bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitory Effect of Pregnancy on Counterregulatory Hormone Responses to Hypoglycemia in Awake Rat
Authors: Rossi Gabriele , Lapaczewski Przemyslaw , Diamond Michael P , Jacob Ralph J , Shulman Gerald I , Sherwin Robert S
Published: September 2013

Intensive insulin treatment during diabetic pregnancy is complicated by maternal hypoglycemia. To investigate whether pregnancy may contribute as an independent hypoglycemia risk factor, awake pregnant rats that were near term underwent stepped insulin hypoglycemic (3.4 and 2.3 mM) clamp studies in the fasted and nonfasted states. In the fasted state, the glucagon response to hypoglycemia was completely suppressed in the pregnant rats (P < 0.01). Epinephrine, but not norepinephrine, was also diminished by ∼70–75% at both hypoglycemic steps, and more exogenous glucose was needed to maintain hypoglycemia during pregnancy. To avoid the potential confounding effect of increased ketone levels (β-hydroxybutyrate was ∼ 170% higher in the pregnant rats), experiments were repeated in the nonfasting state when ketosis was eliminated in both groups. The nonfasted pregnant rats continued to show near complete suppression of the glucagon response, even at glucose levels of 2.3 mM. In contrast, a brisk response occurred in nonpregnant controls when glucose fell to 3.4 mM. Although epinephrine levels in the pregnant rats were also markedly suppressed during the milder hypoglycemic stimulus, they approached values seen in nonpregnant controls when glucose was lowered further to 2.3 mM. We concluded that in the rat, pregnancy markedly suppresses glucagon responses to hypoglycemia. The release of epinephrine, but not norepinephrine, is also blunted, especially during mild hypoglycemia. These findings suggest that pregnancy may impair glucose counterregulation by inhibiting glucagon and epinephrine release during hypoglycemia.
GABA Production in Rat Islets of Langerhans
Authors: Michalik Mariusz , Nelson June , Erecińska Maria
Published: September 2013

Homogenates of pancreatic islets catalyzed breakdown of L-glutamate to GABA with a rate of 0.24 ± 0.04 nmol · min−1 · mg−1 protein at 37°C. The formation of GABA was stimulated by addition of pyridoxal phosphate in the range 0.05–1 microM (0.97 ± 0.02 nmol · min−1 · mg protein-1 at a saturating cofactor concentration), which indicates that the process was catalyzed by glutamic acid decarboxylase. The half-maximal effect was obtained with 0.1 microM PLP. Kinetic analyses of the results showed that the Vmax and Km for the reaction were 1.12 nmol · min−1 · mg protein−1 and 0.66 mM, respectively. The pH optimum was 7.0. Subcellular fractionation revealed that 51% of GAD activity was present in the cytosol, 17% in microsomes, 9% in secretory granules, 5% in mitochondria, and 11% in cell debris. Comparison of the kinetic properties of the cytosolic and microsomal forms of the enzyme showed that their Km for glutamate was the same, but that the cytosolic GAD had a lower Km for PLP. GABA synthesis in the nominal absence of PLP was enhanced by malate (twofold increase at 5 mM) and citrate (threefold increase at 5 mM), but was unaffected by ATP and chloride. However, if the islet homogenate was prepared and incubated in the presence of PLP, neither malate nor citrate influenced enzyme activity. Aspartate and AOA were powerful inhibitors of glutamate breakdown. Freshly isolated islets contained ∼4 mM GABA, whereas the concentration was < 0.1 mM in whole pancreas. Perifusion of islets in the absence of amino acids decreased GABA concentration by ∼40% in 60 min; this decline could be prevented by addition of either GABA itself or gabaculine, an inhibitor of GABA-transaminase. Inclusion of 5 mM GABA in the perifusion buffer raised the tissue ATP/A DP beyond the value observed in the absence of any external oxidizable substrate; the effect was eliminated by gabaculine. It is concluded that pancreatic islets contain active GAD, an enzymeresponsible for GABA formation; malate and citrate may act as physiological stimulators of GAD, whereas aspartate may function as its inhibitor; and GABA serves as a source of fuel for generation of intracellular ATP in islets perifused in the absence of glucose.
Availability of Type II Diabetic Families for Detection of Diabetes Susceptibility Genes
Authors: Cook Joanne T E , Page Renée C L , O'Rahilly Steven , Levy Jonathan , Holman Rury , Barrow Beryl , Hattersley Andrew T , Shaw J Andrew G , Wainscoat James S , Turner Robert C
Published: September 2013

Type II diabetes is a familial disorder, as evidenced by the increased prevalence in monozygotic cotwins and first-degree relatives of affected subjects; however, its genetic etiology is largely unknown. Well-characterized pedigrees are an essential resource for the study of susceptibility genes for type II diabetes. This study describes a 5-yr search for type II diabetic families in Oxfordshire, U.K. We interviewed 950 type II diabetic subjects concerning the availability of first-degree relatives; 127 Caucasian families ascertained through a proband with type II diabetes were studied, and 589 first-degree relatives were characterized. Three large pedigrees with maturity-onset diabetes of the young, and 8 multiplex multigenerational type II diabetic pedigrees were identified. We identified 12 sib-pairs in which both siblings had type II diabetes; however, only 7 sib-pairs had both parents alive, and 2 of these had both parents affected. If one also considers one sib having diabetes and one sib having glucose intolerance as being an affected sib-pair, we identified 30 sib-pairs of which 7 had both parents affected and probably had bilineal inheritance. We identified 76 complete nuclear families with both parents and offspring available for study, but only 6 were of optimal structure for linkage analysis. In conclusion, multiplex pedigrees and type II diabetic sib-pairs with living parents are uncommon, and their ascertainment requires a substantial investment of resources. Large-scale collaborative multicenter initiatives would be needed to collect a large resource of family material for the study of susceptibility genes for type II diabetes.
Dietary Carbohydrate Enhances Intestinal Sugar Transport in Diabetic Mice
Authors: Ferraris Ronaldo P , Casirola Donatella M , Vinnakota Ravi R
Published: September 2013

The rate of intestinal absorption of sugars and their site of absorption determine postprandial plasma glucose concentrations. Does chronic consumption of high-carbohydrate, high-fiber, low-fat diets of the type recommended by many diabetes associations induce adaptive changes in transport and metabolism of sugars in the small intestine? Control and STZ-induced diabetic (> 60 days diabetic) mice were fed high-carbohydrate or no-carbohydrate rations for 7 days. Brush-border glucose and fructose uptake per milligram increased 2 times with dietary carbohydrate in both diabetic and control mice; uptake, however, did not differ between diabetic and control mice. Compared with the distal small intestine, glucose uptake per milligram was 2 to 6 times higher in the proximal and middle regions, and enhancement of uptake by diet was limited to these regions. Changes in site density of intestinal glucose transporters as determined by specific phlorizin binding were tightly correlated with changes in brush-border glucose uptake per milligram. There were neither diabetes- nor diet-induced changes in the Kd of specific phlorizin binding, in the amount of glucose absorbed per transporting site, or in passive glucose permeability. Intestinal weights, wt/cm, intestinal length, and mucosal mass increased significantly with diabetes, and sugar transport per centimeter and per small intestine was up to 60% greater in diabetic mice. Dietary carbohydrate stimulated specific sucrase activity in the proximal small intestine of both diabetic and control mice. Chronic diabetes enhances sugar transport by nonspecific increases in intestinal mass. in diabetic mice by specific increases in site density of sugar transporters, resulting in dramatic increases in total sugar absorptive capacity of the diabetic small intestine.
The Role of Glucose-6-Phosphatase in the Action of Insulin on Hepatic Glucose Production in the Rat
Authors: Gardner Lawrence B , Liu Zhenqi , Barrett Eugene J
Published: September 2013

It has been suggested that regulation of glucose-6-phosphatase by insulin plays a role in the suppression of hepatic glucose production during feeding. We used hepatic glucose production (measured with the D-[3-3H]glucose infusion method) as an indicator of substrate flux through glucose-6-phosphatase in vivo. Compared with saline controls, insulin (7 mU · min−1 · kg−1, euglycemic clamp) suppressed hepatic glucose production virtually completely in both fasted (32.4 ± 2.4 vs. –6.1 ± 14 μmol · min−1 · kg−1) and fed (64.6 ± 6.4 vs. 5.5 ± 5.2 μmol · min−1 · kg−1) rats. Whereas hepatic glucose production was totally suppressed, [glucose-6-phosphate] in liver cytosol declined by only 27 and 35% in fasted and fed rats, respectively. Addition of hyperglycemia (10 mM) to the insulin infusion likewise fully suppressed hepatic glucose production (26.9 ± 1.4 vs. −9 ± 10 μmol · min−1 · kg−1 and 80.8 ± 10.1 vs. −3.6 ± 12.6 μmol · min−1 · kg−1 in fasted and fed rats, respectively), but [glucose-6-phosphate] again declined only modestly (21 and 27% in fasted and fed rats, respectively). This disproportionate suppression of hepatic glucose production could not be explained by cooperative substrate effects inasmuch as microsomal glucose-6-phosphatase isolated from saline- and insulin-treated rats followed Michaelis-Menten kinetics (Hill coefficient approximated 1). Acute insulin treatment of fasted rats in vivo did not reproducibly inhibit glucose-6-phosphatase activity assayed subsequently in isolated microsomes incubated in the absence of insulin. However, addition of insulin (0.6–600 nM) to either crude liver homogenates or to isolated, concentrated hepatic membranes (containing both microsomes and plasma membranes) inhibited glucose-6-phosphatase (P < 0.005 for each preparation). These findings indicate that inhibition of glucose-6-phosphatase is involved in the action of insulin to control hepatic glucose production.
Reduced Sampling Protocols in Estimation of Insulin Sensitivity and Glucose Effectiveness Using the Minimal Model in NIDDM
Authors: Coates Philip A , Ollerton Richard L , Luzio Stephen D , Ismail Ikram S , Owens David R
Published: September 2013

Recent work in healthy subjects, the aged, and subjects with gestational diabetes or drug-induced insulin resistance using minimal model analysis of the tolbutamide-modified frequently sampled intravenous glucose tolerance test suggested that a reduced sampling regimen of 12 time points produced unbiased and generally acceptable estimates of insulin sensitivity (SI) and glucose effectiveness (SG) compared with a full sampling schedule of 30 time points. We have used data from 26 insulin-modified frequently sampled intravenous glucose tolerance tests in 21 subjects with NIDDM to derive and compare estimates of SI and SG from the full sampling schedule (SI(30), SG(30)) with those estimated from the suggested 12 time points (SI(12), SG(12)) and those estimated with the addition of a 25-min time point (SI(13), SG(13)). Percentage relative errors were calculated relative to the corresponding 30 time-point values. A statistically significant bias of 15% (97% confidence interval from 7.4 to 25.6%, interquartile range 25%) was introduced by the estimation of SI(12) but not SI(13) (1%, 97% confidence interval from –9.4 to 9.3%, interquartile range 21%). Results for SG(12) (–12%, 97% confidence interval from –46.7 to 1.2%, interquartile range 49%) and SG(13) (–5%, 97% confidence interval from –27.8 to 6.8%, interquartile range 37%) were statistically equivocal. The precision of estimation of SI(12), SG(12), and SG(13) measured by the interquartile range of the percentage relative errors was poor. The precision of determination measured by the median minimal model coefficient of variation was 18, 29, and 27% for SI(30), SI(12), and SI(13) and 9, 11, and 11% for SG(30), SG(12), and SG(13), respectively. Thus, the application of minimal model analysis to the 12 time-point protocol of the insulin-modified IVGTT for the estimation of SI and SG in NIDDM may necessitate an inordinately large number of subjects. Although the 13 time-point protocol may be more acceptable for the assessment of SG in population studies, we recommend retention of the full sampling schedule where feasible.
Sulfonylurea-Binding Sites and ATP-Sensitive K+ Channels in α-TC Glucagonoma and β-TC Insulinoma Cells
Authors: Ronner Peter , Matschinsky Franz M , Le Hang Tu , Epstein Andrew J , Buettger Carol
Published: September 2013

α-Cells secrete glucagon in a fuel-dependent fashion. We tested the hypothesis that α-cells contain sulfonylurea- and ATP-sensitive K+ channels. We studied two clonal lines of α-TC cells (simian virus 40 T-antigen induced glucagonoma cells) and for reference purposes, similarly transformed β-TC insulinoma cells. α-TC cells each contained ∼3000 high-affinity binding sites for the sulfonylurea [3H]glyburide. Whole-cell ATP- and tolbutamide-sensitive K+ currents of α-TC and β-TC cells, relative to cell surface area, were comparable. In cell-attached membrane patches of α-TC cells, two types of K+ channels were observed. They had slope conductances of ∼63 and 33 pS when the electrode contained 151 mM K+. Tolbutamide and diazoxide decreased and enhanced, respectively, the open probability of these channels. The membrane of α-TC cells depolarized periodically. This electrical activity was inhibited by diazoxide. A physiological mixture of amino acids enhanced glucagon release, and high glucose partially inhibited this release. Tolbutamide also enhanced glucagon release, whereas diazoxide inhibited it. Thus, α-TC glucagonoma cells contain ATP-sensitive K+ channels that regulate glucagon release, yet allow inhibition of hormone release by glucose.
Treatment With an Interleukin-1 Receptor Antagonist Protein Prolongs Mouse Islet Allograft Survival
Authors: Sandberg Jan-Olov , Eizirik Decio L , Sandler Stellan , Tracey Daniel E , Andersson Arne
Published: September 2013

An interleukin-1 receptor antagonist protein was evaluated with regard to its efficacy in allogeneic and xenogeneic islet transplantation. Alloxan-induced diabetic C57BL/6 (H-2b) mice were transplanted under the kidney capsule with 500 C57BL/Ks (H-2d) mouse islets. Alzet osmotic pumps, which release their content over an 11- to 13-day period, were implanted subcutaneously for continuous infusion of interleukin-1 receptor antagonist protein (1.0, 5.0, 8.0 mg · kg−1 · day−1) or phosphate-buffered saline. Blood glucose determinations were performed every second or third day; at death, the islet-bearing kidneys were morphologically evaluated. Mice treated initially with the higher interleukin-1 receptor antagonist protein concentrations were followed for an additional period after cessation of the drug release to evaluate whether a transitory interleukin-1 receptor antagonist protein treatment would induce tolerance to the graft. All phosphate-buffered saline–treated mice were hyperglycemic 11 days after islet allotransplantation. Most of their grafts were heavily infiltrated with mononuclear cells. In the various interleukin-1 receptor antagonist protein–treated groups, 60–80% of the mice were normoglycemic after 11 days. Moreover, light microscopic examinations showed that most mice treated with interleukin-1 receptor antagonist protein had normal islet grafts or grafts infiltrated with only a few mononuclear cells. After interruption of interleukin-1 receptor antagonist protein infusion (8.0 mg · kg−1 · day−1), all animals developed hyperglycemia within 2–9 days. In xenogeneic experiments, 500–750 fetal porcine islet-like cell clusters were transplanted under the kidney capsule of normoglycemic C57BL/6 mice. These animals were treated either with interleukin-1 receptor antagonist protein (8.0 mg · kg−1 · day−1) or phosphate-buffered saline. Examination of graft morphology on day 11 showed similarly rejected grafts in both groups. In conclusion, these data suggest that interleukin-1 receptor antagonist protein protects islet allograft, but not xenograft, against acute rejection. Interleukin-1 receptor antagonist protein may become useful as an adjuvant immunosuppressive drug after human islet transplantation.
G-protein Specificity in Signaling Pathways That Mobilize Calcium in Insulin-Secreting β-TC3 Cells
Authors: Baffy György , Yang Lijun , Wolf Bryan A , Williamson John R
Published: September 2013

Fuel- and receptor-induced signal transduction pathways were investigated in β-TC3 cells, an insulin-secreting cell line. An increase of glucose concentration from 0 to 15 mM and stimulation with 0.5 mM carbachol resulted in up to a twofold increase in insulin secretion by β-TC3 cells, and their simultaneous addition increased insulin release eightfold. In single fura 2–loaded cells, a potentiating effect of carbachol was also observed on glucose-induced intracellular Ca2+ mobilization. Immunoblotting with antibodies raised to the COOH-terminal of G-protein α-subunits showed that Gαi, Gαo, and Gαq are present in β-TC3 cells in commensurable quantities. The novel technique of microinjection of anti-Gα antibodies into single β-cell was used to probe the functional role of these G-proteins. Microinjection of anti-Gαi antibodies into β-TC3 cells had no effect on glucose- and carbachol-induced Ca2+ mobilization. However, anti-Gαq completely inhibited the Ca2+-mobilizing effect of carbachol, but not of glucose, within 5 min. Microinjection of anti-Gαo antibodies had no effect on carbachol-induced Ca2+ mobilization. Microinjection of anti-Gαi and anti-Gαq antibodies had a minimal effect on glucose-induced Ca2+ mobilization (<8% of cells nonresponding), but microinjection of anti-Gαo increased the proportion of nonresponding cells to 37%. The results suggest that, in β-TC3 cells, distinct signal transduction pathways with specific G-protein involvement may interact with secretagogue-induced Ca2+ mobilization and, ultimately, with insulin secretion.
Prevention of Diabetic Vascular Dysfunction by Guanidines: Inhibition of Nitric Oxide Synthase Versus Advanced Glycation End-Product Formation
Authors: Tilton Ronald G , Chang Katherine , Hasan Khalid S , Smith Samuel R , Petrash J Mark , Misko Thomas P , Moore William M , Currie Mark G , Corbett John A , McDaniel Michael L , Williamson Joseph R
Published: September 2013

This study was undertaken to compare the ability of two guanidine compounds (aminoguanidine and methylguanidine), with different in vitro effects on NO synthase activity and AGE formation, to inhibit diabetic vascular dysfunction developing early after the onset of diabetes. In rats with STZ-induced diabetes of 5-wk duration, regional vascular [125I]albumin permeation was increased about two- to threefold in ocular tissues, sciatic nerve, and aorta; in general, both guanidine compounds normalized albumin permeation in diabetic rats without affecting it in controls. Methylguanidine was only ∼7% as effective as aminoguanidine as an inhibitor of AGE formation from L-lysine and G6P; both compounds were poor inhibitors of AR. Methylguanidine was ∼1–5% as potent as aminoguanidine and L-NMMA as an inhibitor of the cytokine- and endotoxin-inducible isoform of NO synthase. In contrast, the potency of methylguanidine as an inhibitor of the constitutive isoform of NO synthase was comparable to that of aminoguanidine, and both guanidine compounds were much less effective than L-NMMA. These observations suggest a role for a relative or absolute increase in NO production in the pathogenesis of early diabetic vascular dysfunction and raise the possibility that inhibition of diabetic vascular functional changes by aminoguanidine may reflect inhibition of NO synthase activity rather than, or in addition to, prevention of AGE formation.
Sustained Recovery of Na+-K+-ATPase Activity in Sciatic Nerve of Diabetic Mice by Administration of H7 or Calphostin C, Inhibitors of PKC
Authors: Hermenegildo Carlos , Felipo Vicente , Miñana Maria-Dolores , Romero Francisco J , Grisolía Santiago
Published: September 2013

We have previously shown that intraperitoneal injection of H-7, an inhibitor of PKC, restores completely the activity of Na+-K+-ATPase in sciatic nerve of diabetic mice; however, the effect was transient, with a half-life of ∼1 h under the conditions used. This work assessed whether calphostin C, a new more potent and specific inhibitor of PKC, is also able to restore the activity of Na+-K+-ATPase in sciatic nerve of ALX-induced diabetic mice and also assessed if continuous administration of H-7 or calphostin C can afford sustained recovery of the ATPase. Small amounts of calphostin C (i.e., 2 μg/kg) restore entirely the activity of the enzyme. Larger doses (e.g., 30 μg/kg) can be administered with equal results. The ED50 was ∼0.5 μg/kg. This indicates that calphostin C is ∼20,000 times more potent than H-7 in restoring the ATPase activity in diabetic mice. A single intraperitoneal injection of 1 or 10 μg/kg of calphostin C maintains the enzyme for 4 and 8 h, respectively. Administration of H-7 by continuous delivery from micro-osmotic pumps implanted in the back of the mice maintains the Na+-K+-ATPase for 24 h, although the activity decreases thereafter. This is the result of instability of H-7 in solution. Continuous administration of calphostin C maintains the activity of the ATPase at nearly normal values for at least 2 wk. The results support the hypothesis that, in sciatic nerve tissue of diabetic animals, the activity of PKC is increased, leading to higher phosphorylation of Na+-K+-ATPase, which results in the decreased activity observed. Also, the results show that inhibitors of PKC such as calphostin C can be of therapeutic interest.
No Effect of Naloxone on Ventilatory Response to Progressive Hypercapnia in IDDM Patients
Authors: Wanke Theodor , Abrahamian Heidemarie , Lahrmann Heinz , Formanek Dieter , Merkle Monika , Auinger Martin , Zwick Hartmut , Irsigler Karl
Published: September 2013

The ventilatory response to hyperoxic progressive hypercapnia was examined by comparing 3 test groups: 7 diabetic patients with AN, 8 diabetic patients without AN, and 8 normal control subjects. In each group, a significant linear correlation was found between PaCO2 and VE. The slopes of the regression curves relating PaCO2 to VE were significantly steeper in the healthy control subjects and diabetic patients without AN than in those with AN (P < 0.01). We conclude that the ventilatory response to progressive hypercapnia is reduced in diabetic patients with AN. By analyzing the power spectrum and the amplitude behavior of the diaphragmatic EMG (calculated from the fc and RMS, respectively), we could exclude a disturbance of neural descending pathways and respiratory muscle dysfunction as possible causal mechanisms for the impaired ventilatory response to increasing CO2. By using lung function analysis, causal factors such as alterations in respiratory system mechanics also could be excluded. As diabetes is known to affect the endogenous opioid system, which, in turn, affects the ventilatory response to CO2, naloxone, as a specific opioid antagonist, was administered in all 3 test groups. Naloxone produced a significant increase of ventilatory response to hypercapnia in the healthy control subjects (P < 0.01), but produced no effect in either of the diabetic groups. We conclude that the ventilatory response to hypercapnia is impaired in diabetic patients with AN, that lung function alterations and diaphragmatic muscle dysfunction are not responsible for this impairment, and that endogenous opioids produce an effect on the response to CO2 in healthy subjects, but they have no effect on CO2 response in diabetic patients with or without AN. These results suggest that the central control of respiration is pathologically altered in diabetic patients with AN.
Influence of Islet Amyloid Polypeptide and the 8–37 Fragment of Islet Amyloid Polypeptide on Insulin Release From Perifused Rat Islets
Authors: Wang Zhi-Li , Bennet William M , Ghatei Mohammad A , Byfield Peter G H , Smith David M , Bloom Stephen R
Published: September 2013

IAPP, or amylin, is a 37-amino acid peptide that is co-secreted with insulin from the pancreatic β-cells. We have determined the effects of IAPP and the antagonist 8–37 fragment of IAPP on the secretion of insulin from isolated rat islets studied in a perifusion system. Insulin secretion was stimulated by 8 mM glucose and 0.2 μM carbachol. IAPP at 10−7 M reduced insulin release by 32% from 7.1 (95% Cl 5.8–8.6) to 4.8 (3.0–7.5) fmol · min−1 · islet−1 (P = 0.046, n = 7). IAPP at 1.5 · 10−6 M reduced insulin release by 62% from 6.5 (3.4–12.3) to 2.5 (1.4–4.4) fmol · min−1 · islet−1 (P = 0.001, n = 6). IAPP at 10−5 M decreased insulin release by 70% (P < 0.001, n = 6). When IAPP(8–37) at 10−5 M was added to IAPP at 1.5 · 10−6 M, there was only a 22% reduction of insulin release (P = 0.06, n = 6) compared with control chambers with ho peptide added. This reduction was less (P = 0.002) than observed with IAPP (1.5 · 10−6 M) alone. IAPP(8–37) at 4 · 10−5 M in the absence of exogenously added IAPP increased insulin secretion by 48% (P = 0.01, n = 6), but IAPP(8–37) at 10−5 M did not alter insulin secretion. These findings demonstrate that IAPP decreases insulin secretion from islet β-cells, an effect that can be antagonized by the 8–37 fragment of IAPP. The ability of the 8–37 fragment of IAPP alone to increase insulin secretion suggests the possibility that endogenously released IAPP has a tonic inhibitory effect on insulin secretion under the conditions tested.
Determination of Diabetogenicity Attributable to a Single Amino Acid, Ala776, on the Polyprotein of Encephalomyocarditis Virus
Authors: Bae Yong-Soo , Yoon Ji-Won
Published: September 2013

The best experimental evidence indicating that viruses have an etiological role in the pathogenesis of diabetes comes from studies of mice infected with EMC virus. For this study we generated mutant viruses from stocks of diabetogenic EMC-D and nondiabetogenic EMC-B viruses by serial passages of the viruses in L-cell cultures at high MOI. The genomic sequence information and the biological activities of three different plaque-purified diabetogenic variants of EMC virus (EMC-D, EMC-D1/6A, EMC-D2/4) and six different plaque-purified nondiabetogenic variants (EMC-B, EMC-BS, EMC-B1/G, EMC-DV1, EMC-D4/1B, EMC-D3/1) revealed that only one amino acid, Ala (776th amino acid on the polyprotein), is critical for the diabetogenicity of EMC virus. The G base at the nucleotide position 3155 (Ala[GCC]776 in the polyprotein) is unique to all diabetogenic variants, whereas the A base at the same position (Thr[ACC]776 in the polyprotein) is identical to all nondiabetogenic variants. A single-point mutation (G to A; Ala to Thr) results in the conversion of the diabetogenic variant into a nondiabetogenic variant of EMC virus. On the basis of these observations, we conclude that a single amino acid, Ala776, on the polyprotein of EMC virus appears responsible for the inducement of diabetes in susceptible mice. Conversion of Ala776 into Thr776 on the polyprotein by a point mutation, G to A at the nucleotide position 3155, results in the loss of diabetogenicity.
Nonenzymatic Glycosylation of Laminin and the Laminin Peptide CIKVAVS Inhibits Neurite Outgrowth
Authors: Federoff Howard J , Lawrence Donald , Brownlee Michael
Published: September 2013

Nerve regeneration in diabetic animals is delayed and qualitatively impaired, but the mechanisms responsible for these defects have not been elucidated. The extracellular matrix protein laminin promotes the extension of neuronal processes, and recent studies have localized neurite-promoting activity to a lysine-containing sequence (IKVAV) within the laminin molecule. Because long-lived molecules such as laminin are likely to accumulate excessive amounts of nonenzymatic glycosylation products in diabetic subjects, we have investigated whether such adduct formation on laminin or the IKVAV peptide affects their neurite-promoting properties. These studies used the murine neuroblastoma cell line NB2a, which extends neurites on laminin when differentiated by cAMP. Neurite outgrowth in NB2a cells plated on glycosylated laminin was significantly decreased from that occurring on unmodified laminin. Similarly, neurite outgrowth in NB2a cells plated on glycosylated IKVAV peptide was inhibited compared with that observed on native IKVAV. These data suggest that nonenzymatic glycosylation of a biologically active domain within laminin may contribute to impaired nerve regeneration in diabetes.
Linkage of Chromosomal Markers on 4q With a Putative Gene Determining Maximal Insulin Action in Pima Indians
Authors: Prochazka Michal , Lillioja Stephen , Tait Jonathan F , Knowler William C , Mott David M , Spraul Maximilian , Bennett Peter H , Bogardus Clifton
Published: September 2013

Insulin action in vivo varies widely in nondiabetic Pima Indians. Not all of this variance is attributable to individual differences in obesity, physical fitness, sex, or age, and after correcting for these co-variates, measures of insulin action aggregate in families. Insulin action at maximally stimulating insulin concentrations has a trimodal frequency distribution, particularly among obese individuals. This is consistent with the hypothesis that a codominantly inherited autosomal gene, unrelated to obesity, determines MaxM in the population. Preliminary sib-pair linkage analyses indicated the possibility of linkage between MaxM and the GYPA/B locus (encoding the MNSs red cell surface antigens) on chromosome 4q. To confirm and extend these findings, 10 additional loci on 4q were typed in 123 siblings and many of their parents from 46 nuclear families. The results indicate significant (P < 0.001) linkage of the FABP2 and ANX5 loci on 4q with MaxM, and of FABP2 with fasting insulin concentration. No linkage was found between the 4q markers and obesity. Our findings indicate that a gene on 4q, near the FABP2 and ANX5 loci, contributes to in vivo insulin action in Pima Indians.
In Vivo Glucose Utilization by Individual Tissues in Virgin and Pregnant Offspring of Severely Diabetic Rats
Authors: Holemans Kathleen , Van Bree Rita , Verhaeghe Johan , Aerts Leona , Van Assche F Andre
Published: September 2013

Adult offspring of diabetic rats or SDF rats are characterized by insulin resistance in the liver and extrahepatic tissues; this insulin resistance does not worsen during pregnancy. In this study, we determined the glucose metabolic index in tissues of anesthetized virgin and pregnant control and SDF rats in basal conditions and during a euglycemic hyperinsulinemic clamp. Tissues comprised insulin-sensitive tissues (five skeletal muscles, diaphragm, and periovarian white adipose tissue) and control tissues (duodenum and cerebrum). In addition, this study measured the GMI of placenta and fetuses. In basal conditions, SDF rats showed a slight decrease (9–29%) in the GMI of skeletal muscles compared with control rats; it was not altered by pregnancy in any of the tissues. During physiological hyperinsulinemia, virgin SDF rats exhibited a 25–70% decrease in the GMI of skeletal muscles compared with control rats; this decrease was not observed in diaphragm, or in adipose tissue in which the GMI was found to be increased. During pregnancy, SDF rats did not show an additional drop in the GMI of skeletal muscles, whereas the GMI of both skeletal muscles and adipose tissue was clearly diminished (25–60%) in control rats. The GMI of skeletal muscles was therefore comparable in pregnant control rats and SDF rats. The placental, but not fetal, GMI was increased by 24% during hyperinsulinemia in control rats; the placental and fetal GMIs, in basal and hyperinsulinemic conditions, were similar in control rats and SDF rats. In conclusion, skeletal muscles, but not white adipose tissue, are involved in the peripheral insulin resistance of the SDF rats. However, pregnancy does not induce a further decrease in glucose utilization by skeletal muscles in SDF rats.
High Risk of Progression to NIDDM in South-African Indians with Impaired Glucose Tolerance
Authors: Motala Ayesha A , Omar Mahomed A K , Gouws Eleanor
Published: September 2013

A four-yr prospective study was undertaken to examine the natural history of IGT in 128 South-African Indians classified as such at year 0 of the study, based on WHO criteria. Subjects were reexamined at year 1 and year 4. Of the 113 subjects who completed the study, 50.4% progressed to NIDDM (rate of progression 12.6%/yr), 24.8% persisted with IGT, and 24.8%, reverted to NGT. The majority (72%) who progressed to NIDDM did so in year 1. At year 1, 47 subjects were still classified as IGT; of the 40 subjects completing the study, 16 subjects (40%) progressed to NIDDM, 17 subjects (42.5%) persisted with IGT, and 7 subjects (17.5%) reverted to NGT. Examination of risk factors predictive of subsequent progression to NIDDM was undertaken by analysis of baseline variables in two ways: When year 0 was used as baseline (in 113 IGT0 subjects), significant predictive risk factors were the FPG and 2-h plasma glucose concentrations. All subjects who at year 0 had 2-h plasma glucose ≥ 10.2 and < 11.1 mM or FPG ≥ 7.3 but < 7.8 mM, subsequently progressed to NIDDM. When year 1 was used as baseline (40 IGT1 subjects), 90-min plasma glucose concentration (midtest level) was found to be a significant risk factor for development of NIDDM. In conclusion, this study has demonstrated that in South-African Indians with IGT, the majority (50.4%) progress to NIDDM within 4 yr; significant predictors of subsequent diabetes are the baseline fasting and 2-h plasma glucose concentration. The midtest plasma glucose also may be a useful predictor of clinical outcome. Moreover, the study highlighted the rapid decompensation to NIDDM in the first year and the demonstration of cut-off levels of plasma glucose above which the risk of development of NIDDM is total.
Oxidative Deamination of Methylamine by Semicarbazide-Sensitive Amine Oxidase Leads to Cytotoxic Damage in Endothelial Cells: Possible Consequences for Diabetes
Authors: Yu Peter H , Zuo Dong-Mei
Published: September 2013

Methylamine was observed to be deaminated by several semicarbazide-sensitive amine oxidases, which were prepared from blood and vascular tissues of various species, including humans. Although methylamine itself is relatively nontoxic toward endothelial cells obtained from both human umbilical vein and calf pulmonary artery, it becomes very toxic in the presence of SSAO. SSAO inhibitors (i.e., MDL-72974A) effectively protected the cells from methylamine-SSAO–induced damage. The cytotoxicity seems, therefore, to be a consequence of the deamination of methylamine. Our findings suggest that formaldehyde, the deaminated product of methylamine, may be responsible for these toxic effects. Human serum, which also contains SSAO, was also capable of deaminating methylamine and causing cytotoxicity to cultured endothelial cells. Both methylamine and SSAO circulate in human blood, and their concentrations in the blood of normal healthy subjects are quite close to those required to induce cytotoxicity in tissue-cultured cells. Both SSAO activity and methylamine levels have been reported to be increased in the blood of diabetic individuals. Blood SSAO activity also has been reported to be elevated in the blood of STZ-induced diabetic rats. It is possible, therefore, that an abnormal metabolism of methylamine may be involved in endothelial injury, and that it may subsequently induce atherosclerotic plaque formation and thus be involved in the cardiovascular disorders seen in diabetes.
Transcription Factor jun-B is Target of Autoreactive T-Cells in IDDM
Authors: Honeyman Margo C , Cram David S , Harrison Leonard C
Published: September 2013

Target antigens defined by autoantibodies in IDDM include insulin, a putative glycolipid that reacts with islet cell antibodies, and a 64,000-Mr) protein recently identified as glutamic acid decarboxylase. In addition, some IDDM sera that contain antibodies to glutamic acid decarboxylase also coprecipitate a 38,000–Mr) protein from islets. This study used a high titer anti-38,000-Mr) serum to screen bacteriophage λ cDNA expression libraries and identified human islet and placental clones encoding jun-B, the nuclear transcription protein, of predicted 38,000 Mr). Peripheral blood T-cells exhibited significant proliferation in response to a recombinant fragment of jun-B (amino acids 1–180) in 12 of 17 (71%) recent-onset IDDM subjects, 8 of 16 (50%) ICA-positive first-degree relatives of IDDM subjects who were at risk, 3 of 12 (25%) other autoimmune disease subjects, and 0 of 10 healthy control subjects. Proliferation to tetanus toxoid did not differ significantly between the groups. Responses to jun-B were not related to age, sex, or human leukocyte antigen status. Thus, autoreactive T-cells identify a novel antigen, p38 jun-B, in IDDM and appear to indicate subjects at risk for the development of clinical disease.
Effect of Dehydroepiandrosterone on Neurotransmitter Levels and Appetite Regulation of the Obese Zucker Rat: The Obesity Research Program
Authors: Abadie Jude M , Wright Bruce , Correa Gonzalo , Browne Elizabeth S , Porter Johnny R , Svec Frank
Published: September 2013

The obese Zucker rat is a model of youth-onset obesity associated with hyperphagia. In this study, dehydroepiandrosterone's effect at decreasing food intake and body weight in the obese Zucker rat was investigated. Rats were treated with a dehydroepiandrosterone-supplemented diet (0.0, 0.06, 0.15, 0.3, or 0.6%) for 7 days. The 0.3 and 0.6% treatment groups showed a dramatic decrease in daily food intake, which was evident the 1st day. In addition to the reduction in food intake, body weight changes also were affected significantly in the high-dose treatment groups. The possibility that these dehydroepiandrosterone-induced changes were correlated to perturbations in central neurotransmitter levels associated with appetite control was investigated. The hypothalamus, frontal cortex, striatum, and hippocampus of dehydroepiandrosterone-treated animals were assayed for neurotransmitters known to have inhibitory or stimulatory effects on feeding behavior (serotonin, dopamine, norepinephrine, and epinephrine). Significant differences from steroid-free controls were noted only in the levels of hypothalamic serotonin in animals treated with dehydroepiandrosterone. Serotonin in the hypothalamus has been shown to decrease feeding behavior. The magnitude of dehydroepiandrosterone's effect on hypothalamic serotonin correlated with its effect on feeding behavior. Thus, dehydroepiandrosterone may reduce hyperphagia by altering hypothalamic levels of serotonin.
Predicting Diabetes: Moving Beyond Impaired Glucose Tolerance
Authors: Stern Michael P , Morales Philip A , Valdez Rodolfo A , Monterrosa Ana , Haffner Steven M , Mitchell Braxton D , Hazuda Helen P
Published: September 2013

We developed predictive models for type II diabetes using stepwise multiple logistic regression analyses of a cohort of 844 Mexican Americans and 641 non-Hispanic whites who were nondiabetic at baseline and who were then followed for 8 yr. Models were developed for the overall population and separately for each sex and ethnic group. For optimal models, the multiple logistic regression program selected potential risk factors from a panel of 5 categorical and 14 continuous demographic, anthropometric, metabolic, and hemodynamic variables. For reduced models, the list of candidate variables was restricted to those commonly used in ordinary clinical practice, i.e., skinfolds, and serum insulin and postural glucose load variables were excluded. For all models, the stepwise process selected a mixture of anthropometric, glucose, lipid, and hemodynamic variables. The top 15% of the risk continuum for each model was defined as high risk to compare the performance of the models with the performance of impaired glucose tolerance (15% prevalence) as a predictor of diabetes. The relative risk of being high risk ranged from 12.16 to 35.29, whereas the relative risk of having impaired glucose tolerance ranged from 7.11 to 10.0. The sensitivity of the multiple logistic regression models ranged from 67.7 to 83.3% compared with 56.5 to 62.1% for impaired glucose tolerance. The results indicate that multivariate predictive models perform at least as well, if not better than impaired glucose tolerance in predicting type II diabetes but need not require an oral glucose load. Moreover, the models highlight the complex metabolic and hemodynamic syndrome that precedes diabetes.
Impact of Arterial Blood Pressure and Albuminuria on the Progression of Diabetic Nephropathy in IDDM Patients
Authors: Rossing Peter , Hommel Eva , Smidt Ulla M , Parving Hans-Henrik
Published: September 2013

To evaluate the impact of systemic blood pressure and albuminuria on the progression of diabetic nephropathy, we followed 41 IDDM patients with persistent albuminuria (> 300 mg/24 h) by measuring glomerular filtration rate (51Cr-EDTA technique), blood pressure, and albuminuria. None of the patients were taking drugs other than insulin. Arterial blood pressure, albuminuria, and blood glucose were measured four to eight times/yr, whereas glomerular filtration rate was determined twice yearly. During the median investigation period of 36 (15–66) mo, glomerular filtration rate decreased from 102 ± 23 to 83 ± 27 ml · min−1 · 1.73 m−2 (P < 0.001), albuminuria increased from 633 to 1435 μg/min (P < 0.001), and blood pressure rose from 133/85 ± 10/9 to 149/93 ± 8/11 mmHg (P < 0.001). Univariate analysis revealed a significant correlation between the rates of decline in glomerular filtration rate and diastolic blood pressure (r = 0.52, P < 0.01) and glomerular filtration rate and albuminuria (r = 0.34, P < 0.02). But stepwise multiple linear regression analysis only showed a significant correlation between the rate of decline in glomerular filtration rate and diastolic blood pressure (P < 0.01). In patients with diastolic blood pressure below the mean value of 89 mmHg, stepwise multiple regression analysis showed that albuminuria and not blood pressure was correlated significantly with rate of decline in glomerular filtration rate. Patients were stratified by average value of diastolic blood pressure measured during the investigation period. Patients in the lowest fertile had a rate of decline in glomerular filtration rate of 4.3 ± 4 ml · min−1 · yr−1 compared with the middle and the highest tertiles of 7.7 ± 5 and 10.1 ± 5 ml · min−1 · yr−1, respectively (P < 0.01). The average diastolic blood pressure in the three groups was 81, 89, and 98 mmHg, respectively. This study suggests that systemic blood pressure elevation and albuminuria accelerate the progression of diabetic nephropathy. The latter progression promoter seems only to play a role in patients with normotension (diastolic blood pressure <89 mmHg).
Immunochemical Detection of Advanced Glycation End Products in Renal Cortex From STZ-Induced Diabetic Rat
Authors: Mitsuhashi Tomoko , Nakayama Hidetaka , Itoh Tomohiro , Kuwajima Satoru , Aoki Shin , Atsumi Toshiya , Koike Takao
Published: September 2013

To reassess the accumulation of advanced glycation end products in diabetic renal cortex, we used a newly developed enzyme-linked immunosorbent assay to measure AGEs in renal cortex from STZ-induced diabetic and age-matched control rats. Kidneys and aortas were obtained from rats after 5 and 20 wk of STZ injection. At 5 wk of diabetes, the mean AGE content in collagenase-digested materials of renal cortex was > 16-fold higher in diabetic animals compared with controls (1044.4 ± 151.8 vs. 64.3 ± 5.7 arbitrary units, P < 0.01). At 20 wk of diabetes, it was > 45-fold higher in diabetic compared with control animals (3841.0 ± 1077.3 vs. 83.8 ± 12.8 AUs, P < 0.01). These increases were surprisingly large compared with the < 1.5-fold increase in the fluorescence levels both after 5 and 20 wk of diabetes. In control animals, neither the AGE content nor the fluorescence level increased during this period. Moreover, at 20 wk of diabetes, the AGE content was 39-fold higher in renal cortex compared with aorta. This study provided the first immunochemical evidence that collagenase-digested materials of renal cortex, as well as aorta, contained AGE products and that these products were present in much higher levels in diabetic animals than in control animals. With duration of diabetes, the AGE contents increased significantly both in renal cortex and aorta. The excessive accumulation of AGEs was most apparent in the diabetic kidney. These findings suggest that the actual level of AGEs, in particular, in diabetic renal cortex is much higher than previously anticipated, and a newly developed enzyme-linked immunosorbent assay may be a powerful tool for investigating the role of the advanced Maillard reaction in the development of diabetic nephropathy.
Comparative Effects of Monomethylsuccinate and Glucose on Insulin Secretion from Perifused Rat Islets
Authors: Zawalich Walter S , Zawalich Kathleen C , Cline Gary , Shulman Gerald , Rasmussen Howard
Published: September 2013

In the absence of any other exogenously added fuel, monomethylsuccinate, the methyl ester of succinic acid, at 10–20 mM stimulates insulin release in a biphasic pattern. In quantitative terms, first-phase release evoked by 20 mM MMSucc was comparable to that observed with 20 mM glucose but second-phase release was only 20% of the glucose-induced response. Secretion to both MMSucc and glucose was virtually abolished by the calcium channel antagonist nitrendipine (0.5 μM). In islets that had phosphoinositide pools labeled with [3H]inositol for 2 h, subsequent stimulation with 20 mM MMSucc results in dramatic and sustained increases in [3H]inositol efflux rates. Inositol phosphate levels are also increased. In contrast to secretion, the increase in phosphoinositide hydrolysis caused by MMSucc was largely resistant to nitrendipine, whereas significant reductions in glucose-induced phosphoinositide hydrolysis were observed in the presence of the calcium channel antagonist. MMSucc (2.75–10 mM) substitutes for glucose in that MMSucc supported the insulinotropic effects of the sulfonylurea tolbutamide (200 μM) and the gut hormone cholecystokinin (200 nM). A prior 15-min exposure to 20 mM MMSucc also sensitized islets to the stimulatory effects of 7.5 mM glucose. Finally, a 2-h exposure to 20 mM MMSucc desensitized the islet, in terms of both phosphoinositide hydrolysis and insulin secretion, to a subsequent exposure to 10 mM glucose. Thus, appropriate concentrations of MMSucc can cause qualitatively many of the effects induced by glucose. However, striking differences exist in the quantitative effects that are most simply explained by assuming that MMSucc is able to stimulate phosphoinositide hydrolysis to the same extent as glucose but is less able to enhance Ca2+ influx than glucose. If this view is correct, it leads to the conclusion that an intracellular calcium-independent signal derived from the metabolism of MMSucc is able to activate phosphoinositide hydrolysis in islets.