David G. Maggs

Efficacy and Metabolic Effects of Metformin and Troglitazone in Type II Diabetes Mellitus

BACKGROUND Combination therapy is logical for patients with non-insulin-dependent (type 2) diabetes mellitus, because they often have poor responses to single-drug therapy. We studied the efficacy and physiologic effects of metformin and troglitazone alone and in combination in patients with type 2 diabetes. METHODS We randomly assigned 29 patients to receive either metformin or troglitazone for three months, after which they were given both drugs for another three months. Plasma glucose concentrations during fasting and postprandially and glycosylated hemoglobin values were measured periodically during both treatments. Endogenous glucose production and peripheral glucose disposal were measured at base line and after three and six months. RESULTS During metformin therapy, fasting and postprandial plasma glucose concentrations decreased by 20 percent (58 mg per deciliter [3.2 mmol per liter], P<0.001) and 25 percent (87 mg per deciliter [4.8 mmol per liter], P<0.001), respectively. The corresponding decreases during troglitazone therapy were 20 percent (54 mg per deciliter [2.9 mmol per liter], P=0.01) and 25 percent (83 mg per deciliter [4.6 mmol per liter], P<0.001). Endogenous glucose production decreased during metformin therapy by a mean of 19 percent (P=0.001), whereas it was unchanged by troglitazone therapy (P=0.04 for the comparison between groups). The mean rate of glucose disposal increased by 54 percent during troglitazone therapy (P=0.006) and 13 percent during metformin therapy (P= 0.03 for the comparison within the group and between groups). In combination, metformin and troglitazone further lowered fasting and postprandial plasma glucose concentrations by 18 percent (41 mg per deciliter [2.3 mmol per liter], P=0.001) and 21 percent (54 mg per deciliter [3.0 mmol per liter], P<0.001), respectively, and the mean glycosylated hemoglobin value decreased 1.2 percentage points. CONCLUSIONS Metformin and troglitazone have equal and additive beneficial effects on glycemic control in patients with type 2 diabetes. Metformin acts primarily by decreasing endogenous glucose production, and troglitazone by increasing the rate of peripheral glucose disposal.

Metabolic Effects of Troglitazone Monotherapy in Type 2 Diabetes Mellitus: A Randomized, Double-Blind, Placebo-Controlled Trial

Type 2 diabetes mellitus is characterized by two major pathophysiologic defects: insulin resistance and impaired capacity to secrete insulin [1, 2]. A major component of insulin resistance exists in peripheral tissues, where insulins ability to stimulate glucose uptake from the circulation is blunted. During the past three decades, treatment of hyper-glycemia in patients with type 2 diabetes mellitus who do not respond to such behavioral modifications as diet and exercise has focused on improving the relative insulin deficiency through therapy with sulfonylurea drugs to stimulate endogenous insulin secretion or through administration of insulin itself. Two additional drugs have recently become available: metformin, which seems to exert much of its glucose-lowering effect by suppressing hepatic glucose production [3], and acarbose, which changes the pattern of glucose absorption from the gastrointestinal tract [4]. Thus, no pharmacologic intervention for type 2 diabetes mellitus has had a major effect on improving insulin resistance in peripheral tissues. New compounds, the thiazolidinediones, have recently been developed as glucose-lowering agents. Early studies showed that the glucose-lowering effect of thiazolidinediones was evident in animal models of type 2 diabetes mellitus but not those of type 1 diabetes mellitus [5, 6], suggesting that some endogenous insulin secretion is needed for these agents to act. Troglitazone has been shown to decrease levels of not only plasma glucose and glycosylated hemoglobin [7-13] but also insulin and C-peptide. These observations, coupled with direct measures of whole-body insulin sensitivity in a small number of patients with type 2 diabetes mellitus [7], suggest that troglitazone exerts its major glucose-lowering effect by ameliorating insulin resistance. However, it is not clear whether troglitazone exerts its major insulin-sensitizing effect predominantly in the liver or in peripheral tissues. We studied this issue using detailed metabolic measurements in a large group of patients with type 2 diabetes mellitus. Methods This multicenter study was conducted at six sites: University of Chicago, Chicago, Illinois; University of Southern California, Los Angeles, California; University of Rochester, Rochester, New York; University of Pittsburgh, Pittsburgh, Pennsylvania; University of California, San Diego, San Diego, California; and Yale University, New Haven, Connecticut. Sample size was projected on the basis of study design, major end points, and standard power analysis. Each center enrolled patients while adhering to a common protocol with the same inclusion and exclusion criteria. At each center, patients gave written informed consent to participate in the study, which was approved by the respective university human investigation committees. All patients were studied in a 6-month, randomized, placebo-controlled, double-blind protocol. Patients were randomly assigned to treatment according to a blocked randomization code (block size, five) that was generated by a central computer. In each center, study personnel (executors of treatment assignment) and patients were blinded to the treatment code. Patients were consecutively assigned to treatments; equal numbers of troglitazone or matching placebo tablets were dispensed in a double-blind fashion. Patients Patients had to have type 2 diabetes mellitus according to the criteria of the National Diabetes Data Group [14], HbA1c levels above the upper limit of normal, and fasting C-peptide levels of 0.49 nmol/L or greater. Therapy with oral antidiabetic medication was discontinued before randomization. Patients were excluded if they had clinically symptomatic heart disease, had had a vascular occlusive event in the previous 3 months, had had cancer in the past 5 years, had a serum creatinine level greater than 176.8 mol/L, or had serum amino-transferase levels above the upper limit of normal. Study Design After medical screening, a 2-week wash-out period was allowed for discontinuation of therapy with oral antidiabetic medication in patients who were taking such medication. Metabolic studies were done before patients were randomly assigned to one of five treatment groups: 100, 200, 400, or 600 mg of troglitazone daily or placebo. At 6 months, follow-up metabolic studies were repeated 24 hours after patients received the last troglitazone or placebo tablet. At baseline and 6 months, patients were hospitalized and fasted overnight before a meal tolerance test (day 1) and a euglycemic-hyperinsulinemic clamp procedure (day 2) [15]. During the study, patients were prescribed a diet designed to maintain baseline body weight. Dietary assessment at the time of enrollment determined the patients caloric needs [16]. The prescribed diet consisted of 50% carbohydrates, 34% fat (ratio of saturated fat to polyunsaturated fat, 1:4) and 16% protein. Patients were seen at monthly outpatient visits between the baseline and 6-month metabolic studies so that their clinical condition could be monitored. Meal Tolerance Test At approximately 7:00 a.m., patients were placed on bed rest and an intravenous catheter was inserted into an antecubital vein for blood sampling. A small volume of normal saline (0.9%) was infused to maintain patency. At approximately 8:00 a.m., patients ingested a liquid formula meal (Sustacal-HC [Mead Johnson & Co., Evansville, Indiana], which contained 33% of total daily caloric requirements); this was followed 4 hours later by an identical meal. Fasting blood samples were drawn, and additional samples were obtained every hour thereafter for 8 hours. Samples were processed immediately and stored at 80C for measurement of serum levels of glucose, insulin, free fatty acids, and triglycerides and plasma levels of C-peptide. Fasting blood was also drawn for measurement of HbA1c. After completing the test, patients received an evening meal according to their prescribed diet. They then fasted until the end of the euglycemic-hyperinsulinemic clamp procedure the following day. The intravenous line was left in situ for the clamp procedure. Euglycemic-Hyperinsulinemic Clamp Procedure At 6:00 a.m., a 4-hour primed (corrected for ambient fasting plasma glucose level), continuous (2 mg/m2 body surface area per minute) infusion of [6,6- 2H]-glucose (di-deuterated glucose) isotope into the antecubital vein began. During the third hour of infusion, a retrograde cannula was inserted into a contralateral hand vein. The hand was warmed for sampling of arterialized venous blood. A small volume of normal saline (0.9%) was infused through the sampling cannula to maintain patency. Blood samples were drawn at 10-minute intervals during the final 40 minutes of the fourth hour for measurement of plasma glucose and insulin levels and glucose isotope enrichment. After 4 hours of isotope infusion, a two-step priming dose of insulin was administered (480 mU/m2 per minute followed by 240 mU/m2 per minute; each lasted 5 minutes); this was followed by a continuous infusion of insulin (120 mU/m2 per minute) that lasted 300 minutes (total, 5 hours). The plasma glucose level was allowed to decrease to 5.5 mmol/L; exogenous glucose (dextrose, 20 g/100 mL of water enriched to approximately 2.5% with di-deuterated glucose) was then infused to maintain the plasma glucose level, measured every 5 minutes, at 5.5 mmol/L. The basal isotope infusion was stopped when the exogenous glucose infusion began. Patients also received a continuous infusion of potassium (KCl and KPo 4), 0.105 mmol/L per minute, during the insulin infusion to maintain the serum potassium level between 3.5 and 4.5 mmol/L. During the final hour of the clamp procedure, blood samples were drawn every 10 minutes for measurement of plasma insulin levels and steady-state glucose isotope enrichment. For comparison with diabetic patients, eight persons without diabetes (mean age SD, 46 6 years; mean fasting plasma glucose level, 5.3 0.2 mmol/L; mean body mass index, 29 3 kg/m2) were also studied on one occasion under basal and clamped conditions after an identical hyperinsulinemic clamp protocol. Substrate and Hormone Measurements Serum and plasma samples were shipped frozen to Corning Nichols Institute for chemical analysis and to Yale University for measurement of isotope enrichment. Serum total triglyceride levels (Boehringer Mannheim Diagnostics, Indianapolis, Indiana) and plasma free fatty acid levels (NEFA C-test, Wako Chemicals, Richmond, Virginia) were determined enzymatically; interassay coefficients of variation were 2% and 3.6%, and intraassay coefficients of variation were 1.6% and 1%, respectively. Insulin and C-peptide levels were measured by radioimmunoassay (Corning Nichols Institute); the interassay coefficients of variation were 12.3% and 12.0%, and the intraassay coefficients of variation were 7.4% and 6.5%, respectively. Levels of HbA1c were measured by high-performance liquid chromatography using BioRad (Hercules, California) equipment (Corning Nichols Institute), with a normal reference range of 0.045 to 0.059. At each center, plasma glucose levels were measured at the bedside by using a Beckman glucose analyzer (Fullerton, California). Glucose Isotope Data Gas chromatography mass spectrometer analysis of enrichment of di-deuterated glucose in plasma and infusates was done at one center (Yale Stable Isotope Core Facility, New Haven, Connecticut) by using the penta-acetate derivative of glucose [17]. Calculations Basal hepatic glucose production was calculated as follows: Basal hepatic glucose production = (f/sa) x ([enrichmentinf/enrichmentplasma] 1), where f = basal [6,6- 2H] glucose infusion rate (mg/min), sa = body surface area (m2), enrichmentinf = [6,6- 2H] glucose infusate enrichment (%), and enrichmentplasma = steady-state basal plasma [6,6- 2H] glucose enrichment (%). The term enrichment refers to the fraction of isotope of glucose to naturally occurring (native) glucose,

Striking Differences in Glucose and Lactate Levels between Brain Extracellular Fluid and Plasma in Conscious Human Subjects: Effects of Hyperglycemia and Hypoglycemia

Brain levels of glucose and lactate in the extracellular fluid (ECF), which reflects the environment to which neurons are exposed, have never been studied in humans under conditions of varying glycemia. The authors used intracerebral microdialysis in conscious human subjects undergoing electro-physiologic evaluation for medically intractable epilepsy and measured ECF levels of glucose and lactate under basal conditions and during a hyperglycemia–hypoglycemia clamp study. Only measurements from nonepileptogenic areas were included. Under basal conditions, the authors found the metabolic milieu in the brain to be strikingly different from that in the circulation. In contrast to plasma, lactate levels in brain ECF were threefold higher than glucose. Results from complementary studies in rats were consistent with the human data. During the hyperglycemia–hypoglycemia clamp study the relationship between plasma and brain ECF levels of glucose remained similar, but changes in brain ECF glucose lagged approximately 30 minutes behind changes in plasma. The data demonstrate that the brain is exposed to substantially lower levels of glucose and higher levels of lactate than those in plasma; moreover, the brain appears to be a site of significant anaerobic glycolysis, raising the possibility that glucose-derived lactate is an important fuel for the brain.

Interstitial fluid concentrations of glycerol, glucose, and amino acids in human quadricep muscle and adipose tissue. Evidence for significant lipolysis in skeletal muscle.

To determine the relationship between circulating metabolic fuels and their local concentrations in peripheral tissues we measured glycerol, glucose, and amino acids by microdialysis in muscle and adipose interstitium of 10 fasted, nonobese human subjects during (a) baseline, (b) euglycemic hyperinsulinemia (3 mU/kg per min for 3 h) and, (c) local norepinephrine reuptake blockade (NOR). At baseline, interstitial glycerol was strikingly higher (P < 0.0001) in muscle (3710 microM) and adipose tissue (2760 microM) compared with plasma (87 microM), whereas interstitial glucose (muscle 3.3, fat 3.6 mM) was lower (P < 0.01) than plasma levels (4.8 mM). Taurine, glutamine, and alanine levels were higher in muscle than in adipose or plasma (P < 0.05). Euglycemic hyperinsulinemia did not affect interstitial glucose, but induced a fall in plasma glycerol and amino acids paralleled by similar changes in the interstitium of both tissues. Local NOR provoked a fivefold increase in glycerol (P < 0.001) and twofold increase in norepinephrine (P < 0.01) in both muscle and adipose tissues. To conclude, interstitial substrate levels in human skeletal muscle and adipose tissue differ substantially from those in the circulation and this disparity is most pronounced for glycerol which is raised in muscle as well as adipose tissue. In muscle, insulin suppressed and NOR increased interstitial glycerol concentrations. Our data suggest unexpectedly high rates of intramuscular lipolysis in humans that may play an important role in fuel metabolism.

Counterregulation in Peripheral Tissues: Effect of Systemic Hypoglycemia on Levels of Substrates and Catecholamines in Human Skeletal Muscle and Adipose Tissue

We used microdialysis to distinguish the effects hyperinsulinemia of and hypoglycemia on glucose, gluconeogenic substrate, and catecholamine levels in adipose and muscle extracellular fluid (ECF). Ten lean humans (six males and four females) were studied during baseline and hyperinsulinemic (3 mU · kg−1 · min−1 · for 3 h) euglycemia (5.0 mmol/l) and hypoglycemia (2.8 mmol/l). In muscle and adipose, basal ECF glucose was lower (muscle, 3.5 ± 0.2 mmol/l; adipose tissue, 3.3 ± 0.2 mmol/l) and lactate was higher (muscle, 2.2 ± 0.2 mmol/l; adipose, 1.5 ± 0.3 mmol/l) than respective plasma values (glucose, 4.9 ±0.1 mmol/l; lactate, 0.7 ± 0.1 mmol/l), whereas alanine was higher in muscle ECF (379 ± 22 μmol/l) than adipose tissue (306 ± 22 μmol/l) and plasma (273 ± 33 μmol/l). Plasma catecholamines (unchanged during euglycemia) rose during hypoglycemia with epinephrine, increasing approximately fivefold more than norepinephrine. In contrast, the hypoglycemia-induced increments in muscle dialysate norepinephrine and epinephrine were similar, suggesting local generation of norepinephrine. Compared with euglycemia, hypoglycemia produced a greater increase in lactate and a smaller reduction in alanine in muscle ECF, whereas hypoglycemia caused a greater relative fall in ECF glucose concentrations in muscle (72 ± 16%) and adipose tissue (69 ±9%) than in plasma (42 ± 3%) (P < 0.05). We conclude that hypoglycemia increases the generation of norepinephrine and gluconeogenic substrates in key target tissues, while increasing the plasma-tissue concentration gradient for glucose. These changes suggest the stimulation of glucose extraction by peripheral tissues, despite systemic counterregulatory hormone release and local sympathetic activation.

Effect of insulin on glycerol production in obese adolescents

Impaired stimulation of glucose metabolism and reduced suppression of lipolytic activity have both been suggested as important defects related to the insulin resistance of adolescent obesity. To further explore the relationship between these abnormalities, we studied seven obese [body mass index (BMI) 35 ± 2 kg/m2] and seven lean (BMI 21 ± 1 kg/m2) adolescents aged 13-15 yr and compared them with nine lean adults (aged 21-27 yr, BMI 23 ± 1 kg/m2) during a two-step euglycemic-hyperinsulinemic clamp in combination with 1) a constant [2H5]glycerol (1.2 mg ⋅ m-2 ⋅ min-1) infusion to quantify glycerol turnover and 2) indirect calorimetry to estimate glucose and net lipid oxidation rates. In absolute terms, basal glycerol turnover was increased and suppression by insulin was impaired in obese adolescents compared with both groups of lean subjects ( P < 0.01). However, when the rates of glycerol turnover were adjusted for differences in body fat mass, the rates were similar in all three groups. Basal plasma free fatty acid (FFA) concentrations were significantly elevated, and the suppression by physiological increments in plasma insulin was impaired in obese adolescents compared with lean adults ( P < 0.05). In parallel with the high circulating FFA levels, net lipid oxidation in the basal state and during the clamp was also elevated in the obese group compared with lean adults. Net lipid oxidation was inversely correlated with glucose oxidation ( r = -0.50, P < 0.01). In conclusion, these data suggest that lipolysis is increased in obese adolescents (vs. lean adolescents and adults) as a consequence of an enlarged adipose mass rather than altered sensitivity of adipocytes to the suppressing action of insulin.Impaired stimulation of glucose metabolism and reduced suppression of lipolytic activity have both been suggested as important defects related to the insulin resistance of adolescent obesity. To further explore the relationship between these abnormalities, we studied seven obese [body mass index (BMI) 35 +/- 2 kg/m2] and seven lean (BMI 21 +/- 1 kg/m2) adolescents aged 13-15 yr and compared them with nine lean adults (aged 21-27 yr, BMI 23 +/- 1 kg/m2) during a two-step euglycemic-hyperinsulinemic clamp in combination with 1) a constant [2H5]glycerol (1.2 mg.m-2.min-1) infusion to quantify glycerol turnover and 2) indirect calorimetry to estimate glucose and net lipid oxidation rates. In absolute terms, basal glycerol turnover was increased and suppression by insulin was impaired in obese adolescents compared with both groups of lean subjects (P < 0.01). However, when the rates of glycerol turnover were adjusted for differences in body fat mass, the rates were similar in all three groups. Basal plasma free fatty acid (FFA) concentrations were significantly elevated, and the suppression by physiological increments in plasma insulin was impaired in obese adolescents compared with lean adults (P < 0.05). In parallel with the high circulating FFA levels, net lipid oxidation in the basal state and during the clamp was also elevated in the obese group compared with lean adults. Net lipid oxidation was inversely correlated with glucose oxidation (r = -0.50, P < 0.01). In conclusion, these data suggest that lipolysis is increased in obese adolescents (vs. lean adolescents and adults) as a consequence of an enlarged adipose mass rather than altered sensitivity of adipocytes to the suppressing action of insulin.

Depressed levels in plasma insulin-growth factor-1 (IGF-1) and binding protein-1 (IGFBP-1) in adolescence obesity. 372

Depressed levels in plasma insulin-growth factor-1 (IGF-1) and binding protein-1 (IGFBP-1) in adolescence obesity. 372