bluuebunny/crossref_metadata_2025 huggingface dataset:
Early development of beta-cells is impaired in the GK rat model of type 2 diabetes.
Authors: Miralles F , Portha B
Published: March 2007

The Goto-Kakisaki (GK) rat is a genetic model of type 2 diabetes obtained by selective inbreeding of mildly glucose-intolerant Wistar rats. Previous studies have shown that at birth, the beta-cell mass of the GK rat is severely reduced compared with that of the Wistar rat. Therefore, beta-cell deficit could be the primary defect leading to type 2 diabetes in this model. To identify the abnormality at the origin of the beta-cell mass deficit, we compared the fetal development of GK and Wistar rats. Our study reveals that during early development (embryonic day 12-14 [E12-14]), GK fetuses present a delayed global growth that progressively recovers: at birth, no size or weight difference persists. However, from E18 onward, the weight and DNA content of the pancreas and liver are reduced by 30% in the GK fetuses. Cell proliferation is reduced in the GK pancreas from E16 to E20. Whereas apoptotic cells are scarce in the Wistar fetal pancreas, a wave of apoptosis from E16 to E18 was detected in the GK pancreas. Analysis of pancreas differentiation revealed that from E12 to E14, there are no significant differences in the number of alpha- and beta-cells between the GK and Wistar pancreas. However, by E16, the average number of beta-cells in the GK pancreas represents only 50% that of the Wistar pancreas, and this difference persists until birth. The number of alpha-cells was reduced by 25% from E18 to E21. To determine whether the defect in GK pancreas development depends on intrinsic pancreatic factors or on endocrine extrapancreatic factors, we performed in vitro cultures of E12 pancreatic rudiments. The cultures show that in vitro, the growth and endocrine differentiation of the GK and Wistar pancreatic rudiments are identical. Thus, impaired development of the GK pancreas probably results from insufficiency of extrapancreatic factor(s) necessary for the growth and survival of fetal pancreatic cells.
Measurements of Cytoplasmic Ca2+ in Islet Cell Clusters Show That Glucose Rapidly Recruits β-Cells and Gradually Increases the Individual Cell Response
Authors: Jonkers Françoise C. , Henquin Jean-Claude
Published: March 2007

The proportion of isolated single β-cells developing a metabolic, biosynthetic, or secretory response increases with glucose concentration (recruitment). It is unclear whether recruitment persists in situ when β-cells are coupled. We therefore measured the cytoplasmic free Ca2+ correction ([Ca2+]i) (the triggering signal of glucose-induced insulin secretion) in mouse islet single cells or clusters cultured for 1–2 days. In single cells, the threshold glucose concentration ranged between 6 and 10 mmol/l, at which concentration a maximum of ∼65% responsive cells was reached. Only 13% of the cells did not respond to glucose plus tolbutamide. The proportion of clusters showing a [Ca2+]i rise increased from ∼20 to 95% between 6 and 10 mmol/l glucose, indicating that the threshold sensitivity to glucose differs between clusters. Within responsive clusters, 75% of the cells were active at 6 mmol/l glucose and 95–100% at 8–10 mmol/l glucose, indicating that individual cell recruitment is not prominent within clusters; in clusters responding to glucose, all or almost all cells participated in the response. Independently of cell recruitment, glucose gradually augmented the magnitude of the average [Ca2+]i rise in individual cells, whether isolated or associated in clusters. When insulin secretion was measured simultaneously with [Ca2+]i, a good temporal and quantitative correlation was found between both events. However, β-cell recruitment was maximal at 10 mmol/l glucose, whereas insulin secretion increased up to 15–20 mmol/l glucose. In conclusion, β-cell recruitment by glucose can occur at the stage of the [Ca2+]i response. However, this type of recruitment is restricted to a narrow range of glucose concentrations, particularly when β-cell association decreases the heterogeneity of the responses. Glucose-induced insulin secretion by islets, therefore, cannot entirely be ascribed to recruitment of β-cells to generate a [Ca2+]i response. Modulation of the amplitude of the [Ca2+]i response and of the action of Ca2+ on exocytosis (amplifying actions of glucose) may be more important.
Effect of Vagal Cooling on the Counterregulatory Response to Hypoglycemia Induced by a Low Dose of Insulin in the Conscious Dog
Authors: Cardin Sylvain , Jackson Patricia A. , Edgerton Dale S. , Neal Doss W. , Coffey Christopher S. , Cherrington Alan D.
Published: March 2007

We previously demonstrated, using a nerve-cooling technique, that the vagus nerves are not essential for the counterregulatory response to hypoglycemia caused by high levels of insulin. Because high insulin levels per se augment the central nervous system response to hypoglycemia, the question arises whether afferent nerve fibers traveling along the vagus nerves would play a role in the defense of hypoglycemia in the presence of a more moderate insulin level. To address this issue, we studied two groups of conscious 18-h–fasted dogs with cooling coils previously placed on both vagus nerves. Each study consisted of a 100-min equilibration period, a 40-min basal period, and a 150-min hypoglycemic period. Glucose was lowered using a glycogen phosphorylase inhibitor and a low dose of insulin infused into the portal vein (0.7 mU · kg−1 · min−1). The arterial plasma insulin level increased to 15 ± 2 μU/ml and the plasma glucose level fell to a plateau of 57 ± 3 mg/dl in both groups. The vagal cooling coils were perfused with a 37°C (SHAM COOL; n = 7) or a −20°C (COOL; n = 7) ethanol solution for the last 90 min of the study to block parasympathetic afferent fibers. Vagal cooling caused a marked increase in the heart rate and blocked the hypoglycemia-induced increase in the arterial pancreatic polypeptide level. The average increments in glucagon (pg/ml), epinephrine (pg/ml), norepinephrine (pg/ml), cortisol (μg/dl), glucose production (mg · kg–1 · min–1), and glycerol (μmol/l) in the SHAM COOL group were 53 ± 9, 625 ± 186, 131 ± 48, 4.63 ± 1.05, −0.79 ± 0.24, and 101 ± 18, respectively, and in the COOL group, the increments were 39 ± 7, 837 ± 235, 93 ± 39, 6.28 ± 1.03 (P < 0.05), −0.80 ± 0.20, and 73 ± 29, respectively. Based on these data, we conclude that, even in the absence of high insulin concentrations, afferent signaling via the vagus nerves is not required for a normal counterregulatory response to hypoglycemia.
GLUT4 Overexpression in db/db Mice Dose-Dependently Ameliorates Diabetes But Is Not a Lifelong Cure
Authors: Brozinick Joseph T. , McCoid Scott C. , Reynolds Thomas H. , Nardone Nancy A. , Hargrove Diane M. , Stevenson Ralph W. , Cushman Samuel W. , Gibbs E. Michael
Published: March 2007

We previously reported that overexpression of GLUT4 in lean, nondiabetic C57BL/KsJ-leprdb/+ (db/+) mice resulted in improved glucose tolerance associated with increased basal and insulin-stimulated glucose transport in isolated skeletal muscle. We used the diabetic (db/db) litter mates of these mice to examine the effects of GLUT4 overexpression on in vivo glucose utilization and on in vitro glucose transport and GLUT4 translocation in diabetic mice. We examined in vivo glucose disposal by oral glucose challenge and hyperinsulinemic-hyperglycemic clamps. We also evaluated the in vitro relationship between glucose transport activity and cell surface GLUT4 levels as assessed by photolabeling with the membrane-impermeant reagent 2-N-(4-(1-azi-2,2,2-trifluoroethyl)benzoyl)-1,3-bis(d-mannose-4-yloxy)-2-propylamine in extensor digitorum longus (EDL) muscles. All parameters were examined as functions of animal age and the level of GLUT4 overexpression. In young mice (age 10–12 weeks), both lower (two- to threefold) and higher (four- to fivefold) levels of GLUT4 overexpression were associated with improved glucose tolerance compared to age-matched nontransgenic (NTG) mice. However, glucose tolerance deteriorated with age in db/db mice, although less rapidly in transgenic mice expressing the higher level of GLUT4. Glucose infusion rates during hyperinsulinemic-hyperglycemic clamps were increased with GLUT4 overexpression, compared with NTG mice in both lower and higher levels of GLUT4 overexpression, even in the older mice. Surprisingly, isolated EDL muscles from diabetic db/db mice did not exhibit alterations in either basal or insulin-stimulated glucose transport activity or cell surface GLUT4 compared to nondiabetic db/+ mice. Furthermore, both GLUT4 overexpression levels and animal age are associated with increased basal and insulin-stimulated glucose transport activities and cell surface GLUT4. However, the observed increased glucose transport activity in older db/db mice was not accompanied by an equivalent increase in cell surface GLUT4 compared to younger animals. Thus, although in vivo glucose tolerance is improved with GLUT4 overexpression in young animals, it deteriorates with age; in contrast, insulin responsiveness as assessed by the clamp technique remains improved with GLUT4 overexpression, as does in vitro insulin action. In summary, despite an impairment in whole-body glucose tolerance, skeletal muscle of the old transgenic GLUT4 db/db mice is still insulin responsive in vitro and in vivo.
A Genome-Wide Scan for Abdominal Fat Assessed by Computed Tomography in the Québec Family Study
Authors: Pérusse Louis , Rice Treva , Chagnon Yvon C. , Després Jean-Pierre , Lemieux Simone , Roy Sonia , Lacaille Michel , Ho-Kim My-Ann , Chagnon Monique , Province Michael A. , Rao D.C. , Bouchard Claude
Published: March 2007

To identify chromosomal regions harboring genes influencing the propensity to store fat in the abdominal area, a genome-wide scan for abdominal fat was performed in the Québec Family Study. Cross-sectional areas of the amount of abdominal total fat (ATF) and abdominal visceral fat (AVF) were assessed from a computed tomography scan taken at L4-L5 in 521 adult subjects. Abdominal subcutaneous fat (ASF) was obtained by computing the difference between ATF and AVF. The abdominal fat phenotypes were adjusted for age and sex effects as well as for total amount of body fat (kilogram of fat mass) measured by underwater weighing, and the adjusted phenotypes were used in linkage analyses. A total of 293 microsatellite markers spanning the 22 autosomal chromosomes were typed. The average intermarker distance was 11.9 cM. A maximum of 271 sib-pairs were available for single-point (SIBPAL) and 156 families for multipoint variance components (SEGPATH) linkage analyses. The strongest evidence of linkage was found on chromosome 12q24.3 between marker D12S2078 and ASF (logarithm of odds [LOD] = 2.88). Another marker (D12S1045) located within 2 cM of D12S2078 also provided evidence of sib-pair linkage with ASF (P = 0.019), ATF (P = 0.015), and AVF (P = 0.0007). Other regions with highly suggestive evidence (P < 0.0023 or LOD ≥1.75) of multipoint linkage and evidence (P < 0.05) of single-point linkage, all for ASF, included chromosomes 1p11.2, 4q32.1, 9q22.1, 12q22-q23, and 17q21.1. Three of these loci (1p11.2, 9q22.1, and 17q21.1) are close to genes involved in the regulation of sex steroid levels, whereas two others (4q32.1 and 17q21.1) are in the proximity of genes involved in the regulation of food intake. This first genome-wide scan for abdominal fat assessed by computed tomography indicates that there may be several loci determining the propensity to store fat in the abdominal depot and that some of these loci may influence the development of diabetes in obese subjects.
Autoantibody Response to CD38 in Caucasian Patients With Type 1 and Type 2 Diabetes
Authors: Mallone Roberto , Ortolan Erika , Baj Germano , Funaro Ada , Giunti Sara , Lillaz Emma , Saccucci Franca , Cassader Maurizio , Cavallo-Perin Paolo , Malavasi Fabio
Published: March 2007

Insulin secretion is one of the functions mediated by CD38, a nonlineage pleiotropic cell surface receptor. The molecule is the target of an autoimmune response, because serum autoantibodies (aAbs) to CD38 have been detected in diabetic patients. In the healthy Caucasian population, the CD38 gene is bi-allelic (86% CD38*B and 14% CD38*A), whereas an Arg140Trp mutation has been identified in Japanese diabetic patients. We investigated the relationship between CD38 and diabetes in Caucasian patients by characterizing anti-CD38 aAbs in terms of prevalence and function (agonistic/nonagonistic activity) and by exploring the potential influence of the CD38 genetic background. A novel enzymatic immunoassay, using recombinant soluble CD38 as the target antigen, was developed for the analysis of anti-CD38 aAb titers. Sera from 19.15% of type 1 and 16.67% of type 2 diabetic patients were positive. The majority of anti-CD38 aAbs (57.14%) displayed agonistic properties, i.e., they demonstrated the capability to trigger Ca2+ release in lymphocytic cell lines. In agreement with these functional features, the presence of anti-CD38 aAbs in type 2 diabetic patients was associated with significantly higher levels of fasting plasma C-peptide and insulin, as compared with anti-CD38– counterparts. No diabetic subject carrying the Arg140Trp mutation and no preferential association between diabetes or aAb status and the CD38*A allele was found in the study population. These results show the significance of anti-CD38 aAbs as a new diagnostic marker of β-cell autoimmunity in diabetes. Moreover, the prevalent agonistic activity of these aAbs suggests that they could mediate relevant effects on target cells by means of Ca2+ mobilization.
Uncoupling Protein 2: A Possible Link Between Fatty Acid Excess and Impaired Glucose-Induced Insulin Secretion?
Authors: Lameloise Nathalie , Muzzin Patrick , Prentki Marc , Assimacopoulos-Jeannet Françoise
Published: March 2007

The mechanism by which long-term exposure of the β-cell to elevated concentrations of fatty acid alters glucose-induced insulin secretion has been examined. Exposure of INS-1 β-cells to 0.4 mmol/l oleate for 72 h increased basal insulin secretion and decreased insulin release in response to high glucose, but not in response to agents acting at the level of the KATP channel (tolbutamide) or beyond (elevated KCl). This also suppressed the glucose-induced increase in the cellular ATP-to-ADP ratio. The depolarization of the plasma membrane promoted by glucose was decreased after oleate exposure, whereas the response to KCl was unchanged. Cells exposed to free fatty acids displayed a lower mitochondrial membrane potential and a decreased glucose-induced hyperpolarization. The possible implication of uncoupling protein (UCP)-2 in the altered secretory response was examined by measuring UCP2 gene expression after chronic exposure of the cells to fatty acids. UCP2 mRNA and protein were increased twofold by oleate. Palmitate and the nonoxidizable fatty acid bromopalmitate had similar effects on UCP2 mRNA, suggesting that UCP2 gene induction by fatty acids does not require their metabolism. The data are compatible with a role of UCP2 and partial mitochondrial uncoupling in the decreased secretory response to glucose observed after chronic exposure of the β-cell to elevated fatty acids, and suggest that the expression and/or activity of the protein may modulate insulin secretion in response to glucose.
A Novel Small Molecule That Directly Sensitizes the Insulin Receptor In Vitro and In Vivo
Authors: Manchem Vara Prasad , Goldfine Ira D. , Kohanski Ronald A. , Cristobal Cristina P. , Lum Robert T. , Schow Steven R. , Shi Songyuan , Spevak Wayne R. , Laborde Edgardo , Toavs Deborah K. , Villar Hugo O. , Wick Michael M. , Kozlowski Michael R.
Published: March 2007

Insulin resistance, an important feature of type 2 diabetes, is manifested as attenuated insulin receptor (IR) signaling in response to insulin binding. A drug that promotes the initiation of IR signaling by enhancing IR autophosphorylation should, therefore, be useful for treating type 2 diabetes. This report describes the effect of a small molecule IR sensitizer, TLK16998, on IR signaling. This compound activated the tyrosine kinase domain of the IR β-subunit at concentrations of 1 μmol/l or less but had no effect on insulin binding to the IR α-subunit even at much higher concentrations. TLK16998 alone had no effect on IR signaling in mouse 3T3-L1 adipocytes but, at concentrations as low as 3.2 μmol/l, enhanced the effects of insulin on the phosphorylation of the IR β-subunit and IR substrate 1, and on the amount of phosphatidylinositol 3-kinase that coimmunoprecipitated with IRS-1. Phosphopeptide mapping revealed that the effect of TLK16998 on the IR was associated with increased tyrosine phosphorylation of the activation loop of the β-subunit tyrosine kinase domain. TLK16998 also increased the potency of insulin in stimulating 2-deoxy-d-glucose uptake in 3T3-L1 adipocytes, with a detectable effect at 8 μmol/l and a 10-fold increase at 40 μmol/l. In contrast, only small effects were observed on IGF-1–stimulated 2-deoxy-d-glucose uptake. In diabetic mice, TLK16998, at a dose of 10 mg/kg, lowered blood glucose levels for up to 6 h. These results suggest, therefore, that small nonpeptide molecules that directly sensitize the IR may be useful for treating type 2 diabetes.
Familial Aggregation of Coronary Artery Calcium in Families With Type 2 Diabetes
Authors: Wagenknecht Lynne E. , Bowden Donald W. , Carr J. Jeffrey , Langefeld Carl D. , Freedman Barry I. , Rich Stephen S.
Published: March 2007

Type 2 diabetes is widely recognized as a major risk factor for atherosclerotic cardiovascular disease, including subclinical atherosclerosis as measured by noninvasive procedures. However, the role of genetic factors that contribute to various measures of subclinical atherosclerosis is largely unknown. We hypothesize that subclinical atherosclerosis, measured as coronary artery calcification (CAC), will be extensive in individuals with type 2 diabetes and that its presence depends on both genetic and environmental factors. The genetic factors should result in the familial aggregation of CAC. To determine the extent of familial aggregation of CAC in the presence of type 2 diabetes, we studied 122 individuals with type 2 diabetes (mean age 60 years) and 13 individuals without diabetes in 56 families. CAC was measured by fast-gated helical computed tomography. Other measured factors included blood pressure, body size, lipids, HbA1c, and self-reported medical history. To test for an association between CAC and these factors while accounting for the potential familial correlation of CAC, generalized estimating equations were used. CAC was detectable in 80% of individuals with diabetes (median score 84, range 0–5,776). Extent of CAC, adjusted for age, was positively associated with male sex (P = 0.0003), reduced HDL (P = 0.02), albumin-to-creatinine ratio (P = 0.008), and cigarette pack–years (P = 0.03). CAC was also positively associated with a history of angina, myocardial infarction, stroke, and vascular procedures (all P < 0.01). HbA1c and fasting glucose were positively, but nonsignificantly, associated with the extent of CAC (P = 0.14 and 0.08, respectively). CAC, adjusted for age, sex, race, and diabetes status, was heritable (h2 = 0.50; P = 0.009). In multivariate analysis with additional adjustment for HDL, BMI, hypertension, and smoking, h2 = 0.40 (P = 0.038). These results suggest that strong (independent) genetic factors as well as environmental factors contribute to the variance of CAC in individuals with type 2 diabetes. In these data, CAC seems heritable and may serve as an important feature in designing studies to map genes contributing to both atherosclerosis and type 2 diabetes.
Differential Splicing of the IA-2 mRNA in Pancreas and Lymphoid Organs as a Permissive Genetic Mechanism for Autoimmunity Against the IA-2 Type 1 Diabetes Autoantigen
Authors: Diez Juan , Park Yongsoo , Zeller Markus , Brown Douglas , Garza David , Ricordi Camillo , Hutton John , Eisenbarth George S. , Pugliese Alberto
Published: March 2007

Type 1 diabetes results from the autoimmune destruction of pancreatic β-cells in genetically susceptible individuals. Growing evidence suggests that genetically determined variation in the expression of self-antigens in thymus may affect the shaping of the T-cell repertoire and susceptibility to autoimmunity. For example, both allelic variation and parent-of-origin effects influence the thymic expression of insulin (a known type 1 diabetes autoantigen), and insulin gene transcription levels in thymus inversely correlate with susceptibility in both humans and transgenic models. It is unclear why patients lose tolerance to IA-2 (insulinoma-associated tyrosine phosphatase-like protein, or islet cell antigen 512 [ICA512]), especially because IA-2 polymorphisms are not associated with type 1 diabetes. We report that alternative splicing determines differential IA-2 expression in islets compared with thymus and spleen. Islets express full-length mRNA and two alternatively spliced transcripts, whereas thymus and spleen exclusively express an alternatively spliced transcript lacking exon 13. This encodes for the transmembrane (TM) and juxta-membrane (JM) domains that comprise several type 1 diabetes target epitopes, supporting the concept that tolerance to IA-2 epitopes not expressed in lymphoid organs may not be achieved. We propose differential splicing as a regulatory mechanism of gene expression playing a permissive role in the development of autoimmune responses to IA-2. Our findings also show that candidate gene expression studies can help in dissecting the complex genetic determinants of a multifactorial disease such as type 1 diabetes.
The Cephalic Insulin Response to Meal Ingestion in Humans Is Dependent on Both Cholinergic and Noncholinergic Mechanisms and Is Important for Postprandial Glycemia
Authors: Ahrén Bo , Holst Jens J.
Published: March 2007

We studied the mechanisms and physiological relevance of the cephalic insulin response to meal ingestion in 12 healthy women (age 63 ± 0.4 years; BMI 27.7 ± 1.7 kg/m2). The ganglionic antagonist, trimethaphan, which impairs neurotransmission across parasympathetic and sympathetic autonomic ganglia, or atropine or saline was given intravenously during the first 15 min after ingestion of a standard meal (350 kcal). During saline infusion, insulin levels increased during the first 10 min after meal ingestion, whereas the first increase in glucose was evident at 15 min. The preabsorptive 10-min insulin response was reduced by 73 ± 11% by trimethaphan (P = 0.009), accompanied by impaired reduction of glucose levels from 25 to 60 min after meal ingestion (Δglucose = –1.27 ± 0.5 [with saline] vs. 0.1 ± 0.4 mmol/l [with trimethaphan]; P = 0.008). This reduction at 25–60 min in glucose levels correlated significantly to the 10-min insulin response (r = 0.65, P = 0.024). The 10-min insulin response to meal ingestion was also reduced by atropine, but only by 20 ± 9% (P = 0.045), which was lower than the reduction with trimethaphan (P = 0.004). The preabsorptive insulin response was not accompanied by any increase in circulating levels of gastric inhibitory polypeptide (GIP) or glucagon-like peptide 1 (GLP-1). In conclusion, 1) the early preabsorptive insulin response to meal ingestion in humans can be largely attributed to autonomic activation mediated by noncholinergic and cholinergic mechanisms, 2) this cephalic insulin response is required for a normal postprandial glucose tolerance, and 3) GIP and GLP-1 do not contribute to the preabsorptive cephalic phase insulin response to meal ingestion.
Increased Intracellular Calcium Is Required for Spreading of Rat Islet β-Cells on Extracellular Matrix
Authors: Bosco Domenico , Gonelle-Gispert Carmen , Wollheim Claes B. , Halban Philippe A. , Rouiller Dominique G.
Published: March 2007

Rat islet β-cells spread in response to glucose when attached on the matrix produced by a rat bladder carcinoma cell line (804G). Furthermore, in a mixed population of cells, it has been observed previously that spread cells secrete more insulin acutely in response to glucose, compared with cells that remain rounded. These results suggest bi-directional signaling between the islet β-cell and the extracellular matrix. In the present study, the role of increased intracellular free Ca2+ concentration [Ca2+]i as an intracellular step linking glucose stimulation and β-cell spreading (inside-out signaling) was investigated. Purified rat β-cells were attached to this matrix and incubated under various conditions known to affect [Ca2+]i. The effect of glucose on β-cell spreading was mimicked by 25 mmol/l KCl (which induces calcium influx) and inhibited by diazoxide (which impairs depolarization and calcium entry) and by the l-type Ca2+ channel blocker SR-7037. When a 24-h incubation at 16.7 glucose was followed by 24 h at 2.8 mmol/l, β-cells that had first spread regained a round phenotype. In the presence of thapsigargin, spreading progressed throughout the experiment, suggesting that capture of calcium by the endoplasmic reticulum is involved in the reversibility of spreading previously induced by glucose. Spreading was still observed in degranulated β-cells and in botulinum neurotoxin E–expressing β-cells when exocytosis was prevented. In summary, the results indicate that increased [Ca2+]i is required for the glucose-induced spreading of β-cells on 804G matrix and that it is not a consequence of exocytotic processes that follow elevation of [Ca2+]i.
Effect of 5-Aminoimidazole-4-Carboxamide-1-β-d-Ribofuranoside Infusion on In Vivo Glucose and Lipid Metabolism in Lean and Obese Zucker Rats
Authors: Bergeron Raynald , Previs Stephen F. , Cline Gary W. , Perret Pascale , Russell III Raymond R. , Young Lawrence H. , Shulman Gerald I.
Published: March 2007

Activation of AMP-activated protein kinase (AMPK) with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) increases glucose transport in skeletal muscle via an insulin-independent pathway. To examine the effects of AMPK activation on skeletal muscle glucose transport activity and whole-body carbohydrate and lipid metabolism in an insulin-resistant rat model, awake obese Zucker fa/fa rats (n = 26) and their lean (n = 23) littermates were infused for 90 min with AICAR, insulin, or saline. The insulin infusion rate (4 mU · kg−1 · min−1) was selected to match the glucose requirements during AICAR (bolus, 100 mg/kg; constant, 10 mg · kg−1 · min−1) isoglycemic clamps in the lean rats. The effects of these identical AICAR and insulin infusion rates were then examined in the obese Zucker rats. AICAR infusion increased muscle AMPK activity more than fivefold (P < 0.01 vs. control and insulin) in both lean and obese rats. Plasma triglycerides, fatty acid concentrations, and glycerol turnover, as assessed by [2-13C]glycerol, were all decreased in both lean and obese rats infused with AICAR (P < 0.05 vs. basal), whereas insulin had no effect on these parameters in the obese rats. Endogenous glucose production rates, measured by [U-13C]glucose, were suppressed by >50% during AICAR and insulin infusions in both lean and obese rats (P < 0.05 vs. basal). In lean rats, rates of whole-body glucose disposal increased by more than twofold (P < 0.05 vs. basal) during both AICAR and insulin infusion; [3H]2-deoxy-d-glucose transport activity increased to a similar extent, by >2.2-fold (both P < 0.05 vs. control), in both soleus and red gastrocnemius muscles of lean rats infused with either AICAR or insulin. In the obese Zucker rats, neither AICAR nor insulin stimulated whole-body glucose disposal or soleus muscle glucose transport activity. However, AICAR increased glucose transport activity by ∼2.4-fold (P < 0.05 vs. control) in the red gastrocnemius from obese rats, whereas insulin had no effect. In summary, acute infusion of AICAR in an insulin-resistant rat model activates skeletal muscle AMPK and increases glucose transport activity in red gastrocnemius muscle while suppressing endogenous glucose production and lipolysis. Because type 2 diabetes is characterized by diminished rates of insulin-stimulated glucose uptake as well as increased basal rates of endogenous glucose production and lipolysis, these results suggest that AICAR-related compounds may represent a new class of antidiabetic agents.
Cardiomyocyte Dysfunction in Sucrose-Fed Rats Is Associated With Insulin Resistance
Authors: Dutta Kaushik , Podolin Deborah A. , Davidson Michael B. , Davidoff Amy J.
Published: March 2007

Diabetes is associated with impaired cardiac dysfunction in both humans and animals. Specific phenotypic changes—prolonged action potentials, slowed cytosolic Ca2+ clearing, and slowed relaxation—that contribute to this whole heart dysfunction occur in isolated ventricular myocytes. The present study was designed to determine whether cardiomyocyte abnormalities occur early in the development of type 2 diabetes (in this case, insulin resistance) and whether an insulin-sensitizing drug (metformin) is cardioprotective. In the study, high-sucrose feeding was used to induce whole-body insulin resistance. Wistar rats were maintained for 7–10 weeks on a starch (ST) diet, sucrose (SU) diet, or diet supplemented with metformin (SU + MET). Whole-body insulin resistance was measured in SU and SU + MET rats by performing euglycemic-hyperinsulinemic clamps. Mechanical properties of isolated ventricular myocytes were measured by high-speed video edge detection, and [Ca2+]i transients were evaluated with Fura-2 AM. Untreated SU rats were insulin-resistant (glucose infusion rate [GIR] = 14.5 ± 1.1 mg · kg−1 · min−1); metformin treatment in SU + MET rats prevented this metabolic abnormality (GIR = 20.0 ± 2.2 mg · kg−1 · min−1). Indexes of myocyte shortening and relengthening were significantly longer in SU rats (area under the relaxation phase [AR/peak] = 103 ± 3 msec) when compared to ST and SU + MET rats (AR/peak = 73 ± 2 and 80 ± 1 msec, respectively). The rate of intracellular Ca2+ decay and the integral of the Ca2+ transient through the entire contractile cycle were significantly longer in myocytes from SU than from ST rats (Ca2+ signal normalized to peak amplitude = 152 ± 8 vs. 135 ± 5 msec, respectively). Collectively, our data showed the presence of cardiomyocyte abnormalities in an insulin-resistant stage that precedes frank type 2 diabetes. Furthermore, metformin prevented the development of sucrose-induced insulin resistance and the consequent cardiomyocyte dysfunction.
Modest Overexpression of Neuropeptide Y in the Brain Leads to Obesity After High-Sucrose Feeding
Authors: Kaga Toshihiro , Inui Akio , Okita Minoru , Asakawa Akihiro , Ueno Naohiko , Kasuga Masato , Fujimiya Mineko , Nishimura Noriyasu , Dobashi Rika , Morimoto Yasuo , Liu I-Min , Cheng Juei-Tang
Published: March 2007

Neuropeptide Y (NPY), one of the most abundant peptide transmitters in the mammalian brain, is assumed to play an important role in feeding and body weight regulation. However, there is little genetic evidence that overexpression or knockout of the NPY gene leads to altered body weight regulation. Previously, we developed NPY-overexpressing mice by using the Thy-1 promoter, which restricts NPY expression strictly within neurons in the central nervous system, but we failed to observe the obese phenotype in the heterozygote. Here we report that in the homozygous mice, overexpression of NPY leads to an obese phenotype, but only after appropriate dietary exposure. NPY-overexpressing mice exhibited significantly increased body weight gain with transiently increased food intake after 50% sucrose–loaded diet, and later they developed hyperglycemia and hyperinsulinemia without altered glucose excursion during 1 year of our observation period.
Polymorphism Screening of Four Genes Encoding Advanced Glycation End-Product Putative Receptors
Authors: Poirier Odette , Nicaud Viviane , Vionnet Nathalie , Raoux Ségolène , Tarnow Lise , Vlassara Helen , Parving Hans-Henrik , Cambien François
Published: March 2007

Advanced glycation end-products (AGEs) may play an important role in the pathogenesis and progression of cardiovascular and renal complications of diabetes. Four putative AGE receptors (RAGEs), AGE-R1, AGE-R2, and AGE-R3 have been described. In this study, we scanned the sequence of the genes encoding these AGE receptors in 48 patients with type 1 diabetes and investigated the identified polymorphisms (n = 19) in 199 type 1 diabetic patients with nephropathy and 193 type 1 diabetic patients without nephropathy. Overall, none of the polymorphisms was strongly associated with nephropathy. The minor allele of a polymorphism located in the promoter region of the RAGE gene (C-1152A) conferred a weak protective effect (P < 0.05) and was associated with a longer duration of nephropathy-free diabetes (P = 0.08).
Identification of Novel Cytokine-Induced Genes in Pancreatic β-Cells by High-Density Oligonucleotide Arrays
Authors: Cardozo Alessandra K. , Kruhøffer Mogens , Leeman Ruth , Ørntoft Torben , Eizirik Décio L.
Published: March 2007

Type 1 diabetes is an autoimmune disease resulting from the selective destruction of insulin-producing β-cells. Cytokines may contribute to pancreatic β-cell death in type 1 diabetes. β-cell exposure to interleukin (IL)-1β induces functional impairment, whereas β-cell culture for 6–9 days in the presence of IL-1β and interferon (INF)-γ leads to apoptosis. To clarify the mechanisms involved in these effects of cytokines, we studied the general pattern of cytokine-induced gene expression in β-cells. Primary rat β-cells were fluorescence-activated cell sorter–purified and exposed for 6 or 24 h to control condition, IL-1β + INF-γ, or IL-1β alone (24 h only). Gene expression profile was analyzed in duplicate by oligonucleotide arrays. Nearly 3,000 transcripts were detected in controls and cytokine-treated β-cells. Of these, 96 and 147 displayed changes in expression after 6 and 24 h, respectively, of exposure to IL-1β + INF-γ, whereas 105 transcripts were modified after a 24-h exposure to IL-1β. The cytokine-responsive genes were clustered according to their biological functions. The major clusters observed were metabolism, signal transduction, transcription factors, protein synthesis/processing, hormones, and related receptors. These modifications in gene expression may explain some of the cytokine effects in β-cells, such as decreased protein biosynthesis and insulin release. In addition, there was induction of diverse cytokines and chemokines; this suggests that β-cells may contribute to mononuclear cell homing during insulitis. Several of the cytokine-induced genes are potentially regulated by the transcription factor NF-κB. Clarification of the function of the identified cytokine-induced gene patterns may unveil some of the mechanisms involved in β-cell damage and repair in type 1 diabetes.
Regulation of the Pancreatic Pro-Endocrine Gene Neurogenin3
Authors: Lee Jane C. , Smith Stewart B. , Watada Hirotaka , Lin Joseph , Scheel David , Wang Juehu , Mirmira Raghavendra G. , German Michael S.
Published: March 2007

Neurogenin3 (ngn3), a basic helix-loop-helix (bHLH) transcription factor, functions as a pro-endocrine factor in the developing pancreas: by itself, it is sufficient to force undifferentiated pancreatic epithelial cells to become islet cells. Because ngn3 expression determines which precursor cells will differentiate into islet cells, the signals that regulate ngn3 expression control islet cell formation. To investigate the factors that control ngn3 gene expression, we mapped the human and mouse ngn3 promoters and delineated transcriptionally active sequences within the human promoter. Surprisingly, the human ngn3 promoter drives transcription in all cell lines tested, including fibroblast cell lines. In contrast, in transgenic animals the promoter drives expression specifically in regions of ngn3 expression in the developing pancreas and gut; and the addition of distal sequences greatly enhances transgene expression. Within the distal enhancer, binding sites for several pancreatic transcription factors, including hepatocyte nuclear factor (HNF)-1 and HNF-3, form a tight cluster. HES1, an inhibitory bHLH factor activated by Notch signaling, binds to the proximal promoter and specifically blocks promoter activity. Together with previous genetic data, these results suggest a model in which the ngn3 gene is activated by the coordinated activities of several pancreatic transcription factors and inhibited by Notch signaling through HES1.
BB Rat Thymocytes Cultured in the Presence of Islets Lose Their Ability to Transfer Autoimmune Diabetes
Authors: Whalen Barbara J. , Marounek Jan , Weiser Peter , Appel Michael C. , Greiner Dale L. , Mordes John P. , Rossini Aldo A.
Published: March 2007

Thymocytes from adult BB rats can adoptively transfer autoimmune diabetes to athymic recipients. It is also known that the development of BB rat T-cells is recapitulated in adult thymus organ cultures (ATOCs). Based on these observations, we tested the hypothesis that cells capable of the adoptive transfer of diabetes would be present in long-term ATOCs but could be rendered nondiabetogenic by co-culture with appropriate antigens. We observed that cells recovered from adult diabetes-resistant BB (BBDR) rat thymi cultured for up to 14 days can adoptively transfer disease to athymic WAG-rnu/rnu rats treated with polyinosinic:polycytidylic acid and a monoclonal antibody to preclude development of ART2a+ regulatory T-cells. Co-culture of adult BBDR thymi in the presence of BBDR thyrocytes had no effect on the ability of recovered cells to induce diabetes in 70–80% of adoptive recipients. In contrast, co-culture in the presence of islets prevented transfer of diabetes, on average, in >90% of recipients. Fresh islets, frozen islets, and islets pretreated with streptozotocin to deplete insulin were equally effective in preventing diabetes, but none prevented insulitis in nondiabetic recipients. Co-culture in the presence of islets was not associated with detectable alterations in phenotype or in the secretion of γ-interferon or interleukin-4, either in cultures or in cells recovered from adoptive recipients. We conclude that islet antigens involved in the initiation of autoimmune diabetes in BB rats may be absent or deficient in BB rat thymi. Exposure of ATOCs to exogenous islets may lead to deletion or anergy of diabetogenic T-cells or to the positive selection of regulatory T-cells.
Successful Reversal of Streptozotocin-Induced Diabetes With Stable Allogeneic Islet Function in a Preclinical Model of Type 1 Diabetes
Authors: Thomas Judith M. , Contreras Juan L. , Smyth Cheryl A. , Lobashevsky Andrew , Jenkins Stacie , Hubbard William J. , Eckhoff Devin E. , Stavrou Scott , Neville Jr. David M. , Thomas Francis T.
Published: March 2007

The recent focus on islet transplantation as primary therapy for type 1 diabetes has heightened interest in the reversal of type 1 diabetes in preclinical models using minimal immunosuppression. Here, we demonstrated in a preclinical rhesus model a consistent reversal of all measured glycemic patterns of streptozotocin-induced type 1 diabetes. The model used single-donor islet transplantation with induction of operational tolerance. The term “operational tolerance” is used to indicate durable survival of single-donor major histocompatibility complex (MHC)-mismatched islet allografts without maintenance immunosuppressive therapy and without rejection or loss of functional islet mass or insulin secretory reserve. In this operational tolerance model, all immunosuppression was discontinued after day 14 posttransplant, and recipients recovered with excellent health. The operational tolerance induction protocol combined peritransplant anti-CD3 immunotoxin to deplete T-cells and 15-deoxyspergualin to arrest proinflammatory cytokine production and maturation of dendritic cells. T-cell deficiency was specific but temporary, in that T-cell–dependent responses in long-term survivors recovered to normal, and there was no evidence of increased susceptibility to infection. Anti-donor mixed lymphocyte reaction responses were positive in the long-term survivors, but all showed clear evidence of systemic T-helper 2 deviation, suggesting that an immunoregulatory rather than a deletional process underlies this operational tolerance model. This study provides the first evidence that operational tolerance can protect MHC nonhuman primate islets from rejection as well as loss of functional islet mass. Such an approach has potential to optimize individual recipient recovery from diabetes as well as permitting more widespread islet transplantation with the limited supply of donor islets.
Peptide and Major Histocompatibility Complex–Specific Breaking of Humoral Tolerance to Native Insulin With the B9-23 Peptide in Diabetes-Prone and Normal Mice
Authors: Abiru Norio , Maniatis Aristides K. , Yu Liping , Miao Dongmei , Moriyama Hiroaki , Wegmann Dale , Eisenbarth George S.
Published: March 2007

NOD mice spontaneously develop anti-insulin autoantibodies and diabetes. A dominant peptide recognized by T-cell clones from NOD mice is insulin B-chain peptide B9-23. When administered subcutaneously to NOD mice, this peptide decreases the development of diabetes. In this study, we evaluated the autoantibody response to native insulin after administration of the B9-23 peptide. In NOD mice, administration of the B9-23 peptide in incomplete Freund’s adjuvant enhanced their insulin autoantibody response with a higher level and longer persistence. Induction of insulin autoantibodies with the B9-23 peptide was observed in non–diabetes-prone BALB/c mice and NOR mice within 2 weeks of administration, but this was not observed in C57BL/6 mice. A series of A-chain, other B-chain, and proinsulin peptides did not induce insulin autoantibodies. Induced anti-insulin autoantibodies could not be absorbed with the peptide alone but could be absorbed with native insulin. The B13-23 peptide (one of two identified epitopes within B9-23) when administered to BALB/c mice, induced autoantibodies, whereas peptide B9-16 did not. Induction of autoantibodies mapped to the major histocompatibility complex (MHC) rather than to the background genes. Both splenocytes with I-Ad/I-Ed or I-Ag7/I-Enull presented the B9-23 peptide to NOD islet-derived T-cell clones. Finally, administration of the B9-23 peptide to BALB/c mice, even without adjuvant, could induce insulin autoantibodies. Our results indicate that B-cell tolerance to intact insulin is readily broken with the presentation of the B9-23 insulin peptide, depending on the host’s specific MHC.
Ezetimibe, a Potent Cholesterol Absorption Inhibitor, Normalizes Combined Dyslipidemia in Obese Hyperinsulinemic Hamsters
Authors: van Heek Margaret , Austin Theodore M. , Farley Constance , Cook John A. , Tetzloff Glen G. , Davis Harry R.
Published: March 2007

Ezetimibe potently and selectively inhibits cholesterol absorption in the intestine, thereby reducing plasma cholesterol in preclinical models of hypercholesterolemia. Clinical trials have demonstrated that ezetimibe lowers LDL cholesterol and raises HDL cholesterol in humans. The effect of ezetimibe on other dyslipidemias, particularly hypertriglyceridemia, is not yet known. In the present studies, we assessed the effect of ezetimibe on combined hypercholesterolemia and hypertriglyceridemia in obese hyperinsulinemic hamsters. Hamsters were fed chow, chow with cholesterol (0.12%), or the same cholesterol diet containing different dietary triglycerides (15%) in the absence or presence of 1 mg/kg ezetimibe (in diet) for up to 84 days. Body weight, serum insulin, leptin, glucose, cholesterol, and triglyceride levels were analyzed. Cholesterol and triglyceride levels were also determined in VLDL+IDL, LDL, and HDL. Hamsters maintained on high-fat diets became obese, hyperinsulinemic, hyperleptinemic, hypercholesterolemic, and hypertriglyceridemic. Ezetimibe did not affect body weight, insulin, or leptin, but ablated the combined hypercholesterolemia and hypertriglyceridemia induced by high-fat diets. Ezetimibe normalized VLDL+IDL cholesterol and triglyceride and significantly decreased LDL cholesterol to below chow-fed levels. The ratio of HDL to LDL cholesterol increased significantly with the addition of ezetimibe. Ezetimibe completely eliminated the accumulation of cholesteryl ester and free cholesterol in liver that was induced under the various dietary conditions in the absence of drug. In conclusion, ezetimibe is very effective in correcting the combined dyslipidemia in diet-induced obese hyperinsulinemic hamsters and may be an effective therapy for ameliorating combined dyslipidemia in obese insulin-resistant and/or type 2 diabetic humans.
Assessment of Hepatic Insulin Action in Obese Type 2 Diabetic Patients
Authors: Staehr Peter , Hother-Nielsen Ole , Levin Klaus , Holst Jens Juul , Beck-Nielsen Henning
Published: March 2007

Defects in hepatic insulin action in type 2 diabetes and its possible underlying mechanisms were assessed in euglycemic-hyperinsulinemic clamp studies, using improved tracer methods (constant specific activity technique). Ten obese diabetic patients (age 54 years, BMI 29 ± 0.5 kg/m2) and ten matched control subjects were studied at baseline (after an overnight fast) and during insulin infusions of 20- and 40-mU · m–2 · min–1. In the diabetic patients, plasma glucose levels were normalized overnight before the studies by low-dose insulin infusion. Hepatic sinusoidal insulin levels were estimated, and plasma levels of free fatty acids (FFAs) and glucagon were determined to assess the direct and indirect effects of insulin on hepatic glucose production (HGP) in type 2 diabetes. Baseline rates of HGP (86 ± 3 vs. 76 ± 3 mg · m–2 · min–1, P < 0.05) were slightly elevated in the diabetic patients compared with control subjects, despite much higher hepatic sinusoidal insulin levels (26 ± 3 vs. 12 ± 2 mU/l, P < 0.001). Consequently, a marked defect in the direct (hepatic) effect of insulin on HGP appeared to be present at low insulin levels. However, in response to a small increase in baseline hepatic sinusoidal insulin levels of 11 mU/l (26 ± 3 to 37 ± 3 mU/l, P < 0.05) in the 20-mU clamp, a marked suppression of HGP was observed in the diabetic patients (86 ± 3 to 32 ± 5 mg · m–2 · min–1, P < 0.001), despite only minimal changes in FFAs (0.33 ± 0.05 to 0.25 ± 0.05 mmol/l, NS) and glucagon (14 ± 1 to 11 ± 2 pmol/l, P < 0.05) levels, suggesting that the impairment in the direct effect of insulin can be overcome by a small increase in insulin levels. Compared with control subjects, suppression of HGP in the diabetic patients was slightly impaired in the 20-mU clamp (32 ± 5 vs. 22 ± 4 mg · m–2 · min–1, P < 0.05) but not in the 40-mU clamp (25 ± 2 vs. 21 ± 3 mg · m–2 · min–1, NS). In the 20-mU clamp, hepatic sinusoidal insulin levels in the diabetic patients were comparable with control subjects (37 ± 3 vs. 36 ± 3 mU/l, NS), whereas both FFA and glucagon levels were higher (i.e., less suppressed) and correlated with the rates of HGP (R = 0.71, P < 0.02; and R = 0.69, P < 0.05, respectively). Thus, at this insulin level impaired indirect (extrahepatic) effects of insulin seemed to prevail. In conclusion, hepatic insulin resistance is present in obese type 2 diabetic patients but is of quantitative significance only at low physiological insulin levels. Defects in both the direct and the indirect effects of insulin on HGP appear to contribute to this resistance.