Adiponectin Synthesis Research Articles

Pediatric Obesity and Vitamin D Deficiency: A Proteomic Approach Identifies Multimeric Adiponectin as a Key Link between These Conditions

Key circulating molecules that link vitamin D (VD) to pediatric obesity and its co-morbidities remain unclear. Using a proteomic approach, our objective was to identify key molecules in obese children dichotomized according to 25OH-vitamin D (25OHD) levels. A total of 42 obese children (M/F = 18/24) were divided according to their 25OHD3 levels into 25OHD3 deficient (VDD; n = 18; 25OHD<15 ng/ml) or normal subjects (NVD; n = 24; >30 ng/ml). Plasma proteomic analyses by two dimensional (2D)-electrophoresis were performed at baseline in all subjects. VDD subjects underwent a 12mo treatment with 3000 IU vitamin D3 once a week to confirm the proteomic analyses. The proteomic analyses identified 53 “spots” that differed between VDD and NVD (p<0.05), amongst which adiponectin was identified. Adiponectin was selected for confirmational studies due to its tight association with obesity and diabetes mellitus. Western Immunoblot (WIB) analyses of 2D-gels demonstrated a downregulation of adiponectin in VDD subjects, which was confirmed in the plasma from VDD with respect to NVD subjects (p<0.035) and increased following 12mo vitamin D3 supplementation in VDD subjects (p<0.02). High molecular weight (HMW) adiponectin, a surrogate indicator of insulin sensitivity, was significantly lower in VDD subjects (p<0.02) and improved with vitamin D3 supplementation (p<0.042). A direct effect in vitro of 1α,25-(OH)2D3 on adipocyte adiponectin synthesis was demonstrated, with adiponectin and its multimeric forms upregulated, even at low pharmacological doses (10−9 M) of 1α,25-(OH)2D3. This upregulation was paralleled by the adiponectin interactive protein, DsbA-L, suggesting that the VD regulation of adiponectin involves post-transciptional events. Using a proteomic approach, multimeric adiponectin has been identified as a key plasma protein that links VDD to pediatric obesity.

Adipose tissue depot and cell size dependency of adiponectin synthesis and secretion in human obesity

Adiponectin is an insulin sensitizing fat cell (FC) hormone whose levels are related to adipose tissue (AT) mass and depot distribution. We hypothesized that the nature of AT expansion (hypertrophy vs. hyperplasia) contributes to obesity-related reductions in serum adiponectin and that this effect is influenced by the regional distribution of AT to subcutaneous (S) and visceral (V) depots. Thirteen obese subjects provided paired AT biopsies. Serum total and high molecular weight (HMW) adiponectin levels were determined by ELISA. Secretion was quantified following 24-h explant culture. FC size, number, % large, and % small FC were determined by microscopic analysis. Secretion of total adiponectin was highest by SAT (P = 0.008) and correlated more strongly with serum adiponectin (total: P = 0.015, r = 0.77; HMW: P = 0.005, r = 0.83) than did secretion by VAT (P = 0.05, r = 0.66 for both). FC size was greatest in SAT and correlated negatively with both serum (total: P = 0.01, r = −0.74; HMW: P = 0.03, r = −0.69) and secreted (total: P = 0.05, r = −0.72; HMW: P = 0.02, r = −0.87) adiponectin. The % small FC in SAT correlated positively with both serum (total: P = 0.006, r = 0.87; HMW: P = 0.009, r = 0.79) and secreted (total: P = 0.03, r = 0.75; HMW: P = 0.01, r = 0.92) adiponectin. VAT FC size correlated negatively with serum HMW adiponectin (P = 0.01, r = −0.76) but not with any measure of secretion. VAT had the greatest % small FC, which related positively to serum HMW (P = 0.004, r = 0.81) and to secreted total adiponectin (P = 0.02, r = 0.78). These studies indicate that differences in fat cell size and depot distribution of AT expansion are important influences on adiponectin in obesity.

Serum under-carboxylated osteocalcin levels in women with polycystic ovary syndrome: weight-dependent relationships with endocrine and metabolic traits

BackgroundUnder-carboxylated osteocalcin (ucOC), the precursor substrate of bone biomarker OC is a potent regulator of energy metabolism by promoting insulin production and adiponectin synthesis and decreasing fat stores. The aim of the present study was to point out the potential role of ucOC in the physiopathology of polycystic ovary syndrome (PCOS), a common disorder defined by the constellation of anovulation, insulinresistance, hyperinsulinemia, obesity and androgen excess.MethodsIn this prospective case–control investigation, 78 young premenopausal women, i.e. 52 PCOS patients and 26 age- and body mass index (BMI)-matched healthy controls, were successively enrolled. Recruitment of PCOS patients was performed according to Androgen Excess-Polycystic Ovary Syndrome (AE-PCOS) Society 2006 criteria. All study participants were subjected to clinical examination, whole-body composition assessment and measurements of serum ucOC, OC (1-49), glucose and lipids, insulin, total testosterone (TT), estradiol, sex-hormone binding globulin (SHBG), high-sensitivity C-reactive protein (Hs-CRP) and β-CrossLaps.ResultsBMI-stratified multivariate analysis revealed significantly higher ucOC levels in PCOS vs. controls in lean (p = 0.001) but not overweight and obese study participants (p = 0.456). Notably, a positive correlation between ucOC and TT (p = 0.018), calculated free testosterone (cFT, p = 0.028) and serum insulin (p = 0.036), respectively, was found to be confined to the lean analysis subgroup. Furthermore, in stepwise multiple regression models, β-CrossLaps and cFT were able to predict 46.71% of serum ucOC variability. (1-43/49)OC failed to be significantly associated to any PCOS trait.ConclusionsCirculating ucOC concentration is related to key endocrine PCOS characteristics in a weight-dependent manner. Within the bone-pancreas loop, high ucOC may favor insulin release in lean hyperandrogenic women to compensate for impaired insulin sensitivity.

Abstract 74: Heme Oxygenase-PPARα Induction of FGF21 in Hepatocytes Recruits pAMPK/pAKT and Attenuates Insulin Resistance in Obese Mice

The peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor that is known to regulate glucose and lipid homeostasis and increases expression of the hormone fibroblast growth factor 21 (FGF21), which is secreted to blood, increases phosphorylation of AMPK and AKT in adipocytes and has been purported to stimulate adiponectin synthesis. The heme oxygenase (HO) system is functionally important for reducing ROS and inflammation whose induction has been shown to increase serum adiponectin levels and affect lowering of blood glucose and fatty acids. This study was designed to examine the hypothesis that a HO-1-PPARα crosstalk could elevate hepatic FGF21 thus leading to enhanced adiponectin secretion and abatement of metabolic imbalance. Preliminary experiments were conducted in human hepatocellular carcinoma cells (Hep G2) cultured in the absence or presence of the HO-1 inducer, cobalt protoporphyrin (CoPP, 2μmoles/l). A 2.0 fold induction of HO-1 in Hep G2 cells by CoPP (p&lt;0.05) increased expression of FGF21 1.5 fold (n=3, p=0.0119 vs. vehicle control) without affecting PPARα expression. Additional experiments in obese (ob/ob) mice treated with CoPP (5mg/kg/day) resulted in 2 fold (p&lt;0.05) increase of FGF21 mRNA expression in liver as compared to mice treated with the vehicle. These observations were complemented by blood glucose measurements, which showed significant attenuation in obese mice treated with CoPP (vehicle: 285.0±24 vs. CoPP: 160.0±27.3, p&lt;0.05, n= 5), along with enhancement of adiponectin levels in CoPP treated obese mice (p&lt;0.05). Taken together these results demonstrate that HO-1 could increase serum adiponectin levels and insulin sensitivity by elevating hepatic FGF21 levels. Our overall hypothesis is that the HO-1 can stimulate PPARα activity with resultant activation of hepatic FGF21 secretion and that this HO-PPARα-FGF21 axis could work in concert to modulate the development of insulin resistance and diabetes.

Resveratrol prevents TNFα-induced suppression of adiponectin expression via PPARγ activation in 3T3-L1 adipocytes

Tumor necrosis factor α (TNFα) is an adipokine, whose increase is known to suppress the expression and secretion of adiponectin in adipocytes. Resveratrol has been ever reported to recover the suppression of adiponectin by TNFα, but the underlying mechanism remains poorly understood. In this study, we validated the roles of resveratrol in the inhibition of the adiponectin by TNFα in 3T3-L1 cells. Exposure to TNFα for 24 h inhibited adiponectin synthesis and secretion, but the inhibitions were partially recovered by resveratrol treatment in 3T3-L1 adipocytes. Furthermore, we found that resveratrol improved the expression of adiponectin by the increase of PPARγ DNA-binding activity. Our results suggest that resveratrol may attenuate the inhibition of adiponectin expression by TNFα via activation of PPARγ, thereby possibly improving insulin resistance. However, significant preventive effects of resveratrol were only observed when it was administrated before TNFα increase, limiting its use as preventive strategy for insulin resistance.

C-Reactive Protein in the Inflammatory Scene: Still a Long and Winding Road

Over the last decades, inflammation and obesity have deserved particular attention to explain the etiopathogenesis of atherosclerosis. (1, 2) White adipose tissue produces tumor necrosis factor-alpha and specific neutralizing antibodies reverse insulin resistance in obese animals. (3) Therefore, the study of adipose tissue has attracted great interest, as it has been recognized as a real endocrine organ. (2) In most subjects, obesity is associated with changes in adipocytes and macrophage function, expressing a “chronic low-grade inflammation” which leads to insulin resistance (IR) and to the development of diabetes mellitus (DM). (4) Adipose tissue is the main source of interleukin-6 (IL-6), a potent marker of inflammation. This cytokine is also associated with IR and increases the risk of DM, independently of body weight. (5) Adipocytes also produce adiponect in, a cytokine with anti-atherosclerotic effects that increases insulin sensitivity by inhibiting the production of hepatic glucose, improves peripheral glucose utilization and increases fatty acid oxidation. Adiponect in synthesis is reduced in IR, obesity, metabolic syndrome (MS) and DM. Recently, the key role of IR has lost importance while more attention is being paid to the theory of lipotoxicity due to leptin resistance and ectopic lipid deposition.(6) Finally, truncal obesity is related to IR, determining a pro inflammatory condition with increased levels of adipocytokines as IL-6 and tumor necrosis factor-alpha and adiponect in reduction. Prothrombotic mechanisms, such as increase in fibrinogen levels and PAI-1 with subsequent inhibition of fibrinolysis, are also activated. All these factors predispose to other major risk factors as DM, hypertension and dyslipidemia. Under this scenario, the development of atherosclerotic disease may be just a matter of time. Obesity predisposes to the development of DM, one of the main risk factors for cardiovascular disease. In a multicenter study, Yusuf et al,(7) have insisted on the importance of truncal obesity as a major cardiovascular risk factor. The MS is a possible intermediary between risk factors and the likelihood of presenting cardiovascu-

Ascorbic acid‐induced reduction in adiponectin is not improved by alpha tocopherol and is accompanied by a small reduction in protein levels of Peroxisome Proliferator‐ Activated Receptor Gamma in 3T3‐L1 adipocytes

Adipocytes play a role in metabolism and regulation of homeostasis through the release of cytokines such as adiponectin. Adiponectin is an anti‐inflammatory adipokine and has been shown to improve insulin sensitivity and enhance beta‐oxidation of fatty acids. A reduction in adiponectin has been associated with increased inflammation, metabolic syndrome and chronic diseases. Since oxidative stress has been known to regulate metabolic disease, we previously investigated the role of ascorbic acid and found that it abolished adiponectin expression in adipocytes. We hypothesized that treatment with ascorbic acid may cause a reduction in PPARgamma protein levels and that alpha tocopherol may mitigate the effect of ascorbic acid on adiponectin synthesis. We used 3T3‐L1 cells to monitor adiponectin protein levels. Differentiated 3T3‐L1 (Day 8), cells were treated with ascorbic acid, with and without alpha tocopherol. We found that the reduction of adiponectin in cell lysates was not overcome with the addition of alpha tocopherol. Further, we also observed a small reduction in PPARgamma in adipocytes treated with ascorbic acid. These results suggest that adipocyte metabolism may be influenced by dietary antioxidants. This research was funded by Florida International University.

Effects of bioactive salmon peptides and fatty acids on cellular transport and secretion of adiponectin

Plasma adiponectin levels are inversely related to several components of the metabolic syndrome and therapeutics that target adiponectin secretion are emerging. Consumption of fish oil is associated with improved metabolic profile, however, there is additional evidence that the protein component of fish may also have metabolic benefits. The aim of this study was to examine the effects of fish fatty acids and peptides on adiponectin assembly and secretion in differentiated 3T3‐L1 adipocytes. Treatment with polyunsaturated fatty acids (PUFAs) increased total cellular adiponectin levels but did not increase adiponectin secretion. In addition, two fractions of molecular weight fractionated (&lt;1000 dalton) salmon peptides, but not cod peptides, stimulated adiponectin synthesis and secretion. Subcellular fractionation on sucrose density gradients revealed that the majority of cellular adiponectin is located in the endoplasmic reticulum (ER). Treatment of the cells with Brefeldin A prevented adiponectin secretion and caused cellular accumulation of the high molecular weight adiponectin oligomers. These data suggest that the ER is an important site of cellular adiponectin accumulation and that treatment with PUFAs or salmon peptides can increase adiponectin in this adipocyte model.Grant Funding Source: Heart and Stroke Foundation of Canada and Natural Sciences and Engineering Research Council of Canada

Mitochondrial dysfunction and activation of iNOS are responsible for the palmitate-induced decrease in adiponectin synthesis in 3T3L1 adipocytes

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress are considered the key determinants of insulin resistance. Impaired mitochondrial function in obese animals was shown to induce the ER stress response, resulting in reduced adiponectin synthesis in adipocytes. The expression of inducible nitric oxide synthase (iNOS) is increased in adipose tissues in genetic and dietary models of obesity. In this study, we examined whether activation of iNOS is responsible for palmitate-induced mitochondrial dysfunction, ER stress, and decreased adiponectin synthesis in 3T3L1 adipocytes. As expected, palmitate increased the expression levels of iNOS and ER stress response markers, and decreased mitochondrial contents. Treatment with iNOS inhibitor increased adiponectin synthesis and reversed the palmitate-induced ER stress response. However, the iNOS inhibitor did not affect the palmitate-induced decrease in mitochondrial contents. Chemicals that inhibit mitochondrial function increased iNOS expression and the ER stress response, whereas measures that increase mitochondrial biogenesis (rosiglitazone and adenoviral overexpression of nuclear respiratory factor-1) reversed them. Inhibition of mitochondrial biogenesis prevented the rosiglitazone-induced decrease in iNOS expression and increase in adiponectin synthesis. These results suggest that palmitate-induced mitochondrial dysfunction is the primary event that leads to iNOS induction, ER stress, and decreased adiponectin synthesis in cultured adipocytes.

Glucose-Based Peritoneal dialysis solution suppresses adiponectin synthesis through oxidative stress in an experimental model of peritoneal dialysis

Accumulation of visceral fat is one of the major risk factors for the development of cardiovascular disease in peritoneal dialysis (PD) patients. Adiponectin, an adipokine commonly regarded as a negative indicator of metabolic disease, is reported to be downregulated in its gene level in end-stage renal disease patients. Since excessive fat deposit is involved in increased reactive oxygen species (ROS), PD solution (PDS) may contribute to ROS production, resulting in dysregulation of adiponectin. In this study, we tested our hypothesis that oxidative stress induced by PDS may play a role in the regulation of adiponectin. Commercial PDS containing 3.86% glucose (20 - 30 mL) was administered to SD rats for 12 weeks with and without N-acetylcysteine (NAC; 10 mmol/L). ELISA was used to quantify adiponectin in plasma and spent dialysate. For in vitro studies, fully differentiated 3T3-L1 adipocytes and adipocytes isolated from abdominal fat were treated with a high glucose solution, PDS, and H(2)O(2). Adiponectin levels in the conditioned media were measured by ELISA and immunoblot assays. The mRNA levels of adiponectin in mature adipocytes were examined using real-time RT-PCR. The levels of adiponectin in plasma and spent dialysate were significantly downregulated by PDS and this effect was suppressed by NAC. In 3T3-L1 adipocytes, adiponectin secretion was inhibited by 50 mmol/L glucose, PDS diluted 2-fold, and H(2)O(2) (200 μmol/L). In addition, H(2)O(2) downregulated expression of adiponectin mRNA and secretion of adiponectin oligomer complexes. Our data suggest that ROS induced by conventional glucose-based PDS may contribute to pathophysiological changes in abdominal fat and downregulate adiponectin secreted from adipocytes during long-term PD.

Mitochondria and Endoplasmic Reticulum in Diabetes and Its Complications

Mitochondria and Endoplasmic Reticulum in Diabetes and Its Complications

Carbohydrate Metabolism and the Skeleton: Picking a Bone with the β-Cell

Novel data show that the bone protein, undercarboxylated osteocalcin, increases islet cell growth and insulin and adiponectin synthesis and enhances glucose metabolism in mice. Human studies, in which undercarboxylated osteocalcin concentrations have been perturbed, show no effect (or a very minor effect) on fasting glucose or insulin concentrations. More extensive and detailed experiments in humans are needed to definitively establish the role of undercarboxylated osteocalcin in human carbohydrate metabolism.

Adiponectin Resistance in Heart Failure and the Emerging Pattern of Metabolic Failure in Chronic Heart Failure

In recent years, an interesting shift in our understanding of chronic heart failure (CHF) pathophysiology may be observed. Beyond the traditional concepts of hemodynamic failure and neuroendocrine activation, it is increasingly appreciated that CHF is a much more complex and truly systemic disease including the peripheral organs and whole body regulatory processes as well. Novel concepts such as the systemic inflammatory immune activation and the skeletal muscle hypothesis have emerged.1 These concepts recognize the significant contribution of peripheral changes to symptomatic status, disease progression, and outcome in CHF. Article see p 185 A further novel facet in heart failure pathophysiology has emerged recently because impaired regulation of systemic metabolic balance is increasingly in the focus of clinical research. The emerging picture suggests a complex but characteristic pattern of metabolic pathways that are imbalanced, attenuated, or abnormally activated. Hormonal imbalances have been previously observed as common features in CHF, such as insulin resistance2 and growth hormone resistance3 that contribute to both morbidity and mortality of patients. The metabolic interaction of neuroendocrine activation and immune activation such as from cytokines and oxygen radical accumulation add to the complexity of these interrelated processes. In general, the findings repeat, again, the classical concept of short-term beneficial adaptive responses on acute injury or disease may eventually turn into harmful maladaptive signals on prolonged and chronic activation. The overall clinical effects that may be observed from impaired energy metabolic efficacy contribute to impaired exercise capacity, muscle fatigue, and early exhaustion—key symptoms in heart …

ENOS plays a major role in adiponectin synthesis in adipocytes

Nitric oxide (NO) stimulates mitochondrial biogenesis. We recently reported that adiponectin synthesis is regulated by mitochondrial function in adipocytes. This study was undertaken to test the hypothesis that endothelial NO synthase (eNOS) plays an important role in adiponectin synthesis by producing NO and enhancing mitochondrial function in adipocytes. We examined the effects of eNOS knockdown on adiponectin synthesis in 3T3-L1 adipocytes and also examined plasma adiponectin levels and the mitochondria in adipose tissue of eNOS knockout (eNOS(-/-)) mice with and without chronic administration of a NO donor. In cultured 3T3-L1 adipocytes, eNOS siRNA decreased rosiglitazone-induced adiponectin secretion, which was associated with decreases in mitochondrial proteins and biogenesis factors. Plasma adiponectin concentrations were reduced in adult eNOS(-/-) mice compared with age-matched wild-type mice. Mitochondrial contents in adipose tissue were reduced in eNOS(-/-) mice, and this was associated with decreased expression of mitochondrial biogenesis factors, increased levels of 8-hydroxyguanosine, a biomarker of oxidative stress, and morphological abnormalities in mitochondria. Rosiglitazone-induced increases in adiponectin expression and mitochondrial content were also reduced significantly in eNOS(-/-) mice. Chronic administration of a NO donor reversed mitochondrial abnormalities and increased adiponectin expression in adipose tissue of eNOS(-/-) mice. eNOS plays an important role in adiponectin synthesis in adipocytes by increasing mitochondrial biogenesis and enhancing mitochondrial function.

Cellular hypoxia and adipose tissue dysfunction in obesity

Expansion of adipose tissue mass, the distinctive feature of obesity, is associated with low-grade inflammation. White adipose tissue secretes a diverse range of adipokines, a number of which are inflammatory mediators (such as TNFalpha, IL-1beta, IL-6, monocyte chemoattractant protein 1). The production of inflammatory adipokines is increased with obesity and these adipokines have been implicated in the development of insulin resistance and the metabolic syndrome. However, the basis for the link between increased adiposity and inflammation is unclear. It has been proposed previously that hypoxia may occur in areas within adipose tissue in obesity as a result of adipocyte hypertrophy compromising effective O2 supply from the vasculature, thereby instigating an inflammatory response through recruitment of the transcription factor, hypoxic inducible factor-1. Studies in animal models (mutant mice, diet-induced obesity) and cell-culture systems (mouse and human adipocytes) have provided strong support for a role for hypoxia in modulating the production of several inflammation-related adipokines, including increased IL-6, leptin and macrophage migratory inhibition factor production together with reduced adiponectin synthesis. Increased glucose transport into adipocytes is also observed with low O2 tension, largely as a result of the up-regulation of GLUT-1 expression, indicating changes in cellular glucose metabolism. Hypoxia also induces inflammatory responses in macrophages and inhibits the differentiation of preadipocytes (while inducing the expression of leptin). Collectively, there is strong evidence to suggest that cellular hypoxia may be a key factor in adipocyte physiology and the underlying cause of adipose tissue dysfunction contributing to the adverse metabolic milieu associated with obesity.

The relationship between adipokines, osteocalcin and bone quality in chronic kidney disease

Osteocalcin, a small peptide secreted by osteoblasts, has been recently described as a circulating hormone involved in the regulation of energy metabolism. In addition, experimental data suggest a regulation of adipocytes by bone, with a stimulation of adiponectin synthesis by osteocalcin and an inverse relationship between serum adiponectin level and bone mineral density (BMD). However, this relationship has not been explored during chronic kidney disease (CKD). Osteocalcin, adiponectin and leptin were prospectively measured in a cohort of 61 CKD patients. A new non-invasive 3D bone imaging technique was performed (high-resolution peripheral quantitative computed tomography, HR-pQCT), measuring volumetric BMD (vBMD) and microarchitecture parameters at the distal tibia. Patients' mean age was 67.2 +/- 13.9 years and mean GFR 33 +/- 12 mL/min/1.73 m(2). We found a positive association between serum osteocalcin and adiponectin (r = 0.29, P = 0.021). Univariate analysis showed inverse correlations between serum adiponectin and total vBMD (r = -0.33, P = 0.01), cortical thickness (r = -0.34, P = 0.008) and trabecular vBMD (r = -0.27, P = 0.04). These associations remained significant in multivariate analysis between serum adiponectin and total vBMD, cortical vBMD and cortical thickness. We report for the first time an inverse relationship between bone density and adiponectin, as well as a positive association between osteocalcin and adiponectin in CKD II-IV patients.

The cannabinoid CB1 receptor antagonist, rimonabant, protects against acute myocardial infarction

CB1 antagonism is associated with reduced doxorubicin-induced cardiotoxicity and decreased cerebrocortical infarction. Rimonabant, a selective CB1 receptor antagonist, was, before it was withdrawn, proposed as a treatment for obesity and reported to reduce cardiovascular risk by improving glucose and lipid profiles and raising adiponectin levels. The cardioprotective actions of rimonabant in 6-week-old C57BL/6J mice fed either high-fat (HFD) or standard diets (STD) for 8 weeks were investigated. At 14 weeks, mice received rimonabant (10 mg/kg/day, i.p.) or vehicle for 1 week and were then subjected to an in vivo acute myocardial infarction. The influence of rimonabant on infarct size (IS) in CB1 knockout (CB1-/-) and wild-type (CB1+/+) mice was also examined. C57BL/6J mice that had been maintained on STD or HFD exhibited 4.3 and 21.4% reductions in body weight following 7 days rimonabant treatment. Rimonabant reduced IS in both STD (29.6 +/- 3.5% vs. 49.8 +/- 6.9% in control, P < 0.05) and HFD (26.9 +/- 1.5% vs. 48.7 +/- 7% in control, P < 0.05) mice. In CB1-/- mice rimonabant failed to reduce body weight or IS (51.0 +/- 5.3% vs. 49.7 +/- 4.7% in control, P > 0.05), although significant reductions were seen in CB1+/+ mice (IS, 48.9 +/- 4.6% control vs. 30.5 +/- 3.1% rimonabant, P < 0.05). To exclude the possibility that weight loss alone induced cardioprotection, HFD mice were switched to STD for 7 days (HFD-STD), resulting in an 11.3 +/- 1.0% decrease in body weight compared to control (+2.1 +/- 1.1% in HFD). This, however, was not associated with IS reduction (39.1 +/- 3.9% HFD-STD vs. 40.0 +/- 5.3% HFD, P > 0.05). Serum and cardiac adiponectin levels were unaltered by rimonabant treatment. HL-1 cell death was not prevented by 1 or 7 days treatment with rimonabant. We conclude that rimonabant-induced infarct limitation may involve the CB1 receptor, although not necessarily cardiac CB1 receptors, and is unrelated to weight loss or altered adiponectin synthesis.

Adiponectin translation is increased by the PPARγ agonists pioglitazone and ω-3 fatty acids

Adiponectin, made exclusively by adipocytes, is a 30-kDa secretory protein assembled posttranslationally into low-molecular weight, middle-molecular weight, and high-molecular weight homo-oligomers. PPARgamma ligand thiozolidinediones, which are widely used in the treatment of type II diabetes, increase adiponectin levels. PPARgamma also has several putative ligands that include fatty acid derivatives. Overnight treatment of rat adipocytes with pioglitazone, docosahexaenoic acid (DHA), or eicosapentaenoic acid (EPA) triggered a twofold increase in the synthesis and secretion of HMW adiponectin, and this increase was blocked by the addition of PPARgamma inhibitor GW-9662. Inhibition of glycosylation using 2,2'-dipyridyl decreased the synthesis of high-molecular weight adiponectin by pioglitazone, EPA, and DHA, but there was increased secretion of trimeric adiponectin resulting from increased translation. Although pioglitazone, DHA, and EPA increased adiponectin synthesis by more than 60%, there was no increase in total protein synthesis and no corresponding change in adiponectin mRNA expression, indicating the upregulation of translation. We examined the possibility of transacting factors in the cytoplasmic extracts from adipocytes treated with pioglitazone or DHA. In vitro translation of adiponectin mRNA was inhibited by S-100 fraction of control adipocytes and increased by S-100 extracts from adipocytes treated with pioglitazone or DHA. Consistent with this observation, both pioglitazone and DHA treatments increased the association of adiponectin mRNA with the heavier polysome fractions. Together, these data suggest that pioglitazone and the fish oils DHA or EPA are PPARgamma agonists in adipocytes with regard to adiponectin expression, and the predominant mode of adiponectin stimulation is via an increase in translation.

Regulation of adiponectin secretion by insulin and amino acids in 3T3-L1 adipocytes

Adiponectin is a fat cell–derived hormone with insulin-sensitizing properties. Low plasma adiponectin levels are associated with insulin resistance as found in obesity. One of the mechanisms for this finding is hampered insulin signaling via phosphatidylinositol 3-kinase (PI3K) with concomitant decreased adiponectin secretion. Because insulin can also stimulate signaling at the level of mammalian target of rapamycin (mTOR) by a mechanism that is dependent on the presence of amino acids, the role of mTOR signaling in adiponectin secretion was studied. In view of the vesicular nature of adiponectin secretion, the role of lysosomes was explored as well. In 3T3-L1 adipocytes, both insulin and amino acids stimulated adiponectin secretion. The stimulation by insulin was PI3K dependent but mTOR independent. The stimulation by amino acids was independent of both PI3K and mTOR. Whereas the effect of insulin via PI3K was mainly on adiponectin secretion from adipocytes, the effect of amino acids was predominantly due to their role as substrates for adiponectin synthesis. The acidotropic agents ammonia and methylamine, but not the lysosomal protease inhibitor leupeptin and the autophagy inhibitor 3-methyladenine, strongly inhibited adiponectin secretion and increased the intracellular adiponectin pool. In conclusion, adiponectin production is substrate driven. Phosphatidylinositol 3-kinase and an acidic lysosomal pH, but not amino acid–mediated mTOR signaling or lysosomal breakdown, are involved in adiponectin secretion.

Pioglitazone acutely stimulates adiponectin secretion from mouse and human adipocytes via activation of the phosphatidylinositol 3′-kinase

Aims Thiazolidinediones increase circulating adiponectin. We have previously demonstrated the involvement of the phosphatidylinositol 3-kinase (PI3K) signaling pathway in insulin-stimulated adiponectin secretion. We therefore investigated the effects of the thiazolidinedione pioglitazone on acute adiponectin secretion, and the involvement of the PI3K signaling pathway in this action. Main methods We treated murine 3T3-L1 and human primary adipocytes with 1–10 uM pioglitazone for 2 h, +/− PI3K inhibition by Wortmannin (WT). Secreted adiponectin was measured by Western blot. PI3K activity following 15-minute treatments with 1–10 uM pioglitazone was measured by thin layer chromatography. Pioglitazone's effect on adiponectin synthesis and on secretion of newly synthesized adiponectin was studied in 3T3-L1 adipocytes using a pulse-chase technique. Key findings Pioglitazone was found to increase adiponectin secretion and PI3K activity in a dose-dependent manner from 3T3-L1 and human adipocytes. In 3T3-L1 adipocytes, 10 uM pioglitazone increased adiponectin secretion by 84 +/− 14% ( p < 0.0001) at 2 h. Similarly, in human adipocytes there was a 56 +/− 18% ( p < 0.02) increase in secretion. WT blocked the pioglitazone effect and decreased adiponectin secretion at 2 h (47% of pioglitazone treated, p < 0.006). Pioglitazone increased PI3K activity in a dose-dependent manner in both 3T3-L1 (1.7 vs. 2.7-fold increase over control at 2 uM vs. 10 uM dose, p = 0.02) and human adipocytes. Significance Our data show that pioglitazone acutely stimulates adiponectin secretion from both 3T3-L1 and human adipocytes. This acute effect of pioglitazone is PI3K-dependent.