Robert C. McEvoy

Targeted Deletion of the Vgf Gene Indicates that the Encoded Secretory Peptide Precursor Plays a Novel Role in the Regulation of Energy Balance

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Metabolic Control and Quality-of-Life Self-Assessment in Adolescents With IDDM

OBJECTIVE To examine the relation between metabolic control and self-assessed quality of life in adolescents with IDDM. RESEARCH DESIGN AND METHODS The Diabetes Quality of Life (DQOL) questionnaire for youths was given to 69 subjects with IDDM aged 10-20 years at the time of their outpatient visit. Subjects with IDDM of < 1 years duration or with documented psychotic disorder or mental retardation were excluded. Metabolic control was assessed by the mean HbA1c during the preceding year (long-term), by a single HbA1c at the time of the visit (short-term), and by the number of acute events related to IDDM in the preceding year. RESULTS The DQOL score correlated with mean HbA1c (β = 6.13, R2 = 0.22, P = 0.0122) and single HbA1c (β = 3.94, R2 = 0.18, P = 0.05). Self-health assessment was the best predictor of DQOL score (β = −44.42, R2 = 0.45, P < 0.0001). The Worries subscale score on DQOL correlated with the occurrence of acute events (β = 6.97, R2 = 0.2, P = 0.006), but did not correlate with either HbA1c level. Correlations of mean HbA1c with the predictors were stronger than the correlations of single HbA1c with the same predictors. CONCLUSIONS Metabolic control and quality of life are two important outcomes of IDDM care. In our study, adolescents in better metabolic control report better quality of life. Both components need to be addressed in developing successful diabetes treatment strategies for adolescents with IDDM.

Pancreatic Insulin-, Glucagon-, and Somatostatin-Positive Islet Cell Populations during the Perinatal Development of the Rat

Insulin, glucagon and somatostatin concentration were determined in the developing pancreas between 16 days post coitum and 10 days postnatal. Plasma insulin and glucagon concentrations were also measured. Pancreatic insulin content increased rapidly during the perinatal period and peaked in concentration on day 6 postnatal. Glucagon content also increased rapidly with the largest percentage increase occurring around the time of birth. The largest increase in somatostatin also occurred postnatally. Plasma insulin and glucagon concentrations were very high during the late fetal period. the insulin concentraton fell immediately after birth and rose slowly toward adult levels during the remainder of the period examined. Plasma glucagon was high during the entire neonatal period. The degree of differentiation of the islet cells, as estimated by their content of specific hormone products, is discussed in relation to the changes in cell volume reported n the preceding paper.

Anti-Insulin Antibodies in Children With Type I Diabetes Mellitus: Genetic Regulation of Production and Presence at Diagnosis Before Insulin Replacement

We evaluated the production of antibodies against insulin in a genetically well-defined population. In the first study, 124 young patients with type I diabetes for longer than 6 mo were included. Anti-insulin antibodies were detected by polyethyleneglycol (PEG) precipitation after incubation of acidified, charcoal-stripped sera with 125I-labeled pork insulin andwere expressed as microunits insulin bound per milliliter whole serum. For comparison, the patients were divided into six groups based on HLA DR antigens: 3/3, 3 /−, 4/4, 4 /− , 3/4, and − / − (– is non-DR3 or -DR4). The mean age of the patients was 14.7 ± 0.5 yr; the duration of diabetes was 5.8 ± 0.4 yr; and the glucose control, as measured byhemoglobin A1c was average (7.6 ± 0.2%). There were no significant differences in any of these parameters among the patients in any of the HLA DR groups. Patients expressing DR3/3 had significantly lower insulin binding than the rest of the groups (2.5 ± 0.4 vs. 13.6 ± 1.4 μU/ml, P < 0.0001). Patients with DR3/− did not differ in insulin-binding capacity from the other groups. The type of insulin used for replacement was not correlated with the serum insulin-binding capacity. In a second study, sera from 48 children, newlydiagnosed with type I diabetes, were examined for the presence of insulin binding before treatment with exogenous insulin and compared with sera from 80 children without diabetes or a family history of diabetes and from 103 unaffected HLA-identical or haploidentical siblings of a child withtype I diabetes. The sera from the newly diagnosed diabetics bound significantly more insulin in the assay (1.02 ± 0.06 μUsol;ml than the sera from either of the two control groups (0.52 ± 0.01 and 0.51 ± 0.02%, respectively, P = 0.0012). Sera from the newly diagnosed girls bound significantly more insulin than did those from the boys (1.13 ± 0.09 vs. 0.89 ± 0.07%, P < 0.02), and there was a highly significant (P = 0.0017) negative correlation between the age at onset of diabetes and the insulin bound by serum before insulin treatment. Finally, sera from six siblings of type I diabetics who became diabetic themselves were obtained when these children were metabolically normal and again just before insulin treatment was begun. Four of these children had a significant increase in their serum insulin binding from normal values to clearly elevated levels over intervals before metabolic decompensation. These data support the following conclusions. The reduced insulin binding previously observed in type I diabetics expressing HLA DR3 can be entirely attributed to the very low binding in individuals homozygous for this antigen. Thus, human antibody response to exogenous insulin is probably regulated by a dominant gene in negative linkage disequilibrium with HLA DR3. Most younger patients have detectable insulin binding at diagnosis of type I diabetes. The level of binding is reduced in older patients, suggesting either that younger children have a better response to insulin released from injured β-cells in a form that is antigenic or that younger children have animmaturity in the clearance of antigen-antibody complexes. Our preliminary data suggest that the elevation in insulin binding is a late event in the pathogenesis of type I diabetes, occurring soon before the symptoms are present.

Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells

The liver is an attractive target organ for insulin gene expression in type 1 diabetes as it contains appropriate cellular mechanisms of regulated gene expression in response to blood glucose and insulin. We hypothesize that insulin production regulated by both glucose and insulin may be achieved using the promoter of the glucose 6-phosphatase gene (G6Pase), the expression of which in the liver is induced by glucose and suppressed by insulin. Recombinant adenoviral vectors expressing the reporter gene CAT or insulin under transcriptional direction of the G6Pase promoter were constructed. Glucose-stimulated as well as self-limiting insulin production was achieved in vector-transduced hepatoma cells in which expression of the insulin gene was controlled by the G6Pase promoter. While insulin strongly inhibited the G6Pase promoter activity under low glucose conditions, its inhibitory capacity was attenuated when glucose levels were elevated. At the physiologic glucose level of 5.5 mM glucose, vector-transduced hepatoma cells produced a self-limited level of insulin at approximately 0.2–0.3 ng/ml, which is within the range of fasting levels of insulin in normal animals. These results indicate that the G6Pase promoter possesses desirable features and may be developed for regulated hepatic insulin gene expression in type 1 diabetes.

Hepatic insulin expression improves glycemic control in type 1 diabetic rats

Low levels of hepatic insulin production have been shown to prevent lethal ketoacidosis associated with type 1 diabetes. To assess the beneficial effects of sustained hepatic production of insulin on glycemic control in type 1 diabetes, we have employed the adenovirus-mediated gene delivery system to transfer an engineered rat preproinsulin gene to the livers of streptozotocin-induced diabetic nude rats. Hepatic insulin production resulted in the reduction of blood glucose in treated diabetic rats, the degree of blood glucose reduction correlated with both the vector dose and the level of hepatic insulin expression. At moderate vector doses, 0.3-0.7 ng/ml of plasma insulin was produced in treated diabetic animals, resulting in significant reduction of nonfasting hyperglycemia and improvement in glucose tolerance. Furthermore, these animals maintained euglycemia after 12-h fast. At higher vector doses, greater than 1 ng/ml of plasma insulin was produced, completely reversing nonfasting hyperglycemia in treated rats. However, all of the treated animals developed severe hypoglycemia upon fasting. This study has defined the maximal tolerable level of hepatic insulin production that is sufficient to reduce the degree and ameliorate the adverse effects of nonfasting hyperglycemia without risk of fasting hypoglycemia in type 1 diabetic rats.

Adenovirus-Mediated Expression of Glucokinase in the Liver as an Adjuvant Treatment for Type 1 Diabetes

Glucokinase (GK) plays a crucial role in hepatic glucose disposal. Its activity is decreased in patients with maturity-onset diabetes of the young and in some animal models of diabetes. We investigated the feasibility of manipulating GK expression as an adjuvant treatment for type 1 diabetes, using an E1/E3-deleted adenoviral vector (Ad.EF1(alpha)GK) delivered to the liver of streptozotocin-induced type 1 diabetic rats. First, we studied the metabolic impact of constitutive glucokinase expression in the absence of insulin. Normal blood glucose levels were observed after gene transfer, and glucose tolerance was substantially enhanced compared with diabetic control animals, suggesting that hepatic GK expression is a feasible mechanism to enhance glucose disposal. In a second study we administered Ad.EF1(alpha)GK together with subcutaneous insulin injections to determine whether the combined action of insulin plus GK activity would provide better glucose homeostasis than insulin treatment alone. This combination approach resulted in constant, near-normal glucose values under fed conditions. Furthermore, the animals stayed in the normoglycemic range after an overnight fast, indicating that the risk to develop hypoglycemia is not increased by expression of GK. Alterations of other metabolic routes were observed, suggesting that insulin-regulated expression of GK may be necessary to use the strategy as a treatment of type 1 diabetes.

Children at High Risk of Diabetes Mellitus: New York Studies of Families with Diabetes and of Children with Congenital Rubella Syndrome

The frequency of Type I, or insulin-dependent mellitus (IDDM) in the general population is approximately 1 in 600 individuals. By contrast, in families where one child has IDDM, the likelihood that another child will develop IDDM is about 100 times higher. This population, at high risk for the development of IDDM, seemed an ideal group in which to identify some of the factors which might precede the development of clinically apparent IDDM. IDDM is clearly a multifactorial disease. The existence of a gene or genes predisposing to diabetes and mapping within or very near the HLA locus on chromosome six has been suggested by several different investigators. In addition, environmental agents also appear to be needed to “trigger” the predisposition towards overt disease since concordance for monozygotic twins is estimated at only approximately 30%. Convincing evidence has accumulated over the last several years that the mechanism of the expression of the genetic susceptibility is an autoimmune destruction of the pancreatic beta cells.

Human insulin B chain but not A chain decreases the rate of diabetes in BB rats

The autoimmune response seen in insulin-dependent diabetes mellitus (IDDM) includes a humoral immune response against human insulin. Early insulin treatment has been used to prevent IDDM in the rodent models of IDDM, and a prevention trial is underway in humans. The metabolic effects of insulin may not be involved in this prevention since, in NOD mice, the use of metabolically inert human insulin B chain was effective. We wished to ascertain whether immunization of diabetes-prone BB/W rats with insulin B chain, A chain, or both could alter the incidence of diabetes. Immunizations began by 30 days of age and the rats were followed until 120 days of age. Only immunization with insulin B chain plus adjuvant was effective in reducing the rate of diabetes. All immunization frequencies were effective, but a significantly lower rate of diabetes was achieved with injections every week. All of the doses tested resulted in significantly lower rates of diabetes. These data confirm in the BB rat model that immunization with insulin B chain in the presence of adjuvant can reduce diabetes incidence. The absence of any metabolic effect of B chain and the requirement for adjuvant suggest that this effect is mediated via modulation of the autoimmune response.

Transplantation of Fetal Rat Islets Into the Cerebral Ventricles of Alloxan-Diabetic Rats: Amelioration of Diabetes by Syngeneic but not Allogeneic Islets

Syngeneic fetal rat islets were isolated and transplanted into alloxan-diabetic inbred male Lewis rats. The effect of transplantation of islets into the cerebral ventricles on the diabetic state of the recipients was compared with that of the conventional transplantation of islets homeotypically into the liver via the portal vein. Fourteen of fourteen rats surviving after stereo-taxic implantation of islets into the ventricles returned to normoglycemia; normoglycemia has been maintained for up to 34 wk. Glucose tolerance tests revealed an improved, although not completely normalized, pattern. Histologie examination of the brains of these recipients revealed clusters of intact islets in the ventricle. These data provided a physiologic basis for further investigation of the immunologically privileged nature of the intraventricular space as a site for implantation of allogeneic pancreatic islets. Islets from Wistar-Furth rats (major histocompatibility difference) or Fischer 344 rats (minor histocompatibility difference) were transplanted into the ventricles of alloxan-diabetic Lewis rats. There were only small and unsus-tained changes in body weight and blood and urine glucose of any of the rats receiving the allogeneic islets. Histologie examination of the ventricles of these rats 3 wk after transplantation revealed only glial scar tissue. These data suggest that the cerebral ventricles cannot serve as a privileged site for islet transplantation, at least using the type of islet preparation employed in these experiments.