Anna Foryst-Ludwig

T-lymphocyte Infiltration in Visceral Adipose Tissue. A Primary Event in Adipose Tissue Inflammation and the Development of Obesity-Mediated Insulin Resistance

Background—Adipose tissue inflammation may play a critical role in the pathogenesis of insulin resistance (IR). The present study examined the role of lymphocytes in adipose tissue inflammation and IR. Methods and Results—In a mouse model of obesity-mediated IR, high-fat diet (HFD) induced IR already after 5 weeks, which was associated with a marked T-lymphocyte infiltration in visceral adipose tissue. In contrast, recruitment of macrophages was delayed with an increase of MAC3-positive staining and F4/80 mRNA expression after 10 weeks of HFD, suggesting a dissociation of macrophage invasion into adipose tissue and IR initiation. In patients with type 2 diabetes, lymphocyte content in adipose tissue biopsies significantly correlated with waist circumference, a marker of IR. Immunohistochemical staining of human adipose tissue revealed the presence of mainly CD4-positive lymphocytes as well as macrophage infiltration. Most macrophages were HLA-DR–positive, reflecting activation through IFN&ggr;, a cytokine released from CD4-positive lymphocytes. Conclusions—Proinflammatory T-lymphocytes are present in visceral adipose tissue and may contribute to local inflammatory cell activation before the appearance of macrophages, suggesting that these cells could play an important role in the initiation and perpetuation of adipose tissue inflammation as well as the development of IR.

PPARγ-Activating Angiotensin Type-1 Receptor Blockers Induce Adiponectin

The adipose-specific protein adiponectin has been recently discovered to improve insulin sensitivity. Angiotensin type-1 receptor (AT1R) blockers (ARBs) reduce the incidence of type 2 diabetes mellitus by mostly unknown molecular mechanisms. To identify new antidiabetic mechanisms of ARBs, we studied the regulation of adiponectin by angiotensin II (Ang II) and different ARBs in murine 3T3-L1 adipocytes and obese Zucker rats. Adiponectin protein expression was markedly stimulated by Ang II (5 nmol/L), which was inhibited by blockade of the AT2R, and further enhanced by the ARB irbesartan. Irbesartan-mediated adiponectin upregulation started beyond the concentrations needed for AT1R blockade and was also present in the absence of Ang II, implicating an AT1R-independent mechanism of action. Recently, certain ARBs (irbesartan, telmisartan) were identified as ligands of the peroxisome proliferator-activated receptor (PPAR)&ggr;. Telmisartan also stimulated adiponectin protein expression, whereas the non-PPAR&ggr;-activating ARB eprosartan had no effect. Blockade of PPAR&ggr; activation by the PPAR&ggr; antagonist GW9662 markedly inhibited irbesartan-induced adiponectin expression. Cognate mRNA levels of adiponectin were not affected by ARBs. Kinetic studies using the protein synthesis inhibitor cycloheximide showed that irbesartan prevented the cellular depletion of adiponectin protein. Finally, administration of irbesartan to obese Zucker rats improved insulin sensitivity and attenuated adiponectin serum depletion. The present study demonstrates that AT2R activation and certain ARBs induce adiponectin in adipocytes, which was associated with an improvement of parameters of insulin sensitivity in vivo. ARB-induced adiponectin stimulation is likely to be mediated via PPAR&ggr; activation involving a post-transcriptional mechanism.

Angiotensin II Type 2 Receptor Stimulation A Novel Option of Therapeutic Interference With the Renin-Angiotensin System in Myocardial Infarction?

Background— This study is the first to examine the effect of direct angiotensin II type 2 (AT2) receptor stimulation on postinfarct cardiac function with the use of the novel nonpeptide AT2 receptor agonist compound 21 (C21). Methods and Results— Myocardial infarction (MI) was induced in Wistar rats by permanent ligation of the left coronary artery. Treatment with C21 (0.01, 0.03, 0.3 mg/kg per day IP) was started 24 hours after MI and was continued until euthanasia (7 days after MI). Infarct size was assessed by magnetic resonance imaging, and hemodynamic measurements were performed via transthoracic Doppler echocardiography and intracardiac Millar catheter. Cardiac tissues were analyzed for inflammation and apoptosis markers with immunoblotting and real-time reverse transcription polymerase chain reaction. C21 significantly improved systolic and diastolic ventricular function. Scar size was smallest in the C21-treated rats. In regard to underlying mechanisms, C21 diminished MI-induced Fas-ligand and caspase-3 expression in the peri-infarct zone, indicating an antiapoptotic effect. Phosphorylation of the p44/42 and p38 mitogen-activated protein kinases, both involved in the regulation of cell survival, was strongly reduced after MI but almost completely rescued by C21 treatment. Furthermore, C21 decreased MI-induced serum monocyte chemoattractant protein-1 and myeloperoxidase as well as cardiac interleukin-6, interleukin-1&bgr;, and interleukin-2 expression, suggesting an antiinflammatory effect. Conclusions— Direct AT2 receptor stimulation may be a novel therapeutic approach to improve post-MI systolic and diastolic function by antiapoptotic and antiinflammatory mechanisms.

Metabolic Actions of Estrogen Receptor Beta (ERβ) are Mediated by a Negative Cross-Talk with PPARγ

Estrogen receptors (ER) are important regulators of metabolic diseases such as obesity and insulin resistance (IR). While ERα seems to have a protective role in such diseases, the function of ERβ is not clear. To characterize the metabolic function of ERβ, we investigated its molecular interaction with a master regulator of insulin signaling/glucose metabolism, the PPARγ, in vitro and in high-fat diet (HFD)-fed ERβ -/- mice (βERKO) mice. Our in vitro experiments showed that ERβ inhibits ligand-mediated PPARγ-transcriptional activity. That resulted in a blockade of PPARγ-induced adipocytic gene expression and in decreased adipogenesis. Overexpression of nuclear coactivators such as SRC1 and TIF2 prevented the ERβ-mediated inhibition of PPARγ activity. Consistent with the in vitro data, we observed increased PPARγ activity in gonadal fat from HFD-fed βERKO mice. In consonance with enhanced PPARγ activation, HFD-fed βERKO mice showed increased body weight gain and fat mass in the presence of improved insulin sensitivity. To directly demonstrate the role of PPARγ in HFD-fed βERKO mice, PPARγ signaling was disrupted by PPARγ antisense oligonucleotide (ASO). Blockade of adipose PPARγ by ASO reversed the phenotype of βERKO mice with an impairment of insulin sensitization and glucose tolerance. Finally, binding of SRC1 and TIF2 to the PPARγ-regulated adiponectin promoter was enhanced in gonadal fat from βERKO mice indicating that the absence of ERβ in adipose tissue results in exaggerated coactivator binding to a PPARγ target promoter. Collectively, our data provide the first evidence that ERβ-deficiency protects against diet-induced IR and glucose intolerance which involves an augmented PPARγ signaling in adipose tissue. Moreover, our data suggest that the coactivators SRC1 and TIF2 are involved in this interaction. Impairment of insulin and glucose metabolism by ERβ may have significant implications for our understanding of hormone receptor-dependent pathophysiology of metabolic diseases, and may be essential for the development of new ERβ-selective agonists.

Metabolic impact of estrogen signalling through ERalpha and ERbeta.

Estrogens, acting on both estrogen receptors alpha (ERalpha) and beta (ERbeta) are recognized as important regulators of glucose homeostasis and lipid metabolism. ERs belong to the family of nuclear hormone receptors which mainly act as ligand activated transcription factors. Both ERs are expressed in metabolic tissue such as adipose tissue, skeletal muscle, liver and pancreas, as well as in the central nervous system. Expression pattern of both ERs differ between species, sexes, and specific tissues. The present review will focus on the key effects of ERs on glucose- and lipid metabolism. It appears that ERalpha mainly mediates beneficial metabolic effects of estrogens such as anti-lipogenesis, improvement of insulin sensitivity and glucose tolerance, and reduction of body weight/fat mass. In contrast, ERbeta activation seems to be detrimental for the maintenance of regular glucose and lipid homeostasis. Metabolic actions of both receptors in relevant tissues will be discussed.

Liver-Specific Peroxisome Proliferator-Activated Receptor α Target Gene Regulation by the Angiotensin Type 1 Receptor Blocker Telmisartan

OBJECTIVE—The angiotensin type 1 receptor blocker (ARB) and peroxisome proliferator–activated receptor (PPAR) γ modulator telmisartan has been recently demonstrated to reduce plasma triglycerides in nondiabetic and diabetic hypertensive patients. The present study investigates the molecular mechanisms of telmisartans hypolipidemic actions, in particular its effect on the PPARα pathway. RESEARCH DESIGN AND METHODS—Regulation of PPARα target genes by telmisartan was studied by real-time PCR and Western immunoblotting in vitro and in vivo in liver/skeletal muscle of mice with diet-induced obesity. Activation of the PPARα ligand binding domain (LBD) was investigated using transactivation assays. RESULTS—Telmisartan significantly induced the PPARα target genes carnitine palmitoyl transferase 1A (CPT1A) in human HepG2 cells and acyl-CoA synthetase long-chain family member 1 (ACSL1) in murine AML12 cells in the micromolar range. Telmisartan-induced CPT1A stimulation was markedly reduced after small interfering RNA–mediated knockdown of PPARα. Telmisartan consistently activated the PPARα-LBD as a partial PPARα agonist. Despite high in vitro concentrations required for PPARα activation, telmisartan (3 mg · kg−1 · day−1) potently increased ACSL1 and CPT1A expression in liver from diet-induced obese mice associated with a marked decrease of hepatic and serum triglycerides. Muscular CPT1B expression was not affected. Tissue specificity of telmisartan-induced PPARα target gene induction may be the result of previously reported high hepatic concentrations of telmisartan. CONCLUSIONS—The present study identifies the ARB/PPARγ modulator telmisartan as a partial PPARα agonist. As a result of its particular pharmacokinetic profile, PPARα activation by telmisartan seems to be restricted to the liver. Hepatic PPARα activation may provide an explanation for telmisartans antidyslipidemic actions observed in recent clinical trials.

Impact of HDL on adipose tissue metabolism and adiponectin expression

OBJECTIVE The objective of the current study was to investigate the hypothesis that high-density lipoprotein (HDL) influences adipocyte metabolism and adiponectin expression. Therefore, HDL was increased in vivo via apolipoprotein (apo) A-I gene transfer and in vitro via supplementation of HDL to partly differentiated adipocytes, in the presence or absence of lipopolysaccharide (LPS), known to decrease HDL cholesterol and adiponectin levels in vivo. METHODS AND RESULTS Apo A-I transfer resulted in a significant increase of HDL cholesterol in control and LPS-injected C57BL/6 mice, which was paralleled by an increase in plasma adiponectin levels and adiponectin expression in abdominal fat. Triglyceride and free fatty acids levels after LPS administration were 2.2-fold (p<0.05) and 1.3-fold (p<0.05) lower, respectively, in Ad.hapoA-I-LPS than in Ad.Null-LPS mice. In parallel, the LPS-induced mRNA expression of hormone sensitive lipase was 3.5-fold (p=0.05) decreased in the Ad.hapoA-I-LPS group. On the other hand, apo A-I transfer abrogated the LPS-mediated reduction in lipin-1 and CD36 mRNA expression by 8.2-fold (p<0.05) and 18-fold (p<0.05), respectively. Concomitantly, the phosphorylation state of Akt was 2.0-fold (p<0.05) increased in the Ad.hapoA-I-LPS compared to the Ad.Null-LPS group. Pre-incubation of partly differentiated adipocytes with HDL (50 microg protein/ml) increased adiponectin expression by 1.5-fold under basal conditions (p<0.05) and could abrogate LPS-induced down-regulation of adiponectin, both in a phosphatidylinositol-3-kinase-dependent manner. CONCLUSIONS HDL affects adipocyte metabolism and adiponectin expression.

Histone Deacetylase 6 (HDAC6) Is an Essential Modifier of Glucocorticoid-Induced Hepatic Gluconeogenesis

In the current study, we investigated the importance of histone deacetylase (HDAC)6 for glucocorticoid receptor–mediated effects on glucose metabolism and its potential as a therapeutic target for the prevention of glucocorticoid-induced diabetes. Dexamethasone-induced hepatic glucose output and glucocorticoid receptor translocation were analyzed in wild-type (wt) and HDAC6-deficient (HDAC6KO) mice. The effect of the specific HDAC6 inhibitor tubacin was analyzed in vitro. wt and HDAC6KO mice were subjected to 3 weeks’ dexamethasone treatment before analysis of glucose and insulin tolerance. HDAC6KO mice showed impaired dexamethasone-induced hepatic glucocorticoid receptor translocation. Accordingly, dexamethasone-induced expression of a large number of hepatic genes was significantly attenuated in mice lacking HDAC6 and by tubacin in vitro. Glucose output of primary hepatocytes from HDAC6KO mice was diminished. A significant improvement of dexamethasone-induced whole-body glucose intolerance as well as insulin resistance in HDAC6KO mice compared with wt littermates was observed. This study demonstrates that HDAC6 is an essential regulator of hepatic glucocorticoid-stimulated gluconeogenesis and impairment of whole-body glucose metabolism through modification of glucocorticoid receptor nuclear translocation. Selective pharmacological inhibition of HDAC6 may provide a future therapeutic option against the prodiabetogenic actions of glucocorticoids.

PPARgamma activation attenuates T-lymphocyte-dependent inflammation of adipose tissue and development of insulin resistance in obese mice.

BackgroundInflammation of adipose tissue (AT) has been recently accepted as a first step towards obesity-mediated insulin resistance. We could previously show that mice fed with high fat diet (HFD) develop systemic insulin resistance (IR) and glucose intolerance (GI) associated with CD4-positive T-lymphocyte infiltration into visceral AT. These T-lymphocytes, when enriched in AT, participate in the development of fat tissue inflammation and subsequent recruitment of proinflammatory macrophages. The aim of this work was to elucidate the action of the insulin sensitizing PPARgamma on T-lymphocyte infiltration during development of IR, and comparison of the PPARgamma-mediated anti-inflammatory effects of rosiglitazone and telmisartan in diet-induced obesity model (DIO-model) in mice.MethodsIn order to investigate the molecular mechanisms underlying early development of systemic insulin resistance and glucose intolerance male C57BL/6J mice were fed with high fat diet (HFD) for 10-weeks in parallel to the pharmacological intervention with rosiglitazone, telmisartan, or vehicle.ResultsBoth rosiglitazone and telmisartan were able to reduce T-lymphocyte infiltration into AT analyzed by quantitative analysis of the T-cell marker CD3gamma and the chemokine SDF1alpha. Subsequently, both PPARgamma agonists were able to attenuate macrophage infiltration into AT, measured by the reduction of MCP1 and F4/80 expression. In parallel to the reduction of AT-inflammation, ligand-activated PPARgamma improved diet-induced IR and GI.ConclusionTogether the present study demonstrates a close connection between PPARgamma-mediated anti-inflammation in AT and systemic improvement of glucose metabolism identifying T-lymphocytes as one cellular mediator of PPARgamma´s action.

Angiotensin II induces peroxisome proliferator‐activated receptor gamma in PC12W cells via angiotensin type 2 receptor activation

The angiotensin type 2 (AT2) receptor has been previously demonstrated to exert neuroprotective actions possibly by inducing neuronal cell differentiation involving neurite outgrowth. The nuclear hormone receptor peroxisome proliferator‐activated receptor gamma (PPARγ) is an important transcriptional regulator of cell differentiation. The aim of the present study was to clarify whether PPARγ is involved in AT2‐receptor‐mediated morphological neuronal cell differentiation. To investigate AT2‐receptor‐mediated morphological neuronal cell differentiation, rat pheochromocytoma cells (PC12W cells) expressing AT2 but not AT1 receptors, were stimulated with angiotensin II (Ang II, 100 nmol/L) ± the PPARγ antagonists GW9662 (3 µmol/L) and bisphenol A diglycidyl ether (BADGE, 1 µmol/L), and neurite outgrowth of these cells was assessed. Ang II induced neurite outgrowth by 19 ± 1.6‐fold (p < 0.01). Antagonizing PPARγ activity by GW9662 or BADGE potently blocked Ang II‐induced neurite outgrowth (Ang II + GW9662: 6.6 ± 1.5‐fold, p < 0.05; Ang II + BADGE: 1.3 ± 0.7‐fold, p < 0.01). AT2 receptor activation by Ang II markedly induced mRNA and protein expression of the PPARγ2 isoform and enhanced ligand‐induced PPARγ activity in transactivation assays. In conclusion, the present study demonstrates that Ang II induces PPARγ expression and ligand‐mediated PPARγ activity via AT2 receptor activation, which appears to be a crucial process in AT2 receptor mediated neurite outgrowth. AT2 receptor/PPARγ‐dependent neurite outgrowth may play an important role during neuroprotective processes.