David Silver

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,

POSTTRANSPLANT BONE DISEASE: EVIDENCE FOR A HIGH BONE RESORPTION STATE

Loss of bone is a significant problem after renal transplant. Although bone loss in the first post transplant year has been well documented, conflicting data exist concerning bone loss after this time.It is equally unclear whether bone loss in long-term renal transplant recipients correlates with bone turnover as it does in postmenapausal osteoporosis. To examine these issues, we conducted a cross-sectional study to define the prevalence of osteoporosis in long-term (>1 year) renal transplant recipients with preserved renal function (mean creatinine clearance 73±23 ml/min). Bone mineral density (BMD) was measured at the hip, spine and wrist by DEXA in 69 patients. Markers for bone formation (serum osteocalcin) and bone resorption [urinary levels of pyridinoline (PYD) and deoxypyridinoline (DPD)] were also measured as well as parameters of calcium metabolism. Correlations were made between these parameters and BMD at the various sites. The mean age of the patients was 45±11 years. Eighty eight percent of patients were on cyclosporine (12% on tacrolimus) and all but 2 were on prednisone [mean dose 9±2 mg/day)]. Osteoporosis (BMD more than 2.5 SD below peak adult BMD) at the spine or hip was diagnosed in 44% of patients and osteopenia was present in an additional 44%. Elevated levels of intact parathyroid hormone (i PTH) were observed in 81% of patients. Elevated urinary levels of PYD or DPD were present in 73% of patients and 38% had elevated serum levels of osteocalcin. Levels of calcium, and of 25(OH) and 1,25(OH)2 vitamin D were normal. In a stepwise multiple regression model that included osteocalcin, PYD, DPD, intact PTH, age, years posttransplant, duration of dialysis, cumulative prednisone dose, smoking, and diabetes: urinary PYD was the strongest predictor of bone mass. These results demonstrate that osteoporosis is common in long-term renal transplant recipients. The data also suggest that elevated rates of bone resorption contribute importantly to this process.

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.

A pilot study of soluble adhesion molecules as surrogate markers for acute liver allograft rejection.

BACKGROUND Tissue endothelial adhesion molecule expression is increased during acute liver allograft rejection. Soluble forms exist, but their correlation to rejection and their clinical value have not been determined. METHODS We studied endothelial adhesion molecule tissue expression and soluble levels in blood and bile and correlated these to the clinical course of 11 adult patients followed for 30 consecutive days after liver transplantation. RESULTS Three biopsies showing acute rejection demonstrated increased intercellular adhesion molecule (ICAM)-1 but not endothelial leukocyte adhesion molecule-1 expression on hepatic endothelial cells during rejection. Vascular cell adhesion molecule (VCAM)-1 staining was focally increased in two of three specimens. Levels of soluble ICAM-1 or soluble VCAM-1 by ELISA did not distinguish acute rejection from nonrejection conditions. CONCLUSION We confirmed that endothelial ICAM-1 expression is more intense in rejecting allografts and is more sensitive than changes in VCAM-1 or endothelial leukocyte adhesion molecule-1. However, soluble adhesion molecule determination was not useful in the accurate detection of acute rejection.

The use of argon beam coagulation in treating vulvar intraepithelial neoplasia III: A retrospective review

UNLABELLED Argon beam coagulation (ABC) has unique properties which make it suitable for the local treatment of superficial epithelial disorders such as vulvar intraepithelial neoplasia (VIN III). OBJECTIVE To evaluate argon beam coagulation in treating multifocal VIN III. METHODS Argon beam coagulation was used in twenty-nine patients. ABC was set at 80 W, 7 L/min. All patients were given 1% silvadene cream to apply to vulva. Patients had follow-up appointments two weeks and six weeks postoperatively. Patients were followed every three to six months for the subsequent year. RESULTS 2 of 29 (6.8%) experienced moderate pain within the first two weeks postoperatively requiring prescriptions for perocet. 2 of 29 (6.8%) had yeast infection requiring diflucan. Mean follow-up time was 34.9 months (11.7-37.4). 15 of 29 (51.7%) had no recurrence within the follow-up period. 14 of 29 (48.3%) recurred within the follow-up period. The mean time to recurrence is 23.2 months. CONCLUSION This small retrospective review is the first to evaluate argon beam coagulation in treating multifocal VIN III. This review indicates that ABC is comparable to other vulva organ conserving therapies. ABC retains cosmesis, and form of the vulva. This is a major advantage over surgery. Repeat treatments are also possible, which is important in a condition such as VIN, which tends to be multifocal and recurrent.