Takako Kikuchi

Xanthine Oxidoreductase Is Involved in Macrophage Foam Cell Formation and Atherosclerosis Development

Objective—Hyperuricemia is common in patients with metabolic syndrome. We investigated the role of xanthine oxidoreductase (XOR) in atherosclerosis development, and the effects of the XOR inhibitor allopurinol on this process. Methods and Results—Oral administration of allopurinol to ApoE knockout mice markedly ameliorated lipid accumulation and calcification in the aorta and aortic root. In addition, allopurinol treatment or siRNA-mediated gene knockdown of XOR suppressed transformation of J774.1 murine macrophage cells, treated with acetylated LDL or very low density lipoprotein (VLDL) into foam cells. This inhibitory effect of allopurinol was also observed in primary cultured human macrophages. In contrast, overexpression of XOR promoted transformation of J774.1 cells into foam cells. Interestingly, SR-A1, SR-B1, SR-B II, and VLDL receptors in J774.1 cells were reduced by XOR knockdown, and increased by XOR overexpression. Conversely, expressions of ABCA1 and ABCG1 were increased by XOR knockdown and suppressed by XOR overexpression. Finally, productions of inflammatory cytokines accompanied by foam cell formation were also reduced by allopurinol administration. Conclusion—These results strongly suggest XOR activity and/or its expression level to contribute to macrophage foam cell formation. Thus, XOR inhibitors may be useful for preventing atherosclerosis.

Peptidyl-prolyl Cis/Trans Isomerase NIMA-interacting 1 Associates with Insulin Receptor Substrate-1 and Enhances Insulin Actions and Adipogenesis

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.

Resistin-Like Molecule β Is Abundantly Expressed in Foam Cells and Is Involved in Atherosclerosis Development

Objective—Resistin-like molecule (RELM) &bgr; is a secretory protein homologous to resistin and reportedly contributes to local immune response regulation in gut and bronchial epithelial cells. However, we found that activated macrophages also express RELM&bgr; and thus investigated the role of RELM&bgr; in the development of atherosclerosis. Approach and Results—It was demonstrated that foam cells in atherosclerotic lesions of the human coronary artery abundantly express RELM&bgr;. RELM&bgr; knockout (−/−) and wild-type mice were mated with apolipoprotein E–deficient background mice. RELM&bgr;−/− apolipoprotein E–deficient mice exhibited less lipid accumulation in the aortic root and wall than RELM&bgr;+/+ apolipoprotein E–deficient mice, without significant changes in serum lipid parameters. In vitro, RELM&bgr;−/− primary cultured peritoneal macrophages (PCPMs) exhibited weaker lipopolysaccharide-induced nuclear factor-&kgr;B classical pathway activation and inflammatory cytokine secretion than RELM&bgr;+/+, whereas stimulation with RELM&bgr; upregulated inflammatory cytokine expressions and increased expressions of many lipid transporters and scavenger receptors in PCPMs. Flow cytometric analysis revealed inflammatory stimulation–induced RELM&bgr; in F4/80(+) CD11c(+) PCPMs. In contrast, the expressions of CD11c and tumor necrosis factor were lower in RELM&bgr;−/− PCPMs, but both were restored by stimulation with recombinant RELM&bgr;. Conclusions—RELM&bgr; is abundantly expressed in foam cells within plaques and contributes to atherosclerosis development via lipid accumulation and inflammatory facilitation.

Valsartan, independently of AT1 receptor or PPARγ, suppresses LPS-induced macrophage activation and improves insulin resistance in cocultured adipocytes

Macrophages are integrated into adipose tissues and interact with adipocytes in obese subjects, thereby exacerbating adipose insulin resistance. This study aimed to elucidate the molecular mechanism underlying the insulin-sensitizing effect of the angiotensin II receptor blocker (ARB) valsartan, as demonstrated in clinical studies. Insulin signaling, i.e., insulin receptor substrate-1 and Akt phosphorylations, in 3T3-L1 adipocytes was impaired markedly by treatment with tumor necrosis factor-α (TNFα) or in the culture medium of lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages, and valsartan had no effects on these impairments. However, in contrast, when cocultured with RAW 264.7 cells using a transwell system, the LPS-induced insulin signaling impairment in 3T3-L1 adipocytes showed almost complete normalization with coaddition of valsartan. Furthermore, valsartan strongly suppressed LPS-induced productions of cytokines such as interleukin (IL)-1β, IL-6, and TNFα with nuclear factor-κB activation and c-Jun NH(2)-terminal kinase phosphorylation in RAW 264.7 and primary murine macrophages. Very interestingly, this effect of valsartan was also observed in THP-1 cells treated with angiotensin II type 1 (AT1) siRNA or a peroxisome proliferator-activated receptor-γ (PPARγ) antagonist as well as macrophages from AT1a receptor-knockout mice. We conclude that valsartan suppresses the inflammatory response of macrophages, albeit not via PPARγ or the AT1a receptor. This suppression appears to secondarily improve adipose insulin resistance.

Hepatic overexpression of a dominant negative form of raptor enhances Akt phosphorylation and restores insulin sensitivity in K/KAy mice

Several serine/threonine kinases reportedly phosphorylate serine residues of IRS-1 and thereby induce insulin resistance. In this study, to investigate the effect of mTOR/raptor on insulin signaling and metabolism in K/KAy mice with genetic obesity-associated insulin resistance, a dominant negative raptor, COOH-terminally deleted raptor (raptor-DeltaC(T)), was overexpressed in the liver via injection of its adenovirus into the circulation. Hepatic raptor-DeltaC(T) expression levels were 1.5- to 4-fold that of endogenously expressed raptor. Glucose tolerance in raptor-DeltaC(T)-overexpressing mice improved significantly compared with that of LacZ-overexpressing mice. Insulin-induced activation of p70S6 kinase (p70(S6k)) was significantly suppressed in the livers of raptor-DeltaC(T) overexpressing mice. In addition, insulin-induced IRS-1, Ser(307), and Ser(636/639) phosphorylations were significantly suppressed in the raptor-DeltaC(T)-overexpressing liver, whereas tyrosine phosphorylation of IRS-1 was increased. PI 3-kinase activation in response to insulin stimulation was increased approximately twofold, and Akt phosphorylation was clearly enhanced under both basal and insulin-stimulated conditions in the livers of raptor-DeltaC(T) mice. Thus, our data indicate that suppression of the mTOR/p70(S6k) pathway leads to improved glucose tolerance in K/KAy mice. These observations may contribute to the development of novel antidiabetic agents.

Twenty‐year trend of increasing obesity in young patients with poorly controlled type 2 diabetes at first diagnosis in urban Japan

To investigate trends over the past 20 years for the prevalence of obesity and glycemic control in association with a patients first hospital visit for type 2 diabetes mellitus.

Decreased SIRT1 expression and LKB1 phosphorylation occur with long-term high-fat diet feeding, in addition to AMPK phosphorylation impairment in the early phase

SUMMARY AIMS Energy sensing systems including AMPK and SIRT1 play important roles in the regulation of hepatic gluconeogenesis and fatty acid oxidation. In this study, we investigated how hepatic LKB1-AMPK signaling and SIRT1 expression are altered after 2 or 8 weeks of HFD feeding. METHODS The livers of male mice fed a HFD or a standard diet for 2 or 8 weeks were removed. The expression and phosphorylation levels of LKB1, AMPK, ACC and TORC2, and SIRT1 expression levels were examined by immunoblotting. RESULTS In mice fed a HFD for 2 weeks, the phosphorylations of AMPKα and ACC were decreased without significant alterations in LKB1 phosphorylation or SIRT1 protein levels, while TORC2 protein levels were increased. In mice fed a HFD for 8 weeks, marked reductions in LKB1 phosphorylation and SIRT1 protein amount were observed in addition to the decreased phosphorylations of AMPKα and ACC. CONCLUSIONS The mechanisms underlying impaired energy sensing signaling differ with the duration of HFD feeding. In the early phase of HFD feeding, LKB1 and SIRT1 were not impaired, while in the later phase of HFD feeding, decreased SIRT1 expression and LKB1 phosphorylation may be involved in the development of severe glucose and lipid intolerance.

Macrophage foam cell formation is augmented in serum from patients with diabetic angiopathy

The differentiation of macrophages into cytokine-secreting foam cells plays a critical role in the development of diabetic angiopathy. J774.1, a murine macrophage cell line, reportedly differentiates into foam cells when incubated with oxidized LDL, ApoE-rich VLDL or WHHLMI (myocardial infarction-prone Watanabe heritable hyperlipidemic) rabbit serum. In this study, serum samples from Type 2 diabetic patients were added to the medium with J774.1 cells and the degree of foam cell induction was quantified by measuring lipid accumulation. These values were calculated relative to the activities of normal and WHHLMI rabbit sera as 0% and 100%, respectively, and termed the MMI (Macrophage Maturation Index). These MMI values reflected intracellular lipids, including cholesteryl ester assayed by GC/MS. Statistical analysis revealed MMI to correlate positively and independently with serum triglycerides, the state of diabetic retinopathy, nephropathy and obesity, but negatively with administration of alpha-glucosidase inhibitors or thiazolidinediones. Taken together, our results suggest that this novel assay may be applicable to the identification of patients at risk for rapidly progressive angiopathic disorders.

C-Peptide Level in Fasting Plasma and Pooled Urine Predicts HbA1c after Hospitalization in Patients with Type 2 Diabetes Mellitus

In this study, we investigate how measures of insulin secretion and other clinical information affect long-term glycemic control in patients with type 2 diabetes mellitus. Between October 2012 and June 2014, we monitored 202 diabetes patients who were admitted to the hospital of Asahi Life Foundation for glycemic control, as well as for training and education in diabetes management. We measured glycated hemoglobin (HbA1c) six months after discharge to assess disease management. In univariate analysis, fasting plasma C-peptide immunoreactivity (F-CPR) and pooled urine CPR (U-CPR) were significantly associated with HbA1c, in contrast to ΔCPR and C-peptide index (CPI). This association was strongly independent of most other patient variables. In exploratory factor analysis, five underlying factors, namely insulin resistance, aging, sex differences, insulin secretion, and glycemic control, represented patient characteristics. In particular, insulin secretion and resistance strongly influenced F-CPR, while insulin secretion affected U-CPR. In conclusion, the data indicate that among patients with type 2 diabetes mellitus, F-CPR and U-CPR may predict improved glycemic control six months after hospitalization.

Glial fibrillary acidic protein (GFAP) is a novel biomarker for the prediction of autoimmune diabetes

Glial fibrillary acidic protein (GFAP) is expressed in peri‐islet Schwann cells, as well as in glia cells, and has been reported to be an autoantigen candidate for type 1 diabetes mellitus (T1DM). We confirmed that the production of the autoantibodies GFAP and glutamic acid decarboxylase 65 (GAD65) was increased and inversely correlated with the concentration of secreted C peptide in female nonobese diabetic mice (T1DM model). Importantly, the development of T1DM in female nonobese diabetic mice at 30 wk of age was predicted by the positive GFAP autoantibody titer at 17 wk. The production of GFAP and GAD65 autoantibodies was also increased in KK‐Ay mice [type 2 diabetes mellitus (T2DM) model]. In patients with diabetes mellitus, GFAP autoantibody levels were increased in patients with either T1DM or T2DM, and were significantly associated with GAD65 autoantibodies but not zinc transporter 8 autoantibodies. Furthermore, we identified a B‐cell epitope of GFAP corresponding to the GFAP autoantibody in both mice and patients with diabetes. Thus, these results indicate that autoantibodies against GFAP could serve as a predictive marker for the development of overt autoimmune diabetes.—Pang, Z., Kushiyama, A., Sun, J., Kikuchi, T., Yamazaki, H., Iwamoto, Y., Koriyama, H., Yoshida, S., Shimamura, M., Higuchi, M., Kawano, T., Takami, Y., Rakugi, H., Morishita, R., Nakagumi, H. Glial fibrillary acidic protein (GFAP) is a novel biomarker for the prediction of autoimmune diabetes. FASEB J. 31, 4053–4063 (2017). www.fasebj.org—Pang, Zhengda, Kushiyama, Akifumi, Sun, Jiao, Kikuchi, Takako, Yamazaki, Hiroki, Iwamoto, Yasuhiko, Koriyama, Hiroshi, Yoshida, Shota, Shimamura, Munehisa, Higuchi, Masayoshi, Kawano, Tomohiro, Takami, Yoichi, Rakugi, Hiromi, Morishita, Ryuichi, Nakagami, Hironori Glial fibrillary acidic protein (GFAP) is a novel biomarker for the prediction of autoimmune diabetes. FASEB J. 31, 4053–4063 (2017)