseases is that children born early in the 21st century will have a lower life expectancy than their parents . One of the most common diseases related to obesity is type 2 diabetes, the prevalence of which is also increasing at an alarming rate . The key metabolic defect predisposing to the development of type 2 diabetes is insulin resistance, which is the relative failure of insulin to exert its multiple biological effects on carbohydrate and lipid metabolism . Insulin resistance is also an underlying defect common to several other components of the metabolic syndrome, including obesity, hypertension, dyslipidaemias and cardiovascular disease. Therefore, unravelling the complex molecular mechanisms responsible for the development of insulin resistance represents a major priority in the fight against the rising prevalence of type 2 diabetes and other metabolic disorders.
Skeletal muscle represents approximately 40% of body mass and accounts for 70–80% of postprandial insulin-stimulated glucose uptake [5, 6]. Defects in insulin-stimulated glucose uptake and glycogen synthesis in muscle have been suggested to be important early events in the pathogenesis of insulin resistance . It is also well established that regular physical exercise improves insulin action in skeletal muscle [7, 8]. The beneficial effects of exercise include acute events regulating signalling pathways to increase glucose metabolism, as well as chronic adaptations of skeletal muscle involving coordinated expression of glucose metabolic genes [9, 10].
Recently, it was reported that the expression of Nur77 (also known as Nr4a1) was induced in human and rodent skeletal muscle following exercise [11, 12]. We have also shown in skeletal muscle cells that the expression of Nur77 can be induced by two exercise-activated pathways, beta-adrenergic stimulation and calcium influx . Furthermore, Nur77 expression is reduced in animal models of obesity and insulin resistance, where responsiveness to beta-adrenergic stimulation is diminished [13, 14]. Nuclear receptor subfamily 4, group A, member 1 (NUR77) has been shown to be a transcriptional regulator of genes linked to glucose as well as lipid metabolism in muscle [15, 16]; however, little is known regarding the functional consequences of altered NUR77 production in skeletal muscle on glucose and/or lipid metabolism. The aim of this study was to therefore investigate the effect of Nur77 overexpression on lipid and glucose metabolism in skeletal muscle.
L6 rat skeletal muscle myoblasts were cultured in six well plates and maintained in alpha-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with antibiotics and 10% (vol./vol.) FCS (Sigma-Aldrich, St Louis, MO, USA). At 70–80% confluence the medium was changed to alpha-MEM supplemented with 2% (vol./vol.) FCS to initiate differentiation. At 7 days post-differentiation cells were incubated for 24 h with adenoviruses (5 × 106 plaque-forming units [PFU]/ml) coding for NUR77 or green fluorescent protein (GFP) as control (generously provided by S. Tetradis, UCLA School of Dentistry, Los Angeles, CA, USA) and used for experiments 48 h after infection.
Male Wistar rats weighing approximately 250 g were purchased from the Animal Resources Centre (Perth, WA, Australia). The animals were kept in a temperature-controlled room (22 ± 1°C) on a 12 h light/dark cycle with free access to food and water. Rats were fed ad libitum for a period of 3 weeks with a standard laboratory diet or a high-fat diet as previously described . All experiments were carried out with the approval of the Garvan Institute/St Vincent's Hospital Animal Experimentation Ethics Committee, following guidelines issued by the National Health and Medical Research Council of Australia.
In vivo electroporation and euglycaemic–hyperinsulinaemic clamp studies with glucose tracers were performed as described previously [18, 19]. For the in vivo electroporation experiments, Nur77 was expressed in the right tibialis anterior (TA) and extensor digitorum longus (EDL) muscle and empty vector (pCDNA3.1, Invitrogen) was expressed in the left leg as an internal control. One week before the tracer studies, the jugular vein and carotid artery of rats designated to undergo clamp studies, or the jugular vein alone for rats designated to undergo basal tracer uptake studies, were cannulated. Rats were singly housed and handled daily for the following week to minimise stress. Body weight was recorded daily, and only rats that had fully recovered to their pre-surgery weight were subsequently studied. Animals were food restricted overnight (65% of normal intake) before the tracer experiments were performed. At the end of each study, rats were killed by intravenous injection of pentobarbitone sodium (Nembutal; Abbott Laboratories, Sydney, NSW, Australia) and muscles rapidly dissected and freeze-clamped using liquid nitrogen-cooled tongs. Plasma tracer disappearance curves and the counts of phosphorylated 2-deoxy-[3H]glucose and [14C]glucose in glycogen extracts from individual muscles were used to calculate tissue-specific glucose uptake and glycogen synthesis, respectively .
Sedentary, non-smoking, non-diabetic obese and lean men (age 21–49 years) were recruited. The study protocol was approved by the Human Research Ethics Committee at St Vincent’s Hospital, Sydney, and participants provided informed written consent. Participants were tested after a 10 h overnight fast. Weight was measured in light clothing and height was assessed by a stadiometer. Following this, a vastus lateralis muscle biopsy was performed and the samples were blotted for blood and any visible fat was removed before they were snap frozen at −80°C. A standard 120 min hyperinsulinaemic–euglycaemic clamp was then performed with indirect calorimetry (Deltatrac; Datex, Helsinki, Finland) according to previously described studies . Immediately following the clamp body fat was measured by dual-energy X-ray absorptiometry (Lunar DPX-Lunar Radiation, Madison, WI, USA).
RNA isolation and quantitative real-time PCR
For cells and tissue samples RNA was isolated according to the manufacturer's instructions using TRI-reagent (Sigma-Aldrich). RNA integrity was evaluated on agarose gels and concentration was determined spectrophotometrically using a Nanodrop spectrophotometer (Biolab, Scoresby, VIC, Australia). RNA was converted into cDNA using random primers (New England Biolabs, Ipswich, MA, USA) and the cDNA synthesis kit from Qiagen (Mississauga, ON, USA). Taqman assays for Nur77 (Rn00577766_m1, Hs00374230_m1; Applied Biosystems, Foster City, CA, USA) and Universal Probe Library assays (Roche Diagnostics, Indianapolis, IN, USA) for glycogenin (5′-tcttgtggcttctgtagaaagga-3′; 5′-gaggatacaaacggctgtgc-3′) and Glut4 (also known as Slc2a4) (5′-ctgtgccatcctgatgactg-3′; 5′-cgtagctcatggctggaact-3′) were employed to quantify mRNA expression. PCR runs were performed on an ABI 9600HT cycler (Applied Biosystems, Foster City, CA, USA) or Roche LC480 (Roche Diagnostics, Indianapolis, IN, USA). A standard curve, consisting of serial dilutions of pooled cDNA samples, was measured with every PCR run to quantify the respective transcripts. Expression data for Nur77, glycogenin and Glut4 were normalised to 18S rRNA expression.
Measurement of fatty acid and glucose oxidation
For fatty acid oxidation, L6 cells transfected with adenovirus coding for NUR77 or GFP were incubated with alpha-MEM medium containing 0.5 mmol/l oleate (Sigma-Aldrich) coupled to 2% BSA (wt/vol.; Sigma-Aldrich) and [1-14C]oleate (GE Healthcare, Rydalmere, NSW, Australia) at a final concentration of 37 kBq/ml. After 1 h of incubation at 37°C in a sealed plate the medium was transferred to a sealed glass vial, acidified with 1 mol/l perchloric acid. Liberated 14CO2 was captured in 1 mol/l NaOH and measured by liquid scintillation counting. To determine the 14C counts present in the acid-soluble metabolites we performed a ‘Folch extraction’ of the cells, measured the radioactivity in the aqueous phase and combined these values with the 14CO2 values to give the total fatty acid oxidation rate. Fatty acid oxidation in EDL strips was conducted as described in detail elsewhere .
Glucose oxidation and glycogen synthesis rates in Nur77- and Gfp-transfected L6 cells were assessed at 37°C by incubating cells for 1 h in alpha-MEM containing 37 kBq/ml [U-14C]glucose (GE Healthcare) and, where indicated, 100 nmol/l insulin. At the completion of the incubation the medium was transferred to a sealed glass vial and acidified with 1 mol/l perchloric acid. Liberated 14CO2 was captured in 1 mol/l NaOH and measured by liquid scintillation counting. Glycogen was extracted from the cells after dissolving tissue with 1 mol/l KOH and precipitating glycogen with 95% (vol./vol.) ethanol. [14C]Glucose incorporation into precipitated glycogen was measured by liquid scintillation counting. Furthermore, the total glycogen amount was determined after digestion with amyloglucosidase and colorimetric determination of glucose content.
Frozen muscle tissue was powdered and solubilised in radioimmunoprecipitation assay (RIPA) buffer supplemented with complete protease inhibitor cocktail and PhosStop phosphatase inhibitor (Roche Diagnostics, Indianapolis, IN, USA). Protein concentration was determined using the method of Bradford (BioRad Laboratories, Gladesville, NSW, Australia). Protein, 50 µg, was diluted in Laemmli SDS buffer and separated on an SDS gel. After transfer to a polyvinylidene fluoride (PVDF) membrane, antibodies against GLUT1, GLUT4 (both generously provided by D. James, Garvan Institute, Sydney, NSW, Australia), glycogen synthase (kindly provided by J. Lawrence, University of Virginia, Charlottesville, VA, USA) and glycogenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for the immunodetection of respective proteins.
Measurements of hexokinase and phosphofructokinase activity in homogenates of electroporated tibialis muscles were performed as described in detail elsewhere .
Values are given as mean ± SEM. Significance was tested for by Student’s t test if comparisons between two groups were made or two-way ANOVA followed by the Holm–Sidak post hoc test if multiple treatments were compared. Where appropriate, paired t tests or ANOVA on repeated measures were used. Calculations were performed with SigmaStat 3.5 or Graphpad Prism 5, and p values less than 0.05 were considered to be significant.
Effect of Nur77 on lipid and glucose metabolism
Effect of NUR77 on glucose uptake and glucose incorporation into glycogen in vivo
Considering the impressive changes observed under basal conditions, we next measured the effect of Nur77 overexpression on glucose metabolism in muscle under insulin-stimulated euglycaemic conditions (∼5 mmol/l blood glucose and 4 mU kg−1 min−1 insulin infusion). Insulin-stimulated glucose uptake was slightly elevated in muscle overexpressing Nur77 compared with the control muscle, but this failed to reach statistical significance (Fig. 3a). Glucose incorporation into glycogen was also not different between Nur77-overexpressing and control muscles following insulin stimulation (Fig 3b).
Consequences of Nur77 overexpression on glucose metabolism in insulin-resistant rats
Physiological characteristics of rats undergoing glucose tracer studies
Basal animals (n = 9)
Clamp animals (n = 8)
Basal animals (n = 9)
Clamp animals (n = 7)
Body mass (g)
284.2 ± 5.9
266.8 ± 4.7
345.0 ± 10.9***
315.3 ± 6.3***
Fasting blood glucose (mmol/l)
4.51 ± 0.1
4.50 ± 0.15
4.61 ± 0.1
4.94 ± 0.25
Glucose infusion rate (mg kg−1 min−1)
31.6 ± 1.1
23.1 ± 1.94***
Plasma insulin (pg/ml)
1,418 ± 220
Pre: 950 ± 139
2,116 ± 122**
Pre: 1,729 ± 578
Post: 4,085 ± 420
Post: 3,913 ± 160
Downstream targets of NUR77
In order to investigate the mechanisms that might underpin the observed increase in glucose uptake under basal conditions, we examined known and putative downstream targets of NUR77. Glut4 expression has been reported to be regulated by NUR77  and analysis of several promoter regions of genes related to glucose metabolism revealed the sequence of putative NUR77 response elements in the promoter of glycogen synthase and glycogenin.
Nur77 expression in skeletal muscle is reduced in obese humans
The nuclear orphan receptor NUR77 has been reported to regulate the transcription of genes linked to lipid and glucose metabolism in skeletal muscle cells [15, 16]. This leads to speculation that this transcription factor may be important in regulating substrate metabolism in the context of insulin resistance and obesity. We recently showed  that two exercise-activated pathways increase Nur77 expression in a skeletal muscle cell line and other studies have reported that physical exercise in humans and rodents increases Nur77 expression in muscle [11, 12]. Therefore, NUR77 may be an important link between the transcriptional regulation of lipid and glucose metabolism and physical exercise, which is known to improve substrate metabolism in insulin-resistant animals and humans [8, 26, 27]. A caveat to many of the previous studies is that the conclusions have been largely based on NUR77-mediated changes in gene expression. However, whether changes in NUR77 impact functional measures of glucose and lipid metabolism in vitro or in vivo has not previously been assessed.
In this study, we overexpressed Nur77 in L6 myotubes and in rat skeletal muscle and measured the effect on lipid and glucose oxidation in vitro. The magnitude of increase in Nur77 expression achieved is relatively similar to that reported following adrenergic stimulation of both muscle cells and skeletal muscle in vivo [13, 16, 28]. Nur77 overexpression did not significantly change lipid oxidation, whereas two endpoints of glucose metabolism (glucose oxidation and glycogen synthesis) were significantly elevated. These findings suggest that NUR77 is a functional regulator of glucose metabolism in muscle, but is not involved in the regulation of fatty acid oxidation. These results might initially seem contradictory to the findings of Maxwell et al. , who used C2C12 cells with a stable knockdown of Nur77 and identified differentially expressed genes linked to lipid and glucose metabolism. However, the loss of Nur77 expression in Nur77-knockout mice results in a compensatory increase of another member of nuclear receptor subfamily 4, group A (NR4A), Nor1 (also known as Nr4a3) [15, 29]. This partial redundancy between NR4A receptors makes it difficult to study the direct effects of gene deletion. Therefore, a rise in Nor1 expression in response to NUR77 knockdown could potentially explain the increase in genes linked to lipid metabolism. Indeed, a recent study showed that NOR1 regulates palmitate oxidation in skeletal muscle cells . Further evidence against a direct role for NUR77 in the regulation of fatty acid metabolism comes from a study by Chao and colleagues  who overexpressed Nur77 in C2C12 cells and did not observe any differential expression of genes linked to fatty acid metabolism, but did see alterations in genes linked to glucose metabolism. These apparent differences in NUR77 function highlight the fact that data obtained from overexpression is not necessarily the opposite of knockdown.
To assess the functional role of NUR77 on glucose metabolism in vivo, we measured glucose uptake and glycogen synthesis under basal and insulin-stimulated conditions in control and transfected TA muscle in rats. Consistent with our findings in vitro, we found that basal glucose uptake and incorporation into glycogen was increased in muscle overexpressing Nur77 and this was associated with an increase in GLUT4 and glycogenin content, as well as elevated activity of the glycolytic enzymes, hexokinase and phosphofructokinase. Interestingly, under insulin-stimulated conditions, we observed only a very minor effect of Nur77 overexpression on glucose uptake and glycogen synthesis, despite the fact that GLUT4 content was increased. These findings of significant NUR77 effects on glucose metabolism in the basal but not insulin-stimulated state were observed in both chow- and fat-fed animals. There are several reasons why Nur77 overexpression might result in significant effects on glucose metabolism in the basal but not the insulin-stimulated state even though GLUT4 protein was increased in both situations.
First, it might be difficult to observe an increase in glucose uptake in insulin-stimulated muscle of a similar magnitude to that observed in muscle in the basal state because such an increase (approximately 4 µmol 100 g−1 min−1) falls within the margins of variation normally observed in determining glucose metabolism in muscle from hyperinsulinaemic–euglycaemic clamped rats [18, 19].
Second, the transport of glucose across the membrane by GLUT4 might not be the rate-limiting step under insulin-stimulated conditions. There is increasing evidence that under basal conditions the transport of glucose across the membrane is the rate-limiting step in muscle glucose uptake but under insulin-stimulated conditions or during exercise the membrane has increased permeability to glucose and this step is no longer rate limiting [24, 25, 30–34]. Under these conditions delivery of glucose via the bloodstream, phosphorylation inside the cells as well as usage further down the glycolytic pathway become increasingly more important. This is also evident in Glut4 transgenic mice where the amount of increased GLUT4 content (fourfold) does not directly translate to an increase in muscle glucose uptake of a similar magnitude (1.9-fold) [35, 36].
The improvement in glucose incorporation into glycogen under basal conditions could also be the result of increased GLUT4 content and increased availability of intracellular glucose. However, putative NUR77 response elements have been revealed by sequence analysis in the promoter regions of genes encoding other important proteins in glycogen synthesis. Glycogenin protein content was increased in response to Nur77 overexpression in chow-fed animals, suggesting that it is indeed a NUR77 downstream target. Both glycogenin and Glut4 expression are consistently upregulated in muscle after exercise in humans , and exercise is also known to increase Nur77 expression [11, 12]. The activity of hexokinase, another exercise-induced gene [9, 10], was also significantly increased in response to NUR77 overproduction. Collectively these findings suggest that NUR77 may mediate some of the beneficial effects of exercise on glucose metabolism. The attenuation of NUR77-induced changes in several of the above proteins in high-fat-fed rats might have contributed to the smaller increase in basal glucose uptake observed. Why high-fat feeding blunted the increase in glycogenin content as well as hexokinase and phosphofructokinase activity in response to Nur77 overexpression is not clear, but this may represent another manifestation of the effects of a high-fat diet on muscle glucose metabolism.
We also observed significantly reduced expression of NUR77 in the muscles of insulin-resistant obese men, as well as a lower expression of glycogenin. NUR77 expression was also closely correlated with body-fat content and insulin sensitivity, suggesting a possible functional role for NUR77 in glucose metabolism in humans. Our current findings, and those from a recent report showing increased NUR77 expression in human muscles following exercise , suggest that NUR77 may be a useful target for treating disorders of glucose metabolism.
The data presented here demonstrate that acute overexpression of the nuclear hormone transcription factor Nur77 in skeletal muscle can increase the capacity for glucose uptake and glycogen synthesis. In a physiological context this might be of relevance in the post-exercise state where Nur77 as well as Glut4 and glycogenin expression are increased to presumably meet the demand for replenishing energy stores [37, 38]. Increased expression of Nur77 after exercise [11, 12] and a decrease in insulin-resistant rats  and humans (Fig. 7) suggest that this regulator of glucose metabolism might be an interesting target in the context of increasing glucose metabolism in muscle. Although overexpression of Nur77 did not improve insulin-stimulated glucose metabolism after high-fat-diet feeding in rats, the augmentation in the basal state would suggest an overall benefit to glucose metabolism by increasing NUR77 activity in insulin-resistant muscle.
In summary, we provide evidence for a functional role of the orphan nuclear receptor NUR77 in glucose metabolism in skeletal muscle. The beneficial effects on glucose metabolism are associated with alterations in GLUT4, glycogenin, hexokinase and phosphofructokinase. These new data support previous studies showing that NUR77 is induced after exercise bouts in skeletal muscle and is reduced in obesity and suggest that manipulation of NUR77 could provide beneficial effects on muscle glucose metabolism.
This study was supported by Deutsche Forschungsgemeinschaft (DFG), Diabetes Australia Research Trust and the National Health and Medical Research Council (NHMRC) of Australia (Project grant 276419). T. Kanzleiter is supported by a DFG Research Fellowship, J. Reznick is supported by an Australian Postgraduate Award, N. Turner is supported by a Career Development Award and G. J. Cooney by a Research Fellowship from the NHMRC of Australia. The authors are grateful to G. Frangioudakis and F. Hochgraefe for their critical comments on this manuscript.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.