Pharmacological modulation of RORα controls fat browning, adaptive thermogenesis, and body weight in mice.

Beiging is an attractive therapeutic strategy to fight against obesity and its side metabolic complications. The loss of function of the nuclear transcription factor RORα has been related to a lean phenotype with higher thermogenesis in sg/sg mice lacking this protein. Here we show that pharmacological modulation of RORα activity exerts reciprocal and cell-autonomous effect on UCP1 expression ex vivo, in cellulo, and in vivo. The RORα inverse-agonist SR3335 upregulated UCP1 expression in brown and subcutaneous white adipose tissue (scWAT) explants of wild-type (WT) mice, whereas the RORα agonist SR1078 had the opposite effect. We confirmed the reciprocal action of these synthetic RORα ligands on gene expression, mitochondrial mass, and uncoupled oxygen consumption rate in cultured murine and human adipocytes. Time course analysis revealed stepwise variation in gene expression, first of TLE3, an inhibitor of the thermogenic program, followed by a reciprocal effect on PRDM16 and UCP1. Finally, RORα ligands were shown to be useful tools to modulate in vivo UCP1 expression in scWAT with associated changes in this fat depot mass. SR3335 and SR1078 provoked the opposite effects on the WT mice body weight, but without any effect on sg/sg mice. This slimming effect of SR3335 was related to an increased adaptive thermogenesis of the mice, as assessed by the rectal temperature of cold-stressed mice and induction of UCP1 in scWAT, as well as by indirect calorimetry in presence or not of a ß3-adrenoceptor agonist. These data confirmed that RORα ligands could be useful tools to modulate thermogenesis and energy homeostasis.NEW & NOTEWORTHY The regulation of adipose tissue browning was not fully deciphered and required further studies explaining how the regulation of this process may be of interest for tackling obesity and related metabolic disorders. Our data confirmed the involvement of the transcription factor RORα in the regulation of nonshivering thermogenesis, and importantly, revealed the possibility to in vivo modulate its activity by synthetic ligands with beneficial consequences on fat mass and body weight of the mice.

Adipocyte Glucocorticoid Receptor Deficiency Promotes Adipose Tissue Expandability and Improves the Metabolic Profile Under Corticosterone Exposure.

Widely used for their anti-inflammatory and immunosuppressive properties, glucocorticoids are nonetheless responsible for the development of diabetes and lipodystrophy. Despite an increasing number of studies focused on the adipocyte glucocorticoid receptor (GR), its precise role in the molecular mechanisms of these complications has not been elucidated. In keeping with this goal, we generated a conditional adipocyte-specific murine model of GR invalidation (AdipoGR knockout [KO] mice). Interestingly, when administered a corticosterone treatment to mimic hypercorticism conditions, AdipoGR-KO mice exhibited an improved glucose tolerance and insulin sensitivity. This was related to the adipose-specific activation of the insulin-signaling pathway, which contributed to fat mass expansion, as well as a shift toward an anti-inflammatory macrophage polarization in adipose tissue of AdipoGR-KO animals. Moreover, these mice were protected against ectopic lipid accumulation in the liver and displayed an improved lipid profile, contributing to their overall healthier phenotype. Altogether, our results indicate that adipocyte GR is a key factor of adipose tissue expansion and glucose and lipid metabolism control, which should be taken into account in the further design of adipocyte GR-selective modulators.

The nuclear retinoid-related orphan receptor RORα controls circadian thermogenic programming in white fat depots.

The RORα-deficient staggerer (sg/sg) mouse is lean and resistant to diet-induced obesity. Its thermogenic activity was shown to be increased not only in brown adipose tissue (BAT), but also in subcutaneous white adipose tissue (WAT) where UCP1 content was enhanced, however, without Prdm16 coexpression. Our observation of partial multilocular lipid morphology of WAT in sg/sg mice both in the inguinal and perigonadal sites led us to focus on the phenotype of both fat depots. Because RORα is a nuclear factor acting in the clock machinery, we looked at the circadian expression profile of genes involved in thermogenesis and browning in WAT and BAT depots of sg/sg and WT mice, through real-time quantitative PCR and western blotting. This 24-h period approach revealed both a rhythmic expression of thermogenic genes in WAT and an increased browning of all the WAT depots tested in sg/sg mice that indeed involved the canonical browning process (through induction of Pgc-1α and Prdm16). This was associated with an enhanced isoproterenol-induced oxygen consumption rate of WAT explants from sg/sg mice, which was reproducible in WT explants by treatment with a RORα inverse agonist SR 3335, that induced a parallel increase in the UCP1 protein. Inhibitors of browning differentiation, such as TLE3 and RIP140, could be new targets of RORα that would be rather implicated in the whitening of adipocytes. Our study showed the pivotal role of RORα as an inhibitor of the thermogenic program in WAT, the role that could be counteracted in vivo with the RORα antagonists currently in development.

Depot-Specific Response of Adipose Tissue to Diet-Induced Inflammation: The Retinoid-Related Orphan Receptor α (RORα) Involved?

OBJECTIVE: Epididymal adipose tissue (EAT), a visceral fat depot, is more closely associated with metabolic dysfunction than inguinal adipose tissue (IAT), a subcutaneous depot. This study evaluated whether the nuclear receptor RORα, which controls inflammatory processes, could be implicated. METHODS: EAT and IAT were compared in a RORα loss-of-function mouse (sg/sg) and in wild-type (WT) littermates, fed a standard diet (SD) or a Western diet (WD), to evaluate the impact of RORα expression on inflammatory status and on insulin sensitivity (IS) of each fat depot according to the diet. RESULTS: Sg/sg mice fed the SD exhibited a decreased inflammatory status and a higher IS in their fat depots than WT mice. WD-induced obesity had distinct effects on the two fat depots. In WT mice, EAT exhibited increased inflammation and insulin resistance while IAT showed reduced inflammation and improved IS, together with a depot-specific increase of RORα, and its target gene IκBα, in the stroma vascular fraction (SVF). Conversely, in sg/sg mice, WD increased inflammation and lowered IS of IAT but not of EAT. CONCLUSIONS: These findings suggest an anti-inflammatory role for RORα in response to WD, which occurs at the level of SVF of IAT, thus possibly contributing to the "healthy" expansion of IAT.

NOV/CCN3: A New Adipocytokine Involved in Obesity-Associated Insulin Resistance.

Identification of new adipokines that potentially link obesity to insulin resistance represents a major challenge. We recently showed that NOV/CCN3, a multifunctional matricellular protein, is synthesized and secreted by adipose tissue, with plasma levels highly correlated with BMI. NOV involvement in tissue repair, fibrotic and inflammatory diseases, and cancer has been previously reported. However, its role in energy homeostasis remains unknown. We investigated the metabolic phenotype of NOV(-/-) mice fed a standard or high-fat diet (HFD). Strikingly, the weight of NOV(-/-) mice was markedly lower than that of wild-type mice but only on an HFD. This was related to a significant decrease in fat mass associated with an increased proportion of smaller adipocytes and to a higher expression of genes involved in energy expenditure. NOV(-/-) mice fed an HFD displayed improved glucose tolerance and insulin sensitivity. Interestingly, the absence of NOV was associated with a change in macrophages profile (M1-like to M2-like), in a marked decrease in adipose tissue expression of several proinflammatory cytokines and chemokines, and in enhanced insulin signaling. Conversely, NOV treatment of adipocytes increased chemokine expression. Altogether, these results show that NOV is a new adipocytokine that could be involved in obesity-associated insulin-resistance.

The nuclear retinoid-related orphan receptor-α regulates adipose tissue glyceroneogenesis in addition to hepatic gluconeogenesis.

Circadian rhythms have an essential role in feeding behavior and metabolism. RORα is a nuclear receptor involved in the interface of the circadian system and metabolism. The adipocyte glyceroneogenesis pathway derives free fatty acids (FFA) liberated by lipolysis to reesterification into triglycerides, thus regulating FFA homeostasis and fat mass. Glyceroneogenesis shares with hepatic gluconeogenesis the key enzyme phosphoenolpyruvate carboxykinase c (PEPCKc), whose gene is a RORα target in the liver. RORα-deficient mice (staggerer, ROR(sg/sg)) have been shown to exhibit a lean phenotype and fasting hypoglycemia for unsolved reasons. In the present study, we investigated whether adipocyte glyceroneogenesis might also be a target pathway of RORα, and we further evaluated the role of RORα in hepatocyte gluconeogenesis. In vivo investigations comparing ROR(sg/sg) mice with their wild-type (WT) littermates under fasting conditions demonstrated that, in the absence of RORα, the release of FFA into the bloodstream was altered and the rise in glycemia in response to pyruvate reduced. The functional analysis of each pathway, performed in adipose tissue or liver explants, confirmed the impairment of adipocyte glyceroneogenesis and liver gluconeogenesis in the ROR(sg/sg) mice; these reductions of FFA reesterification or glucose production were associated with decreases in PEPCKc mRNA and protein levels. Treatment of explants with RORα agonist or antagonist enhanced or inhibited these pathways, respectively, in tissues isolated from WT but not ROR(sg/sg) mice. Our results indicated that both adipocyte glyceroneogenesis and hepatocyte gluconeogenesis were regulated by RORα. This study demonstrates the physiological function of RORα in regulating both glucose and FFA homeostasis.

Plasma NOV/CCN3 levels are closely associated with obesity in patients with metabolic disorders.

OBJECTIVE: Evidence points to a founder of the multifunctional CCN family, NOV/CCN3, as a circulating molecule involved in cardiac development, vascular homeostasis and inflammation. No data are available on the relationship between plasma NOV/CCN3 levels and cardiovascular risk factors in humans. This study investigated the possible relationship between plasma NOV levels and cardiovascular risk factors in humans. METHODS: NOV levels were measured in the plasma from 594 adults with a hyperlipidemia history and/or with lipid-lowering therapy and/or a body mass index (BMI) >30 kg/m(2). Correlations were measured between NOV plasma levels and various parameters, including BMI, fat mass, and plasma triglycerides, cholesterol, glucose, and C-reactive protein. NOV expression was also evaluated in adipose tissue from obese patients and rodents and in primary cultures of adipocytes and macrophages. RESULTS: After full multivariate adjustment, we detected a strong positive correlation between plasma NOV and BMI (r = 0.36 p<0.0001) and fat mass (r = 0.33 p<0.0005). According to quintiles, this relationship appeared to be linear. NOV levels were also positively correlated with C-reactive protein but not with total cholesterol, LDL-C or blood glucose. In patients with drastic weight loss induced by Roux-en-Y bariatric surgery, circulating NOV levels decreased by 28% (p<0.02) and 48% (p<0.0001) after 3 and 6 months, respectively, following surgery. In adipose tissue from obese patients, and in human primary cultures NOV protein was detected in adipocytes and macrophages. In mice fed a high fat diet NOV plasma levels and its expression in adipose tissue were also significantly increased compared to controls fed a standard diet. CONCLUSION: Our results strongly suggest that in obese humans and mice plasma NOV levels positively correlated with NOV expression in adipose tissue, and support a possible contribution of NOV to obesity-related inflammation.

Glyceroneogenesis is inhibited through HIV protease inhibitor-induced inflammation in human subcutaneous but not visceral adipose tissue.

Glyceroneogenesis, a metabolic pathway that participates during lipolysis in the recycling of free fatty acids to triglycerides into adipocytes, contributes to the lipid-buffering function of adipose tissue. We investigated whether glyceroneogenesis could be affected by human immunodeficiency virus (HIV) protease inhibitors (PIs) responsible or not for dyslipidemia in HIV-infected patients. We treated explants obtained from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) depots from lean individuals. We observed that the dyslipidemic PIs nelfinavir, lopinavir and ritonavir, but not the lipid-neutral PI atazanavir, increased lipolysis and decreased glyceroneogenesis, leading to an increased release of fatty acids from SAT but not from VAT. At the same time, dyslipidemic PIs decreased the amount of perilipin and increased interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secretion in SAT but not in VAT. Parthenolide, an inhibitor of the NFκB pathway, counteracted PI-induced increased inflammation and decreased glyceroneogenesis. IL-6 (100 ng) inhibited the activity of phosphoenolpyruvate carboxykinase, the key enzyme of glyceroneogenesis, in SAT but not in VAT. Our data show that dyslipidemic but not lipid-neutral PIs decreased glyceroneogenesis as a consequence of PI-induced increased inflammation in SAT that could have an affect on adipocytes and/or macrophages. These results add a new link between fat inflammation and increased fatty acids release and suggest a greater sensitivity of SAT than VAT to PI-induced inflammation.

Adipose tissue as a target of HIV-1 antiretroviral drugs. Potential consequences on metabolic regulations.

Adipose tissue redistribution occurred at first in HIV-infected patients about 15 years ago after initiation of combination antiretroviral treatment (ART) and the responsibility of drugs was rapidly considered. This lipodystrophic syndrome can associate lipoatrophy, affecting subcutaneous adipose tissue in priority with fat hypertrophy, in particular in the upper part of the body, and metabolic alterations, dyslipidemia and altered glucose tolerance with insulin resistance. The primary role of thymidine analogue reverse transcriptase inhibitors (tNRTI) in peripheral lipoatrophy has been clearly shown in vitro and in vivo, these drugs inducing a severe mitochondrial dysfunction and an increased oxidative stress together with fat inflammation leading to fat loss. In vitro and in vivo studies suggest that some protease inhibitors (PI) or non-NRTIs also exert adverse effects on adipocytes and could act in synergy to amplify the effect of tNRTI. While severe lipoatrophy is now less prevalent in HIV-infected patients, fat hypertrophy is frequently observed: a role for drugs from the different classes acting in synergy to induce fat hyperplasia and hypertrophy is suggested, with milder mitochondrial dysfunction but increased inflammation and activation of the cortisol system. In addition, it is now considered that long-term viral infection, even if controlled, could induce low-grade inflammation and prepare fat to the deleterious effect of ART. Both lipoatrophy and lipohypertrophy are involved in metabolic disorders and increased cardio-metabolic risk that likely participate to early aging reported in these patients. ART can also be directly responsible for metabolic alterations. Strategies to revert or reduce lipodystrophy are important to consider in these patients in addition to the required control of the metabolic disorders.

Human lipodystrophies: genetic and acquired diseases of adipose tissue.

Human lipodystrophies represent a heterogeneous group of diseases characterized by generalized or partial fat loss, with fat hypertrophy in other depots when partial. Insulin resistance, dyslipidemia and diabetes are generally associated, leading to early complications. Genetic forms are uncommon: recessive generalized congenital lipodystrophies result in most cases from mutations in the genes encoding seipin or the 1-acyl-glycerol-3-phosphate-acyltransferase 2(AGPAT2). Dominant partial familial lipodystrophies result from mutations in genes encoding the nuclear protein lamin A/C or the adipose transcription factor PPARgamma. Importantly, lamin A/Cmutations are also responsible for metabolic laminopathies, resembling the metabolic syndrome and progeria, a syndrome of premature aging. A number of lipodystrophic patients remain undiagnosed at the genetic level. Acquired lipodystrophy can be generalized, resembling congenital forms, or partial, as the Barraquer-Simons syndrome, with loss of fat in the upper part of the body contrasting with accumulation in the lower part. Although their etiology is generally unknown, they could be associated with signs of autoimmunity. The most common forms of lipodystrophies are iatrogenic. In human immunodeficiency virus-infected patients, some first-generation antiretroviral drugs were strongly related with peripheral lipoatrophy and metabolic alterations. Partial lipodystrophy also characterize patients with endogenous or exogenous long-term corticoid excess. Treatment of fat redistribution can sometimes benefit from plastic surgery. Lipid and glucose alterations are difficult to control leading to early occurrence of diabetic, cardiovascular and hepatic complications.

Cytosolic aspartate aminotransferase, a new partner in adipocyte glyceroneogenesis and an atypical target of thiazolidinedione.

We show that cytosolic aspartate aminotransferase (cAspAT) is involved in adipocyte glyceroneogenesis, a regulated pathway that controls fatty acid homeostasis by promoting glycerol 3-phosphate formation for fatty acid re-esterification during fasting. cAspAT activity, as well as the incorporation of [(14)C]aspartate into the neutral lipid fraction of 3T3-F442A adipocytes was stimulated by the thiazolidinedione (TZD) rosiglitazone. Conversely, the ratio of fatty acid to glycerol released into the medium decreased. Regulation of cAspAT gene expression was specific to differentiated adipocytes and did not require any peroxisome proliferator-activated receptor gamma (PPARgamma)/retinoid X receptor-alpha direct binding. Nevertheless, PPARgamma is indirectly necessary for both cAspAT basal expression and TZD responsiveness because they are, respectively, diminished and abolished by ectopic overexpression of a dominant negative PPARgamma. The cAspAT TZD-responsive site was restricted to a single AGGACA hexanucleotide located at -381 to -376 bp whose mutation impaired the specific RORalpha binding. RORalpha ectopic expression activated the cAspAT gene transcription in absence of rosiglitazone, and its protein amount in nuclear extracts is 1.8-fold increased by rosiglitazone treatment of adipocytes. Finally, the amounts of RORalpha and cAspAT mRNAs were similarly increased by TZD treatment of human adipose tissue explants, confirming coordinated regulation. Our data identify cAspAT as a new member of glyceroneogenesis, transcriptionally regulated by TZD via the control of RORalpha expression by PPARgamma in adipocytes.

Rosiglitazone controls fatty acid cycling in human adipose tissue by means of glyceroneogenesis and glycerol phosphorylation.

Control of fatty acid homeostasis is crucial to prevent insulin resistance. During fasting, the plasma fatty acid level depends on triglyceride lipolysis and fatty acid re-esterification within fat cells. In rodents, Rosiglitazone controls fatty acid homeostasis by stimulating two pathways in the adipocytes, glyceroneogenesis and glycerol phosphorylation, that provide the glycerol 3-phosphate necessary for fatty acid re-esterification. Here, we analyzed the functionality of both pathways for controlling fatty acid release in subcutaneous adipose tissue samples from lean and overweight women before and after Rosiglitazone ex vivo treatment. In controls, pyruvate, used as a substrate of glyceroneogenesis, could contribute to the re-esterification of up to 65% of the fatty acids released after basal lipolysis, whereas glycerol phosphorylation accounted for only 14 +/- 9%. However, the efficiency of glyceroneogenesis diminished as body mass index (BMI) of women increased. After Rosiglitazone treatment, increase of either pyruvate- or glycerol-dependent fatty acid re-esterification was strictly correlated to that of phosphoenolpyruvate carboxykinase and glycerol kinase, the key enzymes of each pathway, but depended on BMI of the women. Whereas the Rosiglitazone responsiveness of glyceroneogenesis was rather constant according to the BMI of the women, glycerol phosphorylation was mostly enhanced in lean women (BMI < 27). Overall, these data indicate that, whereas glyceroneogenesis is more utilized than glycerol phosphorylation for fatty acid re-esterification in human subcutaneous adipose tissue in the physiological situation, both are solicited in response to Rosiglitazone but with lower efficiency when BMI is increased.

Proposed involvement of adipocyte glyceroneogenesis and phosphoenolpyruvate carboxykinase in the metabolic syndrome.

Elevated concentration of plasma non-esterified fatty acids (NEFA) is now recognized as a key factor in the onset of insulin-resistance and type 2 diabetes mellitus. During fasting, circulating NEFAs arise from white adipose tissue (WAT) as a consequence of lipolysis from stored triacylglycerols. However, a significant part of these FAs (30-70%) is re-esterified within the adipocyte, so that a recycling occurs and net FA output is much less than << true >> lipolysis. Indeed, a balance between two antagonistic processes, lipolysis and FA re-esterification, controls the rate of net FA release from WAT. During fasting, re-esterification requires glyceroneogenesis defined as the de novo synthesis of glycerol-3-P from pyruvate, lactate or certain amino acids. The key enzyme in this process is the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C; EC 4.1.1.32). Recent advance has stressed the role of glyceroneogenesis and of PEPCK-C in FA release from WAT. Results indicate that glyceroneogenesis is indeed important to lipid homeostasis and that a disregulation in this pathway may have profound pathophysiological effects. The present review focuses on the regulation of glyceroneogenesis and of PEPCK-C gene expression and activity by FAs, retinoic acids, glucocorticoids and the hypolipidemic class of drugs, thiazolidinediones.

Disregulated glyceroneogenesis: PCK1 as a candidate diabetes and obesity gene.

Genetics and diet interact to cause type 2 diabetes mellitus and obesity. PCK1 has been implicated as one of many genes associated with type 2 diabetes mellitus. The common assumption is that mutations in PCK1 lead to excessive glucose production through hepatic gluconeogenesis. However, there is an alternative explanation, wherein mutations at the PCK1 locus could selectively affect PCK1 expression in adipose tissue. The result would be changes in glyceroneogenesis that would affect the storage and release of fatty acids. Here, we present the novel hypothesis that a variety of phenotypes could arise from mutations of the various tissue-specific control elements of PCK1. We also suggest specific quantitative metabolic traits that would accompany mutations that selectively affect PCK1 expression in adipose tissue.

Thiazolidinediones block fatty acid release by inducing glyceroneogenesis in fat cells.

Thiazolidinediones are used to treat type 2 diabetes mellitus because they decrease plasma glucose, insulin, triglyceride, and fatty acid levels. Thiazolidinediones are agonists for peroxisome proliferator-activated receptor gamma, a nuclear receptor that is highly expressed in fat tissue. We identify glyceroneogenesis as a target of thiazolidinediones in cultured adipocytes and fat tissues of Wistar rats. The activation of glyceroneogenesis by thiazolidinediones occurs mainly in visceral fat, the same fat depot that is specifically implicated in the progression of obesity to type 2 diabetes. The increase in glyceroneogenesis is a result of the induction of its key enzyme, phosphoenolpyruvate carboxykinase, whose gene expression is peroxisome proliferator-activated receptor gamma-dependent in adipocytes. The main role of this metabolic pathway is to allow the re-esterification of fatty acids via a futile cycle in adipocytes, thus lowering fatty acid release into the plasma. The importance of such a fatty acid re-esterification process in the control of lipid homeostasis is highlighted by the existence of a second thiazolidinedione-induced pathway involving glycerol kinase. We show that glyceroneogenesis accounts for at least 75% of the whole thiazolidinedione effect. Because elevated plasma fatty acids promote insulin resistance, these results suggest that the glyceroneogenesis-dependent fatty acid-lowering effect of thiazolidinediones could be an essential aspect of the antidiabetic action of these drugs.

Regulation of glyceroneogenesis and phosphoenolpyruvate carboxykinase by fatty acids, retinoic acids and thiazolidinediones: potential relevance to type 2 diabetes.

Recent studies brought adipocyte glyceroneogenesis back to the fore as an important pathway in fatty acid homeostasis and underlined the key role played by cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in this pathway. The present review analyses the mechanisms by which a series of hormones and nutrients affect PEPCK-C gene transcription and glyceroneogenesis and describes evidence for disregulation of this pathway in type 2 diabetes.

Glyceroneogenesis comes of age.

Glyceroneogenesis is a generally ignored metabolic pathway that occurs in adipose tissues and liver of mammalian species. This short review highlights a series of recent discoveries showing that glyceroneogenesis is important in lipid homeostasis.