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Dysregulated production of adipocytokines may be involved in the development of atherosclerotic cardiovascular disease in metabolic syndrome and chronic kidney disease (CKD) associated with metabolic syndrome. The aim of this study was to determine the effects of treatment with angiotensin II (Ang II) type-1 receptor blocker (ARB) on the regulation of adipocytokines. Olmesartan, an ARB, significantly blunted the age- and body weight-associated falls in plasma adiponectin both in genetically and diet-induced obese mice, without affecting body weight, but had no effect on plasma adiponectin levels in lean mice. Olmesartan also ameliorated dysregulation of adipocytokines in obesity, such as tumor necrosis factor-alpha, plasminogen activator inhibitor-1, monocyte chemotactic protein-1, and serum amyloid A3. Olmesartan significantly reduced reactive oxygen species originating from accumulated fat and attenuated the expression of nicotinamide adenine dinucleotide phospho hydrogenase oxidase subunits in adipose tissue. In cultured adipocytes, olmesartan acted as an antioxidant and improved adipocytokine dysregulation. Our results indicate that blockade of Ang II receptor ameliorates adipocytokine dysregulation and that such action is mediated, at least in part, by targeting oxidative stress in obese adipose tissue. Ang II signaling and subsequent oxidative stress in adipose tissue may be potential targets for the prevention of atherosclerotic cardiovascular disease in metabolic syndrome and also in metabolic syndrome-based CKD.

Here we investigated the biological functions of adiponectin, a fat-derived hormone, by disrupting the gene encodes it in mice. Adiponectin knockout mice (KO) exhibited severe diet-induced insulin resistance with reduced IRS-1-associated P13-kinase activity in muscle. KO also revealed severe neo-intimal thickening in response to vascular-injury and hypertension induced by salt diet. Carbon-tetrachloride induced severe liver fibrosis in KO with the elevated gene expression of growth factors. These phenotypes in KO were reversed by viral-mediated production of adiponectin. Our results indicate that adiponectin should be one of key molecule of the metabolic syndrome and may be a new therapeutic target for the metabolic syndrome.

Background— Vascular inflammation and subsequent matrix degradation play an important role in the development of atherosclerosis. We previously reported that adiponectin, an adipose-specific plasma protein, accumulated to the injured artery and attenuated vascular inflammatory response. Clinically, high plasma adiponectin level was associated with low cardiovascular event rate in patients with chronic renal failure. The present study was designed to elucidate the effects of adiponectin on matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in human monocyte-derived macrophages. Methods and Results— Human monocyte-derived macrophages were incubated with the physiological concentrations of human recombinant adiponectin for the time indicated. Adiponectin treatment dose-dependently increased TIMP-1 mRNA levels without affecting MMP-9 mRNA levels. Adiponectin also augmented TIMP-1 secretion into the media, whereas MMP-9 secretion and activity were unchanged. Time course experiments indicated that TIMP-1 mRNA levels started to increase at 24 hours of adiponectin treatment and were significantly elevated at 48 hours. Adiponectin significantly increased interleukin-10 (IL-10) mRNA expression at the transcriptional level within 6 hours and significantly increased IL-10 protein secretion within 24 hours. Cotreatment of adiponectin with anti–IL-10 monoclonal antibody completely abolished adiponectin-induced TIMP-1 mRNA expression. Conclusions— Adiponectin selectively increased TIMP-1 expression in human monocyte-derived macrophages through IL-10 induction. This study identified, for the first time, the adiponectin/IL-10 interaction against vascular inflammation.

Endothelial dysfunction is a crucial feature in the evolution of atherosclerosis. Adiponectin is an adipocyte-specific plasma protein with antiatherogenic and antidiabetic properties. In the present study, we investigated the relation between adiponectin and endothelium-dependent vasodilation. We analyzed endothelial function in 202 hypertensive patients, including those who were not taking any medication. Forearm blood flow was measured by strain-gauge plethysmography. Plasma adiponectin level was highly correlated with the vasodilator response to reactive hyperemia in the total ( r =0.257, P r =0.296, P =0.026) groups but not with nitroglycerin-induced hyperemia, indicating that adiponectin affected endothelium-dependent vasodilation. Multiple regression analysis of data from all hypertensive patients revealed that plasma adiponectin level was independently correlated with the vasodilator response to reactive hyperemia. Vascular reactivity was also analyzed in aortic rings from adiponectin-knockout (KO) and wild-type (WT) mice. Adiponectin-KO mice showed obesity, hyperglycemia, and hypertension compared with WT mice after 4 weeks on an atherogenic diet. Endothelium-dependent vasodilation in response to acetylcholine was significantly reduced in adiponectin-KO mice compared with WT mice, although no significant difference was observed in endothelium-independent vasodilation in response to sodium nitroprusside. Our observations suggest that hypoadiponectinemia is associated with impaired endothelium-dependent vasorelaxation and that the measurement of plasma adiponectin level might be helpful as a marker of endothelial dysfunction.

Adiponectin, an adipocyte-derived protein, consists of collagen-like fibrous and complement C1q-like globular domains, and circulates in human plasma in a multimeric form. The protein exhibits anti-diabetic and anti-atherogenic activities. However, adiponectin plasma concentrations are low in obese subjects, and hypoadiponectinemia is associated with the metabolic syndrome, which is a cluster of insulin resistance, type 2 diabetes mellitus, hypertension, and dyslipidemia. We have recently reported a missense mutation in the adiponectin gene, in which isoleucine at position 164 in the globular domain is substituted with threonine (I164T). Subjects with this mutation showed markedly low level of plasma adiponectin and clinical features of the metabolic syndrome. Here, we examined the molecular characteristics of the mutant protein associated with a genetic cause of hypoadiponectinemia. The current study revealed (1) the mutant protein showed an oligomerization state similar to the wild-type as determined by gel filtration chromatography and, (2) the mutant protein exhibited normal insulin-sensitizing activity, but (3) pulse-chase study showed abnormal secretion of the mutant protein from adipose tissues. Our results suggest that I164T mutation is associated with hypoadiponectinemia through disturbed secretion into plasma, which may contribute to the development of the metabolic syndrome.

OBJECTIVES This study examined the association of mutations in adiponectin gene with the prevalence of coronary artery disease (CAD). BACKGROUND Coronary artery disease is a major cause of mortality in the industrial countries. Adiponectin gene locus, chromosome 3q27, is the candidate site for CAD. We have reported that adiponectin has antiatherogenic and antidiabetic properties, and that the plasma levels negatively correlated with body mass index (BMI) are significantly low in patients with CAD or type 2 diabetes. METHODS The study subjects were 383 consecutive patients with angiographically confirmed CAD and 368 non-CAD subjects adjusted for age and BMI in the Japanese population. Single nucleotide polymorphisms (SNPs) in the adiponectin gene were determined by Taqman polymerase chain reaction (PCR) method or a PCR-based assay for the analysis of restriction fragment length polymorphism. The plasma adiponectin concentration was measured by enzyme-linked immunosorbent assay. RESULTS Among SNPs, the frequency of I164T mutation was significantly higher in CAD subjects (2.9%) than in the control (0.8%, p < 0.05). The plasma adiponectin levels in subjects carrying the I164T mutation were significantly lower than in those without the mutation, and were independent of BMI. In contrast, SNP94 and SNP276, which are reported to be associated with an increased risk of type 2 diabetes, were associated neither with CAD prevalence nor with plasma adiponectin level. Subjects with I164T mutation exhibited a clinical phenotype of the metabolic syndrome. CONCLUSIONS The I164T mutation in the adiponectin gene was a common genetic background associated with the metabolic syndrome and CAD in the Japanese population.

Many studies have reported the cholesterol-lowering, anti-lipogenic, anti-obesity and anti-hypertensive effects of soy protein. Adipose tissue-specific plasma protein, adiponectin, has anti-atherogenic and anti-insulin-resistance properties. Here, we investigated the effects of soy protein diet on body fat composition, plasma glucose, lipid and adiponectin levels and expression of genes involved in glucose and fatty acid metabolism in obese KK-A y mice. Body weights and adipose tissue weights of mesenteric, epididymal, and brown fat were lower in mice on calorie-restricted diet containing soy protein isolate. Plasma cholesterol, triglyceride, free fatty acid, and glucose levels were also decreased by this diet. Body fat content and plasma glucose levels in mice on a soy protein isolate diet were still lower than those treated with an isocaloric casein-protein-diet. Among the genes related to glucose and fatty acid metabolism, adiponectin mRNA levels in adipose tissue and adiponectin plasma concentrations were elevated in mice on a calorie-restricted diet, although there were no significant differences between soy protein and casein protein groups. Our results indicate that that soy protein diet decreased body fat content and plasma glucose levels more effectively than isocaloric casein-protein diet in obese mice.

Background— High-sensitive C-reactive protein (hs-CRP) is a well-known risk factor for coronary artery disease (CAD). Recently, we have demonstrated that adiponectin served as an antiatherogenic plasma protein which was secreted specifically from adipocytes. The present study investigated the association between adiponectin and CRP in the blood stream and adipose tissue. Methods and Results— We studied a total of 101 male patients, 71 of whom had angiographically documented coronary atherosclerosis. As a control group, 30 patients with normal coronary angiogram were included. The plasma hs-CRP levels were negatively correlated with the plasma adiponectin levels ( r =−0.29, P r =−0.89, P Conclusions— The reciprocal association of adiponectin and CRP levels in both human plasma and adipose tissue might participate in the development of atherosclerosis.

Plasma glycerol is a major substrate for hepatic gluconeogenesis. Aquaporin adipose (AQPap/7), an adipose-specific glycerol channel, provides fat-derived glycerol into plasma. In the present study, we cloned the coding and promoter regions of mouse aquaporin 9 (AQP9), a liver-specific glycerol channel. Fasting and refeeding of mice increased and decreased hepatic AQP9 mRNA levels, respectively. Insulin deficiency induced by streptozotocin resulted in increased hepatic AQP9 mRNA. These changes in hepatic AQP9 mRNA were accompanied by those of hepatic gluconeogenic mRNAs and plasma glycerol levels. In cultured hepatocytes, insulin downregulated AQP9 mRNA. The AQP9 promoter contained the negative insulin response element TGTTTTC at −496/−502, similar to the promoter of the AQPap/7 gene. In contrast, in insulin-resistant db+/db+ mice, AQPap/7 mRNA in fat and AQP9 mRNA in liver were increased, despite hyperinsulinemia, with high plasma glycerol and glucose levels. Glycerol infusion in the db+/db+ mice augmented hepatic glucose output. Our results indicate that coordinated regulations of fat-specific AQPap/7 and liver-specific AQP9 should be crucial to determine glucose metabolism in physiology and insulin resistance.

Obesity is more linked to vascular disease, including atherosclerosis and restenotic change, after balloon angioplasty. The precise mechanism linking obesity and vascular disease is still unclear. Previously we have demonstrated that the plasma levels of adiponectin, an adipose-derived hormone, decreases in obese subjects, and that hypoadiponectinemia is associated to ischemic heart disease. In current the study, we investigated the in vivo role of adiponectin on the neointimal thickening after artery injury using adiponectin-deficient mice and adiponectin-producing adenovirus. Adiponectin-deficient mice showed severe neointimal thickening and increased proliferation of vascular smooth muscle cells in mechanically injured arteries. Adenovirus-mediated supplement of adiponectin attenuated neointimal proliferation. In cultured smooth muscle cells, adiponectin attenuated DNA synthesis induced by growth factors including platelet-derived growth factor, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), basic fibroblast growth factor, and EGF and cell proliferation and migration induced by HB-EGF. In cultured endothelial cells, adiponectin attenuated HB-EGF expression stimulated by tumor necrosis factor alpha. The current study suggests an adipo-vascular axis, a direct link between fat and artery. A therapeutic strategy to increase plasma adiponectin should be useful in preventing vascular restenosis after angioplasty.

Aquaporin adipose (AQPap), which we identified from human adipose tissue, is a glycerol channel in adipocyte [Kishida et al. (2000) J. Biol. Chem. 275, 20896-20902]. In the current study, we determined the genomic structure of the human AQPap gene, and identified three AQPap-like genes that resembled (approximately 95%) AQPap, with little expression in human tissues. The AQPap promoter contained a putative peroxisome proliferator response element (PPRE) at -46 to -62, and a putative insulin response element (IRE) at -542/-536. Deletion of the PPRE abolished the pioglitazone-mediated induction of AQPap promoter activity in 3T3-L1 adipocytes. Deletion and single base pair substitution analysis of the IRE abolished the insulin-mediated suppression of the human AQPap gene. Analysis of AQPap sequence in human subjects revealed three missense mutations (R12C, V59L and G264V), and two silent mutations (A103A and G250G). The cRNA injection of the missense mutants into Xenopus oocytes revealed the absence of the activity to transport glycerol and water in the AQPap-G264V protein. In the subject homozygous for AQPap-G264V, exercise-induced increase in plasma glycerol was not observed in spite of the increased plasma noradrenaline. We suggest that AQPap is responsible for the increase of plasma glycerol during exercise in humans.

The mechanism by which the obese subjects are more associated with vascular disease remains unclear. We reported that the adipose tissues produce and secrete many bioactive molecules, conceptualized as adipocytokines. Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), produced locally by vascular macrophages and smooth muscle cells, has been suggested to induce the migration and proliferation of vascular smooth muscle cells. The current study reveals that (1) HB-EGF mRNA is abundantly expressed in human adipose tissue, (2) HB-EGF mRNA increases in the fat tissues of obese mice, (3) plasma HB-EGF levels increase in parallel with fat accumulation in human, and (4) the subjects with coronary artery disease have higher plasma HB-EGF levels, associated with fat accumulation. These results suggest that increased plasma HB-EGF derived from the accumulated fat contributes to the higher incidence of vascular disease in obesity, proposing HB-EGF as an adipocytokine directly linking adipovascular axis.

Background The dipeptidyl-peptidase-IV (DPP-4) inhibitors, including sitagliptin, are used for the treatment of type 2 diabetes mellitus (T2DM). Adiponectin, an adipocyte-derived circulating protein, has anti-atherosclerotic and anti-diabetic properties and is effectively elevated in bloodstream by thiazolidinediones, an insulin sensitizer. However, the effect of sitagliptin treatment on serum adiponectin level in T2DM has not fully elucidated in Japanese T2DM patients. The aim of the present study was to examine the effect of sitagliptin treatment on serum adiponectin levels in T2DM subjects. Methods Twenty-six consecutive Japanese T2DM outpatients were recruited between April 2011 and March 2013, and randomized into the control (conventional treatment, n = 10) group and sitagliptin treatment group (n = 16). Serum adiponectin was measured by enzyme-linked immunosorbent assay. Results Indices of glycemic control, such as hemoglobin A1c, glycated albumin, and 1.5-anhydro-D-glucitol, were significantly improved after the three-month treatment in both the control and sitagliptin groups. Serum adiponectin level was significantly increased in sitagliptin group from 6.7 ± 0.8 to 7.4 ± 1.0 μg/mL without change of body mass index (p = 0.034), while serum adiponectin level was not altered in the control group (p = 0.601). Conclusion In Japanese T2DM patients, serum adiponectin level was elevated by three-month treatment with sitagliptin without change of body weight. Trial registration UMIN000004721

Insulin resistance and its dreaded consequence, type 2 diabetes, are major causes of atherosclerosis. Adiponectin is an adipose-specific plasma protein that possesses anti-atherogenic properties, such as the suppression of adhesion molecule expression in vascular endothelial cells and cytokine production from macrophages. Plasma adiponectin concentrations are decreased in obese and type 2 diabetic subjects with insulin resistance. A regimen that normalizes or increases the plasma adiponectin might prevent atherosclerosis in patients with insulin resistance. In this study, we demonstrate the inducing effects of thiazolidinediones (TZDs), which are synthetic PPARγ ligands, on the expression and secretion of adiponectin in humans and rodents in vivo and in vitro. The administration of TZDs significantly increased the plasma adiponectin concentrations in insulin resistant humans and rodents without affecting their body weight. Adiponectin mRNA expression was normalized or increased by TZDs in the adipose tissues of obese mice. In cultured 3T3-L1 adipocytes, TZD derivatives enhanced the mRNA expression and secretion of adiponectin in a dose- and time-dependent manner. Furthermore, these effects were mediated through the activation of the promoter by the TZDs. On the other hand, TNF-α, which is produced more in an insulin-resistant condition, dose-dependently reduced the expression of adiponectin in adipocytes by suppressing its promoter activity. TZDs restored this inhibitory effect by TNF-α. TZDs might prevent atherosclerotic vascular disease in insulin-resistant patients by inducing the production of adiponectin through direct effect on its promoter and antagonizing the effect of TNF-α on the adiponectin promoter.

Background We recently reported that short-term treatment with liraglutide (20.0 ± 6.4 days) reduced body weight and improved some scales of eating behavior in Japanese type 2 diabetes inpatients. However, it remained uncertain whether such liraglutide-induced improvement is maintained after discharge from the hospital. The aim of the present study was to determine the long-term effects of liraglutide on body weight, glycemic control, and eating behavior in Japanese obese type 2 diabetics. Methods Patients with obesity (body mass index (BMI) >25 kg/m2) and type 2 diabetes were hospitalized at Osaka University Hospital between November 2010 and December 2011. BMI and glycated hemoglobin (HbA1c) were examined on admission, at discharge and at 1, 3, and 6 months after discharge. For the liraglutide group (BMI; 31.3 ± 5.3 kg/m2, n = 29), patients were introduced to liraglutide after correction of hyperglycemic by insulin or oral glucose-lowering drugs and maintained on liraglutide after discharge. Eating behavior was assessed in patients treated with liraglutide using The Guideline For Obesity questionnaire issued by the Japan Society for the Study of Obesity, at admission, discharge, 3 and 6 months after discharge. For the insulin group (BMI; 29.1 ± 3.0 kg/m2, n = 28), each patient was treated with insulin during hospitalization and glycemic control maintained by insulin after discharge. Results Liraglutide induced significant and persistent weight loss from admission up to 6 months after discharge, while no change in body weight after discharge was noted in the insulin group. Liraglutide produced significant improvements in all major scores of eating behavior questionnaire items and such effect was maintained at 6 months after discharge. Weight loss correlated significantly with the decrease in scores for recognition of weight and constitution, sense of hunger, and eating style. Conclusion Liraglutide produced meaningful long-term weight loss and significantly improved eating behavior in obese Japanese patients with type 2 diabetes.

To examine the effects of adiponectin on the functions of Kupffer cells, key modulators of lipopolysaccharide (LPS) -induced liver injury.D-galactosamine (GalN) and LPS were injected intraperitoneally into adiponectin-/- mice and wild type mice. Kupffer cells, isolated from Sprague-Dawley rats, were preincubated with or without adiponectin, and then treated with LPS.In knockout mice, GalN/LPS injection significantly lowered the survival rate, significantly raised the plasma levels of alanine transaminase and tumor necrosis factor-alpha (TNF-alpha) and significantly reduced IL-10 levels compared with wild type mice. TNF-alpha gene expression in the liver was which higher and those of IL-10 were lower in knockout mice than in wild type mice. In cultured adiponectin-pre-treated Kupffer cells, LPS significantly lowered TNF-alpha levels and raised IL-10 levels in the culture media and their respective gene expression levels, compared with Kupffer cells without adiponectin-pre-treatment.Adiponectin supresses TNF-alpha production and induces IL-10 production by Kupffer cells in response to LPS stimulation, and a lack of adiponectin enhances LPS-induced liver injury.

Chick embryo epiphyseal cartilage has been shown to contain three different proteoglycan species (PG-H, PG-Lb, and PG-Lt). This report is concerned with the purification and characterization of the third proteoglycan, PG-Lt. The proteoglycan can be separated from the other two by virtue of its low buoyant density in a CsCl density gradient and further purified by consecutive ion exchange and gel chromatography. The final preparation is composed of PG-Lt monomer and PG-Lt oligomer. The amino acid composition of PG-Lt is quite different from that of PG-H and PG-Lb and rather resembles that of collagens with respect to high content of glycine and high degrees of hydroxylation of proline and lysine. PG-Lt monomer is composed of disulfide-bonded subunits of Mr congruent to 120,000 and 190,000 as demonstrated by its gel electrophoretic behavior after reduction with 2-mercaptoethanol. The latter, but not the former, contains dermatan sulfate chains with glucuronic acid/iduronic acid residues and yields a protein-enriched core molecule of Mr congruent to 100,000 after digestion with chondroitinase ABC. Both of the protein subunits are completely digestible with bacterial collagenase. Immunofluorescence microscopic examination of cartilage tissues, using an antibody against PG-Lt, shows that this proteoglycan exists in both the cartilage matrix and perichondrial noncartilagenous region. When chondrocytes are plated onto tissue culture dishes, the antibody stains strands found on the cell surfaces and in the intercellular space of substrate-attached cell layers, suggesting that PG-Lt mediates cell-to-cell and cell-to-substrate contacts.

Background To examine the effects of liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, on visceral fat adiposity, appetite, food preference, and biomarkers of cardiovascular system in Japanese patients with type 2 diabetes. Methods The study subjects were 20 inpatients with type 2 diabetes treated with liraglutide [age; 61.2 ± 14.0 years, duration of diabetes; 16.9 ± 6.6 years, glycated hemoglobin (HbA1c); 9.1 ± 1.2%, body mass index (BMI); 28.3 ± 5.2 kg/m2, mean ± SD]. After improvement in glycemic control by insulin or oral glucose-lowering agents, patients were switched to liraglutide. We assessed the estimated visceral fat area (eVFA) by abdominal bioelectrical impedance analysis, glycemic control by the 75-g oral glucose tolerance test (OGTT) and eating behavior by the Japan Society for the Study of Obesity questionnaire. Results Treatment with liraglutide (dose range: 0.3 to 0.9 mg/day) for 20.0 ± 6.4 days significantly reduced waist circumference, waist/hip ratio, eVFA. It also significantly improved the scores of eating behavior, food preference and the urge for fat intake and tended to reduce scores for sense of hunger. Liraglutide increased serum C-peptide immunoreactivity and disposition index. Conclusions Short-term treatment with liraglutide improved visceral fat adiposity, appetite, food preference and the urge for fat intake in obese Japanese patients with type 2 diabetes.