1 Diabetes Division, Department of Medicine, the University of Texas Health Science Center at San Antonio, San Antonio, Texas
2 Audie L. Murphy Veterans Administration Medical Center, San Antonio, Texas
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ABSTRACT |
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Type 2 diabetes is characterized by defects in both insulin secretion and insulin action (1). Subtle defects in ß-cell function and insulin resistance precede the development of hyperglycemia in individuals at high risk of developing type 2 diabetes, including subjects with a strong family history of type 2 diabetes (25), obese subjects (6,7), individuals with impaired glucose tolerance (IGT) (2,4,5), and women with polycystic ovary syndrome (PCOS) (8,9) or a history of gestational diabetes (10). In these groups hyperglycemia develops as a result of a progressive decline in ß-cell function, a finding reported across multiple ethnic populations (4,1012).
Adipose tissue insulin resistance is believed to play an important role in the development of type 2 diabetes (1,1316). Insulin resistance in adipose tissue is characterized by excessive rates of lipolysis, increased plasma free fatty acid (FFA) levels despite hyperinsulinemia, and impaired suppression of plasma FFA levels by insulin (1,1416). Excessive rates of lipid turnover have been shown to precede the development of type 2 diabetes in subjects with a family history of type 2 diabetes (35) and nondiabetic obese individuals (6,7). The negative effect on glucose homeostasis of an elevation in plasma FFA concentration has been referred to as "lipotoxicity" (13). Elevation in the plasma FFA causes hepatic and skeletal muscle insulin resistance in healthy individuals (1,1418). However, the effect of increased plasma FFA on insulin secretion has been less well studied, and it remains unknown whether a chronic elevation in plasma FFA concentration can impair ß-cell function in subjects genetically predisposed to type 2 diabetes.
Under fasting conditions, mild elevations in plasma FFAs play an important role in sustaining basal insulin secretion and in maintaining a normal insulin secretory response to glucose (16). In contrast, exposure to pharmacological levels of lipids for >2448 h have been shown to impair ß-cell function in vitro (19) and in vivo (2022).
In humans, an acute elevation in plasma FFA either has no effect (23,24) or enhances (25,26) glucose-induced insulin secretion, but the effect of a more prolonged increase in plasma FFA on glucose-stimulated insulin secretion has yielded more variable results. In lean healthy subjects, a 24- to 48-h lipid infusion has been reported to increase (27,28), not significantly change (26), or decrease (25) insulin secretion. In obese insulin-resistant individuals, a 48-h lipid infusion has been reported to reduce insulin secretion by 20%, but plasma insulin concentration was increased because of a
50% reduction in insulin clearance (29). In type 2 diabetes subjects, who already have a severe impairment in ß-cell function, an increase in plasma FFA for 2 days with lipid infusion did not further worsen insulin secretion (29). These conflicting results may be explained, in part, by differences in study populations, plasma FFA levels achieved, variable duration of lipid infusion, or concomitant glucose infusion in some studies (27). At present, it remains unclear whether a prolonged (>48 h) physiological increase in plasma FFA concentration (
500800 µmol/l) impairs insulin secretion in healthy glucose-tolerant subjects, and whether the response to elevated plasma FFA would differ in subjects who are genetically predisposed to develop type 2 diabetes. Of note, a reduction in plasma FFA concentration with the antilipolytic agent acipimox enhanced first-phase insulin secretion in nondiabetic patients with a family history of type 2 diabetes (30).
The aim of the present study was to test the hypothesis that normal glucose-tolerant individuals with a strong family history of type 2 diabetes (genetically predisposed to develop diabetes) might have a defective adaptation to elevated plasma FFA and be more susceptible to lipotoxicity compared with individuals without a family history of type 2 diabetes.
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RESEARCH DESIGN AND METHODS |
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Hyperglycemic clamp.
After an overnight fast, subjects underwent a hyperglycemic clamp on day 3 (31). Briefly, after collection of baseline samples, plasma glucose was acutely raised and maintained (±5%) by 125 mg/dl above baseline for 120 min by periodic adjustment of a 20% dextrose infusion based on the negative feedback principle. Plasma samples were obtained every 2 min from 0 to 10 min (first-phase insulin) and every 5 min from 10 to 120 min (second phase). Subjects voided immediately before and at the end of the study for measurement of urinary glucose loss.
Euglycemic insulin clamp.
At 0730 on day 4, a primed (20 µCi/min x FPG/100) continuous (0.2 µCi/min) infusion of [3-3H]glucose (DuPont-New England Nuclear, Boston, MA) was started and continued until the end of the study. After allowing 120 min for isotopic equilibration, a 2-h euglycemic insulin (40 mU · m-2 · min-1) clamp was performed using a primed-continuous insulin infusion, and the plasma glucose concentration was maintained constant at each subjects fasting level by periodic adjustment of a 20% dextrose infusion as previously described (31). Continuous indirect calorimetry (Deltatrac; Sensormedics, Anaheim, CA) was performed during the last 40 min of the baseline (-40 to 0 min) and insulin clamp (80120 min) periods. Patients were fed at the conclusion of the study and discharged from the hospital.
Analytical determinations.
The plasma glucose concentration was determined by the glucose oxidase method with a Beckman Glucose Analyzer II (Beckman Instruments, Fullerton, CA). Plasma insulin and C-peptide concentrations were determined by radioimmunoassays. The plasma FFA concentration was measured by standard colorimetric methods. Plasma glucose radioactivity was determined on barium hydroxide/zinc sulfate-precipitated plasma extracts.
Calculations.
The C-peptide areas under the curve after breakfast, lunch, and dinner were calculated using the trapezoidal method. During the hyperglycemic and insulin clamp studies, basal (-30 to 0 min) and steady-state (80120 min) plasma glucose, FFA, insulin, and C-peptide represent the mean of values drawn at 10-min intervals. The steady-state glucose infusion rate during the hyperglycemic clamp represents the mean glucose infusion rate from 80 to 120 min, corrected for urinary glucose losses.
Estimation of insulin secretion rates.
Insulin secretion rates (ISRs) were estimated from peripheral plasma C-peptide levels by deconvolution analysis and linear regularization using a two-compartment model with standard parameters for C-peptide kinetics (32). As validated by Van Cauter et al. (32), use of standard parameters for C-peptide clearance and distribution results in ISRs that differ only slightly from those obtained with individual parameters. There is no evidence that differences in plasma FFA levels or lipid infusion affect C-peptide kinetics in humans during acute (2 h) (24) or prolonged (2448 h) lipid infusion (26,27,29). Insulin sensitivity differed significantly between FH+ and control subjects before and after lipid infusion. Diamond et al. (33) and others (10,11,3437) have shown that the ß-cell responds to the presence of insulin resistance with a compensatory increase in insulin secretion. Therefore, to assess ISR between groups with different insulin sensitivities, we related the ISR to the severity of insulin resistance (quantified with the insulin clamp). During the insulin clamp, insulin resistance is the inverse of the glucose disposal rate (Rd), i.e., the lower the Rd, the greater the insulin resistance. Thus, ISR related to peripheral insulin resistance (ISRRd), was expressed as:
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Estimation of endogenous insulin clearance.
Hepatic insulin clearance was calculated by dividing the mean prehepatic ISRs obtained by deconvolution analysis of plasma C-peptide concentration (pmol · min-1 · m-2) by the mean peripheral plasma insulin concentration (pmol · ml-1) during the 20120 min time period of the hyperglycemic clamp (38).
Endogenous glucose production and insulin-stimulated Rd.
During the basal period, the rate of endogenous plasma glucose appearance (which primarily represents hepatic glucose production) (39) equals the rate of plasma glucose disappearance and was calculated by dividing the [3-3H]glucose infusion rate (dpm/min) by the steady-state plasma-tritiated glucose specific activity (dpm/mg) during the last 30 min of tracer equilibration. Because the infusion of insulin results in non-steady-state conditions, the rate of plasma glucose appearance was calculated using Steeles non-steady-state equation (40). During the euglycemic insulin infusion period, endogenous glucose production (EGP) was computed as the difference between the exogenous glucose infusion rate and the isotopically measured rate of plasma glucose appearance. The rate of total-body insulin-mediated glucose disposal was calculated by adding the residual rate of EGP to the rate of exogenous glucose infusion. Net glucose and lipid oxidation rates were calculated from oxygen consumption (VO2) and carbon dioxide production (VCO2) using standard calorimetric equations. Nonoxidative glucose disposal, which primarily represents glycogen synthesis (1), was calculated by subtracting the rate of glucose oxidation (measured by indirect calorimetry) from the rate of total-body glucose disposal. Assessment of lean body mass (LBM) was done by dual-energy X-ray absorptiometry scanning (Hologic Delphi-A; Hologic, Bedford, MA).
Hepatic insulin sensitivity index.
To better describe the effect of an elevation in plasma FFA concentration on hepatic insulin sensitivity, we report EGP in relation to the prevailing fasting plasma insulin (FPI) levels (EGP x FPI), using a validated index of hepatic insulin resistance (41). Under basal conditions, the majority (8590%) of EGP is derived from the liver (39), and there is a linear relationship between the rise in FPI and the decline in EGP over the range of plasma insulin concentrations from
5 to 25 µU/ml (42).
Statistical analysis.
All values represent the mean ± SE. Within-group differences were determined by the paired two-tailed Students t test. Differences between basal and insulin clamp periods and between groups (the FH+ group vs. control subjects) were tested by two-way ANOVA for repeated measures. Normal distribution was checked before all analyses, and nonparametric estimates were used when appropriate. Comparisons were considered statistically significant if the P value was <0.05.
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RESULTS |
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Metabolic profile (48 h): plasma glucose, hormone, and FFA concentrations.
During saline infusion, both groups had similar FPG (93 ± 2 vs. 93 ± 1 mg/dl for control vs. FH+ subjects, respectively; P = NS) and mean 48-h glucose concentrations (95 ± 3 vs. 96 ± 3 mg/dl, P = NS) (Fig. 2). In both groups lipid infusion caused a modest but significant (P < 0.05 vs. baseline, NS between groups) rise in FPG (98 ± 3 vs. 100 ± 2 mg/dl for control vs. FH+ subjects, respectively) and mean 48-h plasma glucose levels (101 ± 3 vs. 103 ± 3 mg/dl).
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The mean fasting C-peptide level increased with lipid infusion in control subjects from 0.8 ± 0.2 to 1.2 ± 0.3 ng/ml but diminished in the FH+ group from 1.5 ± 0.3 to 1.2 ± 0.3 ng/ml (P < 0.05 for the difference from saline infusion between groups). The mean C-peptide concentration during the 48-h metabolic profile (Fig. 2 and 3) increased 28% with lipid infusion in control subjects (1.7 ± 0.4 vs. 2.2 ± 0.6 ng/ml for saline vs. lipid infusion, respectively; P = NS), whereas it decreased by 30% in the FH+ group (2.8 ± 0.6 vs. 2.0 ± 0.4 ng/ml, P < 0.05; P < 0.01 control subjects vs. the FH+ group). In the FH+ group, C-peptide concentration was markedly reduced after each meal (Fig. 3). The deleterious effect of lipid on C-peptide secretion was more pronounced with increasing duration of lipid infusion exposure (Fig. 2). The deleterious effect of lipid infusion on ß-cell function after each meal in the FH+ group is more clearly appreciated by comparing the change with lipid infusion relative to the saline infusion study, as summarized Fig. 3C and for the overall 48-h metabolic profile in Fig. 6.
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Hyperglycemic clamp (day 3) (Figs. 4 and 5).
Fasting plasma FFA concentrations were not significantly different between groups, although they were slightly higher in the FH+ group (saline infusion: 543 ± 54 vs. 651 ± 32 µmol/l for control vs. FH+ subjects, respectively; P = 0.15; lipid infusion: 701 ± 67 vs. 785 ± 57 µmol/l, P = 0.31). During the hyperglycemic clamp studies, the decrease from baseline in plasma FFA concentration was similar in both groups during saline infusion (-461 ± 52 vs. -509 ± 64 µmol/l for control vs. FH+ subjects, respectively; P = NS) and lipid infusion (-380 ± 34 vs. -404 ± 29 µmol/l, P = NS) studies.
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Plasma insulin and C-peptide concentrations and ISR during the hyperglycemic clamp are shown in Figs. 4 and 5. During saline infusion, the incremental first-phase (72 ± 14 vs.
43 ± 6 µU/ml, P < 0.08) and second-phase (
44 ± 4 vs.
33 ± 5 µU/ml, P < 0.13) insulin responses to hyperglycemia tended to be higher in FH+ versus control subjects. During lipid infusion, the incremental first-phase insulin response increased by 63% in control subjects (
43 ± 6 to
70 ± 15 µU/ml, P < 0.01) but decreased by 12% in the FH+ group (
70 ± 17 to
62 ± 14 µU/ml, P < 0.01 control vs. FH+ subjects) (Figs. 4 and 5). Lipid infusion in control subjects enhanced the incremental second-phase insulin response by 50% (
33 ± 5 to
50 ± 8 µU/ml, P < 0.01). In the FH+ group, the incremental second-phase insulin response increased by only 38% (P < 0.05 vs. control subjects). The plasma insulin response to hyperglycemia represents the composite of both insulin secretion and insulin clearance. It is well established that increased plasma FFA levels inhibit hepatic insulin clearance (29,4345). Before lipid infusion, the FH+ group had slightly lower insulin clearance compared with control subjects (0.72 ± 0.09 vs. 1.01 ± 0.13 l · min-1 · m-2, P < 0.11), most likely caused by insulin resistance and chronic hyperinsulinemia (8,35,38,4653). Lipid infusion caused a marked 42% decrease in insulin clearance in the FH+ group (from 0.72 ± 0.09 to 0.42 ± 0.07 l · min-1 · m-2, P < 0.002) but had no effect on insulin clearance in control subjects (1.01 ± 0.13 to 0.96 ± 0.08 l · min-1 · m-2, NS; P < 0.01 vs. the FH+ group). A strong correlation was noted between plasma FFA and insulin concentrations (r = 0.57, P < 0.01) but not between plasma FFA and C-peptide levels (r = 0.23, P > 0.5). If the increase in plasma FFA concentration had not markedly reduced insulin degradation in the FH+ group, the plasma insulin concentrations during the hyperglycemic clamp would have been much lower.
In control subjects, lipid infusion caused a significant increase above baseline in first-phase (010 min) C-peptide concentration compared with saline infusion (2.1 ± 0.3 to
2.6 ± 0.3 ng/ml, P < 0.02) (Fig. 4B and 5B), particularly at 2 min (P < 0.002) and 4 min (P < 0.006) (Fig. 4B). There was also a 17% increase in second-phase (10120 min) insulin response that did not reach statistical significance but that was progressively greater with the duration of the glucose infusion (90 min: P < 0.056; 100 min: P < 0.008; 110 min: P < 0.004; and 120 min: P < 0.002) (Fig. 4B). In marked contrast, lipid infusion in the FH+ group led to a 56% reduction in incremental first-phase C-peptide response to hyperglycemia (
2.7 ± 0.6 to
1.2 ± 0.4 ng/ml, P < 0.02; P < 0.01 vs. control subjects) and a 33% decrease in incremental second-phase C-peptide response to hyperglycemia (
3.6 ± 0.8 to
2.4 ± 0.5 ng/ml, P = 0.06; P < 0.05 vs. control subjects) (Figs. 4 and 5).
In control subjects, the increase above baseline in first-phase ISR with lipid infusion was 75% greater than on saline infusion (512 ± 71 vs.
897 ± 174 pmol/min for saline vs. lipid infusion, respectively; P < 0.01) (Fig. 4C and 5C). A similar trend was observed in second-phase ISR (
287 ± 64 vs.
357 ± 81 pmol/min for saline vs. lipid infusion, respectively; P = 0.16). This contrasted with the marked reduction in the FH+ group during lipid infusion versus saline infusion studies: first-phase ISR was reduced 60% (
818 ± 162 vs.
311 ± 86 pmol/min, P < 0.003) and second-phase ISR decreased by 35% (
327 ± 63 vs.
208 ± 49 pmol/min, P < 0.04) (Fig. 5C). The reduction in second-phase ISR response in the FH+ group compared with the increase in control subjects was most evident toward the last 40 min of the hyperglycemic clamp, during which time ISR increased by 25% in control subjects (P < 0.05 vs. saline infusion), compared with a 50% reduction in the FH+ group (P < 0.01 vs. saline infusion; P < 0.01 between groups). Figure 6 summarizes the striking differences in ß-cell response to lipid infusion between control and FH+ subjects.
Because basal and glucose-stimulated insulin secretion are increased in the presence of insulin resistance (10,11,3337), we related the insulin secretory rate to the severity of the prevailing insulin resistance (ISRRd). As shown in Fig. 7, first-phase ISR (ISRRd 010) was similar in both groups on saline infusion: 37 ± 5 vs. 35 ± 8 pmol/min per (mg · kg LBM-1 · min-1) · 10-2 for control vs. FH+ subjects, respectively; P = NS. However, it diverged significantly in response to an elevation in plasma FFA: control subjects experienced a 48% increase in ISRRd 010 (P < 0.01 vs. saline infusion), whereas it decreased by 63% in the FH+ group: from 37 ± 5 to 54 ± 12 vs. from 35 ± 8 to 13 ± 4 pmol/min per (mg · kg LBM-1 · min-1) · 10-2 for control vs. FH+ subjects, respectively; P < 0.01 vs. saline infusion; P < 0.001 between groups) (Fig. 7A). In control subjects, second-phase ISR (ISRRd 10120) increased in direct proportion to the lipid-induced increase in insulin resistance, and ISRRd 10120 remained constant: 20 ± 5 vs. 22 ± 6 pmol/min per (mg · kg LBM-1 · min-1) · 10-2 for saline vs. lipid infusion, respectively; P = NS. In contrast, lipid infusion in the FH+ group led to a 32% reduction in ISRRd 10120: 14 ± 3 vs. 9 ± 3 pmol/min per (mg · kg LBM-1 · min-1) · 10-2 for saline vs. lipid infusion, respectively (P < 0.05; P < 0.05 between groups) (Fig. 7B).
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During saline infusion, insulin-stimulated glucose disposal (Rd) was reduced by 47% in the FH+ group compared with control subjects (4.3 ± 0.3 vs. 8.1 ± 0.8 mg · kg LBM-1 · min-1, P < 0.0001) (Fig. 8). Lipid infusion decreased Rd by 24% in control subjects (P < 0.001), but it caused no further deterioration in the FH+ group (P < 0.03 vs. control subjects during lipid infusion) (Fig. 8). Basal glucose oxidation was similar between groups during saline infusion, and it was unchanged by lipid infusion. During the insulin clamp, glucose oxidation increased more in control subjects compared with the FH+ group during saline infusion (P < 0.05) (Fig. 8), whereas lipid infusion impaired glucose oxidation similarly in both groups by 15% (P < 0.01 vs. saline infusion) (Fig. 8). Nonoxidative glucose disposal was greatly reduced in response to insulin stimulation in the FH+ group compared with control subjects during saline infusion and lipid infusion studies (both P < 0.01) (Fig. 8).
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DISCUSSION |
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The most striking finding is that, in FH+ subjects, a sustained physiological increase in plasma FFA concentration for 3 days markedly impaired both acute (first-phase) and second-phase insulin secretion by 60 and 35%, respectively. To fully appreciate the severity of FFA-induced impairment in ß-cell function, ISRs were examined in the context of the prevailing insulin resistance. ISR was assessed with the hyperglycemic clamp (day 3) and insulin resistance (inverse of Rd) was assessed with the euglycemic insulin clamp technique (day 4). These studies were performed on separate days because it is not feasible to perform both tests on the same day. It should be noted that the measurement of insulin sensitivity (M/I) from the hyperglycemic clamp correlated very closely (r = 0.90, P < 0.0001) with the measure of insulin sensitivity (Rd) from the euglycemic insulin clamp, indicating that lipid-induced insulin resistance was already established by day 3 and was no different between the hyperglycemic clamp and euglycemic insulin clamp days.
Using the euglycemic and hyperglycemic clamp techniques, we previously have documented a strong positive correlation between the severity of insulin resistance and the increase in insulin secretion in healthy subjects without a family history of type 2 diabetes (33). Using the same techniques, a close relationship between increased insulin secretion and decreased insulin sensitivity has been reported in insulin-resistant subjects genetically predisposed to develop type 2 diabetes (2,3,5) and in insulin-resistant states such as puberty (46,47), obesity (7,47), and PCOS (9). Similar results have been reported by Reaven and colleagues (34,35), who evaluated insulin secretion and insulin resistance with the OGTT and steady-state plasma glucose technique, respectively, as well as by other investigators using the minimal model approach (10,36,37).
In healthy control subjects, when the ISR was related to the prevailing insulin resistance (ISRRd), insulin secretion adapted to FFA-induced insulin resistance with a compensatory increase in first-phase (010 min) and second-phase (10120 min) insulin secretion, and ISRRd either increased (ISRRd 010) or remained constant (ISRRd 10120) (Fig. 7). Note that ISRRd 010 was nearly identical in FH+ and control subjects during saline infusion. However, lipid infusion decreased first-phase ISRRd 010 in the FH+ group to 25% of the control subjects response: 13 vs. 54 pmol/min per (mg · kg LBM-1 · min-1) · 10-2 for FH+ vs. control subjects, respectively; P < 0.001 (Fig. 7). It also reduced second-phase ISRRd 10120 in FH+ to just 42% of that in control subjects: 9 vs. 22 pmol/min per (mg · kg LBM-1 · min-1) · 10-2, P < 0.05 (Fig. 7). It is noteworthy that in control subjects, the 20% decrease in Rd was closely matched by a 20% increase in second-phase insulin secretion, so that the ISRRd 10120 was unchanged. Thus, enhanced ß-cell response in healthy subjects during lipid infusion was tightly coupled with the decrease in insulin sensitivity. This observation is in agreement with recent 48-h lipid infusion studies by Boden et al. (27) and Magnan et al. (28), although this was not seen in the study by Carpentier et al. (26).
McGarry (16) has demonstrated the important role of plasma FFA in the regulation of pancreatic ß-cell function. In the fasting state, FFAs sustain basal insulin secretion and assure efficient nutrient-stimulated insulin secretion when the fast is terminated. Elevated plasma FFAs have been reported to play an important role in maintaining chronic hyperinsulinemia in insulin-resistant obese subjects, and removal of this FFA stimulus by overnight reduction of plasma FFAs with nicotinic acid impairs glucose-induced insulin secretion (48). In normal glucose-tolerant obese individuals (1,14,15), fasting plasma FFA levels are comparable to those achieved during lipid infusion in the control subjects, in whom the induction of insulin resistance was offset by an increase in insulin secretion. These results suggest that elevated plasma FFAs in obesity may represent a compensatory response to augment pancreatic ß-cell function to offset insulin resistance. Our observations in control subjects stand in contrast to those in insulin-resistant FH+ subjects, who already manifested inadequate ß-cell compensation to insulin resistance. During the saline infusion, second-phase ISRRd 10120 was reduced already by 32% compared with control subjects (Fig. 7B), although this difference did not reach statistical significance (P = 0.13). Adipose tissue insulin resistance was evident in FH+ because the plasma FFA level was "normal" (rather than suppressed) in the presence of marked hyperinsulinemia; however, insulin secretion was increased sufficiently to maintain normal glucose tolerance and prevent a further deleterious rise in the plasma FFA concentration. These results suggest that ß-cell lipotoxicity may develop very early in individuals genetically predisposed to develop type 2 diabetes, although one cannot rule out that ß-cell dysfunction was caused by other factors. When the plasma FFA in FH+ subjects was increased by lipid infusion to levels seen in obesity (600 µmol/l), there was a marked reduction in both first- and second-phase insulin secretion (Figs. 4 and 5). When expressed on a percentage basis (60 vs. 35%) (Fig. 6), the reduction in first-phase insulin secretion was approximately twice that of second-phase insulin secretion. These observations are consistent with previous studies demonstrating that first-phase insulin secretion is highly predictive of the development of type 2 diabetes across all populations studied (1,2,4,5,10,11,37). Our results suggest that FH+ subjects may have a unique genetic susceptibility to the deleterious effect of elevated plasma FFAs on insulin secretion.
Lipid infusion caused a 42% decrease in the metabolic clearance rate of insulin in the FH+ group but not in control subjects. A similar finding has been reported by Carpentier et al. (29) in insulin-resistant nondiabetic subjects using the same methodology as in this study, i.e., by dividing the mean prehepatic ISR obtained by deconvolution analysis of plasma C-peptide concentration by the mean peripheral plasma insulin concentration during the hyperglycemic clamp. Reduced hepatic insulin clearance has been previously reported to contribute to the hyperinsulinemia of insulin-resistant subjects who are genetically predisposed to develop type 2 diabetes (35,50), as well as in subjects who are overweight (38,51) and in individuals with IGT (52) or PCOS (8,53).
In FH+ subjects, hyperinsulinemia caused by a reduction of hepatic insulin clearance during lipid infusion was more evident when the portal plasma insulin levels were higher, i.e., in the postprandial periods and during the last hour of the hyperglycemic clamp studies. However, when insulin clearance was estimated during the euglycemic insulin clamps, no change in the metabolic clearance of insulin was observed. Three potential explanations can be offered. First, lipid infusion may have induced an increase in C-peptide clearance and led to an underestimation of the ISR. This is an unlikely explanation because it has been shown that neither acute (24) nor chronic (48 h) (26,27,29) pharmacological increases in plasma FFA concentration by lipid infusion alter C-peptide kinetics or clearance. Furthermore, because the plasma FFA concentration achieved during lipid infusion was similar in FH+ and control subjects, an alteration in C-peptide clearance by increased plasma FFA levels would have underestimated ISRs similarly in both groups and cannot explain the contrasting effect of FFAs on ß-cell function. Second, it is possible that lipid infusion impaired first-pass hepatic insulin clearance of secreted insulin but did not alter the insulin levels during exogenous insulin administration (euglycemic insulin clamp), which measures the posthepatic systemic insulin clearance. Insulin clearance is mediated through receptor-mediated internalization, primarily by the liver (49), but also by the kidney and muscle, which, in contrast to the liver, are not known to be affected by elevated plasma FFA. Posthepatic estimation of insulin clearance by the euglycemic insulin clamp is more likely to reflect insulin clearance by extra-hepatic tissues and is less likely to provide information on first-pass hepatic insulin extraction, in particular at the lower plasma insulin levels observed during the euglycemic insulin clamp compared with the hyperglycemic clamp.
A third explanation for a modest increase in plasma insulin despite a marked reduction in insulin secretion with lipid in FH+ subjects could be the combined effect of a dose-dependent impairment of hepatic insulin clearance mediated by elevated plasma FFAs, in the setting of chronic hyperinsulinemia and very high portal insulin levels after meals or glucose infusion. Exposure to elevated FFAs in vitro (43,54) and in vivo (28,44,45) reduces insulin clearance in a dose-dependent manner with maximal inhibition (4050% reduction) within the physiological range. Moreover, insulin clearance in FH+ subjects was already 29% lower during saline control studies, although it did not reach statistical significance (P = 0.11). Thus, one could speculate that in insulin-resistant FH+ subjects, chronic exposure to increased plasma insulin levels would saturate, or nearly saturate, insulin uptake and degradation by hepatocytes. An additional insult, either directly from FFA-induced inhibition of hepatic insulin degradation and/or indirectly through FFA-induced insulin resistance with a further stimulation of hyperinsulinemia, would exceed the livers ability to clear insulin, leading to an "escape" of insulin toward peripheral tissues.
Because prehepatic insulin levels are usually two- to threefold higher than peripheral plasma insulin concentrations (49), we estimated that during lipid infusion, portal insulin levels in FH+ subjects were 150250 µU/ml during the last hour of the hyperglycemic clamp (measured peripheral plasma insulin levels were
8090 µU/ml during this period) (Fig. 4). This is very different than during the euglycemic insulin clamp, when the portal plasma insulin concentration closely approximates the peripheral insulin levels (5054 µU/ml) because endogenous insulin secretion is inhibited. Thus, during the euglycemic insulin clamp, one would not expect an inhibition of insulin clearance by a modest increase in the plasma FFA concentration, as in our study, because the portal insulin concentration would be well below the level for insulin clearance saturation.
Under postabsorptive conditions, the liver of FH+ individuals demonstrated marked insulin resistance, as evidenced by a 73% increase in the hepatic insulin resistance index (EGP x FPI) (19 ± 2 vs. 11 ± 2 mg · kg LBM-1 · min-1 x µU/ml, P < 0.04). This observation is consistent with previous reports (1,1416). No previous study has examined the effect of a chronic physiological increase in plasma FFAs on EGP in humans. In lean healthy volunteers, an acute pharmacological elevation in plasma FFAs (1,000 µmol/l) causes hepatic insulin resistance and inhibits insulin-mediated suppression of EGP (17,18,55,56). In the present study, 4 days of lipid infusion in the FH+ group increased the hepatic insulin resistance index by 2.3-fold (19 ± 2 vs. 44 ± 2 mg · kg LBM-1 · min-1 x µU/ml, P < 0.001 vs. saline infusion; P < 0.004 vs. control subjects). The worsening hepatic resistance, combined with ß-cell lipotoxicity, resulted in portal insulin levels that were inadequate to prevent a rise in basal EGP (from 2.4 to 2.9 mg · kg LBM-1 · min-1, P < 0.001). In control subjects, lipid infusion did not significantly increase the hepatic insulin resistance index, and a small increase in the FPI from 4 to 6 µU/ml was sufficient to keep EGP from rising. Taken together, these findings indicate that the liver of FH+ individuals is more susceptible to the "lipotoxic" effect of elevated plasma FFAs compared with control subjects. Consistent with this conclusion, the suppression of EGP during the insulin clamp was significantly impaired by lipid infusion in FH+ individuals but unchanged in control subjects.
In agreement with previous studies from our laboratory (3,57) and others (2,4,5), FH+ individuals were insulin resistant compared with matched control subjects without a family history of diabetes. The 47% reduction in insulin-mediated Rd was accounted for primarily by decreased nonoxidative glucose disposal (glycogen synthesis) and, to a lesser extent, by impaired glucose oxidation. After lipid infusion, Rd was reduced by 24% (P < 0.01) in control subjects but not further decreased in the FH+ group (Fig. 8). These results can be interpreted in one of two ways: 1) the "lipotoxic" effect of elevated plasma FFA on peripheral tissues is fully established in normal glucose-tolerant FH+ subjects, so a further elevation in plasma FFA by lipid infusion causes no further reduction in Rd, or 2) peripheral insulin resistance in FH+ is unrelated to "lipotoxicity" but is near maximally established in healthy FH+ subjects, thus lipid infusion cannot further impair insulin-stimulated glucose disposal. Whatever the explanation, the lack of worsening in peripheral insulin resistance by lipid infusion indicates that the deterioration in insulin secretion with lipid infusion resulted from a deleterious effect of elevated plasma FFAs on insulin secretion, and not from worsening insulin resistance and increased ß-cell demand. Chronic exposure to elevated plasma FFA may impair the conversion of proinsulin to insulin (22) and disrupt the physiological glucose-fatty acid cross-talk by altering ß-cell gene expression and signaling pathways (16,58).
The FFA-induced increase in hepatic insulin resistance and impairment in ß-cell function contrasts with the lack of worsening of insulin resistance in skeletal muscle. This may be the result of different thresholds to the lipotoxic effect of plasma FFA on these target tissues. Thus, muscle might be particularly susceptible to even a modest chronic increase in plasma FFA concentration, whereas liver and ß-cells have better adaptive mechanisms and might be less sensitive. It also is possible that mechanisms unrelated to increased FFA availability are responsible for muscle insulin resistance in FH+ individuals. If so, lipotoxicity could play a key role to induce hepatic insulin resistance and increase basal EGP and to impair insulin secretion in individuals genetically predisposed to develop type 2 diabetes, but it could be of less pathophysiological importance in inducing peripheral insulin resistance in FH+ subjects.
In the present study, we infused Liposyn III, composed largely of unsaturated long-chain fatty acids (55% linoleate, 22% oleate, 11% palmitate, and 4% stearate), which is different from the higher saturated fatty acid composition of plasma (11% linoleate, 38% oleate, 28% palmitate, and 12% stearate). One study in rats (59) indicated that the insulinotropic effect of FFAs increases with chain length and degree of saturation, with the saturated fatty acids palmitate and stearate being more potent to stimulate insulin secretion. However, discrepant results regarding the insulinotropic effects of various fatty acids have been reported by others (60,61). In the only study in humans, Stefan et al. (62) reported no difference in either first- or second-phase insulin secretion after 24-h exposure to lipids containing various proportions of saturated versus unsaturated fatty acids.
In conclusion, we have demonstrated for the first time the deleterious effect of a sustained increase of plasma FFA concentration on insulin secretion in nondiabetic subjects who are genetically predisposed to develop type 2 diabetes. In addition, lipid infusion caused a mild increase in basal EGP and impaired the suppression of EGP by insulin, changes characteristic of the early stages of type 2 diabetes. In contrast, healthy control subjects adapted to FFA-induced insulin resistance by mounting an adequate compensatory ß-cell response. We hypothesize that chronically elevated plasma FFA concentrations may contribute to progressive ß-cell failure in at least some individuals who are genetically predisposed to develop type 2 diabetes. This hypothesis emphasizes the important role of adipose tissue insulin resistance in the natural history of progressive ß-cell failure leading to type 2 diabetes. From a therapeutic perspective, future interventions to prevent the development of type 2 diabetes may target insulin resistance in adipose tissue in individuals genetically predisposed to develop type 2 diabetes.
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ACKNOWLEDGMENTS |
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We would like to thank all volunteers and the invaluable efforts of the General Clinical Research Center nursing staff, Celia Darland (research dietitian), and associated personnel. We also thank the skilled nursing assistance of James King, John Kincade, Norma Diaz, and Tricia Wolff, who performed the metabolic studies. We are grateful to Kathy Camp, Sheila Taylor, and Cindy Muñoz for their skilled laboratory work. We are indebted to Shuko Lee, MS, for her expert statistical assistance. Elva Gonzales and Lorrie Albarado contributed with outstanding secretarial support.
Address correspondence and reprint requests to Kenneth Cusi, University of Texas H.S.C. at San Antonio, Diabetes Division, Room 3.380S, 7703 Floyd Curl Dr., San Antonio, TX 78284-3900. E-mail: cusi{at}uthscsa.edu
Received for publication November 27, 2002 and accepted in revised form July 9, 2003
EGP, endogenous glucose production; FFA, free fatty acid; FPG, fasting plasma glucose; FPI, fasting plasma insulin; IGT, impaired glucose tolerance; ISR, insulin secretion rate; LBM, lean body mass; PCOS, polycystic ovary syndrome; Rd, glucose disposal rate
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