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Metabolism Unit (E.F., A.G., M.P.), National Research Council Institute of Clinical Physiology and Department of Internal Medicine, University of Pisa School of Medicine, Pisa 56100, Italy; and Diabetes Division (E.F., A.G., M.M., Y.M., L.G., R.A.D.), University of Texas Health Sciences Center at San Antonio, San Antonio, Texas 78229
Address all correspondence and requests for reprints to: E. Ferrannini, M.D., National Research Council Institute of Clinical Physiology, Via Savi, 8, 56100 Pisa, Italy. E-mail: ferranni{at}ifc.cnr.it.
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Abstract |
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We conclude that the primary physiological target of the propensity to diabetes of Mexican-Americans is insulin resistance of glucose uptake.
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Introduction |
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Although in several studies the presence of diabetes in one or more first-degree relatives was associated with some degree of insulin resistance in the offspring (10, 11), the influence of familial diabetes (FHD) on insulin resistance is still somewhat controversial (12, 13). In any event, FHD is a significant confounder in interethnic comparisons of insulin sensitivity, particularly in ethnic groups such as Mexican-Americans in whom the prevalence of FHD is increased. Finally, the impact of either ethnicity or FHD on endogenous glucose production has not been previously analyzed. The present study was therefore undertaken to compare insulin sensitivity (by the euglycemic clamp technique), endogenous glucose production, and the relationship between insulin resistance and insulin secretion in diabetic and nondiabetic Mexican-Americans and Caucasian subjects living in the same area. Sufficient observations were collected to allow a separate analysis of FHD while taking adequate account of relevant confounders such as sex, age, and obesity.
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Subjects and Methods |
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The study group included 232 subjects (172 Mexican-Americans and 60 Caucasians) recruited at the Clinical Research Center of the University of Texas Health Sciences Center at San Antonio, TX, through advertising within the Medical Center and in local newspapers. Subjects responding to the advertisement were screened by a 75-g oral glucose tolerance test (OGTT), and were invited to participate in the clamp study and to refer their relatives to the Clinical Research Center. The studies were conducted over a period of 5 yr, during which nondiabetic and diabetic subjects, Mexican-American or Caucasian, were studied in no particular order. The diagnosis of diabetes was based on the OGTT according to the criteria of the American Diabetes Association (14). History of diabetes in first-degree relatives (FHD) was obtained from all subjects, and categorized as positive if one or more family members were reported diabetic. The clinical and metabolic characteristics of the study group are given in Table 1
. None of the diabetic patients was treated with insulin, metformin, or troglitazone. Other than sulfonylureas, patients were not taking any other drugs known to affect glucose tolerance; the proportion of Caucasians and Mexican-American on sulfonylureas was 17% and 15%, respectively (P = ns by 
2), and sulfonylureas were withdrawn 2 d before the metabolic studies. The study protocol was approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio, and informed written consent was obtained from each subject before participation.
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Height was measured to the nearest centimeter, and weight to the nearest 0.5 kg. The waist-to-hip circumference ratio (WHR) was determined by measuring the waist circumference at the narrowest part of the torso, and the hip circumference in a horizontal plane at the level of the maximal extension of the buttocks. Fat-free mass (FFM) was measured using the tritiated water technique (in 41 subjects), underwater weighing (in 75 subjects) or by electrical bioimpedance (in 75 subjects); the equivalence of these methods has been documented (15). In the remaining 41 subjects, FFM was estimated using Hume’s formula in its gender-specific version, which has previously been shown to have comparable accuracy as the direct methods (16). In the present series of subjects, the values of FFM calculated by Hume’s formula were strongly correlated with the corresponding values directly measured by each of the three direct methods (with r values of 0.7–0.9). For uniformity purposes, Hume’s FFM was calculated and used for the whole group. For the OGTT, subjects were fasted overnight, and blood samples were collected at -30, -15, 0, 30, 60, 90, and 120 min for the measurement of plasma glucose, free fatty acids (FFA), and insulin concentrations. For the euglycemic insulin clamp, subjects were admitted to the Clinical Research Center at 0700 h, after an overnight fast. A polyethylene cannula was inserted into an antecubital vein for the infusion of all test substances. A second catheter was inserted retrogradely into an ipsilateral wrist vein on the dorsum of the hand for blood sampling and the hand was kept in a heated box at 65 C. A primed (20 µCi)-continuous (0.20 µCi/min) infusion of 3-[3H]glucose (NEN Life Science Products, Boston, MA) was administered for 180 min in diabetic patients and for 120 min in control and IGT subjects. In diabetic subjects, the prime was adjusted to the elevation of fasting plasma glucose (FPG) as follows: 20 µCi·FPG/90 mg/dl. During the last 30 min of the basal equilibration period, plasma samples were taken at 5- to 10-min intervals for the determination of plasma glucose, FFA, and insulin concentration, and tritiated glucose specific activity. After the basal equilibration period, insulin was administered as a primed-continuous infusion at the rate of 40 mU·min-1·m-2 for 120 min. The plasma glucose concentration was measured every 5 min after the start of the insulin infusion, and a variable infusion of 20% glucose was adjusted based on the negative feedback principle to maintain the plasma glucose level at 90–100 mg/dl with a coefficient of variation less than 5%. After the start of insulin in the diabetic subjects, the plasma glucose concentrations was allowed to decrease to approximately 100 mg/dl, at which level it was then maintained. Plasma samples were collected every 15 min from 0–90 min and every 5–10 min from 90–120 min for the determination of plasma glucose, FFA, and insulin concentration, and tritiated glucose specific activity (13).
Analytical techniques
Plasma glucose was measured by the glucose oxidase reaction (Glucose Analyzer, Beckman, Fullerton, CA). Plasma insulin concentrations were measured by RIA using a specific kit (Linco Research, Inc., St. Louis, MO). Plasma FFA were assayed spectrophotometrically. Plasma 3-[3H]glucose levels were measured in Somogyi precipitates as previously described (13).
Data analysis
Insulin-mediated glucose uptake (M value) was expressed per FFM because this normalization has been shown to minimize differences due to sex, obesity, and age (17). During the baseline period, both the plasma glucose concentrations and plasma 3-[3H]glucose specific activity were stable during the last 30 min of tracer infusion in all subjects. Therefore, total endogenous glucose production (EGP) was calculated as the ratio of the 3-[3H]glucose infusion rate to the plasma 3-[3H]glucose specific activity (mean of four to five determinations). During the insulin clamp, total glucose rates of appearance and disappearance were calculated using Steele’s equation. EGP was then obtained as the difference between total glucose rate of appearance and the exogenous glucose infusion rate. To correct for small interindividual differences in steady-state plasma glucose and insulin concentrations, we calculated insulin sensitivity of glucose uptake (ISGU) as the insulin-mediated glucose clearance (= the ratio of total glucose rate of disappearance to steady-state plasma glucose concentrations during the last 40 min of the clamp) normalized at a plasma insulin concentration of 75 µU/ml (i.e. the average steady-state plasma insulin concentration for the whole group). Glucose and insulin areas under the OGTT curve were calculated by the trapezium rule. Because fasting insulin concentration is a strong inhibitory stimulus for EGP (18), and fasting hyperinsulinemia may therefore mask hepatic insulin resistance, an index of the insulin resistance of EGP (IRGP) was calculated as the product of fasting EGP and fasting plasma insulin (FPI) concentration. According to the same rationale, an index of the resistance of lipolysis to insulin inhibition (IRLL) was calculated as the product of fasting FFA (normalized by fat mass to account for individual differences in fat stores) and FPI concentration.
Data are given as the mean ± SE. A preliminary analysis of the separate influence of ethnicity and diabetes on metabolic variables was performed by two-way ANOVA. Assessment of the contribution of multiple factors to the measured variables was carried out by multivariate analysis by using mixed models with both continuous [e.g. age and body mass index (BMI)] and categorical variables (e.g. sex, FHD, diabetes status, and ethnicity); in these models, categorical differences were tested as contrasts.
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Results |
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). Independently of diabetes status, Mexican-Americans were more obese (BMI and percent fat mass) and had a stronger FHD than Caucasians. Response to oral glucose
Figure 1 plots the plasma glucose, insulin, and FFA responses to oral glucose in the NGT and diabetic groups, whereas the corresponding statistical analysis is reported in Table 2. In comparison with NGT, diabetes was characterized by higher fasting plasma glucose and insulin concentrations, a reduced total (as the area-under-curve) and early (as the insulin/glucose ratio at 30 min) plasma insulin response, and a less suppressed plasma FFA response (in Mexican-Americans only). Both FPI levels and the insulin/glucose ratio at 30' were significantly higher in Mexican-Americans than in Caucasians. BMI was a strong positive correlate of both insulin area and the insulin/glucose ratio at 30' (P < 0.0001 for both); when simultaneously accounting for sex, age, BMI, and diabetic status, neither ethnicity nor FHD had any independent influence on these indices of insulin release (Table 3).
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Both M value and ISGU were reduced by approximately 50% on average in diabetics vs. NGT, and both were significantly lower in Mexican-Americans than in Caucasians (Table 2). For M, the interethnic difference was larger in NGT (24%) than in diabetic subjects (13%). In a multiple regression model including family history of diabetes (in addition to sex, age, and BMI) (Table 3), the significant independent correlates of ISGU were age, BMI, diabetes status, and ethnicity, which together explained 48% of the observed variance of ISGU. In particular, the model predicted that the strongest independent effect on ISGU was that of diabetes, which was associated with an average decrement in ISGU of 3.1 ± 0.5 ml·min-1·kgffm-1 (mean ± SE regression coefficient); Mexican-American ethnicity was independently associated with a further decrement of 1.4 ± 0.6 ml·min-1·kgffm-1.
In both NGT and diabetics, ISGU was inversely related to the insulin area as well as the insulin/glucose ratio at 30' (Fig. 2). In each group, this relationship could be described by a power function. In the diabetic groups, the functions were shifted downward relative to the NGT groups (P < 0.0001), indicating that at any given level of insulin sensitivity the insulin responses to glucose, both the total and early one, were markedly depressed in diabetics despite their higher BMI. The slopes of the relationships, however, were not significantly different between NGT and diabetics; this indicates that, within each group a more severe degree of insulin resistance was associated with a higher insulin response to oral glucose. Moreover, in both NGT and diabetic subjects, the slopes were similar in Mexican-Americans and Caucasians, suggesting an equivalent degree of compensatory hyperinsulinemia (early-phase and total) in both ethnic groups.
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Fasting EGP was higher in diabetics than NGT (P = 0.003) and slightly lower in Mexican-Americans than in Caucasians (P = 0.02) (Table 2). In the whole dataset, EGP was positively related to FPG (r = 0.34, P < 0.0001) as well as to FPI concentrations (r = 0.16, P = 0.01). IRGP was still markedly elevated in diabetics vs. NGT (indicating hepatic insulin resistance), but no longer different between Mexican-Americans and Caucasians, suggesting that the lower EGP values in the former were due to their higher FPI concentrations. In a multivariate model accounting for sex, age, ethnicity, BMI, FHD, and diabetes status, the male sex, BMI, and diabetes status were significant independent correlates of IRGP, together accounting for 36% of its observed variability (Table 3). During the clamp, suppression of EGP was 70 ± 4% in NGT and 51 ± 4% in diabetic subjects (P = 0.01 for the effect of diabetes, with no difference between Mexican-Americans and Caucasians). IRGP and ISGU were reciprocally related to one another, with controls and diabetics falling on the same regression line (Fig. 3), indicating that insulin resistance in peripheral tissues is coupled with insulin resistance in the liver.
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In a complete regression model, ISGU, IRGP, IRLL, and the insulin/glucose ratio at 30' were independent determinants of the glucose area (Table 4), accounting for 64% of its observed variability (sex, age, BMI, ethnicity, and FHD being no longer significant correlates).
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Discussion |
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Other physiological determinants of glucose homeostasis were not affected by ethnicity or FHD. Thus, neither the insulin response to oral glucose (insulin area) nor the early insulin release (insulin/glucose ratio at 30') differed significantly between Mexican-Americans and Caucasians—or between individuals with or without diabetes in their family—when accounting for obesity, i.e. the strongest determinant of these insulin secretory indices. Moreover, when the ability of the ß-cell to cope with the prevailing degree of insulin resistance was analyzed by regressing the insulin area or the insulin/glucose ratio at 30' on insulin sensitivity (19), there was no evidence that in Mexican-Americans compensatory hyperinsulinemia was any different from that of Caucasians, nor was there any detectable change in slope according to presence of FHD. Even among diabetic patients, more insulin-resistant subjects had relatively higher insulin responses (early and total) despite the fact that both their insulin sensitivity and insulin responses were severely impaired in comparison with NGT subjects (Fig. 2). Thus, our analysis, while clearly bringing out the adaptive coupling of insulin release to insulin action in both NGT and diabetes, does not show a direct, independent influence of either ethnicity or FHD on the insulin response to oral glucose (early or total).
Fasting EGP was elevated in diabetics vs. NGT but was significantly lower in Mexican-Americans compared with Caucasians (Table 2). In the diabetic groups, FPI concentrations were increased by 50% compared with NGT subjects. We have previously shown that such an increase would per se reduce EGP detectably in normal subjects (19). Therefore, to account for the tonic inhibition of fasting EGP by fasting insulin levels, we used an index of insulin resistance of glucose production, i.e. the product of EGP and fasting insulin. This index was still markedly higher in diabetic subjects than NGT but no longer differed by ethnicity. Thus, IRGP accompanied ISGU across diabetes status; the two defects were similar in degree (Fig. 3), and BMI was a common deteriorating circumstance (Table 3). However, both in univariate and multivariate analysis, neither ethnicity nor FHD had any influence on IRGP. Likewise, when assessing insulin resistance of lipolysis (through IRLL as well as the extent of FFA suppression during euglycemic hyperinsulinemia), there was a clear impairment in this action of insulin in diabetics as compared with NGT, but no independent influence of ethnicity or FHD was evident in the data. These findings support the conclusion that the primary physiological target of the propensity to diabetes of Mexican-Americans is insulin resistance of glucose uptake in peripheral (muscle) tissues. In contrast, in this ethnic group insulin sensitivity of glucose production and insulin secretion (both in amount and time dynamics) only show adaptive changes, i.e. secondary to the insulin resistance and obesity. Although our study cohort was not a population-based sample, the frequency of FHD was higher among Mexican-Americans. This may partially reflect sampling from greater family size. In addition, the higher rate of insulin resistance in previous generations of Mexican-Americans may be responsible for their greater family history.
With regard to obesity, it should be mentioned that in the present dataset fat distribution, estimated as the WHR, had little additional impact on the metabolic variables whenever BMI was included in the analysis. On the other hand, plentiful evidence shows that central fat accumulation has a negative impact on insulin sensitivity and glucose tolerance that is independent of overall fatness. In our own laboratory, a case-control study comparing Mexican-American with Caucasian women with NGT found no difference in insulin sensitivity when subjects were matched for visceral fat mass (measured by magnetic resonance imaging) (20). Thus, WHR, if a useful indicator of fat distribution in epidemiological surveys (6), is a rather poor correlate of visceral fat mass (21). Furthermore, we have previously shown that WHR increases linearly with BMI in the low range (below 
28 kg/m2) and levels off in the obese range (22). This suggests that fat distribution carries more impact in lean individuals than it does in obese subjects. The series of subjects here reported had a mean BMI of 31 kg/m2 (only 13% of subjects had a BMI 
25 kg/m2), and this may have overshadowed any independent effect of WHR.
Of interest is the fact that IRGP, despite being an estimate of insulin inhibition of fasting glucose output, was as strongly related to oral glucose tolerance (as the glucose area) as was ISGU (Table 4); this presumably reflects the circumstance that, during an OGTT, the residual rate of EGP is closely related to the basal rate of EGP, in nondiabetic as well as diabetic subjects (23, 24). Thus, ISGP may also reflect the contribution of residual EGP to the glucose excursion following oral glucose.
Finally, in the complete multivariate model (Table 4), ISGU, IRGP, IRLL, and early insulin response were each an independent correlate of glucose area, together explaining two thirds of its observed variability. Thus, only the indices of insulin action and early release were independent predictors of glucose tolerance, whereas anthropometric characteristics (sex, age, BMI) and ethno-familial traits had little residual effect. Therefore, from the present analysis we can conclude that ethnicity (Mexican-American vs. Caucasian) is an intermediate phenotype, which exerts its influence on glucose tolerance through insulin resistance of glucose uptake. From the physiological standpoint, however, this conclusion is itself partial. The data in fact admit the more general conclusion, that tolerance to oral glucose is quantitatively explained by a metabolic phenotype, consisting of a mix of inefficient disposition and excessive production of glucose, unrestrained lipolysis, and a deficient early insulin response to glucose. Stated otherwise, insulin actions and insulin release alone appear to underlie or mediate much of the influence that constitutive (sex, ethnicity, FHD) or acquired (age and obesity) factors exert on glucose tolerance in humans.
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Acknowledgments |
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Footnotes |
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Abbreviations: BMI, Body mass index; DM, type 2 diabetes (diabetes mellitus); EGP, endogenous glucose production; FFA, free fatty acids; FFM, fat-free mass; FHD, familial diabetes; FPI, fasting plasma insulin; IRGP, insulin resistance of EGP (EGP x FPI); IRLL, insulin resistance of lipolysis (fasting FFA-kgfm x FPI); ISGU, insulin sensitivity of glucose uptake; M value, insulin-mediated glucose uptake; NGT, normal glucose tolerance; OGTT, oral glucose tolerance test; WHR, waist-to-hip circumference ratio.
Received November 26, 2002.
Accepted March 12, 2003.
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