Hiroshi Maegawa

Autophagy regulates inflammation in adipocytes

Autophagy is an essential process for both the maintenance and the survival of cells, with homeostatic low levels of autophagy being critical for intracellular organelles and proteins. In insulin resistant adipocytes, various dysfunctional/damaged molecules, organelles, proteins, and end-products accumulate. However, the role of autophagy (in particular, whether autophagy is activated or not) is poorly understood. In this study we found that in adipose tissue of insulin resistant mice and hypertrophic 3T3-L1 adipocytes autophagy was suppressed. Also in hypertrophic adipocytes, autophagy-related gene expression, such as LAMP1, LAMP2, and Atg5 was reduced, whereas gene expression in the inflammatory-related genes, such as MCP-1, IL-6, and IL-1β was increased. To find out whether suppressed autophagy was linked to inflammation we used the autophagy inhibitor, 3-methyladenine, to inhibit autophagy. Our results suggest that such inhibition leads to an increase in inflammatory gene expression and causes endoplasmic reticulum (ER) stress (which can be attenuated by treatment with the ER stress inhibitor, Tauroursodeoxycholic Acid). Conversely, the levels of inflammatory gene expression were reduced by the activation of autophagy or by the inhibition of ER stress. The results indicate that the suppression of autophagy increases inflammatory responses via ER stress, and also defines a novel role of autophagy as an important regulator of adipocyte inflammation in systemic insulin resistance.

Omega-3 polyunsaturated fatty acid has an anti-oxidant effect via the Nrf-2/HO-1 pathway in 3T3-L1 adipocytes

Oxidative stress is produced in adipose tissue of obese subjects and has been associated with obesity-related disorders. Recent studies have shown that omega-3 polyunsaturated fatty acid (ω3-PUFA) has beneficial effects in preventing atherosclerotic diseases and insulin resistance in adipose tissue. However, the role of ω3-PUFA on adipocytes has not been elucidated. In this study, 3T3-L1 adipocytes were treated with ω3-PUFA and its metabolites, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or 4-hydroxy hexenal (4-HHE). ω3-PUFA and its metabolites dose-dependently increased mRNA and protein levels of the anti-oxidative enzyme, heme oxygenase-1 (HO-1); whereas no changes in the well-known anti-oxidant molecules, superoxide dismutase, catalase, and glutathione peroxidase, were observed. Knockdown of nuclear factor erythroid 2-related factor 2 (Nrf-2) significantly reduced EPA, DHA or 4-HHE-induced HO-1 mRNA and protein expression. Also, pretreatment with ω3-PUFA prevented H(2)O(2)-induced cytotoxicity in a HO-1 dependent manner. In conclusion, treatment with EPA and DHA induced HO-1 through the activation of Nrf-2 and prevented oxidative stress in 3T3-L1 adipocytes. This anti-oxidant defense may be of high therapeutic value for clinical conditions associated with systemic oxidative stress.

Regulation of Mitochondrial Biogenesis by Lipoprotein Lipase in Muscle of Insulin-Resistant Offspring of Parents With Type 2 Diabetes

Recent studies reveal a strong relationship between reduced mitochondrial content and insulin resistance in human skeletal muscle, although the underlying factors responsible for this association remain unknown. To address this question, we analyzed muscle biopsy samples from young, lean, insulin resistant (IR) offspring of parents with type 2 diabetes and control subjects by microarray analyses and found significant differences in expression of ∼512 probe pairs. We then screened these genes for their potential involvement in the regulation of mitochondrial biogenesis using RNA interference and found that mRNA and protein expression of lipoprotein lipase (LPL) in skeletal muscle was significantly decreased in the IR offspring and was associated with decreased mitochondrial density. Furthermore, we show that LPL knockdown in muscle cells decreased mitochondrial content by effectively decreasing fatty acid delivery and subsequent activation of peroxisome proliferator–activated receptor (PPAR)-δ. Taken together, these data suggest that decreased mitochondrial content in muscle of IR offspring may be due in part to reductions in LPL expression in skeletal muscle resulting in decreased PPAR-δ activation.

4-Hydroxy Hexenal Derived from Docosahexaenoic Acid Protects Endothelial Cells via Nrf2 Activation

Recent studies have proposed that n-3 polyunsaturated fatty acids (n-3 PUFAs) have direct antioxidant and anti-inflammatory effects in vascular tissue, explaining their cardioprotective effects. However, the molecular mechanisms are not yet fully understood. We tested whether n-3 PUFAs showed antioxidant activity through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcriptional factor for antioxidant genes. C57BL/6 or Nrf2−/− mice were fed a fish-oil diet for 3 weeks. Fish-oil diet significantly increased the expression of heme oxygenase-1 (HO-1), and endothelium-dependent vasodilation in the aorta of C57BL/6 mice, but not in the Nrf2−/− mice. Furthermore, we observed that 4-hydroxy hexenal (4-HHE), an end-product of n-3 PUFA peroxidation, was significantly increased in the aorta of C57BL/6 mice, accompanied by intra-aortic predominant increase in docosahexaenoic acid (DHA) rather than that in eicosapentaenoic acid (EPA). Human umbilical vein endothelial cells were incubated with DHA or EPA. We found that DHA, but not EPA, markedly increased intracellular 4-HHE, and nuclear expression and DNA binding of Nrf2. Both DHA and 4-HHE also increased the expressions of Nrf2 target genes including HO-1, and the siRNA of Nrf2 abolished these effects. Furthermore, DHA prevented oxidant-induced cellular damage or reactive oxygen species production, and these effects were disappeared by an HO-1 inhibitor or the siRNA of Nrf2. Thus, we found protective effects of DHA through Nrf2 activation in vascular tissue, accompanied by intra-vascular increases in 4-HHE, which may explain the mechanism of the cardioprotective effects of DHA.

A high-fiber, low-fat diet improves periodontal disease markers in high-risk subjects: a pilot study

Periodontal disease is related to aging, smoking habits, diabetes mellitus, and systemic inflammation. However, there remains limited evidence about causality from intervention studies. An effective diet for prevention of periodontal disease has not been well established. The current study was an intervention study examining the effects of a high-fiber, low-fat diet on periodontal disease markers in high-risk subjects. Forty-seven volunteers were interviewed for recruitment into the study. Twenty-one volunteers with a body mass index of at least 25.0 kg/m(2) or with impaired glucose tolerance were enrolled in the study. After a 2- to 3-week run-in period, subjects were provided with a test meal consisting of high fiber and low fat (30 kcal/kg of ideal body weight) 3 times a day for 8 weeks and followed by a regular diet for 24 weeks. Four hundred twenty-five teeth from 17 subjects were analyzed. Periodontal disease markers assessed as probing depth (2.28 vs 2.21 vs 2.13 mm; P < .0001), clinical attachment loss (6.11 vs 6.06 vs 5.98 mm; P < .0001), and bleeding on probing (16.2 vs 13.2 vs 14.6 %; P = .005) showed significant reductions after the test-meal period, and these improvements persisted until the follow-up period. Body weight (P < .0001), HbA1c (P < .0001), and high-sensitivity C-reactive protein (P = .038) levels showed improvement after the test-meal period; they returned to baseline levels after the follow-up period. In conclusion, treatment with a high-fiber, low-fat diet for 8 weeks effectively improved periodontal disease markers as well as metabolic profiles, at least in part, by effects other than the reduction of total energy intake.

A fish-based diet intervention improves endothelial function in postmenopausal women with type 2 diabetes mellitus: a randomized crossover trial.

OBJECTIVEnThe beneficial effects of fish and n-3 polyunsaturated fatty acids (PUFAs) consumption on atherosclerosis have been reported in numerous epidemiological studies. However, to the best of our knowledge, the effects of a fish-based diet intervention on endothelial function have not been investigated. Therefore, we studied these effects in postmenopausal women with type 2 diabetes mellitus (T2DM).nnnMATERIALS/METHODSnTwenty-three postmenopausal women with T2DM were assigned to two four-week periods of either a fish-based diet (n-3 PUFAs ≧ 3.0 g/day) or a control diet in a randomized crossover design. Endothelial function was measured with reactive hyperemia using strain-gauge plethysmography and compared with the serum levels of fatty acids and their metabolites. Endothelial function was determined with peak forearm blood flow (Peak), duration of reactive hyperemia (Duration) and flow debt repayment (FDR).nnnRESULTSnA fish-based dietary intervention improved Peak by 63.7%, Duration by 27.9% and FDR by 70.7%, compared to the control diet. Serum n-3 PUFA levels increased after the fish-based diet period and decreased after the control diet, compared with the baseline (1.49 vs. 0.97 vs. 1.19 mmol/l, p < 0.0001). There was no correlation between serum n-3 PUFA levels and endothelial function. An increased ratio of epoxyeicosatrienoic acid/dihydroxyeicosatrienoic acid was observed after a fish-based diet intervention, possibly due to the inhibition of the activity of soluble epoxide hydrolase.nnnCONCLUSIONSnA fish-based dietary intervention improves endothelial function in postmenopausal women with T2DM. Dissociation between the serum n-3 PUFA concentration and endothelial function suggests that the other factors may contribute to this phenomenon.

Ezetimibe prevents hepatic steatosis induced by a high-fat but not a high-fructose diet

Nonalcoholic fatty liver disease is the most frequent liver disease. Ezetimibe, an inhibitor of intestinal cholesterol absorption, has been reported to ameliorate hepatic steatosis in human and animal models. To explore how ezetimibe reduces hepatic steatosis, we investigated the effects of ezetimibe on the expression of lipogenic enzymes and intestinal lipid metabolism in mice fed a high-fat or a high-fructose diet. CBA/JN mice were fed a high-fat diet or a high-fructose diet for 8 wk with or without ezetimibe. High-fat diet induced hepatic steatosis accompanied by hyperinsulinemia. Treatment with ezetimibe reduced hepatic steatosis, insulin levels, and glucose production from pyruvate in mice fed the high-fat diet, suggesting a reduction of insulin resistance in the liver. In the intestinal analysis, ezetimibe reduced the expression of fatty acid transfer protein-4 and apoB-48 in mice fed the high-fat diet. However, treatment with ezetimibe did not prevent hepatic steatosis, hyperinsulinemia, and intestinal apoB-48 expression in mice fed the high-fructose diet. Ezetimibe decreased liver X receptor-α binding to the sterol regulatory element-binding protein-1c promoter but not expression of carbohydrate response element-binding protein and fatty acid synthase in mice fed the high-fructose diet, suggesting that ezetimibe did not reduce hepatic lipogenesis induced by the high-fructose diet. Elevation of hepatic and intestinal lipogenesis in mice fed a high-fructose diet may partly explain the differences in the effect of ezetimibe.

4-Hydroxy hexenal derived from dietary n-3 polyunsaturated fatty acids induces anti-oxidative enzyme heme oxygenase-1 in multiple organs.

It has recently been reported that expression of heme oxygenase-1 (HO-1) plays a protective role against many diseases. Furthermore, n-3 polyunsaturated fatty acids (PUFAs) were shown to induce HO-1 expression in several cells in vitro, and in a few cases also in vivo. However, very few reports have demonstrated that n-3 PUFAs induce HO-1 in vivo. In this study, we examined the effect of fish-oil dietary supplementation on the distribution of fatty acids and their peroxidative metabolites and on the expression of HO-1 in multiple tissues (liver, kidney, heart, lung, spleen, intestine, skeletal muscle, white adipose, brown adipose, brain, aorta, and plasma) of C57BL/6 mice. Mice were divided into 4 groups, and fed a control, safflower-oil, and fish-oil diet for 3 weeks. One group was fed a fish-oil diet for just 1 week. The concentration of fatty acids, 4-hydroxy hexenal (4-HHE), and 4-hydroxy nonenal (4-HNE), and the expression of HO-1 mRNA were measured in the same tissues. We found that the concentration of 4-HHE (a product of n-3 PUFAs peroxidation) and expression of HO-1 mRNA were significantly increased after fish-oil treatment in most tissues. In addition, these increases were paralleled by an increase in the level of docosahexaenoic acid (DHA) but not eicosapentaenoic acid (EPA) in each tissue. These results are consistent with our previous results showing that DHA induces HO-1 expression through 4-HHE in vascular endothelial cells. In conclusion, we hypothesize that the HO-1-mediated protective effect of the fish oil diet may be through production of 4-HHE from DHA but not EPA in various tissues.

Association between serum soluble TNFα receptors and renal dysfunction in type 2 diabetic patients without proteinuria

AIMnThe aim of our study was to investigate whether serum levels of soluble tumor necrosis factor α receptor (sTNFR) 1 and 2 are markers for renal dysfunction in type 2 diabetic patients without overt proteinuria.nnnMETHODSnJapanese type 2 diabetic patients without overt proteinuria (n = 168) enrolled in the prospective observational follow-up study in 2001 were retrospectively analyzed. At baseline, the serum levels of sTNFR1 and sTNFR2 were measured by sandwich ELISA. The associations between these markers and change in estimated glomerular filtration rate (eGFR) after 5 years were evaluated.nnnRESULTSnThe levels of sTNFR1 and sTNFR2 closely correlated. At baseline, sTNFR1 and sTNFR2 associated inversely with eGFR. After 5 years, patients with high level of both sTNFR1 and sTNFR2 showed a greater decline in eGFR (-13.8 ± 15.5% versus -8.5 ± 11.8%, P = 0.027) and a 4-fold higher risk for a GFR decline of ≥ 25% than those with high level of only one receptor or low level of both receptors. These associations were enhanced in diabetic women.nnnCONCLUSIONSnThe higher levels of sTNFR1 and sTNFR2 were associated with a greater decline in eGFR in type 2 diabetic patients without proteinuria, especially in diabetic women.

Transcription factor AP-2β: A negative regulator of IRS-1 gene expression

Down-regulation of insulin receptor substrate-1 (IRS-1) expression could modify the ability of IRS-1 to fulfill its functions. It has been proposed that the phosphorylation of IRS-1 on serine residues could promote its degradation. However, few studies have investigated the transcriptional regulation of IRS-1 in the pathogenesis of insulin resistance. Genotyping for genome-wide single nucleotide polymorphisms revealed that the transcription factor activating enhancer-binding protein-2beta (AP-2beta) is a novel candidate gene for conferring susceptibility to obesity and type 2 diabetes. AP-2beta is expressed in adipose tissue and its expression is increased during the maturation of adipocytes. Overexpression of AP-2beta leads to adipocyte hypertrophy, directly inhibits adiponectin expression, and enhanced the expression of inflammatory adipokines such as IL-6 and MCP-1. In this study, we found that overexpression of AP-2beta in 3T3-L1 adipocytes impaired the promoter activity of IRS-1, and subsequently decreased mRNA and protein expression. Electrophoretic mobility shift assays showed that AP-2beta bound specifically to the IRS-1 promoter region. Furthermore, site-directed mutagenesis of the AP-2 binding site located at -362 to -351, relative to the transcription start site, markedly decreased AP-2-induced suppression of IRS-1 promoter activity, whereas other putative AP-2 binding sites did not. Our results clearly showed that AP-2beta directly decreased IRS-1 expression by binding to its promoter. Based on these findings, we speculate that the AP-2beta transcriptional factor is a unique regulator of IRS-1 and a candidate gene for insulin resistance.