Muscle Glucocorticoid Receptor Research Articles

Intracellular negative feedback mechanisms in blubber and muscle moderate acute stress responses in fasting seals.

Animals may limit the cost of stress responses during key life history stages such as breeding and molting by reducing tissue sensitivity to energy-mobilizing stress hormones (e.g. cortisol). We measured expression of genes encoding glucocorticoid receptor (GR, NR3C1), GR inhibitor (FKBP5) and cortisol-inactivating enzyme (HSD11B2) in blubber and muscle of northern elephant seals before and after stress axis stimulation by adrenocorticotropic hormone (ACTH) early and late in a fasting period associated with molting. ACTH elevated cortisol levels for >24 h and increased FKBP5 and HSD11B2 expression while downregulating NR3C1 expression in blubber and muscle, suggesting robust intracellular negative feedback in peripheral tissues. This feedback was maintained over prolonged fasting, despite differences in baseline cortisol and gene expression levels between early and late molt, suggesting that fasting-adapted animals use multiple tissue-specific, intracellular negative feedback mechanisms to modulate downstream impacts of acute stress responses during key life history stages.

OR33-04 Mechanistic Dissection Of Circadian-Specific Glucocorticoid Effects On Exercise Tolerance And Muscle Glucose Utilization

Abstract Disclosure: A. Prabakaran: None. M. Wintzinger: None. K. Piczer: None. K. Miz: None. A. Walton: None. H. Durumutla: None. M. Quattrocelli: None. Glucocorticoids (GCs) are circadian hormones regulating metabolism by activating the glucocorticoid receptor (GR) as pleiotropic transcription factor. Timing of exogenous GC drug regimens is emerging as a key determinant of their metabolic effects. This was shown by the surprising finding that once-weekly GC intermittence reverses many of the pro-obesogenic side effects of once-daily GC dosing in mice and humans. However, despite the intrinsic circadian nature of this signaling, the impact of time-of-day of exogenous GC dosing, i.e. chrono-pharmacology, remains remarkably unknown for metabolic physiology and exercise tolerance. Relevance of this question for humans is supported by the initial findings of increased lean mass and motor function with circadian-restricted prednisone intermittence in a recent pilot clinical trial in patients with genetic myopathies. However, mechanisms to support significance of these effects for metabolic diseases and aging remain unknown. We studied 12-week-long intermittent regimens of the exogenous GC prednisone in mice in conditions of diet-induced obesity or advanced aging, comparing dosing at light-phase start (ZT0) versus dark-phase start (ZT12). We found that, compared to dark-phase, light-phase dosing enhanced the regimen-driven increase in body-wide lean mass, muscle mass and muscle function in both obese and aged mice. Intriguingly, experiments with adiponectin-knockout mice showed that these effects were dependent on adiponectin, which promotes muscle insulin sensitivity. We then used tissue-specific inducible knockout models to dissect non-muscle versus muscle-autonomous effects of GC chrono-pharmacology. We found that the GC effects on total and high-molecular-weight adiponectin upregulation were dependent on regimen-specific engagement of the adipocyte-specific GR on the adiponectin gene promoter. Complementary to adipose-driven adiponectin production, we found that adiponectin receptor AdipoR1 upregulation in muscle depended on circadian-specific engagement of the myocyte-specific GR on the AdipoR1 promoter. Consistent with the adiponectin action on muscle insulin sensitivity, ablation of myocyte-specific GR blocked the regimen-driven effects on insulin-sensitive muscle glucose uptake and muscle mass. Furthermore, we found that light-phase GC dosing engaged the muscle GR for a non-canonical interaction with the clock factor BMAL1 to upregulate the mitochondrial regulator PGC1alpha. Indeed, we found that the regimen-driven increase in glucose oxidation and amino acid biogenesis from TCA cycle intermediates in muscle was dependent on the myocyte-specific PGC1alpha. In summary, we present here novel circadian and molecular mechanisms reconverting glucocorticoid drugs from deleterious to re-energizing agents for potential chrono-treatment of metabolic conditions and aging. Presentation: Sunday, June 18, 2023

The crucial role of muscle glucocorticoid signaling in accelerating obesity and glucose intolerance via hyperinsulinemia.

Metabolic crosstalk from skeletal muscle to multiple organs is important for maintaining homeostasis, and its dysregulation can lead to various diseases. Chronic glucocorticoid administration often induces muscle atrophy and metabolic disorders such as diabetes and central obesity; however, the detailed underlying mechanism remains unclear. We previously reported that the deletion of glucocorticoid receptor (GR) in skeletal muscle increases muscle mass and reduces fat mass through muscle-liver-fat communication under physiological conditions. In this study, we show that muscle GR signaling plays a crucial role in accelerating obesity through the induction of hyperinsulinemia. Fat accumulation in liver and adipose tissue, muscle atrophy, hyperglycemia, and hyperinsulinemia induced by chronic corticosterone (CORT) treatment improved in muscle-specific GR knockout (GRmKO) mice. Such CORT-induced fat accumulation was alleviated by suppressing insulin production (streptozotocin injection), indicating that hyperinsulinemia enhanced by muscle GR signaling promotes obesity. Strikingly, glucose intolerance and obesity in ob/ob mice without CORT treatment were also improved in GRmKO mice, indicating that muscle GR signaling contributes to obesity-related metabolic changes, regardless of systemic glucocorticoid levels. Thus, this study provides new insight for the treatment of obesity and diabetes by targeting muscle GR signaling.

OR26-3 USP19 Deubiquitinating Enzyme Functions as a Novel Co-Chaperone of Hsp90 to Regulate Glucocorticoid Receptor Levels

Abstract Ubiquitin specific protease 19 (USP19) is a deubiquitinating enzyme involved in metabolism. Its expression is upregulated in skeletal muscle in many conditions of muscle wasting. Genetic inactivation of USP19 (USP19KO) in mice protects from muscle atrophy and diet induced obesity and insulin resistance. We previously reported that the protection against muscle atrophy in USP19KO is due at least in part to reduction in muscle glucocorticoid receptor (GR) levels and signaling. Here, we report that USP19KO mice show > 50% decrease in GR protein level in multiple tissues with no observed changes in GR mRNA levels. Overexpression of the endoplasmic reticulum or cytosol-localized isoforms of USP19 in cells resulted in increased GR protein levels, while knockdown by siRNA or knockout by CRISPR-Cas9 resulted in decreased GR protein levels. Intriguingly, GR was not more ubiquitinated in the absence of USP19. Furthermore, expression of a catalytically inactive form of USP19 also increased GR protein levels and exposure of cells to a small molecule inhibitor of USP19 enzymatic activity had no effects on levels of GR. Deletion analysis revealed that the N-terminal CS domains of USP19, which are structurally similar to the p23 co-chaperone of HSP90, were required for increasing levels of GR. Since HSP90 is known to regulate GR folding and stability, this suggested that USP19 may function as a p23 like co-chaperone. Indeed, overexpression of USP19 not only increased GR levels, but translocation to the nucleus and transcriptional activity of a reporter gene. Using a novel BRET assay, we observed that USP19 interacts indirectly with GR through binding with HSP90. This interaction with HSP90 is critically dependent on the CS2 domain of USP19 and can be competed by expression of the p23 co-chaperone of HSP90. We have therefore identified a novel co-chaperone-like mechanism through which USP19 regulates GR protein stability independent of its catalytic activity. Targeting the interaction of USP19 with the GR-HSP90 complex could be a strategy to decrease GR levels, reduce glucocorticoid signalling and prevent muscle atrophy. Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 a.m.

Conditional deletion of the glucocorticoid receptor in vascular smooth muscle disrupts the circadian rhythm of vascular reactivity and reduces blood pressure

Glucocorticoids synchronise peripheral clocks with the master clock in the suprachiasmatic nucleus in response to the ambient light cycle. In humans and mice, arrhythmic glucocorticoids induce non‐dipping blood pressure and vascular dysfunction. The mechanisms of this are poorly understood. We hypothesize that arrhythmic activation of the glucocorticoid receptor attenuates the circadian clock signalling in the vasculature leading to changes in blood pressure. To test this hypothesis, we assessed vascular function and blood pressure rhythms in control mice and mice with smooth muscle specific deletion of the glucocorticoid receptor (SMGRKO).Renal and mesenteric arteries were isolated at 7am (ZT0; lights on) and 7pm (ZT12; lights off), mounted on a wire myograph and the responses to increasing doses of phenylephrine or sodium nitroprusside (SNP) assessed. In wild‐type mice, the sensitivity of the renal artery to phenylephrine‐mediated contraction was greater at ZT12 than at ZT0, by comparing the half maximal effective concentration of each group, (logEC50: 7.54±0.26 versus 7.29±0.17 respectively; p=0.04). The sensitivity to SNP‐mediated relaxation was also greater when measured at ZT12 compared to ZT0 (7.64±0.3 versus 7.09±0.5; p=0.03). Similar responses were also detected in the mesenteric arteries. In SMGRKO mice, the temporal differences in renal and mesenteric artery vascular reactivity were absent. We used radiotelemetry to assess the effect of this altered rhythm of vascular reactivity on blood pressure. SMGRKO had significantly lower systolic blood pressure throughout the 24h cycle compared to wild‐type littermates, both when lights were off (107.9 ± 22.13 versus 136.3 ± 17.17 mmHg respectively; p=0.015) and during the lights on period (99.6 ± 13.16 versus 118 ± 15.9 mmHg; p=0.014). Blood pressure rhythmicity was not altered in SMGRKO mice.These data show that the glucocorticoid receptor signalling in vascular smooth muscle contributes both to the diurnal variation of vascular reactivity and to blood pressure, but loss of the receptor does not affect blood pressure rhythm. Whether glucocorticoid receptor deletion in smooth muscle protects against non‐dipping blood pressure induced by glucocorticoids needs further investigation.

Betaine could help ameliorate transport associated water deprivation stress in broilers by reducing the expression of stress-related transcripts and modulating water channel activity

This study evaluated the effects of betaine and pre-slaughter transport of broilers on meat quality, blood parameters, selected water channels (AQP) and stress-related transcripts. Ross 308 broilers (n = 336) were housed in 56-floor pens with seven replicates per treatment. In a 4 × 2 design, broilers were either supplemented with betaine in the feed (0.2%), in drinking water (1 or 2 mL/L), or un-supplemented, and then were transported for 5-min or 4-h pre-slaughter. The results revealed that broilers transported for long-distance had elevated blood glucose pre-slaughter, and their breast meat was characterised with lower initial pH, higher ultimate pH, higher 0-h post-mortem temperature, and lower water holding capacity than those transported for short-distance. Moreover, there was a two-way interaction effect between betaine and transport on the expression of brain genes (Proopiomelanocortin, AQP1, AQP4), muscle glucocorticoid receptor (GR), hepatic AQP3 and AQP9 and intestinal AQP3. Long-distance transport upregulated the relative expression of the muscle AQP9 and downregulated the hepatic GR. Furthermore, betaine in the water at 2 mL upregulated the muscle AQP9 and hepatic GR. In conclusion, betaine could ameliorate the water-deprivation stress associated with transportation by reducing the expression of stress-related transcripts and modulate water channels without improving broiler meat quality. HIGHLIGHTS Long-distance transport of broilers is stressful, as evidenced by rising blood glucose, high temperature of fresh meat, and expression of stress-related transcripts. Betaine could ameliorate the water-deprivation stress associated with transport by reducing the expression of stress-related transcripts and modulating water channels.

The role of striated muscle Pik3r1 in glucose and protein metabolism following chronic glucocorticoid exposure

Chronic glucocorticoid exposure causes insulin resistance and muscle atrophy in skeletal muscle. We previously identified phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1) as a primary target gene of skeletal muscle glucocorticoid receptors involved in the glucocorticoid-mediated suppression of insulin action. However, the in vivo functions of Pik3r1 remain unclear. Here, we generated striated muscle-specific Pik3r1 knockout (MKO) mice and treated them with a dexamethasone (DEX), a synthetic glucocorticoid. Treating wildtype (WT) mice with DEX attenuated insulin activated Akt activity in liver, epididymal white adipose tissue, and gastrocnemius (GA) muscle. This DEX effect was diminished in GA muscle of MKO mice, therefore, resulting in improved glucose and insulin tolerance in DEX-treated MKO mice. Stable isotope labeling techniques revealed that in WT mice, DEX treatment decreased protein fractional synthesis rates in GA muscle. Furthermore, histology showed that in WT mice, DEX treatment reduced GA myotube diameters. In MKO mice, myotube diameters were smaller than in WT mice, and there were more fast oxidative fibers. Importantly, DEX failed to further reduce myotube diameters. Pik3r1 knockout also decreased basal protein synthesis rate (likely caused by lower 4E-BP1 phosphorylation at Thr37/Thr46) and curbed the ability of DEX to attenuate protein synthesis rate. Finally, the ability of DEX to inhibit eIF2α phosphorylation and insulin-induced 4E-BP1 phosphorylation was reduced in MKO mice. Taken together, these results demonstrate the role of Pik3r1 in glucocorticoid-mediated effects on glucose and protein metabolism in skeletal muscle.

Sex-based differences in resting MAPK, androgen, and glucocorticoid receptor phosphorylation in human skeletal muscle

PurposeTo determine if there is differential expression and phosphorylation of the androgen receptor (AR), glucocorticoid receptor (GR), and mitogen-activated protein kinases (MAPK) in skeletal muscle at rest between males and females. MethodsTen college-aged males (mean ± SD; age = 22 ± 2.4 yrs, ht = 175 ± 7 cm, body mass = 84.1 ± 11.8 kg) and ten females (mean ± SD; age = 20 ± 0.9 yrs; ht = 169 ± 7 cm; body mass = 67.1 ± 8.7 kg) reported to the laboratory following an overnight fast. Resting muscle biopsies were collected from the vastus lateralis and analyzed for total and phosphorylated GR (ser134, ser211, and ser226), total and phosphorylated AR (ser81, ser213, ser515, ser650), and total and phosphorylated MAPK (ERK, JNK, p38) via western blotting. A phosphorylation index (PI) was calculated to determine phosphorylated receptor expression after accounting for differences in total receptor content. ResultsMales had more total AR compared to females (+42 ± 4%; p < 0.001). Females had higher phosphorylation of ARser81 (+87 ± 11%; p = 0.001) and ser515 (+55 ± 13%; p = 0.019). However, when the phosphorylated ratios were corrected for differences in total AR expression (i.e. our PI), the overall phosphorylation at these sites were similar between sexes (ser515, males = 100% vs females 92%; ser81, males = 100% vs females = 107%). pGRser134 was higher in males compared to females (+50 ± 15%; p = 0.016). Phospho-p38 was higher in females compared to males (+5050 ± 16%; p < 0.001). ConclusionAt rest, ARs and GRs are differentially phosphorylated at some, but not all sites when comparing males and females. Differential regulation of phosphorylated AR, GR, and p38 between males and females may have implications for the degree of muscle adaptations observed following resistance or endurance training.

Glucocorticoid receptor activation is associated with increased resistance to heat‐induced hyperthermia and injury

Anti-inflammatory mediators likely play a key role in maintaining thermal homeostasis and providing protection against heat stress. The aim of this study was to investigate the association between activation of the glucocorticoid receptor (GR) and resistance to heat-induced hyperthermia and injury. Effects of heat exposure on core body temperature, muscle GR phosphorylation status and subcellular expression were examined in control mice and thermal acclimation (TA)-exposed mice. In addition, effects of TA and corticosterone on C2C12 mouse myoblast viability and subcellular GR were assessed during heat exposure. Phosphorylated, nuclear and mitochondrial GR levels were significantly higher in the gastrocnemius muscles of mice with mild hyperthermia (tolerant), compared to mice with severe hyperthermia (intolerant) during a heat exposure test. Similar changes were found in mice after TA, compared to non-TA-exposed controls. Additional groups of TA and non-TA-exposed mice underwent a heat exposure test. TA mice presented a significantly lower hyperthermic response during heat exposure than non-TA-exposed control. C2C12 cells exposed to TA incubation had higher viability against heat shock and showed higher GR levels in their mitochondria and nuclei detected by Western blot analysis and fluorescence microscopy, compared to cells exposed to normal incubation. Furthermore, pre-incubation with 0.1 μM corticosterone increased C2C12 cell viability during heat exposure and mitochondrial and nuclear GR expression. The results of these in vivo and in vitro studies suggest that GR activation is associated with increased resistance against heat-induced hyperthermia and injury.

P362Pathological cardiac remodeling caused by cardiomyocyte/vascular smooth muscle glucocorticoid receptor deficiency

Variation in the human glucocorticoid receptor (GR) gene associates with relative glucocorticoid resistance, hypertension and increased cardiovascular disease risk. Here, the contribution of cardiovascular GR to disease risk was assessed in male and female mice with cardiomyocyte and vascular smooth muscle deletion of GR. SMGRKO mice, generated by crossing GR “floxed” mice (congenic on C57BL/6J) with SM22α-Cre mice, have reduced cardiac GR protein and mRNA levels (by 52% and 57%, respectively), compared to Cre-negative littermate controls. Loss of cardiovascular GR impairs viability. There are fewer SMGKRO mice than controls at weaning with a greater effect in females [SMGRKO:Control; females 27:79(25% vs expected 50%); males 37:68(35%) p<0.0001]. Doppler measurements (Visualsonics Vevo770 ultrasound) of blood flow within the left ventricle show a detrimental increase in the myocardial performance index, which measures combined systolic and diastolic function, in both sexes of adult SMGRKO mice. This is primarily due to prolonged isovolumetric contraction time (p<0.05) indicating impairment of the initial left ventricular contractile phase. In male SMGRKO mice (but not female) heart weight (% body weight) was increased (Control:0.50±0.02%; SMGRKO:0.55±0.01%, p<0.05) as was cardiomyocyte cross sectional area (Control:180±4μm2; SMGRKO:194±4μm2, p<0.01). Furthermore, cardiac levels of mRNA encoding myosin heavy chain-β, a marker of pathological cardiac hypertrophy, were elevated in male SMGRKO mice only (p<0.05). Both sexes showed left ventricular fibrosis (histopathology) and elevated levels of cardiac mRNA encoding pro-fibrotic factors. Thus, cardiomyocyte/smooth muscle GR-deficiency causes the same functional impairment of isovolumetric contraction but differential pathological changes in the left ventricle of surviving male and female mice. Whether pathology is worse in neonatal or fetal SMGRKO females is under investigation. These findings support a role for cardiovascular GR in determination of cardiovascular disease risk, though without overt signs of cardiac hypertrophy in females.

19 Sex Differences in Pathological Remodelling Caused by Cardiomyocyte/Vascular Smooth Muscle Glucocorticoid Receptor Defficiency

<h3></h3> Variation in the human glucocorticoid receptor (GR) gene associates with relative glucocorticoid resistance, hypertension and increased cardiovascular disease risk. Here, the contribution of cardiac GR to disease risk was assessed in male and female “SMGRKO” mice with cardiomyocyte and vascular smooth muscle deletion of GR. SMGRKO mice, generated from GR “floxed” (congenic on C57BL/6J) x SM22α-Cre crosses, have reduced cardiac GR protein and mRNA levels (by 52% and 57%, respectively), compared to Cre-negative littermate controls. Interestingly, fewer female SMGKRO mice survive to weaning [SMGRKO: Control; females 23:68(25%); males 42:52(45%)]. Doppler measurements (Visual Sonics Vevo770 ultrasound) of blood flow within the left ventricle show a detrimental increase in the myocardial performance index, which represents combined systolic and diastolic function, in both sexes of adult SMGRKO mice, primarily due to greater isovolumetric contraction time (p &lt; 0.05) indicating impairment of the initial left ventricular contractile phase. In males only, cardiomyocyte size and heart weight (% body weight) are increased (Control: 0.5 ± 0.02%; SMGRKO: 0.55 ± 0.01%, p &lt; 0.05), as are levels of mRNA encoding myosin heavy chain-β, a marker of pathological cardiac hypertrophy (p &lt; 0.05). Both sexes showed left ventricular fibrosis (histopathology) and elevated levels of mRNA encoding pro-fibrotic factors. Thus, cardiomyocyte/smooth muscle GR-deficiency causes the same functional impairment of isovolumetric contraction but differential pathological changes in the left ventricle of male and surviving female mice. Whether pathology is worse in females that died before weaning is under investigation. Nevertheless, these findings support a role for cardiomyocyte GR in determination of cardiovascular disease risk, though without overt signs of cardiac hypertrophy in females.

Increased Skeletal Muscle 11βHSD1 mRNA Is Associated with Lower Muscle Strength in Ageing

BackgroundSarcopenia, the loss of muscle mass and function with age, is associated with increased morbidity and mortality. Current understanding of the underlying mechanisms is limited. Glucocorticoids (GC) in excess cause muscle weakness and atrophy. We hypothesized that GC may contribute to sarcopenia through elevated circulating levels or increased glucocorticoid receptor (GR) signaling by increased expression of either GR or the GC-amplifying enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11βHSD1) in muscle.MethodsThere were 82 participants; group 1 comprised 33 older men (mean age 70.2years, SD 4.4) and 19 younger men (22.2years, 1.7) and group 2 comprised 16 older men (79.1years, 3.4) and 14 older women (80.1years, 3.7). We measured muscle strength, mid-thigh cross-sectional area, fasting morning plasma cortisol, quadriceps muscle GR and 11βHSD1 mRNA, and urinary glucocorticoid metabolites. Data were analysed using multiple linear regression adjusting for age, gender and body size.ResultsMuscle strength and size were not associated with plasma cortisol, total urinary glucocorticoids or the ratio of urinary 5β-tetrahydrocortisol +5α-tetrahydrocortisol to tetrahydrocortisone (an index of systemic 11βHSD activity). Muscle strength was associated with 11βHSD1 mRNA levels (β -0.35, p = 0.04), but GR mRNA levels were not significantly associated with muscle strength or size.ConclusionAlthough circulating levels of GC are not associated with muscle strength or size in either gender, increased cortisol generation within muscle by 11βHSD1 may contribute to loss of muscle strength with age, a key component of sarcopenia. Inhibition of 11βHSD1 may have therapeutic potential in sarcopenia.

Cancer‐ and endotoxin‐induced cachexia require intact glucocorticoid signaling in skeletal muscle

Cachexia is a wasting condition defined by skeletal muscle atrophy in the setting of systemic inflammation. To explore the site at which inflammatory mediators act to produce atrophy in vivo, we utilized mice with a conditional deletion of the inflammatory adaptor protein myeloid differentiation factor 88 (MyD88). Although whole-body MyD88-knockout (wbMyD88KO) mice resist skeletal muscle atrophy in response to LPS, muscle-specific deletion of MyD88 is not protective. Furthermore, selective reexpression of MyD88 in the muscle of wbMyD88KO mice via electroporation fails to restore atrophy gene induction by LPS. To evaluate the role of glucocorticoids as the inflammation-induced mediator of atrophy in vivo, we generated mice with targeted deletion of the glucocorticoid receptor in muscle (mGRKO mice). Muscle-specific deletion of the glucocorticoid receptor affords a 71% protection against LPS-induced atrophy compared to control animals. Furthermore, mGRKO mice exhibit 77% less skeletal muscle atrophy than control animals in response to tumor growth. These data demonstrate that glucocorticoids are a major determinant of inflammation-induced atrophy in vivo and play a critical role in the pathogenesis of endotoxemic and cancer cachexia.

Pulmonary inflammation‐induced muscle atrophy involves a GSK‐3beta‐GR signaling axis

Skeletal muscle atrophy is induced by raised glucocorticoid (GC) levels and reduced insulin/IGF‐1‐Akt signaling. Glycogen synthase kinase‐3 beta (GSK‐3b) is a downstream kinase of Akt involved in regulation of muscle protein turnover.We hypothesized that muscle GC receptor (GR) and GSK‐3b are required for muscle atrophy in response to pulmonary inflammation.Control, muscle specific‐GR (mGRKO) or ‐GSK‐3b (mGSK‐3bKO) mice were subjected to intra‐tracheal LPS instillation to induce acute pulmonary inflammation.Increases in circulating corticosterone levels following IT‐LPS were similar in control, mGRKO and mGSK‐3KO mice, but accompanied by substantial muscle atrophy in control mice only. Accordingly, increases in mRNA transcripts encoding effectors of the Ub 26S‐proteasome (MuRF1 and Atrogin‐1) and autophagy‐lysosomal (Bnip3, LC3b) systems were strongly attenuated in muscle of mGRKO or mGSK‐3KO mice. Interestingly, increased expression of GC‐sensitive genes (Glul, KLF15 and REDD1) and FOXO1 and ‐3a following IT‐LPS, was suppressed in skeletal muscle of GRKO and GSK‐3KO mice. This dependency of GR signaling on GSK‐3b was confirmed in cultured muscle cells.We conclude that pulmonary inflammation‐induced muscle atrophy is dependent on an intact GSK‐3b‐GR signaling axis.Support: Dutch Astma Foundation (3.2.07.017) and Dutch Top Institute Pharma (T1–201)

Metabolic functions of glucocorticoid receptor in skeletal muscle

Glucocorticoids (GCs) exert key metabolic influences on skeletal muscle. GCs increase protein degradation and decrease protein synthesis. The released amino acids are mobilized from skeletal muscle to liver, where they serve as substrates for hepatic gluconeogenesis. This metabolic response is critical for mammals’ survival under stressful conditions, such as fasting and starvation. GCs suppress insulin-stimulated glucose uptake and utilization and glycogen synthesis, and play a permissive role for catecholamine-induced glycogenolysis, thus preserving the level of circulating glucose, the major energy source for the brain. However, chronic or excess exposure of GCs can induce muscle atrophy and insulin resistance. GCs convey their signal mainly through the intracellular glucocorticoid receptor (GR). While GR can act through different mechanisms, one of its major actions is to regulate the transcription of its primary target genes through genomic glucocorticoid response elements (GREs) by directly binding to DNA or tethering onto other DNA-binding transcription factors. These GR primary targets trigger physiological and pathological responses of GCs. Much progress has been made to understand how GCs regulate protein and glucose metabolism. In this review, we will discuss how GR primary target genes confer metabolic functions of GCs, and the mechanisms governing the transcriptional regulation of these targets. Comprehending these processes not only contributes to the fundamental understanding of mammalian physiology, but also will provide invaluable insight for improved GC therapeutics.

Crosstalk between Glucocorticoid Receptor and Nutritional Sensor mTOR in Skeletal Muscle

Maintenance of skeletal muscle mass relies on the dynamic balance between anabolic and catabolic processes and is important for motility, systemic energy homeostasis, and viability. We identified direct target genes of the glucocorticoid receptor (GR) in skeletal muscle, i.e., REDD1 and KLF15. As well as REDD1, KLF15 inhibits mTOR activity, but via a distinct mechanism involving BCAT2 gene activation. Moreover, KLF15 upregulates the expression of the E3 ubiquitin ligases atrogin-1 and MuRF1 genes and negatively modulates myofiber size. Thus, GR is a liaison involving a variety of downstream molecular cascades toward muscle atrophy. Notably, mTOR activation inhibits GR transcription function and efficiently counteracts the catabolic processes provoked by glucocorticoids. This mutually exclusive crosstalk between GR and mTOR, a highly coordinated interaction between the catabolic hormone signal and the anabolic machinery, may be a rational mechanism for fine-tuning of muscle volume and a potential therapeutic target for muscle wasting.

Age and tissue specific differences in the development of acute insulin resistance following injury

Injuries, hemorrhage, sepsis, burn, and critical illnesses all induce insulin resistance, and insulin resistance is strongly associated with advancing age. However, the effect of age on injury induced insulin resistance is not well studied. We performed surgical trauma in male rats of three different ages (3-, 6-, and 10-weeks old). Rats were either hemorrhaged to a mean arterial pressure of 35-40 mmHg and subsequently maintained at that pressure for up to 90 min, or maintained without hemorrhage as controls. Results indicate that insulin-induced intracellular signaling was diminished in liver and skeletal muscle of 6- and 10-week old rats following trauma and hemorrhage. In even younger rats, immediately post-weaning ( approximately 3 weeks of age), insulin signaling was lost in liver, but not in skeletal muscle. Glucocorticoids can play a role in the chronic development of insulin resistance. Our results demonstrate that corticosterone levels were increased in 6- and 10-week old animals following hemorrhage, but little change was measured in 3-week old animals. Blockade of glucocorticoid synthesis prevented the development of insulin resistance in skeletal muscle, but not in liver of 6- and 10-week old rats. Moreover, skeletal muscle glucocorticoid receptor levels increased dramatically between 3 and 6 weeks of age. These results indicate that trauma and hemorrhage-induced hepatic insulin resistance occurs at all ages tested. However, there is no development of insulin resistance following trauma and hemorrhage in skeletal muscle of post-weaning rats. In skeletal muscle of 6- and 10-week old rats, inhibition of glucocorticoid levels prevents the development of insulin resistance.

Alteration of 11β-hydroxysteroid dehydrogenase type 1 in skeletal muscle in a rat model of type 2 diabetes

Recent investigations have demonstrated that activation of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in liver and adipose tissue is closely related to the pathogenesis of obesity and diabetes. However, the relationship between alteration of 11beta-HSD1 and the pathogenesis of type 2 diabetes in skeletal muscle is still unclear. A rat model of Type 2 diabetes was developed by high fat diet feeding combined with multiple low dose streptozotocin injection (30 mg/kg, i.p. twice). Intraperitoneal glucose tolerance test, insulin tolerance test were performed. Fasting blood glucose, fasting insulin, total cholesterol, triglyceride were measured. The protein and mRNA level of 11beta-HSD1 and glucocorticoid receptor in gastrocnemius muscle were determined. The alteration of insulin signaling pathway related protein was investigated. We found that the protein levels of 11beta-HSD1 and glucocorticoid receptor were significantly increased (P < 0.05); the mRNA level of 11beta-HSD1 was also elevated (P < 0.05); the mRNA level of glucocorticoid receptor was decreased (P < 0.05). After insulin stimulation, diabetic rats had no significant changes in the level of the insulin receptor beta-subunit (IR-beta), AKT, as in phosphorylated AKT in the gastrocnemius muscle compared to its basal state. Similar results were observed in the protein expression level of glucose transporter 4 (GLUT4). Our data indicate that the alteration of 11beta-HSD1 at protein and mRNA level may be related to the abnormality of insulin signal pathway in skeletal muscle, this effect may be mediated by glucocorticoid receptor.

Modeling Circadian Rhythms of Glucocorticoid Receptor and Glutamine Synthetase Expression in Rat Skeletal Muscle

The circadian rhythm of endogenous corticosterone (CS) may produce fluctuations of downstream gene expression in normal rats. This study examined changes in glucocorticoid receptor (GR) and glutamine synthetase (GS) expression in rat skeletal muscle in relation to plasma CS over a 24-h period. Fifty-four normal male Wistar rats were sacrificed at 18 time points (n = 3) over 24 h. Plasma CS concentrations and gastrocnemius muscle GR and GS mRNA and GS activity were measured. The circadian rhythm of plasma CS was captured by a two-harmonic function. The expression of GR and GS mRNA and GS activity follow a circadian rhythm in normal rat skeletal muscle. GR mRNA reaches a trough at 4 h after the peak of plasma CS and it fluctuates between 0.55 and 0.9 fmol g tissue(-1). GS mRNA and activity reach peaks at 6 and 12 h after the endogenous CS peak. GS mRNA oscillates between 3 and 6 fmol g tissue(-1), whereas GS activity fluctuates between 17 and 23 micromol min(-1) g protein(-1). Mechanistic receptor/gene-mediated pharmacodynamic models were applied to describe the temporal patterns of GR mRNA, GS mRNA, and GS activity within the circadian cycle. The integrated models were able to capture the circadian expression patterns of plasma CS, and GR and GS in normal rat skeletal muscle showing a dependence of tissue gene expression on plasma CS.

Expression and binding activity of the glucocorticoid receptor are upregulated in septic muscle.

We examined the influence of sepsis, induced by cecal ligation and puncture in rats, on the protein and gene expression and hormone binding activity of the glucocorticoid receptor (GR) in skeletal muscle. Sepsis resulted in increased GR mRNA and protein levels and upregulated hormone binding activity in extensor digitorum longus and soleus muscles. Scatchard analysis suggested that the increased GR hormone binding activity reflected an increased number of hormone binding sites, whereas receptor affinity for glucocorticoids was unchanged. The GR antagonist RU-38486 blocked the sepsis-induced increase in GR expression and hormone binding activity, implicating a positive regulatory effect of glucocorticoids on GR expression and binding activity under the present experimental conditions. The results suggest that glucocorticoid-dependent metabolic changes in skeletal muscle during sepsis may reflect not only high circulating glucocorticoid levels but increased amounts and hormone binding activity of the GR as well.