Differential gene expression between visceral and subcutaneous fat depots.

Abdominal obesity has been linked to the development of insulin resistance and Type 2 diabetes mellitus (DM2). By surgical removal of visceral fat (VF) in a variety of rodent models, we prevented insulin resistance and glucose intolerance, establishing a cause-effect relationship between VF and the metabolic syndrome. To characterize the biological differences between visceral and peripheral fat depots, we obtained perirenal visceral (VF) and subcutaneous (SC) fat from 5 young rats. We extracted mRNA from the fat tissue and performed gene array hybridization using Affymetrix technology with a platform containing 9 000 genes. Out of the 1 660 genes that were expressed in fat tissue, 297 (17.9 %) genes show a two-fold or higher difference in their expression between the two tissues. We present the 20 genes whose expression is higher in VF fat (by 3 - 7 fold) and the 20 genes whose expression is higher in SC fat (by 3 - 150 fold), many of which are predominantly involved in glucose homeostasis, insulin action, and lipid metabolism. We confirmed the findings of gene array expression and quantified the changes in expression in VF of genes involved in insulin resistance (PPARgamma leptin) and its syndrome (angiotensinogen and plasminogen activating inhibitor-1, PAI-1) by real-time PCR (qRT-PCR) technology. Finally, we demonstrated increased expression of resistin in VF by around 12-fold and adiponectin by around 4-fold, peptides that were not part of the gene expression platform. These results indicate that visceral fat and subcutaneous fat are biologically distinct.     

Reduced response to adiponectin and lower abundance of adiponectin receptor proteins in type 2 diabetic monocytes

The abundance of the adiponectin receptors, AdipoR1 and AdipoR2, and the effects of the antidiabetic adipokine adiponectin in monocytes of normal-weight and overweight controls and type 2 diabetic patients (T2D) were analyzed. AdipoR1 and AdipoR2 mRNAs were increased in monocytes of obese controls and T2D patients when compared to normal-weight controls, and AdipoR1 mRNA positively correlated to AdipoR2 mRNA, the waist to hip ratio and systemic adiponectin. However, AdipoR1 and AdipoR2 proteins were lower in monocytes of T2D compared to normal-weight donors. Induction of IL-6 and IL-8 by adiponectin, an effect involving p38 MAPK, was also reduced in T2D monocytes.     

Effects of dietary Korean proso-millet protein on plasma adiponectin, HDL cholesterol, insulin levels, and gene expression in obese type 2 diabetic mice

We investigated the effect of dietary Korean proso-millet protein concentrate (PMP) on glycemic responses, plasma lipid levels, and the plasma level and gene expression of adiponectin in obese type 2 diabetic mice under normal and high-fat feeding conditions. The findings were that the feeding of PMP clearly elevated plasma high-density lipoprotein cholesterol (HDL cholesterol) and adiponectin levels and brought about effective reduction in the levels of glucose and insulin in mice under high-fat diet conditions as compared with a control diet. Gene expression study revealed that the diet up-regulated expression of adiponectin and down-regulated tumor necrosis factor-alpha (TNF-alpha). Considering the central role of adiponectin and HDL cholesterol in improving and ameliorating type 2 diabetes, obesity, and cardiovascular disease, our findings imply that PMP may have potential for therapeutic intervention in type 2 diabetes.     

Dynamic alteration of adiponectin/adiponectin receptor expression and its impact on myocardial ischemia/reperfusion in type 1 diabetic mice

The present study determined the dynamic change of adiponectin (APN, a cardioprotective adipokine), its receptor expression, and their impact upon myocardial ischemia/reperfusion (MI/R) injury during type 1 diabetes mellitus (T1DM) progression, and involved underlying mechanisms. Diabetic state was induced in mice via multiple intraperitoneal injections of low-dose streptozotocin. The dynamic change of plasma APN concentration and cardiac APN receptor-1 and -2 (AdipoR1/2) expression were assessed immediately after diabetes onset (0 wk) and 1, 3, 5, and 7 wk thereafter. Indicators of MI/R injury (infarct size, apoptosis, and LDH release) were determined at 0, 1, and 7 wk of DM duration. The effect of APN on MI/R injury was determined in mice subjected to different diabetic durations. Plasma APN levels (total and HMW form) increased, whereas cardiac AdipoR1 expression decreased early after T1DM onset. With T1DM progression, APN levels were reduced and cardiac AdipoR1 expression increased. MI/R injury was exacerbated with T1DM progression in a time-dependent manner. Administration of globular APN (gAD) failed to attenuate MI/R injury in 1-wk T1DM mice, while an AMP-activated protein kinase (AMPK) activator (AICAR) reduced MI/R injury. However, administration of gAD (and AICAR) reduced infarct size and cardiomyocyte apoptosis in 7-wk T1DM mice. In conclusion, our results demonstrate a dynamic dysfunction of APN/AdipoR1 during T1DM progression. Reduced cardiac AdipoR1 expression and APN concentration may be responsible for increased I/R injury susceptibility at early and late T1DM stages, respectively. Interventions bolstering AdipoR1 expression during early T1DM stages and APN supplementation during advanced T1DM stages may potentially reduce the myocardial ischemic injury in diabetic patients.     

Oxidative protein damage in plasma of type 2 diabetic patients.

In this study, we evaluated protein oxidation in 84 patients with Type 2 diabetes with no complications and in 61 healthy volunteers who formed the control group, whose ages matched those of the patients. We determined plasma carbonyl and plasma thiol levels as markers of oxidative protein damage and erythrocyte glutathione, plasma ceruloplasmin and transferrin as markers of free radical scavengers. The concentrations (mean +/- SD) of both of plasma carbonyl (1.24 +/- 0.46 vs. 0.72 +/- 0.17 nmole/mg protein; p < 0.0001) and lipid hydroperoxides (1.8 +/- 0.63 vs. 1.3 +/- 0.21 micromole/l; p < 0.0001) were increased, and the concentration of plasma transferrin (3.85 +/- 0.65 vs. 4.59 +/- 0.79 g/l; p < 0.05) was decreased, respectively, in Type 2 diabetic patients compared with those of the controls. There were no significant differences in the concentrations of plasma thiol (0.0064 +/- 0.001 vs. 0.0068 +/- 0.001 micromole/mg protein), erythrocyte glutathione (2.54 +/- 0.57 vs. 2.65 +/- 0.56 mg/g Hb), plasma ceruloplasmin (548 +/- 107.30 vs. 609 +/- 93.34 mg/l) between the patients and the controls. These changes observed in diabetic patients contribute to the imbalance in the redox status of the plasma. We attribute this imbalance to oxidative protein damage in Type 2 diabetic patients clinically free of complications.     

Novel adiponectin variants identified in type 2 diabetic patients reveal multimerization and secretion defects

ADIPOQ, encoding adiponectin, is a candidate gene for type 2 diabetes (T2D) identified by genome-wide linkage analyses with supporting evidence showing the protein function in sensitizing insulin actions. In an endeavor to characterize candidate genes causing T2D in Thai patients, we identified 10 novel ADIPOQ variations, several of which were non-synonymous variations observed only in the patients. To examine the impact of these non-synonymous variations on adiponectin structure and biochemical characteristics, we conducted a structural analysis of the wild-type and variant proteins by in silico modeling and further characterized biochemical properties of the variants with predicted structural abnormalities from the modeling by molecular and biochemical studies. The recombinant plasmids containing wild-type and variant ADIPOQ cDNAs derived from the variations identified by our study (R55H, R112H, and R131H) and previous work (G90S and R112C) were constructed and transiently expressed and co-expressed in cultured HEK293T cells to investigate their oligomerization, interaction, and secretion. We found that the novel R55H variant impaired protein multimerization but it did not exert the effect over the co-expressed wild-type protein while novel R131H variant impaired protein secretion and also affected the co-expressed wild-type protein in a dominant negative fashion. The R131H variant could traffic from the endoplasmic reticulum to the Golgi, trans-Golgi network, and early endosome but could not be secreted. The R131H variant was likely to be degraded through the lysosomal system and inhibition of its degradation rescued the variant protein from secretion defect. We have shown the possibility of using in silico modeling for predicting the effect of amino acid substitution on adiponectin oligomerization. This is also the first report that demonstrates a dominant negative effect of the R131H variant on protein secretion and the possibility of using protein degradation inhibitors as therapeutic agents in the patients carrying adiponectin variants with secretion defect.     

Regulation of adiponectin receptor gene expression in diabetic mice

Adiponectin is an adipocyte-derived factor that plays pivotal roles in lipid and glucose metabolism in muscle and liver. The following two adiponectin receptor types were recently identified: AdipoR1 is abundantly expressed in muscle, whereas AdipoR2 is predominantly expressed in the liver. To clarify the regulation of adiponectin receptor gene expression in diabetic states, we examined mRNA levels of AdipoR1 in the muscles of diabetic animals by Northern blotting. The level of AdipoR1 mRNA was increased approximately 2.5-fold in muscle of streptozotocin (STZ) diabetic mice, but the normal level was restored by insulin administration, indicating that insulin has an inhibitory effect on AdipoR1 expression. To confirm this inhibitory effect of insulin, we performed in vitro experiments using C2C12 skeletal muscle cells. Insulin treatment for 24 h decreased AdipoR1 expression by approximately 60% in C2C12 cells. In addition, this effect was mediated by the phosphatidylinositol 3-kinase-dependent pathway rather than the mitogen-activated protein kinase pathway. AdipoR1 expression in insulin-resistant diabetic mice was also investigated. AdipoR1 expression was decreased by 36% in type 2 diabetic obese db/db mice compared with lean mice. In contrast, hepatic AdipoR2 expression was not significantly changed in either STZ mice or genetically obese mice. Our results indicate that regulation of AdipoR1, but not that of AdipoR2, may be involved in glucose and lipid metabolism in diabetic states.     

Effects of dietary protein of Korean foxtail millet on plasma adiponectin, HDL-cholesterol, and insulin levels in genetically type 2 diabetic mice

We examined the effects of intake of Korean foxtail millet protein (FMP) on plasma levels of lipid, glucose, insulin, and adiponectin in genetically type 2 diabetic KK-Ay mice. When mice were fed a normal FMP diet or a high-fat-high-sucrose diet containing FMP for 3 weeks, in both experiments plasma concentrations of high-density lipoprotein cholesterol (HDL-cholesterol) and adiponectin increased remarkably in comparison with a casein diet group, whereas concentrations of insulin decreased greatly and that of plasma glucose was comparable to that in the casein diet group. Considering the role of adiponectin, insulin, and HDL-cholesterol in diabetes, atherosclerosis, and obesity, it appears likely that FMP may improve insulin sensitivity and cholesterol metabolism through an increase in adiponectin concentration. Therefore, FMP would serve as another beneficial food component in obesity-related diseases such as type 2 diabetes and cardiovascular diseases.     

Uncoupling protein 2 G(-866)A polymorphism: a new gene polymorphism associated with C-reactive protein in type 2 diabetic patients C-reactive protein in type 2 diabetic patients

Objective

To evaluate glycemic variability associated with two different premixed insulin analogue formulations when used in a twice-daily regimen.

Patients and Methods

Subjects comprised type 2 diabetic patients aged 20-79 years, treated with twice daily premixed insulin or insulin analogue formulations. All subjects were hospitalized for 6 days and randomized to receive either Humalog Mix 25 (Mix 25) or Humalog Mix 50 (Mix 50). They were then crossed over to the other arm between day 3 and day 4 of the study. Continuous glucose monitoring (CGM) was performed on all subjects to examine the differences in glycemic variability.

Results

Eleven type 2 diabetic patients were enrolled. No significant difference was found in 24-hour mean glucose values and their SDs, pre-meal glucose values, increases from pre-meal to peak glucose values, or time to peak glucose levels between either group. However, the mean glucose values observed during 0-8 hrs were significantly lower with Mix 25 compared to Mix 50 (128 vs. 147 mg/dL; p = 0.024).

Conclusions

The twice-daily Mix 25 regimen provided superior overnight glycemic control compared to the Mix 50 regimen in Japanese patients with type 2 diabetes. However, both twice-daily regimens with either Mix 25 or Mix 50 provided inadequate post-lunch glycemic control.

Trial Registration

Current Controlled Trials UMIN000001327     

The effect of etomoxir on insulin sensitivity in type 2 diabetic patients.

Oral application of 50 mg Etomoxir caused a significant rise (33.1%) of insulin-mediated glucose uptake. This was shown in a placebo-controlled, double-blind randomized study in 8 type 2 diabetic patients by using the euglycemic clamp technique. The mean metabolic clearance rate of glucose (MCR) was raised from 4.1 +/- 0.9 mg/(kg.min) to 5.4 +/- 1.2 mg/(kg.min) (x +/- SEM, P = 0.039). Plasma levels of free fatty acids (FFA), glucose counterregulatory hormones, lipids and C-peptide values during the clamps were not different after verum and placebo. We conclude that Etomoxir improves insulin sensitivity in type 2 diabetic patients.     

Effect of telmisartan on the expression of adiponectin receptors and nicotinamide adenine dinucleotide phosphate oxidase in the heart and aorta in type 2 diabetic rats

Background

Although the Fat Mass and Obesity (FTO) and Melanocortin-4 Receptor (MC4R) genes have been consistently associated with obesity risk, the association between the obesity-risk alleles with type 2 diabetes is still controversial. In some recent meta-analyses in which significant results have been reported, the associations disappeared after adjustment for body mass index (BMI). However gene-diet interactions with dietary patterns have not been investigated. Our main aim was to analyze whether these associations are modulated by the level of adherence to the Mediterranean Diet (MedDiet).

Methods

Case-control study in 7,052 high cardiovascular risk subjects (3,430 type 2 diabetes cases and 3,622 non-diabetic subjects) with no differences in BMI. Diet was assessed by validated questionnaires. FTO-rs9939609 and MC4R-rs17782313 were determined. An aggregate genetic score was calculated to test additive effects. Gene-diet interactions were analyzed.

Results

Neither of the polymorphisms was associated with type 2 diabetes in the whole population. However, we found consistent gene-diet interactions with adherence to the MedDiet both for the FTO-rs9939609 (P-interaction=0.039), the MC4R-rs17782313 (P-interaction=0.009) and for their aggregate score (P-interaction=0.006). When adherence to the MedDiet was low, carriers of the variant alleles had higher type 2 diabetes risk (OR=1.21, 95%CI: 1.03-1.40; P=0.019 for FTO-rs9939609 and OR=1.17, 95%CI:1.01-1.36; P=0.035 for MC4R-rs17782313) than wild-type subjects. However, when adherence to the MedDiet was high, these associations disappeared (OR=0.97, 95%CI: 0.85-1.16; P=0.673 for FTO-rs9939609 and OR=0.89, 95%CI:0.78-1.02; P=0.097 for MC4R-rs17782313). These gene-diet interactions remained significant even after adjustment for BMI. As MedDiet is rich in folate, we also specifically examined folate intake and detected statistically significant interaction effects on fasting plasma glucose concentrations in non-diabetic subjects. However these findings should be interpreted with caution because folate intake may simply reflect a healthy dietary pattern.

Conclusions

These novel results suggest that the association of the FTO-rs9939609 and the MC4R-rs17782313 polymorphisms with type 2 diabetes depends on diet and that a high adherence to the MedDiet counteracts the genetic predisposition.     

Effect of telmisartan on the expression of adiponectin receptors and nicotinamide adenine dinucleotide phosphate oxidase in the heart and aorta in type 2 diabetic rats

ABSTRACT: BACKGROUND: Diabetic cardiovascular disease is associated with decreased adiponectin and increased oxidative stress. This study investigated the effect of telmisartan on the expression of adiponectin receptor 2 (adipoR2) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits in the heart and the expression of adiponectin receptor 1 (adipoR1) in aorta in type 2 diabetic rats. METHODS: Type 2 diabetes was induced by high-fat and high-sugar diet and intraperitoneal injection of a low dose of streptozotocin (STZ). Heart function, adipoR2, p22phox, NOX4, glucose transporter 4(GLUT4), monocyte chemoattractant protein-1(MCP-1) and connective tissue growth factor CTGFin the heart, and adipoR1, MCP-1 and nuclear factor kappa B (NF-kappaB) in aorta were analyzed in controls and diabetic rats treated with or without telmisartan (5mg/kg/d) by gavage for 12 weeks. RESULTS: Heart function, plasma and myocardial adiponectin levels, the expression of myocardial adipoR2 and GLUT4 were significantly decreased in diabetic rats (P<0.05). The expression of myocardial p22phox, NOX4, MCP-1, and CTGF was significantly increased in diabetic rats (P<0.05). The expression of adipoR1 was decreased and the expression of MCP-1 and NF-kappaB was increased in the abdominal aorta in diabetic rats (P<0.05). Telmisartan treatment significantly attenuated these changes in diabetic rats (P<0.05). CONCLUSIONS: Our results suggest that telmisartan upregulates the expression of myocardial adiponectin, its receptor 2 and GLUT4. Simultaneously, it downregulates the expression of myocardial p22phox, NOX4, MCP-1, and CTGF, contributing so to the improvement of heart function in diabetic rats. Telmisartan also induces a protective role on the vascular system by upregulating the expression of adipoR1 and downregulating the expression of MCP-1 and NF-kappaB in the abdominal aorta in diabetic rats. KEYWORDS: Telmisartan; Adiponectin receptor; NADPH oxidase; Type 2 diabetic; Cardiac; Aorta.     

TNF-alpha alters visfatin and adiponectin levels in human fat.

Adiponectin and visfatin are newly discovered adipokines that are strongly expressed in human visceral adipose tissue. To identify new regulatory mechanisms in fat, the effect of TNF-alpha (TNF) on adiponectin, on its two receptors, and on visfatin was investigated by incubating human visceral adipose tissue from patients without diabetes mellitus with TNF for 24, 48 and 72 hours. The mRNA expression of visfatin, adiponectin, and its two receptors, as well as the protein expression of adiponectin were determined. A decrease of adiponectin mRNA expression of 97% after incubation with TNF (5.75 nmol/l) for 24 hours, a decrease of 91% after 48 hours, and a decrease of 96% after 72 hours were measured. The reduction of protein expression was measured to be 42% after 24 hours, 28% after 48 hours, and 39% after 72 hours of incubation with TNF (5.75 nmol/l). The mRNA level of adiponectin receptor 1 (AdipoR1) was elevated about 72% after 48 hours of incubation and 67% after 72 hours of incubation, whereas the mRNA expression of adiponectin receptor 2 (AdipoR2) was not altered significantly. The visfatin mRNA level was found to be highly increased by 255% after 24 hours and 335% after 48 hours and 341% after 72 hours of incubation with TNF (5.75 nmol/l). Our results support the concept of visceral adipose tissue as an endocrine organ. We demonstrate that TNF has regulatory functions on adiponectin, AdipoR1 and on visfatin in human visceral adipose tissue. TNF levels are elevated in states of obesity and insulin resistance. Due to this fact TNF could be the reason that there is a decrease in the level of adiponectin, whereas there is an increase in the level of visfatin in states of obesity and insulin resistance.     

Beneficial effect of heme oxygenase-1 expression on myocardial ischemia-reperfusion involves an increase in adiponectin in mildly diabetic rats

Transient reduction in coronary perfusion pressure in the isolated mouse heart increases microvascular resistance (paradoxical vasoconstriction) by an endothelium-mediated mechanism. To assess the presence and extent of paradoxical vasoconstriction in hearts from normal and diabetic rats and to determine whether increased heme oxygenase (HO)-1 expression and HO activity, using cobalt protoporphyrin (CoPP), attenuates coronary microvascular response, male Wistar rats were rendered diabetic with nicotinamide/streptozotocin for 2 wk and either CoPP or vehicle was administered by intraperitoneal injection weekly for 3 wk (0.5 mg/100 g body wt). The isolated beating nonworking heart was submitted to transient low perfusion pressure (20 mmHg), and coronary resistance (CR) was measured. During low perfusion pressure, CR increased and was associated with increased lactate release. In diabetic rats, CR was higher, HO-1 expression and endothelial nitric oxide synthase were downregulated, and inducible nitric oxide synthase and O(2)(-) were upregulated. After 3 wk of CoPP treatment, HO activity was significantly increased in the heart. Upregulation of HO-1 expression and HO activity by CoPP resulted in the abolition of paradoxical vasoconstriction and a reduction in oxidative ischemic damage. In addition, there was a marked increase in serum adiponectin. Elevated HO-1 expression was associated with increased expression of cardiac endothelial nitric oxide synthase, B-cell leukemia/lymphoma extra long, and phospho activator protein kinase levels and decreased levels of inducible nitric oxide synthase and malondialdehyde. These results suggest a critical role for HO-1 in microvascular tone control and myocardial protection during ischemia in both normal and mildly diabetic rats through the modulation of constitutive and inducible nitric oxide synthase expression and activity, and an increase in serum adiponectin.     

Hepatic expression of cytochrome P450 in type 2 diabetic Goto-Kakizaki rats

Although hepatic expression of cytochrome P450 (CYP) changes markedly in diabetes, the role of ketone bodies in the regulation of CYP in diabetes is controversial. The present study was performed to determine the expression and activity of CYP in non-obese type II diabetic Goto-Kakizaki (GK) rats with normal levels of ketone bodies. In the present study, basal serum glucose levels increased 1.95-fold in GK rats, but acetoacetate and β-hydroxybutyrate levels were not significantly different. Hepatic expression of CYP reductase and CYP3A2 was up-regulated in the GK rats, and consequently, activities of CYP reductase and midazolam 4-hydroxylase, mainly catalyzed by CYP3A2, increased. In contrast, hepatic expression of CYP1A2 and CYP3A1 was down-regulated and the activities of 7-ethoxyresorufin-O-deethylase and 7-methoxyresorufin-O-demethylase, mainly catalyzed by CYP1A, also decreased in GK rats. Hepatic levels of microsomal protein and total CYP and hepatic expression of cytochrome b(5), CYP1B1, CYP2B1 and CYP2C11 were not significantly different between the GK rats and normal Wistar rats. Moreover, the expression and activity of CYP2E1, reported to be up-regulated in diabetes with hyperketonemia, were not significantly different between GK rats and control rats, suggesting that elevation of ketone bodies plays a critical role in the up-regulation of hepatic CYP2E1 in diabetic rats. Our results showed that the expression of hepatic CYP is regulated in an isoform-specific manner. The present results also show that the GK rat is a useful animal model for the pathophysiological study of non-obese type II diabetes with normal ketone body levels.