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A new meal tolerance test (MTT) using a 75 g glucose- and high fat-containing meal was applied to classify glucose intolerance in morbidly obese patients. According to the MTT data, the concordance rate of diagnosis was 82.5% compared to the 75 g oral glucose tolerance test (OGTT) in patients with normal glucose tolerance (NGT, n = 40). In the NGT patients, the insulinogenic index (r = 0.833), Matsuda index (r = 0.752), and disposition index (r = 0.845) calculated from the MTT data were each significantly (p0.001) correlated with those derived from the OGTT data. However, in patients with impaired glucose tolerance (IGT, n = 23) or diabetes mellitus (DM, n = 17), the postprandial glucose levels post-MTT were significantly lower than those post-OGTT, without increases in the postprandial insulin levels post-MTT. Thus, the severity of glucose intolerance measured by the MTT was milder than that indicated by the OGTT. Plasma levels of both glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) were increased at the postprandial state, but only the GIP levels post-MTT were significantly higher than those post-OGTT. The enhancement of glucose disposal rates in patients with NGT or IGT after the MTT was associated with increased GIP levels. The postprandial hypertriglyceridemia induced by the MTT was associated with insulin resistance, but it was not associated with the impaired insulinogenic index or the disposition index. These results indicate that the new MTT is clinically useful to evaluate both abnormal glucose and triglyceride excursions caused by abnormal insulin sensitivity and secretions of insulin and gut hormones in morbidly obese patients.

Introduction Various types of skin lesions with pruritus have been reported in participants of Asian clinical trials on sodium-glucose cotransporter-2 (SGLT2) inhibitors. The aim of this study was to determine whether the diuretic effect of a SGLT2 inhibitor could modify skin hydration status in patients with type 2 diabetes mellitus. Methods A prospective, short-term, open-label, two-parallel-arm, pilot study was conducted. Eligible patients were assigned to either a SGLT2 inhibitor (50 mg ipragliflozin once daily) group or to a dipeptidyl peptidase-4 inhibitor (50 mg sitagliptin once daily) group (control). The biophysical characteristics of the skin were measured and blood chemistry tests were run in all participants 1 day prior to medication initiation (pre-treatment values) and 14 days thereafter (post-treatment values). Results Fourteen patients were enrolled in the study, of whom eight were in the ipragliflozin group and six in the sitagliptin group. Compared to the pre-treatment values, the glycated hemoglobin (HbA1c) levels were slightly but significantly reduced in the ipragliflozin group (p = 0.02), but the changes in HbA1c from the pre-treatment to post-treatment time points did not significantly differ between the two treatment groups. Serum 3-hydroxy butyrate levels were significantly higher in the ipragliflozin group than in the sitagliptin group (p

Aims/Introduction Preprandial metformin administration significantly reduces postprandial plasma triglyceride levels in animal studies by reducing intestinal absorption through delayed gastric emptying. However, this effect has not been shown in a clinical study. Therefore, we planned to investigate the efficacy of preprandial metformin administration on postprandial hypertriglyceridemia and the related gastrointestinal effects in patients with type 2 diabetes mellitus. Materials and Methods A total of 11 patients taking single‐dose metformin at 500–1,000 mg, with non‐fasting plasma triglyceride levels of 150–1,000 mg/dL, were recruited at a single university hospital. The difference between preprandial and postprandial metformin administration on postprandial hypertriglyceridemia was examined by a meal test. The gastrointestinal effects of metformin, including stomach heaviness, heartburn and satiety, were also assessed using a visual analog scale. Results The mean bodyweight of patients was 80.6 kg (body mass index 27.9 kg/m2), and the mean non‐fasting plasma triglyceride level was 275.9 ± 57.0 mg/dL. The area under the curve of triglyceride during the meal test was significantly lower in the preprandial protocol than in the postprandial protocol (P

Mitochondria are critical in heat generation in brown and beige adipocytes. Mitochondrial number and function are regulated in response to external stimuli, such as cold exposure and β3 adrenergic receptor agonist. However, the molecular mechanisms regulating mitochondrial biogenesis during browning, especially by microRNAs, remain unknown. We investigated the role of miR-494-3p in mitochondrial biogenesis during adipogenesis and browning. Intermittent mild cold exposure of mice induced PPARγ coactivator1-α (PGC1-α) and mitochondrial TFAM, PDH, and ANT1/2 expression along with uncoupling protein-1 (Ucp1) in inguinal white adipose tissue (iWAT). miR-494-3p levels were significantly downregulated in iWAT upon cold exposure (p p p p

Aim/Introduction Diabetes and obesity are important health and economic concerns. We investigated the influence of obesity on diabetes control, the annual medical expenditures, and medications in Japanese patients with type 2 diabetes who were relatively lean in comparison to those in Western countries. Materials and Methods A total of 402 Japanese patients with type 2 diabetes were enrolled and their annual medical expenditures investigated. Obesity was defined as body mass index over 25 kg/m2, according to the obesity classifications from the Japan Society for the Study of Obesity. Results A total of 165 patients (41.0%) were classified as obese. The obese group was younger, had poor glycemic control, and higher frequency of hypertension than the non-obese group. The median total annual medical expenditures for all participants was 269,333 Japanese yen (interquartile: 169,664 to 437,437 Japanese yen), which was equivalent to approximately 2,450 US dollars. The annual medical expenditure was significantly higher in patients with obesity than in non-obese patients (P

Adipose tissues considerably influence metabolic homeostasis, and both white (WAT) and brown (BAT) adipose tissue play significant roles in lipid and glucose metabolism. O-linked N-acetylglucosamine (O-GlcNAc) modification is characterized by the addition of N-acetylglucosamine to various proteins by O-GlcNAc transferase (Ogt), subsequently modulating various cellular processes. However, little is known about the role of O-GlcNAc modification in adipose tissues. Here, we report the critical role of O-GlcNAc modification in cold-induced thermogenesis. Deletion of Ogt in WAT and BAT using adiponectin promoter–driven Cre recombinase resulted in severe cold intolerance with decreased uncoupling protein 1 (Ucp1) expression. Furthermore, Ogt deletion led to decreased mitochondrial protein expression in conjunction with decreased peroxisome proliferator–activated receptor γ coactivator 1-α protein expression. This phenotype was further confirmed by deletion of Ogt in BAT using Ucp1 promoter–driven Cre recombinase, suggesting that O-GlcNAc modification in BAT is responsible for cold-induced thermogenesis. Hypothermia was significant under fasting conditions. This effect was mitigated after normal diet consumption but not after consumption of a fatty acid–rich ketogenic diet lacking carbohydrates, suggesting impaired diet-induced thermogenesis, particularly by fat. In conclusion, O-GlcNAc modification is essential for cold-induced thermogenesis and mitochondrial biogenesis in BAT. Glucose flux into BAT may be a signal to maintain BAT physiological responses.

Background O-linked β- N -acetylglucosamine modification O-GlcNAcylation) is a post-translational modification of intracellular proteins, serving as a nutrient sensor. Growing evidence has demonstrated its physiological and pathological importance in various mammalian tissues. This study examined the physiological role of O-GlcNAcylation in podocyte function and development. Methods O-GlcNAc transferase (Ogt) is a critical enzyme for O-GlcNAcylation and resides on the X chromosome. To abrogate O-GlcNAcylation in podocytes, we generated congenital and tamoxifen (TM)-inducible podocyte-specific Ogt knockout mice (Podo-Ogt y/- and TM-Podo-Ogt y/- , respectively) and analyzed their renal phenotypes. Results Podo-Ogt y/- mice showed normal podocyte morphology at birth. However, they developed albuminuria at 8 weeks of age, increasing progressively until age 32 weeks. Glomerular sclerosis, proteinuria-related tubulointerstitial lesions and markedly altered podocyte foot processes, with decreased podocin expression, were observed histologically in 32-week-old Podo-Ogt y/- mice. Next, we induced adult-onset deletion of the Ogt gene in podocytes by TM injection in 8-week-old TM-Podo-Ogt y/- mice. In contrast to Podo-Ogt y/- mice, the induced TM-Podo-Ogt y/- mice did not develop albuminuria or podocyte damage, suggesting a need for O-GlcNAcylation to form mature foot processes after birth. To test this possibility, 3-week-old Podo-Ogt y/- mice were treated with Bis-T-23, which stimulates actin-dependent dynamin oligomerization, actin polymerization and subsequent foot process elongation in podocytes. Albuminuria and podocyte damage in 16-week-old Podo-Ogt y/- mice were prevented by Bis-T-23 treatment. Conclusions O-GlcNAcylation is necessary for maturation of podocyte foot processes, particularly after birth. Our study provided new insights into podocyte biology and O-GlcNAcylation.

Laparoscopic sleeve gastrectomy has been proven effective in treating obesity-associated type 2 diabetes mellitus (T2DM). However, reports of the effect of laparoscopic sleeve gastrectomy on glucose metabolism in Japanese obese patients with T2DM are rare. The aim of this study was to evaluate the effects of laparoscopic sleeve gastrectomy on glucose tolerance in Japanese obese patients with T2DM, and to analyze factors influencing diabetes remission after surgery. This was a retrospective analysis of data for 24 consecutive patients with T2DM who underwent laparoscopic sleeve gastrectomy. We investigated weight loss and its impact on T2DM 1 year postoperatively. We also compared baseline characteristics and postoperative factors between patients who achieved diabetes remission and patients without remission. Mean body weight loss and percent total weight loss were 23.9 kg and 23.3%, respectively. Mean hemoglobin A1c levels dropped from 7.3 ± 0.3% to 6.1 ± 0.2%, and 18 patients (75%) achieved diabetes remission 1 year postoperatively. Patients achieving remission had significantly lower hemoglobin A1c levels (p = 0.026), higher fasting C-peptide values (p < 0.001), shorter diabetes duration (p < 0.001), lower insulin requirement (p = 0.002), and higher area under the insulin response curve (p < 0.001) and insulinogenic index (p < 0.001) during oral glucose tolerance testing. In conclusion, laparoscopic sleeve gastrectomy is an effective treatment for Japanese obese patients with T2DM. Preserving insulin secretion is the major determinant of diabetes remission.

We herein report a case involving a woman with type 1 diabetes and a history of metal allergy who developed a local delayed-type (type IV) allergy to zinc-containing insulin. She had been treated by continuous subcutaneous insulin infusion, but her glycemic control was poor, and she developed diabetic ketoacidosis. Her plasma insulin concentration was unexpectedly low during use of insulin lispro, but it was recovered by changing from the zinc-containing insulin lispro to the zinc-free insulin glulisine. Intradermal tests showed no reactions to various insulins except for zinc chloride. A skin biopsy at the injection site of insulin lispro showed invasion of lymphocytes, neutrophils, and eosinophils, but a skin biopsy at the injection site of insulin glulisine showed invasion of only lymphocytes. A drug lymphocyte stimulation test against polaprezinc, an antiulcer drug containing zinc, was positive. Therefore, we diagnosed the patient with local delayed allergy to zinc-containing insulin. Insulin allergy should be considered as a possible cause of poor glycemic control and diabetic ketoacidosis in patients with type 1 diabetes.

We assessed the association between smoking status and mild cognitive impairment (MCI) in Japanese diabetic patients. This cross-sectional study included 323 diabetic patients, aged 40–79 years, who were referred to an outpatient diabetic clinic between January and July 2013 at Shiga University of Medical Science Hospital (Otsu, Japan). Cognitive function was assessed using the mini-mental state examination (MMSE), and patients were classified into two categories: normal cognitive function (MMSE score ≥27) and MCI (MMSE score 22–26). Logistic regression analysis was used to estimate the multivariable-adjusted odds ratios (ORs) and 95 % confidence intervals (CIs) for MCI in current smokers and ex-smokers compared with never-smokers. Of the 323 patients, 55 (17.0 %), 134 (41.5 %), and 134 (41.5 %) were current smokers, ex-smokers, and never-smokers, respectively. Of these, 68 (21.0 %) patients had MCI. After adjusting for age, sex, systolic blood pressure, body mass index, high-density lipoprotein cholesterol, estimated glomerular filtration rate, hemoglobin A1c, insulin therapy, sulfonylurea, history of coronary heart disease, exercise habit, drinking status, and education, the OR for MCI was 3.62 (95 % CI 1.26–10.40) in current smokers compared with never-smokers. In addition, the multivariable-adjusted ORs for MCI were 3.02 (95 % CI 0.64–14.32) in current smokers

Objective: Weight gain is a common problem in the insulin treatment. SGLT2 inhibitors are expected to reduce body weight (BW) due to negative energy balance. Previous reports suggested that BW reduction is achieved mainly by loss of body fat, and ∼20% of reduction is by lean mass. However, the efficacy of SGLT2 inhibitor on BW and body composition remains unclear. We examined these in the Japanese T2DM treated with insulin. Methods: The study was an open label randomized controlled trial. Primary endpoint was the change in BW from baseline to week 24 between the two groups. Fifty overweight patients (BMI > 23) who had inadequate glycemic control with insulin treatment were randomly assigned to ipragliflozin (Ipra) or standard treatment (Con) and followed for 24 weeks. Body composition was assessed by DEXA and impedance method. Results: The change in BW was significantly larger in Ipra compared to Con. Ipra showed significant larger reduction of HbA1c. Total fat mass was reduced in the Ipra compared to Con mainly due to lower limb fat and trunk fat. Total muscle mass was maintained at lower limbs and trunk but tended to be reduced at arm in Ipra. Conclusion: Ipragliflozin treatment for 24 weeks resulted in reduction of BW mainly due to fat mass at lower limb and trunk. Reduction of the muscle mass at lower limb may be protected by anti-gravity effect. Disclosure K. Morino: Research Support; Self; Astellas Pharma US, Inc., AstraZeneca, Sunstar Inc., CMIC Pharmascience, Kowa Pharmaceutical. H. Inoue: None. K. Fuse: None. S. Tanaka: None. K. Kondo: None. H. Arima: Advisory Panel; Self; Kyowa Kirin. Speaker's Bureau; Self; Takeda Japan, Daiichi Sankyo Company, Limited. K. Miura: None. D. Sato: None. N. Ohashi: None. S. Ida: None. I. Miyazawa: None. O. Sekine: None. S. Ugi: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., MSD K.K. H. Maegawa: Speaker's Bureau; Self; Astellas Pharma US, Inc.. Research Support; Self; Astellas Pharma US, Inc.. Speaker's Bureau; Self; Mitsubishi Tanabe Pharma Corporation. Research Support; Self; Mitsubishi Tanabe Pharma Corporation. Speaker's Bureau; Self; Sanofi. Research Support; Self; Sanofi. Speaker's Bureau; Self; Nippon Boehringer Ingelheim Co. Ltd.. Research Support; Self; Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Daiichi Sankyo Company, Limited. Research Support; Self; Daiichi Sankyo Company, Limited, Takeda Pharmaceutical Company. Speaker's Bureau; Self; Takeda Pharmaceutical Company, Novo Nordisk A/S, Eli Lilly and Company.

Background and Aim: Fatty liver is reported to be associated with type 2 diabetes mellitus. Most of the previous studies are cross-sectional and liver fat was evaluated using ultrasound echography or plasma levels of liver enzyme. We assessed liver fat content in a semi-quantitative method using computed tomographic (CT) images and verified the hypothesis that the L/S ratio for CT attenuation value was associated with the onset of diabetes in longitudinal observational cohort study of Japanese men. Methods: Participants were randomly selected men from Kusatsu City, Japan. We analyzed 622 participants (63.5±9.9 years) without diabetes at baseline. We calculated odds ratio (OR) and 95% confidence interval (CI) of the presence of fatty liver (defined by L/S ratio Results: During 5.1±1.4 years of follow-up period, 36 participants (5.8%) developed diabetes. Although the presence of fatty liver was not significantly associated with the development of diabetes (OR 1.77, 95% CI 0.81-3.85), OR for those of per 1 SD (0.18) decrease in L/S ratio was 1.40 (95% CI 1.02-1.92). We still found the significant results that L/S ratio was associated with the development of diabetes in the stratified analysis. Conclusions: The ratio of liver to spleen for CT attenuation value was associated with the development of diabetes during 5 years regardless of BMI in Japanese community-based study. Disclosure K. Fuse: None. A. Kadota: None. K. Kondo: None. A. Fujiyoshi: None. T. Hisamatsu: None. S. Kadowaki: None. I. Miyazawa: None. K. Morino: Research Support; Self; Astellas Pharma US, Inc., AstraZeneca, Sunstar Inc., CMIC Pharmascience, Kowa Pharmaceutical. O. Sekine: None. S. Ugi: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., MSD K.K. H. Maegawa: Speaker's Bureau; Self; Astellas Pharma US, Inc.. Research Support; Self; Astellas Pharma US, Inc.. Speaker's Bureau; Self; Mitsubishi Tanabe Pharma Corporation. Research Support; Self; Mitsubishi Tanabe Pharma Corporation. Speaker's Bureau; Self; Sanofi. Research Support; Self; Sanofi. Speaker's Bureau; Self; Nippon Boehringer Ingelheim Co. Ltd.. Research Support; Self; Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Daiichi Sankyo Company, Limited. Research Support; Self; Daiichi Sankyo Company, Limited, Takeda Pharmaceutical Company. Speaker's Bureau; Self; Takeda Pharmaceutical Company, Novo Nordisk A/S, Eli Lilly and Company. K. Miura: None. H. Ueshima: None.

Background Teff is a staple food in Ethiopia that is rich in dietary fiber. Although gaining popularity in Western countries because it is gluten-free, the effects of teff on glucose metabolism remain unknown. Aim To evaluate the effects of teff on body weight and glucose metabolism compared with an isocaloric diet containing wheat. Results Mice fed teff weighed approximately 13% less than mice fed wheat (p < 0.05). The teff-based diet improved glucose tolerance compared with the wheat group with normal chow but not with a high-fat diet. Reduced adipose inflammation characterized by lower expression of TNFα, Mcp1, and CD11c, together with higher levels of cecal short chain fatty acids such as acetate, compared with the control diet containing wheat after 14 weeks of dietary treatment. In addition, beige adipocyte formation, characterized by increased expression of Ucp-1 (~7-fold) and Cidea (~3-fold), was observed in the teff groups compared with the wheat group. Moreover, a body-weight matched experiment revealed that teff improved glucose tolerance in a manner independent of body weight reduction after 6 weeks of dietary treatment. Enhanced beige adipocyte formation without improved adipose inflammation in a body-weight matched experiment suggests that the improved glucose metabolism was a consequence of beige adipocyte formation, but not solely through adipose inflammation. However, these differences between teff- and wheat-containing diets were not observed in the high-fat diet group. Conclusions Teff improved glucose tolerance likely by promoting beige adipocyte formation and improved adipose inflammation.

O-GlcNAcylation is a post-translational modification that is characterized by the addition of N-acetylglucosamine (GlcNAc) to proteins by O-GlcNAc transferase (Ogt). The degree of O-GlcNAcylation is thought to be associated with glucotoxicity and diabetic complications, because GlcNAc is produced by a branch of the glycolytic pathway. However, its role in skeletal muscle has not been fully elucidated. In this study, we created skeletal muscle-specific Ogt knockout (Ogt-MKO) mice and analyzed their glucose metabolism. During an intraperitoneal glucose tolerance test, blood glucose was slightly lower in Ogt-MKO mice than in control Ogt-flox mice. High fat diet-induced obesity and insulin resistance were reversed in Ogt-MKO mice. In addition, 12-month-old Ogt-MKO mice had lower adipose and body mass. A single bout of exercise significantly reduced blood glucose in Ogt-MKO mice, probably because of higher AMP-activated protein kinase α (AMPKα) protein expression. Furthermore, intraperitoneal injection of 5-aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, resulted in a more marked decrease in blood glucose levels in Ogt-MKO mice than in controls. Finally, Ogt knockdown by siRNA in C2C12 myotubes significantly increased protein expression of AMPKα, glucose uptake and oxidation. In conclusion, loss of O-GlcNAcylation facilitates glucose utilization in skeletal muscle, potentially through AMPK activation. The inhibition of O-GlcNAcylation in skeletal muscle may have an anti-diabetic effect, through an enhancement of glucose utilization during exercise.

N-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have protective effects against atherosclerosis. Monocyte chemotactic protein (MCP)-1 is a major inflammatory mediator in the progression of atherosclerosis. However, little is known about the regulation of MCP-1 by DHA and EPA in vessels and vascular smooth muscle cells (VSMCs). In this study, we compared the effect of DHA and EPA on the expression of Mcp-1 in rat arterial strips and rat VSMCs. DHA, but not EPA, suppressed Mcp-1 expression in arterial strips. Furthermore, DHA generated 4-hydroxy hexenal (4-HHE), an end product of n-3 polyunsaturated fatty acids (PUFAs), in arterial strips as measured by liquid chromatography-tandem mass spectrometry. In addition, 4-HHE treatment suppressed Mcp-1 expression in arterial strips, suggesting 4-HHE derived from DHA may be involved in the mechanism of this phenomenon. In contrast, Mcp-1 expression was stimulated by DHA, EPA and 4-HHE through p38 kinase and the Keap1-Nuclear factor erythroid-derived 2-like 2 (Nrf2) pathway in VSMCs. In conclusion, there is a dual effect of n-3 PUFAs on the regulation of Mcp-1 expression. Further study is necessary to elucidate the pathological role of this phenomenon.

Postprandial hypertriglyceridemia is a potential target for cardiovascular disease prevention in patients with diabetic dyslipidemia. Metformin has been reported to reduce plasma triglyceride concentrations in the postprandial states. However, little is known about the mechanisms underlying the triglyceride-lowering effect of metformin. Here, we examined the effects of metformin on lipid metabolism after olive oil-loading in 129S mice fed a high fat diet for three weeks. Metformin administration (250 mg/kg) for one week decreased postprandial plasma triglycerides. Pre-administration (250 mg/kg) of metformin resulted in a stronger triglyceride-lowering effect (approximately 45% lower area under the curve) than post-administration. A single administration (250 mg/kg) of metformin lowered plasma postprandial triglycerides comparably to administration for one week, suggesting an acute effect of metformin on postprandial hypertriglyceridemia. To explore whole body lipid metabolism after fat-loading, stomach size, fat absorption in the intestine, and fat oxidation (13C/12C ratio in expired CO₂ after administration of glyceryl-1-13C tripalmitate) were measured with and without metformin (250 mg/kg) pre-treatment. In metformin-treated mice, larger stomach size, lower fat oxidation, and no change in lipid absorption were observed. In conclusion, metformin administration before fat loading reduced postprandial hypertriglyceridemia, most likely by delaying gastric emptying.

N-3 polyunsaturated fatty acids (PUFAs) improve endothelial function. The arachidonic acid-derived metabolites (epoxyeicosatrienoic acids (EETs)) are part of the endothelial hyperpolarization factor and are vasodilators independent of nitric oxide. However, little is known regarding the regulation of EET concentration by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in blood vessels. Sprague-Dawley rats were fed either a control or fish oil diet for 3 weeks. Compared with the control, the fish oil diet improved acetylcholine-induced vasodilation and reduced the protein expression of soluble epoxide hydrolase (sEH), a key EET metabolic enzyme, in aortic strips. Both DHA and EPA suppressed sEH protein expression in rat aorta endothelial cells (RAECs). Furthermore, the concentration of 4-hydroxy hexenal (4-HHE), a lipid peroxidation product of n-3 PUFAs, increased in n-3 PUFA-treated RAECs. In addition, 4-HHE treatment suppressed sEH expression in RAECs, suggesting that 4-HHE (derived from n-3 PUFAs) is involved in this phenomenon. The suppression of sEH was attenuated by the p38 kinase inhibitor (SB203580) and by treatment with the antioxidant N-acetyl-L-cysteine. In conclusion, sEH expression decreased after n-3 PUFAs treatment, potentially through oxidative stress and p38 kinase. Mild oxidative stress induced by n-3 PUFAs may contribute to their cardio-protective effect.

The 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).Twenty-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).A 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, p0.0001). 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.A 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.

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.

O-GlcNAcylation is characterised by the addition of N-acetylglucosamine to various proteins by O-GlcNAc transferase (OGT) and serves in sensing intracellular nutrients by modulating various cellular processes. Although it has been speculated that O-GlcNAcylation is associated with glucose metabolism, its exact role in whole body glucose metabolism has not been fully elucidated. Here, we investigated whether loss of O-GlcNAcylation globally and in specific organs affected glucose metabolism in mammals under physiological conditions. Tamoxifen-inducible global Ogt-knockout (Ogt-KO) mice were generated by crossbreeding Ogt-flox mice with R26-Cre-ERT2 mice. Liver, skeletal muscle, adipose tissue and pancreatic beta cell-specific Ogt-KO mice were generated by crossbreeding Ogt-flox mice with Alb-Cre, Mlc1f-Cre, Adipoq-Cre and Pdx1 PB-CreER™ mice, respectively. Glucose metabolism was evaluated by i.p. glucose and insulin tolerance tests. Tamoxifen-inducible global Ogt-KO mice exhibited a lethal phenotype from 4 weeks post injection, suggesting that O-GlcNAcylation is essential for survival in adult mice. Tissue-specific Ogt deletion from insulin-sensitive organs, including liver, skeletal muscle and adipose tissue, had little impact on glucose metabolism under physiological conditions. However, pancreatic beta cell-specific Ogt-KO mice displayed transient hypoglycaemia (Ogt-flox 5.46 ± 0.41 vs Ogt-βKO 3.88 ± 0.26 mmol/l) associated with about twofold higher insulin secretion and accelerated adiposity, followed by subsequent hyperglycaemia (Ogt-flox 6.34 ± 0.32 vs Ogt-βKO 26.4 ± 2.37 mmol/l) with insulin depletion accompanied by beta cell apoptosis. These findings suggest that O-GlcNAcylation has little effect on glucose metabolism in insulin-sensitive tissues but plays a crucial role in pancreatic beta cell function and survival under physiological conditions. Our results provide novel insight into O-GlcNAc biology and physiology in glucose metabolism.

Adipose tissues considerably influence metabolic homeostasis, and both white (WAT) and brown (BAT) adipose tissue play significant roles in lipid and glucose metabolism.

Background & Aims A fiber-rich diet has a cardioprotective effect, but the mechanism for this remains unclear. We hypothesized that a fiber-rich diet with brown rice improves endothelial function in patients with type 2 diabetes mellitus. Methods Twenty-eight patients with type 2 diabetes mellitus at a single general hospital in Japan were randomly assigned to a brown rice (n = 14) or white rice (n = 14) diet and were followed for 8 weeks. The primary outcome was changes in endothelial function determined from flow debt repayment by reactive hyperemia using strain-gauge plethysmography in the fasting state. Secondary outcomes were changes in HbA1c, postprandial glucose excursions, and markers of oxidative stress and inflammation. The area under the curve for glucose after ingesting 250 kcal of assigned rice was compared between baseline (T0) and at the end of the intervention (T1) to estimate glucose excursions in each group. Results Improvement in endothelial function, assessed by fasting flow debt repayment (20.4% vs. −5.8%, p = 0.004), was significantly greater in the brown rice diet group than the white rice diet group, although the between-group difference in change of fiber intake was small (5.6 g/day vs. −1.2 g/day, p

Objective The aim of this study was to search for novel markers of visceral adiposity. Methods Visceral (omental) and subcutaneous adipose tissues were obtained from 43 Japanese men. Microarray analysis using total RNA from visceral and subcutaneous adipose tissues obtained from five men with abdominal obesity and five nonobese men was first conducted. Then the expression pattern of candidate genes identified in the human study in mouse models of adiposity was examined. Results Among 30,500 genes evaluated, the mRNA expression of CCDC3 (encoding coiled-coil domain-containing protein 3) was upregulated in omental adipose tissues from abdominally obese subjects (3.07-fold) but not in subcutaneous adipose tissues (0.89-fold). Similar expression patterns were found in two distinct mouse models of obesity. In the analysis of all 43 men, CCDC3 mRNA levels in omental, but not in subcutaneous adipose tissue, were positively correlated with waist circumference and body mass index. CCDC3 was predicted to be a secretory protein, which was confirmed by western blotting, as overexpressed CCDC3 was secreted into the culture media. Conclusions The expression of CCDC3 is specifically increased in visceral adipose tissues in abdominally obese subjects. These results suggest that CCDC3 is a potential biomarker for estimating visceral adiposity.

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.

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.

Inflammatory signaling via Toll-like receptor 4 (TLR4) has been shown to facilitate atherogenesis. Recent lines of evidence show that saturated fatty acids (SFAs) induce the inflammatory response via the TLR4 pathway in macrophages and adipocytes. The aims of this study are to confirm the role of SFAs in TLR4-mediated inflammatory signaling in vascular cells and to propose soy phosphatidylcholine (SPC) as an effective inhibitor against TLR4-mediated agonists. SFAs such as palmitate and stearate increased the expression and secretion of MCP-1 in human umbilical vein endothelial cells (HUVECs) and rat vascular smooth muscle cells (VSMCs). SFAs up-regulated the activity of MCP-1 promoter through the activation of NF-kappaB. Knockdown of TLR4 using siRNA diminished the SFA-induced MCP-1 expression in HUVECs and rat VSMCs, while PKC or ceramide signal inhibitor did not inhibit the expression. Furthermore, we found that SPC effectively inhibited the MCP-1 expression induced by palmitate or LPS in a dose-dependent manner. However, SPC did not inhibit the mRNA expression of MCP-1 induced by cytokines such as TNF-alpha and IL-1beta, or by agonists binding to TLRs other than TLR4. In addition, SPC did not affect the activity of LPS assessed by clotting activity of the Limulus amoebocyte lysate. These results clearly show that SPC specifically inhibits the inflammatory responses induced by the TLR4-dependent signal. In conclusion, we have demonstrated a role of SFAs for inflammatory response via TLR4-NF-kappaB signaling in vascular cells. Moreover, we propose that SPC can be useful as a selective inhibitor to suppress the TLR4-mediated inflammatory signaling.

Protein-tyrosine phosphatase 1B (PTP1B) is a major regulator of insulin sensitivity. We have described a novel action of PTP1B in the induction of sterol regulatory element-binding protein-1 (SREBP-1) gene expression through activation of protein phosphatase 2A (PP2A). PTP1B is anchored to the endoplasmic reticulum membrane via its C-terminal tail. We have previously reported that membrane localization of PTP1B is essential for PP2A activation, which is crucial for enhancing SREBP-1 gene expression in in vitro experiments. In this study, we further investigated the physiological importance of membrane localization of PTP1B in vivo. We found that transient liver-specific overexpression of wild-type PTP1B (PTP1B-WT) using adenovirus-mediated gene transfer was associated with hypertriglyceridaemia and enhanced hepatic SREBP-1 gene expression in mice. However, overexpression of the C-terminal truncated PTP1B (PTP1BDeltaCT) failed to increase hepatic SREBP-1 expression or serum triglyceride levels, despite causing insulin resistance. Our results indicate that activation of PTP1B in the liver could induce hypertriglyceridaemia and that anchoring of PTP1B to the membrane is crucial for its action.

Sterol regulatory element-binding protein (SREBP)-1c plays a crucial role in the regulation of lipogenic enzymes in the liver. We previously reported that an X-chromosome-linked RNA binding motif (RBMX) regulates the promoter activity of Srebp-1c. However, still unknown was how it regulates the gene expression. To elucidate this mechanism, we screened the cDNA library from mouse liver by yeast two-hybrid assay using RBMX as bait and identified scaffold attachment factor B1 (SAFB1). Immunoprecipitation assay demonstrated binding of SAFB1 to RBMX. Chromatin immunoprecipitation assay showed binding of both SAFB1 and RBMX to the upstream region of Srebp-1c gene. RNA interference of Safb1 reduced the basal and RBMX-induced Srebp-1c promoter activities, resulting in reduced Srebp-1c gene expression. The effect of SAFB1 overexpression on Srebp-1c promoter was found only in the presence of RBMX. These results indicate a major role for SAFB1 in the activation of Srebp-1c through its interaction with RBMX.

Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor in stimulating lipogenesis in the liver. Protein-tyrosine phosphatase 1B (PTP1B) induces SREBP-1 gene expression via protein phosphatase 2A (PP2A) activation. PTP1B is reported to be anchored on the endoplasmic reticulum (ER) via its C-terminal tail, and change in intracellular localization of PTP1B by C-terminal-truncation did not alter its inhibitory effects on insulin signaling. In this study, we investigated whether the change in intracellular localization of PTP1B could influence SREBP-1 gene expression. Overexpression of C-terminal truncated PTP1B (PTP1BdeltaCT) in rat Fao cells did not induce SREBP-1 gene expression. Furthermore, PTP1BdeltaCT failed to bind PP2A, resulting in impaired PP2A activation, whereas overexpression of wild-type PTP1B (PTP1BWT) associated with PP2A. Moreover, a membrane-targeted PTP1BDeltaCT activated PP2A with restored PP2A binding, despite the absence of its C-terminal region. Finally, overexpression of PTP1BdeltaCT into mouse primary cultured hepatocytes failed to enhance SREBP-1c mRNA, whereas membrane-targeted PTP1BdeltaCT led to enhanced SREBP-1c mRNA in hepatocytes as well as PTP1BWT. In conclusion, membrane localization of PTP1B is essential for PP2A activation, which is crucial for its enhancement of SREBP-1 gene expression.

This study assessed the association between symptoms of bilateral numbness and/or paresthesia in the feet and postural instability in Japanese patients with diabetes. This cross-sectional study included 303 patients with diabetes, aged 40–88 years, who were referred to an outpatient diabetic clinic between January and July 2013 at Shiga University of Medical Science Hospital. A posturography test was used to evaluate postural sway in patients. Indices of postural sway were the total length and the enveloped area. Analysis of covariance was used to estimate the multivariable-adjusted means of indices of postural sway according to the presence or absence of symptoms of bilateral numbness and/or paresthesia in the feet. Of 303 patients, 35 (11.6 %) had symptoms of bilateral numbness and/or paresthesia in the feet. After adjustment for age, sex, diabetic retinopathy, regular exercise, body mass index, hemoglobin A1c level, and quadriceps’ strength, patients with symptoms had higher levels of postural sway length and an enveloped area in the posturography test than those without symptoms. In addition, we observed similar results when we analyzed 234 patients aged ≥60 years. Our findings suggest that patients who had symptoms of bilateral numbness and/or paresthesia in the feet may have more postural instability than those without symptoms.

The objective of this study was to explore predictors, including social factors, lifestyle factors, and factors relevant to glycemic control and treatment, for mild and severe hypoglycemia in insulin-treated Japanese diabetic patients. This study included 123 insulin-treated diabetic patients who were referred to the diabetes clinic between January and July 2013 at Shiga University of Medical Science Hospital. After a survey examining the various factors, patients were followed for 6 months. During the follow-up period, blood glucose was self-monitored. Mild hypoglycemia was defined as blood glucose level 50–69 mg/dl, and severe hypoglycemia was defined as blood glucose level ≤49 mg/dl. Multinomial logistic regression was used to estimate the adjusted odds ratio (OR) and 95% confidence interval (CI) of each factor for mild and severe hypoglycemia. During the 6-month follow-up period, 41 (33.3%) patients experienced mild hypoglycemia, and 20 (16.3%) experienced severe hypoglycemia. In multivariable-adjusted analyses, assistance from family members at the time of the insulin injection [presence/absence, OR (95% CI): 0.39 (0.16–0.97)] and drinking [current drinker/non- and ex-drinker, OR (95% CI): 4.89 (1.68–14.25)] affected mild hypoglycemia. Assistance from family members at the time of insulin injection [presence/absence, OR (95% CI): 0.19 (0.05–0.75)] and intensive insulin therapy [yes/no, OR (95% CI): 3.61 (1.06–12.26)] affected severe hypoglycemia. In conclusion, our findings suggest that not only a factor relevant to glycemic control and treatment (intensive insulin therapy) but also a social factor (assistance from family members) and a lifestyle factor (current drinking) were predictors for mild or severe hypoglycemia in Japanese insulin-treated diabetic patients.

To evaluate the genetic susceptibility to metabolic disorders induced by high fructose diet, we investigated the metabolic characteristics in 10 strains of inbred mice and found that they were separated into CBA and DBA groups according to the response to high fructose diet. The hepatic mRNA expression of the sterol regulatory element-binding protein-1 (SREBP-1) in CBA/JN was remarkably enhanced by high fructose diet but not in DBA/2N. Similar results were observed in primary hepatocytes after exposure to fructose. The nucleotide sequence at -468 bp from the putative starting point of the SREBP-1c gene was adenine in the DBA group while it was guanine in the CBA group. In hepatocytes from CBA/JN, the activity of CBA-SREBP-1c promoter was significantly increased by 2.4- and 2.2-fold, in response to 30 mm fructose or 10 nm insulin, respectively, whereas the activity of DBA-SREBP-1c promoter responded to insulin but not to fructose. In hepatocytes from DBA/2N, both types of SREBP-1c promoter activities in response to insulin were attenuated. Furthermore, electrophoretic mobility shift assay revealed an unidentified nuclear protein bound to the oligonucleotides made from the region between -453 to -480 bp of the SREBP-1c promoter of CBA/JN but not to the probe from DBA/2N. Thus, in DBA/2N, the reduced mRNA expression of SREBP-1 after fructose refeeding appeared to associate with two independent mechanisms, 1). loss of binding of unidentified proteins to the region between -453 to -480 bp of the SREBP-1c promoter and 2). impaired insulin stimulation of SREBP-1c promoter activity.

Steric hindrance by the backbone of extra oligosaccharides at gamma-Asn 308 may cause the repulsive force to widen the junction at the D:D interface, and thus, interfere with the longitudinal elongation and lateral association of protofibrils.

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 dynamic cycle of O-linked GlcNAc (O-GlcNAc) addition and removal is catalyzed by O-GlcNAc transferase and O-GlcNAcase, respectively, in a process that serves as the final step in a nutrient-driven “hexosamine-signaling pathway.” Evidence points to a role for O-GlcNAc cycling in diabetes and insulin resistance. We have used Drosophila melanogaster to determine whether O-GlcNAc metabolism plays a role in modulating Drosophila insulin-like peptide (dilp) production and insulin signaling. We employed transgenesis to either overexpress or knock down Drosophila Ogt(sxc) and Oga in insulin-producing cells (IPCs) or fat bodies using the GAL4-UAS system. Knockdown of Ogt decreased Dilp2, Dilp3, and Dilp5 production, with reduced body size and decreased phosphorylation of Akt in vivo. In contrast, knockdown of Oga increased Dilp2, Dilp3, and Dilp5 production, increased body size, and enhanced phosphorylation of Akt in vivo. However, knockdown of either Ogt(sxc) or Oga in the IPCs increased the hemolymph carbohydrate concentration. Furthermore, phosphorylation of Akt stimulated by extraneous insulin in an ex vivo cultured fat body of third instar larvae was diminished in strains subjected to IPC knockdown of Ogt or Oga. Knockdown of O-GlcNAc cycling enzymes in the fat body dramatically reduced neutral lipid stores. These results demonstrate that altered O-GlcNAc cycling in Drosophila IPCs modulates insulin production and influences the insulin responsiveness of peripheral tissues. The observed phenotypes in O-GlcNAc cycling mimic pancreatic β-cell dysfunction and glucose toxicity related to sustained hyperglycemia in mammals.

Horseback riding therapy is popularly used as one of the physiotherapy in Europe. Dr. Kimura hit upon Horseback Riding Robot, which can precisely simulate horseback motion ; as a result, he has developed and completed the Horseback Riding Robot, which can be worked by six motors. The Horseback Riding Robot, however, is very expensive. For that reason we have decided to turn over a new leaf and have developed a home machine by realizing one motor type. We proved the muscle strength trainings by use of Horse Riding Robot and a home machine, unlike a existing monotonous and boring muscle training, to be worthwhile and effective.

We evaluated whether hyperinsulinaemia stimulates the expression of transcription factor CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP-delta and leads to the induction of the chemokine (C-C motif) ligand 2 gene (Ccl2, also known as MCP-1) expression in aortas.Hyperinsulinaemia was induced by feeding rats a high-fructose diet. CCL2 production was analysed by ELISA. The expression of Ccl2, Cebpb and Cebpd mRNAs was investigated by quantitative RT-PCR. The binding of C/EBP-beta to Ccl2 was assessed by chromatin immunoprecipitation (ChIP) assay.Insulin at a concentration of 10 nmol/l significantly stimulated the expression of Cebpb, Cebpd and Ccl2 mRNAs, depending on activation of phosphatidylinositol 3-kinase (PI3K) in cultured vascular smooth muscle cells. The knock-down of C/EBP-beta with siRNA abolished the insulin-induced Ccl2 mRNA expression. In the aortas from fructose-fed rats, the levels of phosphorylation of Akt/protein kinase B, a downstream effector of PI3K, were also increased. The expression of Cebpb, Cebpd and Ccl2 mRNAs in the aortas from fructose-fed rats were significantly elevated, by 330, 300 and 300%, respectively, compared with those of control-fed rats. The induction Ccl2 mRNA expression in the aortas was significantly correlated with the expression of Cebpb and Cebpd mRNAs in the aortas. Furthermore, the ChIP assay showed elevated binding of C/EBP-beta to the 5' upstream region of Ccl2 in the aortas from fructose-fed rats.These findings clearly indicate the role of C/EBPs in the mechanism of upregulation of CCL2, an inflammation-related protein, observed in the hyperinsulinaemic state, which may initiate the process of atherosclerosis.