1Wallenberg Laboratory, Department of Endocrinology, Lund University, S-20502 Malmo, Sweden; 2Jakobstad Hospital, 68601 Jakobstad; and 3Department of Medicine, University Hospital, 00029 Helsinki, Finland
Submitted 18 March 2003 ; accepted in final form 14 July 2003
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ABSTRACT |
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insulin secretion; insulin sensitivity; metabolic syndrome; genetics of type 2 diabetes
Significant heterogeneity is observed in the clinical phenotypes of the different types of diabetes in Scandinavia, where 15% have classical early-onset type 1 diabetes, 10% have latent autoimmune diabetes in adults (LADA), and <5% have maturity onset diabetes of the young (MODY). Also, type 1 and type 2 diabetes occur together in 10% of all diabetic families (17). The etiology of the remaining 6065% of the common form of type 2 diabetes remains a topic of controversy (2, 5, 10). To circumvent the problems of secondary defects due to chronic hyperglycemia, several groups have studied normoglycemic relatives of patients with type 2 diabetes (6, 13, 18, 25, 31, 34). Considerable controversy still exists regarding the primacy of insulin resistance or defects in insulin secretion in the pathogenesis of the disease (9, 11, 21, 22, 24). In earlier studies, however, type 2 diabetes in the proband was considered to be homogenous, and the proband was not studied.
Recently, Vauhkonen et al. (32) reported that the metabolic defects in offspring of diabetic parents resemble those in the parents. However, in that study, 50% of the offspring had impaired glucose tolerance at the time of the study. Another problem with the earlier studies is that they did not take into account the dependence of insulin secretion on insulin sensitivity (3, 15, 25) or slight elevations in blood glucose concentrations. This is important, because the presence of insulin resistance could act as a compensatory stimulus for insulin secretion and thereby attenuate putative differences.
The Botnia study was designed to characterize early metabolic defects in first-degree relatives of diabetic subjects and to identify a genetic basis for these defects (9). Here, we have studied 2,100 glucose-tolerant first-degree relatives of patients with type 2 diabetes (FH+) and 388 spouses with no family history of diabetes (FH) after exclusion of families with both type 1 and type 2 diabetes, LADA, and MODY. The specific aim was to determine 1) whether a family history of the common form of type 2 diabetes has an impact on insulin sensitivity and/or insulin secretion adjusted for insulin sensitivity and 2) whether the phenotype of the diabetic proband is reflected in the phenotype of the nondiabetic relative.
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METHODS |
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To study the influence of the specific phenotype of the proband on the metabolic phenotype of the relative, diabetic probands (n = 516) were grouped by their WHR and the age of onset of diabetes.
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We thereby identified 399 first-degree relatives of diabetic subjects with low WHR (1st quartile, males: <0.95 and females: <0.84) and 396 relatives with high WHR (4th quartile, males: >1.02 and females: >0.95). Likewise, the numbers of first-degree relatives of diabetic subjects with early (1st quartile, <44 yr) and late (4th quartile, >65 yr) age of diabetes onset were 310 and 353, respectively.
Body weight and height were measured with subjects in light clothing without shoes. Fat-free mass (FFM) was measured with infrared spectroscopy from the outer layer of the biceps on the dominant arm with a Futrex-5000 device (Futrex, Gaithersburg, MD). The coefficient of variation (CV) of repeated measurements by the same investigator was <1%. Waist circumference was measured with a soft tape on standing subjects midway between the lower rib and iliac crest. Hip circumference was measured over the widest part of the gluteal region, and WHR as a measure of central obesity was accordingly calculated. Three blood pressure recordings were obtained from the right arm of each seated person at 5-min intervals after 30 min of rest, and the mean values were calculated. Fasting blood samples were drawn for the measurement of GADAb, serum total cholesterol, HDL2 and HDL3 cholesterol, triglyceride, and free fatty acid (FFA) concentrations.
All subjects participated in an oral glucose tolerance test (OGTT) by ingesting 75 g of glucose in a volume of 300 ml (Glucodyn; Leiras, Turku, Finland) after a 12-h overnight fast. Samples for the measurement of glucose and insulin were drawn at 10, 0, 30, 60, and 120 min. Several indexes of insulin sensitivity and insulin secretion were calculated from the OGTT. The homeostasis model assessment (HOMA) of insulin resistance (IR) index was calculated by using the fasting plasma glucose and insulin concentrations (19).
As measures of insulin secretion we used the incremental 30-min insulin response (I30), as well as the ratio of 30-min insulin and glucose during the OGTT (I/G30) (12). As a surrogate of the disposition index (DI), we considered the product of insulin sensitivity (1/HOMA) and insulin secretion (I/G30) (14, 27).
Insulin action was also measured in a subset of subjects by a hyperinsulinemic euglycemic clamp. Subjects in different subgroups in which euglycemic clamps were performed numbered 54 for FH+ and 21 for FH. After a priming dose of insulin, an infusion (infusion rate 45 mU/m2) of short-acting human insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) was started and continued until 120 min. Blood samples for the measurement of blood glucose were obtained at 5-min intervals throughout the clamp. A variable glucose infusion of 20% glucose was started to maintain blood glucose concentration unchanged at 5.5 mmol/l, with a CV of 6%. Insulin sensitivity was calculated from the glucose infusion rates during the last 60 min of the euglycemic clamp (M value) and expressed as glucose uptake per lean body mass.
A subset of subjects underwent an intravenous glucose tolerance test (IVGTT; n = 300: FH+ = 278, FH = 22). Briefly, 0.3 g/kg body weight of a 50% glucose solution was given at time 0. Blood samples for the measurement of insulin and blood glucose were obtained at 10, 0, 2, 4, 6, 8, 10, 20, 40, 50, and 60 min. The first-phase insulin response (FPIR) was calculated as the incremental insulin response during the first 10 min of the study. To quantify the relation between insulin secretion and insulin sensitivity, we measured the DI (3, 15), the product of insulin sensitivity (M value) and insulin secretion (FPIR).
Assays. Plasma glucose during the clamp was measured with a glucose oxidation method by use of a Beckman Glucose Analyzer II (Beckman Instruments, Fullerton, CA). Serum insulin concentrations were measured with a specific radioimmunoassay (Pharmacia, Uppsala, Sweden), with an interassay CV of 5%. Serum C-peptide concentrations were measured with radioimmunoassay (Linco Research, St. Charles, MO) with an interassay CV of 9%. FFA were measured by an enzymatic colorimetric method (Wako Chemicals, Neuss, Germany). Serum total cholesterol, HDL subfractions (after precipitation), and triglyceride concentrations were measured on a Cobas Mira analyzer (Hoffman-La Roche, Basel, Switzerland). GAD65 antibodies were determined by a modified radiobinding assay employing 35S-labeled recombinant human GAD65 (10a). Screening for MODY mutations was carried out as described earlier (15a).
Statistical analysis. All data are expressed as means ± SE. The significance of difference between groups was tested with Student's t-test and Mann-Whitney's test when applicable. The average number of individuals from each family was 1.5, with a range from 1 to 6 members in a family. Therefore, the subjects did not completely represent independent observations. To avoid this problem, we reanalyzed the data by using weighted family-specific means from different families for all variables that showed a significant difference in the first analysis. While comparing relatives of diabetic subjects from different phenotypes, we also analyzed the data by limiting individuals to one from each family. Measured variables were log transformed when they were not normally distributed. Computation was performed using the NCSS statistical package (Statistical Solutions, Cork, Ireland). Spearman's correlations were used for univariate correlation between the variables, and least square regression analysis was carried out to test differences in slopes from the regressions.
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RESULTS |
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Insulin sensitivity. FH+ subjects were more insulin resistant, as reflected by higher fasting insulin (P = 0.03) and higher HOMA (P = 0.006) values compared with FH subjects. This was further supported by a reduced rate of glucose uptake (8.11 ± 0.34 vs. 10.3 ± 0.5 mg · FFM kg1 · min1, P = 0.001) in FH+ compared with FH subjects. The insulin area under the OGTT curve adjusted for glucose was also higher in the FH+ than in the FH subjects (P = 0.04), suggesting impaired insulin action.
Insulin secretion. There was no difference in insulin secretion measured as FPIR between FH+ and FH groups (250 ± 35 vs. 258 ± 10 mU · l1 · 10 min1, P = not significant). However, the insulin secretion adjusted for insulin sensitivity (DI) was lower in FH+ than in FH subjects (1,805 ± 155 vs. 2,715 ± 422, P = 0.04).
Comparison of clinical and metabolic characteristics between relatives of type 2 diabetic probands with high and low WHR and early and late age of onset of diabetes. The probands were divided into quartiles of WHR and age of onset of diabetes. The relatives of diabetic subjects with high WHR had higher BMI (P < 0.005), and the females had higher WHR compared with relatives of probands with a low WHR (Table 2). The relatives of probands with a high WHR were more insulin resistant, as manifested by a lower rate of insulin-stimulated glucose uptake (8.1 ± 0.6 vs. 10.6 ± 0.7, P = 0.02) and higher lnHOMA IR (0.40 ± 0.02 vs. 0.48 ± 0.02, P = 0.02) compared with relatives of probands from the lowest WHR quartile (Table 2). In addition, they had higher total cholesterol and triglyceride concentrations than relatives of probands with low WHR. The waist circumference in relatives of probands with high (r = 0.526, P < 0.005) and low WHR (r = 0.299, P < 0.005) correlated with the lnHOMA IR. However, the slopes were significantly different (Fig. 1, P = 0.0002); e.g., for the same WHR there was a steeper increase in insulin resistance in those from the high compared with the low WHR. No difference was observed in insulin secretion adjusted for insulin sensitivity between the two groups.
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Relatives of probands with age at onset before 44 yr (lowest quartile) were, as expected, younger than relatives of probands with onset of diabetes >65 yr (highest quartile; age 42.9 ± 0.9 vs. 48.3 ± 0.7, P < 0.005) (Table 3). Despite this, they were more insulin resistant (HOMA IR 1.85 ± 0.05 vs. 1.59 ± 0.05, P < 0.005) and had significantly reduced -cell function, lnI/G30 (1.15 ± 0.02 vs. 1.24 ± 0.02, P = 0.001), particularly after adjustment for insulin sensitivity (lnI/G30/HOMA, 0.94 ± 0.001 vs. 1.08 ± 0.01, P < 0.0005) and lower HDL2 and HDL3 concentrations compared with subjects whose relatives had onset of diabetes after 65 yr (Fig. 2).
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DISCUSSION |
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Impairment in insulin action has been reported in nondiabetic relatives of patients with diabetes (6, 11, 13, 34). On the other hand, there have been conflicting reports regarding the impact of a family history of diabetes on insulin secretion (1, 18, 21, 24). Compared with these earlier studies, the present study is unique in several aspects. First, we have a truly homogeneous population of a large number of individuals without confounding factors affecting pancreatic -cell function, like the presence of GAD antibodies or known MODY mutations. Second, we have included only subjects with normal glucose tolerance. Third, insulin secretion has been adjusted for the degree of glycemia and insulin sensitivity. Fourth, we have also taken into account the possible heterogeneity in the phenotype of the diabetic probands.
Insulin sensitivity was clearly impaired in FH+ subjects, reflected by lower insulin-stimulated glucose uptake and higher HOMA IR. Also, insulin secretion (FPIR) adjusted for insulin sensitivity (M value) was reduced, suggesting that the FH+ subjects were not able to compensate for their degree of insulin resistance. This measure (DI) is an important test of the degree of metabolic derangement, because subjects with low DI have been shown to have an increased risk of developing future diabetes (36).
A key finding was also that relatives of diabetic probands with abdominal obesity were more insulin resistant and showed more features of the metabolic syndrome than relatives of nonobese probands. Interestingly, in siblings of probands with high WHR, a small increase in abdominal obesity had a greater impact on insulin resistance than in siblings of probands with low WHR. Several studies have shown that measures of abdominal obesity show strong heritability (16, 29). It has been suggested that genetic factors account for 40% of total body fat but that genetic factors contribute more to abdominal than to subcutaneous fat (4, 23, 28). Familiality does not a priori mean that a trait is inherited; twin studies have shown that both genetic and environmental factors contribute to insulin resistance, whereas genetic factors are more likely to determine insulin secretion (16). These findings may seem at variance with studies suggesting greater recurrence risk of diabetes in first-degree relatives of nonobese than obese type 2 diabetic patients (35). It should, however, be kept in mind that we did not estimate risk of diabetes in the relatives, only the presence of metabolic abnormalities.
Relatives of diabetic probands with early-onset disease were more insulin resistant and had lower insulin secretion compared with relatives of subjects with late-onset diabetes. Our results agree with similar observations in the Pima Indians, where the offspring of mothers with early-onset diabetes (<35 yr) had a reduced -cell function but unchanged insulin sensitivity (8). These data suggest not only that the probands with early-onset diabetes had a more severe form of diabetes but also that the metabolic abnormalities showed a greater degree of familiality than in relatives of type 2 diabetic patients with late-onset disease. Of importance, this is not due to admixture of patients with MODY and LADA, who were excluded by genetic and antibody testing.
In conclusion, this study clearly reinforces the importance of age at diabetes onset when patients are selected for genetic studies, because the metabolic phenotype of the proband is shared by relatives of probands with early- but not of late-onset diabetes. The study further emphasizes the value of stratifying for a phenotype characterized by abdominal obesity, as this characteristic also shows a high degree of familiality.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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