INSULIN-LIKE growth factor I (IGF-I) is a 70-amino acid

Transcription

1 X/00/$03.00/0 Vol. 85, No. 9 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 2000 by The Endocrine Society Recombinant Human Insulin-Like Growth Factor I Treatment for 1 Week Improves Metabolic Control in Type 2 Diabetes by Ameliorating Hepatic and Muscle Insulin Resistance* KENNETH CUSI AND RALPH DEFRONZO Diabetes Division, Department of Medicine, University of Texas Health Science Center, San Antonio, Texas INSULIN-LIKE growth factor I (IGF-I) is a 70-amino acid peptide that plays an important role in the regulation of growth and fuel metabolism (1). The broad range of biological actions of IGF-I are modulated by several binding proteins (IGFBPs) (2). The IGF/IGFBPs system has been proposed as a complementary system to insulin and its counterregulatory hormones in the regulation of carbohydrate metabolism, and its function is disturbed in diabetes mellitus and other insulin-resistant states (3). Early studies showed that recombinant human IGF-I (rhigf-i) reduced plasma glucose and insulin levels in syndromes of severe insulin resistance (4 7). rhigf-i also has been shown to improve glycemic control in patients with type 1 (8, 9) and type 2 diabetes mellitus (T2DM) (10 14). However, the initial enthusiasm about the use of rhigf-i as a treatment modality in T2DM patients has been tempered by the occurrence of adverse effects, particularly at high doses (6, 15, 16), and by concern about its potential to worsen diabetic complications during long-term treatment (17). Received December 2, Revision received April 10, Accepted June 15, Address all correspondence and requests for reprints to: Kenneth Cusi, M.D., Diabetes Division, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas cusi@uthscsa.edu. * This work was supported by General Clinical Research Center Grant RR-01346, a V.A. Merit Award (to R.A.D.), and the V.A. Medical Research Fund. ABSTRACT The administration of recombinant human insulin-like growth factor I (rhigf-i) reduces hyperglycemia and insulin requirements in subjects with severe insulin resistance syndromes and in patients with type 2 diabetes mellitus (T2DM). However, the mechanisms responsible for the improved metabolic control are incompletely understood. One proposed mechanism is that rhigf-i therapy in T2DM may bypass early defects in insulin action (i.e. signal transduction), leading to improved hepatic and/or peripheral insulin sensitivity. To test this hypothesis, we used the euglycemic insulin clamp to measure the response to 7 days of rhigf-i therapy (80 g/kg, sc, twice daily) in eight poorly controlled T2DM subjects. rhigf-i significantly improved fasting ( vs mg/dl; P 0.01) and day-long ( h; vs mg/dl; P 0.01) plasma glucose levels. Basal endogenous glucose production decreased from to mg/kg lean body mass min (P 0.03) despite a concomitant decline in the fasting plasma insulin concentration from 13 5 to 5 1 U/mL (P 0.01). The decrement in basal endogenous glucose production was closely correlated with the decrement in fasting plasma glucose concentration (r 0.78; P 0.01). Whole body insulinstimulated glucose disposal increased by 27% (from to mg/kg lean body mass min; P 0.01), but remained well below that observed in age- and weight-matched healthy subjects. The effects of rhigf-i on endogenous glucose production and peripheral insulin sensitivity resemble those observed with intensified insulin regimens in T2DM. We conclude that 7 days of sc rhigf-i improves glucose control by improving hepatic and muscle insulin sensitivity, but it remains markedly abnormal. This indicates that an intrinsic defect(s) responsible for insulin resistance in T2DM cannot be overcome by rhigf-i treatment. (J Clin Endocrinol Metab 85: , 2000) Nonetheless, rhigf-i may provide an important tool to understand the mechanism(s) of insulin resistance and the function of the IGF-I/IGFBPs system in T2DM. In nondiabetic subjects, iv rhigf-i stimulates whole body glucose uptake in a dose-dependent fashion (18 20). However, the available data on the effects of sc rhigf-i on insulin action in T2DM are limited. An early study in six type 2 diabetics suggested that sc rhigf-i treatment improved insulin-stimulated glucose disposal (12). More recently, the same group reported similar findings with the frequently sampled iv glucose tolerance test (FSIVGTT) (13). However, the index of insulin sensitivity from the FSIVGTT correlates poorly with more direct measurements of insulin sensitivity (i.e. the euglycemic insulin clamp), especially in insulinresistant T2DM individuals (21, 22). In addition, neither the steady state plasma glucose (12) nor the FSIVGTT (13), used to measure insulin sensitivity in these studies, distinguishes between the hepatic and peripheral actions of insulin. In healthy nondiabetic subjects, it is believed that the liver is less sensitive than muscle to the action of IGF-I (20, 23, 24). However, from the few studies available, inhibition of endogenous glucose production (EGP) by rhigf-i appears to be comparable to that observed with insulin during acute administration (23, 24). Nevertheless, the response of the liver and peripheral tissues to prolonged sc rhigf-i in patients with T2DM is difficult to predict from these experiments, because 1) the metabolic response to rhigf-i is strongly de- 3077

2 3078 CUSI AND DEFRONZO JCE&M 2000 Vol. 85 No. 9 termined by the plasma free IGF-I levels achieved, and these, in turn, depend on the dose, route, and duration of rhigf-i administration (25); and 2) T2DM individuals have multiple abnormalities in the IGFBP system, an important determinant of IGF-I action. This may explain why in animal models of obesity and T2DM, suppression of EGP by rhigf-i is markedly blunted compared to that in nondiabetic control animals (26, 27). Therefore, the effect of rhigf-i on hepatic glucose production in T2DM subjects will depend upon the complex interaction between the dose and duration of rhigf-i administration and the adaptation of the IGFBPs system to treatment (2, 3). The purpose of the present study was to examine whether 1) a 7-day sc rhigf-i treatment can bypass early defects in insulin action and improve hepatic and/or peripheral insulin sensitivity in T2DM, and 2) to define the relative contributions of the liver vs. muscle to the observed improvement in glycemic control in insulin-resistant T2DM patients treated with rhigf-i. Subjects Subjects and Methods Eight subjects (four men and four women) with a mean ( sem) age of 48 4 yr participated in the study. The subjects were modestly obese with a mean body mass index of kg/m 2, body weight of kg, and height of cm. Body weight was stable for at least 3 months before participation. The duration of diabetes was 7 2 yr. Subjects were poorly controlled with a fasting plasma glucose concentration of mg/dl, and hemoglobin A 1c of % (normal, %). No subject was excessively sedentary or participated in any unusual or strenuous exercise. Other than diabetes, no subject had any clinical or laboratory evidence of renal, hepatic, or any other organ system disease, as determined by a complete medical history, physical examination, electrocardiogram, routine blood chemistries, and urinalysis. None of the volunteers had any evidence of clinically significant coronary artery or peripheral vascular disease, autonomic or peripheral neuropathy, or proliferative retinopathy. Participants were allowed to continue taking sulfonylureas (all eight subjects) and angiotensinconverting enzyme inhibitors (three of eight subjects) as long as the dose had been stable for at least 3 months before enrollment and remained unchanged during the study. Otherwise, participants were not receiving any medications known to affect glucose metabolism. Sulfonylureas were discontinued only on the morning of the day on which the oral glucose tolerance test (OGTT) and insulin clamp were performed (see below). The study protocol was approved by the Institutional Review Board of the University of Texas Health Science Center (San Antonio, TX), and each subject gave written informed consent before participation. Study design After the initial screening visit, eligible subjects were instructed by a dietician to ingest a weight-maintaining American Dietary Association diet and were seen weekly for 4 weeks. During this period they were encouraged to maintain their dietary intake and physical activity constant. If after the 4-week run-in period participants were considered to be compliant with the diet and if their weight had remained stable, they were admitted to the Clinical Research Center for three baseline metabolic studies, which were carried out at 0730 h after a 12 h overnight fast: 1) a day-long metabolic profile from h, 2) 75-g OGTT, and 3) a euglycemic insulin (40 mu/m 2 min) clamp, as described below. Within 7 14 days after completion of the three baseline studies all participants were admitted to the Clinical Research Center and treated with rhigf-i for 7 days. rhigf-i was given sc in the abdomen at a dose of 80 g/kg twice daily at 0700 and 1800 h (total daily dose, 160 g/kg). The plasma glucose concentration was determined from an indwelling antecubital venous catheter in the fasting state and hourly during the first h; thereafter, plasma glucose was measured every 2 3 h. The indwelling catheter was changed every 48 h. During the last 3 days of the in-hospital stay, the day-long profile, the OGTT, and the euglycemic insulin clamp were repeated in the same order that they were performed prior to treatment. During the 7-day in-hospital stay, subjects were encouraged to remain physically active and ingested a weight-maintaining diet containing 55% carbohydrate, 25% fat, and 20% protein. During the in-hospital stay, blood pressure was measured every 4 h. Plasma IGF-I levels were measured under fasting conditions before each study and hourly from 0730 h until noon on the day the metabolic profile was carried out. Description of metabolic studies Metabolic profile. On the evening prior to study, subjects were fasted after supper (1800 h) until the next morning, when they participated in a 9-h ( h) metabolic profile. A research dietician administered a weight-maintaining diet, which consisted of 55% carbohydrate, 25% fat, and 20% protein. Meals were given at 0800, 1200, and 1800 h with a caloric distribution of 30%, 30%, and 40% of total daily calories in each meal, respectively. Complete food intake was confirmed by a research nurse after each meal. The same diet was consumed on the metabolic profile day before and after treatment with rhigf-i. From h blood was withdrawn through an indwelling venous catheter every hour for the determination of plasma glucose, insulin, C peptide, and free fatty acid (FFA) concentrations. OGTT. After an overnight fast, three blood samples were drawn at 30, 15, and 0 min through an iv catheter placed into an antecubital vein, and subjects then ingested a 75-g orange-flavored glucose drink. Blood was drawn every 30 min for the next 2 h for measurement of plasma glucose, insulin, and C peptide levels. Euglycemic insulin clamp. Insulin sensitivity was measured before and after rhigf-i treatment in all participants with a 40 mu/m 2 min euglycemic insulin clamp (28). Briefly, a 20-gauge Teflon catheter was inserted into an antecubital vein for the infusion of test substances ([3-3 H]glucose, insulin, and 20% dextrose). In addition, a second catheter was placed retrogradely into a vein on the dorsum of the hand, and the hand was placed in a thermoregulated box at 65 C for arterialization of the venous blood. Both iv lines were kept patent with a slow infusion of normal saline. At 0730 h, a primed continuous infusion of [3-3 H]glucose (Du Pont-NEN Life Science Products, Boston, MA) was started and continued until the end of the study. The [3-3 H]glucose constant infusion rate was 0.2 mci/min, and the tritiated glucose prime was calculated as follows: 20 Ci fasting plasma glucose/100. After 180 min to allow for isotopic equilibration, four baseline blood samples were taken at 10-min intervals for the determination of plasma tritiated glucose specific activity and plasma glucose, FFA, and hormone concentrations. After the determination of basal glucose turnover, a 3-h euglycemic insulin (40 mu/m 2 min) clamp was performed using a primed continuous insulin infusion (28). After the start of insulin infusion, no glucose was infused until the plasma glucose concentration had declined to 100 mg/dl, at which level it was maintained by adjustment of a variable glucose infusion based upon the negative feedback principle (28). Blood for determination of plasma tritiated glucose specific activity was obtained every min during the insulin clamp. After 150 min, four blood samples were drawn at 10-min intervals for measurement of plasma glucose, FFA, hormone concentrations and tritiated glucose specific activity. Continuous indirect calorimetry (29) was performed for measurement of oxygen consumption and carbon dioxide production during the last 40 min of the baseline ( 40 and 0 minutes) and euglycemic insulin clamp ( min) periods (Deltatrac, Sensormedics, Anaheim, CA). Timed urine collections were performed during the 3-h baseline and 3-h insulin clamp periods for the measurement of urinary nitrogen excretion. Analytical procedures The plasma glucose concentration was measured in duplicate using the glucose oxidase method with a Beckman Glucose Analyzer II (Beckman Instruments, Inc., Fullerton, CA). Plasma insulin (Coat-A-Count Insulin, Diagnostic Products, Los Angeles, CA), C peptide (Diagnostics

3 rhigf-i AND TYPE 2 DIABETES MELLITUS 3079 Systems Laboratories, Inc., Webster, TX), and glucagon (Double Antibody Glucagon, Diagnostic Products Corp., Los Angeles, CA) concentrations were determined by RIA. The plasma FFA concentration was measured using standard colorimetric methods (Wako Chemicals USA, Inc., Richmond, VA). Intra- and interassay coefficients of variation (CVs) were: insulin, 4.0% and 4.9%; C peptide, 4.3% and 2.4%; glucagon, 4.4% and 6.5%; and FFA, 1.1% and 3.3%. IGF-I (total and free) and IGFBP-3 plasma concentrations were measured by RIA at Genentech, Inc. (South San Francisco, CA). Total and free plasma IGF-I concentrations were determined by RIA after acid-ethanol extraction, with a recovery greater than 80% (30). There were no binding proteins detectable by high performance liquid chromatography (HPLC) in the acid-ethanol supernatant after extraction. Free IGF-I was separated from the IGF/IGFBP complex using size-exclusion HPLC. Following chromatography, the IGF-I concentration was determined by RIA in the fractions in which standard IGF-I would elute. The sum of these fractions was considered to be the free IGF-I for a given sample. The RIA used to measure total and free IGF-I had maximum intra- and interassay CVs of 10% and 17%, respectively. The maximum intra- and interassay CVs for total IGF-I concentration (extraction procedure and RIA) were 15% and 19%, respectively. Free IGF-I measurement (size-exclusion HPLC procedure and RIA) had maximum intra- and interassay CVs of 8% and 8%, whereas for IGFBP-3 the CVs were 9% and 12%, respectively. Values were expressed as nanograms per ml. For the determination of plasma glucose radioactivity, plasma was deproteinized (31) and centrifuged for 30 min at 3500 g, and the clear supernatant was evaporated to dryness at 55 C in a Speed-Vac evaporator (Savant Instruments, Farmingdale, NY). The pellet was resuspended in 1 ml distilled water, mixed with 5 ml Scintiverse II (Fischer Scientific, Pittsburgh, PA), and counted in a scintillation counter (LS 6000IC, Beckman Instruments, Inc., Fullerton, CA). Calculations During the basal period, the rate of endogenous plasma glucose appearance [which primarily represents hepatic glucose production after an overnight fast (32)] equals the rate of plasma glucose disappearance and was calculated by dividing the [3-3 H]glucose infusion rate [disintegrations per min (dpm)] by the steady state plasma tritiated glucose specific activity (dpm/ mol) during the last 40 min of tracer equilibration. A steady state plateau of plasma [3-3 H]glucose was achieved in all subjects during the last 40 min of the basal period. As the infusion of insulin results in nonsteady state conditions, the rate of plasma glucose appearance was calculated using Steele s nonsteady state equation (33), as modified by DeBodo et al. (34), using a pool fraction of 0.65 (35) and a volume of distribution of 200 ml/kg (34). During the euglycemic insulin infusion period, EGP was computed as the difference between the exogenous glucose infusion rate and the isotopically measured rate of plasma glucose appearance. Negative numbers for EGP were not observed in any study. 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. Glucose oxidation was calculated from the nonprotein respiratory quotient as previously described (29). Nonoxidative glucose disposal, which primarily represents glycogen synthesis (36), was calculated by subtracting the rate of glucose oxidation (as measured by indirect calorimetry) from the rate of total body glucose disposal. All turnover rates are expressed as milligrams per kg lean body mass (LBM). Determination of LBM was calculated from the iv bolus injection of tritiated water ( 3 H 2 O) as follows. At 0800 h, after a 10-h overnight fast, subjects received an iv bolus of 3 H 2 O, and plasma samples were obtained at 90, 105, and 120 min for determination of tritiated water radioactivity. A steady state plateau of tritiated water radioactivity was achieved during the 90- to 120-min period in all subjects. The ratio between the total amount of radioactivity administered (dpm) and steady state 3 H 2 O radioactivity (dpm/ml) gives a measure of total body water (37). LBM was estimated by dividing total body water (milliliters) by 0.73, assuming that total body water is 73% of the LBM (37). Statistical analysis All data are presented as the mean sem. Statistical significance was determined by paired two-tailed Student s t test, using the JMP statistical package (SAS Institute, Inc., Cary, NC). Comparisons were considered statistically significant if P Where appropriate, regressions were calculated by least squares linear correlation coefficients analysis. Results Plasma IGF-I and IGFBP-3 concentrations The mean fasting plasma IGF-I concentration was significantly higher after IGF-I treatment [OGTT study day, vs ng/ml (P 0.05); metabolic profile day, vs ng/ml (P 0.01); insulin clamp study day, vs ng/ml (P 0.05); before vs. after treatment]. The mean total IGF-I plasma concentration measured from h during the metabolic profile increased significantly after the administration of rhigf-i (from to ng/ml; P 0.01). Consistent with the total IGF-I results, fasting free IGF-I plasma levels were 3-fold higher after 1 week of treatment with rhigf-i during the insulin clamp day (from to ng/ml; P 0.01) and the day-long profile (from to ng/ml; P 0.01). After the 80 mg/kg rhigf-i dose given during the day-long profile (rhigf-i was not given on the insulin clamp day), free IGF-I peaked about 2 h after sc administration at ng/ml and returned to near predose levels ( ng/ml) by 4 h. Plasma IGFBP-3 levels were significantly lower after rhigf-i treatment: OGTT study day, vs ng/ml (P 0.01; 23% vs. pretreatment); metabolic profile day, vs ng/ml (P 0.01; 29% vs. pretreatment); and insulin clamp study day, vs ng/ml (P 0.01; 22% vs. pretreatment). Plasma glucose, hormone, FFA, and lactate concentrations during the day-long metabolic profile (Fig. 1) rhigf-i treatment reduced the plasma glucose concentration by mg/dl at all time points. The fasting (from to mg/dl; P 0.001) and mean day-long ( vs mg/dl; P 0.001; h) plasma glucose concentrations were significantly reduced by rhigf-i treatment (Fig. 1). Most of the improvement in mean daylong plasma glucose profile resulted from the decline in fasting plasma glucose concentration (Fig. 1). Mean fasting and day-long plasma insulin (fasting, from 10 1to5 1 U/mL; h, from 25 3to13 2 U/mL; both P 0.01) and plasma C peptide (fasting, from to ng/ml; h, from to ng/ml; both P 0.01) concentrations were significantly reduced by rhigf-i treatment (Fig. 1). A reduction in plasma insulin and C peptide concentrations was observed in every subject (data not shown). After rhigf-i treatment, plasma insulin and C peptide increments above baseline ( ) were reduced to a greater extent after the noon (1200 h) meal compared to those after breakfast (0800 h). rhigf-i treatment had no effect on the fasting plasma FFA concentration ( vs mol/l; P NS) or on the h plasma FFA levels ( vs mol/l; P NS). Both the fasting plasma lactate concentration (from to mmol/l) and the h plasma lactate concentration (from to mmol/l) fell slightly, but significantly (P 0.05), after 7 days of rhigf-i administration.

4 3080 CUSI AND DEFRONZO JCE&M 2000 Vol. 85 No. 9 Plasma glucose and hormone concentrations during the OGTT (Fig. 2) During the OGTT, the fasting plasma glucose concentration ( vs ; P 0.001) and the area under the plasma glucose concentration curve (37,236 2,452 vs. 29,619 2,530 mg/dl per 120 min; P 0.01) were significantly reduced following rhigf-i treatment (Fig. 2). This improvement was primarily accounted for by a reduction in the fasting plasma glucose concentration, as the increments in postprandial glucose above baseline were similar before and after rhigf-i treatment (13,236 1,738 vs. 14,169 1,246 mg/dl per 120 min, respectively; P NS). rhigf-i reduced the fasting plasma insulin concentration from 11 1to6 2 U/mL (P 0.01) and the C peptide concentration from to ng/ml (P 0.01). The area under the insulin (from to U/mL per 120 min; P 0.04) and C peptide ( vs ng/ml per 120 min; P 0.04) concentration curves (Fig. 2) also were reduced by rhigf-i treatment. The insulin secretory response to glucose after rhigf-i treatment, expressed as the incremental area under the curve for insulin, was similar in the pre- and posttreatment studies (before vs. after, vs U/mL per 120 min; P NS), indicating that 7 days of rhigf-i therapy did not inhibit glucose-stimulated insulin secretion even though the basal insulin secretory rate was diminished. Consistent with this, the incremental area under the plasma C peptide concentration curve was significantly greater after rhigf-i treatment (before vs. after, vs ng/ml per 120 min; P 0.05). The greatest increments in plasma C peptide concentration after rhigf-i administration were observed at 90 and 120 min (P 0.05), suggesting improved second phase -cell secretion in response to glucose. Plasma glucose, hormone, and FFA concentrations during the euglycemic insulin clamp During the euglycemic insulin clamp studies performed before and after rhigf-i treatment the steady state plasma glucose concentrations were similar (98 2 vs mg/ dl), and the coefficient of variation in plasma glucose was less than 5% in every study. The fasting plasma insulin concentration was lower after rhigf-i treatment (13 5 vs. 5 1 U/mL; P 0.01) during the insulin clamp performed after rhigf-i treatment. The increments in plasma insulin concentration were similar in the insulin clamp studies performed before and after rhigf-i (64 5 vs U/mL; P NS). The fasting plasma FFA concentration ( vs mol/l; P NS) and the decrease in plasma FFA concentration during insulin infusion ( vs mol/l; P NS) were not different before vs. after treatment. Plasma glucagon levels were similar in the basal state (pre- vs. posttreatment, 82 9 vs pg/ml; P NS) and during the euglycemic insulin clamp (pre- vs. posttreatment, 72 6 vs pg/ml; P NS). FIG. 1. Effect of rhigf-i on plasma glucose, insulin, C peptide, and FFA concentrations during a 9-h ( h) metabolic profile in T2DM patients. Subjects were studied before (E) and after (F) treatment with rhigf-i at a dose of 80 g/kg twice daily for 1 week. Results represent the mean SE. Endogenous glucose production (Figs. 3 and 4) Basal EGP decreased in all but one patient (who had a near-normal fasting plasma glucose of 121 mg/dl at baseline) from to mg/kg LBM min (P 0.03) following treatment with rhigf-i. EGP during the euglycemic insulin clamp was suppressed by over 90% before and FIG. 2. Effect of rhigf-i on plasma glucose, insulin, and C peptide concentrations during an OGTT in T2DM patients. All subjects were studied before (E) and after (F) treatment with rhigf-i at a dose of 80 g/kg twice daily for 1 week. Results represent the mean SE.

5 rhigf-i AND TYPE 2 DIABETES MELLITUS 3081 FIG. 3. EGP in the basal state ( ) and during a 40 mu/m 2 min insulin clamp (1), performed before (left) and after (right) treatment with rhigf-i at a dose of 80 g/kg twice daily for 1 week. Results represent the mean SEM. FIG. 5. Whole body insulin-mediated glucose disposal (total column height), glucose oxidation (shaded part of the column), and nonoxidative glucose disposal (open part of the column) during the 40 mu/ m 2 min insulin clamp, performed before (left) and after (right) treatment with rhigf-i at a dose of 80 g/kg twice daily for 1 week. Results represent the mean SEM. *,P 0.01 vs. pretreatment. ( vs mg/kg LBM min; P NS) and improved significantly during the euglycemic insulin clamp after rhigf-i administration (from to mg/kg LBM min; P 0.01). Basal lipid oxidation ( vs mg/kg LBM min; P NS) as well as lipid oxidation during the insulin clamp ( vs mg/kg LBM min; P NS) were not significantly different after rhigf-i treatment. FIG. 4. Correlation between the decrement in EGP, which primarily represents liver, and the decrement in fasting plasma glucose concentration (r 0.78; P 0.01) after rhigf-i treatment at a dose of 80 g/kg twice daily for 1 week. Results represent the mean SEM. after treatment with rhigf-i ( vs mg/kg LBM min; P NS; Fig. 3). There was a significant correlation between the decrement in fasting plasma glucose and the decrement in basal EGP after rhigf-i treatment (r 0.78; P 0.01; Fig. 4). There was no correlation between the decrement in fasting plasma glucose (r 0.28; P NS) or EGP (r 0.34; P NS) and the change in whole body insulin-mediated glucose disposal. Whole body glucose disposal (Fig. 5) Under basal conditions, steady state conditions prevail, and the rate of glucose disappearance is identical to the rate of endogenous glucose appearance. After 1 week of rhigf-i treatment, whole body insulin-mediated glucose disposal during the euglycemic insulin clamp increased by 27%, from to mg/kg LBM min (P 0.01). Basal glucose oxidation was similar before and after treatment ( vs mg/kg LBM min, respectively; P NS). However, during the euglycemic insulin clamp there was a significant enhancement of glucose oxidation after rhigf-i from to mg/kg LBM min (P 0.01). The basal rate of nonoxidative glucose disposal was not significantly different before and after rhigf-i treatment Effect of rhigf-i on the cardiovascular system One week of rhigf-i administration resulted in a small, but statistically insignificant, decrease in blood pressure (average of the 3 days on which the metabolic testing was performed; pre- vs. post-rhigf-i, 129 9/75 5 vs /72 2mmHg;P NS). However, the resting heart rate increased from 72 4to88 5 beats/min (P 0.01). Two subjects had asymptomatic orthostatic hypotension (decrement in standing vs. supine blood pressure, 15/10 mm Hg), and one had an increase in resting heart rate to 100 beats/min. Adverse effects of rhigf-i All patients completed the study. Side-effects of treatment with rhigf-i were assessed by a standardized daily questionnaire, physical examination, and laboratory tests. Adverse events were of mild to moderate intensity and selflimited, with no patient requiring a reduction or discontinuation of study drug. The most common sideeffects were discomfort at the injection site of rhigf-i (three of eight patients) and parotid gland tenderness, either spontaneous or induced by palpation (four of eight patients). These symptoms were mild and dissipated during the course of rhigf-i treatment. There were two episodes of migraine headache in one subject. This subject had a prior history of migraine headaches. Two patients had mild facial and peripheral edema. No patient experienced hypoglycemia or arthralgias, myalgias, flushing, rash, fatigue, dyspnea, or neurologic complications, side-effects previously reported with the use of rhigf-i at higher doses (6, 11, 13, 15, 16). Fundoscopic examinations, electrocardiograms, and routine

6 3082 CUSI AND DEFRONZO JCE&M 2000 Vol. 85 No. 9 laboratory blood tests were unchanged after rhigf-i treatment. Weight remained stable in all subjects throughout the study ( vs kg, respectively; P NS). Discussion The present study represents the first comprehensive examination of the effect of 7 days of sc administration of rhigf-i on the three major mechanisms responsible for impaired glucose homeostasis in T2DM patients: EGP, which primarily reflects liver (32); insulin-stimulated glucose disposal, which primarily reflects muscle (38); and insulin secretion. Earlier studies in small numbers of subjects provided useful, but indirect, measures of the effects of rhigf-i on peripheral glucose disposal (12, 13). However, as isotopic tracers were not employed in these studies, lack of a simultaneous measurement of EGP has limited our ability to fully understand the effects of treatment with sc rhigf-i in T2DM. Our results suggest that 1 week of treatment with rhigf-i suppresses basal EGP despite a decrease in fasting insulin concentration and enhances insulin-mediated muscle glucose disposal in T2DM patients. In T2DM patients, fasting hyperglycemia is largely the result of an increased rate of EGP (39), whereas decreased insulin-mediated glucose disposal and impaired suppression of EGP contribute approximately equally to postprandial glycemia (40). In the present study rhigf-i reduced fasting and postprandial glucose levels despite a decline in fasting plasma insulin and C peptide concentrations (Figs. 1 and 2). These observations are consistent with previously published results suggesting that rhigf-i enhances insulin action (10, 11, 13). Although there is no previous information on EGP, Moses et al. reported improved peripheral insulinstimulated glucose uptake in T2DM patients treated with sc rhigf-i administration, as measured using the steady state plasma glucose (12) and the minimal model technique (13). In the later study, this group reported an improvement in insulin sensitivity (S I ) without any change in glucose effectiveness (S G ). However, the number of diabetic patients was small (n 8), and in two of the eight subjects an accurate measure of S I could not be determined before treatment. Moreover, assessment of insulin sensitivity without the use of isotopic measurements of glucose turnover correlates poorly with the insulin clamp, and in particular, the measurement of S G with the minimal model has been questioned (21, 22). Our results show that 7 days of sc rhigf-i treatment improves insulin-mediated glucose uptake in T2DM by 27% by augmenting both glucose oxidation and nonoxidative glucose metabolism (Fig. 5). Stimulation of both pathways has been described with acute iv infusion of rhigf-i in healthy volunteers (18, 19, 23, 24). The magnitude of improvement in insulin sensitivity is similar or only slightly greater than the approximately 10 20% improvement achieved with intensified insulin regimens in T2DM (41) (Cusi, K., and R. DeFronzo, unpublished data) and is about 50% lower than that in healthy individuals studied in our laboratory (42). This supports the concept that the IGF-I and insulin metabolic pathways converge early in the insulin receptor signal transduction cascade, consistent with in vitro reports involving insulin receptor substrate-1 (IRS-1) as the predominant substrate for both receptors (43, 44). Despite the improvement in whole body insulin sensitivity, the increment in plasma glucose above baseline was unchanged after an oral glucose load and improved only modestly in the morning after a mixed meal. Thus, most of the improvement in day-long plasma glucose levels was explained by the decrease in fasting plasma glucose concentration, which correlated strongly with the reduction in EGP (r 0.78; P 0.01). No correlation was observed between the improvement in insulin-mediated glucose disposal and the reduction in fasting or postprandial or post-ogtt plasma glucose concentrations. A number of mechanisms could be advanced to explain the suppressive effect of rhigf-i on EGP: 1) direct effect on the liver mediated via the IGF-I receptor or hybrid IGF-I/insulin receptors; 2) cross-over binding to the insulin receptor; 3) indirect effect on EGP secondary to a decrease in plasma FFA concentration or to a decrease in gluconeogenic supply, i.e. glycerol or lactate; 4) reduction in plasma GH or glucagon concentrations; and 5) amelioration of glucose toxicity. As fasting plasma insulin and C peptide levels declined by 50% and 23%, respectively, enhanced insulin secretion cannot account for the suppression of basal EGP observed with rhigf-i administration. It is generally believed that IGF-I receptors in the liver are scarce. However, in man this observation is based upon a single report (46), which has yet to be confirmed. Therefore, a direct effect of rhigf-i on EGP mediated via the hepatic IGF-I receptor cannot be excluded at the present time. Although another possibility is that IGF-I may have suppressed hepatic glucose production by cross-over binding to the insulin receptor, in vitro studies have shown that IGF-I binds to the insulin receptor with an affinity only approximately 5% that of insulin (1 3). Moreover, the plasma IGF-I concentrations achieved in the present study were relatively low, making this an unlikely explanation. Finally, one may consider whether IGF-I exerts its hepatic effect via binding to hybrid IGF-I/insulin receptors, which behave more like IGF-I receptors (48) and are increased in muscle of insulinresistant type 2 diabetic subjects (49) and in patients with chronic hyperinsulinemia from insulinomas (50). If present in liver tissue of T2DM patients, they could mediate the effect of rhigf-i on hepatic glucose production. Circulating plasma FFA and excessive rates of FFA/lipid oxidation influence hepatic glucose production (51). Acute iv administration of rhigf-i has been shown to have no effect (19, 20, 52) or to reduce (18, 23, 53) plasma FFA concentrations. However, when rhigf-i is given sc during 7 days, plasma FFA levels have been reported to be slightly increased in nondiabetic subjects (54) or unchanged in patients with T2DM (11, 55). We observed no effect of rhigf-i on the fasting or postprandial plasma FFA concentration or in response to an insulin infusion during euglycemia. Thus, alterations in plasma FFA or lipid oxidation could not explain the inhibitory effect of IGF-I treatment on EGP. Another explanation for a reduction in EGP by rhigf-i is a decrease in gluconeogenic substrate availability (56). We observed a slight decrease in fasting plasma lactate levels after 7 days of rhigf-i therapy, but this was most likely secondary to the decrease in fasting plasma glucose concentration, leading to

7 rhigf-i AND TYPE 2 DIABETES MELLITUS 3083 a diminished flux of glucose into the cell and a reduction in anaerobic glycolysis. Hepatic glucose production is also strongly influenced by circulating hormone levels, of which insulin and glucagon are paramount (42). Both fasting and postprandial insulin concentrations fell significantly, whereas plasma glucagon concentrations remained unchanged during rhigf-i treatment. GH levels, which were not measured in the present study, have been shown to decline during rhigf-i administration in man (3). However, a decrease in plasma GH cannot explain the prompt reduction in EGP observed during an acute infusion of rhigf-i (20, 23, 24). Moreover, the effect of GH on EGP is rather weak, as suggested by recent studies in GH-deficient adults (57, 58). Indeed, when GH-deficient subjects receive physiological replacement with rhgh, there is no increase in basal EGP (58). These results suggest that a potential decrease in plasma GH concentration by rhigf-i is unlikely to explain the reduction in basal EGP in our study. Lastly, it is possible that the initial glucose-lowering effect during rhigf-i therapy is enhanced glucose uptake by skeletal muscle, where IGF-I receptors are abundant (47), reducing glucose toxicity and progressively improving liver (and muscle) insulin sensitivity (41). A direct effect of rhigf-i on muscle, independent of amelioration of hyperglycemia, is suggested by the improvement in insulin-mediated glucose uptake in individuals with type A phenotype of severe insulin resistance (7) and in some type 2 diabetic patients with normal or minimally elevated fasting plasma glucose concentrations (12, 13). Chronic hyperglycemia per se may contribute to excessive rates of EGP in T2DM by up-regulating the glucose-6-phosphatase complex, the final step involved in the release of glucose by the liver (59, 60). Alternatively, it is possible that the primary effect of IGF-I is on the liver and that the improvement in peripheral tissue sensitivity to insulin results from amelioration of glucose toxicity on muscle (41). Therefore, and also based upon studies of acute rhigf-i administration (18 20, 23, 24, 52, 53), we believe that the initial improvement in glycemic control results from direct effects on both liver and muscle, with secondary improvements due to the removal of glucose toxicity. Patients tolerated rhigf-i with minimal side-effects, possibly because of the short duration of treatment and the relatively low dose of rhigf-i employed. However, most patients had a small ( 10 20%) increase in resting heart rate, and two had asymptomatic orthostatic hypotension. These effects are consistent with the known effect of rhigf-i to decrease peripheral vascular resistance (61). No patient experienced hypoglycemia or significant side-effects previously reported with the use of rhigf-i at high doses in diabetic subjects, i.e. arthralgias, myalgias, flushing, rash, fatigue, dyspnea, severe peripheral edema, or neurological complications (6, 13, 15, 16). However, in a 3-month clinical trial, a dose of 80 g/kg rhigf-i given twice daily (as in the present study) was reported to cause significant side-effects (16). In summary, 7 days of treatment with rhigf-i causes a decrease in EGP (primarily hepatic glucose output) and an improvement in whole body (primarily muscle) sensitivity to insulin in patients with T2DM. The decline in fasting plasma glucose concentration is closely correlated with the decrease in HGP, but not with the improvement in whole body insulin sensitivity. Whether the reduction in HGP is directly mediated via the IGF-I receptor, hybrid IGF-I/insulin receptors, or, less likely, the insulin receptor or indirectly through its effect on skeletal muscle deserves further investigation. Acknowledgments We thank the nurses of the General Clinical Research Center for the diligent care of our volunteers, especially Sylvia Smith, R.N., and Deanna Juarez, R.N., for their expert assistance in carrying out the insulin clamp studies. We are also grateful for the enriching discussion about the manuscript with Martin Adamo, Ph.D., and Lawrence Mandarino, Ph.D. Mrs. Lorrie Albarado provided skilled secretarial assistance in preparation of the manuscript. Genentech, Inc. kindly supplied the rhigf-i and determined the plasma IGF-I and IGFBP-3 concentrations. References 1. Froesch E, Hussain M, Schmid C, Zapf J Insulin-like growth factor I: physiology, metabolic effects and clinical uses. Diabetes Metab Rev. 12: Clemmons D Insulin-like growth factor binding proteins and their role in controlling IGF actions. Cytokine Growth Factor Rev. 8: Cusi K, DeFronzo R Treatment of NIDDM, IDDM and other insulin resistant states with IGF-I. Diabetes Rev. 3: Quin J, Fisher B, Paterson K, Inoue A, Beastall G, MacCuish A Acute response to recombinant insulin-like growth factor I in a patient with Mendenhall s syndrome. N Engl J Med. 323: Kuzuya H, Matsuura N, Sakamoto M, et al Trial of insulinlike growth factor I therapy for patients with extreme insulin resistance syndromes. Diabetes. 42: Usala A-L, Madigan T, Burguera B, et al High dose intravenous, but not low dose subcutaneous, insulin-like growth factor-i therapy induces sustained insulin sensitivity in severely resistant type I diabetes mellitus. J Clin Endocrinol Metab. 79: Morrow L, O Brien M, Moller D, Flier J, Moses A Recombinant human insulin-like growth factor-i therapy improves glycemic control and insulin action in the type A syndrome of severe insulin resistance. J Clin Endocrinol Metab. 79: Acerini C, Patton C, Savage M, Kernell A, Westphal O, Dunger D Randomised placebo-controlled trial of human recombinant insulin-like growth factor I plus intensive insulin therapy in adolescents with insulindependent diabetes mellitus. Lancet. 350: Carroll P, Umpleby M, Ward G, et al rhigf-i administration reduces insulin requirements, decreases growth hormone secretion, and improves the lipid profile in adults with IDDM. Diabetes. 46: Zenobi P, Jaeggi-Groisman S, Riesen W, Roder M, Froesch E Insulinlike growth factor-i improves glucose and lipid metabolism in type 2 diabetes mellitus. J Clin Invest. 90: Schalch D, Turman N, Marcsisin V, Heffernan M, Guler H-P Short-term effects of recombinant human insulin-like growth factor I on metabolic control in patients with type II diabetes mellitus. J Clin Endocrinol Metab. 77: Moses A, Morrow L, O Brien M, Moller D, Flier J Insulin-like growth factor I (rhigf-i) as a therapeutic agent for hyperinsulinemic insulin-resistant diabetes mellitus. Diabetes Res Clin Pract. 28(Suppl):S185 S Moses A, Young S, Morrow L, O Brien M, Clemmons D Recombinant human insulin-like growth factor I increases insulin sensitivity and improves glycemic control in type II diabetes. Diabetes. 45: rhigf-i in NIDDM Study (RINDS) Group Evidence from a doseranging study that recombinant insulin-like growth factor I (rhigf-i) effectively and safely improves glycemic control in non-insulin dependent diabetes mellitus [Abstract]. Diabetes. 45(Suppl 1):27A. 15. Jabri N, Schalch D, Schwartz S, et al Adverse effects of recombinant human insulin-like growth factor I in obese insulin-resistant type II diabetic patients. Diabetes. 43: rhigf-i in NIDDM Study (RINDS) Group Safety profiling of rhigf-i therapy in patients with NIDDM: a dose-ranging, placebo controlled trial [Abstract]. Diabetes 45(Suppl 1):71A. 17. Kolaczynski J, Caro J Insulin-like growth factor I therapy for diabetes mellitus? Diabetes Care. 17: Turkalj I, Keller U, Ninnis R, Vosmeer S, Stauffacher W Effect of inscreasing doses of recombinant human insulin-like growth factor-i on glucose, lipid, and leucine metabolism in man. J Clin Endocrinol Metab. 75: Elahi D, McAloon-Dyke M, Fukagawa N, et al Effects of recombinant

8 3084 CUSI AND DEFRONZO JCE&M 2000 Vol. 85 No. 9 human IGF-I on glucose and leucine kinetics in men. Am J Physiol 265:E831 E Laager R, Ninnis R, Keller U Comparison of the effects of rhigf-i and insulin on glucose and leucine kinetics in man. J Clin Invest. 92: Saad M, Anderson R, Laws A, et al A comparison between the minimal model and the glucose clamp in the assessment of insulin sensitivity across the spectrum of glucose tolerance. Insulin Resistance Atherosclerosis Study. Diabetes. 43: Avogaro A, Vicini P, Valerio A, Caumo A, Cobelli C The hot but not the cold minimal model allows precise assessment of insulin sensitivity in NIDDM subjects. Am J Physiol. 270:E532 E Boulware S, Tamborlane W, Mathews L, Sherwin R Diverse effects of insulin-like growth factor I on glucose, lipid, and amino acid metabolism. Am J Physiol. 262:E130 E Boulware S, Tamborlane W, Rennert N, Gesunheidt N, Sherwin R Comparison of metabolic effects of recombinant human insulin-like growth factor-i and insulin. J Clin Invest. 93: Mauras N, Martha P, Quarmby V, Haymond M rhigf-i administration in humans: differential metabolic effects of bolus vs. continuous subcutaneous delivery. Am J Physiol. 272:E628 E Cascieri M, Slater E, Vicario P, Green B, Bayne M, Sperstein R Impaired insulin growth factor I-mediated stimulation of glucose incorporation into glycogen in vivo in the ob/ob mouse. Diabetologia. 32: Jacob R, Sherwin R, Greenawalt K, Shulman G Simultaneous insulinlike growth factor I and insulin resistance in obese Zucker rats. Diabetes. 41: DeFronzo RA, Tobin JD, Andres R Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol. 237:E214 E Simonson D, DeFronzo R Indirect calorimetry: methodological and interpretative problems. Am J Physiol. 258:E399 E Davenport M, Svoboda M, Koerber K, Van Wyk J, Clemmons D, Underwood L Serum concentrations of insulin-like growth factor II are not changed by short term fasting and refeeding. J Clin Endocrinol Metab. 67: Somogyi M Determination of blood sugar. J Biol Chem. 160: Ekberg K, Landau B, Wajngot A, et al Contributions by kidney and liver to glucose production in the postabsorptive state and after 60h of fasting. Diabetes. 48: Steele R, Wall J, De Bodo R, Altszuler N Measurement of size and turnover rate of body glucose pool by the isotope dilution method. Am J Physiol. 187: DeBodo R, Steele R, Altszuler N, Dunn A, Bishop J On the hormonal regulation of carbohydrate metabolism: studies with 14 C glucose. Recent Prog Horm Res 19: Cowan J, Hetenyi G Glucoregulatory responses in normal and diabetic dogs recorded by a new tracer method. Metabolism. 20: Shulman GI, Rothman DL, Jue T, Stein P, DeFronzo RA, Shulman RG Quantitation of muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent diabetes by 13 C nuclear magnetic resonance spectroscopy. N Engl J. Med. 322: Sheng H, Huggins R A review of body composition studies with emphasis on total body water and fat. Am J Clin Nutr. 32: DeFronzo RA Lilly Lecture The triumvirate: -cell, muscle, liver. A collusion responsible for NIDDM. Diabetes. 37: DeFronzo RA, Simonson DC Fasting hyperglycemia in non-insulindependent diabetes mellitus; contributions of excessive hepatic glucose production and impaired tissue glucose uptake. Metabolism. 38: Ferrannini E, Simonson DC, Katz LD, et al The disposal of an oral glucose load in patients with non-insulin-dependent diabetes. Metabolism. 37: Rossetti L, Giaccari A, DeFronzo RA Glucose toxicity. Diabetes Care. 13: DeFronzo R Pathogenesis of type 2 diabetes: metabolic and molecular implications of identifying diabetes genes. Diabetes Rev. 5: Adamo M, Roberts CJ, LeRoith D How distinct are the insulin and insulin-like growth factor-i signalling systems? Biofactors. 3: Myers M, Sun X, Cheatham B, Glasheen E, Backer J, White M IRS-1 is a common element in insulin and insulin-like growth factor-i signaling to the phosphatidylinositol 3-kinase. Endocrinology. 132: Le Roith D, Werner H, Beitner-Johnson D, Roberts C Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev. 16: Caro J, Poulos J, Ittoop O, Flickinger E, Sinha M Insulin-like growth factor I binding in hepatocytes from human liver, human hepatome, and normal, regenerating, and fetal rat liver. J Clin Invest. 81: Dohm G, Elton W, Raju M, et al IGF-I stimulated glucose transport in human skeletal muscle and IGF-I resistance in obesity and NIDDM. Diabetes. 39: Langlois W, Sasaoka T, Yip C, Olefsky J Functional characterization of hybrid receptors composed of a truncated insulin receptor and wild type insulin-like growth factor 1 or insulin receptor. Endocrinology. 136: Federici M, Zucaro L, Porzio O, et al Increased expresion of insulin/ insulin-like growth factor-i hybrid receptors in skeletal muscle of noninsulindependent diabetes mellitus subjects. J Clin Invest. 98: Federici M, Lauro D, D Adamo M, et al Expresion of insulin/igf-i hybrid receptors is increased in skeletal muscle of patients with chronic primary hyperinsulinemia. Diabetes. 47: Boden G Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes. 46: Mauras N, Horber F, Haymond M Low dose recombinant human insulin-like growth factor-i fails to affect protein anabolism but inhibits islet cell secretion in humans. J Clin Endocrinol Metab. 75: Guler H, Zapf J, Froesch E Short-term metabolic effects of recombinant human insulin-like growth factors I in healthy adults. N Engl J Med. 317: Hussain M, Schmitz O, Mengel A, et al Insulin-like growth factor I stimulates lipid oxidation, reduces protein oxidation, and enhances insulin sensitivity in humans. J Clin Invest. 92: Zenobi P, Holzmann P, Glatz Y, Riesen W, Froesch E Improvement of lipid profile in type 2 (non-insulin-dependent) diabetes mellitus by insulin-like growth factor I. Diabetologia. 36: Consoli A Role of liver in the pathophysiology of NIDDM. Diabetes Care. 15: Weaver J, Monson J, Noonan K, et al The effect of low dose recombinant human growth hormone replacement on regional fat distribution, insulin sensitivity, and cardiovascular risk factors in hypopituitary adults. J Clin Endocrinol Metab. 80: Christopher M, Hew F, Oakley M, Rantzau C, Alford F Defects of insulin action and skeletal muscle glucose metabolism in growth hormonedeficient adults persist after 24 months of recombinant human growth hormone therapy. J Clin Endocrinol Metab. 83: Massillon D, Barzilai N, Chen W, Hu M, Rossetti L Glucose regulates in vivo glucose-6-phosphatase gene expression in the liver of diabetic rats. J Biol Chem. 271: Massillon D, Chen W, Barzilai N, et al Carbon flux via the pentose phosphate pathway regulates the hepatic expression of the glucose-6-phosphatase and phosphoenolpyruvate carboxykinase genes in conscious rats. J Biol Chem. 273: Hussain M, Studer K, Messmer E, Froesch E Treatment with insulin-like growth factor-i alters capillary permeability in skin and retina. Diabetes. 44: