Masaharu Urakaze

Regulatory Mechanisms for Adipose Tissue M1 and M2 Macrophages in Diet-induced Obese Mice

OBJECTIVE To characterize the phenotypic changes of adipose tissue macrophages (ATMs) under different conditions of insulin sensitivity. RESEARCH DESIGN AND METHODS The number and the expressions of marker genes for M1 and M2 macrophages from mouse epididymal fat tissue were analyzed using flow cytometry after the mice had been subjected to a high-fat diet (HFD) and pioglitazone treatment. RESULTS Most of the CD11c-positive M1 macrophages and the CD206-positive M2 macrophages in the epididymal fat tissue were clearly separated using flow cytometry. The M1 and M2 macrophages exhibited completely different gene expression patterns. Not only the numbers of M1 ATMs and the expression of M1 marker genes, such as tumor necrosis factor-α and monocyte chemoattractant protein-1, but also the M1-to-M2 ratio were increased by an HFD and decreased by subsequent pioglitazone treatment, suggesting the correlation with whole-body insulin sensitivity. We also found that the increased number of M2 ATMs after an HFD was associated with the upregulated expression of interleukin (IL)-10, an anti-inflammatory Th2 cytokine, in the adipocyte fraction as well as in adipose tissue. The systemic overexpression of IL-10 by an adenovirus vector increased the expression of M2 markers in adipose tissue. CONCLUSIONS M1 and M2 ATMs constitute different subsets of macrophages. Insulin resistance is associated with both the number of M1 macrophages and the M1-to-M2 ratio. The increased expression of IL-10 after an HFD might be involved in the increased recruitment of M2 macrophages.

Adiponectin Inhibits Endothelial Synthesis of Interleukin-8

Adiponectin is an antiatherogenic adipokine that inhibits inflammation by mechanisms that are not completely understood. We explored the effect of adiponectin on endothelial synthesis of interleukin-8 (IL-8), a pro-inflammatory chemokine that plays a role in atherogenesis. Adiponectin decreased the secretion of IL-8 from human aortic endothelial cells (HAEC) stimulated with tumor necrosis factor-&agr; (TNF-&agr;). Adiponectin also inhibited IL-8 mRNA expression induced by TNF-&agr;. Phosphorylation of I&kgr;B-&agr; was decreased by adiponectin, but phosphorylation of ERK, SAPK/JNK, and p38MAPK were unaffected. Adiponectin increased intra-cellular cAMP levels in HAEC in a dose-dependent manner; PKA activity was also increased. The inhibitory effect of adiponectin on TNF-&agr;–induced IL-8 synthesis was inhibited by pretreatment with Rp-cAMP, a PKA inhibitor. These observations suggest that adiponectin inhibits IL-8 synthesis through inhibition of a PKA dependent NF-&kgr;B signaling pathway. We also showed that adiponectin enhances Akt phosphorylation. The inhibitory effect of adiponectin on TNF-&agr;–induced IL-8 synthesis was abrogated in part by pretreatment with the PI3 kinase inhibitor LY294002 or by Akt siRNA transfection, suggesting that Akt activation might inhibit IL-8 synthesis induced by TNF-&agr;. We conclude that inhibition of NF-&kgr;B and activation of Akt phosphorylation may mediate adiponectin inhibition of atherosclerosis.

Treatment with SRT1720, a SIRT1 activator, ameliorates fatty liver with reduced expression of lipogenic enzymes in MSG mice

Nonalcoholic fatty liver disease (NAFLD) is an abnormal liver metabolism often observed with insulin resistance and metabolic syndrome. Calorie restriction is a useful treatment for NAFLD and reportedly prolongs the life spans of several species in which sirtuin plays an important role. In this study, we examined whether the activation of SIRT1, a mammalian ortholog of sirtuin, may ameliorate the development of NAFLD. Monosodium glutamate (MSG) mice, which exhibited obesity and insulin resistance, were treated with SRT1720, a specific SIRT1 activator from the age of 6-16 wk. Sixteen-week-old MSG mice exhibited increased liver triglyceride content and elevated levels of aminotransferase. SRT1720 treatment significantly reduced these levels without affecting body weight or food intake. These results suggested that the administration of SRT1720 ameliorated the development of NAFLD in MSG mice. The expressions of lipogenic genes, such as sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase, and the serum lipid profiles, including free fatty acids, were elevated in MSG mice and were reduced by SRT1720 treatment. SRT1720 treatment also reduced the expressions of lipogenic genes in cultured HepG2 cells. Furthermore, SRT1720 treatment decreased the expressions of marker genes for oxidative stress and inflammatory cytokines in the liver of MSG mice. Taken together, SRT1720 treatment may reduce liver lipid accumulation, at least in part, by directly reducing the expressions of lipogenic genes. The reduction of oxidative stress and inflammation may also be involved in the amelioration of NAFLD.

Effects of Pioglitazone on Suppressor of Cytokine Signaling 3 Expression: Potential Mechanisms for Its Effects on Insulin Sensitivity and Adiponectin Expression

Pioglitazone is widely used for the treatment of diabetic patients with insulin resistance. The mechanism of pioglitazone to improve insulin sensitivity is not fully understood. Recent studies have shown that the induction of suppressor of cytokine signaling 3 (SOCS3) is related to the development of insulin resistance. Here, we examined whether the insulin-sensitizing effect of pioglitazone affects the SOCS induction. In db/db mice and high-fat–fed mice, expression of SOCS3 mRNA in fat tissue was increased compared with that in lean control mice, and pioglitazone suppressed SOCS3 levels. In 3T3-L1 adipocytes, mediators of insulin resistance such as tumor necrosis factor-α (TNF-α), interleukin-6, growth hormone, and insulin increased SOCS3 expression, which was partially inhibited by pioglitazone. The ability of pioglitazone to suppress SOCS3 induction by TNF-α was greatly augmented by peroxisome proliferator–activated receptor γ overexpression. SOCS3 overexpression and tyrphostin AG490, a Janus kinase 2 inhibitor, or dominant-negative STAT3 expression partially inhibited adiponectin secretion and was accompanied by decreased STAT3 phosphorylation. Conversely, pioglitazone increased adiponectin secretion and STAT3 phosphorylation in fat tissue of db/db mice and in 3T3-L1 adipocytes. These results suggest that pioglitazone exerts its effect to improve whole-body insulin sensitivity in part through the suppression of SOCS3, which is associated with the increase in STAT3 phosphorylation and adiponectin production in fat tissue.

SRT1720, a SIRT1 activator, promotes tumor cell migration, and lung metastasis of breast cancer in mice

Silent information regulator 2 (SIR2) is a highly conserved protein, the mammalian orthologue of which, SIRT1, exhibits histone deacetylase activity. SIRT1 is involved not in only longevity due to caloric restriction but in a variety of diseases such as diabetes, cardiovascular dysfunction and neurodegeneration. However, accumulating evidence shows that SIRT1 is overexpressed in various types of malignant cells, and its inhibitors suppress the growth of tumor cells. The relationship between SIRT1 and metastasis remains to be clarified. Here, we examined the effect of SRT1720, a SIRT1 activator, on lung metastasis of breast cancer cells. 4T1 breast cancer cells were subcutaneously implanted into syngeneic BALB/c mice and SRT1720 was administered alone or with an antitumor agent, cisplatin. As expected, cisplatin decreased the lung metastasis score, whereas SRT1720 increased metastasis irrespective of cisplatin. In the primary tumors, cisplatin suppressed the mRNA level of angiopoietin-like protein 4 (angptl4), a lung metastasis-promoting gene product of breast cancer, but SRT1720 reduced the effectiveness of cisplatin. The results obtained with animal experiments were in accordance with those in human cancer cells; SRT1720 significantly increased the amount of VEGF secreted from MDA-MB-231 cells. Moreover, a transendothelial cell migration assay showed that SRT1720 promotes the migration of MDA-MB-231 cells across an endothelial cell layer despite the presence of cisplatin. These findings imply that SRT1720 promotes the pulmonary metastasis of breast cancer cells and SIRT1 may be an important target for suppressing metastasis to the lung.

Association of the polymorphisms in the 5′-untranslated region of PTEN gene with type 2 diabetes in a Japanese population

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is known to act as a lipid phosphatase hydrolyzing phosphatidylinositol (PI)(3,4,5)P3 to PI(4,5)P2. Since the PI3‐kinase product, PI(3,4,5)P3, is an important second messenger leading to the metabolic action of insulin, PTEN functions as a potent negative regulator of insulin signaling and its gene is one of the possible candidates involved in susceptibility to the development of type 2 (non‐insulin‐dependent) diabetes. In the present study, we investigated the polymorphisms of the PTEN gene in Japanese patients with type 2 diabetes and non‐diabetic control subjects. We identified three mutations of the gene in the type 2 diabetes patients. Among these mutations, the frequency of the substitution of C with G at position −9 (−9C→G) (SNP1), located in the untranslated region of exon 1, was significantly higher in type 2 diabetic patients than in control subjects. In addition, transfection of the PTEN gene with SNP1 resulted in a significantly higher expression level of PTEN protein compared with that of the wild‐type PTEN gene in Cos1 and Rat1 cells. Furthermore, insulin‐induced phosphorylation of Akt in HIRc cells was decreased more greatly by transfection of SNP1 PTEN gene than that of wild‐type PTEN gene. These findings suggest that the change of C to G at position −9 of the PTEN gene is associated with the insulin resistance of type 2 diabetes due possibly to a potentiated hydrolysis of the PI3‐kinase product.

High glucose enhances the gene expression of interleukin-8 in human endothelial cells, but not in smooth muscle cells: possible role of interleukin-8 in diabetic macroangiopathy

Summary We examined the effect of high glucose concentrations on the production of interleukin(IL)-8, which seems to be important for the development of atherosclerosis, in cultured human aortic endothelial cells (AoEC) and smooth muscle cells (AoSMC). After incubating these cells with various concentrations of glucose for 2 days or 7 days, the IL-8 concentration in cell lysates was measured by enzyme-linked immunosorbent assay and the IL-8 mRNA expression was examined by Northern analysis. After 2 days culture, 42.5 mmol/l glucose enhanced IL-8 mRNA expression in AoEC, but not in AoSMC, compared to 4 mmol/l glucose. After 7 days culture, the IL-8 concentration in AoEC lysate and the expression of IL-8 mRNA were significantly increased by 20.5 mmol/l glucose, or 42.5 mmol/l glucose compared to 4 mmol/l glucose. On the other hand, the IL-8 concentration in AoSMC lysate was not affected by any glucose concentration and the expression of IL-8 mRNA in AoSMC was diminished by high glucose. These results suggest that the chemotactic gradient by IL-8 is established between arterial intima and media in response to high glucose levels in diabetic patients, and that it may be one of the key factors for SMC migration to the intima leading to diabetic macroangiopathy. [Diabetologia (1997) 40: 610–613]

Inhibitory effect of IL-8 on insulin action in human adipocytes via MAP kinase pathway

BackgroundVarious cytokines and other compounds are produced in human adipose tissue and might have functions in the adipose tissue. They might be involved in complications associated with obesity and diabetes. Recently, interleukin-8 (IL-8) has been shown to be produced and released from human adipose tissue and/or adipocytes, suggesting IL-8 involvement in some obesity-related health complications. Therefore, we found it of interest to investigate whether IL-8 is involved in the insulin action in human adipocytes.MethodsThe IL-8 levels in the medium were measured using ELISA. The IL-8 mRNA expression was analyzed using Northern blot analysis. The phosphorylation of Akt was analyzed using Western blot analysis. Furthermore, we examined the effect of IL-8 on the phosphorylation of Akt induced by insulin.ResultsThe level of IL-8 in the medium and the IL-8 mRNA expression after stimulation with either TNF-α, IL-1β, or CRP was significantly enhanced in human adipocytes. It is particularly interesting that IL-8 per se also enhanced IL-8 mRNA expression. The IL-8 induced-IL-8 mRNA expression was inhibited by PD98059 (a MEK inhibitor) or SB203580 (a p38 MAPK inhibitor). The IL-8 inhibited insulin-induced Akt phosphorylation. The inhibitory effect of IL-8 was eliminated by either PD 98059 or SB203580.ConclusionThese data suggest that IL-8 is a main adipocytokine producing insulin resistance via the inhibition of insulin-induced Akt phosphorylation in adipocytes. The attenuation of IL-8 action might be a target for prevention of diabetes and its complications.

Effects of cilostazol on serum lipid concentrations and plasma fatty acid composition in type 2 diabetic patients with peripheral vascular disease.

Abstract. Cilostazol is an anti-thrombotic and vasodilating agent, reported to have both anti-thrombotic and cerebral vasodilating effects. We investigated the effects of cilostazol on serum lipid concentrations and plasma fatty acid composition in type 2 diabetic patients with peripheral vascular disease. The serum concentrations of total cholesterol, triglycerides, high-density lipoprotein-cholesterol, lipoprotein (a), remnant-like particles-cholesterol, apolipoproteins, and plasma fatty acid composition were measured in 17 diabetic patients with peripheral vascular disease before and 1, 3, and 6 months after administration of cilostazol (200 mg/day). Serum triglyceride concentrations were significantly decreased after cilostazol (from 1.31±0.17 mmol/l to 0.86±0.07 mmol/l at 6 months, P<0.01). Plasma docosahexaenoic acid levels were significantly increased after cilostazol (4.11±0.26% to 4.94±0.26% at 6 months, P<0.01). Our findings show that cilostazol can induce some beneficial changes in serum lipid profile and plasma fatty acid composition.

Deletion of platelet-derived growth factor receptor-β improves diabetic nephropathy in Ca2+/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice

Aims/hypothesisThe activation of platelet-derived growth factor receptor-β (PDGFR-β) signalling is increased in the glomeruli and tubules of diabetic animals. In this study, we examined the role of PDGFR-β signalling during the development of diabetic nephropathy.MethodsWe recently generated pancreatic beta cell-specific Ca2+/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice (CaMKIIα mice), which show very high plasma glucose levels up to 55.5xa0mmol/l and exhibit the features of diabetic nephropathy. These mice were crossed with conditional knockout mice in which Pdgfr-β (also known as Pdgfrb) was deleted postnatally. The effect of the deletion of the Pdgfr-β gene on diabetic nephropathy in CaMKIIα mice was evaluated at 10 and 16xa0weeks of age.ResultsThe plasma glucose concentrations and HbA1c levels were elevated in the CaMKIIα mice from 4xa0weeks of age. Variables indicative of diabetic nephropathy, such as an increased urinary albumin/creatinine ratio, kidney weight/body weight ratio and mesangial area/glomerular area ratio, were observed at 16xa0weeks of age. The postnatal deletion of the Pdgfr-β gene significantly decreased the urinary albumin/creatinine ratio and mesangial area/glomerular area ratio without affecting the plasma glucose concentration. Furthermore, the increased oxidative stress in the kidneys of the CaMKIIα mice as shown by the increased urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion and the increased expression of NAD(P)H oxidase 4 (NOX4), glutathione peroxidase 1 (GPX1) and manganese superoxide dismutase (MnSOD) was decreased by Pdgfr-β gene deletion.Conclusions/interpretationThe activation of PDGFR-β signalling contributes to the progress of diabetic nephropathy, with an increase in oxidative stress and mesangial expansion in CaMKIIα mice.