bluuebunny/crossref_metadata_2025 huggingface dataset:
Ductal Cyst Formation in Collagen-Embedded Adult Human Islet Preparations: A Means to the Reproduction of Nesidioblastosis In Vitro
Authors: Kerr-Conte Julie , Pattou François , Lecomte-Houcke Martine , Xia YingJian , Boilly Benoni , Proye Charles , Lefebvre Jean
Published: September 2013

Neogenesis of endocrine islets from ductal epithelium termed nesidioblastosis has been described in vivo after various experimental conditions (90% pancreatectomy or pancreas wrapping in the rodent) and in clinical pathologies. In the adult regenerating pancreas, a proliferation and organization of ductal epithelium into tubular structures precedes its differentiation into endocrine cells. Reproduction of nesidioblastosis in vitro may provide a novel approach to human islet propagation in vitro. With this aim, adult human islet preparations were cultured in diverse three-dimensional (3D) gels in the presence of serum. After 3–5 days in rat tail collagen gels, proliferating (bromodeoxyuridine-positive) cystic structures appeared associated with islets and as isolated spheres. Percentage labeling indexes of the cysts were 4.1, 18.7, 15.4, and 13.3% after 3, 5, 7, and 10 days of culture, respectively. Immunohistochemistry confirmed the ductal (carbohydrate antigen 19–9) and epithelial (keratin-1) nature of the cysts. No cysts were formed in agarose gels or Vitrogen 100, whereas the cyst number was increased by the quantity of serum (20% > 10%) and gels rich in extracellular matrix components and growth factors (Matrigel). The latter lead to tubular networks. Single endocrine islet cells were observed in the ductal cysts after 7 (2.8%) to 10 (5.6%) days in rat tail collagen. Our observations paralleled the changes characteristic of the regenerating pancreas in vivo. 3D culture may permit the identification of matrix and media constituents promoting the neogenesis of islets and may be the means to increase the mass of endocrine tissue obtained from adult cadaveric pancreases for transplantation.
Major Histocompatibility Complex Class I–Restricted Infiltration and Destruction of Pancreatic Islets by NOD Mouse-Derived β-Cell Cytotoxic CD8+ T-Cell Clones In Vivo
Authors: Utsugi Toshihiro , Yoon Ji-Won , Park Byung-Ju , Imamura Minoru , Averill Nuzhat , Kawazu Shoji , Santamaria Pere
Published: September 2013

NOD mouse-derived β-cell-specific cytotoxic T-cell (β-CTL) clones are diabetogenic in adult NOD mice, but only if co-injected with splenic CD4+ T-cells from diabetic animals. This investigation was initiated to determine whether infiltration of pancreatic islets by β-CTL is a major histocompatibility complex (MHC) class I-restricted response, and whether β-CTL has a direct cytopathic effect on β-cells in vivo. Pancreatic islets from BALB/c (H-2d) or B6 (H-2b) mice were transplanted under the renal capsule of streptozotocin (STZ)-induced diabetic (NOD × BALB/c) Fl (H-2Kd, H-2Dd,b) or (NOD × B6) Fl (H-2Kd,b, H-2Db) mice, respectively. H-2Kd-restricted β-CTL clones from NOD mice were transfused into euglycemic mice within 3 days after transplantation. In all of the H-2d islet-grafted (NOD × BALB/c) Fl mice that received the β-CTL clones, the β-CTLs homed into the grafts, recruited host Mac-1+ cells and CD4+ and CD8+ T-cells, and caused diabetes within 7 days. In contrast, none of the H-2b islet-grafted (NOD × B6) Fl mice who received the β-CTL clones and none of the H-2d islet-grafted (NOD × BALB/c) Fl mice who received a non-β-cell cytotoxic CTL clone (Nβ-CTL) developed graft inflammation or diabetes. Depletion of CD4+ T-cells in H-2d islet-grafted (NOD × BALB/c) Fl mice did not prevent β-CTL clone-induced diabetes but reduced its severity. In contrast, when the β-CTL clones were injected >8 days after transplantation, none of the H-2d islet-grafted (NOD × BALB/c) Fl mice became diabetic or developed graft inflammation. We conclude that (1) isletderived β-CTLs can destroy β-cells in vivo; (2) infiltration of grafted islets by β-CTLs is an MHC class I-restricted response; (3) β-CTLs can recruit naive CD4+ T-cells to the site, leading to further β-cell damage; and (4) revascularized islet grafts are, like pancreatic islets of irradiated adult NOD mice, “sequestered” from circulating β-CTLs.
T-Cell Responses to Autoantigens in IDDM: The Search for the Holy Grail
Authors: Roep Bart O
Published: September 2013

IDDM (type I diabetes) is generally believed to result from T-cell-mediated autoimmune destruction of the insulin-producing β-cells in the pancreatic islets of Langerhans. In the last few years, considerable progress has been made with regard to the identification and characterization of candidate autoantigens recognized by autoantibodies; several of these candidate autoantigens are recognized by T-cells, including insulin, GAD65 and GAD67, heat-shock protein 65 (hsp65), and islet-cell antigen 69 (ICA69). In addition to these, a number of unidentified β-cell antigens, including insulin-secretory granule membrane proteins and a 38-kDa protein, have been shown to stimulate T-cells of IDDM patients. However, T-cell autoreactivity to islet antigens is not specific for IDDM, and the T-cell target antigens are not specific for β-cells. Moreover, the autoantigens involved in the initiation of the insulitis must be defined, and the mechanism of the T-cell-dependent β-cell destruction remains to be unraveled. This review focuses on T-cell autoreactivity in IDDM in humans and the implications of the present knowledge for immunointervention and monitoring of immunotherapeutic trials.
Absence of Linkage of Obesity and Energy Metabolism to Markers Flanking Homologues of Rodent Obesity Genes in Pima Indians
Authors: Norman Rod A , Leibel Rudolph L , Chung Wendy K , Power-Kehoe Loraine , Chua Streamson C , Knowler William C , Thompson D Bruce , Bogardus Clifton , Ravussin Eric
Published: September 2013

The homologues of single genes that cause obesity in rodents are suggested as candidate genes for modulation of body composition in humans. Among these genes are the four mouse mutations—diabetes (db), obesity (ob), tubby (tub), and yellow agouti (AY). Variation in the human counterparts to these genes (OB, DB, TUB, and ASP, respectively) may contribute to human obesity, which is thought to have a substantial genetic component. To initially assess the potential contribution of these genes to human obesity, we examined polymorphic DNA markers that, by virtue of syntenic relationships to appropriate regions of the mouse genome, should be closely linked to the human counterparts of these genes. Using combined data from 716 Pima Indians comprising 217 nuclear families, we have tested a number of polymorphic microsatellite markers (three at DB, two at OB, five at TUB, and three at ASP) for sib-pair linkage to BMI, percentage body fat, resting metabolic rate, 24-h energy expenditure, and 24-h respiratory quotient. No significant linkages were found in an analysis of all sibships or in an analysis restricted to discordant sib pairs.
Contribution of Amino Acids and Insulin to Protein Anabolism During Meal Absorption
Authors: Volpi Elena , Lucidi Paola , Cruciani Guido , Monacchia Francesca , Reboldi Gianpaolo , Brunetti Paolo , Bolli Geremia B , De Feo Pierpaolo
Published: September 2013

The contribution of dietary amino acids and endogenous hyperinsulinemia to prandial protein anabolism still has not been established. To this end, leucine estimates ([1- 14C]leucine infusion, plasma α-ketoisocaproic acid [KIC] specific activity [SA] as precursor pool SA) of whole-body protein kinetics and fractional secretory rates (FSRs) of albumin, fibrinogen, antithrombin III, and immunoglobulin G (IgG) were measured in three groups of healthy volunteers during intragastric infusion of water (controls, n = 5), liquid glucose–lipid–amino acid (AA) meal (meal+AA, n = 7), or isocaloric glucose–lipid meal (meal-AA, n = 7) that induced the same insulin response as the meal+AA. The results of this study demonstrate that 1) by increasing (P < 0.01) whole-body protein synthesis and decreasing (P < 0.01) proteolysis, dietary amino acids account for the largest part (∼90%) of postprandial protein anabolism; 2) the ingestion of an isocaloric meal deprived of amino acids exerts a modest protein anabolic effect (10% of postprandial protein anabolism) by decreasing amino acid oxidation and increasing (P < 0.01) albumin synthesis; 3) albumin FSR is increased (∼20%) by postprandial hyperinsulinemia (meal–AA) and additionally increased (∼50%) by amino acid intake (meal+AA); 4) IgG FSR is stimulated (∼40%) by amino acids, not by insulin; and 5) fibrinogen and antithrombin III FSR are not regulated by amino acids or insulin.
Effect of Troglitazone on Leptin Production: Studies In Vitro and in Human Subjects
Authors: Nolan John J , Olefsky Jerrold M , Nyce Mark R , Considine Robert V , Caro Jose F
Published: September 2013

Leptin, the product of the ob gene, is a hormone secreted by adipocytes. Animals with mutations in the ob gene are obese and lose weight when given leptin, but little is known about the physiological role of leptin in humans. Obese subjects have higher concentrations of leptin than lean subjects, the strongest correlation being with percentage body fat. Thus, it appears that obese subjects are resistant to the effects of endogenously secreted leptin. We have also shown that insulin stimulates leptin production, chronically but not acutely, presumably through its trophic effect on adipocytes. Troglitazone is an insulinsensitizing thiazolidinedione, which improves hepatic and skeletal muscle insulin resistance in NIDDM and obesity. This study was undertaken to investigate the effects of troglitazone on leptin production in vitro and in vivo. In the presence and absence of 100 nmol/l insulin and 10 umol/L troglitazone, 72-h primary cultures of isolated abdominal adipocytes were studied. Insulin led to an almost twofold increase in leptin in vitro, and this increase was completely abolished by coincubation with troglitazone. Incubation with troglitazone alone led to a 40% decrease in leptin production. In obese patients administered troglitazone 200 mg twice daily for 12 weeks, there was no significant change in fasting plasma leptin concentrations, despite a 40–50% reduction in fasting and postmeal plasma insulin concentrations. Troglitazone treatment led to a significant increase in insulin sensitivity, and there was a positive correlation between the change in insulin sensitivity and the change in plasma leptin concentration in these subjects. In conclusion, troglitazone treatment had no net effect on plasma leptin concentrations, possibly because of improvement in insulin sensitivity and reduction in plasma insulin concentrations.
Defective Glucagon Secretion During Sustained Hypoglycemia Following Successful Islet Allo- and Autotransplantation in Humans
Authors: Kendall David M , Teuscher Adrian U , Robertson R Paul
Published: September 2013

Defective glucagon secretion during hypoglycemia is characteristic of long-standing type I diabetes. To determine whether this defect can be corrected by successful intrahepatic islet transplantation, we performed studies of hypoglycemia in four nondiabetic patients with chronic pancreatitis who had undergone total pancreatectomy and successful intrahepatic islet autotransplantation, in two type I diabetic recipients of successful intrahepatic islet allotransplantation, and in matched control subjects. We examined 1) whether intrahepatic islet autotransplantation provides glucagon secretion during prolonged periods of hypoglycemia and 2) whether intrahepatic islet allotransplantation in type I diabetic patients and consequent long-term normoglycemia reestablishes native α-cell responses to hypoglycemia. Glucagon secretion was assessed during 3-h hypoglycemic hyperinsulinemic clamp studies. The islet autograft recipients were studied 63 ± 19 months posttransplant, and all were insulinindependent and normoglycemic (HbA1c, 5.8 ±± 0.2%). Neither allograft recipient required exogenous insulin and maintained HbA1c levels of 5.7 and 6.4% 30 and 34 months posttransplant, respectively. All recipients were normoglycemic (fasting glucose: autograft recipients, 5.6 ± 0.1 mmol/l; allograft recipient #1, 6.3 mmol/l; allograft recipient #2, 5.8 mmol/l) at the time of study. During hypoglycemia, no increase in glucagon secretion was observed in either the auto- or allotransplant recipients, whereas healthy control subjects and recipients of kidney transplantation had significant increases in glucagon. In contrast, both allo- and autograft recipients had glucagon responses to intravenous arginine. These data uniquely demonstrate that: 1) intrahepatic islet transplant grafts secrete glucagon in response to arginine, but fail to secrete glucagon in response to sustained hypoglycemia; and 2) the restoration of sustained normoglycemia for over 2 years in type I diabetic patients may not reestablish glucagon responses from the native pancreas during hypoglycemia. Transplantation sites other than the liver may be required to achieve normal glucagon secretion from the transplanted islets.
Moving GLUT4: The Biogenesis and Trafficking of GLUT4 Storage Vesicles
Authors: Rea Shane , James David E
Published: September 2013

The GLUT4 system in muscle and fat cells plays an important role in whole-body glucose homeostasis. Insulin stimulates the translocation of GLUT4 from an intracellular storage compartment to the cell surface. The nature of this compartment remains largely unknown. We review recent studies describing the biogenesis and molecular constituents of the GLUT4 storage compartment and conclude that it is segregated from the endosomal and biosynthetic pathways. Further, we present evidence to suggest that the GLUT4 storage compartment moves directly to the plasma membrane in response to insulin and, hence, is analogous to small synaptic vesicles in neurons. We propose that the GLUT4 storage compartment be referred to as GLUT4 storage vesicles or GSVs.
A Nonsense Mutation in the Inward Rectifier Potassium Channel Gene, Kir6.2, Is Associated With Familial Hyperinsulinism
Authors: Nestorowicz Ann , Inagaki Nobuya , Gonoi Tohru , Schoor Kathleen P , Wilson Beth Anne , Glaser Benjamin , Landau Heddy , Stanley Charles A , Thornton Paul S , Seino Susumu , Permutt M Alan
Published: September 2013

ATP-sensitive potassium (KATP) channels are an essential component of glucose-dependent insulin secretion in pancreatic islet β-cells. These channels comprise the sulfonylurea receptor (SUR1) and Kir6.2, a member of the inward rectifier K+ channel family. Mutations in the SUR1 subunit are associated with familial hyperinsulinism (HI) (MIM:256450), an inherited disorder characterized by hyperinsulinism in the neonate. Since the Kir6.2 gene maps to human chromosome 11p15.1 (1,2), which also encompasses a locus for HI, we screened the Kir6.2 gene for the presence of mutations in 78 HI probands by single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses. A nonsense mutation, Tyr→Stop at codon 12 (designated Y12X) was observed in the homozygous state in a single proband. 86Rb+ efflux measurements and single-channel recordings of COS-1 cells co-expressing SUR1 and either wild-type or Y12X mutant Kir6.2 proteins confirmed that KATP channel activity was abolished by this nonsense mutation. The identification of an HI patient homozygous for the Kir6.2/Y12X allele affords an opportunity to observe clinical features associated with mutations resulting in an absence of Kir6.2. These data provide evidence that mutations in the Kir6.2 sub-unit of the islet β-cell KATP channel are associated with the HI phenotype and also suggest that the majority of HI cases are not attributable to mutations in the coding region of the Kir6.2 gene.
Diminished Insulin and Glucagon Secretory Responses to Arginine in Nondiabetic Subjects With a Mutation in the Hepatocyte Nuclear Factor–4α/MODY1 Gene
Authors: Herman William H , Fajans Stefan S , Smith Maria J , Polonsky Kenneth S , Bell Graeme I , Halter Jeffrey B
Published: September 2013

Nondiabetic subjects with the Q268X mutation in the hepatocyte nuclear factor (HNF)-4α/MODY1 gene have impaired glucose-induced insulin secretion. To ascertain the effects of the nonglucose secretagogue arginine on insulin and glucagon secretion in these subjects, we studied 18 members of the RW pedigree: 7 nondiabetic mutation negative (ND[−]), 7 nondiabetic mutation positive (ND[+]), and 4 diabetic mutation positive (D[+]). We gave arginine as a 5-g bolus, followed by a 25-min infusion at basal glucose concentrations, and after glucose infusion to clamp plasma glucose at ∼200 mg/dl. The acute insulin response (AIR), the 10–60 min insulin area under the curve (AUC), and the insulin secretion rate (ISR) were compared, as were the acute glucagon response (AGR) and glucagon AUC. The ND[+] and D[+] groups had decreased insulin AUC and ISR and decreased glucose potentiation of AIR, insulin AUC, and ISR to arginine administration when compared with the ND[−] group. At basal glucose concentrations, glucagon AUC was greatest for the ND[−] group, intermediate for the ND[+] group, and lowest for the D[+] group. During the hyperglycemic clamp, there was decreased suppression of glucagon AUC for both ND[+] and D[+] groups compared with the ND[−] group. The decreased ISR to arginine in the ND[+] group compared with the ND[−] group, magnified by glucose potentiation, indicated that HNF-4α affects the signaling pathway for arginine-induced insulin secretion. The decrease in glucagon AUC and decreased suppression of glucagon AUC with hyperglycemia suggest that mutations in HNF-4α may lead to α-cell as well as β-cell secretory defects or a reduction in pancreatic islet mass.
Insulin Resistance of Muscle Glucose Transport in Rats Fed a High-Fat Diet: A Reevaluation
Authors: Han Dong-Ho , Hansen Polly A , Host Helen H , Holloszy John O
Published: September 2013

Rats fed a high-fat diet develop skeletal muscle insulin resistance. There is disagreement regarding whether a decrease in the GLUT4 isoform of the glucose transporter is responsible. We found that feeding rats a high-fat diet that reduced the responsiveness of glucose transport to insulin in skeletal muscles by ∼25–45% in 4 weeks, had no significant effect on muscle GLUT4 content. There is also controversy regarding whether the contraction/anoxia activated pathway of glucose transport stimulation is affected by fat feeding. We found that stimulation of muscle glucose transport by either swimming, in situ contractions, or anoxia was depressed to a similar extent as insulin responsiveness in high-fat–fed rats. It has been suggested that the muscle insulin resistance caused by a high-fat diet is due to increased fat oxidation and glucose-fatty acid cycle activity. However, we found that insulin-stimulated glucose transport was reduced by ∼40% when muscles of fat-fed rats were incubated under anoxic conditions under which fatty acid oxidation should not occur. Rats maintained on the high-fat diet up to 32 weeks developed the characteristics of the abdominal obesity syndrome, including insulin resistance, hyperinsulinemia, hyperglycemia, elevated LDL cholesterol and VLDL triglycerides, and marked visceral obesity. We conclude that 1) in rats fed a high-fat diet the muscle insulin resistance is not due to a decrease in total GLUT4 content or to increased fat oxidation, 2) fat feeding also results in resistance of muscle glucose transport to stimulation via the contraction/anoxia pathway, and 3) rats fed a high-fat diet may be a useful model of the abdominal obesity syndrome.
Inhibition of Food Intake by Neuropeptide Y Y5 Receptor Antisense Oligodeoxynucleotides
Authors: Schaffhauser A O , Stricker-Krongrad A , Brunner L , Cumin F , Gerald C , Whitebread S , Criscione L , Hofbauer K G
Published: September 2013

The recently discovered rat neuropeptide Y (NPY) receptor, the Y5 subtype, has been proposed to mediate the NPY-induced feeding response and therefore plays a central role in the regulation of food intake. These conclusions were based on studies with peptidic agonists. We now report studies in which phosphothioate end-protected antisense oligodeoxynucleotides (ODNs) targeted to prepro NPY (prepro NPY antisense ODNs) or to the Y5 receptor (Y5 antisense ODNs) were used to assess the functional importance of this novel receptor subtype in vivo. NPY antisense ODNs given intracere-broventricularly to rats prevented the increase in hypothalamic NPY levels during food deprivation and inhibited fasting-induced food intake. Likewise, repeated intracerebroventricular injections of Y5 antisense ODNs prevented fasting-induced food intake in rats. Moreover, two Y5 antisense ODNs, targeted to different sequences of the receptor, significantly decreased basal food intake and inhibited the increase in food intake after intracerebroventricular injection of NPY. These effects proved to be selective, since the feeding response to galanin was not affected. Analysis of the structure of feeding behavior revealed that prepro NPY and Y5 receptor antisense ODNs reduced food intake by inducing decreases in meal size and meal duration analogous to the orexigenic effects of NPY that are mediated by increases in these parameters. Although changes in Y5 receptor density could not be measured, the results with Y5 antisense ODNs strongly suggest that this receptor subtype mediates the feeding response to exogenous and endogenous NPY. Selective Y5 antagonists may therefore be of therapeutic value for the treatment of obesity and eating disorders.
Reassessment of Glucose Effectiveness and Insulin Sensitivity From Minimal Model Analysis: A Theoretical Evaluation of the Single-Compartment Glucose Distribution Assumption
Authors: Ni Ta-Chen , Ader Marilyn , Bergman Richard N
Published: September 2013

Minimal model analysis with the frequently sampled intravenous glucose tolerance test provides an effective way to measure two important metabolic parameters in vivo under non-steady-state conditions: glucose effectiveness (SG) and insulin sensitivity (SI). Two questions regarding the validity of SG and SI have recently emerged. First, SG from the minimal model is suspected to be overestimated. Second, the occurrence of SI values indistinguishable from zero (“zero-SI”) is not negligible in large clinical studies, and its physiological meaning is uncertain. In this study, we examined the significance of the assumed single-compartment glucose distribution embedded in the minimal model on the estimation of SG and SI. A more accurate two-compartment model was constructed by incorporating insulin action on hepatic glucose output and uptake into a previously validated construction. The two-compartment results were compared with the one-compartment minimal model results. It was shown that the one-compartment assumption contributes to a systematic deviation of SG (slope = 0.54, y-intercept = 0.014 min−1; n = 195 simulations). However, SG from the minimal model was linearly correlated to SG determined from the two-compartment model (r = 0.996). The one-compartment assumption also contributed to the occurrence of zero S1 values for insulin-resistant subjects. A similar linear relationship was found between S1 estimated by both the minimal model and the two-compartment model (slope = 0.58, y-intercept = −0.57 × 10−4 min−1 per μU/ml, r = 0.998). In conclusion, SG and SI from the minimal model are not necessarily equivalent to values emanating from the more accurate two-compartment model. However, the very high correlation between one- and two-compartment results suggests that the minimal model-derived SG and SI are dependable indexes of in vivo glucose effectiveness and insulin sensitivity. Minimal model analysis' advantages of simplicity, minimal invasiveness, reasonable reflection of non-steady-state glucose kinetics, and cost-effectiveness could in many cases outweigh the structural bias introduced by the model simplification.
Immunohistochemical Quantification of Heparan Sulfate Proteoglycan and Collagen IV in Skeletal Muscle Capillary Basement Membranes of Patients With Diabetic Nephropathy
Authors: Yokoyama Hiroki , Høyer Poul E , Hansen Pernille M , Born Jacob van den , Jensen Tonny , Berden Jo H M , Deckert Torsten , Garbarsch Charly
Published: September 2013

In IDDM patients, an increased permeability of the glomerular capillaries has been associated with a general loss of negatively charged heparan sulfate proteo-glycans (HSPGs) within basement membranes (BMs). In the present study, we used immunohistochemical staining to quantify heparan sulfate (HS), HSPG core protein, and collagen IV in capillary basement membranes of skeletal muscle biopsies taken from 9 healthy control subjects (C) and 20 IDDM patients: 7 with normal albumin excretion rate (<30 mg/24 h) (D0), 5 with incipient nephropathy (albumin excretion rate 30–300 mg/24 h) (Dx), and 8 with clinical nephropathy (albumin excretion rate >300 mg/24 h) (D2). In the capillaries, staining was measured by a scanning and integrating microspectrophotometer. A significant difference in the absorbance of HS was found among the four subgroups (means ± SD): 0.477 ± 0.082 (C), 0.627 ± 0.031 (D0), 0.542 ± 0.098 (D1, and 0.371 ± 0.118 (D2) (P = 0.006). Similarly, an overall significant difference in the absorbance of collagen IV was demonstrated (means ± SD): 0.836 ± 0.111 (C), 0.838 ± 0.300 (D0), 0.970 ± 0.173 (D1), and 0.512 ± 0.248 (D2) (P = 0.02). No statistical difference in the absorbance of core protein was demonstrated among the groups. Within the diabetic groups, HS was inversely correlated to albumin-uria (r = −0.76, P = 0.003) and albuminuria corrected for creatinine clearance (r = −0.69, P = 0.008). Because, in IDDM patients with albuminuria, alterations of the content of HS and collagen IV within the capillary BM have been demonstrated immunohistochemically, not only in the glomerular filtration barrier, but also in the skeletal muscle capillary BM, we suggest that these changes reflect universal quantitative or qualitative alterations within the capillary filtration barrier.
Reduction in Diabetes Incidence in an I-Ag7 Transgenic Nonobese Diabetic Mouse Line
Authors: Wherrett Diane K , Singer Steven M , McDevitt Hugh O
Published: September 2013

Susceptibility to IDDM is strongly associated with major histocompatibility complex (MHC) class II genotypes. Nonobese diabetic (NOD) mice develop a similar autoimmune diabetes and have a unique MHC class IIIA allele that is required for the development of diabetes. A number of groups have shown that the introduction of resistant MHC class II alleles as transgenes into the NOD mouse protects from diabetes. We made control transgenic NOD mice, expressing their own Iβg7 molecule as a transgene. One of two lines of these mice showed a reduced incidence of diabetes, without any change in T-cell proliferative response to a number of diabetes autoantigens or any change in insulitis severity. This line developed a subtle decrease in the percentage of splenic B-cells that progressed with age. This defect was not associated with any other phenotypic abnormalities. Our findings suggest that assessment of splenic B-cell number is necessary in interpretation of the effects of MHC class II transgenes on the development of diabetes in the NOD mouse.
Localization and Functional Role of Synaptotagmin III in Insulin Secretory Vesicles in Pancreatic β-Cells
Authors: Mizuta Masanari , Kurose Takeshi , Miki Takashi , Shoji-Kasai Yoko , Takahashi Masami , Seino Susumu , Matsukura Shigeru
Published: September 2013

Pancreatic β-cells secrete insulin by Ca2+-triggered exocytosis of insulin-containing large dense-core vesicles. Synaptotagmin is a Ca2+/phospholipid-binding protein and is a good candidate for the Ca2+ sensor for exocytosis of synaptic vesicles in neurons. In the present study, we generated a polyclonal antibody against synaptotagmin III, and found that synaptotagmin III immunoreactivity was present at high levels in insulincontaining pancreatic islet cells and insulin-secreting clonal MIN6 cells. In subcellular fractionations of MIN6 cells, synaptotagmin III was recovered in the vesicular fractions containing both insulin and vesicleassociated membrane protein-2 (VAMP-2), but not in synaptophysin-positive fractions. The secretory vesicles immunoprecipitated by anti-VAMP-2 antibody contained synaptotagmin III and insulin. In addition, treatment of streptolysin-O-permeabilized MIN6 cells with anti-synaptotagmin III antibody significantly inhibited Ca2+-triggered insulin secretion. These results indicate that synaptotagmin III is localized in insulin-containing dense-core vesicles in pancreatic β-cells, and further strongly suggest that synaptotagmin III is the Ca2+ sensor in the exocytosis of insulin secretory vesicles.
Induction of Renal Kallikrein and Renin Gene Expression by Insulin and IGF-I in the Diabetic Rat
Authors: Jaffa Ayad A , Vio Carlos , Velarde Victoria , LeRoith Derek , Mayfield Ronald K
Published: September 2013

The renal kallikrein-kinin system and the reninangiotensin system are implicated in the pathogenesis of diabetic nephropathy. We have shown that renal kallikrein and renin gene expression are altered by diabetes. To investigate the cellular mechanisms responsible for these changes, we examined the effects of acute insulin and insulin-like growth factor I (IGF-I) treatment on renal kallikrein-kinin and renin-angiotensin system components. Three weeks after induction of diabetes, we measured renal kallikrein and renin mRNA levels, renal kallikrein and renal renin activity, and plasma renin activity in control and diabetic rats and diabetic rats treated with insulin or IGFI for 2 or 5 h. In diabetic rats, kallikrein and renin mRNA levels were reduced >50% compared with control rats. Renal tissue kallikrein levels and plasma renin activity were decreased, whereas renal renin content was unchanged. Insulin increased kallikrein and renin mRNA levels after 2 h. IGF-I, at a dosage that stimulated kallikrein mRNA levels in control rats, had no effect on renal kallikrein and renin content or mRNA levels in diabetic rats. However, infusion of a fivefold higher IGF-I dosage resulted in a two- to threefold increase in kallikrein and renin mRNA levels in 2 h. These data suggest that 1) diabetes suppresses kallikrein and renin gene expression, and these abnormalities are reversed by insulin or IGF-I; and 2) the diabetic state produces resistance to IGF-I induction of kallikrein and renin gene expression. These changes in regulated synthesis of kallikrein and renin in the kidney may underlie renal vascular changes that develop in diabetes.
Hyperinsulinemia and Abdominal Obesity Affect the Expression of Hypertriglyceridemia in Heterozygous Familial Lipoprotein Lipase Deficiency
Authors: Julien Pierre , Void Marie-Claude , Gaudet Daniel , Gagné Claude , Lévesque Georges , Després Jean-Pierre , Cadelis François , Brun Louis Daniel , Nadeau André , Ven Murthy M R
Published: September 2013

We have reported three missense mutations (G188E, P207L, and D250N) in the lipoprotein lipase (LPL) gene among French-Canadians, resulting in the absence of measurable postheparin plasma LPL activity in homozygotes. Presence of triglyceride- and cholesterolrich VLDL, as well as cholesterol-poor HDL particles, has been shown in heterozygotes affected by partial reduction in postheparin LPL activity. However, significant heterogeneity in their plasma triglyceride levels has been found, even among individuals carrying the same LPL gene mutation, indicating that factors other than LPL deficiency could affect the phenotypic expression of hypertriglyceridemia in the heterozygous state. The aim of the present study was to examine the combined effects of abdominal fat accumulation and hyperinsulinemia on plasma triglyceride levels among heterozygous patients for familial LPL deficiency. Based on sex and BMI, 43 heterozygotes (25 women and 18 men) were matched with noncarrier control subjects. Our data indicate that heterozygotes with higher abdominal fat deposition, as defined as waist girth values above the 50th percentile, had higher plasma triglyceride levels than nonobese heterozygotes. However, an important proportion of male heterozygote subjects were hypertriglyceridemic, even in absence of abdominal obesity, suggesting that another factor(s) was involved in the modulation of hypertriglyceridemia in these subjects. Indeed, multivariate analyses revealed that fasting hyperinsulinemia was a significant correlate of hypertriglyceridemia among these heterozygotes. Results of the present study indicate that abdominal obesity and hyperinsulinemia both have deleterious effects on plasma triglyceride levels in familial LPL deficiency. It is suggested that heterozygotes with moderate obesity and/or insulin resistance may be at higher risk of coronary artery disease because of the expression of an atherogenic lipoprotein phenotype among these patients.
Increased Expression of Intercellular Adhesion Molecule-1 (ICAM-1) in Diabetic Rat Glomeruli: Glomerular Hyperfiltration Is a Potential Mechanism of ICAM-1 Upregulation
Authors: Sugimoto Hikaru , Shikata Kenichi , Hirata Kyoji , Akiyama Kenji , Matsuda Mitsuhiro , Kushiro Masahiko , Shikata Yasushi , Miyatake Nobuyuki , Miyasaka Masayuki , Makino Hirofumi
Published: September 2013

Mononuclear cells, including monocytes/macrophages and T-cells, are considered to be involved in the progression of diabetic nephropathy, although the mechanism of their recruitment into diabetic glomeruli is unclear. The intercellular adhesion molecule-1 (ICAM-1) promotes the infiltration of leukocytes into atherosclerotic lesions as well as inflammatory tissues. In the present study, we investigated the expression of ICAM-1 in the glomeruli of streptozotocin-induced diabetic rats. The expression of ICAM-1 was increased significantly during the early stage of diabetes. The number of mononuclear cells, primarily monocytes/macrophages and lymphocytes, was significantly increased in diabetic glomeruli. Mononuclear cell infiltration into diabetic glomeruli was prevented by anti-ICAM-1 monoclonal antibody. Insulin treatment decreased ICAM-1 expression and mononuclear cell infiltration. The ICAM-1 expression on cultured human umbilical vein endothelial cells was not induced under high glucose culture conditions. Glomerular hyperfiltration is a characteristic change in the early stage of diabetic nephropathy. Treatment with aldose reductase inhibitor, which prevented glomerular hyperfiltration without changes in blood glucose levels, decreased ICAM-1 expression and mononuclear cell infiltration. Moreover, we examined the ICAM-1 expression in the glomeruli of the 5/6 nephrectomized rat, which is a model for glomerular hyperfiltration without hyperglycemia. The ICAM-1 expression and infiltration of mononuclear cells was significantly increased in the glomeruli of 5/6 nephrectomized rats. We conclude that ICAM-1 is upregulated and promotes the recruitment of mononuclear cells in diabetic glomeruli. Moreover, glomerular hyperfiltration that occurs in the early stage of diabetic glomeruli may be one of the potential mechanisms of ICAM-1 upregulation in diabetic nephropathy.
Thiazolidinediones Downregulate Stearoyl-CoA Desaturase 1 Gene Expression in 3T3-L1 Adipocytes
Authors: Kurebayashi Shogo , Hirose Takahisa , Miyashita Yoshihiro , Kasayama Soji , Kishimoto Tadamitsu
Published: September 2013

Thiazolidinediones (TZDs) are known to have potent increases of insulin sensitivity. Because peroxisome proliferator-activated receptor-γ (PPAR-γ), a receptor for TZDs, is mainly expressed in adipocytes, we tried to search the TZD-targeted genes in mouse 3T3-L1 adipocytes. By the mRNA differential display method, one band repressed by troglitazone was obtained, which corresponded to the partial sequences of the stearoyl-CoA desaturase 1 (SCD1) gene. Troglitazone dramatically decreased SCD1 mRNA levels in 3T3-L1 adipocytes in a dose-dependent manner. Pioglitazone also repressed the SCD1 mRNA expression, whereas WY-14,643 had no apparent effect. Both troglitazone and pioglitazone raised the composition (weight percentage) of myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0), but lowered the composition of the Δ9-cis desaturated fatty acids such as myristoleic acid (C14:l, Δ9), palmitoleic acid (C16:l, Δ9), oleic acid (C18:l, Δ9), and linoleic acid (C18:2, Δ9,12). These results indicate that TZDs repress SCD1 activity in 3T3-L1 adipocytes via downregulating SCD1 enzyme gene expression.
Characterization of the Insulin Inhibition of the Peptidolytic Activities of the Insulin-Degrading Enzyme–Proteasome Complex
Authors: Bennett Robert G , Hamel Frederick G , Duckworth William C
Published: September 2013

Insulin-degrading enzyme (IDE) is a component of a cytosolic complex that includes multicatalytic proteinase (MCP), the major cytoplasmic proteolytic activity. Insulin, the primary substrate for IDE, inhibits the proteolytic activity of the IDE-MCP complex but not of purified MCP. This provides a regulatory role for IDE in cellular proteolysis and a potential mechanism for intracellular insulin action. To examine the specificity and to explore the mechanisms for the IDE-MCP interaction, we studied the functional interaction of a variety of peptides with the complex. Atrial natriuretic peptide (ANP), relaxin, glucagon, proinsulin, and insulin-like growth factor II (IGF-II) bind to and are degraded by IDE. These peptides have significant inhibitory effects on the chymotrypsin-like and trypsinlike MCP catalytic activities but not the peptidyl-glutamyl hydrolyzing activity. A panel of peptides that are not ligands of IDE had no effect. To explore the potential mechanism for the IDE control of MCP activity, dose response curves for insulin-like growth factor I (IGF-I) and IGF-II effects on MCP chymotrypsin-like activity were determined. IGF-II, which (similar to insulin) is a good substrate for IDE, had a substantial inhibitory effect, whereas IGF-I, which is bound but poorly degraded, had little inhibitory activity on MCP. Proinsulin, another ligand of IDE that is tightly bound but poorly degraded, had a partial effect on MCP activity, but inhibited the full insulin effect. These data suggest a requirement for both the binding and degradation of IDE ligands for the full inhibition of MCP. Insulinsized degradation products, substrates of IDE, also inhibited MCP activity. Further examination of the insulin effect on MCP included kinetic studies. Insulin produced a noncompetitive inhibition of both the chymotrypsin-like and trypsin-like activities of MCP. These data suggest that the insulin-IDE effect on MCP is due to conformational changes in the IDE-MCP complex and provide an intracellular mechanism of action for insulin.
Adrenalectomy Prevents the Obesity Syndrome Produced by Chronic Central Neuropeptide Y Infusion in Normal Rats
Authors: Sainsbury Amanda , Cusin Isabelle , Rohner-Jeanrenaud Françoise , Jeanrenaud Bernard
Published: September 2013

Neuropeptide Y (NPY) in the hypothalamus plays an important role in the regulation of food intake and body weight and seems to be implicated in the etiology of obesity. When intracerebroventricularly (ICV) infused for 6 days in normal rats, NPY resulted in hyperphagia, increased body weight gain, hyperinsulinemia, hypercorticosteronemia, and hypertriglyceridemia compared with vehicle-infused control rats. NPY infusion also resulted in an insulin-resistant state in muscles and in a state of insulin hyperresponsiveness in white adipose tissue, as assessed by the measurement of the in vivo glucose utilization index of these tissues during euglycemic-hyperinsulinemic clamps. All of these hormono-metabolic effects produced by chronic central NPY infusion were completely prevented when rats were adrenalectomized before NPY administration. Adrenalectomy per se had no effect on any of the parameters mentioned above. The levels of mRNA for the obese gene were increased in white adipose tissue after 6 days of ICV NPY infusion in normal rats, and white adipose tissue weight was also increased. These effects of ICV NPY infusion were markedly decreased by prior adrenalectomy, although NPY infusion was able to somewhat enhance the low white adipose tissue obese mRNA levels and tissue weight of adrenalectomized rats. In conclusion, intact adrenal glands, and probably circulating corticosterone in particular, are necessary for the establishment of most of the hormonal and metabolic effects induced by chronic ICV infusion of NPY in normal rats.
The Effect of Pioglitazone on Hepatic Glucose Uptake Measured With Indirect and Direct Methods in Alloxan-Induced Diabetic Dogs
Authors: Matsuhisa Munehide , Shi Z Qing , Wan Calvin , Lekas Michael , Rodgers Carol D , Giacca Adria , Kawamori Ryuzo , Vranic Mladen
Published: September 2013

Pioglitazone, a thiazolidinedione derivative, ameliorates hyperglycemia by augmenting peripheral glucose disposal and suppressing hepatic glucose production in diabetic animals. However, the effect of this agent on hepatic glucose uptake has not been explored. To determine this, experiments were conducted in alloxan-induced diabetic dogs with (pioglitazone group, n = 7) or without (control group, n = 5) a 10-day oral treatment with pioglitazone (1 mg · kg−1 · day−1). A euglycemic-hyperinsulinemic (insulin infusion rate 25.2 pmol · kg−1 · min−1) clamp was maintained by adjusting the peripheral glucose infusion rate (GIR). After a 60-min basal period (period I), portal glucose infusion (Pinf, 33.3 μmol · kg−1 · min−1) was administered for 120 min (period II). This was followed by a 60-min recovery period (period III). Arterial insulin levels were kept stable in the supraphysiological range throughout the experiment (1,623 ± 52, pioglitazone group; 1,712 ± 52 pmol/l, C group). There was no significant difference in whole-body glucose utilization determined by [3-3H]glucose between the pioglitazone and C groups in period I (68.4 ± 2.8 vs. 70.1 ± 2.8 μmol · kg−1 · min , respectively) and period III (81.2 ± 5.0 vs. 74.5 ± 3.3 μmol · kg−1 · min−1, respectively). Net hepatic glucose uptake (NHGU) determined by arteriovenous difference method was approximately zero in the basal period (−0.7 ± 1.1, pioglitazone group; 0.1 ± 1.2 μmol · kg−1 · min−1, C group). In period II, hepatic glucose uptake, determined by the changes in GIR, was significantly higher in the pioglitazone group (6.5 ± 0.6 μmol · kg−1 · min) than in the C group (–0.4 ± 0.6 μmol · k−1 · min−1, P < 0.001). This observation was also confirmed by NHGU during portal glucose infusion (6.9 ± 1.4 vs. 2.1 ±1. 8 μmol · kg−1 · min−1, pioglitazone vs. C, respectively; P < 0.025). We conclude that pioglitazone treatment enhances hepatic glucose uptake during portal glucose loading in alloxan-induced diabetic dogs. However, in hyperinsulinemic conditions, pioglitazone does not enhance the already high peripheral glucose uptake.
Functional β-Cell Mass After Transplantation of Human Fetal Pancreatic Cells: Differentiation or Proliferation?
Authors: Beattie Gillian M , Otonkoski Timo , Lopez Ana D , Alberto Hayek
Published: September 2013

The scarcity of human adult islets available for transplantation in IDDM makes the use of human fetal pancreatic cells desirable. Human fetal pancreatic cells grow and differentiate after transplantation in nude mice. It is unclear whether proliferation of preexisting endocrine cells or differentiation of precursor cells is mainly responsible for the increased islet mass and if β-cell enrichment before transplantation enhances the functional outcome of the graft. To answer these questions, we transplanted purified human fetal islets, isletlike cell clusters (ICCs), and fresh tissue under the kidney capsule of nude mice. Insulin content was highest in the fresh tissue but fell rapidly during culture as either fetal islets or ICCs. Although fetal islets contained fourfold more insulin than ICCs before transplantation, the insulin content of the resulting grafts was the same after 3 months in vivo. The degree of stimulation after glucose challenge was comparable; however, more tissue was needed to generate the fetal islets. Grafts of fresh tissue also had similar total insulin contents, but when normalized to DNA, insulin concentration was significantly higher in the grafts from cultured tissue. Moreover, there were distinct morphological differences; the grafts from fresh tissue were more fibrous, with prominent ductal and cystic elements. Grafts from cultured tissue were two- to threefold enriched in endocrine tissue when compared with grafts originating from fresh tissue. These results suggest that islet cells identified in the grafted ICCs are mainly derived through differentiation of endocrine precursors and that cultured ICCs are more preferable than either fetal islets or uncultured tissue for transplantation.
Pancreas Transplantation Restores Epinephrine Response and Symptom Recognition During Hypoglycemia in Patients With Long-Standing Type I Diabetes and Autonomic Neuropathy
Authors: Kendall David M , Rooney Desmond P , Smets Yves F C , Salazar Bolding Lupe , Robertson R Paul
Published: September 2013

Impaired epinephrine secretion and symptom unawareness are characteristic of severe hypoglycemia in individuals with long-standing type I diabetes. Recently, the avoidance of clinical hypoglycemia has been reported to improve epinephrine and symptom responses to hypoglycemia in type I patients. However, the extent to which these defects can be restored in individuals with long-standing type I diabetes and autonomic neuropathy has not been assessed, nor has it been determined whether pancreas transplantation, which not only obviates hypoglycemia but also prevents hyperglycemia, results in the complete recovery of either epinephrine response or symptom awareness during insulin-induced hypoglycemia. We performed stepped hypoglycemic clamp studies in successful pancreas transplantation recipients to assess epinephrine and other counterregulatory hormone responses during hypoglycemia and to determine the degree to which hypoglycemic symptom recognition could be restored. Thirteen pancreas transplant recipients and matched control subjects were studied utilizing stepped hypoglycemic clamp protocol to achieve target glucose levels of 3.9, 3.3, 2.8, and 2.2 mmol/l (70, 60, 50, and 40 mg/dl, respectively). Plasma epinephrine response was significantly greater in healthy control subjects and pancreas transplant patients compared with type I subjects at the glucose plateaus of 3.9, 3.3, and 2.8 mmol/l. However, epinephrine response in pancreas transplant recipients was significantly less than that seen in either healthy control subjects or nondiabetic kidney transplant recipients at each of these glucose plateaus. The magnitude of the epinephrine response in pancreas transplant type I patients did not correlate with either the duration of diabetes, the duration of transplantation, or the measures of autonomic nerve function. Hypoglycemic symptom recognition was significantly greater in pancreas transplant subjects than type I patients and did not differ between pancreas transplant and control groups. No improvement in norepinephrine response was observed after pancreas transplantation, while glucagon responses to hypoglycemia were normalized in pancreas transplant patients. In conclusion, these studies uniquely demonstrate that successful pancreas transplantation improves epinephrine response and normalizes hypoglycemia symptom recognition in patients with long-standing diabetes and established autonomic neuropathy. No correlation was observed between the severity of autonomic neuropathy or the duration of diabetes and the recovery of either the epinephrine or symptom responses to hypoglycemia.