Roger Assan

Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13.

Congenital generalized lipodystrophy, or Berardinelli–Seip syndrome (BSCL), is a rare autosomal recessive disease characterized by a near-absence of adipose tissue from birth or early infancy and severe insulin resistance. Other clinical and biological features include acanthosis nigricans, hyperandrogenism, muscular hypertrophy, hepatomegaly, altered glucose tolerance or diabetes mellitus, and hypertriglyceridemia. A locus (BSCL1) has been mapped to 9q34 with evidence of heterogeneity. Here, we report a genome screen of nine BSCL families from two geographical clusters (in Lebanon and Norway). We identified a new disease locus, designated BSCL2, within the 2.5-Mb interval flanked by markers D11S4076 and D11S480 on chromosome 11q13. Analysis of 20 additional families of various ethnic origins led to the identification of 11 families in which the disease cosegregates with the 11q13 locus; the remaining families provide confirmation of linkage to 9q34. Sequence analysis of genes located in the 11q13 interval disclosed mutations in a gene homologous to the murine guanine nucleotide-binding protein (G protein), γ3-linked gene (Gng3lg) in all BSCL2-linked families. BSCL2 is most highly expressed in brain and testis and encodes a protein (which we have called seipin) of unknown function. Most of the variants are null mutations and probably result in a severe disruption of the protein. These findings are of general importance for understanding the molecular mechanisms underlying regulation of body fat distribution and insulin resistance.

CYCLOSPORIN INCREASES THE RATE AND LENGTH OF REMISSIONS IN INSULIN-DEPENDENT DIABETES OF RECENT ONSET: Results of a Multicentre Double-blind Trial

In a double-blind trial 122 patients aged 15-40 years with insulin-dependent diabetes of recent onset were randomly assigned to cyclosporin 7.5 mg/kg per day or placebo. At the sixth month 25.4% of the cyclosporin group and 18.6% of the placebo group were in complete remission (not a significant difference). Treatment was continued in those patients with complete or partial remission (insulin requirement less than 0.25 U/kg per day) and 106 patients were followed to nine months, at which stage 24.1% of the original cyclosporin group and 5.8% of the original placebo group were in complete remission (p less than 0.01). For those patients whose whole-blood trough cyclosporin levels in the first three months averaged 300 ng/ml or more, the rates of complete remission at six and nine months were 37.5% and 37%. The rates of partial remission were also higher in the cyclosporin group and at six months the rate of complete or partial remission was 46% in the whole cyclosporin group and 65.6% in those with an average blood level exceeding 300 ng/ml in the first three months, versus 28.8% in the placebo group. The principal side-effect of cyclosporin was a modest and reversible increase in plasma creatinine. These results indicate that cyclosporin promotes the remission of type I diabetes and suggest the need for new controlled protocols aimed at evaluating the length of the effect and selecting the best drug regimen.

Factors Affecting the Secretion of Insulin and Glucagon by the Rat Fetus

Insulin (IRI) and glucagon (IRG) increased in the plasma of the rat fetus from 18½ to 20½ days of gestation and decreased on day 21½. The demonstrated failure of insulin and glucagon to cross the placenta of the rat allowed the conclusion that fetal rat pancreas secreted IRI and IRG in the plasma of the fetus at the end of gestation. Fetal hyperglycemia induced by perfusing pregnant rats with glucose for one hour produced a marked increase in fetal plasma IRI but did not modify plasma IRG. Fetal hypoglycemia induced by perfusing pregnant rats with insulin for one hour produced a fall in fetal plasma IRI but no rise in fetal plasma IRG. Chronic fetal hypoglycemia produced by fasting pregnant rats for ninety-six hours or by intrauterine growth retardation of fetuses increased fetal plasma IRG and decreased fetal plasma IRI. Norepinephrine injection in term rat fetus increased plasma IRG and decreased plasma IRI. Acetylcholine injection increased both plasma IRI and IRG, whereas serotonin remained without effect. These data suggest that IRI and IRG secretion by the fetal rat pancreas is controlled by both the blood glucose level and autonomic nervous system activity. Lower blood glucose levels and higher plasma IRI and IRG levels in pregnant than nonpregnant rats are also reported.

Pentamidine-Induced Derangements of Glucose Homeostasis: Determinant roles of renal failure and drug accumulation: A study of 128 patients

OBJECTIVE To assess the prevalence, presentation, and risk factors of pentami-dine-induced dysglycernia. RESEARCH DESIGN AND METHODS Blood glucose values were screened in 244 consecutive immunocompromised patients with Pneumocystis carinii pneumonia: 116 being treated with cotrimoxazole and 128 others with pentamidine. RESULTS Two cotrimoxazole patients developed diabetes as a result of necrotiz-ing pancreatitis (1.7%); the others remained euglycemic. Forty-eight pentamidine-treated patients (38.5%) developed severe glucose homeostasis disorders: hypoglyce-mia in 7, hypoglycemia and then diabetes in 18, and diabetes alone in 23 (P < 0.001 vs. the cotrimoxazole group). Hypoglycemia was early, sudden, often recurrent, and life-threatening, associated with inappropriately high insulin levels in plasma; the B-cell response to stimuli was poor. Of the 41 diabetic patients, 26 required insulin therapy; their plasma C-peptide levels were lower than normal, and the B-cell secretory responses to stimuli were poor. Islet cell antibodies, insulin antibodies, and insulitis were not detected. The pentamidine-treated dysglycemic patients differed from their euglycemic counterparts by higher pentamidine doses (P < 0.001), higher plasma creatinine levels (P < 0.001), and more severe anoxia (P < 0.05) and shock (P < 0.001). Most of them had received pentamidine mesylate parenterally (n = 36; 75%); six others received the isethionate salt and six exclusively pentamidine aerosols. CONCLUSIONS Pentamidine-induced dysglycemic accidents are primarily due to inappropriate insulin release and toxicity to the islet B-cells. Drug accumulation due to excessive doses, iterative courses, and/or renal impairment is the determining risk factor.

No Major Role for the CTLA-4 Gene in the Association of Autoimmune Thyroid Disease With IDDM

IDDM is a T-cell-mediated autoimmune disease depending on both genetic and environmental susceptibility factors. The HLA class II region (IDDM1) and the insulin promoter region (IDDM2) account for -50 and 10%, respectively, of IDDM genetic risk. At least 15 other IDDM susceptibility markers have been identified by genomewide scanning studies (1). One must consider that IDDM is a heterogeneous disorder that can vary in terms of age at clinical onset, duration of hyperglycemia before strict insulin dependency, existence of familial aggregation, occurrence of complications, and presence of extrapancreatic autoimmune diseases, so that different genes might influence the course or the presentation of the disease. The CTLA-4 gene, which has been mapped to the IDDM12 locus (2q33), is a good candidate gene in IDDM (2). Apart from recognition of major histocompatibility complex (MHC)/peptide complex by the T-cell receptor (TCR), T-cell activation requires a co-stimulatory signal mediated by CD28/B7 interaction. The CTLA-4 gene encodes a T-cell surface molecule whose binding to the B7 molecule on antigen-presenting cell delivers a negative signal to the T-cell and can mediate its apoptosis (3). Thus, CTLA-4 expression on T-cells might well influence the course of an ongoing immune process. CTLA4-deficient mice develop a severe lymphoproliferative disease with multiorgan lymphocytic infiltration and tissue destruction, including the pancreas (4,5). In the same respect, the blockade of the CD28/B7 co-stimulatory signal prevents the occurrence of diabetes but not of insulitis in the NOD mouse model, suggesting that it either promotes a shift of the immune response or makes an additional signal unavailable for the final destructive stage (6). Recently, linkage to IDDM of a point mutation in exon 1 of CTLA-4 (position 49 A/G) leading to a Thr/Ala substitution in the leader peptide has been

Plasma C-peptide levels and clinical remissions in recent-onset type I diabetic patients treated with cyclosporin A and insulin.

Remission from insulin dependency in insulin-treated recent-onset type I (insulin-dependent) diabetic patients can result from a partial recovery of insulin secretion, an improvement in tissue sensitivity to insulin, or both. The same hypothesis must be analyzed when remission occurs in cyclosporin A (CsA)-treated patients. In this study, plasma C-peptide levels were serially measured in the basal state and after stimulation in 219 recent-onset type I diabetic patients; 129 received CsA, and all patients were similarly monitored and insulin treated. The results were analyzed in view of the occurrence of remission. Remission was defined as good metabolic control in the absence of hypoglycemic treatment for ≥1 mo. Remission occurred in 44% of the CsA-treated group and lasted for mean ± SE 10.0 ± 0.9 mo vs. 21.6% in the non-CsA-treated group with a duration of 4.4 ± 0.8 mo. Plasma C-peptide levels were initially dramatically lower than normal in both groups in the basal and stimulated states. C-peptide levels increased significantly later, at 3 and 6 mo, in both groups. C-peptide values were proportional to the rates of remission in both groups. In the non-CsA-treated group, C-peptide levels later decreased, and these patients inexorably relapsed to insulin dependency. In contrast, in the CsA-treated group, the initial recovery in insulin secretory capacity was maintained over the 18–24 mo of the study. Furthermore, higher remission rates and longer-lasting remission were obtained in patients who reached higher C-peptide levels at the 3rd mo of treatment. Conversely, a progressive decrease in C-peptide values followed interuption of CsA treatment in some patients who had previously maintained some C-peptide secretory capacity. Our results support the hypothesis of an efficient suspension by CsA of the progressive deterioration of islets. The maintenance of β-cell secretory capacity by CsA plus insulin was better than by insulin alone. However, C-peptide concentrations remained lower than normal in patients in remission. An improvement in sensitivity to insulin may have also contributed to the development of remission.

Phenformin-induced Lactic Acidosis in Diabetic Patients

Eighteen diabetic patients with lactic acidosis (L.A.) were analyzed for possible causal factors, metabolic changes, and efficacy of treatment. An antecedent phenformin therapy was performed in fifteen cases and was associated with renal insufficiency in ten cases and liver disease in eight cases. Tissular anoxia of primary hemodynamic or respiratory origin was absent in all cases. The severe metabolic acidosis (pH = 6.93 ± 0,03 ; HCO3¯ = 6 ± 1 mM ; PaCO2 = 18 ± 2 mm. Hg) and hyperlactatemia (14.2 ± 0.3 mM) were associated with high lactate/pyruvate ratio (70 ± 22). High alanine levels (up to 4.6 mM) were measured in some of these patients. High beta-hydroxybutyrate levels were sometimes measured (up to 7.6 mM), and substantial amounts of acetoacetate were also detected in twelve cases. Glucagon level was always increased (1,050 ± 240 pg./ml.), and insulin/glucagon ratio was low. Cortisol (49 ± 10 µg./100 ml.) and HGH (10.8 ± 2.6 ng./ml.) were also elevated. Increased plasma levels of phenformin were measured in five L.A. diabetic subjects (50 ± 5 µ./ml.) by comparison with other phenformin-treated diabetic subjects. The specificity of the assay was investigated, and phenformin metabolites were characterized by thin-layer chromatography. For the treatment of L.A., adjunction of dialysis and furosemide improved the efficacy of early and massive sodium bicarbonate infusion. It is suggested that accumulation of phenformin via renal insufficiency plays a determinant role in causing L.A. through an impairment of lactate metabolism in the liver. An accelerated epuration of the drug may be helpful. in therapy of L.A. Phenformin treatment should be avoided in case of renal and/or liver insufficiency.

Role of the Insulin/Glucagon Ratio in the Changes of Hepatic Metabolism During Development of the Rat

The insulin/glucagon ratio was measured during the embryologic development of the rat and its changes were correlated with the variation in the diet and in hepatic metabolism. During late fetal life, the high carbohydrate diet and the high insulin/glucagon ratio are appropriate for an organism whose metabolism is set at a maximally anabolic mode: growth, protein synthesis, liver glycogen storage. After birth and during suckling, rapid growth can coexist with active gluconeogenesis and ketogenesis in the liver and with a low insulin/glucagon ratio, since the mother’s milk is a high fat diet. The weaning period is characterized by a replacement of this high fat by a high carbohydrate diet. Suppression of gluconeogenesis and ketogenesis and appearance of an active lipogenesis in the liver occur, with a rise in insulin/glucagon ratio. These data suggest that glucagon plays a more important role during early life than during adult life.

Anti-islet Cellular and Humoral Immunity, T-Cell Subsets, and Thymic Function in Type I Diabetes

Peripheral lymphocyte subsets were enumerated, using OKT monoclonal sera, in 56 diabetic (43 adults and 13 children) and 20 control subjects. Concomitantly, anti-islet humoral and cellular immunity was tested in vitro and serum thymulin level was measured. In the newly diagnosed patients (<30 days; 18 cases), the percent of OKT4+ and OKT8+ cells was reduced, the OKT8+ depletion being particularly pronounced in children. Tests for cellular immunity were positive in 83% of the newly diagnosed diabetic subjects and anti-islet cytotoxic antibodies were detected in 50%. The serum thymulin level was decreased in 2 children. Later on in the course of the disease, a marked reduction in OKT3+, OKT4+ and OKT8+ cell percentage was observed, the mean OKT4/OKT8 ratio being normal or lower than normal. The percent of antibody-positive sera rose to 64%, while anti-islet cellular immunity was detectable in 54%. When extrapancreatic manifestations of probable autoimmune nature were present, anti-islet cellular immunity was detected in 100% of cases, accompanied by cytotoxic antibodies in 54%. (1) the magnitude of T-cell depletion and/or imbalance in diabetic subjects depended mainly on the duration of the disease, (2) anti-islet cellular immunity was the anomaly most frequently detectable, and (3) a decrease in serum thymulin level was infrequently detected.

Time course of islet cell antibodies in diabetic and nondiabetic BB rats.

The BB rat develops a spontaneous type I diabetic syndrome with anti-islet autoimmunity. Sera from diabetic and nondiabetic BB rats (from diabetes-prone litters), nondiabetic BB rats (from low-risk lines), and nondiabetes-prone Sprague-Dawley rats were collected twice a week from age 40 days to 160 days. Sera were tested for: (1) complement-dependent toxicity to 51Crlabeled islet cells in vitro; (2) immunoglobulin binding to RIN-5 F insulinoma cells; and (3) ability to selectively suppress insulin secretion from normal islets in vitro. All sera from rats that subsequently became diabetic or glucose-intolerant were toxic to islet cells from various rat strains in the presence of complement. They were toxic neither to hepatocytes nor to fibroblasts. The toxic potency was associated with the globulin fraction. It was, in most cases, maximal either before or immediately after the onset of the disease. Sera from the nondiabetes-susceptible BB rats and the rats which, in diabetes-prone litters, died too early to be classified tended toward greater toxicity to islets. Immunoglobulins from diabetic sera bound to RIN-5 F cells more than did the serum globulins from other groups, their maximal binding capacity occurring afterthe onset of diabetes. Furthermore, BB diabetic sera were capable of selectively inhibiting the insulin secretion from normal rat islets in vitro either in the presence or, in some cases, in theabsence of complement. The A- and D-cell functions were not suppressed. The combination of such results suggests the presence of one or more antibodies capable of binding to beta cells, inhibiting their function, and inducing their lysis. These antibodies may contribute to the beta cell disruption in this model of diabetes.