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There is an urgent need for developing effective drugs to combat the obesity and Type 2 diabetes mellitus epidemics. The endocannabinoid system plays a major role in energy homeostasis. It comprises the cannabinoid receptors 1 and 2 (CB1 and CB2), endogenous ligands called endocannabinoids and their metabolizing enzymes. Because the CB1 receptor is overactivated in metabolic alterations, pharmacological blockade of the CB1 receptor arose as a promising candidate to treat obesity. However, because of the wide distribution of CB1 receptors in the central nervous system, their negative central effects halted further therapeutic use. Although the CB2 receptor is mostly peripherally expressed, its role in metabolic homeostasis remains unclear. This review discusses the potential of CB1 and CB2 receptors at the peripheral level to be therapeutic targets in metabolic diseases. We focus on the impact of pharmacological intervention and/or silencing on peripheral cannabinoid receptors in organs/tissues relevant for energy homeostasis. Moreover, we provide a perspective on novel therapeutic strategies modulating these receptors. Targeting CB1 with peripherally restricted antagonists, neutral antagonists, inverse agonists, or monoclonal antibodies could represent successful strategies. CB2 agonism has shown promising results at preclinical level. Beyond classic antagonism and agonism targeting orthosteric sites, the recently described crystal structures of CB1 and CB2 open new possibilities for therapeutic interventions with negative and positive allosteric modulators. The challenge of simultaneously targeting CB1 and CB2 might be possible by developing dual-steric ligands. The future will tell whether these promising strategies result in a renaissance of the cannabinoid receptors as therapeutic targets in metabolic diseases.

The beneficial health-related effects of exercise are well recognized, and numerous studies have investigated underlying mechanism using various in vivo and in vitro models. Although electrical pulse stimulation (EPS) for the induction of muscle contraction has been used for quite some time, its application on cultured skeletal muscle cells of animal or human origin as a model of in vitro exercise is a more recent development. In this review, we compare in vivo exercise and in vitro EPS with regard to effects on signalling, expression level and metabolism. We provide a comprehensive overview of different EPS protocols and their applications, discuss technical aspects of this model including critical controls and the importance of a proper maintenance procedure and finally discuss the limitations of the EPS model.

Background Dipeptidyl peptidase-4 (DPP4) is a key protein in glucose homeostasis and a pharmacological target in type 2 diabetes mellitus. This study explored whether the novel adipokine soluble DPP4 (sDPP4) can cause endothelial dysfunction, an early marker of impaired vascular reactivity. Method Reactivity was studied in mesenteric arteries from 3-month-old female mice, using a small vessel myograph. Thromboxane A2 (TXA2) release was explored in cultured human coronary artery endothelial cells by enzyme immunoassay. Results Neither the contractility to noradrenaline nor the endothelium-independent relaxations induced by sodium nitroprusside were modified by sDPP4. However, sDPP4 impaired in a concentration-dependent manner the endothelium-dependent relaxation elicited by acetylcholine. The DPP4 inhibitors K579 and linagliptin prevented the defective relaxation induced by sDPP4, as did the protease-activated receptor 2 (PAR2) inhibitor GB83. Downstream of PAR2, the cyclooxygenase (COX) inhibitor indomethacin, the COX2 inhibitor celecoxib or the thromboxane receptors blocker SQ29548 prevented the deleterious effects of sDPP4. Accordingly, sDPP4 triggered the release of TXA2 by endothelial cells, whereas TXA2 release was prevented by inhibiting DPP4, PAR2 or COX. Conclusion In summary, these findings reveal sDPP4 as a direct mediator of endothelial dysfunction, acting through PAR2 activation and the release of vasoconstrictor prostanoids. By interfering with these actions, DPP4 inhibitors might help preserving endothelial function in the context of cardiometabolic diseases.

Aims/Hypothesis: Besides a therapeutic target for type 2 diabetes, dipeptidyl peptidase 4 (DPP4) is an adipokine linked to human obesity. We aimed to explore role of adipocyte-derived DPP4 in diet-induced obesity and insulin resistance. Methods: Wild type (WT) and AT-specific KO mouse (AT-DPP4-KO) were fed for 24 weeks with a high fat diet (HFD) and characterized for body weight, glucose tolerance, insulin sensitivity by hyperinsulinemic-euglycemic clamp, and body composition and hepatic fat content. Image and molecular biology analysis of inflammation as well as adipocyte secretion was performed in AT by immunohistochemistry, Western blot, qRT- PCR and ELISA. Incretin levels were determined by multiplex. Results: Under high fat diet (HFD), AT-DPP4-KO displayed markedly reduced circulating DPP4 concentrations, proving AT as a relevant source of DPP4. Independently of glucose-stimulated incretin hormones, AT-DPP4-KO had improved glucose tolerance and hepatic insulin sensitivity. AT-DPP4-KO displayed smaller adipocytes and a shift in the inflammatory phenotype with more crown-like structures. IGF binding protein 3 (IGFBP3) levels were lower in AT and serum. Conclusions: The absence of adipose DPP4 triggers beneficial AT remodeling with decreased production of IGFBP3 during HFD, likely contributing to the observed improved hepatic insulin sensitivity.

The secretory function of adipose tissue (AT) has gained substantial and continuously increasing interest during the last 20–30 years, and it is this tissue that has set the stage for a new understanding of organ communication and crosstalk and its impact for metabolic homeostasis. This crosstalk is mediated by more than 1000 so-called adipocytokines, and future work using computational biology and other advanced approaches will be needed to decipher the complex language of AT. In this chapter, we analyze the basics of adipokine release, the auto- and paracrine crosstalk within AT, and its endocrine function. Finally, we focus on specific fat depots, such as perivascular, epicardial, and brown fat, and describe the current knowledge on the secretome of these tissues, considering their functional impact for health and disease.

The discovery of irisin as an exercise-regulated myokine inducing browning of WAT has gained interest as a potential new strategy to combat obesity and its associated disorders, such as type 2 diabetes. However, there are inconsistencies regarding the relevance of irisin in humans. The regulation of FNDC5 mRNA expression by exercise and contraction could not be reproduced by a number of human studies using several exercise protocols and in vitro approaches. Furthermore, the nature of FNDC5 fragments and the presence of irisin in humans are questionable and probably contribute to conflicting data obtained with commercially available ELISA kits. Most importantly, the information regarding the concentration of circulating irisin in humans is not clear, as different studies using different kits measure irisin levels in a wide range. Data about the role of irisin in states of human obesity and metabolic diseases are conflicting and, in some cases, changes in irisin levels have been observed; they were only moderate in 10–20%. Independent of the presence and regulation of FNDC5/irisin in humans, the application of recombinant irisin could still represent a therapeutic strategy to fight obesity. However, the current data obtained from human cell models reveal that FNDC5/irisin has no effect on browning of the major WAT depots in humans and is likely to selectively target a small subpopulation of adipocytes, which are located in classical BAT regions, such as the supraclavicular adipose tissue. Thus, other candidates, such as BMP7 or CNPs, seem to be more prominent candidates as inducers of browning in humans.

The discovery of adipokines has revealed adipose tissue as a central node in the interorgan crosstalk network, which mediates the regulation of multiple organs and tissues. Adipose tissue is a true endocrine organ that produces and secretes a wide range of mediators regulating adipose tissue function in an auto-/paracrine manner and important distant targets, such as the liver, skeletal muscle, the pancreas and the cardiovascular system. In metabolic disorders such as obesity, enlargement of adipocytes leads to adipose tissue dysfunction and a shift in the secretory profile with an increased release of pro-inflammatory adipokines. Adipose tissue dysfunction has a central role in the development of insulin resistance, type 2 diabetes, and cardiovascular diseases. Besides the well-acknowledged role of adipokines in metabolic diseases, and the increasing number of adipokines being discovered in the last years, the mechanisms underlying the release of many adipokines from adipose tissue remain largely unknown. To combat metabolic diseases, it is crucial to better understand how adipokines can modulate adipose tissue growth and function. Therefore, we will focus on adipokines with a prominent role in auto-/paracrine crosstalk within the adipose tissue such as RBP4, HO-1, WISP2, SFRPs and chemerin. To depict the endocrine crosstalk between adipose tissue with skeletal muscle, the cardiovascular system and the pancreas, we will report the main findings regarding the direct effects of adiponectin, leptin, DPP4 and visfatin on skeletal muscle insulin resistance, cardiovascular function and β-cell growth and function.

Cardiovascular complications are common in patients with type 2 diabetes. Adipokines have been implicated in the induction of proliferative and pro-atherogenic alterations in human vascular smooth muscle cells (hVSMC). Other reports demonstrated the importance of the miRNA cluster miR-143/145 in the regulation of VSMC homeostasis and insulin sensitivity. Here we investigated whether the detrimental effects of adipokines on hVSMC function could be ascribed to alterations in miR-143/145 expression. The exposure of hVSMC to conditioned media (CM) from primary human subcutaneous adipocytes increased the expression of smooth muscle α-actin (SMA), and the miR-143/145 cluster, but markedly impaired the insulin-mediated phosphorylation of Akt and its substrate endothelial nitric oxide synthase (eNOS). Furthermore, CM promoted the phosphorylation of SMAD2 and p38, which have both been linked to miR-143/145 induction. Accordingly, the induction of miR-143/145 as well as the inhibition of insulin-mediated Akt- and eNOS-phosphorylation was prevented when hVSMC were treated with pharmacological inhibitors for Alk-4/5/7 and p38 before the addition of CM. The transfection of hVSMC with precursor miR-143, but not with precursor miR-145, resulted in impaired insulin-mediated phosphorylation of Akt and eNOS. This inhibition of insulin signaling by CM and miR-143 is associated with a reduction in the expression of the oxysterol-binding protein-related protein 8 (ORP8). Finally, the knock-down of ORP8 resulted in impaired insulin-mediated phosphorylation of Akt in hVSMC. Thus, the detrimental effects of adipocyte-derived conditioned media on insulin action in primary hVSMC can be ascribed to the Alk- and p38-dependent induction of miR-143 and subsequent downregulation of ORP8.

Background— Secreted factors from epicardial adipose tissue (EAT) have been implicated in the development of cardiomyocyte dysfunction. This study aimed to assess whether alterations in the secretory profile of EAT in patients with type 2 diabetes mellitus (DM2) affect contractile function and insulin action in cardiomyocytes. Methods and Results— Contractile function and insulin action were analyzed in primary adult rat cardiomyocytes incubated with conditioned media (CM) generated from explants of EAT biopsies obtained from patients without and with DM2. CM from subcutaneous and pericardial adipose tissue biopsies from the same patients served as the control. Cardiomyocytes treated with CM (EAT) from DM2 patients showed reductions in sarcomere shortening, cytosolic Ca 2+ fluxes, expression of sarcoplasmic endoplasmic reticulum ATPase 2a, and decreased insulin-mediated Akt-Ser473-phosphorylation as compared with CM from the other groups. Profiling of the CM showed that activin A, angiopoietin-2, and CD14 selectively accumulated in CM-EAT-DM2 versus CM-EAT in patients without DM2 and CM from the other fat depots. Accordingly, EAT biopsies from DM2 patients were characterized by clusters of CD14-positive monocytes. Furthermore, SMAD2-phosphorylation, a downstream target of activin A signaling, was elevated in cardiomyocytes treated with CM (EAT) from DM2 patients, and the detrimental effects of CM (EAT) from DM2 patients were partially abolished in cardiomyocytes pretreated with a neutralizing antibody against activin A. Finally, both recombinant activin A and angiopoietin-2 reduced cardiomyocyte contractile function, but only activin A reduced the expression of sarcoplasmic endoplasmic reticulum ATPase 2a. Conclusions— Collectively, our data implicate DM2-related alterations in the secretory profile of EAT in the pathogenesis of diabetes mellitus–related heart disease.

It is widely accepted that obesity is a major risk factor for the development of atherosclerosis. In this context, adipose tissue produces a variety of adipokines and releases free fatty acids, contributing to a chronic-low grade inflammation state implicated in vascular complications. In this study, we investigated the role of adipokines, oleic acid (OA), palmitic acid (PA), and the combinations on activation of NF-κB target genes in human vascular smooth muscle cells (SMC) to assess the hypothesis of synergistic interactions between these molecules. Adipocyte-conditioned medium (CM), generated from human adipocytes, in combination with low concentrations of OA, but not PA, induces SMC proliferation and activation of the transcription factor NF-κB in a synergistic way. Combined treatment of CM and OA further regulates a set of downstream NF-κB target genes including angiopoietin-1, activin A, and MMP-1, all critically involved in SMC dysfunction. This suggests that the lipotoxic potential of fatty acids is substantially enhanced by the presence of adipocyte-derived factors.

Obesity is the hallmark of the metabolic syndrome and predisposes patients to the development of major chronic metabolic diseases including type 2 diabetes mellitus. Adipose tissue expansion in obesity is characterized by increasing infiltration of proinflammatory immune cells into adipose tissue causing chronic, low-grade inflammation. Phenotypic switching of macrophages is an important mechanism of adipose tissue inflammation, and there is involvement of cells from the adaptive immune system in this process. T-cell phenotype changes and recruitment of B cells and T cells precedes macrophage infiltration. Cytokines and chemokines produced by immune cells influence localized and systemic inflammation, which is a pathogenic link between obesity and insulin resistance. Antigens absorbed from the gut might contribute to T-cell activation and recruitment into visceral adipose tissue in obesity. This Review summarizes, in the context of obesity, the evidence for infiltration of adipose tissue by cells of the adaptive immune system, how adaptive system cells affect innate cell populations and the influence of adaptive immune cells on the development of insulin resistance.

Low testosterone accompanied by elevated estradiol associates with the development of metabolic dysfunction in men.The aim of the study was to explore the hypothesis that alterations in sex steroid levels induce metabolic dysfunction through adipokines.Circulating levels of sex steroids and 28 adipokines were determined in a cross-sectional study of morbidly obese men and aged-matched controls, as well as in a randomized clinical trial with healthy young men in which obesity-related alterations in sex steroid levels were mimicked by treatment with an aromatase inhibitor plus estradiol patches.Morbidly obese men had lower testosterone levels than normal-weight controls. Estradiol levels were increased in morbidly obese men (without DM2) as compared to normal-weight controls. Circulating levels of multiple proinflammatory cytokines, including IL-1Ra, IL-5, IL-6, IL-10, leptin, monocyte chemoattractant protein 1 (MCP1), and macrophage inflammatory protein 1α, positively associated with estradiol and negatively with testosterone. The associations with estradiol, but not with testosterone, remained significant after adjusting for adipocyte cell size. In a separate clinical trial, the direct adverse effects of lowering testosterone and raising estradiol on MCP1 were substantiated in vivo.Initial alterations in sex steroid levels may contribute to metabolic dysfunction through adverse effects on adipokine levels in obese men. The direct adverse effects on MCP1, a chemokine highly linked to the development of metabolic dysfunction, were substantiated in a trial mimicking obesity-related alterations of sex steroid levels in healthy young males.

Studies have shown the implication of specific adipokines or fatty acids (FA) in the pathogenesis of insulin resistance. However, the interplay of adipokines with FA remains poorly understood. This study aimed to investigate the combined effects of adipokines and low concentrations of palmitic acid (PA, 100 µmol/l) on skeletal muscle metabolism. Human skeletal muscle cells were incubated with adipocyte-conditioned medium (CM), PA or PA+CM, and FA transporter and FA metabolism were analysed. CM-incubation increased CD36 level (1.8 fold) and PA-uptake (1.4 fold). However, only co-application of PA+CM resulted in profound lipid accumulation (5.3 fold), 60% reduction of PA-oxidation and 3.5 fold increased diacylglycerol content. Our results support a novel role for adipokines in the pathogenesis of T2D by increasing the lipotoxic potential of PA, notably of low concentrations. This implies an increased lipotoxic risk already at an early stage of weight gain, when lipolysis has not yet contributed to increased plasma free FA levels.

Abdominal obesity is a major risk factor for cardiovascular disease, and recent studies highlight a key role of adipose tissue dysfunction, inflammation, and aberrant adipokine release in this process. An increased demand for lipid storage results in both hyperplasia and hypertrophy, finally leading to chronic inflammation, hypoxia, and a phenotypic change of the cellular components of adipose tissue, collectively leading to a substantially altered secretory output of adipose tissue. In this review we have assessed the adipo-vascular axis, and an overview of adipokines associated with cardiovascular disease is provided. This resulted in a first list of more than 30 adipokines. A deeper analysis only considered adipokines that have been reported to impact on inflammation and NF-κB activation in the vasculature. Out of these, the most prominent link to cardiovascular disease was found for leptin, TNF-α, adipocyte fatty acid-binding protein, interleukins, and several novel adipokines such as lipocalin-2 and pigment epithelium-derived factor. Future work will need to address the potential role of these molecules as biomarkers and/or drug targets.

Epicardial adipose tissue (EAT) has been implicated in the development of heart disease. Nonetheless, the crosstalk between factors secreted from EAT and cardiomyocytes has not been studied. Here, we examined the effect of factors secreted from EAT on contractile function and insulin signalling in primary rat cardiomocytes. EAT and subcutaneous adipose tissue (SAT) were isolated from guinea pigs fed a high-fat (HFD) or standard diet. HFD feeding for 6 months induced glucose intolerance, and decreased fractional shortening and ejection fraction (all P < 0.05). Conditioned media (CM) generated from EAT and SAT explants were subjected to cytokine profiling using antibody arrays, or incubated with cardiomyocytes to assess the effects on insulin action and contractile function. Eleven factors were differentially secreted by EAT when compared to SAT. Furthermore, secretion of 30 factors by EAT was affected by HFD feeding. Most prominently, activin A-immunoreactivity was 6.4-fold higher in CM from HFD versus standard diet-fed animals and, 2-fold higher in EAT versus SAT. In cardiomyocytes, CM from EAT of HFD-fed animals increased SMAD2-phosphorylation, a marker for activin A-signalling, decreased sarcoplasmic-endoplasmic reticulum calcium ATPase 2a expression, and reduced insulin-mediated phosphorylation of Akt-Ser473 versus CM from SAT and standard diet-fed animals. Finally, CM from EAT of HFD-fed animals as compared to CM from the other groups markedly reduced sarcomere shortening and cytosolic Ca2+ fluxes in cardiomyocytes. These data provide evidence for an interaction between factors secreted from EAT and cardiomyocyte function.

Context: Zinc-α2-glycoprotein (ZAG) induces lipid mobilization in adipose tissue (AT) and stimulates energy utilization in AT and skeletal muscle by up-regulation of UCP isoforms and GLUT4.Objective: Our study aimed to investigate whether ZAG activates AMPKα, an important regulator of energy metabolism, in human skeletal muscle cells (SkMc).Materials and Methods: SkMc were treated with recombinant ZAG, and activation of AMPKα and ACC, protein abundance of GLUT4, and UCP2 and UCP3 gene expression were analysed.Results: Treatment of SkMc with ZAG induced short-time phosphorylation of AMPKα and ACC. Furthermore, AMPKα phosphorylation was elevated after 24 h, while for ACC no activation was observed. GLUT4 level was increased by 1.3-fold. However, UCP2 and UCP3 expression remained unaltered.Discussion and Conclusion: These results show that ZAG leads to phosphorylation of AMPKα and ACC, thereby activating a pathway central to the regulation of energy metabolism. This mechanism may be involved in mediating the...

OBJECTIVE Comprehensive proteomic profiling of the human adipocyte secretome identified dipeptidyl peptidase 4 (DPP4) as a novel adipokine. This study assessed the functional implications of the adipokine DPP4 and its association to the metabolic syndrome. RESEARCH DESIGN AND METHODS Human adipocytes and skeletal and smooth muscle cells were used to monitor DPP4 release and assess the effects of soluble DPP4 on insulin signaling. In lean and obese subjects, depot-specific expression of DPP4 and its release from adipose tissue explants were determined and correlated to parameters of the metabolic syndrome. RESULTS Fully differentiated adipocytes exhibit a substantially higher release of DPP4 compared with preadipocytes or macrophages. Direct addition of DPP4 to fat and skeletal and smooth muscle cells impairs insulin signaling. A fivefold higher level of DPP4 protein expression was seen in visceral compared with subcutaneous fat of obese patients, with no regional difference in lean subjects. DPP4 serum concentrations significantly correlated with adipocyte size. By using adipose tissue explants from lean and obese subjects, we observed a twofold increase in DPP4 release that strongly correlated with adipocyte volume and parameters of the metabolic syndrome and was decreased to the lean level after weight reduction. DPP4 released from adipose tissue correlated positively with an increasing risk score for the metabolic syndrome. CONCLUSIONS DPP4 is a novel adipokine that may impair insulin sensitivity in an autocrine and paracrine fashion. Furthermore, DPP4 release strongly correlates with adipocyte size, potentially representing an important source of DPP4 in obesity. Therefore, we suggest that DPP4 may be involved in linking adipose tissue and the metabolic syndrome.

Obesity, insulin resistance and the metabolic syndrome, are characterized by expansion and inflammation of adipose tissue, including the depots surrounding the heart and the blood vessels. Epicardial adipose tissue (EAT) is a visceral thoracic fat depot located along the large coronary arteries and on the surface of the ventricles and the apex of the heart, whereas perivascular adipose tissue (PVAT) surrounds the arteries. Both fat depots are not separated by a fascia from the underlying tissue. Therefore, factors secreted from epicardial and PVAT, like free fatty acids and adipokines, can directly affect the function of the heart and blood vessels. In this review, we describe the alterations found in EAT and PVAT in pathological states like obesity, type 2 diabetes, the metabolic syndrome and coronary artery disease. Furthermore, we discuss how changes in adipokine expression and secretion associated with these pathological states could contribute to the pathogenesis of cardiac contractile and vascular dysfunction.

Epicardial adipose tissue (EAT) is a visceral adipose tissue in close contact with coronary vessels, the excess of which is associated with coronary artery disease (CAD).Our goal was to identify candidate molecule(s) characterizing EAT that could intervene in the pathogenesis of CAD.An approach combining microarrays and bioinformatic sequence analysis tools for predicting secreted proteins (TargetP) was applied to paired biopsies of sc adipose tissue (SAT) and EAT, obtained from patients with or without CAD (NCAD). RESULTS were validated in three independent groups of subjects by quantitative RT-PCR, Western blot, immunohistochemistry, and explant secretion.Secretory type II phospholipase A2 (sPLA2-IIA) ranked as the highest gene coding for potentially secreted proteins with the highest overexpression in EAT in both CAD and NCAD. Quantitative RT-PCR confirmed its increased expression in EAT (P0.01) as well as EAT from CAD as compared with NCAD (49.3 +/- 13 vs. 17.4 +/- 9.7 P0.01). sPLA2-IIA protein levels were higher in EAT than SAT (P0.001). EAT explants also showed significantly higher sPLA2-IIA secretion levels than SAT ones (4.37 +/- 2.7 vs. 0.67 +/- 0.28 ng/ml to 1 per gram tissue per 24 h, P0.03). sPLA2-IIA labeling was seen in the stroma vascular fraction between adipocytes and in connective capsules in EAT, with no immunostaining of the adipocytes. SAT was weakly labeled following the same process.We have shown for the first time an increased expression of sPLA2-IIA in EAT in patients with CAD. sPLA2-IIA is a phospholipase, which has been shown to be an independent risk factor for CAD. These findings suggest that EAT has a potentially pathophysiological role in CAD.

OBJECTIVE Vascular endothelial cells (VECs) downregulate their rate of glucose uptake in response to hyperglycemia by decreasing the expression of their typical glucose transporter GLUT-1. Hitherto, we discovered critical roles for the protein calreticulin and the arachidonic acid–metabolizing enzyme 12-lipoxygenase in this autoregulatory process. The hypothesis that 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), the peroxidation product of 12-lipoxygenase, mediates this downregulatory mechanism by activating peroxisome proliferator–activated receptor (PPAR) δ was investigated. RESEARCH DESIGN AND METHODS Effects of 4-HDDE and PPARδ on the glucose transport system and calreticulin expression in primary bovine aortic endothelial cells were evaluated by pharmacological and molecular interventions. RESULTS Using GW501516 (PPARδ agonist) and GSK0660 (PPARδ antagonist), we discovered that high-glucose–induced downregulation of the glucose transport system in VECs is mediated by PPARδ. A PPAR-sensitive luciferase reporter assay in VECs revealed that high glucose markedly increased luciferase activity, while GSK0660 abolished it. High-performance liquid chromatography analysis showed that high-glucose incubation substantially elevated the generation of 4-HDDE in VECs. Treatment of VECs, exposed to normal glucose, with 4-HDDE mimicked high glucose and downregulated the glucose transport system and increased calreticulin expression. Like high glucose, 4-HDDE significantly activated PPARδ in cells overexpressing human PPAR (hPPAR)δ but not hPPARα, -γ1, or -γ2. Moreover, silencing of PPARδ prevented high-glucose–dependent alterations in GLUT-1 and calreticulin expression. Finally, specific binding of PPARδ to a PPAR response element in the promoter region of the calreticulin gene was identified by utilizing a specific chromatin immunoprecipitation assay. CONCLUSIONS Collectively, our data show that 4-HDDE plays a central role in the downregulation of glucose uptake in VECs by activating PPARδ.

Insulin resistance in skeletal muscle is an early event in the development of diabetes with obesity being one of the major contributing factors. Conditioned medium (CM) from differentiated human adipocytes impairs insulin signalling in human skeletal muscle cells. Recent data on adipocyte-induced insulin resistance in skeletal muscle cells describes underlying mechanisms of this process. Skeletal muscle insulin resistance involves multiple pathways and irreversible changes in the expression level of critical proteins. Furthermore, the reversibility of insulin resistance could be demonstrated. Several strategies to combat insulin resistance have been developed. One recent approach to treat obesity and the metabolic syndrome is the use of endocannabinoid receptor antagonists such as rimonabant. These compounds might also reduce insulin resistance in type 2 diabetes with effects on adipose tissue and liver and possibly skeletal muscle.

The E23K variant of the Kir6.2 gene has been shown to be associated with type 2 diabetes mellitus in Caucasian subjects. Because offspring of type 2 diabetic patients have a genetically increased risk of developing diabetes, we sought to identify the E23K variant of the Kir6.2 gene in offspring of Caribbean patients with type 2 diabetes and assess the contribution of this variant to impaired glucose tolerance in these subjects. Forty-six offspring of patients with type 2 diabetes and 39 apparently healthy subjects whose immediate parents were not diabetic (‘control’) were studied after an overnight fast. Anthropometric indices were measured and blood samples were collected. Fasting and 2 h plasma glucose, insulin and lipids were subsequently determined. Insulin resistance was calculated using the homeostatic model assessment technique. The offspring and control subjects had similar frequencies of the E23K polymorphism (52.6 vs 45.5%, P>0.05) and the frequency of the E23K variant did not differ significantly between gender and ethnic distributions, irrespectively of a family history of diabetes (P>0.05). There were no significant differences in biochemical risk factors for developing diabetes in offspring carriers of the E23K variant compared with offspring non-carriers of the mutation. Offspring with the E23K mutation had even significantly higher 2 h insulin concentrations when compared with control subjects. It is concluded that the presence of the Kir6.2 E23K genotype in Caribbean subjects with an immediate positive family history of diabetes does not confer significantly higher levels of biochemical risk factors for the development of type 2 diabetes.

Dipeptidyl-peptidase 4 (DPP4) is a glycoprotein of 110 kDa, which is ubiquitously expressed on the surface of a variety of cells. This exopeptidase selectively cleaves N-terminal dipeptides from a variety of substrates, including cytokines, growth factors, neuropeptides, and the incretin hormones. Expression of DPP4 is substantially dysregulated in a variety of disease states including inflammation, cancer, obesity, and diabetes. Since the incretin hormones, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide (GIP), are major regulators of post-prandial insulin secretion, inhibition of DPP4 by the gliptin family of drugs has gained considerable interest for the therapy of type 2 diabetic patients. In this review, we summarize the current knowledge on the DPP4-incretin axis and evaluate most recent findings on DPP4 inhibitors. Furthermore, DPP4 as a type II transmembrane protein is also known to be cleaved from the cell membrane involving different metalloproteases in a cell-type-specific manner. Circulating, soluble DPP4 has been identified as a new adipokine, which exerts both para- and endocrine effects. Recently, a novel receptor for soluble DPP4 has been identified, and data are accumulating that the adipokine-related effects of DPP4 may play an important role in the pathogenesis of cardiovascular disease. Importantly, circulating DPP4 is augmented in obese and type 2 diabetic subjects, and it may represent a molecular link between obesity and vascular dysfunction. A critical evaluation of the impact of circulating DPP4 is presented, and the potential role of DPP4 inhibition at this level is also discussed.

Soluble dipeptidyl peptidase (sDPP)-4 is a novel adipokine whose release is increased in patients with the metabolic syndrome. This study aimed to explore the capacity of sDPP-4 to directly impair vascular reactivity, which is considered as an early hallmark of endothelial dysfunction. As determined using a small vessel myograph, sDPP-4 (20-500 ng/mL) did not affect the contractility to noradrenaline (3 nmol/L to 30 μmol/L) in isolated murine mesenteric microvessels. sDPP-4 did not modify either the endothelium-independent relaxations induced by sodium nitroprusside (1 nmol/L to 100 μmol/L) in microvessels precontracted with 3 μmol/L noradrenaline. However, sDPP-4 impaired in a concentration-dependent manner the endothelium-dependent relaxation elicited by acetylcholine (ACh; 1 nmol/L to 10 μmol/L) with pD2 values of 6.88±0.17, 6.86±0.25, 6.24±0.18, 5.54±0.31 for 0, 20, 100 and 200 ng/mL sDPP-4, respectively. At 500 ng/mL sDPP-4, the maximal relaxation induced by ACh was reduced from 78.20±8.83% to 20.29±3.94%. The inhibition of cyclooxygenase or the blockade of thromboxane TP receptors with indomethacin (10 μmol/L) and SQ29548 (100 nmol/L), respectively, prevented the impaired relaxation to ACh evoked by a submaximal concentration of sDPP-4 (200 ng/mL). In line with these results, sDPP-4 (500 ng/mL) stimulated the release of thromboxane A2 (TXA2) by cultured human coronary artery endothelial cells (HCAEC) from 1.64±0.21 to 3.60±0.70 pg/mL. The DPP-4 inhibitors K579 (100 nmol/L) and linagliptin (10 nmol/L) prevented both TXA2 release and the impaired relaxation caused by sDPP-4. In conclusion, sDPP-4 arises as a causative agent of endothelial dysfunction through the release of COX-derived vasoconstrictor prostanoids. By interfering with such deleterious action of sDPP-4, pharmacological DPP-4 inhibitors might help preventing impaired endothelial function in type 2 diabetes mellitus or the metabolic syndrome.

Adipose tissue (AT) was long perceived as a passive lipid storage depot but it is now considered as an endocrine organ that produces a large number of mediators that affect metabolism, inflammation and coagulation. In obesity, the increased size of adipocytes and chronic low-grade inflammation within AT alter its normal physiological function. AT dysfunction results in altered production and secretion of adipokines, which in turn affect several tissues, e.g. the liver, skeletal muscles and vasculature, in a para- or endocrine manner. Numerous circulating proinflammatory mediators involved in the development of cardiovascular disease (CVD) are directly released from adipocytes, thereby linking obesity to an increased cardiovascular risk. In the current chapter, we focus, on the one hand, on a small selection of novel adipokines with a potentially strong link to CVD: soluble dipeptidyl peptidase-4, visfatin and lipocalin-2. On the other hand, we summarize the most recent findings on the novel cardioprotective adipokines omentin and apelin.

Adipose tissue (AT) was long perceived as a passive lipid storage depot but it is now considered as an endocrine organ that produces a large number of mediators that affect metabolism, inflammation and coagulation. In obesity, the increased size of adipocytes and chronic low-grade inflammation within AT alter its normal physiological function. AT dysfunction results in altered production and secretion of adipokines, which in turn affect several tissues, e.g. the liver, skeletal muscles and vasculature, in a para- or endocrine manner. Numerous circulating proinflammatory mediators involved in the development of cardiovascular disease (CVD) are directly released from adipocytes, thereby linking obesity to an increased cardiovascular risk. In the current chapter, we focus, on the one hand, on a small selection of novel adipokines with a potentially strong link to CVD: soluble dipeptidyl peptidase-4, visfatin and lipocalin-2. On the other hand, we summarize the most recent findings on the novel cardioprotective adipokines omentin and apelin.

Monocyte chemoattractant protein-induced protein 1 (MCPIP1) encoded by the ZC3H12a gene (also known as Regnase-1) is involved in the regulation of degradation of mRNA of inflammatory modulators and for processing of pre-miRNA. These functions depend on the presence of the PIN domain. Moreover, MCPIP1 was described as a negative regulator of NF-κB and AP-1 signaling pathways although mechanisms underlying such activity remain unknown. We aimed at determining the role of MCPIP1 in adipogenesis. Here, we present evidence that Mcpip1 transcription is transiently activated during 3T3-L1 transition from pre- to adipocytes. However Mcpip1 protein expression is also strongly decreased at day one after induction of adipogenesis. Knockdown of Mcpip1 results in an upregulation of C/EBPβ and PPARγ mRNAs, whereas overexpression of MCPIP1 reduces the level of both transcription factors and impairs adipogenesis. MCPIP1-dependend modulation of C/EBPβ and PPARγ levels results in a modulation of the expression of downstream controlled genes. In addition, decreased C/EBPβ, but not PPARγ, depends on the activity of the MCPIP1 PIN domain, which is responsible for RNase properties of this protein. Together, these data confirm that MCPIP1 is a key regulator of adipogenesis.

Obesity is considered a worldwide health concern. Most of obesity therapies are aimed at decreasing energy intake. However, recent data suggest that increasing cellular energy expenditure could be a useful approach to reduce adiposity. Adaptive thermogenesis, a biological process within the brown fat by which energy is dissipated in mitochondria, is a great tool to increase energy expenditure. Several studies have confirmed the presence of brown adipose tissue in adult humans, whose activity may make it a target for the treatment of obesity. Differentiation of brown adipocytes could be a potent tool to promote weight loss by increasing energy expenditure. Here we review the mechanisms potentially associated with expansion and activation of brown adipose tissue, and modulation of adaptive thermogenesis. Controlling one or more of these pathways could induce a positive regulation of brown adipogenesis. A better understanding of these molecular pathways could potentially result in novel anti-obesity ...

Glucose disposal in skeletal muscle is a major target for insulin action and assessment of insulin-regulated glucose uptake under in vitro conditions allows the direct determination of insulin sensitivity in this organ. For this purpose, a variety of muscle preparations from different parts of the body can be used. We describe here a detailed protocol for using epitrochlearis muscle strips and additionally for using primary skeletal muscle cells.

Recent secretome analyses suggest that human fat cells secrete hundreds of proteins (adipokines).We made an overall analysis of their potential functional importance.A secretome of 347 adipokines was evaluated by in silico analysis of their expression during adipocyte differentiation, regulation by obesity and adipose region. The gene expression in human adipose tissue was investigated in microarray studies using samples from different adipose depots from lean or obese patients.60% of the adipokines were regulated by obesity and 50% between visceral and subcutaneous adipose region. Eight adipokines, all novel, scored particularly high in the in silico analysis. Among those, four were both regulated by obesity and adipose region, namely WNT1-inducible-signaling pathway protein 2, transmembrane glycoprotein NMB, inter-alpha-trypsin inhibitor heavy chain H5, and complement C4-A. Furthermore, many adipokines were extracellular matrix proteins.Several novel adipokines have potential important functional features warranting in depth analysis.

To allow early detection and prevention of metabolic disorders, circulating levels of adipokines involved in insulin sensitivity were compared with the hyperinsulinemic-euglycemic clamp. Twenty non-obese normo-glycaemic men (age 32.1 ± 6 years) underwent a clamp procedure. Levels of leptin, adiponectin, resistin, visfatin, omentin and chemerin levels were determined in fasting blood samples. Pearson correlation coefficients between the M-value for insulin sensitivity and fasting levels of chemerin (r = -0.63, P = 0.003) and leptin (r = -0.54, P = 0.013) performed better than conventional surrogate measures of insulin sensitivity (HOMA-IR: r = -0.45, P = 0.048; Quicki: r = 0.36, P = 0.12). However, only the relation between M-value(LBM) and chemerin remained significant when adjusting for BMI and fasting insulin levels (r = -0.559, P = 0.016). In conclusion, fasting levels of chemerin might be used as biomarker to identify insulin resistance in healthy men without typical characteristics of metabolic disorders.

Glucose disposal in skeletal muscle is a major target for insulin action and assessment of insulin-regulated glucose uptake under in vitro conditions allows the direct determination of insulin sensitivity in this organ. For this purpose, a variety of muscle preparations from different parts of the body can be used. We describe here a detailed protocol for using epitrochlearis muscle strips and additionally for using primary skeletal muscle cells.

Summary Aims: Lipotoxicity in obesity might be a failure of adipocytes to respond sufficiently adequate to persistent energy surplus. To evaluate the role of lipolytic enzymes or mitochondria in lipotoxicity, we studied expression levels of genes and proteins involved in lipolysis and mitochondrial DNA (mtDNA) content. Methods: As differences in lipid metabolism between men and women are extremely complex, we recruited only men (lean and morbidly obese) and collected subcutaneous and visceral adipose tissue during abdominal surgery for real-time PCR gene expression, protein expression, and microscopic study. Results: Although mRNA levels of hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) were increased in visceral adipose tissue of morbidly obese men, this was not paralleled by alterations in protein expression and phosphorylation of HSL and ATGL. mtDNA content of visceral adipose tissue was increased in morbidly obese men as compared to lean controls (p < 0.013). Positive correlations were observed between visceral adipocyte size and serum triacylglycerol (r = 0.6, p < 0.007) as well as between visceral adipocyte size and CRP (r = 0.6, p < 0.009) in analyses performed separately in obese men. Conclusion: Lipotoxicity of morbidly obese men might be related to the quantitative impact of the visceral fat depot rather than to important dysregulation of involved lipolytic enzymes or adipocyte mitochondria.

Adipose tissue is a major endocrine organ, releasing signaling and mediator proteins, termed adipokines, via which adipose tissue communicates with other organs. Expansion of adipose tissue in obesity alters adipokine secretion, which may contribute to the development of metabolic diseases. Although recent profiling studies have identified numerous adipokines, the amount of overlap from these studies indicates that the adipokinome is still incompletely characterized. Therefore, we conducted a complementary protein profiling on concentrated conditioned medium derived from primary human adipocytes. SDS-PAGE/liquid chromatography-electrospray ionization tandem MS and two-dimensional SDS-PAGE/matrix-assisted laser desorption ionization/time of flight MS identified 347 proteins, 263 of which were predicted to be secreted. Fourty-four proteins were identified as novel adipokines. Furthermore, we validated the regulation and release of selected adipokines in primary human adipocytes and in serum and adipose tissue biopsies from morbidly obese patients and normal-weight controls. Validation experiments conducted for complement factor H, αB-crystallin, cartilage intermediate-layer protein, and heme oxygenase-1 show that the release and expression of these factors in adipocytes is regulated by differentiation and stimuli, which affect insulin sensitivity, as well as by obesity. Heme oxygenase-1 especially reveals to be a novel adipokine of interest. In vivo, circulating levels and adipose tissue expression of heme oxygenase-1 are significantly increased in obese subjects compared with lean controls. Collectively, our profiling study of the human adipokinome expands the list of adipokines and further highlights the pivotal role of adipokines in the regulation of multiple biological processes within adipose tissue and their potential dysregulation in obesity.