bluuebunny/crossref_metadata_2025 huggingface dataset:
Islet-Infiltrating Lymphocytes from Prediabetic NOD Mice Rapidly Transfer Diabetes to NOD-scid/scid mice
Authors: Rohane Patricia W , Shimada Akira , Kim Dewey T , Edwards Cariel T , Charlton Brett , Shultz Leonard D , Fathman C Garrison
Published: September 2013

In an effort to study the development of diabetes in NOD mice, our laboratory developed a novel adoptive transfer model using NOD-scid/scid (NOD-scid) mice as recipients of islet-infiltrating lymphocytes from donor prediabetic female NOD mice. We first confirmed previous results that demonstrated that splenocytes of diabetic and prediabetic female NOD mice could transfer diabetes to NOD-scid mice. We demonstrated that the kinetics of disease transfer were dependent on the age of transferred lymphocytes and reiterated the kinetics of diabetes in conventional female NOD mice. We then demonstrated that islet-infiltrating lymphocytes from prediabetic female NOD mice could transfer diabetes. In contrast with the age-dependent transfer of diabetes seen using splenocytes, islet-infiltrating lymphocytes obtained from prediabetic female NOD mice aged ≥40 days rapidly transferred diabetes to NOD-scid recipients. The time required to transfer insulin-dependent diabetes mellitus (IDDM) using islet-infiltrating lymphocytes from young prediabetic mice (25 ± 9 days) was not statistically different from the time required to transfer IDDM using splenocytes from overtly diabetic mice (32 ± 5 days). Cotransfer of splenocyte cells or CD4+, but not CD8+ spleen cells, from 60- to 80-day-old prediabetic female NOD mice together with either splenocytes from diabetic mice or islet-infiltrating lymphocytes from prediabetic NOD mice delayed the rapid transfer of IDDM, suggesting that CD4+ cells mediated immunoregulation. Use of the NOD-scid islet-infiltrating lymphocyte-adoptive transfer model should help elucidate the pathophysiology of the early inflammatory events leading to insulitis and subsequent β-cell destruction. Use of the NOD-scid adoptive model in cotransfer experiments should help identify the immunoregulatory cells that delay development of IDDM in conventional NOD mice.
Predicting Future Diabetes in Latino Women With Gestational Diabetes: Utility of Early Postpartum Glucose Tolerance Testing
Authors: Kjos Siri L , Peters Ruth K , Xiang Anny , Henry Olivia A , Montoro Martin , Buchanan Thomas A
Published: September 2013

We tested 32 routine clinical parameters for their ability to discriminate between a high risk and a low risk of non-insulin-dependent diabetes mellitus (NIDDM) within 5–7 years after pregnancies complicated by gestational diabetes mellitus (GDM). Latino women (n = 671) with GDM who did not have diabetes 4–16 weeks after delivery returned for at least one 75-g oral glucose tolerance test (OGTT) within 7.5 years. Multivariate analysis was used to identify parameters ascertained during or immediately after the index pregnancy that were independently associated with the development of diabetes during follow-up. Life table analysis revealed a 47% cumulative incidence rate of NIDDM 5 years after delivery for this cohort of patients who did not have diabetes at the initial postpartum examination. Four variables were identified as independent predictors of NIDDM: the area under the OGTT glucose curve at 4–16 weeks postpartum, the gestational age at the time of diagnosis of GDM, the area under the OGTT glucose curve during pregnancy, and the highest fasting serum glucose concentration during pregnancy. Examination of relative risks (RRs) of NIDDM between the highest and lowest quartiles of the cohort for each variable, adjusted for the other three variables, revealed that the postpartum OGTT provided the best discrimination between high-risk and low-risk individuals (adjusted RR = 11.5 [95% confidence interval 4.5–29.1] compared with adjusted RRs of only 0.5–2.5 for the other three variables). Women who met World Health Organization criteria for impaired glucose tolerance at the early postpartum examination had a 5-year unadjusted 80% risk of diabetes, which was much higher than the risk of NIDDM that has been reported for Latino people with impaired glucose tolerance who were not selected for a history of GDM. Our findings indicate that postpartum glucose tolerance testing is superior to other routine clinical parameters in defining the risk of NIDDM within 5–7 years after pregnancies complicated by GDM. Furthermore, a history of GDM appears to impart a specific risk for NIDDM that cannot be explained by the degree of glucose tolerance observed when patients are not pregnant.
Isolation of a cDNA Clone Encoding a KATP Channel–Like Protein Expressed in Insulin-Secreting Cells, Localization of the Human Gene to Chromosome Band 21q22.1, and Linkage Studies With NIDDM
Authors: Tsaur Meei-Ling , Menzel Stephan , Lai Fang P , Espinosa Rafael , Concannon Patrick , Spielman Richard S , Hanis Craig L , Cox Nancy J , Le Beau Michelle M , German Michael S , Jan Lily Y , Bell Graeme I , Stoffel Markus
Published: September 2013

The metabolism of glucose in insulin-secreting cells leads to closure of ATP-sensitive K+ channels (KATP), an event that initiates the insulin secretory process. Defects in insulin secretion are a common feature of non-insulin-dependent diabetes mellitus (NIDDM), and the β-cell KATP that couples metabolism and membrane potential is a candidate for contributing to the development of this clinically and genetically heterogeneous disorder. We screened a hamster insulinoma cDNA library by low-stringency hybridization with a probe coding for the G-protein-coupled inwardly rectifying K+ channel GIRK1/KGA and isolated clones encoding a protein, KATP-2, whose sequence is 90% similar to that of the recently described KATP-1, an ATP-sensitive K+ channel expressed in heart and other tissues. RNA blotting showed that KATP mRNA was present in insulin-secreting cells and brain but not in heart. To assess the contribution of KATP-2 to the development of NIDDM, the human KATP-2 gene (symbol KCNJ7) was isolated and mapped to chromosome band 21q22.1 by fluorescence in situ hybridization. A simple tandem repeat DNA polymorphism, D21S1255, was identified in the region of the KATP-2 gene, and linkage studies between this marker and NIDDM were carried out in a group of Mexican-American sib pairs with NIDDM. There was no evidence for linkage between D21S1255 and NIDDM, indicating that KATP-2 is not a major susceptibility gene in this population.
Glomerular Mesangial Cell Altered Contractility in High Glucose Is Ca2+ Independent
Authors: Hurst Roger D , Stevanovic Zivojin S , Munk Snezana , Derylo Bogdan , Zhou Xiaopeng , Meer Jehangir , Silverberg Miriam , Whiteside Catharine
Published: September 2013

In diabetes, loss of renal arteriolar smooth-muscle cell contractility leads to intraglomerular hypertension. In glomeruli isolated from streptozotocin (STZ)-induced diabetic rats, the mesangial cells (smooth muscle-like) display loss of contractile responsiveness to angiotensin II. This study examines the mechanistic relationship between altered mesangial cell contractility and vasopressor hormone-stimulated Ca2+ signaling in high glucose. Glomeruli were isolated from normal or STZ-induced diabetic rats to observe ex vivo mesangial cell contractile function. Also, rat mesangial cells were cultured (10–20 passages) in normal (5.6 mmol/1) or high (10–25.6 mmol/1) glucose for 1–5 days. Reduction of glomerular volume and decreased planar surface area of cultured mesangial cells in response to vasoconstrictor stimulation over 60 min were measured by videomicroscopy and personal computer—based morphometry. Contraction of glomeruli isolated from STZ-administered rats in response to endothelin (ET)-1 (0.1 μmol/1) or the Ca2+ ionophore A23187 (5 μmol/l) was impaired significantly compared with that in normal glucose. In the presence of arginine vasopressin (AVP) (1.0 μmol/1) or ET-1 (0.1 μmol/1), mesangial cells demonstrated a dose-dependent loss of contractile response to increasing glucose concentrations (5.6–25.6 mmol/1) within 24 h of high-glucose exposure, which was sustained for 5 days. Mesangial cells in high glucose were consistently smaller in size compared with those in normal glucose. Mesangial cells were preloaded with myo-[2-3H]inositol and intracellular [3H] inositol phosphate release in response to AVP (1.0 μmol/1) was analyzed by Dowex chromatography. Comparing cells in normal (5.6 mmol/1) versus high (25.6 mmol/1) glucose, we observed no significant difference in stimulated inositol phosphate levels from 10 to 60 s. The Ca2+-signaling response of cultured mesangial cells preloaded with Fura 2 or Indo 1 was measured by spectrofluorometry. After culture in 25.6 mmol/1 glucose, 0.1 μmol/l AVP or 0.1 μmol/1 ET-1 stimulated a cytosolic Ca2+ signal, with first and second phases, identical to that exhibited by mesangial cells in normal glucose. Fluorescence imaging of cultured mesangial cell cytoskeletal filamentous actin (F-actin) stained with rhodamine-phalloidin demonstrated reduced F-actin staining in the basal state and loss of the normal F-actin disassembly response to ET-1 in high glucose. Glomerular mesangial cells display glucose-induced altered contraction and F-actin disassembly to vasoactive stimuli, which occur in the presence of normal Ca2+ signaling.
On the Mechanisms of Blunted Nocturnal Decline in Arterial Blood Pressure in NIDDM Patients With Diabetic Nephropathy
Authors: Nielsen Flemming S , Rossing Peter , Bang Lia E , Svendsen Tage L , Gall Mari-Anne , Smidt Ulla M , Parving Hans-Henrik
Published: September 2013

Nondiabetic hypertensive patients lacking the normal nocturnal decline in arterial blood pressure have enhanced cardiovascular complications. Since cardiovascular morbidity and mortality are increased in non-insulindependent diabetes mellitus (NIDDM), we performed a prospective cross-sectional case-controlled study comparing the diurnal variation in arterial blood pressure, prevalence of dippers, cardiac autonomic nervous function (beat-to-beat variation during deep breathing), and extracellular fluid volume (51Cr-labeled EDTA) in 55 NIDDM patients with diabetic nephropathy (group 1), 55 NIDDM patients with normoalbuminuria (group 2), and 22 nondiabetic control subjects (group 3). All antihypertensive treatments were withdrawn at least 2 weeks before the study. The nocturnal blood pressure reduction (daytime-to-nighttime)/daytime (mean ± SE) was impaired in group 1 (6.6 ± 1.5%) and group 2 (11.1 ± 1.4%) as compared with group 3 (17.6 ± 1.7%), and it was impaired in group 1 as compared with group 2 (JP <0.05 for each comparison). The prevalence of dippers (95% confidence interval) was lower in group 1 (42% [29–56]) as compared with group 2 (58% [44–71]; P = 0.08) and group 3 (86% [65+97]; P < 0.001) and in group 2 as compared with group 3 (P < 0.01). Abolished beat-to-beat variation was more prevalent in group 1 (63% [50–76]) as compared with group 2 (15% [7–27]) and with group 3 (5% [0–23]) (P < 0.001). Nocturnal blood pressure reduction was associated with beat-to-beat variation during deep breathing (r = 0.22, P < 0.01). Extracellular fluid volume (mean ± SE) was higher in group 1 (15.9 ± 0.5 1/m2) as compared with group 3 (14.1 ± 0.8 1/m2) (P < 0.05) with group 2 between the two (15.1 ± 0.4 1/m2). Extracellular fluid volume was not associated with the degree of nocturnal blood pressure reduction. In conclusion, NIDDM patients with and without diabetic nephropathy have blunted nocturnal decline in arterial blood pressure, a condition that might enhance the strain on the microvascular and cardiovascular system. The high prevalence of autonomic neuropathy may also contribute to the increased cardiovascular morbidity and mortality characteristically found in these patients.
Very-Low-Dose Streptozotocin Induces Diabetes in Insulin Promoter-mB7-1 Transgenic Mice
Authors: Harlan David M , Barnett Michelle A , Abe Ryo , Pechhold Klaus , Patterson Noelle B , Gray Gary S , June Carl H
Published: September 2013

Transgenic mice that express mouse B7-1 (mB7-1, recently designated CD80) on their pancreatic β-cells maintain normal islet architecture, have normal pancreatic insulin content, and only rarely spontaneously develop insulitis and diabetes. Nevertheless, these mice display an extreme sensitivity to streptozotocin (STZ)-induced diabetes. Female mice were administered two STZ doses intraperitoneally, 20 and 40 mg/kg body wt, each for five consecutive days. Nontransgenic but otherwise syngeneic mice responded to the STZ with a moderate diminution in pancreatic insulin content but not with persistent glycosuria. In striking contrast, STZ administered to transgenic mice resulted in a severe diminution of pancreatic insulin content and in diabetes. Notably, the lower STZ dose resulted in diabetes only after a prolonged (26- to 100-day) latency. STZ-induced diabetes appears to be T-cell dependent, since treatment with T-cell–depleting (and in particular CD8+ subset-depleting) antibodies ameliorated the response. Anti-mB7-1 monoclonal antibody administration also prevented STZ-induced diabetes. Thus, unmasked mB7-1 is a required component in the pathway resulting in β-cell killing. Immunohistological analysis revealed that early after STZ administration, both mB7-1 transgenic and nontransgenic mice developed insulitis. While this insulitis resolved in the nontransgenic mice, the islet-infiltrating CD4+ and CD8+ T-cells in the transgenic mice were associated with complete β-cell destruction. These data suggest that STZ-induced diabetes in mB7-1 transgenic mice is an immune-mediated process with distinct potential advantages over existing insulindependent diabetes models.
Defective Insulin Action on Protein and Glucose Metabolism During Chronic Hyperinsulinemia in Subjects With Benign Insulinoma
Authors: Battezzati Alberto , Terruzzi Ileana , Perseghin Gianluca , Bianchi Elda , Di Carlo Valerio , Pozza Guido , Luzi Livio
Published: September 2013

The ability of chronic endogenous hyperinsulinemia to induce a resistance to insulin action on protein and glucose metabolism was studied in 10 subjects affected by a benign (functioning) insulinoma and 18 healthy subjects by means of infusions of [l-14C]leucine and [3-3H] glucose. The insulinoma subjects were divided into two groups with moderate (139 ± 12 pmol/1) (n = 5) and marked (438 ± 42 pmol/1) (n = 5) hyperinsulinemia and were studied during a euglycemic dextrose infusion. Control subjects were studied postabsorptively and during a low-dose (0.3 mU · kg−1·min−1) (n = 3) and a high-dose (1 mU · kg−1 · min−1) (n = 15) euglycemic insulin clamp to match peripheral insulin concentrations with those of insulinoma subjects. In insulinoma subjects there was no correlation among plasma insulin concentration and leucine concentration (r = 0.05), endogenous leucine flux (r = 0.44), hepatic glucose production (r = 0.47), and glucose uptake (r = 0.05). Insulinoma subjects with marked hyperinsulinemia demonstrated a defective suppression of leucine concentrations (100 ± 11 vs. 65 ± 5 μmol/l,P < 0.01), endogenous leucine flux (50.1 ± 6.3 vs. 27.1 ± 0.9 μmol · m−2 · min−1, P < 0.01), and hepatic glucose production (5.4 ± 2.0 vs. 0.6 ± 0.6 ± mol · kg−1 · min−1, P < 0.05), and a defective stimulation of glucose uptake (13.5 ± 1.6 vs. 41.1 ± 2.8 ± mol · kg−1 · min−1, P <0.001) with respect to normal subjects at a comparable degree of hyperinsulinemia. Our results demonstrate that chronic endogenous hyperinsulinemia causes a defect in insulin action that is generalized to both protein metabolism and hepatic and peripheral glucose metabolism.
Estimation of β-Cell Sensitivity From Intravenous Glucose Tolerance Test C-Peptide Data: Knowledge of the Kinetics Avoids Errors in Modeling the Secretion
Authors: Toffolo Gianna , De Grandi Fabio , Cobelli Claudio
Published: September 2013

Parametric models of insulin secretion are used to measure indexes of β-cell function from plasma C-peptide concentration during an intravenous glucose tolerance test (IVGTT). Since the models have been usually assessed against plasma C-peptide data, both secretory and kinetic parameters need to be simultaneously estimated. However, undesired compensations between the two sets of parameters may arise. In this study, in order to evaluate IVGTT insulin secretion models, we have analyzed IVGTT data from seven normal subjects for whom individual C-peptide kinetics were known from a separate experiment. Three different β-cell models have been examined: the minimal model Ml (Diabetes 37:223–231, 1988); a variation of a published model, M2 (Math Biosci 27:319–332, 1975); and a new one, M3. A two-compartment model was used to describe C-peptide kinetics. The results suggest the inadequacy of Ml since kinetic parameter estimates were consistently biased versus the known individual values, and systematic errors were present in the prediction of C-peptide data when kinetic parameters were fixed to the known individual values. M2 performs better than Ml since it reproduces C-peptide data satisfactorily when the individually known description of the kinetics is adopted. M3 retains the second-phase description of M2 but improves the description of first-phase release. M3 is thus proposed to reconstruct the insulin secretion time course and to estimate parameters of first and second-phase sensitivity to glucose. We also show the robustness of M3, i.e., standard values of C-peptide kinetic parameters can be used when individual values are not available without a loss of accuracy in the estimated secretion parameters. Finally, the shortcomings of using a simplified single-compartment description of C-peptide kinetics are discussed.
Involvement of ATP-Sensitive K+ Channels in Free Radical–Mediated Inhibition of Insulin Secretion in Rat Pancreatic β-Cells
Authors: Nakazaki Mitsuhiro , Kakei Masafumi , Koriyama Nobuyuki , Tanaka Hiromitsu
Published: September 2013

To explore the mechanisms of inhibition of insulin secretion in pancreatic β-cells by oxygen free radicals, we studied the effects of H2O2 on membrane currents using the patch-clamp technique. Exposure of β-cells to H2O2 (≥30 (μmol/1) increased the activity of ATP-sensitive potassium (K+ATP) channels without changing the single channel conductance in cell-attached membrane patches. Action currents observed during superfusion of 11.1 mmol/1 glucose were suppressed. In inside-out membrane patches, the activity of K+ATP channels was not influenced by H2O2. In conventional whole-cell clamp experiments using a pipette solution containing 3 mmol/1 ATP, H2O2 did not influence the membrane currents. However, H2O2 did activate the K+ATP channel current in perforated whole-cell clamp configurations. The increased K+ATP channel current was reversed by subsequent exposure to 11.1 mmol/12-ketoisocaproic acid. In cell-attached membrane patches, the K+ATP channel current evoked by exposure to 30 μmol/l H2O2 was inhibited by exposure to 11.1 mmol/l glyceraldehyde, whereas the channel was again activated by exposure to 0.3 mmol/l H2O2. Subsequent superfusion of 11.1 mmol/l 2-ketoisocaproic acid inhibited the channel; this effect was counteracted by exposure to 10 mmol/l H2O2. Transient inhibition of K+ATP channels with provocation of action potentials was observed after washout of 100 μmol/l H2O2 during superfusion of 2.8 or 11.1 mmol/l glucose. We conclude that H2O2 has no direct effect on the K+ATP channels but that it indirectly activates the channels when it is exposed to β-cells under conditions in which the cellular metabolism is physiologically regulated.
Significance of Glutathione Depletion and Oxidative Stress in Early Embryogenesis in Glucose-Induced Rat Embryo Culture
Authors: Trocino Romulo A , Akazawa Shoichi , Ishibashi Miwa , Matsumoto Kazunari , Matsuo Hiroshi , Yamamoto Hidefumi , Goto Shinji , Urata Yoshishige , Kondo Takahito , Nagataki Shigenobu
Published: September 2013

Recent studies have demonstrated the protective effects of supplementing free oxygen radical scavenging enzymes against hyperglycemia-induced embryonic malformations. In this study, the glutathione (GSH)-dependent protection system in hyperglycemia-induced embryopathy was investigated. Rat embryos at the early head-fold stage (day 9.5) cultured in 66.7 mmol/l glucose for 48 h showed significant growth retardation and an increase in the frequency of malformations. The concentration of GSH and activity of the rate-limiting GSH-synthesizing enzyme, γ-glutamylcysteine synthetase (γ-GCS), significantly decreased in embryos exposed to hyperglycemia compared with controls (7.9 ± 0.6 vs. 12.5 ± 0.9 nmol/mg protein, P < 0.01 and 13.3 ± 1.9 vs. 22.6 ± 1.1 μU/mg protein, P < 0.01, respectively). Decreased activity of γ-GCS in embryos exposed to hyperglycemia was associated with decreased expression of γ-GCS mRNA levels. However, the activities of superoxide dismutase and glutathione peroxidase did not significantly change in these embryos. Extracellular and intracellular free oxygen radical formations estimated by Lucigenin-dependent chemoluminescence and flow cytometric analysis using 2′,7′-dichlorofluorescein diacetate increased in isolated embryonic cells taken from embryos cultured under hyperglycemia. Supplementation of 2 mmol/l GSH ester into the hyperglycemic culture nearly restored GSH concentration in these embryos (11.9 ± 0.5 vs. 12.5 ± 0.9 nmol/mg protein) and reduced the formation of free oxygen radical species leading to almost complete normalization of growth retardation and embryonic dysmorphogenesis. These results indicate that the GSHdependent protection system has a central role against oxidative stress in embryos cultured under hyperglycemia and that GSH depletion in embryonic cells during the critical periods of organogenesis plays a role in hyperglycemia-induced embryopathy.
Evidence for Linkage of Postchallenge Insulin Levels With Intestinal Fatty Acid-Binding Protein (FABP2) in Mexican-Americans
Authors: Mitchell Braxton D , Kammerer Candace M , O'Connell Peter , Harrison Chantal R , Manire Madelene , Shipman Patricia , Moyer Mary Pat , Stern Michael P , Frazier Marsha L
Published: September 2013

Single genes with large effects may contribute to insulin resistance or influence susceptibility to non-insulin-dependent diabetes mellitus (NIDDM). In the Pima Indians, results from sib-pair analysis have suggested that a gene on chromosome 4q influences both fasting insulin levels and maximal insulin action. We conducted sib-pair and logarithm of odds (LOD)-score linkage analysis to seek evidence for linkage between genes influencing insulin levels and chromosome 4q loci. Analyses were conducted on nondiabetic individuals from 28 different families participating in the San Antonio Family Diabetes Study. All subjects received a 2-h oral glucose tolerance test. Fasting insulin levels were measured in 382 nondiabetic individuals, and 2-h insulin levels were measured in 366 individuals. Initial sib-pair linkage analysis revealed a possible association between 2-h post–glucose challenge insulin levels and the intestinal fatty acid–binding protein (FABP2) locus located in the region of chromosome 4q28–31 (P = 0.006). Subsequent sib-pair linkage analysis of 11 additional chromosome 4q markers supported this hypothesis. We next conducted segregation analyses to estimate allele frequencies and other model parameters for the putative locus influencing 2-h insulin levels. Results of LOD-score linkage analysis indicated possible linkage between the major gene described by the segregation model and FABP2. Using combined segregation and linkage analysis, we obtained a LOD-score of 2.80 at recombination frequency of 0.0 between FABP2 and the putative locus influencing 2-h insulin levels. The maximum likelihood estimate of the allele associated with low insulin levels was 0.21. Individuals having one or two copies of this allele had a mean ln(2-h insulin level) equal to 3.484 (back-transformed mean = 298.4 pmol/1), compared with 4.480 (back-transformed mean = 807.8 pmol/1) for individuals in whom this allele was absent. Approximately 32% of the total phenotypic variance in ln(2-h insulin levels) could be attributed to this locus. These results are consistent with the hypothesis that FABP2, or a tightly linked gene, influences 2-h insulin levels. This gene may be associated with insulin resistance.
Processing of Proinsulin by Furin, PC2, and PC3 in (Co) Transfected COS (Monkey Kidney) Cells
Authors: Vollenweider Florence , Kaufmann Jocelyne , Irminger Jean-Claude , Halban Philippe A
Published: September 2013

The enzymology of proinsulin conversion was studied in COS cells by cotransfection of three species of proinsulin and each of three conversion endoproteases (furin, PC2, and PC3). In addition to the pairs of basic residues linking the B-chain to C-peptide (Arg31-Arg32) and C-peptide to the A-chain (Lys64-Arg65), which were present in all three proinsulins studied, human proinsulin presents a P4 basic residue (four residues NH2-terminal to the point of cleavage) only at the former junction (Lys29) and rat proinsulin II only at the latter (Arg62). Human proinsulin Arg62 (prepared by site-directed mutagenesis of human proinsulin) contains a P4 basic residue at both junctions. Transfected cells were incubated for four successive 2-h periods. The media were pooled, and proinsulin, conversion intermediates, and insulin were separated by reverse-phase high-performance liquid chromatography to monitor conversion activity. There was little conversion of any proinsulin in COS cells without cotransfection of an exogenous endoprotease. When furin or PC3 was cotransfected with any of the three proinsulins, there was extensive processing, with insulin as the major conversion product. PC2, by contrast, failed to cleave human proinsulin but was able to cleave both human proinsulin Arg62 and rat proinsulin II. Cleavage by PC2 of these proinsulins was predominantly at the C-peptide–A-chain junction, generating the conversion intermediate des-64,65-split proinsulin as the major product and only very small amounts of insulin itself.
Syngeneic Pancreatic Islet Transplantation Reverses Endothelial Dysfunction in Experimental Diabetes
Authors: Pieper Galen M , Jordan Milan , Adams Mark B , Roza Allan M
Published: September 2013

Diabetes is known to cause impaired endothelium-dependent relaxation of blood vessels. The purpose of this study was to determine whether this endothelial dysfunction is a permanent defect or is reversible after acute arginine supplementation in vitro or by surgical intervention in vivo using syngeneic pancreatic islet transplantation. Lewis rats were injected with streptozotocin to induce diabetes and were studied either 8 or 12 weeks later. Another group received syngeneic islets via intraportal injection at 8 weeks of diabetes and were allowed to become euglycemic for 4 weeksbefore study. Thoracic aortic rings were tethered in isolated muscle baths, contracted with a submaximal concentration of norepinephrine, and challenged with either the endothelium-dependent vasodilator acetylcholine or the endothelium-independent vasodilator nitroglycerin. Relaxation to acetylcholine (but not nitroglycerin) was reduced in both 8- and 12-week diabetic rings compared with age-matched control rings. Preincubation of diabetic rings in vitro with L-arginine (but not D-arginine) restored relaxation to acetylcholine to normal to rings from 8-week but not 12-week diabeticanimals. Plasma basic amino acids (arginine, lysine, and histidine) were reduced by diabetes, whereas other neutral or acidic amino acids were unchanged (phenylalanine, proline, and glutamate), reduced (serine, cysteine, threonine, tyrosine, tryptophan, and aspartate), or elevated (isoleucine,leucine, and valine). Islet transplantation restored to normal the changes in plasma amino acids. Elevation in blood glucose and total glycosylated hemoglobin in diabetic animals was normalized after islet transplantation. Furthermore, islet transplantation completely restored the defective endothelium-dependent relaxation to acetylcholine in diabetic rings. These studies provide evidence for the existence of reversible and irreversible stages of endothelial cell dysfunction that occurin diabetes and that preemptive surgical intervention using pancreatic islet transplantation can completely restore normal endothelial function.
Effects of Treatment of Spontaneously Hypertensive Rats With the Angiotensin-Converting Enzyme Inhibitor Trandolapril and the Calcium Antagonist Verapamil on the Sensitivity of Glucose Metabolism to Insulin in Rat Soleus Muscle In Vitro
Authors: Leighton Brendan , Sanderson Alison L , Young Martin E , Radda George K , Boehm Ernest A , Clark Joseph F
Published: September 2013

We measured the sensitivity of glucose metabolism to insulin in soleus muscle preparations isolated from spontaneously hypertensive (SH) rats and normotensive age-matched Wistar-Kyoto (WKY) rats. SH rats were treated with the angiotensin-converting enzyme (ACE) inhibitor trandolapril (1 mg/kg) and/or a second antihypertensive drug, the calcium antagonist verapamil, alone (100 mg/ kg) or as combination therapy (50 mg/kg). Treatment of SH rats with trandolapril or trandolapril in combination with verapamil for 6 weeks normalized the blood pressure. The estimated concentration of insulin required for half-maximal stimulation of glycogen synthesis (i.e., EC50values) was ∼500 μU/ml for muscles from both WKY and SH rats. This value is five times higher than the value obtained from soleus muscle preparations isolated from insulin-sensitive Wistar rats. This indicates that glycogen synthesis is insensitive to insulin in SH and WKY rat soleus muscle. Treatment of SH rats with trandolapril with or without verapamil improved the sensitivity of glycogen synthesis to insulin in soleus muscle. Further experiments investigated whether acute exposure (1 h) of insulin-sensitive skeletal muscle with either trandolaprilat (the active metabolite of trandolapril) or bradykinin (levels of which may be raised by ACE inhibition) could affect the insulin-stimulated rate of glucose metabolism. These results show that both trandolaprilat and bradykinin caused a small but significant increase in the rates of glucose metabolism. In conclusion, 1) SH and WKY rat skeletal muscle was insulin resistant, 2) chronic treatment of SH rats with trandolapril with or without verapamil normalized blood pressure and improved the response of glycogen metabolism to insulin, and 3) bradykinin and trandolaprilat acutely caused a small but significant increase in the rate of glycogen synthesis to a submaximal physiological concentration of insulin.
Control of Energy Metabolism During Muscle Contraction
Authors: Radda George K
Published: September 2013

Skeletal muscle energetics can be studied noninvasively at rest, during exercise, and in recovery using phosphorus nuclear magnetic resonance (31P-NMR). In resting muscle, inorganic phosphate (Pi) and total cellular phosphate concentration are regulated by Na+-dependent Pi transport. Insulin was shown to stimulate Pi uptake in G-8 muscle cells, in isolated rat soleus muscle, and in human muscle in vivo under conditions of hyperinsulinemic-euglycemic clamp. The relationship between plasma Pi and intracellular muscle Pi was examined in a group of patients with elevated plasma Pi resulting from renal failure. The total creatine content of muscle cells is controlled by an active creatine uptake in which β2-receptor stimulation and the activity of the Na+-K+-ATPase play a significant role. Recovery after exercise is entirely oxidative; the rate of ATP synthesis is largely controlled by ADP, the concentration of which is determined by the creatine kinase equilibrium that includes the concentration of H+. At the onset of aerobic dynamic exercise, ATP is maintained largely by glycolysis, producing lactic acid, and by phosphocreatine breakdown. After vasodilation, ATP synthesis becomes predominantly oxidative. The above processes can be quantitatively evaluated by 31P-NMR.
High D-Glucose–Induced Changes in Endothelial Ca2+/EDRF Signaling are Due to Generation of Superoxide Anions
Authors: Graier Wolfgang F , Simecek Sabine , Kukovetz Walter R , Kostner Gert M
Published: September 2013

Pretreatment of porcine aortic endothelial cells with high D-glucose results in enhanced endothelium-derived relaxing factor (EDRF) formation (39%) due to increased endothelial Ca2+ release (57%) and Ca2+ entry (97%) to bradykinin. This study was designed to investigate the intracellular mechanisms by which high D-glucose affects endothelial Ca2+/EDRF response. The aldose-reductase inhibitors, sorbinil and zopolrestat, failed to diminish high D-glucose-mediated alterations in Ca2+/EDRF response, suggesting that aldose-reductase does not contribute to high D-glucose-initiated changes in Ca2+/EDRF signaling. Pretreatment of cells with the nonmetabolizing D-glucose analog, 3-O-methylglucopyranose (3-OMG), mimicked the effect of high D-glucose on Ca2+ release (41%) and Ca2+ entry (114%) to bradykinin, associated with elevated EDRF formation (26%). High D-glucose and 3-OMG increased superoxide anion (O2−) formation (133 and 293%, respectively), which was insensitive to inhibitors of cyclooxygenase (5,8,11,14-eicosatetraynoic acid [ETYA], indomethacin), lipoxygenase (ETYA, gossypol, nordihydroguaiaretic acid [NDGA]), cytochrome P450 (NDGA, econazole, miconazole), and nitric oxide (NO) synthase (L-omega N-nitroarginine), while it was diminished by desferal, a metal chelator. The gamma-glutamyl-cysteine-synthase inhibitor, buthioninesulfoximine (BSO), also increased formation of O2− by 365% and mimicked the effect of high D-glucose on Ca2+/EDRF signaling. The effects of high D-glucose, 3-OMG, and BSO were abolished by co-incubation with superoxide dismutase. Like high D-glucose, pretreatment with the O2−-generating system, xanthine oxidase/hypoxanthine, elevated bradykinin-stimulated Ca2+ release (+10%), Ca2+ entry (+75%), and EDRF (+73%). We suggest that prolonged exposure to pathologically high D-glucose concentration results in enhanced formation of O2−, possibly due to metal-mediated oxidation of D-glucose within the cells. This overshoot of O2− enhances agonist-stimulated Ca2+/EDRF signaling via a yet unknown mechanism.
The Weight-Reducing Effect of an Intracerebroventricular Bolus Injection of Leptin in Genetically Obese fa/fa Rats: Reduced Sensitivity Compared With Lean Animals
Authors: Cusin I , Rohner-Jeanrenaud Françoise , Stricker-Krongrad Alain , Jeanrenaud Bernard
Published: September 2013

The effect of different doses of leptin, given as an intracerebroventricular (ICV) bolus, on body weight gain and food intake was investigated during refeeding, following a 24-h fast in lean (FA/fa) rats. It was observed that ICV leptin resulted in a dose-dependent decrease in body weight gain, compared with vehicle injection, a difference that persisted for at least 6 days. This was associated with a transient reduction in food intake over the first 2 days after leptin injection. More importantly, the effect of leptin was also observed in genetically obese fa/fa rats but at the expense of two to ten times higher leptin concentrations, indicating the presence of decreased leptin sensitivity. Furthermore, ICV leptin injections were able to decrease neuropeptide Y (NPY) levels in the arcuate and paraventricular hypothalamic nuclei in both lean and genetically obese fa/fa rats, although a higher leptin dose was again needed in the obese group. These observations provide further evidence for the implication of NPY and leptin in a regulatory loop controlling body homeostasis. This loop is functional in lean and genetically obese fa/fa rats, provided that leptin levels in the central nervous system are high enough in the obese group, in particular. Since human obesity is frequently associated with elevated circulating leptin levels, a state of decreased leptin sensitivity (i.e., leptin resistance), similar to that described here in fa/fa rats, could possibly occur in human syndromes as well.
The Human Glucokinase Gene β-Cell-Type Promoter: An Essential Role of Insulin Promoter Factor 1/PDX-1 in Its Activation in HIT-T15 Cells
Authors: Watada Hirotaka , Kajimoto Yoshitaka , Umayahara Yutaka , Matsuoka Taka-aki , Kaneto Hideaki , Fujitani Yoshi , Kamada Takenobu , Kawamori Ryuzo , Yamasaki Yoshimitsu
Published: September 2013

The glycolytic enzyme glucokinase plays a primary role in the glucose-responsive secretion of insulin, and defects of this enzyme can cause NIDDM. As a step toward understanding the molecular basis of glucokinase (GK) gene regulation, we assessed the structure and regulation of the human GK gene β-cell-type promoter. The results of reporter gene analyses using HIT-T15 cells revealed that the gene promoter was comprised of multiple cis-acting elements, including two primarily important cis-motifs: a palindrome structure, hPal-1, and the insulin gene cis-motif A element-like hUPE3. While both elements were bound specifically by nuclear proteins, it was the homeodomain-containing transcription factor insulin promoter factor 1 (IPF1)/STF-1/PDX-1 that bound to the hUPE3 site: IPF1, when expressed in CHO-K1 cells, became bound to the hUPE3 site and activated transcription. An anti-IPF1 antiserum used in gel-mobility shift analysis supershifted the DNA protein complex formed with the hUPE3 probe and nuclear extracts from HIT-T15 cells, thus supporting the involvement of IPF1 in GK gene activation in HIT-T15 cells. In contrast to the insulin gene, however, neither the synergistic effect of the Pan1 expression on the IPF1-induced promoter activation nor the glucose responsiveness of the activity was observed for the GK gene promoter. These results revealed some conservative but unique features for the transcriptional regulation of the β-cell-specific genes in humans. Being implicated in insulin and GK gene regulations as a common transcription factor, IPF1/STF-1/PDX-1 is likely to play an essential role in maintaining normal β-cell functions.
Normalization of Insulin Responses to Glucose by Overnight Infusion of Glucagon-Like Peptide 1 (7–36) Amide in Patients With NIDDM
Authors: Rachman Jonathan , Gribble Fiona M , Barrow Beryl A , Levy Jonathan C , Buchanan Keith D , Turner Robert C
Published: September 2013

Glucagon-like peptide 1 (GLP-1) is a natural enteric incretin hormone, which is a potent insulin secretogogue in vitro and in vivo in humans. Its effects on overnight glucose concentrations and the specific phases of insulin response to glucose and nonglucose secretogogues in subjects with NIDDM are not known. We compared the effects of overnight intravenous infusion of GLP-1 (7-36) amide with saline infusion, on overnight plasma concentrations of glucose, insulin, and glucagon in eight subjects with NIDDM. The effects on basal (fasting) β-cell function and insulin sensitivity were assessed using homeostasis model assessment (HOMA) and compared with seven age- and weight-matched nondiabetic control subjects. The GLP-1 infusion was continued, and the first- and second-phase insulin responses to a 2-h 13 mmol/l hyperglycemic clamp and the insulin response to a subsequent bolus of the nonglucose secretogogue, arginine, were measured. These were compared with similar measurements recorded after the overnight saline infusion and in the control subjects who were not receiving GLP-1. The effects on stimulated β-cell function of lowering plasma glucose per se were assessed by a separate overnight infusion of soluble insulin, the rate of which was adjusted to mimic the blood glucose profile achieved with GLP-1. Infusion of GLP-1 resulted in significant lowering of overnight plasma glucose concentrations compared with saline, with mean postabsorptive glucose concentrations (2400–0800) of 5.6 ± 0.8 and 7.8 ± 1.4 mmol/l, respectively (P < 0.0002). Basal β-cell function assessed by HOMA was improved from geometric mean (1 SD range), 45% β (24–85) to 91% β (55–151) by GLP-1 (P < 0.0004). First-phase incremental insulin response to glucose was improved by GLP-1 from 8 pmol/l (-–33) to 116 pmol/l (12–438) (P < 0.005), second-phase insulin response to glucose from 136 pmol/l (53–352) to 1,156 pmol/l (357–3,748) (P < 0.0002), and incremental insulin response to arginine from 443 pmol/l (172–1,144) to 811 pmol/l (272–2,417) (P < 0.002). All responses on GLP-1 were not significantly different from nondiabetic control subjects. Reduction of overnight glucose by exogenous insulin did not improve any of the phases of stimulated β-cell function. Prolonged intravenous infusion of GLP-1 thus significantly lowered overnight glucose concentrations in subjects with NIDDM and improved both basal and stimulated β-cell function to nondiabetic levels. It may prove to be a useful agent in the reduction of hyperglycemia in NIDDM.
Glucose Transport: Pivotal Step in Insulin Action
Authors: Kahn Barbara B
Published: September 2013

The effect of insulin to acutely stimulate glucose uptake into muscle and adipose tissue is essential for normal glucose homeostasis. The GLUT4 glucose transporter is a major mediator of this action, and insulin recruits GLUT4 from an intracellular pool to the plasma membrane. An important pathologic feature of obesity, NIDDM, and to a lesser extent IDDM is resistance to insulin-stimulated glucose uptake. Investigations of the mechanisms have revealed tissue-specific regulation of GLUT4 with decreased gene expression in adipose cells but not in skeletal muscle. This has led to the hypothesis that alterations in the trafficking of the GLUT4 vesicle or in the exposure or activation of the GLUT4 transporter may cause insulin resistance in skeletal muscle in obesity and diabetes. Exercise training increases GLUT4 expression in muscle in association with enhanced glucose tolerance in vivo. Transgenic mice have been created to investigate other approaches to improve insulin action on glucose transport. Overexpression of GLUT4 in adipocytes of transgenic mice increases the proportion of GLUT4 on the plasma membrane and enhances insulin sensitivity in vivo. Altering insulin signaling by overexpressing p21ras in adipocytes of transgenic mice results in increased GLUT4 on the plasma membrane in the absence of insulin and increases insulin sensitivity in vitro and in vivo. Thus, glucose transport is a pivotal step in whole-body insulin action. Strategies to increase the number of GLUT4 transporters that are functionally inserted in the plasma membrane in muscle and adipocytes may lead to new therapies to treat or prevent NIDDM.
Disease-Associated Antibodies in Offspring of Mothers with IDDM
Authors: Martikainen Anu , Saukkonen Tero , Kulmala Petri K , Reijonen Helena , Ilonen Jorma , Teramo Kari , Koskela Pentti , Knip Mikael , Åkerblom Hans K
Published: September 2013

We studied 20 infants of mothers with IDDM participating in a pilot study for a dietary intervention trial, testing the hypothesis that avoidance of cow's milk proteins early in life will reduce the risk of subsequent IDDM. The aim was to evaluate the elimination of IDDM-associated antibodies from the peripheral circulation of the infants, the possible emergence of autoantibodies indicating β-cell destruction, and the influence of the dietary intervention and genetic disease susceptibility on the development of these autoantibodies. Transplacentally transferred islet cell antibodies (ICAs) and antibodies to the 65-kDa isoform of glutamic acid decarboxylase (GAD65As) disappeared from the peripheral circulation of most infants over the first few months of life and in all infants before the age of 9 months. Insulin antibodies were eliminated before the same age in all cases but one. The higher the initial antibody level was, the longer the time required for elimination. Four infants tested positive for insulin autoantibodies (IAAs) on at least one occasion during the first year of life, and 5 out of 16 unaffected subjects (31%) had IAAs at the age of 2 years. One infant became positive for IAA before the age of 6 months, with increasing levels later, seroconverted to positivity for ICAs and GAD65As between 6 and 9 months and presented with clinical IDDM at the age of 14 months. He had the HLA DQB1*0302/x genotype, which predisposes carriers to IDDM, and had been given the casein hydrolysate formula as supplementary milk. There were no significant differences in the levels of various autoantibodies between two groups of subjects defined either on the type of dietary intervention or the degree of genetic susceptibility. The findings indicate that transplacentally transferred antibodies related to IDDM are usually eliminated from the peripheral circulation of infants before 9 months of age and that IDDM-associated autoantibodies may emerge before the age of 6 months. Our results also illustrate that avoidance of cow's milk proteins over the first 9 months of life does not provide total protection against IDDM.
Increased Hexosamine Availability Similarly Impairs the Action of Insulin and IGF-1 on Glucose Disposal
Authors: Hawkins Meredith , Barzilai Nir , Chen Wei , Angelov Ivo , Hu Meizhu , Cohen Pinchas , Rossetti Luciano
Published: September 2013

Prolonged glucosamine (GlcN) infusion increases the skeletal muscle hexosamine concentration and induces peripheral insulin resistance in conscious rats. IGF-1 and insulin share common steps in signal transduction, and the action of IGF-1 on carbohydrate metabolism is preserved in certain insulin-resistant states. In our study, we attempted to delineate whether increased GlcN availability also impairs the effects of IGF-1 on glucose uptake (Rd), glycolysis, and glycogen synthesis. We performed euglycemic IGF-1 (5 and 15 μg · kg−1 · min−1) and insulin (3 and 18 mU mg · kg−1 · min−1) clamp studies at 0–2 h and 5–7 h in conscious rats (n = 44) during saline or GlcN infusions. GlcN infusion raised plasma GlcN levels to ∼ 2.0 mmol/l and skeletal muscle uridinediphospho-n-acetylglucosamine to 80–150 nmol/g (approximately three- to fivefold over basal). During physiological hyperinsulinemia (3 mU · kg−1 · min−1, plasma insulin ≌ 50 μU/ml), GlcN infusion caused comparable decreases in Rd (15.7 ± 1.0 [5–7 h] vs. 21.7 ± 2.3 [0–2 h] mg · kg−1 · min−1; P < 0.01) and glycogen synthesis (5.4 ± 0.5 [5–7 h] vs. 10.4 ± 1.9 [0–2 h] mg · kg−1 · min−1; P < 0.005). Furthermore, GlcN markedly decreased Rd by 7.8 ± 1.2 mg · kg−1 · min−1 (18.7 ± 0.7 [5–7 h] vs. 26.5 ± 1.3 [0–2 h] mg · kg−1 · min−1; P < 0.001 vs. control) during IGF-1 (5 μg · kg−1 · min−1) clamp studies. This decline was associated with a 26% decrease in the steady-state concentration of skeletal muscle Glc-6-P (286 ± 45 vs. 386 ± 36 nmol/g; P < 0.01) and was primarily caused by impaired glycogen synthesis (6.7 ± 0.5 [5–7 h] vs. 13.9 ± 0.9 [0–2 h] mg · kg−1 · min−1; P < 0.005). The effects of GlcN infusion on glucose disposal (percentage decrease in Rd) were correlated (r2 = 0.803; P < 0.01) with the skeletal muscle concentration of UDP-GlcNAc. To investigate whether IGF-1 can overcome GlcN-induced insulin resistance, GlcN and insulin (18 mU · kg−1 · min−1) were infused for 7 h during euglycemic clamps, and IGF-1 (15 μg · kg−1 · min−1) was superimposed during the final 2 h. GlcN infusion induced severe impairment of insulin action on Rd (39.4 ± 3.2 [4–5 h] vs. 49.8 ± 3.6 [1–2 h] mg · kg−1 · min−1; P < 0.05), which the addition of IGF-1 failed to improve (35.9 ± 2.3 [6–7 h] vs. 39.4 ± 3.2 [4–5 h] mg · kg−1 · min−1; P > 0.1). In summary, GlcN induced severe resistance to the actions of both insulin and IGF-1 on glucose uptake and glycogen synthesis, and IGF-1 was unable to overcome GlcN-induced insulin resistance. Thus, it is likely that GlcN causes peripheral insulin resistance acting at a site common to both IGF-1 and insulin signaling pathways.