ABSTRACT

DESCRIPTION. State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This descnption is meant to serve as a succinct and accurate description of the proposed work when separated from the application. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE PROVIDED.

The increased prevalence of type 2 diabetes among children is attributed to a simultaneous increase in childhood obesity. Most children are diagnosed during puberty. Ethnic minority children, such as Native Americans, African Americans, and Hispanics, are disproportionately affected. Asians represent a rapidly growing minority group in the United States that is poorly represented in epidemiologic research. Based on information from studies of school children in Japan and studies in Japanese-American adults, Japanese American children are also likely to be at high risk due to a tendency toward central adiposity.

The overall aim of this study is to better understand in children the metabolic changes that precede the development of type 2 diabetes, and the influence of Asian ethnicity on diabetes risk. The specific aims of this project are: 1) to describe the metabolic changes and adipose factors that are associated. with the insulin resistance metabolic syndrome in prepubertal children; 2) to describe the relationship between pancreatic islet B-cell function and family history of type 2 diabetes; 3) to describe changes in these factors as children progress through puberty; 4) to describe the relationship of diet and physical activity to the metabolic and adipose factors; and 5) to describe the relationship between Japanese ancestry and metabolic, adipose, and insulin secretion factors. To accomplish these goals, a longitudinal cohort study of 450 prepubertal (8-10 year old) nondiabetic Japanese-American and Caucasian children is proposed. Measurements at baseline and 2 year follow-up will include: lipids and LDL particle size, insulin, C-peptide, proinsulin, glucose tolerance and insulin secretion determined by an intravenous glucose tolerance test, plasminogen activator inhibitor-1, fibrinogen, C-reactive protein, insulin-like growth factor-1 and insulin-like growth factor binding protein-3, body composition by DEXA, and intra-abdominal fat by MRI.

This study will improve the understanding of how pubertal changes in metabolic and adipose factors affect diabetes risk in Asian and Caucasian children.

PERFORMANCE SITE(S) (organization, city, state)

University of Washington (UW), Seattle, WA

Children's Hospital and Regional Medical Center (CHMC), Seattle, WA

Veteran's Administration Medical Center (VAMC), Seattle, WA

KEY PERSONNEL See instructions on Page 11. Use continuation pages as needed to provide the required information in the format shown below.

Name Organization Role on Project

A. SPECIFIC AIMS

The increased prevalence of type 2 diabetes among children is attributed to a simultaneous increase in childhood obesity. Many ethnic minority groups are known to be at increased risk for type 2 diabetes in adulthood, yet relatively little is known about the risk factors that precede this condition among ethnic minority youth. Asians represent a rapidly growing minority group in the United States that is poorly represented in epidemiologic research. Asian adults, despite having a low prevalence of obesity, appear to be at increased risk for diabetes due to a predisposition to accumulate visceral fat. While no data are available on type 2 diabetes among Asian-American children, in Japan type 2 diabetes prevalence among school children has increased markedly in recent years as obesity has become more prevalent. Rates are likely to be even higher among Asian children in the United States than children in Japan, since obesity is more prevalent in this country.

The long-term aim of this study is to better understand in children the metabolic changes that precede the development of type 2 diabetes, and the influence of Asian ethnicity on diabetes risk. This proposal extends the Japanese American Community Diabetes Study to create a separate, longitudinal study of prepubertal children of varying proportions of Japanese ancestry (ranging from 0 to 100%) who will be followed into and through puberty.

Specific Aim 1: To describe in prepubertal (8-10 years), nondiabetic children the metabolic and adipose factors that are associated with the insulin resistance metabolic syndrome. These include fasting plasma lipids (cholesterol, triglycerides, HDL-cholesterol, and LDL-cholesterol), LDL particle size, plasminogen activator inhibitor-1 (PAI-1), fibrinogen, C-reactive protein, glucose, insulin, C-peptide, and proinsulin; glucose tolerance assessed as glucose disappearance rate constant (K_G) during an intravenous glucose tolerance test; body composition by DEXA; body fat distribution by MRI; and body mass index.

Hypothesis 1: Features of the metabolic syndrome are evident in some prepubertal children.

Specific Aim 2: To assess variation in pancreatic islet ß-cell function by measuring fasting plasma insulin, C-peptide, proinsulin, and acute insulin response to glucose (AIR_g) by an intravenous glucose tolerance test.

Hypothesis 2: Glucose-stimulated insulin secretion is lower among children with a family history of type 2 diabetes.

Specific Aim 3: To describe the changes in these factors as children progress through and complete puberty. Tanner staging is used and plasma testosterone, estradiol, DHEA-S, IGF-1, and IGFBP-3 are measured.

Hypothesis 3: Puberty is associated with changes in body fat distribution and metabolic parameters in a direction consistent with higher risk of glucose intolerance and cardiovascular disease.

Specific Aim 4: To describe the relationship of lifestyle factors (diet and physical activity) to the metabolic and adipose factors, and changes therein.

Hypothesis 4: Diet and physical activity are important predictors of adiposity and metabolic changes in children.

Specific Aim 5: To describe the relationship of proportion of Japanese ancestry to the metabolic and adipose factors, and changes therein.

Hypothesis 5: A higher proportion of Japanese ancestry is associated with a greater predisposition to the metabolic syndrome and diminished insulin secretion.

B. BACKGROUND AND SIGNIFICANCE

B1. EPIDEMIOLOGY OF TYPE 2 DIABETES IN CHILDREN

· Increasing Incidence of Childhood Type 2 Diabetes.

The natural history of type 2 diabetes is characterized by both insulin resistance and islet ß-cell dysfunction, and hyperglycemia usually develops gradually. Thus, it is relatively asymptomatic in its early stages. Type 2 diabetes is often associated with obesity. In contrast, the pathophysiology of type 1 diabetes is completely different. Type 1 diabetes results from insulin deficiency due to autoimmune islet ß-cell destruction, and is thus often associated with autoantibodies to islet ß-cell components and contents. Unlike type 2 diabetes, the onset of type 1 diabetes is often precipitous with prominent diabetic symptoms, often including ketoacidosis. The majority of children with diabetes have type 1. Prior to the 1990's, there were only a few reports of childhood type 2 diabetes, which has therefore been considered a disease of adults. However, although population-based data are sparse, there is consensus that the incidence of type 2 diabetes among children and adolescents has increased in recent years [1-4]. This trend is attributed to increasing rates of childhood obesity and physical inactivity.

In Cincinnati, where almost all children (ages 0 to 19) diagnosed with diabetes are referred to a single specialty clinic, the proportion of type 2 diabetes increased from £ 4% prior to 1992 to 16% in 1994 [5]. Of diabetic children aged 10 to 19, the proportion with type 2 diabetes increased from between 3-10% before 1992 to 33% in 1994. The incidence of clinically diagnosed type 2 diabetes among children aged 10 to 19 living in Cincinnati increased 10-fold between 1982 and 1994, from 0.7/100,000 to 7.2/100,000. An 8.5-fold increase in type 2 diabetes was reported by a tertiary pediatric endocrine center in Arkansas between 1988 and 1995 [6]. In Allegheny County, Pennsylvania, a 3-fold increase in newly diagnosed diabetes among African American adolescents was noted between the periods 1985-89 and 1990-94, although the type of diabetes was not confirmed [7]. Subsequent evaluation demonstrated that many diabetic African-American adolescents did not have antibodies associated with type 1 diabetes, suggesting that many of the excess cases were type 2 [8].

· Lifestyle and Childhood Obesity

The prevalence of childhood obesity in the United States is increasing [9], probably as a result of lifestyle changes. Obese children report a higher proportion of total caloric intake as fat than non-obese children [10]. In Japan, increased prevalence of childhood obesity is associated with increased consumption of animal fat and protein [11]. Obese adolescents with type 2 diabetes also report high fat consumption and no regular exercise [12]. A greater amount of time spent in sedentary activities, such as television viewing, is also associated with childhood obesity [13]. Furthermore, obese children who reduce time spent in sedentary activities lose weight, even in the setting of stable food intake [14]. Physical activity is inversely related to obesity [15], and an exercise program has been shown to reduce fat mass and intra-abdominal fat accumulation in obese children [16]. Intra-abdominal fat is strongly associated with the metabolic sequelae of obesity (see section B2c.), yet little is known about the effect of lifestyle on longitudinal changes in intra-abdominal fat accumulation in non-obese children.

· Risk Factors for Childhood Type 2 Diabetes

In general, the risk factors for type 2 diabetes among children are similar to those reported for adults. Adolescents are affected more often than younger children, with an average age at diagnosis of about 13.5 years [2]. This suggests that body composition and/or metabolic changes during puberty play an important role in the onset of diabetes. About 95% of affected children are ³ 85th age- and sex-specific percentile for body mass index (BMI), and most have a family history of type 2 diabetes [2, 5]. A strong association between acanthosis nigricans and childhood type 2 diabetes has also been reported [2, 5, 6]. As with adults, Hispanic [17, 18], African-American [5, 6], and Native-American [19, 20] children appear to be disproportionately affected. Several studies have shown a gender discrepancy, with more girls affected than boys [2], an observation that is consistent with the earlier onset of puberty in girls.

· Lack of Information on Asian-American Children Despite Increased Risk in Asian Adults.

There are little population-based health data available on Asian Americans, and this is especially true for Asian-American children. Yet Asians are the fastest growing ethnic minority population in the United States [21]. Despite having a lower average BMI than Caucasians, South Asian adults living in the United Kingdom are 4 times as likely to have diabetes [22]. The prevalence of self-reported, physician diagnosed diabetes in residents of Hawaii is lowest in Caucasians (2.7%), highest in Japanese Americans (6.4%), and intermediate in those of Chinese (3.5%), Filipino (4.6%), and Native Hawaiian (4.7%) ancestry [23]. The increased risk of diabetes among Asians has been associated with a propensity for central or visceral adiposity [24-26]. Thus, there is reason to suspect that Asian-American children, particularly those who have adopted a western lifestyle, are at increased risk for diabetes.

The only published data on the incidence of type 2 diabetes in Asian children comes from Japan [11]. In a population-based study from Tokyo, asymptomatic schoolchildren were periodically screened for glucosuria, and an oral glucose tolerance test was performed on those who screened positive. Among primary school children, diabetes incidence increased tenfold from 0.2/100,000 in 1976-1980 to 2.0/100,000 in 1991-1995. Among junior high school children, diabetes prevalence increased from 7.3/100,000 to 13.9/100, 000 during the same years. Diabetes trends mirrored upward trends in body mass index and consumption of animal fats. Thus, it appears that vulnerability to diabetes among Asians begins in childhood. It is likely that the problem is even greater in the United States, where the prevalence of childhood obesity exceeds 20% [9].

B2. PATHOPHYSIOLOGY OF TYPE 2 DIABETES

The pathophysiology of hyperglycemia in type 2 diabetes includes both abnormalities in islet ß-cell function and development of insulin resistance. The latter is associated with overall obesity as well as with increased accumulation of body fat centrally.

B2a. ß-cell Dysfunction

· Abnormal Glucose-Stimulated Insulin Secretion

It is well established that even with obesity and insulin resistance, euglycemia is maintained in the presence of normal ß-cells, although at the expense of hyperinsulinemia. As is true for adults, normoglycemic obese children and adolescents are insulin resistant and hypersecrete insulin [27-30]. Japanese adults with impaired glucose tolerance demonstrate both impaired insulin sensitivity and hypersecretion of insulin, particularly if they are obese [31]. Despite hypersecretion of insulin, however, individuals with impaired glucose tolerance exhibit reduced glucose-stimulated insulin secretion relative to the degree of insulin resistance. Furthermore, the defect is even greater in persons who have type 2 diabetes. Thus, in the setting of insulin resistance, plasma glucose levels are more likely to reach values diagnostic of diabetes among individuals with abnormal ß-cell function who are unable to maintain adequate insulin secretion to compensate for insulin resistance. Although there is evidence that insulin resistance precedes the decline in insulin secretion among some individuals at high risk for type 2 diabetes [32], other reports suggest that impaired insulin secretion precedes or accompanies the development of insulin resistance [33]. In Japanese adults with impaired glucose tolerance, low insulin secretion predicts progression to diabetes [34, 35].

The causes of impaired glucose-stimulated insulin secretion are not fully understood. Among adults, aging is associated with a gradual decline in insulin secretion, and may explain the increased incidence of type 2 diabetes in the elderly [27, 36]. Insulin secretion capacity may also be genetically determined. For example, insulin secretion is 65% lower among nondiabetic individuals who have an identical twin with type 2 diabetes, compared to other nondiabetic individuals [37]. Other studies have demonstrated reduced insulin secretion among first-degree relatives of patients with type 2 diabetes compared to individuals of similar age and BMI without a family history of diabetes [38]. Thus, it is plausible that ethnic variation in diabetes prevalence may be partly explained by genetic determinants of insulin secretion.

· Abnormal Processing of Proinsulin to Insulin

Another measure of islet ß-cell dysfunction is incomplete processing of proinsulin to insulin. Within the secretory granules of the ß-cell, two enzymes (prohormone convertases 2 and 3) process proinsulin to intermediate proinsulin split products and then to insulin plus C-peptide [39]. If this process is abnormal, increased amounts of proinsulin and intermediate split products are present in plasma. Depending on the assay used to measure proinsulin, this increase may be measured as the plasma concentration of either proinsulin or of proinsulin plus intermediates. Individuals with type 2 diabetes secrete excess proinsulin [40, 41]. Both the concentration of proinsulin and the proportion of immunoreactive insulin attributable to proinsulin are increased. Moreover, the magnitude of the proinsulin to insulin ratio is inversely correlated with insulin secretion in patients with type 2 diabetes [42]. Since the orderly cleavage of proinsulin appears intact in type 2 diabetes, the excess release of incompletely processed proinsulin seems to be the result of either slower conversion or reduced storage time in the ß-cell [40].

This abnormality of proinsulin secretion precedes the diabetic state. Individuals with impaired glucose tolerance have an elevated proinsulin to insulin ratio compared to normoglycemic individuals [43], and fasting proinsulin levels predict the development of diabetes [44-46]. Among normoglycemic individuals, the proinsulin level and the proinsulin to insulin ratio are inversely correlated with insulin secretion, independent of age, gender, body mass index, waist to hip ratio, and insulin sensitivity [47]. Although it has been reported that proinsulin levels increase following hemipancreatectomy, suggesting that this may be a response to increased ß-cell demand [48], insulin resistance induced by administration of nicotinic acid is not accompanied by a disproportionate increase of proinsulin [49, 50]. Thus elevated proinsulin levels found with type 2 diabetes appear not to be simply a response of the ß-cell to insulin resistance, but probably represents an intrinsic abnormality of the ß-cells.

B2b. Obesity and Insulin Resistance

Increased adiposity, as measured by BMI, triceps skinfold thickness, and dual-energy x-ray absorptiometry (DEXA), is associated with increased fasting insulin levels in prepubertal and postpubertal children [29, 51-53]. As mentioned previously, normoglycemic obese children and adolescents are insulin resistant and hypersecrete insulin [27-30]. Thus, the association between obesity and insulin resistance seems to be well established in children.

· Effect of Pubertal Stage

A recent study demonstrated transient insulin resistance (measured by euglycemic clamp) during early puberty (Tanner stages 2 to 3), returning to prepubertal levels by late puberty [51]. Girls were more insulin resistant than boys regardless of pubertal stage in this study. These findings are consistent with prior studies demonstrating lower insulin levels in prepubertal children compared to midpubertal children [54, 55]. Both sex steroids and growth hormone (and peptides related to growth hormone action) have been implicated as causing insulin resistance during puberty since both rise during puberty [56-62]. Growth hormone effects are now more commonly assessed by measurements of insulin-like growth factor-1 (IGF-1) [63], the peripheral hormone that mediates many of the effects of growth hormone, and insulin-like growth factor binding protein-3 (IGFBP-3) [64].

· Effect of Ethnicity

The effect of ethnicity has been most extensively studied in African-American and Caucasian children. In prepubertal children, insulin sensitivity (determined by a tolbutamide-modified frequently sampled intravenous glucose tolerance test with minimal modeling) was 42% lower among African-American children compared to Caucasian children [52]. This same group reported higher fasting insulin levels in African American prepubertal children [53]. African-American adolescent girls have higher fasting insulin levels and decreased hepatic insulin clearance compared to Caucasians [65]. Arslanian and colleagues also showed decreased insulin sensitivity and increased insulin secretion among African-American adolescents compared to Caucasians using a 2-hour hyperglycemic clamp [66]. In contrast, others reported that insulin resistance (measured by euglycemic clamp) was greater in pubertal Caucasian than African-American boys, but did not differ by ethnicity in pubertal girls [51]. It remains unclear if these discrepant findings are due to differences in methodology or pubertal stage of the subjects.

B2c. Visceral Adiposity and Features of the Insulin Resistance Syndrome.

· Adults

The terms insulin resistance syndrome, metabolic syndrome, and syndrome X refer to a constellation of metabolic findings associated with increased cardiovascular disease risk in adults [67-69]. These metabolic factors include hyperinsulinemia, insulin resistance, hypertension, dyslipidemia (elevated triglycerides, low HDL cholesterol, and increased amounts of small, dense LDL), and obesity. While not part of the original description, increases in hemostatic factors [70-72] and inflammatory markers such as C-reactive protein [73-75] are also associated with the insulin resistance syndrome. In adults, the insulin resistance syndrome is more strongly associated with central adiposity (particularly visceral or intra-abdominal fat) than total body adiposity or subcutaneous fat [76-85]. Since intra-abdominal fat deposition is influenced by gender and menopausal status [86-88], it is presumed that sex hormones are involved in body fat distribution. Thus, puberty may be an important milestone in determining body fat distribution.

· Prepubertal Children

A few research groups have studied the metabolic effects of intra-abdominal (visceral) fat in prepubertal children. Visceral adiposity is associated with elevated fasting insulin and triglycerides in prepubertal children [52, 89, 90]. Incremental 30-minute insulin measured during an oral glucose tolerance test is associated with visceral fat in Caucasian, but not African-American children [53]. Insulin sensitivity (measured by a tolbutamide-modified, frequently sampled intravenous glucose tolerance test with minimal modeling), however, is associated with total fat mass but not visceral fat