Wilfred Y. Fujimoto

A Pro12Ala substitution in PPARgamma2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity

The peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor that has a pivotal role in adipocyte differentiation and expression of adipocyte-specific genes. The PPARγ1 and γ2 isoforms result from alternative splicing and have ligand-dependent and -independent activation domains. PPARγ2 has an additional 28 amino acids at its amino terminus that renders its ligand-independent activation domain 5-10-fold more effective than that of PPARγ1. Insulin stimulates the ligand-independent activation of PPARγ1 and γ2 (ref. 5), however, obesity and nutritional factors only influence the expression of PPARγ2 in human adipocytes. Here, we report that a relatively common Pro12Ala substitution in PPARγ2 is associated with lower body mass index (BMI; P=0.027; 0.015) and improved insulin sensitivity among middle-aged and elderly Finns. A significant odds ratio (4.35, P=0.028) for the association of the Pro/Pro genotype with type 2 diabetes was observed among Japanese Americans. The PPARγ2 Ala allele showed decreased binding affinity to the cognate promoter element and reduced ability to transactivate responsive promoters. These findings suggest that the PPARγ2 Pro12Ala variant may contribute to the observed variability in BMI and insulin sensitivity in the general population.

Evidence of Cosecretion of Islet Amyloid Polypeptide and Insulin by β-Cells

Islet amyloid polypeptide (IAPP) has been identified as the major constituent of the pancreatic amyloid of non-insulin-dependent diabetes mellitus (NIDDM) and is also present in normal β-cell secretory granules. To determine whether IAPP is a pancreatic secretory product, we measured the quantity of lAPP-like immunoreactivity (IAPP-LI), insulin, and glucagon released into 5 ml of incubation medium during a 2-h incubation of monolayer cultures (n = 5) of neonatal (3- to 5-day-old) Sprague-Dawley rat pancreases under three conditions: 1.67 mM glucose, 16.7 mM glucose, and 16.7 mM glucose plus 10 mM arginine and 0.1 mM isobutylmethylxanthine (IBMX). The quantity of IAPP-LI, insulin, and glucagon in the cell extract was also determined. Mean ± SE IAPP-LI in the incubation medium increased from 0.041 ± 0.003 pmol in 1.67 mM glucose to 0.168 ± 0.029 pmol in 16.7 mM glucose (P < 0.05) and 1.02 ± 0.06 pmol in 16.7 mM glucose plus arginine and IBMX (P < 0.05 vs. 1.67 or 16.7 mM glucose). Insulin secretion increased similarly from 4.34 ± 0.27 to 20.2 ± 0.6 pmol (P < 0.05) and then to 135 ± 5 pmol (P < 0.05 vs. 1.67 or 16.7 mM glucose). Glucagon release tended to decrease with the increase in glucose concentration (0.39 ± 0.01 vs. 0.33 ± 0.02 pmol, P < 0.1), whereas with the addition of arginine and IBMX to high glucose, glucagon release increased to 1.32 ± 0.03 pmol (P < 0.05 vs. 1.67 or 16.7 mM glucose). Thus, the molar proportion of IAPP-LI to insulin secreted in low glucose was ∼1% and did not differ significantly with stimulation (0.95 ± 0.08 vs. 0.84 ± 0.15 vs. 0.76 ± 0.05%). In contrast, there was no constant proportional relationship between the release of IAPP-LI and glucagon (10.6 ± 0.8 vs. 51.3 ± 8.7 vs. 77.5 ± 5.2%). After incubation in 1.67 mM glucose, the extracted cells contained 3.7 ± 0.2 pmol IAPP-LI, 944 ± 25 pmol insulin, and 28.2 ±1.5 pmol glucagon. After maximal stimulation, the fractional release of IAPP-LI was 26.7 ± 0.7% vs. 14.7 ± 0.6% of insulin and 4.4 ± 0.2% of glucagon. These data indicate that nondiabetic neonatal rat islet cultures contain IAPP-LI and release it after stimulation by glucose and nonglucose secretagogues. Furthermore, the data suggest that IAPP-LI is a product of the β-cell, which coreleases it with insulin in a molar ratio of ∼1.100.

Oral Disposition Index Predicts the Development of Future Diabetes Above and Beyond Fasting and 2-h Glucose Levels

OBJECTIVE—We sought to determine whether an oral disposition index (DIO) predicts the development of diabetes over a 10-year period. First, we assessed the validity of the DIO by demonstrating that a hyperbolic relationship exists between oral indexes of insulin sensitivity and β-cell function. RESEARCH DESIGN AND METHODS—A total of 613 Japanese-American subjects (322 men and 291 women) underwent a 75-g oral glucose tolerance test (OGTT) at baseline, 5 years, and 10 years. Insulin sensitivity was estimated as 1/fasting insulin or homeostasis model assessment of insulin sensitivity (HOMA-S). Insulin response was estimated as the change in insulin divided by change in glucose from 0 to 30 min (ΔI0–30/ΔG0–30). RESULTS—ΔI0–30/ΔG0–30 demonstrated a curvilinear relationship with 1/fasting insulin and HOMA-S with a left and downward shift as glucose tolerance deteriorated. The confidence limits for the slope of the loge-transformed estimates included −1 for ΔI0–30/ΔG0–30 versus 1/fasting insulin for all glucose tolerance groups, consistent with a hyperbolic relationship. When HOMA-S was used as the insulin sensitivity measure, the confidence limits for the slope included −1 only for subjects with normal glucose tolerance (NGT) or impaired fasting glucose (IFG)/impaired glucose tolerance (IGT) but not diabetes. On the basis of this hyperbolic relationship, the product of ΔI0–30/ΔG0–30 and 1/fasting insulin was calculated (DIO) and decreased from NGT to IFG/IGT to diabetes (P < 0.001). Among nondiabetic subjects at baseline, baseline DIO predicted cumulative diabetes at 10 years (P < 0.001) independent of age, sex, BMI, family history of diabetes, and baseline fasting and 2-h glucose concentrations. CONCLUSIONS—The DIO provides a measure of β-cell function adjusted for insulin sensitivity and is predictive of development of diabetes over 10 years.

Visceral Adiposity Is an Independent Predictor of Incident Hypertension in Japanese Americans

Context Central obesity and hypertension are well-established components of the metabolic syndrome, but what exactly is the relationship between visceral adiposity and hypertension? Contribution This prospective study used computed tomography to measure multiple body fat areas of 300 middle-aged, normotensive Japanese Americans. Ninety-two participants developed hypertension within 10 to 11 years. Greater visceral adiposity, independent of other measures of body fat and other risk factors, such as plasma insulin and glucose levels, was associated with increased risk for hypertension. Cautions Relationships between visceral adiposity and the development of hypertension may vary in different ethnic groups. The Editors A central pattern of body fat distribution is now generally considered to play an important role in the metabolic syndrome, which involves obesity, insulin resistance, hyperinsulinemia, dyslipidemia, glucose intolerance, and hypertension (1, 2). In particular, visceral adiposity rather than regional or generalized obesity appears to play a key role in these diseases (3-7). Several cross-sectional and prospective studies have examined associations between hypertension and greater central obesity, as measured by waist circumference, the ratio of waist-to-hip circumference, or the ratio of subscapular-to-triceps skinfold thickness (8-15). The cross-sectional studies have reported a positive association (8, 9), but the prospective studies have been inconclusive (10-15). These studies have posited that visceral adiposity and insulin resistance are the most important factors linking greater abdominal obesity (as assessed by surface measurements) and hypertension. Although visceral fat is thought to affect the prevalence of hypertension, only 3 cross-sectional studies have suggested a possible association between visceral adiposity (measured by using computed tomography [CT]) and blood pressure (3, 4, 16); however, the results of these studies were inconclusive. No prospective studies have examined whether directly measured visceral fat is associated with an increased risk for incident hypertension. Therefore, we prospectively examined the relationship between directly measured visceral adiposity and the risk for incident hypertension, independent of other measurements of total and regional adiposity and fasting plasma insulin. Methods Study Sample Between 1983 and 1988, we enrolled 658 second- and third-generation Japanese Americans who were between 34 and 76 years of age (mean age, 54.2 years) into the Japanese American Community Diabetes Study (17, 18). Participants were chosen from volunteers through community-wide recruitment and were representative of Japanese-American residents of King County, Washington, in age distribution, residential distribution, and parental immigration pattern. A comprehensive mailing list and telephone directory that included almost 95% of the Japanese-American population of King County, Washington, was used. All participants were of 100% Japanese ancestry. Participants returned for follow-up examinations 5 to 6 and 10 to 11 years after a baseline evaluation. For the current analysis, eligible participants had systolic blood pressure less than 140 mm Hg and diastolic blood pressure less than 90 mm Hg and were not taking antihypertensive or oral hypoglycemic medications or insulin. We excluded 277 of the 658 participants in the original cohort because they did not meet the inclusion criteria. We excluded an additional 67 persons because of death, loss to follow-up, or withdrawal from the study. Another 14 persons who completed follow-up but had missing covariate information were also excluded. The analytic cohort consisted of 300 persons (Figure). The follow-up rate in the present study was 91% (345 of 381) at the 5- to 6-year examination and 80% (304 of 381) at the 10- to 11-year examination (Figure). Figure. Flow of participants through the study. Data Collection All measurements were made in the General Clinical Research Center at the University of Washington, Seattle, Washington. The Human Subjects Review Committee at the University of Washington approved the protocol for this research, and we obtained signed informed consent from all participants. At all examinations, blood pressure was measured to the nearest 2 mm Hg with a mercury sphygmomanometer while the participant was in a recumbent position. Systolic blood pressure was determined by the first perception of sound, and diastolic blood pressure was determined at the disappearance of sounds (fifth-phase Korotkoff). Average blood pressure was calculated from the second and third of 3 consecutive measurements. We diagnosed hypertension at baseline or follow-up if the average systolic blood pressure was 140 mm Hg or greater, the average diastolic blood pressure was 90 mm Hg or greater, or the participant was receiving antihypertensive medications. We classified participants as hypertensive if they met these criteria at the follow-up examination at 5 to 6 years or 10 to 11 years (Figure). All patients received a 75-g oral glucose tolerance test after a 10-hour fast. We then used the American Diabetes Association criteria (19) to classify patients as having normal glucose tolerance, impaired glucose tolerance, or type 2 diabetes mellitus. Blood samples were drawn after an overnight 10-hour fast and during an oral glucose tolerance test for measurement of plasma glucose and insulin levels. We used an automated glucose oxidase method to assay plasma glucose. Fasting plasma insulin was measured by radioimmunoassay, as reported previously (5, 7). We measured triglyceride and high-density lipoprotein cholesterol levels in the Northwest Lipid Research Laboratory, according to modified procedures of the Lipid Research Clinics (20). We calculated body mass index (BMI) as the weight in kilograms divided by height in meters squared. For CT scans, we used single slices of the thorax, abdomen (at the umbilicus), and mid-thigh to measure cross-sectional subcutaneous thoracic, abdominal, and right thigh and intra-abdominal fat areas (measured in cm2), as described elsewhere (21). We directly estimated visceral adiposity from the intra-abdominal fat area. This measurement has been reported to have a high correlation with directly ascertained total visceral fat volume measured by using CT or magnetic resonance imaging (22, 23). We calculated total subcutaneous fat area as the sum of subcutaneous thoracic and abdominal fat areas and twice the right thigh subcutaneous fat area. We defined total fat area as total subcutaneous fat area plus intra-abdominal fat area. Among Japanese Americans, total fat area correlates highly with fat mass, as measured by hydrodensitometry (r= 0.89 to 0.94) (24). Waist circumference was measured at the level of the umbilicus to the nearest tenth of a centimeter. Participants were questioned about current use of cigarettes and daily consumption of alcoholic beverages, which was converted into grams of alcohol consumed per day. Usual weekly energy expenditure in kilocalories was estimated from questionnaire data on work and recreational activities, strenuous exercise, distance walked, and stairs climbed, as described elsewhere (25). Statistical Analysis We used multiple logistic regression analysis to estimate the odds ratio for incident hypertension in relation to an increase of 1 SD in baseline variables. For rare outcomes, the odds ratio will approximately equal the relative risk. For more frequent outcomes, such as hypertension, the odds ratio will overestimate the relative risk (26). We evaluated nonlinear effects of continuous independent variables by using quadratic and log transformations (27). The linear trends in odds were evaluated by using the median value for each quartile category of continuous variables. To assess departure from linearity, we included linear and quadratic terms (the median and the value squared) in the model (28). To determine whether interaction was present (that is, the relationship between the risk factor and the outcome varied depending on the value of a third variable) (27, 29, 30), we inserted first-order interaction terms into appropriate regression models. We assessed interaction to determine whether the relationship between hypertension status at follow-up and baseline adipose variables, such as intra-abdominal fat area, subcutaneous abdominal fat area, total subcutaneous fat area, BMI, or waist circumference, differed according to the level of an additional variable (for example, sex) in the model. We used the likelihood ratio test to determine the statistical significance of nonlinear effects of continuous independent variables and interaction terms in the logistic regression models. Multicollinearity was assessed by using the variance inflation factor (31). A variance inflation factor exceeding 10 is regarded as indicating serious multicollinearity, and values greater than 4.0 may be a cause for concern (31). We calculated the 95% CI for each odds ratio. P values were 2-tailed. We performed statistical analyses using Stata SE, version 8.0 (Stata Corp., College Station, Texas). Role of the Funding Sources The funding sources had no role in the collection, analysis, or interpretation of the data or in the decision to submit the manuscript for publication. Results Among the 300 eligible men and women followed for 10 to 11 years, there were 92 incident cases of hypertension. In univariate logistic regression analysis, intra-abdominal fat area, abdominal subcutaneous fat area, total subcutaneous fat area, total fat area, BMI, and waist circumference were associated with a higher incidence of hypertension. Fasting plasma insulin level, fasting plasma glucose level, 2-hour plasma glucose level, and high-density lipoprotein cholesterol level were also associated with incidence of hypertension (Table 1). Wealso compared the baseline characteristics of participants included in

Detection and characterization of the heterozygote state for lipoprotein lipase deficiency.

Because there are no characteristic clinical or biochemical manifestations, the heterozygote state for lipoprotein lipase (LPL) deficiency has been difficult to detect. Measurements of postheparin plasma LPL activity and of LPL mass were performed In six families of probands with LPL deficiency to characterize the heterozygote state. LPL mass was measured in a sandwich enzyme-linked immunosorbent assay (EUSA) using a monoclonal antibody (5D2) that had been produced against bovine milk LPL Thirteen obligate heterozygotes from these families had reduced LPL activity and mass below the 95th percent confidence limits of 34 normal controls, while one obligate heterozygote had LPL activity and mass between the 90th and 95th percent confidence limits. Potential heterozygotes In these families were Identified as normal (n=8) or heterozygotes (n=6) by comparison to the 95th percent confidence limits of the controls. Some relatives In four of the six families exhibited mild hyperlipidemla, similar to the pattern seen In familial combined hyperilpldemla (FCHL). The hyperilpldemla segregated with the heterozygote state for LPL deficiency In these families (p<0.03). High density lipoprotein (HDL) cholesterol was significantly reduced In the heterozygotes for LPL deficiency (p<0.01). The measurement of LPL activity and mass allows Identification of the heterozygote state for LPL deficiency, which Is characterized by variable expressions of hyperlipidemla and reduced HDL cholesterol. These results suggest that the heterozygote state for LPL deficiency may form one subset of FCHL.

Association of Elevated Fasting C-Peptide Level and Increased Intra-Abdominal Fat Distribution With Development of NIDDM in Japanese-American Men

The Japanese-American population of King County, Washington, is known to have a high prevalence of non-insulin-dependent diabetes mellitus (NIDDM). As part of a community-based study, we reexamined 146 second-generation Japanese-American men who had been initially classified as nondiabetic. At a mean follow-up period of 30 mo, 15 men had developed NIDDM, and 131 remained nondiabetic. The variables measured at the initial visit that distinguished the 15 diabetic men from the 131 nondiabetic men were older age, higher serum glucose level at 2 h after 75 g oral glucose, higher fasting plasma C-peptide level, and increased cross-sectional intra-abdominal fat area as determined by computed tomography. Both older age and higher 2-h glucose levels are variables that have been associated with the development of NIDDM, but the association of higher fasting C-peptide level and greater intra-abdominal fat area with subsequent development of NIDDM were new observations. The elevated fasting C-peptide level persisted after adjustment for fasting serum glucose. The elevated C-peptide level represents hypersecretion of insulin and was interpreted to reflect a compensatory response to an underlying insulin-resistant state that antedates the development of NIDDM. The fasting C-peptide level was correlated with the intra-abdominal fat area, suggesting that the intra-abdominal fat area may be associated with insulin resistance. Thus, in individuals who develop NIDDM, insulin resistance, increased insulin secretion, and increased intra-abdominal fat are present before diabetic glucose tolerance can be demonstrated.

Prevalence of Cardiovascular Disease Risk Factors in U.S. Children and Adolescents With Diabetes The SEARCH for Diabetes in Youth Study

OBJECTIVE—The purpose of this study was to determine the prevalence and correlates of selected cardiovascular disease (CVD) risk factors among youth aged <20 years with diabetes. RESEARCH DESIGN AND METHODS—The analysis included 1,083 girls and 1,013 boys examined as part of the SEARCH for Diabetes in Youth study, a multicenter, population-based study of youth 0–19 years of age with diabetes. Diabetes type was determined by a biochemical algorithm based on diabetes antibodies and fasting C-peptide level. CVD risk factors were defined as follows: HDL cholesterol <40 mg/dl; age- and sex-specific waist circumference >90th percentile; systolic or diastolic blood pressure >90th percentile for age, sex, and height or taking medication for high blood pressure; and triglycerides >110 mg/dl. RESULTS—The prevalence of having at least two CVD risk factors was 21%. The prevalence was 7% among children aged 3–9 years and 25% in youth aged 10–19 years (P < 0.0001), 23% among girls and 19% in boys (P = 0.04), 68% in American Indians, 37% in Asian/Pacific Islanders, 32% in African Americans, 35% in Hispanics, and 16% in non-Hispanic whites (P < 0.0001). At least two CVD risk factors were present in 92% of youth with type 2 and 14% of those with type 1A diabetes (P < 0.0001). In multivariate analyses, age, race/ethnicity, and diabetes type were independently associated with the odds of having at least two CVD risk factors (P < 0.0001). CONCLUSIONS—Many youth with diabetes have multiple CVD risk factors. Recommendations for weight, lipid, and blood pressure control in youth with diabetes need to be followed to prevent or delay the development of CVD as these youngsters mature.

Prevalence of diabetes mellitus and impaired glucose tolerance among second-generation Japanese-American men.

We describe the initial findings from a multidisciplinary, epidemiologic study of diabetes mellitus conducted in a population of secondgeneration Japanese-American (Nisei) men born between 1910 and 1939 who reside in King County, Washington (n = 1746). From this study population, 487 volunteered, and 229 were enrolled to comprise the study sample. A random sample of Nisei men was also drawn from the population to develop a reference sample of 189 men. All subjects participated in a 75-g oral glucose tolerance test; the National Diabetes Data Group (NDDG) and World Health Organization (WHO) diagnostic criteria as well as a modification of the WHO criteria were used to classify individuals with normal glucose tolerance, impaired glucose tolerance (IGT), or diabetes. Within the study sample, 79 men were found to have normal glucose tolerance, 72 had IGT, and 78 had type II diabetes. The mean age of the study sample was 61.4 yr. Based on comparison of the study sample to the reference sample, the study sample was ascertained to be representative of Nisei men in King County. Extrapolating from our observations in the reference sample and in the study sample, we have estimated that ∼56% of Nisei men in the study population have abnormal glucose tolerance. Much of this is undiagnosed because only ∼13% of the reference sample of Nisei men reported a prior diagnosis of diabetes. Of the men who enrolled in the study as nondiabetic subjects, 11.1% had diabetes and 39.2% had IGT; i.e., 50.3% had previously unknown abnormalities in glucose tolerance. We estimate that ∼20% of Nisei men have diabetes (both previously diagnosed and undiagnosed) and ∼36% have IGT.

Gastric Inhibitory Polypeptide Enhanced Lipoprotein Lipase Activity in Cultured Preadipocytes

Fat feeding stimulated the release of gastric inhibitory polypeptide (GIP) without concomitant insulin secretion. Since antilipolytic effects of GIP have been demonstrated and the uptake of triglyceride fatty acid by adipose tissue postprandially is a process reciprocally regulated with lipolysis, a stimulatory role of GIP on adipose tissue lipoprotein lipase activity may be present. After cultured preadipocytes were incubated for 2 h with GIP, the release of lipoprotein lipase activity into the culture medium and the total cellular activity present in acetone-ether powders of cells were measured. GIP stimulated significant increases in the lipoprotein lipase activity released into the culture medium and in cells. A dose response relationship was strongest for the effect of GIP on the enzyme activity in extracts of acetone-ether powders of the cells. The increased lipoprotein lipase activity produced by GIP could provide a mechanism for clearance of chylomicron triglyceride after feeding in man.

Low serum testosterone level as a predictor of increased visceral fat in Japanese-American men.

OBJECTIVE: To examine the association between baseline testosterone levels and changes in visceral adiposity in Japanese-American men.DESIGN: Prospective observational study.SUBJECTS: Second-generation Japanese-American males enrolled in a community-based population study.MEASUREMENTS: At baseline, 110 men received a 75 g oral glucose tolerance test (OGTT), and an assessment of body mass index (BMI); visceral adiposity measured as intra-abdominal fat area (IAF) using computed tomography (CT); fasting insulin and C-peptide levels; and total testosterone levels. IAF was re-measured after 7.5 y. Subcutaneous fat areas were also measured by CT in the abdomen, thorax and thigh. The total fat (TF) was calculated as the sum of IAF and total subcutanous fat areas (SCF).RESULTS: After 7.5 y, IAF increased by a mean of 8.0 cm2 (95% CI: 0.8, 15.3). Baseline total testosterone was significantly correlated with change in IAF (r=−0.26, P=0.006), but not to any appreciable degree with change in BMI, TF, or SCF. In a linear regression model with change in IAF as the dependent variable, baseline testosterone was significantly related to this outcome while adjusting for baseline IAF, SCF, BMI, age, diabetes mellitus status (OGTT by the WHO diagnostic criteria) and fasting C-peptide (regression coefficient for baseline testosterone [nmol/l;[equals;−107.13, P=0.003).CONCLUSIONS: In this Japanese-American male cohort, lower baseline total testosterone independently predicts an increase in IAF. This would suggest that by predisposing to an increase in visceral adiposity, low levels of testosterone may increase the risk of type 2 diabetes mellitus.