Subjects were islet cell antibody–negative women who participated in a longitudinal study of the pathogenesis of type 2 diabetes following GDM. Selection of the original cohort has been described in detail (6
). Briefly, all Latino women referred to Los Angeles County Women's Hospital for management of GDM between August 1993 and March 1995 were asked to participate if they met all of the following criteria: 1
) gestational age between 28 and 34 weeks, 2
) no current or prior insulin therapy, 3
) all fasting serum glucose concentrations <130 mg/dl (7.2 mmol/l) during pregnancy, 4
) otherwise uncomplicated singleton pregnancy, and 5
) both parents and at least three of four grandparents were from Mexico, Guatemala, or El Salvador. All women had detailed metabolic testing during the third trimester (6
). They were asked to return for a 75-g oral glucose tolerance test (oGTT) 6 months postpartum and then for an oGTT, intravenous glucose tolerance test (ivGTT), and glucose clamp at ~15 months postpartum. oGTTs and ivGTTs were scheduled every 15 months thereafter. Height, weight, and information on contraceptive use and pregnancies were collected at each visit. Bioelectrical impedance was measured at each oGTT visit to assess body composition. At the time of diagnosis of impaired glucose tolerance or diabetes, subjects met with a dietitian and received advice on nutrition and daily walking. Subjects remained in follow-up until they withdrew consent, were lost to follow-up, developed a fasting plasma glucose concentration >140 mg/dl, or reached the final scheduled study visit 12 years postpartum. Women who were pregnant at the time of a scheduled battery of tests were studied at least 4 months after pregnancy and at least 1 month after completion of breastfeeding.
All subjects gave written, informed consent for participation in the study, which was approved by the institutional review board of the University of Southern California and the Los Angeles County and the University of Southern California Medical Center.
End point for present analysis.
For the present report, which is focused on physiological changes associated with development of diabetes from the first postpartum visit onward, we analyzed data from all subjects who 1) had baseline oGTT, ivGTT, glucose clamp, and body composition studies without diabetes within 30 months postpartum and 2) returned for at least one additional oGTT to determine diabetes status. Follow-up data were used up to earlier of either the diagnosis of diabetes by American Diabetes Association criteria (fasting ≥126 mg/dl or 2 h ≥200 mg/dl) or the last visit without diabetes.
For the baseline battery of oGTT, ivGTT, glucose clamp, and body composition, subjects came to the general clinical research center on 3 separate days, at least 48 h apart, after 8–12 h overnight fasts and at least 3 days on an unrestricted diet. The order of ivGTTs and clamps was alternated among individuals.
On one day, bioelectrical impedance (BIA) was measured immediately prior to an oGTT. For BIA, subjects lay supine while plastic electrodes were placed on their right hand and foot and a trained technician took dual resistance and reactance readings with a Quantom Impedance Meter (RJL Systems, Clinton Township, MI). For oGTTs, subjects drank 75 g of dextrose. Blood was obtained from an antecubital venous catheter before and 15, 30, 60, 90, 120, and 180 min after the glucose ingestion, placed on ice, and plasma was separated within 20 min and stored at −80°C.
On a separate day, an ivGTT was performed starting between 0700 and 1000 h. Dextrose (300 mg/kg) was injected over 1 min, followed in 20 min by a 5-min infusion of crystalline human insulin (0.03 units/kg). Arterialized venous blood was drawn into iced tubes before (n = 2) and for 240 min after (n = 32) the dextrose injection. Plasma was separated within 20 min and stored at −80°C.
On a third day, a glucose clamp was performed starting between 0600 and 0630 h. A primed (0.035 mmol/kg body wt), continuous (2.5 × 10−4 mmol/min/kg) infusion of 6,6 2H2
d-glucose (“tracer”) was administered through an antecubital vein for 360 min. A nonprimed infusion of crystalline human insulin (40 mU/min per m2 body surface area) was administered during the final 180 min of the tracer infusion. Dextrose (20% wt/vol in water), containing dideutero-glucose (0.021 mmol/cc) to minimize changes in plasma tracer enrichment, was given to maintain arterialized venous plasma glucose concentrations at ~88 mg/dl during the insulin infusion. Blood samples for measurement of tracer, hormone/ and metabolite concentrations were drawn into ice-cold tubes at −90, −50, −30, −10, 30, 60, 90, 120, 160, and 180 min relative to the start of the insulin infusion. Plasma was separated within 20 min and stored at −80°C.
Glucose was measured by glucose oxidase (Beckman Glucose Analyzer II; Beckman, Brea, CA). Insulin was measured by a radioimmunoassay (Novo Pharmaceuticals, Danbury, CT) that measured insulin and proinsulin. Plasma free fatty acids (FFAs) were measured by an enzymatic colorimetric method (WAKO Chemicals, Richmond, VA). Plasma adiponectin and leptin levels were measured using radioimmunoassay kits from Linco Research. Plasma C-reactive protein (CRP) and interleukin (IL)-6 were measured using CRP enzyme-linked immunosorbent assays and ultrasensitive IL-6 enzyme-linked immunosorbent assays kits from ALPCO Diagnostics. 6,6, 2H2-glucose concentrations in infusates and perchloroacetate (PCA) supernatants of plasma were measured by gas chromatography and mass spectrometry after conversion of glucose to its aldonitrile penta-acetate derivative. Anti–pancreatic islet cell antibodies in plasma were measured in the laboratory of Dr. Jerry Palmer by an indirect immunofluorescence assay using human pancreas.
BMI was calculated as weight in kilograms divided by the square of height in meters. Diabetes was diagnosed by oGTTs using the American Diabetes Association criteria (7
). ivGTT results were analyzed using the MINMOD program (8
) to obtain measures of fractional glucose disappearance due to an increase in insulin above basal (insulin sensitivity; SI
). The acute insulin response to intravenous glucose (AIRg
) was calculated by the trapezoid rule as the incremental area under the insulin curve during the first 10 min after the glucose injection. The product of SI
(the disposition index; DI
) was calculated as a measure of acute pancreatic β-cell compensation for insulin resistance (8
). Body fat and fat-free mass were calculated by the formula of Kotler et al. (9
) using height, weight, and bioelectrical impedance analysis measurements.
Follow-up data were used up to the diagnosis of diabetes or the last visit without diabetes, whichever occurred first. Cumulative diabetes incidence rates were estimated using life-table methodology and displayed using Kaplan-Meier plots. Cox proportional hazard regression was used to test for associations between baseline and/or follow-up variables and time to diabetes development. Baseline variables were treated as fixed covariates and follow-up variables were treated as time-dependent covariates. The baseline measures included all variables listed in . Follow-up measures included changes from baseline in body weight and fat and disposition index, the presence or absence of additional pregnancies, and use of hormonal contraception. The proportional hazard assumption was evaluated by testing for interaction between the covariate and time in the model; no significant violation was found for the tested variables.
All statistical tests were two-sided, and statistical significance was defined as P ≤ 0.05.