Alternative mRNA splicing produces a novel biologically active short isoform of PGC-1alpha.

The transcriptional co-activator PGC-1alpha regulates functional plasticity in adipose tissue by linking sympathetic input to the transcriptional program of adaptive thermogenesis. We report here a novel truncated form of PGC-1alpha (NT-PGC-1alpha) produced by alternative 3' splicing that introduces an in-frame stop codon into PGC-1alpha mRNA. The expressed protein includes the first 267 amino acids of PGC-1alpha and 3 additional amino acids from the splicing insert. NT-PGC-1alpha contains the transactivation and nuclear receptor interaction domains but is missing key domains involved in nuclear localization, interaction with other transcription factors, and protein degradation. Expression and subcellular localization of NT-PGC-1alpha are dynamically regulated in the context of physiological signals that regulate full-length PGC-1alpha, but the truncated domain structure conveys unique properties with respect to protein-protein interactions, protein stability, and recruitment to target gene promoters. Therefore, NT-PGC-1alpha is a co-expressed, previously unrecognized form of PGC-1alpha with functions that are both unique from and complementary to PGC-1alpha.

Direct adipotropic actions of atorvastatin: differentiation state-dependent induction of apoptosis, modulation of endocrine function, and inhibition of glucose uptake.

Statins exert anti-inflammatory, anti-atherogenic actions. The mechanisms responsible for these effects remain only partially elucidated. Diabetes and obesity are characterized by low-grade inflammation. Metabolic and endocrine adipocyte dysfunction is known to play a crucial role in the development of these disorders and the related cardiovascular complications. Thus, direct modulation of adipocyte function may represent a mechanism of pleiotropic statin actions. We investigated effects of atorvastatin on apoptosis, differentiation, endocrine, and metabolic functions in murine white and brown adipocyte lines. Direct exposure of differentiating preadipocytes to atorvastatin strongly reduced lipid accumulation and diminished protein expression of the differentiation marker CCAAT/enhancer binding protein-beta (CEBP-beta). In fully differentiated adipocytes, however, lipid accumulation remained unchanged after chronic atorvastatin treatment. Furthermore, cell viability was reduced in response to atorvastatin treatment in proliferating and differentiating preadipocytes, but not in differentiated cells. Moreover, atorvastatin induced apoptosis and inhibited protein kinase B (AKT) phosphorylation in proliferating and differentiating preadipocytes, but not in differentiated adipocytes. On the endocrine level, direct atorvastatin treatment of differentiated white adipocytes enhanced expression of the pro-inflammatory adipokine interleukin-6 (IL-6), and downregulated expression of the insulin-mimetic and anti-inflammatory adipokines visfatin and adiponectin. Finally, these direct adipotropic endocrine effects of atorvastatin were paralleled by the acute inhibition of insulin-induced glucose uptake in differentiated white adipocytes, while protein expression of the thermogenic uncoupling protein-1 (UCP-1) in brown adipocytes remained unchanged. Taken together, our data for the first time demonstrate direct differentiation state-dependent effects of atorvastatin including apoptosis, modulation of pro-inflammatory and glucostatic adipokine expression, and insulin resistance in adipose cells. These differential interactions may explain variable clinical observations.

Adipose tissue as source and target for novel therapies.

The physiology of adipose tissue has a key role in the pathogenesis of the metabolic syndrome and related cardiovascular disorders. Three main functions of adipocytes have been proposed to explain this role: the secretion of adipocyte-derived hormones (so-called adipokines), energy dissipation/thermogenesis, and energy storage. There is mounting evidence that this repertoire of actions and reactions contributes to whole-body glucose and energy homeostasis, the control of blood pressure, immune-system function, haemostasis and atherosclerosis. In this article we highlight the most recent examples of adipocyte-based therapies and discuss future pharmacological options for exploiting this triad of adipocyte functions.

Dopamine directly increases mitochondrial mass and thermogenesis in brown adipocytes.

Brown adipose tissue (BAT) is key to energy homeostasis. By virtue of its thermogenic potential, it may dissipate excessive energy, regulate body weight and increase insulin sensitivity. Catecholamines are critically involved in the regulation of BAT thermogenesis, yet research has focussed on the effects of noradrenaline and adrenaline. Some evidence suggests a role of dopamine (DA) in BAT thermogenesis, but the cellular mechanisms involved have not been addressed. We employed our extensively characterised murine brown adipocyte cells. D1-like and D2-like receptors were detectable at the protein level. Stimulation with DA caused an increase in cAMP concentrations. Oxygen consumption rates (OCR), mitochondrial membrane potential (Δψm) and uncoupling protein 1 (UCP1) levels increased after 24 h of treatment with either DA or a D1-like specific receptor agonist. A D1-like receptor antagonist abolished the DA-mediated effect on OCR, Δψm and UCP1. DA induced the release of fatty acids, which did not additionally alter DA-mediated increases of OCR. Mitochondrial mass (as determined by (i) CCCP- and oligomycin-mediated effects on OCR and (ii) immunoblot analysis of mitochondrial proteins) also increased within 24 h. This was accompanied by an increase in peroxisome proliferator-activated receptor gamma co-activator 1 alpha protein levels. Also, DA caused an increase in p38 MAPK phosphorylation and pharmacological inhibition of p38 MAPK abolished the DA-mediated effect on Δψm In summary, our study is the first to reveal direct D1-like receptor and p38 MAPK-mediated increases of thermogenesis and mitochondrial mass in brown adipocytes. These results expand our understanding of catecholaminergic effects on BAT thermogenesis.

Troglitazone treatment increases protein kinase B phosphorylation in skeletal muscle of normoglycemic subjects at risk for the development of type 2 diabetes.

We investigated whether the effect of troglitazone on glucose disposal is associated with altered insulin signaling. Nondiabetic first-degree relatives of type 2 diabetic patients (age 30 +/- 2 years, BMI 30 +/- 1 kg/m(2); n = 20) were randomized in a double-blind manner to 3 months of troglitazone (200 mg/day) or placebo treatment. Before and after treatment, 3-h euglycemic-hyperinsulinemic glucose clamps (40 mU. m(-2). min(-1)) were performed, and muscle biopsies were obtained immediately before and after the clamps. In the biopsies, insulin receptor kinase (IRK) activity, insulin receptor substrate (IRS)-1-associated phosphatidylinositol 3-kinase (PI3K) activity, Ser(473) and Thr(308) phosphorylation of protein kinase B (PKB), and protein expression of IRS-1, IRS-2, phosphoinositol-dependent kinase-1 (PDK-1), PKB, and GLUT-4 were determined. After troglitazone treatment, insulin-stimulated glucose disposal was increased compared with pretreatment and placebo (279 +/- 37 vs. 211 +/- 26 and 200 +/- 25 mg. m(-2). min(-1); both P < 0.05). IRK and PI3K activities were not altered by troglitazone, but PKB Ser(473) phosphorylation was enhanced compared with pretreatment and placebo at the clamp insulin level (138 +/- 36 vs. 77 +/- 16 and 55 +/- 13 internal standard units; both P < 0.05) and with pretreatment at the basal level (31 +/- 9 vs. 14 +/- 4 internal standard units; P < 0.05). PKB Thr(308) phosphorylation also tended to be higher, but this was not statistically significant. Troglitazone did not alter insulin receptor number or IRS-1, IRS-2, PKB, PDK-1, or GLUT-4 protein expression. We conclude that increased PKB phosphorylation may contribute to the insulin-sensitizing effects of thiazolidinediones in human skeletal muscle.

Metformin inhibits leptin secretion via a mitogen-activated protein kinase signalling pathway in brown adipocytes.

Metformin is an anti-diabetic drug with anorexigenic properties. The precise cellular mechanisms of its action are not entirely understood. Adipose tissue has recently been recognized as an important endocrine organ that is pivotal for the regulation of insulin resistance and energy homeostasis. Due to its thermogenic capacity brown adipose tissue contributes to the regulation of energy metabolism and is an attractive target tissue for pharmacological approaches to treating insulin resistance and obesity. Leptin is the prototypic adipocyte-derived hormone inducing a negative energy balance. We investigated effects of metformin on adipocyte metabolism, signalling, and leptin secretion in a brown adipocyte model. Metformin acutely stimulated p44/p42 mitogen-activated protein (MAP) kinase in a dose- (3.2-fold at 1 mmol/l, P< 0.05) as well as time-dependent (3.8-fold at 5 min, P< 0.05) manner. This stimulation was highly selective since phosphorylation of intermediates in the stress kinase, janus kinase (JAK)-signal transducer and activator of transcription (STAT), and phosphatidylinositol (PI) 3-kinase signalling pathways such as p38 MAP kinase, STAT3, and Akt was unaltered. Furthermore, chronic metformin treatment for 12 days dose-dependently inhibited leptin secretion by 35% and 75% at 500 mumol/l and 1 mmol/l metformin respectively (P< 0.01). This reduction was not caused by alterations in adipocyte differentiation. Moreover, the impairment in leptin secretion by metformin was reversible within 48 h after removal of the drug. Pharmacological inhibition of p44/p42 MAP kinase prevented the metformin-induced negative effect on leptin secretion. Taken together, our data demonstrate direct acute effects of metformin on adipocyte signalling and endocrine function with robust inhibition of leptin secretion. They suggest a selective molecular mechanism that may contribute to the anorexigenic effect of this antidiabetic compound.

Ciliary neurotrophic factor influences endocrine adipocyte function: inhibition of leptin via PI 3-kinase.

Ciliary neurotrophic factor (CNTF), originally known for its involvement in the modulation of neuronal growth, has been discovered to exert anorexigenic effects and is currently being investigated in clinical studies for the treatment of obesity and insulin resistance. This neuropeptide acts on the central nervous system. However, we have recently demonstrated direct peripheral effects on adipocyte signalling and thermogenesis. Given the emerging endocrine role of adipose tissue in the regulation of energy homeostasis and insulin resistance, we investigated potential effects of CNTF on leptin expression and secretion. Our study demonstrates a direct inhibition of leptin expression and secretion by acute and chronic CNTF treatment. Furthermore, we demonstrate a differentiation- and Janus kinase 2 (JAK2)-independent, but phosphatidylinositol 3-kinase-dependent signalling pathway mediating this negative effect. These results provide novel evidence for a role of CNTF in the selective modulation of adipocyte endocrine function which may have important implications for the regulation of energy homeostasis.

Cold-induced alteration of adipokine profile in humans.

Adipose tissue function and sympathetic nervous system (SNS) activity are tightly interconnected. Adipose tissue is densely innervated by the SNS. Adipokines secreted by adipose tissue are implicated in maintaining energy homeostasis, the control of blood pressure, immune system function, hemostasis, and atherosclerosis. Little is known about a direct effect of SNS activation on influencing adipose tissue endocrine function in humans. In 10 lean, healthy male volunteers, SNS was activated by whole-body exposure to cold for 2 hours; a group of 10 subjects served as controls. Vital parameters were evaluated, plasma adipokine levels were measured, and adipokine gene expression in subcutaneous abdominal adipose tissue was determined. Cold exposure caused an increase in cold sensation and a drop in body temperature and heart rate. Norepinephrine, but not epinephrine, plasma levels were elevated. Adiponectin plasma concentrations were acutely and significantly decreased. There was a trend of increased monocyte chemoattractant protein-1 plasma concentrations. Interleukin-6 and leptin levels increased and decreased, respectively, in both groups. Vascular endothelial growth factor plasma levels were unaffected. Subcutaneous adipokine gene expression was unchanged. Cold exposure caused SNS activation and differentially influenced adipokine secretion. Adiponectin levels were acutely reduced, whereas monocyte chemoattractant protein-1 concentrations tended to increase. No specific changes in leptin and IL-6 concentrations were detectable. The observed alterations appeared to be posttranscriptional because adipokine gene expression was found to be unaltered.

Arginine-vasopressin directly promotes a thermogenic and pro-inflammatory adipokine expression profile in brown adipocytes.

Arginine-vasopressin (AVP) - via activation of the hypothalamic-pituitary-adrenal (HPA) axis - may play a role in the regulation of energy homeostasis and related cardiovascular complications. Brown adipose tissue (BAT) - via dissipation of energy in the form of heat - contributes to whole body energy balance. BAT expresses vasopressin receptors. We investigated direct effects of AVP on brown adipose endocrine and metabolic functions. UCP-1 protein expression in differentiated brown adipocytes was induced after acute exposure of adipocytes to AVP. This effect was time-dependent with a maximum increase after 8h. AVP also induced a time- and dose-dependent increase in p38 MAP kinase phosphorylation. Pharmacological inhibition of p38 MAP kinase with SB 202190 abolished the induction of UCP-1 protein expression. Furthermore, while acute AVP treatment enhanced mRNA expression of MCP-1 and IL-6, adiponectin mRNA expression was reduced. Yet, on the level of intracellular glucose uptake, there was no AVP-induced change of adipose insulin-induced glucose uptake. Finally, there was no difference in lipid accumulation between control and AVP-treated cells. Taken together, our data demonstrate direct effects of AVP on thermogenic, inflammatory, and glucoregulatory gene expression in brown adipocytes, thus expanding the hitherto known spectrum of this neuropeptides's biological effects and suggesting a direct adipotropic role as a stress-promoting factor.

Putting fat cells onto the road map to novel therapeutic strategies.

Fat cells are traditionally considered dormant cells, quietly functioning in lipid accumulation and lipolysis. In recent years, fat cells received a glowing appraisal and were found to be an important player in energy homeostasis, tumorigenesis, immunity, and reproduction, via their endocrine and regulatory functions. Fat cells secrete adipokines, many of which are inflammation-related peptides such as cytokines and cytokine-like molecules.