Chemokine-like receptor 1 regulates skeletal muscle cell myogenesis.

The chemokine-like receptor-1 (CMKLR1) is a G protein-coupled receptor that is activated by chemerin, a secreted plasma leukocyte attractant and adipokine. Previous studies identified that CMKLR1 is expressed in skeletal muscle in a stage-specific fashion during embryogenesis and in adult mice; however, its function in skeletal muscle remains unclear. Based on the established function of CMKLR1 in cell migration and differentiation, we investigated the hypothesis that CMKLR1 regulates the differentiation of myoblasts into myotubes. In C(2)C(12) mouse myoblasts, CMKLR1 expression increased threefold with differentiation into multinucleated myotubes. Decreasing CMKLR1 expression by adenoviral-delivered small-hairpin RNA (shRNA) impaired the differentiation of C(2)C(12) myoblasts into mature myotubes and reduced the mRNA expression of myogenic regulatory factors myogenin and MyoD while increasing Myf5 and Mrf4. At embryonic day 12.5 (E12.5), CMKLR1 knockout (CMKLR1(-/-)) mice appeared developmentally delayed and displayed significantly lower wet weights and a considerably diminished myotomal component of somites as revealed by immunolocalization of myosin heavy chain protein compared with wild-type (CMKLR1(+/+)) mouse embryos. These changes were associated with increased Myf5 and decreased MyoD protein expression in the somites of E12.5 CMKLR1(-/-) mouse embryos. Adult male CMKLR1(-/-) mice had significantly reduced bone-free lean mass and weighed less than the CMKLR1(+/+) mice. We conclude that CMKLR1 is essential for myogenic differentiation of C(2)C(12) cells in vitro, and the CMKLR1 null mice have a subtle skeletal muscle deficit beginning from embryonic life that persists during postnatal life.

Chemerin, a novel peroxisome proliferator-activated receptor gamma (PPARgamma) target gene that promotes mesenchymal stem cell adipogenesis.

Chemerin is an adipocyte-secreted protein that regulates adipogenesis and the metabolic function of mature adipocytes via activation of chemokine-like receptor 1 (CMKLR1). Herein we report the interaction of peroxisome proliferator-activated receptor γ (PPARγ) and chemerin in the context of adipogenesis. Knockdown of chemerin or CMKLR1 expression or antibody neutralization of secreted chemerin protein arrested adipogenic clonal expansion of bone marrow mesenchymal stem cells (BMSCs) by inducing a loss of G(2)/M cyclins (cyclin A2/B2) but not the G(1)/S cyclin D2. Forced expression of PPARγ in BMSCs did not completely rescue this loss of clonal expansion and adipogenesis following chemerin or CMKLR1 knockdown. However, forced expression and/or activation of PPARγ in BMSCs as well as non-adipogenic cell types such as NIH-3T3 embryonic fibroblasts and MCA38 colon carcinoma cells significantly induced chemerin expression and secretion. Sequence analysis revealed a putative PPARγ response element (PPRE) sequence within the chemerin promoter. This PPRE was able to confer PPARγ responsiveness on a heterologous promoter, and mutation of this sequence abolished activation of the chemerin promoter by PPARγ. Chromatin immunoprecipitation confirmed the direct association of PPARγ with this PPRE. Treatment of mice with rosiglitazone elevated chemerin mRNA levels in adipose tissue and bone marrow coincident with an increase in circulating chemerin levels. Together, these findings support a fundamental role for chemerin/CMKLR1 signaling in clonal expansion during adipocyte differentiation as well as a role for PPARγ in regulating chemerin expression.

Influence of peroxisome proliferator-activated receptor-alpha (PPARα) activity on adverse effects associated with amiodarone exposure in mice.

Although amiodarone is the most effective antiarrhythmic agent currently available, concerns regarding adverse effects, including liver, lung and thyroid toxicity, often limit its use. Previously, we reported that amiodarone-induced hepatic steatosis in mice was associated with an upregulation of target genes modulated by peroxisome proliferator-activated receptor-alpha (PPARα). Because amiodarone does not directly stimulate PPARα, target gene induction may reflect a compensatory reaction countering some adverse effects of amiodarone. To test this, we examined co-treatment with the PPARα agonist, fenofibrate, and amiodarone in both PPARα(+/+) and PPARα(-/-) mice. Amiodarone treated PPARα(-/-) mice exhibited significantly greater weight loss and higher serum aspartate aminotransferase (AST) compared to PPARα(+/+) mice. Fenofibrate co-treatment reduced weight loss in amiodarone treated PPARα(-/-) mice, but not PPARα(+/+) mice. Fenofibrate stimulation of PPARα reduced serum amiodarone concentrations in normal mice. Serum amiodarone concentrations were higher in mice without PPARα expression given at 40-80 mg/kg amiodarone doses. These results are consistent with a protective influence of PPARα in reducing amiodarone-induced hepatic toxicity. In addition to PPARα-dependent effects, fenofibrate also demonstrated PPARα-independent actions that suggest a complex interaction modulating both hepatic lipid metabolism and amiodarone disposition. Further studies of the beneficial effect of fenofibrate and the interplay between lipid metabolism and amiodarone pharmacokinetics are required.

A procedure for creating a frailty index based on deficit accumulation in aging mice.

This study developed an approach to quantify frailty with a frailty index (FI) and investigated whether age-related changes in contractions, calcium transients, and ventricular myocyte length were more prominent in mice with a high FI. The FI combined 31 variables that reflect different aspects of health in middle-aged (∼12 months) and aged (∼30 months) mice of both sexes. Aged animals had a higher FI than younger animals (FI = 0.43 ± 0.03 vs 0.08 ± 0.02, p < .001, n = 12). Myocyte hypertrophy increased by 30%-50% as the FI increased in aged animals. Peak contractions decreased more than threefold from lowest to highest FI values in aged mice (p < .037), but calcium transients were unaffected. Similar results were seen with an FI based on eight noninvasive variables identified as underlying factors. These results show that an FI can be developed for murine models and suggest that age-associated changes in myocytes are more prominent in animals with a high FI.

Disruption of the chemokine-like receptor-1 (CMKLR1) gene is associated with reduced adiposity and glucose intolerance.

Adipose tissue secretes a variety of bioactive signaling molecules, termed adipokines, which regulate numerous biological functions including appetite, energy balance, glucose homeostasis, and inflammation. Chemerin is a novel adipokine that regulates adipocyte differentiation and metabolism by binding to and activating the G protein-coupled receptor, chemokine like receptor-1 (CMKLR1). In the present study, we investigated the impact of CMKLR1 deficiency on adipose development, glucose homeostasis, and inflammation in vivo. Herein we report that regardless of diet (low or high fat), CMKLR1(-/-) mice had lower food consumption, total body mass, and percent body fat compared with wild-type controls. CMKLR1(-/-) mice also exhibited decreased hepatic and white adipose tissue TNFα and IL-6 mRNA levels coincident with decreased hepatic dendritic cell infiltration, decreased adipose CD3+ T cells, and increased adipose natural killer cells. CMKLR1(-/-) mice were glucose intolerant compared with wild-type mice, and this was associated with decreased glucose stimulated insulin secretion as well as decreased skeletal muscle and white adipose tissue glucose uptake. Collectively these data provide compelling evidence that CMKLR1 influences adipose tissue development, inflammation, and glucose homeostasis and may contribute to the metabolic derangement characteristic of obesity and obesity-related diseases.

Chemerin: at the crossroads of inflammation and obesity.

Chemerin is a secreted protein with a complex but well-established role in immune function. Parallel lines of investigation also support the notion that chemerin is a novel adipokine that regulates adipocyte development and metabolic function as well as glucose metabolism in liver and skeletal muscle tissues. A growing body of human experimental data indicates that serum chemerin levels are elevated in patients with obesity and that they exhibit a positive correlation with various aspects of the metabolic syndrome. Thus, the dual role of chemerin in inflammation and metabolism might provide a link between chronic inflammation and obesity, as well as obesity-related disorders such as type 2 diabetes and cardiovascular disease.

Chemerin exacerbates glucose intolerance in mouse models of obesity and diabetes.

Obesity, characterized by an excess of adipose tissue, is an established risk factor for cardiovascular disease and type 2 diabetes. Different mechanisms linking obesity with these comorbidities have been postulated but remain poorly understood. Adipose tissue secretes a number of hormone-like compounds, termed adipokines, that are important for the maintenance of normal glucose metabolism. Alterations in the secretion of adipokines with obesity are believed to contribute to the undesirable changes in glucose metabolism that ultimately result in the development of type 2 diabetes. In the present study, we have shown that serum levels of the novel adipokine chemerin are significantly elevated in mouse models of obesity/diabetes. The expression of chemerin and its receptors, chemokine-like receptor 1, chemokine (C-C motif) receptor-like 2, and G protein-coupled receptor 1 are altered in white adipose, skeletal muscle, and liver tissue of obese/diabetic mice. Administration of exogenous chemerin exacerbates glucose intolerance, lowers serum insulin levels, and decreases tissue glucose uptake in obese/diabetic but not normoglycemic mice. Collectively, these data indicate that chemerin influences glucose homeostasis and may contribute to the metabolic derangements characteristic of obesity and type 2 diabetes.

Serum chemerin levels vary with time of day and are modified by obesity and tumor necrosis factor-{alpha}.

Chemerin is an adipokine with important regulatory roles in adipogenesis. In humans, serum total chemerin (i.e. prochemerin plus chemerin) levels are positively associated with body mass index and metabolic syndrome. However, the mechanisms that increase serum chemerin concentration are unknown. We hypothesized that chronic low-grade inflammation that occurs in obesity promotes chemerin production by adipocytes. Consistent with this, TNFalpha treatment of 3T3-L1 adipocytes increased bioactive chemerin levels in the cell media as detected using a CMKLR1 cell-based bioassay. This effect was blocked by the protein synthesis inhibitor cycloheximide and protein secretion inhibitor brefeldin A, indicating that TNFalpha may enhance prochemerin synthesis and secretion from adipocytes. In vivo, TNFalpha produced a time-dependent increase in serum total chemerin and bioactive chemerin. Bioactive chemerin was produced by primary mouse adipocytes and hepatocytes. Only primary adipocyte-derived chemerin was responsive to TNFalpha regulation implicating adipocytes as a potential source of elevated serum chemerin after TNFalpha exposure in vivo. In lean mice, serum total chemerin levels oscillated with peak levels occurring during daytime and trough levels at night. Comparatively, leptin- and leptin receptor-deficient obese mice, which have elevated adipose tissue expression of TNFalpha, displayed elevated serum total chemerin levels with an enhanced oscillatory pattern. In summary, our novel results identified TNFalpha as a positive regulator of adipocyte-derived chemerin. We corroborate the finding of elevated chemerin in obese humans by identifying elevated serum levels of total chemerin in two obese mouse models with a corresponding alteration in the rhythmic pattern of serum chemerin levels.