Assaf Rudich

Adolescent BMI trajectory and risk of diabetes versus coronary disease.

BACKGROUND The association of body-mass index (BMI) from adolescence to adulthood with obesity-related diseases in young adults has not been completely delineated. METHODS We conducted a prospective study in which we followed 37,674 apparently healthy young men for incident angiography-proven coronary heart disease and diabetes through the Staff Periodic Examination Center of the Israeli Army Medical Corps. The height and weight of participants were measured at regular intervals, with the first measurements taken when they were 17 years of age. RESULTS During approximately 650,000 person-years of follow-up (mean follow-up, 17.4 years), we documented 1173 incident cases of type 2 diabetes and 327 of coronary heart disease. In multivariate models adjusted for age, family history, blood pressure, lifestyle factors, and biomarkers in blood, elevated adolescent BMI (the weight in kilograms divided by the square of the height in meters; mean range for the first through last deciles, 17.3 to 27.6) was a significant predictor of both diabetes (hazard ratio for the highest vs. the lowest decile, 2.76; 95% confidence interval [CI], 2.11 to 3.58) and angiography-proven coronary heart disease (hazard ratio, 5.43; 95% CI, 2.77 to 10.62). Further adjustment for BMI at adulthood completely ablated the association of adolescent BMI with diabetes (hazard ratio, 1.01; 95% CI, 0.75 to 1.37) but not the association with coronary heart disease (hazard ratio, 6.85; 95% CI, 3.30 to 14.21). After adjustment of the BMI values as continuous variables in multivariate models, only elevated BMI in adulthood was significantly associated with diabetes (β=1.115, P=0.003; P=0.89 for interaction). In contrast, elevated BMI in both adolescence (β=1.355, P=0.004) and adulthood (β=1.207, P=0.03) were independently associated with angiography-proven coronary heart disease (P=0.048 for interaction). CONCLUSIONS An elevated BMI in adolescence--one that is well within the range currently considered to be normal--constitutes a substantial risk factor for obesity-related disorders in midlife. Although the risk of diabetes is mainly associated with increased BMI close to the time of diagnosis, the risk of coronary heart disease is associated with an elevated BMI both in adolescence and in adulthood, supporting the hypothesis that the processes causing incident coronary heart disease, particularly atherosclerosis, are more gradual than those resulting in incident diabetes. (Funded by the Chaim Sheba Medical Center and the Israel Defense Forces Medical Corps.).

Positive and Negative Regulation of Insulin Signaling by Reactive Oxygen and Nitrogen Species

Regulated production of reactive oxygen species (ROS)/reactive nitrogen species (RNS) adequately balanced by antioxidant systems is a prerequisite for the participation of these active substances in physiological processes, including insulin action. Yet, increasing evidence implicates ROS and RNS as negative regulators of insulin signaling, rendering them putative mediators in the development of insulin resistance, a common endocrine abnormality that accompanies obesity and is a risk factor of type 2 diabetes. This review deals with this dual, seemingly contradictory, function of ROS and RNS in regulating insulin action: the major processes for ROS and RNS generation and detoxification are presented, and a critical review of the evidence that they participate in the positive and negative regulation of insulin action is provided. The cellular and molecular mechanisms by which ROS and RNS are thought to participate in normal insulin action and in the induction of insulin resistance are then described. Finally, we explore the potential usefulness and the challenges in modulating the oxidant-antioxidant balance as a potentially promising, but currently disappointing, means of improving insulin action in insulin resistance-associated conditions, leading causes of human morbidity and mortality of our era.

Oxidative Stress Disrupts Insulin-induced Cellular Redistribution of Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes A PUTATIVE CELLULAR MECHANISM FOR IMPAIRED PROTEIN KINASE B ACTIVATION AND GLUT4 TRANSLOCATION

In a recent study we have demonstrated that 3T3-L1 adipocytes exposed to low micromolar H2O2 concentrations display impaired insulin stimulated GLUT4 translocation from internal membrane pools to the plasma membrane (Rudich, A., Tirosh, A., Potashnik, R., Hemi, R., Kannety, H., and Bashan, N. (1998) Diabetes 47, 1562–1569). In this study we further characterize the cellular mechanisms responsible for this observation. Two-hour exposure to ∼25 μm H2O2 (generated by adding glucose oxidase to the medium) resulted in disruption of the normal insulin stimulated insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI) 3-kinase cellular redistribution between the cytosol and an internal membrane pool (low density microsomal fraction (LDM)). This was associated with reduced insulin-stimulated IRS-1 and p85-associated PI 3-kinase activities in the LDM (84 and 96% inhibition, respectively). The effect of this finding on the downstream insulin signal was demonstrated by a 90% reduction in insulin stimulated protein kinase B (PKB) serine 473 phosphorylation and impaired activation of PKBα and PKBγ. Both control and oxidized cells exposed to heat shock displayed a wortmannin insensitive PKB serine phosphorylation and activity. These data suggest that activation of PKB and GLUT4 translocation are insulin signaling events dependent upon a normal insulin induced cellular compartmentalization of PI 3-kinase and IRS-1, which is oxidative stress-sensitive. These findings represent a novel cellular mechanism for the induction of insulin resistance in response to changes in the extracellular environment.

Perilipin Promotes Hormone-sensitive Lipase-mediated Adipocyte Lipolysis via Phosphorylation-dependent and -independent Mechanisms

Hormone-sensitive lipase (HSL) is the predominant lipase effector of catecholamine-stimulated lipolysis in adipocytes. HSL-dependent lipolysis in response to catecholamines is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin A (Peri A), an essential lipid droplet (LD)-associated protein. It is believed that perilipin phosphorylation is essential for the translocation of HSL from the cytosol to the LD, a key event in stimulated lipolysis. Using adipocytes retrovirally engineered from murine embryonic fibroblasts of perilipin null mice (Peri–/– MEF), we demonstrate by cell fractionation and confocal microscopy that up to 50% of cellular HSL is LD-associated in the basal state and that PKA-stimulated HSL translocation is fully supported by adenoviral expression of a mutant perilipin lacking all six PKA sites (Peri AΔ1–6). PKA-stimulated HSL translocation was confirmed in differentiated brown adipocytes from perilipin null mice expressing an adipose-specific Peri AΔ1–6 transgene. Thus, PKA-induced HSL translocation was independent of perilipin phosphorylation. However, Peri AΔ1–6 failed to enhance PKA-stimulated lipolysis in either MEF adipocytes or differentiated brown adipocytes. Thus, the lipolytic action(s) of HSL at the LD surface requires PKA-dependent perilipin phosphorylation. In Peri–/– MEF adipocytes, PKA activation significantly enhanced the amount of HSL that could be cross-linked to and co-immunoprecipitated with ectopic Peri A. Notably, this enhanced cross-linking was blunted in Peri–/– MEF adipocytes expressing Peri AΔ1–6. This suggests that PKA-dependent perilipin phosphorylation facilitates (either direct or indirect) perilipin interaction with LD-associated HSL. These results redefine and expand our understanding of how perilipin regulates HSL-mediated lipolysis in adipocytes.

Altered autophagy in human adipose tissues in obesity.

CONTEXT Autophagy is a housekeeping mechanism, involved in metabolic regulation and stress response, shown recently to regulate lipid droplets biogenesis/breakdown and adipose tissue phenotype. OBJECTIVE We hypothesized that in human obesity autophagy may be altered in adipose tissue in a fat depot and distribution-dependent manner. SETTING AND PATIENTS Paired omental (Om) and subcutaneous (Sc) adipose tissue samples were used from obese and nonobese (n = 65, cohort 1); lean, Sc-obese and intraabdominally obese (n = 196, cohort 2); severely obese persons without diabetes or obesity-associated morbidity, matched for being insulin sensitive or resistant (n = 60, cohort 3). RESULTS Protein and mRNA levels of the autophagy genes Atg5, LC3A, and LC3B were increased in Om compared with Sc, more pronounced among obese persons, particularly with intraabdominal fat accumulation. Both adipocytes and stromal-vascular cells contribute to the expression of autophagy genes. An increased number of autophagosomes and elevated autophagic flux assessed in fat explants incubated with lysosomal inhibitors were observed in obesity, particularly in Om. The degree of visceral adiposity and adipocyte hypertrophy accounted for approximately 50% of the variance in omental Atg5 mRNA levels by multivariate regression analysis, whereas age, sex, measures of insulin sensitivity, inflammation, and adipose tissue stress were excluded from the model. Moreover, in cohort 3, the autophagy marker genes were increased in those who were insulin resistant compared with insulin sensitive, particularly in Om. CONCLUSIONS Autophagy is up-regulated in adipose tissue of obese persons, especially in Om, correlating with the degree of obesity, visceral fat distribution, and adipocyte hypertrophy. This may co-occur with insulin resistance but precede the occurrence of obesity-associated morbidity.

Lipoic acid protects against oxidative stress induced impairment in insulin stimulation of protein kinase B and glucose transport in 3T3-L1 adipocytes

Aims/hypothesis. Oxidative stress has been shown to impair insulin-stimulated glucose transporter 4 translocation in 3T3-L1 adipocytes. This study explores the potential of the antioxidant lipoic acid to protect the cells against the induction of insulin resistance when given before exposure to oxidative stress. Methods. 3T3-LI were exposed for 16 h to lipoic acid after which cells were exposed for 2 h to continuous production of H2O2 by adding glucose oxidase to the culture medium. Results. These conditions resulted in a 50–70 % reduction in insulin-stimulated glucose transport activity associated with a decrease in reduced glutathione content from 37.4 ± 3.1 to 26.4 ± 4.9 nmol/mg protein, (p < 0.005). Lipoic acid pretreatment increased insulin-stimulated glucose transport following oxidative stress, reaching 84.8 ± 4.4 % of the control, associated with an increase in reduced glutathione content. Oxidation impaired the 4.89 ± 0.36-fold insulin-stimulated increase in glucose transporter 4 content in plasma membrane lawns of control cells. Lipoic acid pretreatment was, however, associated with preserved insulin-induced glucose transporter 4 translocation in cells exposed to oxidation, yielding 80 % of its content in controls. Although tyrosine phosphorylation patterns were not affected by lipoic acid pretreatment, insulin-stimulated protein kinase B/Akt serine 473 phosphorylation and activity were considerably impaired by oxidation but protected by lipoic acid pretreatment. A protective effect was not observed with either troglitazone, its isolated vitamin E moiety, or with vitamin C. Conclusion/interpretation. This study shows the ability of lipoic acid to provide partial protection against the impaired insulin-stimulated glucose transporter 4 translocation and protein kinase B/Akt activation induced by oxidative stress, potentially by its capacity to maintain intracellular redox state. [Diabetologia (1999) 42: 949–957]

Ceramide- and Oxidant-Induced Insulin Resistance Involve Loss of Insulin-Dependent Rac-Activation and Actin Remodeling in Muscle Cells

In muscle cells, insulin elicits recruitment of the glucose transporter GLUT4 to the plasma membrane. This process engages sequential signaling from insulin receptor substrate (IRS)-1 to phosphatidylinositol (PI) 3-kinase and the serine/threonine kinase Akt. GLUT4 translocation also requires an Akt-independent but PI 3-kinase–and Rac-dependent remodeling of filamentous actin. Although IRS-1 phosphorylation is often reduced in insulin-resistant states in vivo, several conditions eliciting insulin resistance in cell culture spare this early step. Here, we show that insulin-dependent Rac activation and its consequent actin remodeling were abolished upon exposure of L6 myotubes beginning at doses of C2-ceramide or oxidant-producing glucose oxidase as low as 12.5 μmol/l and 12.5 mU/ml, respectively. At 25 μmol/l and 25 mU/ml, glucose oxidase and C2-ceramide markedly reduced GLUT4 translocation and glucose uptake and lowered Akt phosphorylation on Ser473 and Thr308, yet they affected neither IRS-1 tyrosine phosphorylation nor its association with p85 and PI 3-kinase activity. Small interfering RNA–dependent Rac1 knockdown prevented actin remodeling and GLUT4 translocation but spared Akt phosphorylation, suggesting that Rac and actin remodeling do not contribute to overall Akt activation. We propose that ceramide and oxidative stress can each affect two independent arms of insulin signaling to GLUT4 at distinct steps, Rac–GTP loading and Akt phosphorylation.

Changes in Triglyceride Levels and Risk for Coronary Heart Disease in Young Men

Context Whether the serum triglyceride level is an independent risk factor for coronary heart disease (CHD) is not clear. Contribution The authors measured triglyceride levels and performed stress electrocardiographies 5 years apart on 13593 young Israeli male career soldiers and did coronary angiography if the stress test was abnormal. Triglycerides and change in triglyceride levels strongly predicted incident CHD after adjustment for known CHD risk factors and lifestyle. Decreases in triglyceride levels were associated with adoption of a healthier lifestyle and lower CHD risk. The lowest CHD risk occurred when triglyceride levels remained low. Caution The participants were healthy male soldiers. Implication In healthy young men, triglycerides and changes in triglyceride levels are an independent CHD risk factor. The Editors A recent meta-analysis (1) and most published papers suggest a moderate association between fasting triglyceride levels and coronary heart disease (CHD) (211). Of the lipid fractions, the triglyceride-rich very-low-density lipoprotein particle is probably the most sensitive to lifestyle modification (8). For example, estimates from meta-analyses suggest that for every 4.5 kg (approximately 10 lb) of stable weight reduction, triglyceride levels decrease by at least 0.068 mmol/L (6 mg/dL) (12). Accordingly, a considerable increase in the proportion of hypertriglyceridemic patients accompanies the obesity epidemic (6, 9). In addition, aerobic exercise, independent of weight loss, has been shown to modestly reduce triglyceride levels in a dose-dependent fashion (13). Hence, when assessing the risk associated with triglyceride levels, triglyceride measurement at a single time point (typically at enrollment) may not be a reliable indicator of a persons long-term triglyceridemia during follow-up. Whether changes in triglyceride levels over time can improve cardiovascular risk assessment is largely unknown, particularly in young adults, in whom information on the association between triglycerides and CHD is not available (1). For 13953 apparently healthy young adult men (mean age, 32 years; range, 26 to 45 years) from the MELANY (Metabolic, Lifestyle, and Nutrition Assessment in Young Adults) study (14), we obtained 2 measurements of fasting serum triglycerides and lifestyle variables 5 years apart and followed for incident cases of angiography-proven CHD. Here, we estimate the effect of baseline triglyceride levels (time 1) and changes (between time 1 and time 2) in triglyceride levels on CHD risk. Methods The MELANY Study The MELANY study, which was designed to investigate risk factors for common diseases in young adults, is being conducted at the Israel Defense Forces Staff Periodic Examination Center (SPEC), Zrifin, Israel. All career service personnel are evaluated every 5 years between 25 and 35 years of age and every 3 years thereafter until they are discharged from service. A computerized database established in 1992 is the source of data for the MELANY study. At each SPEC visit, participants complete a detailed questionnaire that assesses demographic, nutritional, lifestyle, and medical factors. Thereafter, blood samples are drawn after a 14-hour fast and analyzed. A trained medical technician measures height, weight, and blood pressure (by mercury sphygmomanometers), and a physician at the center performs a complete physical examination. Inclusion and Exclusion Criteria We included apparently healthy men 26 to 45 years of age who had fasting triglyceride levels less than 3.39 mmol/L (<300 mg/dL) at their initial SPEC visit. We used the cutoff value of 3.39 mmol/L (300 mg/dL) because SPEC persons with greater triglyceride values are subjected to additional interventionsnutritional, pharmacologic, or both. Of 15155 men age 26 to 45 years, 1202 were excluded because they had diabetes (type 1 or 2) (n= 227) or CHD (n= 17) at baseline; had fasting triglyceride levels of 3.39 mmol/L or greater (300 mg/dL) (n= 676); or were receiving long-term medications (n= 282), including lipid-lowering medications. Therefore, for analysis of the association between baseline triglyceride levels and incident CHD, we included 13953 men. In studying the effect of changes in triglyceride levels on CHD, we excluded an additional 413 men who did not have a triglyceride measurement from the second SPEC visit (n= 363) or had received a diagnosis of diabetes (n= 38) or CHD (n= 12) between the first and second SPEC visit or during the second visit. Women were not included because the number of new cases of CHD in women was too small to facilitate meaningful analysis. Outcome Definition The outcome definition of the study was clinically significant CHD (angiography-proven stenosis >50% in at least 1 coronary artery) or fatal or nonfatal myocardial infarction (MI). At each sequential SPEC visit, all Israel Defense Forces military personnel older than 35 years of age who were participating in the current analysis had a treadmill exercise test (Bruce protocol [15]) in the presence of a board-certified cardiologist. End points for the exercise test were clinically significant ST-segment depression (>2 mm in 2 contiguous leads, measured 80 ms after the J point), intolerable symptoms of angina and exhaustion, or achievement of the target heart rate. All cases with a pathologic stress test were referred for coronary angiography. In participants with a borderline stress test, or when participants reported angina symptoms without diagnostic electrocardiographic changes, stress perfusion imaging with thallium-201 was performed. Those with a pathologic thallium-201 cardiac scan underwent coronary angiography. All Israel Defense Forces personnel obtain primary care between scheduled SPEC visits at designated military clinics, and all medical records are stored in the same central database, thereby facilitating ongoing, tight, and uniform follow-up. Individuals presenting with symptoms of angina, MI, or both between SPEC visits were also referred for coronary angiography after consultation with a board-certified cardiologist. Laboratory Methods Investigators performed biochemical analyses of fresh blood samples in an adjacent core laboratory facility that handles 1.2 million samples per year. The laboratory is authorized to perform tests according to the International Organization for Standardization standard 9002. The United Kingdom National External Quality Assessment Service, Sheffield, United Kingdom, performed periodic assessment of quality control on a regular basis. All lipid levels were directly measured, except for low-density lipoprotein (LDL) levels, which were calculated. Investigators measured all biochemical markers by using a BM/Hitachi 917 automated analyzer (Boehringer, Mannheim, Germany). Statistical Analysis For the primary analysis, we included 13953 untreated, apparently healthy young men with triglyceride levels lower than 3.39 mmol/L (<300 mg/dL). We used a general linear model to assess the age-adjusted means and proportions of the populations characteristics across quintiles of triglycerides and to fit the median of the quintiles as a continuous variable to estimate the trend of variables across quintiles (Table 1). We conducted Cox proportional hazards analysis during the 10.5-year follow-up to estimate the hazard ratios and 95% CIs for the development of CHD (the dependent variable) according to triglyceride levels at time 1 (first measurement). In stepwise models (Table 2), we added the values for body mass index (BMI), high-density lipoprotein (HDL) cholesterol, and family history of CHD separately to the age-adjusted model to evaluate their potential role as confounders. In the final multivariate model, we controlled for age, BMI, HDL cholesterol, family history of CHD, fasting plasma glucose, mean arterial blood pressure, physical activity, and smoking status. Because the total cholesterolHDL cholesterol ratio is a predictor of CHD (8), we performed a secondary analysis that included this ratio instead of HDL cholesterol in the multivariate model. Table 1. Baseline (Time 1) Characteristics, by Quintile of Triglyceride Level Table 2. Hazard Ratios for Coronary Heart Disease, by Quintile of Time-1 Triglyceride Level To assess the risk associated with changes in triglyceride levels, we analyzed data from 13540 men who had 2 triglyceride measurements (obtained at time 1 and time 2, 5 years apart) that were available before the end of follow-up or before being censored after a diagnosis of CHD or diabetes. In the model, time 2 was considered the baseline of 5.2 years of subsequent follow-up, whereas time 1 was considered prebaseline. We cross-classified triglyceride levels at each time point by tertiles: median levels of 0.68, 1.18, and 2.08 mmol/L (60, 104, and 184 mg/dL) at time 1 and 0.79, 1.33, and 2.49 mmol/L (70, 118, and 220 mg/dL) at time 2. In parallel, we determined changes in BMI, smoking status, physical activity, and habit of eating breakfast between time 2 and time 1 (Figure 1). Next, we evaluated the joint risk attributed to triglyceride levels at time 1 and time 2, categorized according to low (bottom), intermediate, and high (top) tertiles, and we used men with triglyceride levels in the low tertile at both time 1 and time 2 as a reference group (low/low group; hazard ratio, 1.0). To evaluate the direct association of changes in triglyceride levels (Figure 2), we used a multivariate model to further adjust for the interval between the 2 measurements and for the changes between time 2 and time 1 in BMI and lifestyle variables (physical activity, smoking, and habit of eating breakfast). We included these variables by calculating differences () in BMI and creating 3 groups of each categorical variable (smoking, physical activity, and habit of eating breakfast) based on their status at time 1 and time 2 (yes/yes, yes/no, no/yes, or no/no). Figure 1. Changes in selected lifestyle variables

Dietary Intervention to Reverse Carotid Atherosclerosis

Background— It is currently unknown whether dietary weight loss interventions can induce regression of carotid atherosclerosis. Methods and Results— In a 2-year Dietary Intervention Randomized Controlled Trial–Carotid (DIRECT-Carotid) study, participants were randomized to low-fat, Mediterranean, or low-carbohydrate diets and were followed for changes in carotid artery intima-media thickness, measured with standard B-mode ultrasound, and carotid vessel wall volume (VWV), measured with carotid 3D ultrasound. Of 140 complete images of participants (aged 51 years; body mass index, 30 kg/m2; 88% men), higher baseline carotid VWV was associated with increased intima-media thickness, age, male sex, baseline weight, blood pressure, and insulin levels (P<0.05 for all). After 2 years of dietary intervention, we observed a significant 5% regression in mean carotid VWV (−58.1 mm3; 95% confidence interval, −81.0 to −35.1 mm3; P<0.001), with no differences in the low-fat, Mediterranean, or low-carbohydrate groups (−60.69 mm3, −37.69 mm3, −84.33 mm3, respectively; P=0.28). Mean change in intima-media thickness was −1.1% (P=0.18). A reduction in the ratio of apolipoprotein B100 to apolipoprotein A1 was observed in the low-carbohydrate compared with the low-fat group (P=0.001). Participants who exhibited carotid VWV regression (mean decrease, −128.0 mm3; 95% confidence interval, −148.1 to −107.9 mm3) compared with participants who exhibited progression (mean increase, +89.6 mm3; 95% confidence interval, +66.6 to +112.6 mm3) had achieved greater weight loss (−5.3 versus −3.2 kg; P=0.03), greater decreases in systolic blood pressure (−6.8 versus −1.1 mm Hg; P=0.009) and total homocysteine (−0.06 versus +1.44 &mgr;mol/L; P=0.04), and a higher increase of apolipoprotein A1 (+0.05 versus −0.00 g/L; P=0.06). In multivariate regression models, only the decrease in systolic blood pressure remained a significant independent modifiable predictor of subsequent greater regression in both carotid VWV (β=0.23; P=0.01) and intima-media thickness (β=0.28; P=0.008) levels. Conclusions— Two-year weight loss diets can induce a significant regression of measurable carotid VWV. The effect is similar in low-fat, Mediterranean, or low-carbohydrate strategies and appears to be mediated mainly by the weight loss–induced decline in blood pressure. Clinical Trial Registration— http://www.clinicaltrials.gov. Unique Identifier: NCT00160108.

Deletion of Fas in adipocytes relieves adipose tissue inflammation and hepatic manifestations of obesity in mice.

Adipose tissue inflammation is linked to the pathogenesis of insulin resistance. In addition to exerting death-promoting effects, the death receptor Fas (also known as CD95) can activate inflammatory pathways in several cell lines and tissues, although little is known about the metabolic consequence of Fas activation in adipose tissue. We therefore sought to investigate the contribution of Fas in adipocytes to obesity-associated metabolic dysregulation. Fas expression was markedly increased in the adipocytes of common genetic and diet-induced mouse models of obesity and insulin resistance, as well as in the adipose tissue of obese and type 2 diabetic patients. Mice with Fas deficiency either in all cells or specifically in adipocytes (the latter are referred to herein as AFasKO mice) were protected from deterioration of glucose homeostasis induced by high-fat diet (HFD). Adipocytes in AFasKO mice were more insulin sensitive than those in wild-type mice, and mRNA levels of proinflammatory factors were reduced in white adipose tissue. Moreover, AFasKO mice were protected against hepatic steatosis and were more insulin sensitive, both at the whole-body level and in the liver. Thus, Fas in adipocytes contributes to adipose tissue inflammation, hepatic steatosis, and insulin resistance induced by obesity and may constitute a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes.