Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis.

Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of ß-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.

ILRUN, a Human Plasma Lipid GWAS Locus, Regulates Lipoprotein Metabolism in Mice.

RATIONALE: Single-nucleotide polymorphisms near the ILRUN (inflammation and lipid regulator with ubiquitin-associated-like and NBR1 [next to BRCA1 gene 1 protein]-like domains) gene are genome-wide significantly associated with plasma lipid traits and coronary artery disease (CAD), but the biological basis of this association is unknown. OBJECTIVE: To investigate the role of ILRUN in plasma lipid and lipoprotein metabolism. METHODS AND RESULTS: ILRUN encodes a protein that contains a ubiquitin-associated-like domain, suggesting that it may interact with ubiquitinylated proteins. We generated mice globally deficient for Ilrun and found they had significantly lower plasma cholesterol levels resulting from reduced liver lipoprotein production. Liver transcriptome analysis uncovered altered transcription of genes downstream of lipid-related transcription factors, particularly PPARα (peroxisome proliferator-activated receptor alpha), and livers from Ilrun-deficient mice had increased PPARα protein. Human ILRUN was shown to bind to ubiquitinylated proteins including PPARα, and the ubiquitin-associated-like domain of ILRUN was found to be required for its interaction with PPARα. CONCLUSIONS: These findings establish ILRUN as a novel regulator of lipid metabolism that promotes hepatic lipoprotein production. Our results also provide functional evidence that ILRUN may be the casual gene underlying the observed genetic associations with plasma lipids at 6p21 in human.

FoxO1 inhibits transcription and membrane trafficking of epithelial Na+ channel.

The epithelial Na(+) channel (ENaC), regulated by insulin, is of fundamental importance in the control of Na(+) reabsorption in the distal nephron. The potential role of Forkhead box O1 (FoxO1), downstream of insulin signaling, in the regulation of ENaC remains to be investigated. Here, we found that the overexpression of a constitutively active form of FoxO1 (ADA-FoxO1) suppressed the mRNA level of the ENaC α subunit (α-ENaC; also known as SCCN1A) and the apical density of ENaC in mouse cortical collecting duct (mCCD) cells. Conversely, knockdown of FoxO1 increased the apical membrane levels of α-ENaC and Na(+) transport under basal conditions. Insulin elevated α-ENaC expression and induced FoxO1 phosphorylation; however, the increase in α-ENaC and phosphorylated FoxO1 expression observed with insulin treatment was blunted ∼ 60% in cells expressing ADA-FoxO1. Moreover, insulin induced the interaction between phosphorylated FoxO1 and 14-3-3ε, indicating that FoxO1 phosphorylation promotes ENaC membrane trafficking by binding to 14-3-3ε. FoxO1 also suppressed activity of the α-ENaC promoter, and the putative FoxO1 target site is located in the -500 to -200 nt region of the α-ENaC promoter. These findings indicate that FoxO1 is a key negative regulatory factor in the insulin-dependent control of ENaC expression and forward trafficking in mCCD epithelia.

Inhibitory effects of peroxisome proliferator-activated receptor γ agonists on collagen IV production in podocytes.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists have beneficial effects on the kidney diseases through preventing microalbuminuria and glomerulosclerosis. However, the mechanisms underlying these effects remain to be fully understood. In this study, we investigate the effects of PPAR-γ agonist, rosiglitazone (Rosi) and pioglitazone (Pio), on collagen IV production in mouse podocytes. The endogenous expression of PPAR-γ was found in the primary podocytes and can be upregulated by Rosi and Pio, respectively, detected by RT-PCR and Western blot. PPAR-γ agonist markedly blunted the increasing of collagen IV expression and extraction in podocytes induced by TGF-ß. In contrast, adding PPAR-γ antagonist, GW9662, to podocytes largely prevented the inhibition of collagen IV expression from Pio treatment. Our data also showed that phosphorylation of Smad2/3 enhanced by TGF-ß in a time-dependent manner was significantly attenuated by adding Pio. The promoter region of collagen IV gene contains one putative consensus sequence of Smad-binding element (SBE) by promoter analysis, Rosi and Pio significantly ameliorated TGF-ß-induced SBE4-luciferase activity. In conclusion, PPAR-γ activation by its agonist, Rosi or Pio, in vitro directly inhibits collagen IV expression and synthesis in primary mouse podocytes. The suppression of collagen IV production was related to the inhibition of TGF-ß-driven phosphorylation of Smad2/3 and decreased response activity of SBEs of collagen IV in PPAR-γ agonist-treated mouse podocytes. This represents a novel mechanistic support regarding PPAR-γ agonists as podocyte protective agents.

Hrd1 participates in the regulation of collagen I synthesis in renal fibrosis.

The production and accumulation of collagen-rich extracellular matrix are common hallmarks during the process of renal fibrogenesis. However, the mechanisms of the regulation of collagen synthesis in renal fibrosis are still unclear. Hrd1, an E3 ubiquitin ligase, plays important roles for protein folding in ER and transport to Golgi. Here, we examined the hypothesis that Hrd1 posttranslationally regulates collagen synthesis in renal interstitial fibrogenesis. Unilateral ureteral obstruction induced Hrd1 expression, predominantly in the renal interstitium and tubular epithelium of fibrotic kidneys. Transforming growth factor ß1, as a key mediator in kidney fibrosis, significantly increased the expressions of Hrd1, α-smooth muscle actin, fibronectin as well as procollagen I and mature collagen I in dose-dependent manner in tubular epithelial cells, suggesting that collagen I maturation might be modulated during renal fibrosis. In cultured renal fibroblasts, Hrd1 knockdown decreased secreted collagen I ~60% in the supernatant of NRK-49F cells. Conversely, Hrd1 overexpression increased secreted collagen I ~1.5-fold. Hrd1 overexpression significantly increased the expressions of both procollagen I and mature collagen I, ~2.2-fold and ~1.8-fold, respectively. However, Hrd1 knockdown markedly decreased the expression of mature collagen I ~80%, while procollagen I expression only was decreased ~21%. Moreover, short interfering RNA-induced knockdown of Sec23A blunted the increase in collagen I expression (both immature and mature form) by Hrd1 overexpression and returned collagen I expression toward control levels. These results indicate that Hrd1 plays an important role in the maturation of collagen I in renal fibrosis, and that Sec23A pathway is required for ER-to-Golgi procollagen trafficking to promote collagen synthesis.

Shared PPARα/γ Target Genes Regulate Brown Adipocyte Thermogenic Function.

Brown adipose tissue (BAT) generates heat to maintain body temperature and suppress obesity. Agonists for nuclear receptors PPARα and PPARγ both affect brown adipocyte function, yet the interplay between these factors in BAT is uncertain. Here, we report that PPARα shares most genomic binding sites with PPARγ, and these common binding sites are more related to BAT function than PPARγ-selective sites without PPARα. Integrating PPARα and PPARγ genomic occupancy with cold-responsive BAT transcriptomes identifies a subset of 16 genes with potential relevance to BAT function. Among these, we focused on the lysosomal protease cathepsin Z (CTSZ) and showed it is necessary for mitochondrial respiration in both mouse and human brown adipocytes. Thus, CTSZ is a shared PPARα/γ target gene in BAT and a regulator of brown adipocyte thermogenic function.

Estrogen negatively regulates the renal epithelial sodium channel (ENaC) by promoting Derlin-1 expression and AMPK activation.

The main functions of the epithelial sodium channel (ENaC) in the kidney distal nephron are mediation of sodium and water balance and stabilization of blood pressure. Estrogen has important effects on sodium and water balance and on premenopausal blood pressure, but its role in the regulation of ENaC function is not fully understood. Female Sprague-Dawley rats were treated with 17ß-estradiol for 6 weeks following bilateral ovariectomy. Plasma estrogen, aldosterone, creatinine, and electrolytes were analyzed, and α-ENaC and derlin-1 protein expression in the kidney was determined by immunohistochemistry and western blotting. The expression levels of α-ENaC, derlin-1, AMPK, and related molecules were also examined by western blotting and real-time PCR in cultured mouse renal collecting duct (mpkCCDc14) epithelial cells following estrogen treatment. Immunofluorescence and coimmunoprecipitation were performed to detect α-ENaC binding with derlin-1 and α-ENaC ubiquitination. The results demonstrated that the loss of estrogen elevated systolic blood pressure in ovariectomized (OVX) rats. OVX rat kidneys showed increased α-ENaC expression but decreased derlin-1 expression. In contrast, estrogen treatment decreased α-ENaC expression but increased derlin-1 expression in mpkCCDc14 cells. Moreover, estrogen induced α-ENaC ubiquitination by promoting the interaction of α-ENaC with derlin-1 and evoked phosphorylation of AMPK in mpkCCDc14 cells. Our study indicates that estrogen reduces ENaC expression and blood pressure in OVX rats through derlin-1 upregulation and AMPK activation.

Warming Induces Significant Reprogramming of Beige, but Not Brown, Adipocyte Cellular Identity.

Beige and brown adipocytes generate heat in response to reductions in ambient temperature. When warmed, both beige and brown adipocytes exhibit morphological "whitening," but it is unknown whether or to what extent this represents a true shift in cellular identity. Using cell-type-specific profiling in vivo, we uncover a unique paradigm of temperature-dependent epigenomic plasticity of beige, but not brown, adipocytes, with conversion from a brown to a white chromatin state. Despite this profound shift in cellular identity, warm whitened beige adipocytes retain an epigenomic memory of prior cold exposure defined by an array of poised enhancers that prime thermogenic genes for rapid response during a second bout of cold exposure. We further show that a transcriptional cascade involving glucocorticoid receptor and Zfp423 can drive warm-induced whitening of beige adipocytes. These studies identify the epigenomic and transcriptional bases of an extraordinary example of cellular plasticity in response to environmental signals.

Adipocyte glucocorticoid receptor is important in lipolysis and insulin resistance due to exogenous steroids, but not insulin resistance caused by high fat feeding.

OBJECTIVE: The critical role of adipose tissue in energy and nutrient homeostasis is influenced by many external factors, including overnutrition, inflammation, and exogenous hormones. Prior studies have suggested that glucocorticoids (GCs) in particular are major drivers of physiological and pathophysiological changes in adipocytes. In order to determine whether these effects directly require the glucocorticoid receptor (GR) within adipocytes, we generated adipocyte-specific GR knockout (AGRKO) mice. METHODS: AGRKO and control mice were fed chow or high fat diet (HFD) for 14 weeks. Alternatively, AGRKO and control mice were injected with dexamethasone for two months. Glucose tolerance, insulin sensitivity, adiposity, lipolysis, thermogenesis, and insulin signaling were assessed. RESULTS: We find that obesity, insulin resistance, and dysglycemia associated with high fat feeding do not require an intact GR in the adipocyte. However, exogenous dexamethasone (Dex) promotes metabolic dysfunction in mice, and this effect is reduced in mice lacking GR in adipocytes. The ability of Dex to promote "whitening" of brown fat is also reduced in these animals. We also show that GR is required for ß-adrenergic and cold stimulation-mediated lipolysis via expression of the key lipolytic enzyme ATGL. CONCLUSIONS: Our data suggest that the GR plays a role in normal adipose physiology via effects on lipolysis and mediates at least some of the adverse effects of exogenous steroids on metabolic function. The data also indicate that intra-adipocyte GR plays less of a role than previously believed in the local and systemic pathology associated with overnutrition.

HRD1-Mediated IGF-1R Ubiquitination Contributes to Renal Protection of Resveratrol in db/db Mice.

Many studies have provided evidence to demonstrate the beneficial renal effects of resveratrol (RESV) due to its antioxidant character and its capacity for activation of surtuin 1. However, the molecular mechanisms underlying the protective role of RESV against kidney injury are still incompletely understood. The present study used Lepr db/db (db/db) and Lepr db/m (db/m) mice as models to evaluate the effect of RESV on diabetic nephropathy (DN). RESV reduced proteinuria and attenuated the progress of renal fibrosis in db/db mice. Treatment with RESV markedly attenuated the diabetes-induced changes in renal superoxide dismutase copper/zinc, superoxide dismutase manganese, catalase, and malonydialdehyde as well as the renal expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), α-smooth muscle actin (α-SMA), and E-cadherin in db/db mice. The kidney expression of the IGF-1 receptor (IGF-1R) was increased in db/db mice, but the expression of 3-hydroxy-3-methylglutaryl reductase degradation (HRD1), a ubiquitin E3 ligase, was significantly decreased in the DN model. RESV treatment dramatically decreased IGF-1R and increased HRD1 expressions, consistent with data obtained with HKC-8 cells. HRD1 physically interacted with IGF-1R in HKC-8 cells and liquid chromatography and tandem mass spectrometry (LC-MS/MS) data supported the concept that IGF-1R is one of the HRD1 substrates. HRD1 promoted the IGF-1R ubiquitination for degradation in HKC-8 cells, and the down-regulation of HRD1 reversed the protective effects of RESV in HKC-8 cells. In summary, we have demonstrated that RESV reduces proteinuria and attenuates the progression of renal fibrosis in db/db mice. These protective effects of RESV on DN were associated with the up-regulation of HRD1, induced by RESV, and the promotion of IGF-1R ubiquitination and degradation.

Hyperuricemia induces hypertension through activation of renal epithelial sodium channel (ENaC).

OBJECTIVES: The mechanisms leading to hypertension associated with hyperuricemia are still unclear. The activity of the distal nephron epithelial sodium channel (ENaC) is an important determinant of sodium balance and blood pressure. Our aim was to investigate whether the effect of hyperuricemia on blood pressure is related to ENaC activation. METHODS: A hyperuricemic model was induced in rats by 2% oxonic acid and 6 mg/dl uric acid (UA). The hyperuricemic rats were co-treated with either 10mg/kg/d benzbromarone (Ben) or 1 mg/kg/d amiloride (Ami). Blood pressure was monitored using a tail-cuff, and blood, urine, and kidney samples were taken. Western blotting and immunohistochemical staining were performed to determine the expressions of ENaC subunits and components of the ENaC Regulatory Complex (ERC) in kidney tissue or mCCD cells. RESULTS: Serum uric acid (SUA) was increased 2.5-3.5 times above normal in hyperuricemic rats after 3 weeks and remained at these high levels until 6 weeks. The in vivo rise in SUA was followed by elevated blood pressure, renal tubulointerstitial injury, and increased expressions of ENaC subunits, SGK1, and GILZ1, which were prevented by Ben treatment. The decrease in urinary Na(+) excretion in hyperuricemic rats was blunted by Ami. UA induced the expression of all three ENaC subunits, SGK1, and GILZ1, and increased Na(+) transport in mCCD cells. Phosphorylation of ERK was significantly decreased in both UA-treated mCCD cells and hyperuricemic rat kidney; this effect was prevented by Ben co-treatment. CONCLUSION: Our findings suggest that elevated serum uric acid could induce hypertension by activation of ENaC and regulation of ERC expression.

HRD1 suppresses the growth and metastasis of breast cancer cells by promoting IGF-1R degradation.

HRD1 (3-hydroxy-3-methylglutaryl reductase degradation) is an E3 ubiquitin ligase. We found that HRD1 was significantly downregulated in 170 breast cancer tissues. Low tumoral HRD1 expression was correlated with clinicopathological characteristics and a shorter survival in breast cancer patients. P65 specifically bound to the HRD1 promoter and inhibited HRD1 expression. Suppression of NF-κB activity reversed IL-6-induced downregulation of HRD1 expression. HRD1 interacted with IGF-1R and promoted its ubiquitination and degradation by the proteasome. Overexpression of HRD1 resulted in the inhibition of growth, migration and invasion of breast cancer cells in vitro and in vivo. Furthermore, HRD1 attenuated IL-6-induced epithelial-mesenchymal transition in MCF10A cells. These findings uncover a novel role for HRD1 in breast cancer.

Hyperuricemia causes pancreatic ß-cell death and dysfunction through NF-κB signaling pathway.

Accumulating clinical evidence suggests that hyperuricemia is associated with an increased risk of type 2 diabetes. However, it is still unclear whether elevated levels of uric acid can cause direct injury of pancreatic ß-cells. In this study, we examined the effects of uric acid on ß-cell viability and function. Uric acid solution or normal saline was administered intraperitoneally to mice daily for 4 weeks. Uric acid-treated mice exhibited significantly impaired glucose tolerance and lower insulin levels in response to glucose challenge than did control mice. However, there were no significant differences in insulin sensitivity between the two groups. In comparison to the islets in control mice, the islets in the uric acid-treated mice were markedly smaller in size and contained less insulin. Treatment of ß-cells in vitro with uric acid activated the NF-κB signaling pathway through IκBα phosphorylation, resulting in upregulated inducible nitric oxide synthase (iNOS) expression and excessive nitric oxide (NO) production. Uric acid treatment also increased apoptosis and downregulated Bcl-2 expression in Min6 cells. In addition, a reduction in insulin secretion under glucose challenge was observed in the uric acid-treated mouse islets. These deleterious effects of uric acid on pancreatic ß-cells were attenuated by benzbromarone, an inhibitor of uric acid transporters, NOS inhibitor L-NMMA, and Bay 11-7082, an NF-κB inhibitor. Further investigation indicated that uric acid suppressed levels of MafA protein through enhancing its degradation. Collectively, our data suggested that an elevated level of uric acid causes ß-cell injury via the NF-κB-iNOS-NO signaling axis.