Ana Tereza M.G. Santomauro

Effect of a sulfonylurea (gliclazide) treatment on insulin sensitivity and glucose-mediated glucose disposal in patients with non-insulin-dependent diabetes mellitus (NIDDM)

Five male non-obese newly diagnosed NIDDM and 5 age-, sex- and body mass index (BMI) matched healthy controls without a family history of diabetes were submitted to a frequently sampled intravenous (i.v.) glucose tolerance test modified by exogenous insulin administration for estimation of insulin sensitivity (SI) and glucose-mediated glucose disposal (SG) with Bergmans minimal model computer analysis of glucose kinetics. The tests were repeated after 3 months treatment with a second generation sulfonylurea, gliclazide, in the diabetics subjects. SI and SG were markedly reduced before gliclazide therapy in the diabetics in comparison to the paired controls. After gliclazide, despite significantly lower (almost normal) plasma glucose, normalization of glycosylated hemoglobin and increased fasting insulin levels, there was a slight but significant increase in SI while SG showed a further reduction, the improvement in glucose control being also associated to the significant increased first and 2nd phase insulin release for the first 20 min after glucose infusion.

Insulin resistance in limb and trunk partial lipodystrophy (type 2 Köbberling-Dunnigan syndrome)

We studied insulin action in two patients with limb and trunk partial lipodystrophy with hirsutism and acanthosis nigricans. Glucose was normal in one of the patients and slightly above normal in the other during an oral glucose tolerance test (OGTT). An intravenous glucose tolerance test (IVGTT) was normal in both patients. Basal and glucose-stimulated insulin levels were elevated in both the OGTT and IVGTT in both patients. The response of plasma glucose to exogenously administered insulin was decreased. A euglycemic-hyperinsulinemic clamp performed in patient no. 2 indicated insulin resistance, which was not corrected by reducing the increased basal level of serum free fatty acids (FFAs). Binding of insulin to neck adipocytes was normal in both subjects, but glucose transport and oxidation in these cells was impaired. Insulin binding to abdominal adipocytes was increased in one patient whose adipocytes displayed higher glucose transport at low insulin concentrations. Glucose oxidation was decreased in abdominal adipocytes of both patients. We conclude that insulin resistance in Köbberling-Dunnigan type 2 partial lipodystrophy is not related to an alteration of the insulin molecule or to changes in insulin binding, but is more likely associated with a postreceptor defect, since glucose oxidation was impaired in adipocytes of the neck and abdomen.

A case of congenital generalized lipodystrophy: metabolic effects of four dietary regimens. Lack of association of CGL with polymorphism in the lamin A/C Gene

Sirs, Congenital generalized lipodystrophy (CGL) is an autosomal recessive disorder, characterized by severe metabolic derangement associated with the absence of subcutaneous adipose tissue. The exact genetic defect is unknown, and systematic studies of optimal dietary and pharmacological therapy for CGL have not been performed. We report a case of CGL, in which the patients dietary/pharmacological management was optimized with the help of metabolic testing. In addition, DNA from this patient was screened for mutations in several candidate genes. A 36-year-old female presented for treatment of diabetes mellitus uncontrolled with 0·8 units per kg of insulin per day. The diabetes was diagnosed 10 years earlier and was complicated by macroalbuminuria (533 µg/minutes), retinopathy, neuropathy, and peripheral vascular disease. Her parents were first cousins. She had generalized fat loss, prominent musculature, hepatomegaly, clitoromegaly, mild hirsutism and no acanthosis nigricans. Her height was 151 cm and weight was 49·2 kg (body mass index 21 kg/m2). Tetrapolar bioelectric impedance revealed a fat mass of 3% (1·5 kg). CT demonstrated no subcutaneous fat in the abdominal area, and visceral abdominal fat was approximately one third of that in age, height and body weight matched females. Her blood chemistry profile, pituitary, gonadal, adrenal and thyroid tests were normal. Serum leptin was 0·8 µg/l (10·8–16·6 µg/l is normal for females with BMI 21 ± 5 kg/m2). All research procedures were approved by the Institutional Review Board of the Hospital das Clinicas at the University, Sao Paulo, Brazil. After the patient was hospitalized, baseline studies were performed, and four dietary regimens (Reg. 1–4, see Table 1) varying in energy and fat content, and with the addition of dL-fenfluramine with intensive insulin therapy were Instituted under carefully controlled conditions with a goal to maintain body weight and optimize glycaemic control. After each intervention, energy substrate metabolism was tested with a euglycaemic hyperinsulinaemic clamp (insulin infusion rate 10 mU × m−2 × minutes−1) and indirect calorimetry using a flow-through canopy gas analyser system in the resting state and after a glucose load (Felber et al., 1987). The respiratory exchange ratios (RER) indicate (Table 1) that in all but the hypercaloric moderate-fat diet (Reg. 2), glucose was the primary fuel source after an overnight fast. This finding was supported by the low fasting free fatty acid availability in all regimens except for the hypercaloric moderate-fat regimen. The thermic effect of glucose did not change with any of the interventions, including dL-fenfluramine (Table 1). As measured by euglycaemic hyperinsulinaemic clamp exogenous glucose disposal did not correlate with energy content in the diet. According to the clamp data (Table 1), treatment with a hypercaloric very low-fat diet (Reg. 3) and hypercaloric low-fat diet (Reg. 4) resulted in markedly greater exogenous glucose disposal than treatment with a hypercaloric moderate-fat diet (Reg. 2). However, the total glucose disposal determined by indirect calorimetry was not considerably different in the four regimens, suggesting that the disposal of glucose produced endogenously accounts for the difference between glucose disposal measured by the clamp vs. indirect calorimetry. Indeed, basal plasma glucose was higher after treatment with a moderate-fat diet (Reg. 2 vs. 3). Interestingly, with slightly higher basal plasma glucose levels, basal plasma insulin and C-peptide levels after an overnight fast (Table 1) were almost twice as low in Reg. 2 compared to Reg. 3. The lower endogenous insulin levels in Reg. 2 may be due to suppression by free fatty acids, which were at higher levels in Reg. 2. Table 1 Effects of dietary modifications, insulin therapy and addition of dL-fenfluramine in a patient with CGL Energy restriction in the diet has limitations in patients with CGL because of undesirable weight loss and persistent hunger (Reg. 1). However, as in our patients case, modestly hypercaloric diets (Reg. 2 and 3, see Table 1) add a risk of hyperglycaemia that responds poorly to insulin. The exogenous glucose disposal rate was the highest and basal plasma glucose after an overnight fast normalized after the addition of dL-fenfluramine to a hypercaloric low-fat diet (Reg. 4). In addition, this drug in sharp contrast to dietary treatments alone, seems to be responsible for increased lipid oxidation after an oral glucose load, apparently at the expense of suprabasal glucose oxidation. However, even this decrease in suprabasal glucose oxidation may be a positive phenomenon in a patient with CGL if it reflects an overall decrease in glucose recycling and hepatic glucose production. Our findings are supported by data from Andersen et al. (1993), who have shown that lipid oxidation is increased and glucose oxidation is decreased during dL-fenfluramine treatment in obese nondiabetic patients. We suggest that the improvement in insulin sensitivity, despite no change in total energy intake and virtually the same nutrient composition after transition from dietary Reg. 3–4, is related to a dL-fenfluramine induced augmentation in fat oxidation and diminished cycling of the nutritional substrates. Clinically this positive effect was reflected by achievement of euglycaemia with just 1·1 U/kg of insulin per day. We performed single-stranded conformational polymorphism (SSCP) and DNA analysis to search for mutations in the coding regions, exon-intron junctions and proximal promoter regions of the β3-adrenergic receptor (β3AR) and the peroxisome proliferator-activated receptor-gamma (PPARγ) genes. We did not detected mutations in the β3-AR gene in this patient, similar to studies by Silver et al. (1997) and Vigouroux et al. (1997) in other patients with CGL. The only polymorphism in the PPARγ gene in this patient was a silent nucleotide substitution at the position 1431 (CACHis→CATHis). This variant was also present in several nonlipodystrophic controls. Similarly, we (unpublished) and colleagues (Vigouroux et al., 1998) did not find unique mutations in the PPARγ gene in other patients with CGL. Heterozygosity for several missense mutations in the lamin A/C gene on chromosome 1 has been associated with familial partial lipodystrophy (Cao & Hegele 2000). DNA sequence analysis of the lamin A/C gene revealed that our patient was heterozygous for the previously reported polymorphism at codon 583 [GCCAla→GACAla] (Speckman et al. 2000). The patient was also heterozygous for a C2737T polymorphism in intron 8, which introduces a potential new splice donor site (GC→GT). However, this novel intron variant was also present in three of nine normal controls. In conclusion, we show that a low-fat diet with supplemental dL-fenfluramine and insulin therapy may be an effective method to manage hyperglycaemia in a patient with CGL. Studies of the genes for lamin A/C, PPARγ and β3-AR in our patient failed to detect pathogenic alterations. Uncovering the genetic defect of CGL will allow a better understanding of the pathogenesis of this disease and provide insights into adipocyte development and function, as well as potential treatments for CGL and perhaps other metabolic disorders such as obesity and type 2 diabetes.

Metabolism of chylomicrons in patients with congenital lipoatrophic diabetes: a study with emulsion models of chylomicrons

background  Lipoatrophic diabetes is characterized by the near absence of adipose tissue and the presence of insulin‐resistant diabetes. Fasting hypertriglyceridaemia and increased postprandial lipidaemia are also present, but the metabolism of chylomicrons, the triglyceride‐rich lipoproteins in the circulation that carry the dietary fats absorbed by the intestine, was not specifically investigated. Because both the activity of insulin‐dependent lipoprotein lipase that catalyses the chylomicron lipolysis and the storage of the lipolysis products are affected in the disease, it is important to evaluate how those changes may ultimately affect the chylomicron lipolysis and removal of chylomicron remnants from the circulation.

Erythropoietin reduces the expression of myostatin in mdx dystrophic mice

Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO=0.60±0.11, control=1.07±0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO=0.95±0.14, control=1.05±0.16) and TNF-α (rhEPO=0.73±0.20, control=1.01±0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle.

Short- and long-term gliclazide effects on pancreatic islet cell function and hepatic insulin extraction in non-insulin-dependent diabetes mellitus

Nine non-obese males with non-insulin-dependent diabetes mellitus (NIDDM) were evaluated before and after 3 and 12 months (6 patients) treatment with the second generation hypoglycemic sulfonylurea: gliclazide. They underwent an oral glucose tolerance test, intravenous glucose and arginine tests measuring plasma insulin and C-peptide responses. Pre-hepatic insulin production and insulin delivery to peripheral tissues were calculated by deconvolution techniques and hepatic extraction of insulin estimated. An improvement was observed in the beta-cell function of the patients on gliclazide treatment: reduction of fasting plasma glucose associated with a progressive increase in C-peptide level but insulin levels decreased at 12 months, suggesting an increase in hepatic insulin extraction at this time. In the same way, while plasma glucose values after oral and i.v. glucose were greatly reduced at 3 and 12 months treatment, insulin did not change but C-peptide levels increased significantly at 12 month treatment. While the prehepatic insulin secretion rate increased progressively on gliclazide during all glucose challenges, the fractional hepatic insulin extraction fell after 3 and increased at 12 month treatment, with opposite changes in insulin delivered to peripheral tissues. Thus the insulinogenic effect of gliclazide could be masked during long-term administration by a concomitant effect of gliclazide which increases hepatic extraction of insulin. The maintenance of the responsiveness to the non-glucose secretagogue, arginine, as evaluated by the C-peptide levels, before and after correction of hyperglycemia, suggested improvement of beta-cell sensitivity to glucose after sulfonylurea treatment.(ABSTRACT TRUNCATED AT 250 WORDS)