Mechanisms for the Deterioration in Glucose Tolerance ... - Diabetes

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TABLE 1 Antiviral therapy before and after starting protease inhibitors Subject Pre-PI treatment Post-PI treatment Age Sex Race 1 Nevirapine, stavudine, lamivudine Stavudine, lamivudine, nelfinavir 53 M AA 2 Efavirenz, abacavir, stavudine Lamivudine, zidovudine, nelfinavir 35 F C 3 Nevirapine, stavudine, lamivudine Lamivudine, stavudine, nelfinavir 46 F H 4 Nevirapine, stavudine, lamivudine Stavudine, lamivudine, indinavir, ritonavir 47 F AA 5 Efavirenz, abacavir, stavudine Abacavir, stavudine, saquinavir 38 F AA 6 Stavudine, lamivudine, nevirapine Stavudine, lamivudine, saquinavir, ritonavir 31 M AA 7 Stavudine, lamivudine, efavirenz Stavudine, lamivudine, nelfinavir 40 F C 8 Nevirapine, stavudine, lamivudine Stavudine, lamivudine, nelfinavir 50 M C 9 None Stavudine, lamivudine, nelfinavir 47 M AA 10 None Stavudine, lamivudine, nelfinavir 46 M H 11 None Stavudine, lamivudine, nelfinavir 32 F AA 12 None Zidovudine, lamivudine, nelfinavir 39 M AA 13 Normal volunteers None Lopinavir, efavirenz, lamivudine, ritonavir 38 F C (N 14) — — 42 3 6F/8M 3AA/10C/1H Data are means SD unless otherwise indicated. AA, African-American; C, Caucasian; H, Hispanic; PI, protease inhibitor. initiation of protease inhibitor treatment. All subjects had normal routine laboratory screening results including normal liver function tests and urinalysis. None of the subjects had a family history of diabetes. Normal glucose tolerance had been ascertained by fasting plasma glucose levels 6 mmol/l and plasma glucose levels 7.8 mmol/l 2 h after a 75-g oral glucose load (17). The drug that patients received was based on the clinical judgment of their physician. Table 1 gives the drug regimens of individual patients before and after protease inhibitor treatment. Eight subjects (NRTI pretreated) had been on a combination therapy of nucleoside analogues and a non-NRTI for at least 12 months; the NRTIs were continued throughout the study, the non-NRTI was discontinued, and a protease inhibitor was added. Five subjects (NRTI naïve), who were drug naïve, were started on two NRTIs plus a protease inhibitor. Thus, during the treatment phase all patients were on a combination of a protease inhibitor and NRTIs. None of the subjects experienced any subcutaneous fat wasting before or throughout the study. All were studied on three occasions: once before initiation of protease inhibitor treatment and at 6 and 12 weeks thereafter. Only results of those before and at 12 weeks are reported. Subjects consumed a weight maintenance diet containing 200–300 g carbohydrate at least 3 days before the first hyperglycemic clamp experiment and throughout the whole 12-week study period. On each study occasion subjects were admitted to the clinical research center at 5:00 P.M. the day before the experiment. Between 6:00 P.M. and 7:00 P.M. a standard dinner (10 kcal/kg: 50% carbohydrate, 35% fat, and 15% protein) was given. At 5:00 A.M. the following morning, primed-continuous infusions of [6,6- 2 H 2]glucose (24 mol/kg, 0.24 mol kg 1 min 1 ) and [9,10- 3 H]palmitate (0.8 Ci/min) were started via a forearm vein. At 6:00 A.M. a retrograde venous catheter was inserted into a dorsal hand vein and maintained in a thermoregulated box at 65°C to obtain arterialized blood samplings (18). After allowing at least 2htoachieve isotopic steady state, baseline samples for substrate enrichments, specific activities, plasma glucose, FFAs, glycerol, insulin, glucagon, C-peptide, and proinsulin concentrations were collected at 30, 15, and 0 min. Just before beginning the clamp, subjects took their medications. Subsequently, a primed (150 mg/kg body wt) 20% glucose infusion was given and plasma glucose levels were clamped at 10 mmol/l (180 mg/dl) for 3 h (19). Plasma insulin, proinsulin, and C-peptide concentrations were measured at 2.5, 5.0, 7.5, 10, 15, 30, 60, 90, 120, 140, 160, and 180 min. Samples for plasma glucose, FFA, glucagon, glycerol concentrations, enrichments, and specific activities (SAs) were collected during the last hour of the clamp at 20-min intervals (120, 140, 160, and 180 min). Plasma glucose was measured at 5-min intervals throughout for adjustments of glucose infusion rates using a glucose analyzer (YSI Glucose Analyzer; Yellow Springs Instruments). Plasma insulin (Linco Research), proinsulin (Linco Research), C-peptide (Diagnostic Product), and glucagon (Linco Research) were measured by standard radioimmunoassays. Plasma FFA levels were measured by an enzymatic calorimetric method (NEFAC; Wako Pure Chemical). Plasma glycerol concentrations were determined by standard microfluorometric assays (20). HbA 1c was determined by high-performance liquid chromatography (HPLC). Plasma [6,6- 2 H 2]glucose enrichments were measured by gas chromato- H.J. WOERLE AND ASSOCIATES graphy mass spectroscopy (21), and plasma palmitate concentrations and SAs were determined by a previously described HPLC technique (22) with coefficients of variation of 3.8 and 2.3%, respectively. Total body fat and fat-free mass was determined by bioimpedance measurements (23). Calculations. First-phase insulin release was calculated as the sum of the increments of C-peptide above baseline at 2.5, 5.0, 7.5, and 10 min. Secondphase insulin release was calculated as the average C-peptide concentration during the last hour of the clamp (24). Insulin sensitivity index (25) was calculated by dividing the average glucose infusion rate during the last hour of the clamp (mol kg 1 min 1 ) by the product of the average plasma insulin (pmol/l) and glucose (mmol/l) concentration during the same time interval (25). -Cell function and insulin sensitivity were also assessed by using HOMA as previously described (26). Insulin sensitivity (IS HOMA) was calculated as IS HOMA 1/(135 insulin glucose), i.e., the inverse of insulin resistance. -Cell function (SECR homa) was calculated as SECR homa C-peptide 3.33/(GLUC 3.5), where C-peptide (pmol/l) represents the average fasting plasma C-peptide concentration and GLUC (mmol/l) the average fasting plasma glucose concentration (27). C-peptide was used in place of the originally proposed plasma insulin because of changes in insulin metabolism induced by protease inhibitor treatment as described below. To evaluate the appropriateness of changes in -cell function in relation to changes in insulin sensitivity, the disposition index (28) was calculated as the product of -cell function and insulin sensitivity determined during the clamp experiments. During the basal state before the clamp, rates of appearance and disappearance (turnover) of FFA were calculated using the steady-state equation for the infusion of radioactive isotopes Ra(Rd) F/SA (29), where Ra represents the rate of appearance, Rd the rate of disappearance, F the infusion rate of tracer (dpm min 1 kg 1 ), and SA the specific activity (dpm mol 1 ). For glucose turnover, isotope enrichments (%) were substituted for SAs and corrected for the proportion of infused [6,6- 2 H 2]glucose by subtracting its infusion rate from Ra(Rd). Glucose enrichments and palmitate SAs were not significantly different from one another, indicating that isotopic steady state had been achieved. Because there is evidence that NRTIs may be associated with insulin resistance (30), subjects were divided into two groups. Group 1 (NRTI pretreated, n 8) had been on an NRTI and had a protease inhibitor added; group 2 (NRTI naïve, n 5) had been drug naïve and were started on an NRTI and a protease inhibitor. The data of these groups were compared in an attempt to factor out the effects of protease inhibitors. Furthermore, baseline data of NRTI pretreated volunteers was compared with those of 14 healthy subjects to determine whether NRTI pretreatment had effects on insulin sensitivity and -cell function. Unless specified otherwise, data are given as the mean SE and were initially evaluated by ANOVA for repeated measurements followed by Wilcoxon’s matched pairs test comparing baseline data with results after 12 weeks of treatment. Mann-Whitney tests were used to compare changes between the groups during the treatment period as well as baseline data. Correlations between variables were performed using Spearman’s regression analysis. DIABETES, VOL. 52, APRIL 2003 919
PROTEASE INHIBITOR–INDUCED GLUCOSE INTOLERANCE TABLE 2 Body composition, viral load, and CD 4 counts Baseline 12 weeks P vs. baseline BMI (kg/m 2 ) NRTI pretreated 29.7 2.1 29.9 1.7 0.93 NRTI naïve 26.7 1.6 27.3 2.6 0.92 Both 29.0 1.8 28.9 1.5 0.81 Normal volunteers 28.2 0.8 P vs. groups Lean body mass (kg) 0.34 NRTI pretreated 56.4 3.8 55.9 3.6 NRTI naïve 58.9 3.1 60.5 4.0 Both 57.4 2.6 57.7 2.7 0.69 Normal volunteers 60.1 4.9 P vs. groups Body fat (kg) 0.62 NRTI pretreated 33.0 5.6 33.2 5.5 NRTI naïve 16.5 2.9 18.2 3.0 Both 26.7 4.9 27.4 4.0 0.03 Normal volunteers 23.2 2.6 P vs. groups 0.048 CD4 counts NRTI pretreated 393 50 NRTI naïve 311 51 Both 361 37 P vs. groups Viral load 0.73 NRTI pretreated 3638 1588 256 119 0.001 NRTI naïve 2814 1267 511 205 0.001 Both 3321 1060 355 108 0.001 P vs. groups 0.82 Data are means SD. RESULTS Body composition, waist-to-hip ratio, viral loads, and CD 4 counts Baseline characteristics of the groups did not differ except that the NRTI-naïve subjects had lower initial body fat. During 12 weeks of treatment, BMI, total body fat, and lean body mass did not change in either group; viral load decreased by 80% in both groups (P 0.001) (Table 2). HbA 1c, fasting plasma glucose, insulin, glucagon, Cpeptide, proinsulin, glycerol, FFAs, and plasma lipids Baseline values, which were comparable with those of the normal volunteers, and responses during treatment in NRTI-pretreated and NRTI-naïve groups were not significantly different (Tables 3 and 4). After 12 weeks of treatment, fasting plasma glucose increased in all subjects (P 0.001). Fasting plasma insulin also increased (P 0.001). In contrast, fasting plasma C-peptide and proinsulin did not change (both P 0.61). Consequently, both the plasma C-peptide–to–insulin ratio and the plasma proinsulin–to–insulin ratio decreased (both P 0.007). The plasma C-peptide–to–proinsulin ratio and plasma glucagon remained unchanged (P 0.59 and 0.69, respectively). Fasting plasma glycerol increased (P 0.02), whereas plasma FFAs, triglycerides, and total and HDL cholesterol remained unchanged (all P 0.13). However, plasma LDL cholesterol increased slightly but significantly (P 0.03). -Cell function and insulin sensitivity HOMA. Baseline values and responses to treatment were not significantly different in NRTI-pretreated and NRTI- TABLE 3 HbA 1c, fasting plasma glucose, insulin, glucagon, C-peptide, and proinsulin Before 12 weeks P vs. baseline HbA1c (%) NRTI pretreated 5.5 0.3 — NRTI naïve 5.8 0.3 — Both 5.6 0.2 — Normal volunteers 5.8 0.3 P vs. groups Glucose (mmol/l) 0.91 NRTI pretreated 4.58 0.23 5.36 0.13 NRTI naïve 4.87 0.25 5.69 0.14 Both 4.69 0.11 5.49 0.11 0.001 Normal volunteers 4.96 0.11 P vs. groups Plasma insulin (pmol/l) 0.59 NRTI pretreated 108 17 125 17 NRTI naïve 61 12 87 9 Both 90 13 111 11 0.001 Normal volunteers 72 12 P vs. groups Plasma glucagon (pg/ml) 0.08 NRTI pretreated 80 4 805 NRTI naïve 78 7 713 Both 79 3 7740.69 Normal volunteers 70 6 P vs. groups Plasma C-peptide (nmol/l) 0.98 NRTI pretreated 0.77 0.13 0.73 0.08 NRTI naïve 0.51 0.09 0.61 0.06 Both 0.67 0.09 0.68 0.05 0.84 Normal volunteers 0.54 0.06 P vs. groups Plasma proinsulin (pmol/l) 0.34 NRTI pretreated 13.3 2.1 13.0 1.9 NRTI naïve 11.1 1.6 12.0 1.4 Both 12.4 1.4 12.6 1.2 0.61 Normal volunteers 12.0 1.6 P vs. groups 0.87 Data are means SD. naïve subjects. At baseline, both groups had comparable insulin sensitivity and -cell function with those of normal volunteers. After 12 weeks of treatment, both insulin sensitivity and pancreatic -cell function decreased significantly (P 0.002 and 0.003, respectively) (Tables 5 and 6 and Fig. 1). Hyperglycemic clamp experiments Insulin sensitivity. Plasma glucose, insulin, C-peptide, and proinsulin concentrations and glucose infusion rates are given in Fig. 1. Plasma glucose levels were not significantly different in clamps performed at baseline and 12 weeks in all groups. The insulin sensitivity index calculated by dividing the glucose infusion rates by the product of plasma glucose and insulin concentrations were comparable with those of the normal volunteers, and responses to treatment were not significantly different in NRTI-pretreated and NRTI-naïve subjects. The insulin sensitivity index decreased significantly after treatment (P 0.001). -Cell function. At baseline, both first- and second-phase insulin release was normal in both patient groups. After treatment, first-phase insulin release decreased signifi- 920 DIABETES, VOL. 52, APRIL 2003
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