bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Human Insulin Receptor Gene Mutation Uniquely Inhibits Insulin Binding Without Impairing Posttranslational Processing
Authors: Roach Paris , Zick Yehiel , Formisano Pietro , Accili Domenico , Taylor Simeon I , Gorden Phillip
Published: September 2013

The precise nature of the insulin-binding site of the insulin receptor (IR) has not been determined, although the importance of several regions of the α-subunit in insulin binding has been demonstrated. A naturally occurring mutation in a patient with severe insulin resistance that changes the Ser323 codon in the α-subunit of the IR to a leucine codon is associated with markedly impaired insulin binding to cells from the patient and to transfected cells expressing the mutant receptor. However, unlike other IR α-subunit mutations associated with decreased insulin binding, this mutation does not lead to a defect in posttranslational processing or cell-surface expression of IRs. Thus, the defect in insulin binding associated with the Leu323 mutant IR is a direct result of an alteration in the insulin-binding site. No natural IR mutation described thus far is associated with both decreased insulin binding and normal cell-surface expression of the mutant receptor. This study demonstrates the critical role that Ser323 of the IR α-subunit plays in insulin binding, either by forming part of the binding site or by stabilizing its conformation.
Effects of Glucose on Insulin Secretion, Glucokinase Activity, and Transgene Expression in Transgenic Mouse Islets Containing an Upstream Glucokinase Promoter-Human Growth Hormone Fusion Gene
Authors: Liang Yin , Jetton Thomas L , Zimmerman Elisabeth C , Najafi Habiba , Berner Donna K , Matschinsky Franz M , Magnuson Mark A
Published: September 2013

We have analyzed in organ culture the effects of glucose on glucose-induced insulin secretion, glucokinase (GK) activity, and human growth hormone (hGH) expression in pancreatic islets from transgenic mice containing an upstream GK promoter-hGH fusion gene. Freshly isolated islets from these mice had a normal insulin secretory response to glucose but showed subtle defects after culture in low or high glucose for 4 days that may have been due to the accumulation of hGH in the culture media. Islets cultured from both normal and transgenic mice had approximately a fourfold induction of GK activity in response to an increased concentration of glucose in the culture media, whereas no such change in total islet hGH production was observed. Immunocytochemical localization of hGH in islets cultured in 3 mM glucose showed a pattern similar to that in freshly isolated islets. However, after culture in 30 mM glucose, hGH immunostaining became strikingly more heterogeneous. We conclude 1) that GK-hGH transgene expression does not appear to adversely affect glucose-stimulated insulin secretion in vivo or in freshly isolated islets, 2) that glucose does not induce transgene expression, thus providing additional evidence against an effect of glucose on GK gene transcription in the islet, and 3) that glucose stimulates the co-release of hGH with insulin, thereby enhancing the heterogeneous staining pattern seen among pancreatic β-cells.
3-Deoxyfructose Concentrations are Increased in Human Plasma and Urine in Diabetes
Authors: Wells-Knecht Kevin J , Lyons Timothy J , McCance David R , Thorpe Suzanne R , Feather Milton S , Baynes John W
Published: September 2013

3-Deoxyglucosone (3-DG) is a reactive dicarbonyl sugar thought to be a key intermediate in the nonenzymatic polymerization and browning of proteins by glucose. 3-DG may be formed in vivo from fructose, fructose 3-phosphate, or Amadori adducts to protein, such as NE-fructoselysine (FL), all of which are known to be elevated in body fluids or tissues in diabetes. Modification of proteins by 3-DG formed in vivo is thought to be limited by enzymatic reduction of 3-DG to less reactive species, such as 3-deoxyfructose (3-DF). In this study, we have measured 3-DF, as a metabolic fingerprint of 3-DG, in plasma and urine from a group of diabetic patients and control subjects. Plasma and urinary 3-DF concentrations were significantly increased in the diabetic compared with the control population (0.853 ± 0.189 vs. 0.494 ± 0.072 μM, P < 0.001, and 69.9 ± 44.2 vs. 38.7 ± 16.1 nmol/mg creatinine, P < 0.001, respectively). Plasma and urinary 3-DF concentrations correlated strongly with one another, with HbA1c (P < 0.005 in all cases), and with urinary FL (P < 0.02 and P = 0.005, respectively). The overall increase in 3-DF concentrations in plasma and urine in diabetes and their correlation with other indexes of glycemic control suggest that increased amounts of 3-DG are formed in the body during hyperglycemia in diabetes and then metabolized to 3-DF. These observations are consistent with a role for increased formation of the dicarbonyl sugar 3-DG in the accelerated browning of tissue proteins in diabetes.
Hyperinsulinemia Decreases Second-Phase But Not First-Phase Arginine-Induced Insulin Release in Humans
Authors: Piatti Piero M , Pontiroli Antonio E , Caumo Andrea , Santambrogio Giovanni , Monti Lucilla D , Costa Sabrina , Garbetta Francesca , Baruffaldi Laura , Cobelli Claudio , Pozza Guido
Published: September 2013

The aim of this study was to investigate the effect of hyperinsulinemia on the first and second phase of arginine-induced insulin release in humans. Seven healthy subjects underwent three studies (lasting 360 min): a control study using saline infusion and two euglycemic clamps using a low-dose (0.33 mU · kg−1 · min−1) and a high-dose (1.20 mU · kg−1 · min−1) insulin infusion. After a 3-h equilibration period, arginine (25 g) was infused for 30 min, and insulin and C-peptide responses to arginine were followed for 180 min. At the end of the equilibration period, before arginine administration, steady-state insulin levels were (means ± SE) 60.0 ± 2.4, 165.6 ± 1.8, and 455.4 ± 7.8 pmol/l during saline, low-dose, and high-dose insulin infusions, respectively. The time course of insulin release during the arginine test was calculated from C-peptide concentrations by using C-peptide kinetic modeling and deconvolution. In particular, first-phase and second-phase insulin response was obtained by integrating the time course of the insulin release during either the first 5 min or the following 40 min of the arginine test, respectively. Whereas first-phase insulin release was independent of any effect induced by either insulin infusion, second-phase insulin release was reduced in a similar degree by both insulin infusion doses. First phase was 75.5 ± 10.1, 73.7 ± 12.8, and 73.4 ± 10.3 pmol/kg, whereas second phase was 266.1 ± 46.0, 143.1 ± 33.5, and 133.0 ± 30.2 pmol/kg for saline, low-dose, and high-dose insulin infusions, respectively. We conclude that second-phase, but not first-phase, arginine-induced insulin release is modulated by the pre-stimulus insulin levels. In addition, the inhibitory effect exerted by insulin on second-phase insulin response to arginine appears to be maximized at insulin levels only four times basal.
Type II Diabetes: Clinical Aspects of Molecular Biological Studies
Authors: Turner Robert C , Hattersley Andrew T , Shaw Joanne T , Levy Jonathan C
Published: September 2013

Type II diabetes remains a genetic nightmare. The major problem is identifying suitable pedigrees, sib-pairs, and populations for study. Segregation analysis data suggest that type II diabetes is likely to be polygenic, although one or more major genes could also be involved. This and the high prevalence of diabetes affect the strategies for searching for genetic mutations. Linkage analysis in classical type II diabetes pedigrees is unlikely to be successful. In addition, affected sib-pair analysis is limited because both parents are often affected, leading to bilineal inheritance. Sib-pairs with both parents alive are unusual, so identity by descent analysis is rarely feasible. Strategies to reduce bilineal inheritance by identifying sib-pairs with one known nondiabetic parent or with the second sibling having mild subclinical diabetes may be worthwhile. Identification of individuals or pedigrees with an unusual phenotype that suggests a single gene disorder, such as maturity-onset diabetes of the young, will continue to be important, for this allows linkage analysis with markers near candidate genes and exclusion mapping of chromosomal regions using highly polymorphic markers. Population association studies with candidate genes can detect mutations that have a minor role in the majority proportion of diabetic subjects, but large numbers are required and great care must be taken to exclude ethnic group differences between the diabetic and normoglycemic populations. The study of small inbred communities might be helpful because they may have fewer diabetogenic genes than outbred populations, and this would increase the power of sib-pair and population association studies. Direct screening for mutations in candidate genes (with single-strand conformation polymorphism or heteroduplex screening or with direct sequencing) in patients with the appropriate pathophysiological abnormality can be a successful strategy. The identification of well-defined diabetic pedigrees, sib-pairs, and suitable matched diabetic and nondiabetic populations will be key to the discovery of the genes for diabetes.
Retinopathy, Glucose, and Insulin in an Elderly Population: The Rotterdam Study
Authors: Stolk Ronald P , Vingerling Johannes R , de Jong Paulus T V M , Dielemans Ida , Hofman Albert , Lamberts Stewen W , Pols H A , Grobbee D E
Published: September 2013

We studied the association between retinopathy and glucose metabolism in a population-based study of elderly men and women. Glucose metabolism was assessed by serum fructosamine and a nonfasting oral glucose tolerance test, and retinopathy was evaluated by fundus photography. Retinopathy was present in 296 of 6,191 subjects examined (4.8%; 120 men and 176 women). Serum glucose and fructosamine levels were higher in subjects with retinopathy compared with those without (8.4 vs. 6.8 mmol/l and 329.8 vs. 308.5 γmol/l, respectively, P < 0.001). Two-hour postload insulin levels and insulin resistance, assessed by the ratio of postload insulin over postload glucose level, did not differ between the two groups. These associations were similar in men and women and in subjects with and without diabetes and did not change after adjustment for body mass index or systolic blood pressure. Within the group of subjects who had retinopathy, serum glucose was significantly associated with the number of hemorrhages. These findings suggest that the presence and severity of retinopathy are associated with higher serum glucose levels, both in subjects with and without diabetes.
Insulin Secretagogues, But Not Glucose, Stimulate an Increase in [Ca2+]i in the Fetal Rat β-cell
Authors: Weinhaus Anthony J , Poronnik Philip , Cook David I , Tuch Bernard E
Published: September 2013

Fetal pancreatic islets release insulin poorly in response to glucose; however, the cellular mechanism for this is controversial. By using fura 2 to measure changes in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in β-cells, we have examined islets from fetal, neonatal, and adult rats to determine the ability of glucose and other secretagogues to cause an increase in [Ca2+]i. The effects of glucose (20 mmol/l), glyceraldehyde (20 mmol/l), leucine (20 mmol/l), arginine (20 mmol/l), and the channel effectors glipizide (50 μmol/l), BAY K8644 (2 μmol/l), diazoxide (300 μmol/l), and verapamil (20 μmol/l) on changes in [Ca2+]i were studied. In both the fetal and the mature islet, glyceraldehyde, leucine, arginine, glipizide, and BAY K8644 caused an increase in [Ca2+]i. In mature islets, glucose also increased [Ca2+]i; however, in the fetal islet, glucose had no effect on [Ca2+]i. The stimulus-induced increases in [Ca2+]i in fetal and adult islets were both significantly inhibited by the addition of either diazoxide or verapamil. Similar results were obtained when insulin secretion was measured. Our data show that various secretagogues are able to stimulate fetal islets and cause an increase in [Ca2+]i. Glucose, however, fails to cause an increase in [Ca2+]i in the fetal islet. Hence, the immature insulin secretory response to glucose by the fetal islet is due to the inability of the fetal β-cell to translate glucose stimulation into the increase in [Ca2+]i required for exocytosis of the insulin granule.
Polymorphic Amino Acid Variations in HLA-DQ Are Associated With Systematic Physical Property Changes and Occurrence of IDDM
Authors: Sanjeevi Carani B , Lybrand Terry P , DeWeese Carol , Landin-Olsson Mona , Kockum Ingrid , Dahlquist Gisela , Sundkvist Göran , Stenger David , Lernmark Åke
Published: September 2013

The association between human leukocyte antigen (HLA) and insulin-dependent diabetes was studied in a large population-based investigation using genotyping of 425 new-onset patients, 0–14 years of age, and 367 matched control subjects. As many as 97% of patients compared with 75% of control subjects were positive for one or several of DQA1*0301, DQA1*0501, DQB1*0302, or DQB1*0201. Asp-57 DQB was present among 28% of patients, indicating that this residue alone does not confer protection. Combining Asp-57 DQB1 with either Arg-52 DQA1 or Leu-69 DQA1 did not explain susceptibility or protection either. DQA1*0301-DQB1*0302 (DQ8) and DQA1*0301-DQB1*0301 (DQ7) are identical except for four amino acid substitutions in the β-chain, but DQ8 was positively (odds ratio 8.07; P < 0.001) and DQ7 negatively (odds ratio 0.38; P < 0.001) associated with the disease. Molecular modeling was used to determine whether physiochemical properties such as steric factors and surface electrostatic potentials also differ in a systematic way for various DQ molecules. Amino acids were substituted systematically at the four polymorphic sites, and the solvent-accessible surfaces and electrostatic potentials were computed for each molecule. Dramatic alterations in electrostatic potential were seen for double substitutions at position 45 (G45E) and 57 (A57D) of DQB1. The variation of physicochemical properties due to polymorphic substitutions may be significant to the mechanism of HLA-DQ association with insulin-dependent diabetes, via the effect these property variations have on peptide antigen binding selectivity and subsequent interactions with specific T-cell receptors.
Reduction of the Incretin Effect in Rats by the Glucagon-Like Peptide 1 Receptor Antagonist Exendin (9–39) Amide
Authors: Kolligs Frank , Fehmann Hans-C , Göke Rüdiger , Göke Burkhard
Published: September 2013

Glucagon-like peptide 1 (7–37)/(7–36) amide (GLP-1) is derived from the intestinal proglucagon processing. It is considered an important insulin-releasing gut hormone. This study uses exendin (9–39) amide as a GLP-1 receptor antagonist to evaluate the contribution of GLP-1 to the incretin effect. Anesthetized rats were challenged by an intraduodenal glucose infusion to evaluate maximally occurring GLP-1 and gastric inhibitory polypeptide (GIP) plasma levels. Maximal immunoreactive (IR) GLP-1 plasma levels amounted to 10 pmol/l (IR-GIP 11 pmol/l). Exendin (9–39) amide abolished the insulin-stimulatory effect of 60 pmol of GLP-1 or of the GLP-1 agonist exendin-4 (0.5 nmol) injected as bolus, respectively. An intravenous bolus injection of 5.94 nmol of exendin (9–39) amide 3 min before enteral glucose infusion grossly reduced the total insulin secretory response (by 60%) and significantly increased circulating blood glucose levels (P < 0.05). In contrast, the GLP-1 antagonist left the insulin response after an intravenous glucose or glucose plus GIP (60 pmol) load unaltered. Our data support the concept that GLP-1 is an important incretin factor. Exendin (9–39) amide is a useful GLP-1 antagonist for in vivo studies.
Gender and Insulin Sensitivity in the Heart and in Skeletal Muscles: Studies Using Positron Emission Tomography
Authors: Nuutila Pirjo , Knuuti M Juhani , Mäki Maija , Laine Hanna , Ruotsalainen Ulla , Teräs Mika , Haaparanta Mikko , Solin Olof , Yki-Järvinen Hannele
Published: September 2013

Good insulin sensitivity is independently associated with a low risk for coronary heart disease, but it is unclear whether this risk factor differs between men and women. We compared insulin sensitivity of glucose uptake directly in muscle and heart tissues between healthy women (age 29 ± 2 years, body mass index [BMI] 22 ± 1 kg/m2, VO2max 39 ± 4 ml · kg−1 · min−1) and men matched for age (31 ± 2 years), BMI (23 ± 1 kg/m2), and VO2max (44 ± 3 ml · kg−1 · min−1) using [18F]fluoro-2-deoxy-D-glucose and positron emission tomography under hyperinsulinemic (insulin infusion rate 1 mU · kg−1 · min−1) normoglycemic conditions. Whole body insulin sensitivity was 41% greater in women (52 ± 6 μmol · kg body wt−1 · min−1) than in men (37 ± 3 μmol · kg body wt−1 · min−1, P < 0.05). This difference was explained by a 47% greater rate of glucose uptake by femoral muscles (113 ± 10 vs. 77 ± 7 μmol · kg muscle−1 · min−1, women vs. men, P < 0.01). Insulin-stimulated glucose uptake rates in the heart were similar in women (738 ± 58) and men (749 ± 62 μmol · kg muscle−1 · min−1). Femoral muscle insulin sensitivity was closely correlated with whole body insulin sensitivity (r = 0.84, P < 0.001). Gender and VO2max together explained 68% of the variation in femoral muscle glucose uptake. We conclude that women are more sensitive to insulin than equally fit men because of enhanced muscle but not heart insulin sensitivity.
Determinants of Plasminogen Activator Inhibitor 1 Activity in Treated NIDDM and Its Relation to a Polymorphism in the Plasminogen Activator Inhibitor 1 Gene
Authors: Panahloo Arshia , Mohamed-Ali Vidya , Lane Anne , Green Fiona , Humphries Steve E , Yudkin John S
Published: September 2013

Plasminogen activator inhibitor 1 (PAI-1) activity is increased in patients with non-insulin-dependent diabetes mellitus (NIDDM) and may contribute to their excess risk of cardiovascular disease. We examined the determinants of PAI-1 activity in 146 NIDDM subjects by using specific assays of insulin and intact and des-31,32-proinsulin and measures of insulin resistance, relating these measurements to serum lipids, hypoglycemic therapy, and a common 4G/5G polymorphism in the promoter region of the PAI-1 gene. Subjects were treated with insulin, sulfonylurea, sulfonylurea plus metformin, metformin, and diet alone. In the whole group, PAI-1 activity correlated significantly with serum triglycerides (r = 0.39, P < 0.001), specific insulin (r = 0.29, P < 0.001), intact proinsulin (r = 0.24, P = 0.004), and des-31,32-proinsulin (r = 0.30, P < 0.001) and in subjects not on insulin (n = 110), with insulin sensitivity (r = −0.42, P < 0.001). There was a significant difference in PAI-1 activity among the three genotypic groups (P = 0.016); subjects with the genotype 4G/4G had PAI-1 levels one-third higher than those with the 5G/5G genotype. In the 4G/4G group, PAI-1 activity correlated significantly with triglyceride levels (r = 0.65, P < 0.0001). There was no significant difference in PAI-1 activity in the different treatment groups despite a significant difference in concentrations of intact and des-31,32-proinsulin. In a multiple regression model, insulin sensitivity and the interaction between PAI-1 4G/5G genotype and triglyceride were the strongest determinants of PAI-1 activity.In conclusion, in this sample of NIDDM subjects, the influence of triglyceride on PAI-1 seems to be genotype dependent, and this interaction is a major determinant of PAI-1 activity.
Cloning of a Human Insulin-Stimulated Protein Kinase (ISPK-1) Gene and Analysis of Coding Regions and mRNA Levels of the ISPK-1 and the Protein Phosphatase-1 Genes in Muscle From NIDDM Patients
Authors: Bjørbæk Christian , Vik Terry A , Echwald Søren M , Yang Pei-Yi , Vestergaard Henrik , Wang Jennifer P , Webb Graham C , Richmond Karen , Hansen Torben , Erikson Raymond L , Gabor Miklos George L , Cohen Patricia T W , Pedersen Oluf
Published: September 2013

Complementary DNA encoding three catalytic subunits of protein phosphatase 1 (PP1α, PP1β, and PP1γ) and the insulin-stimulated protein kinase 1 (ISPK-1) was analyzed for variations in the coding regions related to insulin-resistant glycogen synthesis in skeletal muscle of 30 patients with non-insulin-dependent diabetes mellitus (NIDDM). The human ISPK-1 cDNA was cloned from T-cell leukemia and placental cDNA libraries and mapped to the short arm of the human × chromosome. Single-strand conformation polymorphism (SSCP) analysis identified a total of six variations in the coding regions of the PP1 genes: two in PP1α at codons 90 and 255; one in PP1γ at codon 67; and three in PP1γ at codons 11,269, and 273, respectively. All were, however, silent single nucleotide substitutions. SSCP analysis of the ISPK-1 gene identified one silent polymorphism at codon 266 and one amino acid variant at codon 38 (Ile→Ser). This variant was primarily found in one male NIDDM patient. This subject, however, did not exhibit an impairment of muscle insulin-stimulated glycogen synthase activation. No significant differences were found in mRNA levels in muscle of the four genes between 15 NIDDM patients and 14 healthy subjects. Our findings suggest that 1) genetic abnormalities in the coding regions of PP1α, PP1β, PP1γ, and ISPK-1 are unlikely to be frequently occurring causes of the reduced insulin-stimulated activation of the glycogen synthesis in muscle from the analyzed group of NIDDM patients; 2) the mRNA levels of PP1α, PP1β, PP1γ, and ISPK-1 are normal in muscle from the NIDDM patients; and 3) putative inherited defects in insulin-stimulated activation of muscle glycogen synthesis in patients with insulin-resistant NIDDM may be located further upstream of ISPK-1 in the insulin action cascade.
Differential Immunogenetic Determinants of Polyclonal Insulin Autoimmune Syndrome (Hirata's Disease) and Monoclonal Insulin Autoimmune Syndrome
Authors: Uchigata Yasuko , Tokunaga Katsushi , Nepom Gerald , Bannai Makoto , Kuwata Shoji , Dozio Nicoletta , Benson Edward A , Ronningen Kjersti S , Spinas Glatgen A , Tadokoro Kenji , Hirata Yukimasa , Juji Takeo , Omori Yasue
Published: September 2013

The insulin autoimmune syndrome (IAS), or Hirata's disease, is characterized by the combination of fasting hypoglycemia, high concentration of total serum immunoreactive insulin, and presence of autoantibodies to native human insulin in serum. Autoantibody production is classified as monoclonal or polyclonal, with the majority of IAS cases classified as polyclonal. Previously, we observed a striking association between the human leukocyte antigen (HLA) class II alleles DRB1*0406/DQA1* 0301/DQB1*0302 and Japanese IAS patients with polyclonal insulin autoantibodies (IAAs) and T-cell recognition of human insulin in the context of DRB1*0406 molecules. Because of such a strong HLA association in IAS, we performed intra- and interethnic studies on IAS-associated DRB1 alleles and searched for the critical amino acid residue(s) for IAS pathogenesis. Glutamate at position 74 in the HLA-DR4 β1-chain was presumed to be essential to the production of polyclonal IAA in IAS, whereas alanine at the same position of the HLA-DR β1-chain might be important in the production of monoclonal IAA.
A Defective Stimulus-Secretion Coupling Rather Than Glucotoxicity Mediates the Impaired Insulin Secretion in the Mildly Diabetic F1 Hybrids of GK-Wistar Rats
Authors: Abdel-Halim Samy M , Östenson Claes-Göran , Andersson Arne , Jansson Leif , Efendić Suad
Published: September 2013

Adult F1 hybrids of male GK and female Wistar control rats exhibit mild, spontaneous non-insulin-dependent diabetes mellitus characterized by impaired glucose-induced insulin secretion. Using isolated pancreatic islets of hybrid rats, we first studied whether impaired glucose-induced insulin response is present not only in adult but also in neonatal rats. Furthermore, we investigated whether the impaired glucose-induced insulin response can be restored by long-term normalization of glycemia. Both 1-week- and 2- to 3-month-old hybrid rats had similar body weights but increased fed blood glucose levels (P < 0.05) compared with age-matched control rats. At 5.5 mmol/1 glucose, insulin release was two- to threefold lower in isolated islets of hybrid than in control rats of both age groups (P < 0.05). At 16.7 mmol/1 glucose, insulin secretion from hybrid islets was ∼25% of that from control islets of both 1-week- and 2- to 3-month-old rats. For the second objective, batches of 250 islets from hybrid or control rats were transplanted under the kidney capsule of athymic, normoglycemic nude mice and maintained there for 4 weeks. Perfusion of kidneys demonstrated that glucose-induced (16.7 mmol/l) insulin secretion was impaired markedly in hybrid grafts compared with that in control grafts (0.66 ± 0.23 vs. 1.8 ± 0.38 pmol/20 min; P < 0.01), whereas stimulation by 20 mmol/l arginine resulted in similar insulin responses in both groups. The volumes of the grafted islets were similar in kidneys bearing either control or hybrid islets. In conclusion, in hybrid rats the insulin response to glucose is impaired already at neonatal age. This impairment was not restored by long-term maintenance of the hybrid islets in a normoglycemic environment, whereas the arginine effect was intact. These findings suggest that the primary defect resides in the stimulus-secretion coupling for glucose in the β-cell and that hyperglycemia (glucotoxicity) does not play a major role in sustaining this selective β-cell defect.
Phosphofructokinase Isozymes in Pancreatic Islets and Clonal β-Cells (INS-1)
Authors: Yaney Gordon C , Schultz Vera , Cunningham Barbara A , Dunaway George A , Corkey Barbara E , Tornheim Keith
Published: September 2013

Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes. We and others have shown oscillations in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion from the glucose-sensitive clonal β-cell line INS-1. We have proposed that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the β-cell, analogous to those seen in glycolyzing muscle extracts. The mechanism of the latter involves autocatalytic activation of the key regulatory enzyme, phosphofructokinase (PFK), by its product fructose 1,6-bisphosphate (F16BP). However, of the three PFK subunit isoforms (M-[muscle], L-[liver], and C-type, predominant in fibroblasts), only M-type is activated by micromolar F16BP at near-physiological conditions. We therefore studied PFK isoforms in the β-cell. Western analysis of PFK subunits in isolated rat islets and INS-1 cells showed the presence of M-type, as well as C-type and perhaps lesser amounts of L-type. Kinetic studies of PFK activity in INS-1 cell extracts showed strong activation by micromolar concentrations of F16BP at near-physiological concentrations of ATP (several millimolar) and AMP and fructose 6-phosphate (micromolar), indicative of the M-type isoform. Activation by submicromolar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inhibition by citrate were also observed. The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity. These experiments demonstrate that μ-cells contain the M-type isoform of PFK with the requisite regulatory properties for generating glycolytic oscillations that may be the basis of oscillatory insulin secretion.
Emv30null NOD-scid Mice: An Improved Host for Adoptive Transfer of Autoimmune Diabetes and Growth of Human Lymphohematopoietic Cells
Authors: Serreze David V , Leiter Edward H , Hanson Matthew S , Christianson Sherri W , Shultz Leonard D , Hesselton RuthAnn M , Greiner Dale L
Published: September 2013

When used as hosts in passive transfer experiments, a stock of NOD/Lt mice congenic for the severe combined immunodeficiency (scid) mutation have provided great insight to the contributions of various T-cell populations in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM). Moreover, NOD-scid mice support higher levels of human lymphohematopoietic cell growth than the C.B-17-scid strain in which the mutation originated. However, the ability to perform long-term lymphohematopoietic repopulation studies in the NOD-scid stock has been limited by the fact that most of these mice develop lethal thymic lymphomas beginning at 20 weeks of age. These thymic lymphomas are characterized by activation and subsequent genomic reintegrations of Emv30, an endogenous murine ecotropic retrovirus unique to the NOD genome. To test the role of this endogenous retrovirus in thymomagenesis, we produced a stock of Emv30null NOD-scid mice by congenic replacement of the proximal end of chromosome 11 with genetic material derived from the closely related NOR/Lt strain. Thymic lymphomas still initiate in Emv30null NOD-scid females, but their rate of progression is significantly retarded since the frequency of tumors weighing between 170 and 910 mg at 25 weeks of age was reduced to 20.8% vs. 76.2% in Emv30% segregants. The thymic lymphomas that did develop in Emv30null NOD-scid mice were not characterized by a compensatory increase in mink cell focus–forming proviral integrations, which initiate thymomagenesis in other susceptible mouse strains. Significantly, the ability of standard NOD T-cells to transfer IDDM to the Emv30null NOD-scid stock was not impaired. Similarly, the elimination of Emv30 did notabrogate the enhanced ability of NOD-scid mice to support the growth of human peripheral blood leukocytes. These findings coupled with the slowed progression of thymic lymphomas indicate that Emv30null NOD-scid mice are an improved,but not yet optimal, recipient for long-term
Localization of MODY3 to a 5-cM Region of Human Chromosome 12
Authors: Menzel Stephan , Yamagata Kazuya , Trabb Jeffrey B , Nerup Jørn , Permutt M Alan , Fajans Stefan S , Menzel Ruth , Iwasaki Naoko , Omori Yasue , Cox Nancy J , Bell Graeme I
Published: September 2013

Maturity-onset diabetes of the young (MODY) is a heterogeneous disorder that appears to be characterized by a primary defect in insulin secretion. Mutations in an unknown locus (MODY1) on chromosome 20 and the glucokinase gene (MODY2) on chromosome 7 can cause this form of non-insulin-dependent diabetes. Recent genetic studies have identified a third locus on chromosome 12 (MODY3) that is linked to MODY in a group of French families. We have identified three families from Denmark, Germany, and the U.S. (Michigan) showing evidence of linkage with MODY3 and a family from Japan showing suggestive evidence. Analysis of key recombinants in these families localized MODY3 to a 5-cM interval between the markers D12S86 and D12S807/D12S820.
Effects of Diabetes and Hyperglycemia on the Hexosamine Synthesis Pathway in Rat Muscle and Liver
Authors: Robinson Katherine A , Weinstein Mitchell L , Lindenmayer George E , Buse Maria G
Published: September 2013

In vitro studies suggested that increased flux of glucose through the hexosamine biosynthesis pathway (HexNSP) contributes to glucose-induced insulin resistance. Glutamine:fructose-6- phosphateamidotransferase (GFAT) catalyzes glucose flux via HexSNP; its major products are uridine diphosphate (UDP)-N-acetyl hexosamines (UDP-HexNAc). We examined whether streptozotocin (STZ)-induced diabetes (4–10 days) or sustained hyperglycemia (1–2 h) in normal rats alters absolute or relative concentrations of nucleotide-linked sugars in skeletal muscle and liver in vivo. UDP-HexNAc and UDP-hexoses (UDP-Hex) were increased and decreased, respectively, in muscles of diabetic rats, resulting in an ∼ 50% increase in the UDP-HexNAc:UDPHex ratio (P < 0.01). No significant changes in nucleotide sugars were observed in livers of diabetic rats. In muscles of normal rats, UDP-HexNAc concentrations increased (P < 0.01) and UDP-Hex decreased (P < 0.01) during hyperglycemia. The UDP-HexNAc:UDP-Hex ratio increased ∼40% (P < 0.01) and correlated strongly with plasma glucose concentrations. Changes in liver were similar to muscle but were less marked. GFAT activity in muscle and liver was unaffected by 1–2 h of hyperglycemia. GFAT activity decreased 30–50% in muscle, liver, and epididymal fat of diabetic rats, and this was reversible with insulin therapy. No significant change in GFAT mRNA expression was detected, suggesting post-transcriptional regulation. The data suggest that glucose flux via HexNSP increases in muscle during hyperglycemic hyperinsulinemia and that the relative flux of glucose via HexNSP is increased in muscle in STZ-induced diabetes. Since nucleotide sugars are essential substrates for glycoprotein synthesis, changes in their absolute or relative concentrations may affect signal transduction and contribute to insulin resistance.
Evolution of β-Cell Dysfunction in the Male Zucker Diabetic Fatty Rat
Authors: Tokuyama Yoshiharu , Sturis Jeppe , DePaoli Alex M , Takeda Jun , Stoffel Markus , Tang Jiping , Sun Xiaohong , Polonsky Kenneth S , Bell Graeme I
Published: September 2013

The molecular basis for the β-cell dysfunction that characterizes non-insulin-dependent diabetes mellitus (NIDDM) is unknown. The Zucker diabetic fatty (ZDF) male rat is a rodent model of NIDDM with a predictable progression from the prediabetic to the diabetic state. We are using this model to study β-cell function during the development of diabetes with the goal of identifying genes that play a key role in regulating insulin secretion and, thus, may be potential targets for therapeutic intervention aimed at preserving or improving β-cell function. As a first step, we have characterized morphology, insulin secretion, and pattern of gene expression in islets from prediabetic and diabetic ZDF rats. The development of diabetes was associated with changes in islet morphology, and the islets of diabetic animals were markedly hypertrophic with multiple irregular projections into the surrounding exocrine pancreas. In addition, there were multiple defects in the normal pattern of insulin secretion. The islets of prediabetic ZDF rats secreted significantly more insulin at each glucose concentration tested and showed a leftward shift in the dose-response curve relating glucose concentration and insulin secretion. Islets of prediabetic animals also demonstrated defects in the normal oscillatory pattern of insulin secretion, indicating the presence of impairment of the normal feedback control between glucose and insulin secretion. The islets from diabetic animals showed further impairment in the ability to respond to a glucose stimulus. Changes in gene expression were also evident in islets from prediabetic and diabetic ZDF rats compared with age-matched control animals. In prediabetic animals, there was no change in insulin mRNA levels. However, there was a significant 30–70% reduction in the levels of a large number of other islet mRNAs including glucokinase, mitochondrial glycerol-3-phosphate dehydrogenase, voltage-dependent Ca2+ and K+ channels, Ca2+-ATPase, and transcription factor Islet-1 mRNAs. In addition, there was a 40–50% increase in the levels of glucose-6-phosphatase and 12-lipoxygenase mRNAs. There were further changes in gene expression in the islets from diabetic ZDF rats, including a decrease in insulin mRNA levels that was associated with reduced islet insulin levels. Our results indicate that multiple defects in β-cell function can be detected in islets of prediabetic animals well before the development of hyperglycemia and suggest that changes in the normal pattern of gene expression contribute to the development of β-cell dysfunction.
An ATP-Sensitive Cl− Channel Current That Is Activated by Cell Swelling, cAMP, and Glyburide in Insulin-Secreting Cells
Authors: Kinard Tracie A , Satin Leslie S
Published: September 2013

Although chloride ions are known to modulate insulin release and islet electrical activity, themechanism or mechanisms mediating these effects are unclear. However, numerous studies of islet Cl− fluxes have suggested that Cl− movements and glucose and sulfonylurea sensitive and are blocked by stilbene-derivative Cl− channel blockers. We now show for the first time that insulin-secreting cells have a Cl− channel current, which we term ICl,islet· The current is activated by hypotonic conditions, 1–10 μmol/l glyburide and 0.5 mmol/l 8-bromoadenosine 3′:5′-cyclic monophosphate sodium. ICl,islet is mediated by Cl− channels, since replacing [Cl−]o with less permeant aspartate reduces current amplitude and depolarizes its reversal potential. In addition, 100 μmol/l 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) or glyburide, which blocks the Cl− channels of other cell types, block ICl,islet· Reducing [ATP]i reduces the amplitude of the current, suggesting thatit may be under metabolic control. The current is time-independent and shows strong outward-rectification beyond ∼0 mV. At potentials associated with the silent phase of islet electrical activity (approximately −65 mV), ICl,islet mediates a large inward current, which would be expected to depolarize islet membrane potential. Thus, activation of this novel current byincreased intracellular cAMP, sulfonylureas, or ATP may contribute to the well-known depolarizing effects of these agents.
Contribution of Hepatic Glycogenolysis to Glucose Production in Humans in Response to a Physiological Increase in Plasma Glucagon Concentration
Authors: Magnusson Inger , Rothman Douglas L , Gerard David P , Katz Lee D , Shulman Gerald I
Published: September 2013

The contribution of net hepatic glycogenolysis to overall glucose production during a physiological increment in the plasma glucagon concentration was measured in six healthy subjects (18–24 years, 68–105 kg) after an overnight fast. Glucagon (∼3 ng · kg−1 · min−1), somatostatin (0.1 μg · kg−1 · min−1), and insulin (0.9 pmol · kg−1 · min−1) were infused for 3 h. Liver glycogen concentration was measured at 15-min intervals during this period using 13C-labeled nuclear magnetic resonance spectroscopy, and liver volume was assessed from magnetic resonance images. The rate of net hepatic glycogenolysis was calculated from the decrease in liver glycogen concentration over time, multiplied by the liver volume. The rate of glucose appearance (Ra) was calculated from [3-3H]glucose turnover data using a two-compartment model of glucose kinetics. Plasma glucagon concentration rose from 136 ± 18 to 304 ± 57 ng/1 and plasma glucose concentration rose from 5.6 ± 0.1 to 10.4 ± 0.9 mmol/1 on initiation of the infusions. Mean baseline Ra was 11.8 ± 0.4 μmol · kg−1 · min−1, increased rapidly after the beginning of the infusions, reaching its highest value after 20–40 min, and returned to baseline by 140 min. Liver glycogen concentration decreased almost linearly (from 300 ± 19 mmol/1 liver at baseline to 192 ± 20 mmol/1 liver at t = 124 min) during 2 h after the beginning of the infusions, and the calculated mean rate of net hepatic glycogenolysis was 21.7 ± 3.6 μmol · kg−1 · min−1. Mean Ra during the same time period was 22.8 ± 2.3 μmol · kg−1 · min−1. Thus, net hepatic glycogenolysis accounted for 93 ± 9% of Ra. In conclusion, during the initial response to a physiological increment in plasma glucagon, 1) net hepatic glycogenolysis accounts for virtually all of the increase in hepatic glucose production, and 2) glucagon's evanescent effect on hepatic glucose production is not caused by depletion of hepatic glycogen stores.
Inhibition of Sorbitol Dehydrogenase: Effects on Vascular and Neural Dysfunction in Streptozocin-Induced Diabetic Rats
Authors: Tilton Ronald G , Chang Kathy , Nyengaard Jens R , Enden Maria Van den , Ido Yasuo , Williamson Joseph R
Published: September 2013

These experiments were undertaken to assess the role of sorbitol dehydrogenase in mediating sorbitol pathway-linked neural and vascular dysfunction in rats with strep-tozocin-induced diabetes. 2-methyl-4-[N N-dimethylsulfamoyl-piperazino]-pyrimidine (S-0773), a putative inhibitor of sorbitol dehydrogenase, was given in the drinking water to control and diabetic rats. After 5 weeks of diabetes, glycosylated hemoglobin levels were increased twofold and were unaffected by S-0773. Sorbitol levels in diabetic rats were increased 11- to 14-fold in ocular tissues and sciatic nerve; S-0773 increased sorbitol levels another 4-fold or more in these same tissues but had much smaller effects in other tissues. Diabetes-associated increases in fructose levels and lactate:pyruvate ratios in retina and in sciatic nerve were markedly attenuated by S-0773. S-0773 also attenuated, but did not completely normalize, impaired caudal nerve conduction and vascular dysfunction in ocular tissues, sciatic nerve, and aorta in diabetic rats. These observations, together with other evidence, suggest that sorbitol pathway-linked vascular dysfunction (in ocular tissues, peripheral nerve, and aorta) and electrophysiological dysfunction (in peripheral nerve) induced by diabetes are more closely linked to increased oxidation of sorbitol to fructose than to putative osmotic effects of elevated sorbitol levels or redox and metabolic imbalances associated with reduction of glucose to sorbitol by aldose reductase.
Dynamics of β-cell Mass in the Growing Rat Pancreas: Estimation With a Simple Mathematical Model
Authors: Finegood Diane T , Scaglia Luisa , Bonner-Weir Susan
Published: September 2013

The growth and development of the endocrine pancreas has been studied for many years, but questions remain concerning the regulation of the mass of insulin-producing β-cells both in the normal growing pancreas and during the pathogenesis of diabetes. The homeostatic control of β-cell mass in both normal and pathophysiological conditions is based on the balance of cell proliferation, cell growth, and cell death. To gain insight into the relative contribution of each of these dynamic processes, we first mathematically analyzed the data available on the components involved in the maintenance of β-cell mass, including rates of replication, β-cell volume, and the β-cell mass itself, at various ages in normal Sprague-Dawley rats. Then these data were combined in a simple mass balance equation to construct a mathematical model of the dynamics of the β-cell mass in the normal growing rat pancreas. Such a model has allowed us to infer the contributions of fluxes that cannot be measured, i.e., neogenesis and cell death, to the known mass of β-cells. Another important contribution of this model is to raise unanswered questions concerning the control of the balance of cell death and cell renewal in the endocrine pancreas.
Amino Acid Substitutions in Hexokinase II Among Patients With NIDDM
Authors: Laakso Markku , Malkki Mari , Deeb Samir S
Published: September 2013

Hexokinase (HK) II plays an important role in intracellular glucose metabolism by catalyzing the conversion of glucose to glucose-6-phosphate. HKII is considered to be a promising candidate gene for non-insulin-dependent diabetes mellitus (NIDDM) and insulin resistance. Therefore, we investigated the frequency of variants in the coding region of the HKII gene in patients with NIDDM. Initial screening included a population-based sample of 40 Finnish patients with typical NIDDM, and subsequent screening included an additional 72 patients with NIDDM. By applying single-strand conformation polymorphism analysis and direct sequencing, the following amino acid substitutions were found among the 112 NIDDM patients: Ala314Val in one patient (0.9%), Arg353Cys in three patients (2.7%), and Arg775Gln substitution in three patients (2.7%). We also screened 97 subjects with completely normal glucose tolerance and a negative family history of diabetes for these mutations. The Ala314Val and the Arg353Cys substitutions were not found in control subjects, but the Arg775Gln substitution was found in two (2.1%) control subjects. None of these mutations were located close to the glucose- and ATP-binding sites of HKII. We conclude that mutations of the HKII gene are not a major etiological factor for NIDDM in the Finnish population.
Nitric Oxide Synthesis and the Effect of Aminoguanidine and NG-monomethyl-L-Arginine on the Onset of Diabetes in the Spontaneously Diabetic BB Rat
Authors: Wu Guoyao
Published: September 2013

Nitric oxide (NO) synthesis and the effect of aminoguanidine (AG) and NG-monomethyl-L-arginine (NMMA) (inhibitors of NO synthase) on the onset of diabetes were studied in the spontaneously diabetic BB rat. To measure in vivo NO production, 20 male 50-day-old diabetes-prone BB (BBdp) rats and age-matched non-diabetes-prone BB (BBn) rats were individually placed in metabolism cages. The animals had free access to a casein-based semipurified diet and deionized and double-distilled water. Urine excretion was collected every other day for 70 days, and urinary excretion of nitrate was measured as an index of in vivo NO synthesis. The urinary excretion of nitrate was enhanced by 150–200% in BBdp rats 4–6 days before the onset of diabetes, compared with aged-matched BBn rats. There was no difference in urinary excretion of nitrate between BBn rats and those BBdp rats that did not develop diabetes by the age of up to 120 days. To determine a role of NO in the development of spontaneous diabetes, 40-day-old male BBdp rats (30 rats per group) received daily subcutaneous injections of NMMA (acetate salt) (5 mg/kg body wt) or equal amounts of acetate (control) or oral administration of AG (0 or 3 g/l of drinking water) for 80 days. Both NMMA and AG delayed the onset of diabetes in BBdp rats by 13–15 days without altering the rate of incidence of diabetes. The results demonstrate for the first time an enhanced in vivo NO production in insulin-dependent diabetes and provide another line of evidence suggesting an important role of NO in the process of autoimmune destruction of pancreatic β-cells.
Diabetes and Cardiovascular Disease: The “Common Soil” Hypothesis
Authors: Stern Michael P
Published: September 2013

Unlike classical microvascular complications, large-vessel atherosclerosis can precede the development of diabetes, suggesting that rather than atherosclerosis being a complication of diabetes, both conditions have common genetic and environmental antecedents, i.e., they spring from a “common soil.” It is now known that adverse environmental conditions, perhaps related to less-than-optimal nutrition, in fetal and early life are associated with an enhanced risk of both diabetes and cardiovascular disease many decades later. These same adverse environmental conditions are also associated with the development in adult life of abdominal obesity and the insulin-resistance syndrome (IRS). The IRS consists of glucose intolerance, hyperinsulinemia, dyslipidemia (high triglyceride and low high-density lipoprotein [HDL] cholesterol levels), and hypertension. Although the mechanism underlying this cluster is controversial, the statistical association is well established. All of the elements of the IRS have been documented as risk factors for type II diabetes. Some, but not all, of these elements are also cardiovascular disease risk factors, in particular, hypertension and low HDL cholesterol. Other factors associated with the IRS that may enhance cardiovascular disease risk are plasminogen activator inhibitor 1 and small, dense low-density lipoprotein particles. Whether insulin itself is a risk factor remains controversial, but recent epidemiological evidence has been mostly negative. This question has marked clinical relevance because if the IRS enhances cardiovascular disease risk by virtue of its concomitant factors and not the hyperinsulinemia per se, this would tend to alleviate concerns that intensive insulin management of type II diabetic subjects could enhance the risk of large-vessel atherosclerosis. Clinical trials are urgently needed to settle this point.