bluuebunny/crossref_metadata_2025 huggingface dataset:
Proprotein-Processing Endoprotease Furin Controls Growth of Pancreatic β-Cells
Authors: Kayo Tsuyoshi , Sawada Yoshie , Suda Masayuki , Konda Yoshitaka , Izumi Tetsuro , Tanaka Shigeyasu , Shibata Hiroshi , Takeuchi Toshiyuki
Published: September 2013

We have previously reported that in the well-differentiated β-cell line MIN6 cells, theβ-cell–specific differentiated characteristics, such as insulin content, expression of prohormone convertases PC2 and PC3, and glucose-regulated insulin secretion, diminished when the proprotein-processing endoprotease furin was highly expressed. Since furin converts many growth-related protein precursors to their bioactive forms, we compared the four pancreatic islet cell lines RINm5F, βTC3, βHC9, and MIN6 with respect to cell growth rate, furin expression, endoprotease activity, and insulin content. RINm5F cells exhibited the strongest furin expression, higher furin-type endoprotease activity, and the fastest cell growth, but had the least insulin content. In contrast, MIN6 cells exhibited only a weak furin expression, little furin-type endoprotease activity, and the slowest cell growth, but had the highest insulin content. To test whether furin-expressing cells secrete growth-promoting factors cleaved by furin, we prepared conditioned media from RINmSF and furin cDNA–introduced MIN6 (MIN6-F) cells. The conditioned media from RINm5F and MIN6-F induced increased DNA synthesis and promoted the growth of normal MIN6 cells, compared with the medium from the empty vector-introduced MIN6-0 cells. We then examined the effect of the protease inhibitors α1-antitrypsin and its variants by infecting their vaccinia recombinants to the four cell lines. All conditioned media from each cell line expressing the furin-specific α1-antitrypsin variant exhibited the least DNA synthetic capacity on normal MIN6 cells. Furthermore, all three sublines of MIN6-F grew faster than MIN6-0 and MIN6. Thus, we suggest that the islet cells with higher furin expression may induce increased production of growth factors, which result in an increase in cell growth, through an autocrine/paracrine mechanism.
Daily Energy Expenditure Is Related to Plasma Leptin Concentrations in Older African-American Women but Not Men
Authors: Nicklas Barbara J , Toth Michael J , Poehlman Eric T
Published: September 2013

Recent research suggests that leptin may control body weight by regulating energy expenditure and energy intake in mice. To explore the possible role of leptin in the regulation of energy expenditure in humans, we used doubly-labeled water methodology to determine whether fasting plasma leptin concentrations were related to total daily energy expenditure (TEE) and its components, resting energy expenditure (REE) and physical activity energy expenditure (PAEE), in free-living older African-American men (n = 21) and women (n = 25). Plasma leptin concentrations were higher in women than men, even after the adjustment for differences in fat mass (28 ± 3 ng/ml for women vs. 17 ± 3 ng/ml for men; P < 0.01). The logarithm of plasma leptin concentrations correlated with fat mass in both women (r = 0.80) and men (r = 0.78) (P < 0.0001). After statistical adjustment for sex differences in fat-free mass and fat mass, women had lower TEE (22%) and REE (15%) (P < 0.01) and a trend (P = 0.08) toward lower PAEE, compared with men. After controlling for the effects of fat-free mass on energy expenditure, plasma leptin concentrations were related to REE (r = 0.68, P < 0.001) and tended to be related to TEE (r = 0.37, P = 0.07) in African-American women but not men (r = 0.18 and −0.03, respectively). Plasma leptin concentrations were not related to PAEE in either men or women. These results suggest that leptin may contribute to the regulation of TEE in older African-American women through its effects on resting energy metabolism, but the role of leptin in the regulation of energy expenditure is less apparent in older African-American men.
Glucose-Induced Insulin Secretion in INS-1 Cells Depends on Factors Present in Fetal Calf Serum and Rat Islet–Conditioned Medium
Authors: Sekine Nobuo , Fasolato Cristina , Pralong William F , Theler Jean-Marc , Wollheim Claes B
Published: September 2013

To study the regulation of growth and differentiated function of insulin-secreting cells, the rat insulinoma cell line INS-1 was cultured in a defined serum-free medium containing prolactin, IGF-I, and triiodothyronine, which was originally reported to maintain insulin secretion of islet cells. Growth and viability, as well as cellular insulin content of INS-1 cells in the defined medium, were comparable to the control cells cultured in the complete medium containing 10% fetal calf serum. However, after a 3-day culture in this medium, insulin secretion in response to glucose, pyruvate, and leucine was markedly blunted compared with the control cells (−78, −68, and −56%, respectively), whereas the response to 30 mmol/1 K+ was only slightly decreased. In these cells: 1) nutrient metabolism assessed by tetrazolium salt reduction was reduced in response to pyruvate and leucine, which are mainly metabolized in the mitochondria; 2) oxidation of both [3,4-14C]glucose and [1-14C]pyruvate was decreased (−22 and −32%, respectively); 3) glucose failed to depolarize the membrane potential, whereas tolbutamide was fully active; 4) video imaging analysis of cytosolic Ca2+ showed a decrease in the population of glucose-responsive cells, while the response to 30 mmol/1 K+ was preserved; 5) serum replenishment for 3 days restored glucose-induced insulin secretion. Interestingly, conditioned serum-free medium from rat islets maintained the insulin secretory function of INS-1 cells, although glucagon, somatostatin, and some other factors failed to restore the function. In contrast, conditioned media from HepG2, PC12, and human umbilical vein endothelial cells did not substitute for serum. Thus, the impaired insulin secretion of the cells cultured in the defined medium is best explained by defective mito-chondrial metabolism. Islet cells, but not INS-1 cells, produce factors required for normal signal generation by nutrient secretagogues.
Culture of Adult Human Islet Preparations With Hepatocyte Growth Factor and 804G Matrix Is Mitogenic for Duct Cells but Not for β-Cells
Authors: Lefebvre Veronique H , Otonkoski Timo , Ustinov Jarkko , Huotari Mari-Anne , Pipeleers Daniel G , Bouwens Luc
Published: September 2013

It has recently been reported that human adult β-cells proliferate during culture on an extracellular matrix prepared from rat 804G cells and in the presence of hepatocyte growth factor (HGF) (6). The present study compares the mitogenic effect of this condition on human β-cells and on neighboring non-endocrine duct cells. Islet cell-enriched fractions were prepared from adult human organ donors and cultured in suspension or on 804G matrix, with or without HGF. The combination of 804G matrix and HGF increased the number of 5-bromo-2'-deoxyuridine-positive (BrdU+) cells within 48 h reaching a maximum after 4 days. In sections, virtually all BrdU+ cells were negative for insulin or glucagon and for preproinsulin mRNA but expressed the ductal cell markers cytokeratin 19 and 7, carbonic anhydrase-II, and carbohydrate antigen 19-9. After 4 days of culture, the cytokeratin 19+ ductal cells exhibited a BrdU-labeling index of 30% (P < 0.01 vs. 2% without HGF and matrix), whereas <0.1% of insulin-positive and <1% of glucagonpositive cells were labeled. Formation of bilayers with ductal cells covering the endocrine cells may cause erroneous interpretation on double positivity in unsectioned tissue. It is concluded that culture of human islet cell preparations with HGF and 804G matrix stimulates the proliferation of the duct cells but not of the underlying β-cells.
Glucagon-Like Peptide 1 Increases Mass But Not Frequency or Orderliness of Pulsatile Insulin Secretion
Authors: Pørksen Niels , Grøfte Thorbjørn , Nyholm Birgit , Holst Jens Juul , Pincus Steven M , Veldhuis Johannes D , Schmitz Ole , Butler Peter C
Published: September 2013

Glucagon-like peptide 1 (GLP-1) is a peptide hormone that is released from the gut after luminal stimulation. The hormone is a potent insulin secretagogue and is a potential novel pharmaceutical adjuvant in the treatment of NIDDM. Insulin is secreted as a series of punctuated secretory bursts superimposed on a basal insulin release. Recently, the contribution of these secretory bursts to overall insulin secretion has been evaluated, and studies using catheterization across the pancreas in a canine model and studies using deconvolution in humans have revealed that the majority of insulin is released during these secretory bursts. Moreover, the main regulation of insulin secretion is through perturbation of mass and frequency of these secretory bursts. The mode of delivery of insulin into the circulation seems important for insulin action, and it is therefore important to know the impact of a potential therapeutic insulin secretagogue on the mode of insulin secretion. To assess the effects of GLP-1 on the mass, frequency, amplitude, and overall contribution of pulsatile insulin secretion, we used a recently validated deconvolution model to examine these variables before and during infusion of GLP-1 in eight healthy men (age 28 ± 2 years; BMI 24 ± 2 kg/m2). At a constant glucose infusion (2.5 mg · kg−1 · min−1), near-steady state was reached at 75 min, and sampling was performed every minute at t = 75–115 and 145–185 min. At t = 115 min, an infusion of saline or GLP-1 (50 pmol · kg−1 · min−1) was given. The regularity of insulin secretion was measured by approximate entropy, a recently developed mathematical statistic, applied herein to assess the regularity in a hormone concentration time series. After GLP-1 infusion, there was an abrupt increase in the peripheral concentrations of serum Cpeptide (696 ± 65 vs. 1,538 ± 165 pmol/1) and insulin (49 ± 8 vs. 138 ±21 pmolA) concentrations. This increase was mainly due to an increase in the pulsatile component of insulin secretion that was achieved by a fourfold increase in secretory burst mass (28.2 ± 4.4 vs. 100.1 ± 15.8 pmol · .l−1· pulse−1; P < 0.001), and amplitude (12.7 ± 2.2 vs. 4.3 ± 7.7 pmol · l−1 · min−1; P < 0.002), whereas the secretory burst frequency was not affected by GLP-1 (11.5 ± 0.7 vs. 12.6 ± 0.6 pulses/h; P = 0.4). As a consequence, the detected contribution of pulsatile to overall insulin secretion was increased from 56 ± 4 to 77 ± 4% (P < 0.005). The orderliness of the insulin release process was not deteriorated by short-term GLP-1 infusion as assessed by approximate entropy (1.19 ± 0.04 vs. 1.18 ± 0.04; P = 0.7).
Depot-Specific Differences in Adipose Tissue Gene Expression in Lean and Obese Subjects
Authors: Lefebvre Anne-Marie , Laville Martine , Vega Nathalie , Riou Jean Paul , Gaal Luc van , Auwerx Johan , Vidal Hubert
Published: September 2013

Intra-abdominal and subcutaneous adipose tissue display important metabolic differences that underlie the association of visceral, but not subcutaneous, fat with obesity-related cardiovascular and metabolic problems. Because the molecular mechanisms contributing to these differences are not yet defined, we compared by reverse transcription-polymerase chain reaction the expression of 15 mRNAs that encode proteins of known importance in adipocyte function in paired omental and subcutaneous abdominal biopsies. No difference in mRNA expression between omental and subcutaneous adipose tissue was observed for hormone sensitive lipase, lipoprotein lipase, 6-phosphofructo-1-kinase, insulin receptor substrate 1, p85α regulatory subunit of phosphatidylinositol-3-kinase, and Rad. Total amount of insulin receptor expression was significantly higher in omental adipose tissue. Most of this increase was accounted for by expression of the differentially spliced insulin receptor lacking exon 11, which is considered to transmit the insulin signal less efficiently than the insulin receptor with exon 11. Perhaps consistent with a less efficient insulin signaling, a twofold reduction in GLUT4, glycogen synthase, and leptin mRNA expression was observed in omental adipose tissue. Finally peroxisome proliferator activated receptor-γ (PPAR-γ) mRNA levels were significantly lower in visceral adipose tissue in subjects with a BMI <30 kg/m2, but not in obese subjects, indicating that relative PPAR-γ expression is increased in omental fat in obesity. This suggests that altered expression of PPAR-γ might play a role in adipose tissue distribution and expansion.
The B-Subunit of Cholera Toxin Induces Immunoregulatory Cells and Prevents Diabetes in the NOD Mouse
Authors: Sobel Douglas O , Yankelevich Boris , Goyal Deepshikha , Nelson David , Mazumder Amitabha
Published: September 2013

The B-subunit of the cholera toxin molecule (CT-B) has T-cell immunomodulatory properties. Because the pathogenesis of diabetes in the nonobese diabetic (NOD) mouse model of IDDM is thought to be a Tcell-mediated process due to an imbalance of immunoregulatory and anti-islet effector cells, we examined the effect of CT-B administration on the development of diabetes in the NOD mouse and assessed whether this potential diabetes-sparing effect of CT-B is mediated by changes in immunoregulatory and/or anti-islet cytotoxic effector cell activity. The administration of either intravenous or intraperitoneal CT-B decreased the development of diabetes with no apparent drug toxicity. At 6 months of age, only 18% of CT-B vs. 75% of saline-treated animals had diabetes. Histopathological examination revealed less islet atrophy in CT-B-treated animals. The in vitro proliferative responses of mononuclear splenocytes and thymocytes to concanavalin A and lipolysaccharide and the proportion of B-cells and T-cell subsets were not altered by CT-B treatment. CT-B administration did not inhibit the primary immunization of mice to tetanus toxoid. The development of diabetes in irradiated NOD mice was slower in the animals injected with spleen cells (SC) from CT-B–treated than from saline-treated NOD mice, suggesting that CT-B decreases anti-islet effector cell activity. The injection of SC from CT-B–treated mice inhibited the adoptive transfer of diabetes by SC from diabetic mice into irradiated NOD mice, documenting that CT-B administration induces regulatory cell activity. In conclusion, CT-B administration prevents the development of diabetes in NOD mice by inhibiting the immune destruction of islets. This islet-sparing activity appears mediated, at least in part, by the induction of regulatory cells and, in turn, suppression of anti-islet effector cells, which is not associated with generalized immunosuppression or T- or B-cell depletion.
Normoglycemia Restores β-Cell Replicative Response to Glucose in Transplanted Islets Exposed to Chronic Hyperglycemia
Authors: Nacher Victor , Merino Juan F , Raurell Mercè , Soler Joan , Montanya Eduard
Published: September 2013

We studied the effects of chronic hyperglycemia on β-cell replication and mass in transplanted (Tx) islets. Five groups of streptozocin-induced diabetic C57B1/6 mice were transplanted with 100 (Tx-100) syngeneic islets, an insufficient β-cell mass to restore normoglycemia. Groups 1 and 2 remained hyperglycemic throughout the study; after 30 days of hyperglycemia, a second transplantation of 250 islets (Tx-250) restored normoglycemia in groups 3, 4, and 5. Tx-250 was harvested on day 60 in all three groups, and transient mild hyperglycemia developed (10–12 days); thereafter, Tx-100 maintained blood glucose values in the normal range. Tx-100 was harvested 14 (group 1), 60 (groups 2 and 3), 74 (group 4), and 90 (group 5) days after transplantation. Hyperglycemia increased β-cell replication after 14 days (group 1:1.26 ± 0.18%, P < 0.05) but not after 60 days (group 2: 0.59 ± 0.13%) compared with islets exposed to normoglycemia (group 3: 0.51 ± 0.07%) (analysis of variance [ANOVA], P < 0.0002). β-Cell replication in group 4 increased after Tx-250 harvesting (0.94 ± 0.16%, P < 0.05). The initially Tx β-cell mass (0.21 ± 0.014 mg) was progressively reduced in hyperglycemic groups (group 1: 0.13 ± 0.020 mg; group 2: 0.048 ± 0.012 mg; P < 0.05) (ANOVA, P = 0.0001). Restoration of normoglycemia after Tx-250 did not modify β-cell mass in Tx-100 grafts (group 3: 0.076 ± 0.008 mg). However, after Tx-250 harvesting, β-cell mass increased progressively (group 4: 0.11 ± 0.018 mg; group 5: 0.14 ± 0.026 mg, P < 0.05), although it was still reduced compared with the initially Tx β-cell mass (P < 0.05). In summary, Tx islets exposed to severe chronic hyperglycemia showed a limited β-cell replication and a progressive reduction in β-cell mass. With normoglycemia, the Tx β-cells recovered the replicative response to glucose and partially restored the initially Tx β-cell mass, indicating that normoglycemia, even after long-term hyperglycemia, has a beneficial effect in islet transplantation.
Increased Plasma Leptin Levels Are Associated With Fat Accumulation in Japanese Americans
Authors: Chessler Steven D , Fujimoto Wilfred Y , Shofer Jane B , Boyko Edward J , Weigle David S
Published: September 2013

Although the hormone leptin seems to play a role in ensuring the maintenance of adequate energy stores and thereby protects against starvation, its role in the regulation of body weight and adiposity under normal circumstances is unclear. Overweight individuals have markedly elevated circulating leptin levels, suggesting that leptin's effect on food intake and thermogenesis is diminished or absent in obesity. Recent evidence, though, indicates that weight gain in Pima Indians is associated with relatively decreased levels of the hormone. Because it is important to understand whether a deficiency in circulating leptin contributes to the development of obesity, we sought to determine whether there is a relationship between leptin levels and subsequent changes in adiposity in a more typical population. We compared baseline plasma leptin concentrations to changes over 5 years in body weight, BMI, and computed tomography-determined total fat in 492 second- and third-generation Japanese Americans. Subjects were of 100% Japanese ancestry; male subjects had a mean BMI at baseline of 25.4 kg/m2 and a mean age of 54 years; female subjects had a mean BMI of 23.1 kg/m2 and a mean age of 53 years. Changes in weight (men: r = 0.17, P < 0.05; women: r = 0.20, P < 0.05), BMI (men: r = 0.17, P < 0.05; women: r = 0.18, P < 0.05), and total fat (men: r = 0.19, P < 0.05; women: r = 0.20, P < 0.01) were positively correlated with baseline leptin levels adjusted for baseline adiposity, fasting insulin, and age. In Japanese Americans, then, relatively increased leptin levels are associated with greater subsequent gains in weight and adiposity. We concluded that in this population, fat accumulation is associated not with leptin deficiency but possibly with leptin resistance and is preceded by increased leptin levels.
β-Hydroxybutyrate Increases Reactive Oxygen Species in Late but Not in Early Postimplantation Embryonic Cells In Vitro
Authors: Forsberg Henrik , Eriksson Ulf J , Melefors Öjar , Welsh Nils
Published: September 2013

Embryonic dysmorphogenesis has been blocked by antioxidant treatment in vivo and in vitro, suggesting that embryonic excess of reactive oxygen species (ROS) has a role in the teratogenic process of diabetic pregnancy. We report that the basal levels of ROS in dispersed rat embryonic cells in vitro, as determined by fluorescence of dichlorofluorescein (DCF), were not different in cells from control and diabetic pregnancy at day 10 or 12. β-Hydroxybutyrate (β-HB) and succinic acid monomethyl ester both augmented DCF fluorescence in cells from day 12 embryos of normal and diabetic rats but not from day 10 embryos. Cells of day 10 and day 12 embryos from normal and diabetic rats responded to increasing glucose concentrations with a dosage-dependent alleviation of DCF fluorescence. Day 10 embryonic cells exhibited high glucose utilization rates and high pentose phosphate shunt rates, but low mitochondrial oxidation rates. Moreover, in vitro culture of embryos between gestational days 9 and 10 in the presence of 20% oxygen induced an increased and glucose-sensitive oxidation of glucose compared with embryos not cultured in vitro. At gestation day 12, however, pentose phosphate shunt rates showed a decrease, whereas the mitochondrial P-HB oxidation rates were increased compared with those at gestation day 10. This was paralleled by a lower expression of glucose 6-phosphate dehydrogenase- and phosphofructokinase-mRNA levels at day 12 than at day 10. On the other hand, H-ferritin mRNA expression at day 12 was high compared with day 10. None of the mRNA species investigated were affected by the diabetic state of the mother. It was concluded that β-HB-induced stimulation of mitochondrial oxidative events may lead to the generation of ROS at gestational day 12, but probably not at day 10, when only a minute amount of mitochondrial activity occurs. Thus our results do not support the notion of diabetes-induced mitochondrial oxidative stress before the development of a placental supply of oxygen.
Genetic Structure of IDDM1: Two Separate Regions in the Major Histocompatibility Complex Contribute to Susceptibility or Protection
Authors: Moghaddam P Hanifi , de Knijf P , Roep B O , Van der Auwera B , Naipal A , Gorus F , Schuit F , Giphart M J ,   Belgian Diabetes Registry
Published: September 2013

We analyzed 11 markers in the IDDM1 region in 120 IDDM patients and 83 healthy control subjects who were fully matched for the highest risk HLADQA1*O3O1-DQB1*O3O2/DQA1*O5O1-DQB1*O2O1 genotype. Our study provides strong evidence that two regions in the major histocompatibility complex contribute to IDDM susceptibility or protection. First, despite selection for highest IDDM-associated risk DQ genotypes, this region displays extensive linkage disequilibrium (LD) differences between IDDM patients and control subjects. A second critical region was mapped around the microsatellite locus D6S273 centromeric of TNF, and it is ∼200 kb in size. LD analysis shows that “diabetogenic haplotypes” may have resulted from a recombination telomeric of D6S1014 in the region of D6S273 and TNFa. Haplotype analysis using HLA and microsatellite loci refines IDDM risk assessment in carriers of the HLA-DQ highest risk genotype.
Overexpression of Glycogen Phosphorylase Increases GLUT4 Expression and Glucose Transport in Cultured Skeletal Human Muscle
Authors: Baqué Susanna , Montell Eulàlia , Camps Marta , Guinovart Joan J , Zorzano Antonio , Gòmez-Foix Anna M
Published: September 2013

Skeletal muscle glucose utilization, a major factor in the control of whole-body glucose tolerance, is modulated in accordance with the muscle metabolic demand. For instance, it is increased in chronic contraction or exercise training in association with elevated expression of GLUT4 and hexokinase II (HK-II). In this work, the contribution of increased metabolic flux to the regulation of the glucose transport capacity was analyzed in cultured human skeletal muscle engineered to overexpress glycogen phosphorylase (GP). Myocytes treated with an adenovirus-bearing muscle GP cDNA (AdCMVMGP) expressed 10 times higher GP activity and exhibited a twofold increase in the Vmax for 2-deoxy-D-[3H]glucose (2-DG) uptake, with no effect on the apparent Km. The stimulatory effect of insulin on 2-DG uptake was also markedly enhanced in AdCMVMGP- treated cells, which showed maximal insulin stimulation 2.8 times higher than control cells. No changes in HKII total activity or the intracellular compartmentalization were found. GLUT4, protein, and mRNA were raised in AdCMV-MGP-treated cells, suggesting pretranslational activation. GLUT4 was immunodetected intracellularly with a perinuclear predominance. Culture in glucose-free or high-glucose medium did not alter GLUT4 protein content in either control cells or AdCMV-MGP-treated cells. Control and GP-overexpressing cells showed similar autoinhibition of glucose transport, although they appeared to differ in the mechanism(s) involved in this effect. Whereas GLUT1 protein increased in control cells when they were switched from a high-glucose to a glucose-free medium, GLUT1 remained unaltered in GP-expressing cells upon glucose deprivation. Therefore, the increased intracellular metabolic (glycogenolytic-glycolytic) flux that occurs in muscle cells overexpressing GP causes an increase in GLUT4 expression and enhances basal and insulin-stimulated glucose transport, without significant changes in the autoinhibition of glucose transport. This mechanism of regulation may be operative in the postexercise situation in which GLUT4 expression is upregulated in coordination with increased glycolytic flux and energy demand.
Interleukin-4 Deficiency Does Not Exacerbate Disease in NOD Mice
Authors: Wang Bo , Gonzalez Antonio , Höglund Petter , Katz Jonathan D , Benoist Christophe , Mathis Diane
Published: September 2013

To investigate the role of interleukin (IL)-4 in the regulation of autoimmune diabetes, we crossed the IL-4 knock-out mutation onto the NOD genetic background. This experiment was accelerated by typing for microsatellites linked to known diabetes susceptibility (Idd) loci, and included a control backcross of the wildtype 129/SvJ-derived IL-4 gene, the original target locus. We also crossed the mutation into the BDC2.5 transgenic line, a diabetes model that carries the rearranged T-cell receptor genes from a diabetogenic Tcell clone. The IL-4-null mutation did not accelerate or intensify insulitis in regular NOD mice or in the BDC2.5 transgenic model; it also had no effect on the timing or frequency of the transition to overt diabetes. These data indicate that IL-4 plays no required role in controlling the aggressiveness of murine diabetes.
In Situ Characterization of Nonmitochondrial Ca2+ Stores in Individual Pancreatic β-Cells
Authors: Tengholm Anders , Hagman Charlotte , Gylfe Erik , Hellman Bo
Published: September 2013

Free Ca2+ was measured in intracellular stores of individual mouse pancreatic β-cells using dual-wavelength microfluorometry and the low-affinity Ca2+ indicator furaptra. Controlled permeabilization of the plasma membrane with 4 umol/1 digitonin revealed that 22% of the furaptra was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/1 Ca2+ were simultaneously present, this cation rapidly accumulated in the organelle pool, reaching an average concentration of 200–500 umol/1. Whereas agents affecting the mitochondrial function (5 mmol/l succinate, 2 umol/1 ruthenium red, or 10 umol/1 antimycin A + 2 ug/ml oligomycin) had little effects, the Ca2+-ATPase inhibitor thapsigargin released 92% of the Ca2+ mobilizable with the ionophore Br-A23187. Digital imaging revealed regional differences in the organelle Ca2+. The regions with the highest Ca2+ concentration were particularly responsive to inositol 1,4,5-trisphosphate (IP3). IP3 mobilized Ca2+ in a dose-dependent way with half-maximal and maximal effects at about 1 and 5 umol/1, respectively. High concentrations of IP3 released about half of the thapsigargin-sensitive Ca2+, but there were no responses to agents known to activate ryanodine receptors, such as 10 mmol/l caffeine, 0.1–1 umol/1 ryanodine, or 1–5 umol/1 cyclic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca2+ in the pancreatic β-cell is mediated by IP3 receptors rather than ryanodine receptors.
Serum Lipoproteins of Diabetic Rats Fed a High Cholesterol Diet
Authors: Arbeeny Cynthia M , Edelstein Diane , Freedman Sharon R , Eder Howard A
Published: September 2013

Feeding a diet containing 2% cholesterol and 1% cholic acid (wt/wt) to rats made diabetic by administration of streptozotocin (45 mg/kg) produced marked hypercholesterolemia characterized by high concentrations of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) and a reduction in concentration of high density lipoproteins (HDL). The VLDL was unique in that it contained apo A-I and apo A-IV in addition to its usual complement of apoproteins: apo B, apo E, and the C apoproteins. IDL had a similar apoprotein composition. The HDL from these rats was deficient in apo E. Nondiabetic rats fed the same diet exhibited similar qualitative changes in lipoprotein concentration and composition but with lesser increases in VLDL and IDL concentrations. The altered apoprotein composition suggested that the hyperlipoproteinemia associated with cholesterol feeding in the rat is due to an inadequate rate of removal of lipoproteins of intestinal origin, and that this is greatly exacerbated by diabetes.
The Minimal Amino Acid Sequence of the Insulin-potentiating Fragments of Human Growth Hormone: Its Mechanism of Action
Authors: Ng Frank M , Bornstein Joseph , Pullin Claire E , Bromley Janet O , Lance Macaulay S
Published: September 2013

A series of synthetic peptides corresponding to the amino-terminal sequence of human growth hormone (hGH) has been studied for insulin-potentiating effects using three different bioassay systems: (1) intravenous insulin tolerance tests, (2) insulin binding to specific receptors of hepatic plasma membranes and isolated hepatocytes, and (3) modulation of insulindependent glycogen synthase and glycogen phosphorylase in muscle and adipose tissue. The results establish that the minimum active sequence is the hexapeptide (hGH 8-13) containing H2N-Arg-Leu-Phe-Asp-Asn-Ala-COOH and strongly indicate that the insulin- potentiating action of the active peptides is to increase the binding of insulin tospecific receptors and thus modulate the action of glycogen synthase and phosphorylase, producing hypoglycemia as the result of increased glycogen storage in liver, muscle, and adipose tissue.
The Evaluation of the Drug-metabolizing Capacity in Patients with Diabetes Mellitus
Authors: Salmela Pasi I , Sotaniemi Eero A , Olavi Pelkonen R
Published: September 2013

Hepatic drug-metabolizing capacity was investigated in 56 diabetics. The antipyrine test was selected as an in vivo index, since its kinetics indirectly reflect the metabolically active liver mass. Hepatic cytochrome P-450 (P-450), determined from the biopsy samples, was used as an in vitro parameter, since it is a direct measure of microsomal drug-metabolizing enzyme activity. There was a wide interindividual variation in the indexes of drug metabolism in the diabetics: 40 fold in P-450 content and eightfold in antipyrine metabolism. P-450 levels were higher and antipyrine metabolism faster in the subjects with normal liver than in those with fatty liver, parenchymal inflammatory changes, or cirrhosis. Thus the in vivo and in vitro parameters of drug metabolism were related to the alterations in liver histology. On the other hand, the diabetes per se did not seem to alter the drug-metabolizing capacity of the liver. Also, drug metabolism in diabetics classified by treatment regimen did not differ significantly.
In Vivo Incorporation of [3H] Leucine and [3H] Tryptophan into Proinsulin-insulin and Other Islet Cell Proteins in Normoglycemic, Hyperglycemic, and Hypoglycemic Rats
Authors: Logothetopoulos John , Jain Kanti
Published: September 2013

Rats were infused for brief periods with buffer, glucose, or insulin. L-[4,5-3H] leucine (2.5 mCl) or L-[2,3-3H]-tryptophan (0.5 mCl) was quickly injected intravenously 30 min after the onset of the infusion, when marked hyperglycemia or hypoglycemia had been established. Rats remained connected to the infusion system and were killed 30 min after the injection of the labeled amino acid. Pancreatic islets were isolated by enzymatic digestion of the pancreas. They were processed for radioautography or for the measurement of [3H] proinsulin and [3H] insulin by immunoprecipitation and of other islet [3H]proteins by TCA precipitation. Various tissues of the rats were also removed to measure TCA-precipitable-labeled proteins. Incorporation of [3H]-leucine into proinsulin and insulin was 9 to 10 times greater in the hyperglycemic than in the hypoglycemic rats. Incorporation of [3H]-tryptophan into sedentary beta-cell proteins, measured by the density of silver grain in radioautographs, showed a sixfold difference. The great sensitivity of hormonal and nonhormonal protein biosynthesis of the pancreatic beta cell to plasma glucose was unique among tissues and among other pancreatic islet cells we studied.
A Capillary Pressure Disturbance in Young Diabetics
Authors: Tooke John E
Published: September 2013

Finger nailfold capillary pressure (CP) was determined by direct microinjection technique in nine insulin-dependent male diabetic and nine matched control subjects. Mean CP, recorded under resting conditions after acclimatization in a constant temperature room at 24°C, was similar in all parts of the capillary loop in the two groups. Control subjects demonstrated a positive correlation between skin temperature and CP (r = + 0.74 and P < 0.01, arterial limb values; r = +0.61 and P < 0.01, summit and venous limb values). No such relationship was found for diabetics under the initial warm conditions of study (r = −0.15, arterial limb values; r = −0.44, summit and venous limb values).

Mean arterial limb CP, recorded during postocclusive reactive hyperemia, was significantly higher in control subjects than in diabetics (P < 0.001). The diabetic group exhibited no clinical evidence of neuropathy, and their digital vasoconstrictor capacity appeared to be intact.

These findings are interpreted as evidence for an intrinsic microvascular lesion at the precapillary sphincter level in insulin-dependent diabetic patients, which may result in the failure of vasodilatation to respond to physiologic Stress.
Islet Glutathione and Insulin Release
Authors: Ammon Hermann P T , Grimm Andreas , Lutz Susanne , Wagner-Teschner Doris , Handel Monika , Hagenloh Ingrid
Published: September 2013

In isolated rat pancreatic islets, glucose (5.6,11.1, and 16.7 mM) significantly increased reduced glutathione (GSH) and decreased oxidized glutathione (GSSG) levels in a dose-related manner. This was paralleled by a concomitant increase of NADPH and a decrease of NADP. The change of the GSH level occurred as quickly as one minute after addition of glucose.

Exogenous insulin (200, 400, and 800 μU/ml) significantly decreased islet GSH levels in the presence of 5.6 and 16.7 mM glucose and significantly inhibited the insulin-releasing effect of the thiol reagent parachloromercuribenzoate (p-CMB) and tolbutamide.

These data, together with earlier observations, suggest that GSH levels in pancreatic islets are increased by glucose and decreased by exogenous insulin via their effects on the pentose phosphate shunt and NADPH. Our results are compatible with the hypothesis that glucose and exogenous insulin, by modifying the redox state of the NADPH/NADP and GSH/GSSG systems, modulate the sensitivity of the β-cell to the insulin-triggering actions of glucose, p-CMB, and tolbutamide.
Elevated Factor VIII Activity and Factor Vlll-related Antigen in Diabetic Children Without Vascular Disease
Authors: Borkenstein Martin H , Muntean Wolfgang E
Published: September 2013

Factor VIII coagulant activity (VIM C) and factor Vlllrelated antigen (VIII R:Ag) were studied in 86 insulindependent diabetic children. All children were without signs of vascular disease based on a negative funduscopy, negative fluorescein angiography, normal serum creatinine levels, and absence of proteinuria. Age ranged from 4 to 17 yr; duration of clinical diabetes ranged from 1 to 12 yr. The children were grouped according to their urinary sugar excretion, the HbA1 levels, and the duration of clinical diabetes. The group with high urinary sugar excretion and the group withhigh HbA1 levels had a significantly higher VIII C than the group with low urinary sugar excretion and the group with low HbA1 levels. VIII C levels did not differ significantly in the groups with a different duration of clinical diabetes, but VIII R:Ag was significantly higher in the group with the longest duration of diabetes as compared with the group with the shortest duration. VIII R:Ag levels did not differ significantly in the groups with different degrees of urinary sugar excretion or different HbA1 levels.

The results show that in children without vascular disease, and even in children with a short duration of diabetes, alterations of the factor VIII complex can be demonstrated.
Role of Glucose Transport in the Postreceptor Defect of Non-insulin-dependent Diabetes Mellitus
Authors: Ciaraldi T P , Kolterman O G , Scarlett J A , Kao M , Olefsky J M
Published: September 2013

In an attempt to elucidate the cellular mechanism(s) of the insulin resistance associated with impaired glucose tolerance (IGT) and non-insulin-dependent diabetes (NIDDM), insulin-sensitive glucose transport was studied employing isolated adipocytes obtained from these subjects using the nonmetabolized glucose analogue, 3-O-methyl glucose. In the subjects with IGT, basal and maximal rates of 3-O-methyl glucose uptake were normal while the responses at submaximal insulin concentrations were decreased, i.e., the dose-response curves were shifted rightward, indicative of decreased insulin sensitivity. In contrast, the dose-response curves for insulin-stimulated 3-O-methyl glucose uptake in adipocytes obtained from subjects with NIDDM were right-shifted and, in addition, there was a marked decrease in both the basal and maximally stimulated rates of glucose transport (i.e., decreased insulin responsiveness). Thus, the adipocytes from subjects with IGT show decreased insulin sensitivity consistent with decreased insulin binding, whereas the adipocytes from subjects with NIDDM exhibit both decreased insulin sensitivity and decreased insulin responsiveness consistent with a combined receptor and postreceptor defect in cellular insulin action. In the groups as a whole, the magnitude of the rightward shift in the dose-response curve for insulin-stimulated glucose transport correlated with the reduction in adipocyte insulin binding (r = 0.47, P < 0.02). In these subjects, the level of fasting hyperglycemia was correlated (P < 0.01) to the magnitude of the decrease in maximal glucose transport and a highly significant correlation was found between the maximal insulinstimulated rate of glucose transport and the maximal in vivo rate of insulin-stimulated glucose disposal (r = 0.49, P < 0.01). Therefore, we conclude that the insulin resistance in subjects with IGT is due solely to a decrease in insulin binding, whereas subjects with NIDDM exhibit both decreased insulin binding and decreased maximal rates of insulin-stimulated adipocyte glucose transport due to a postreceptor defect at this site in the insulin action pathway. If adipocytes are reflective of changes in glucose transport in other insulin target tissues, then these findings suggest that the cellular lesion responsible for the postreceptor defect in insulin action, previously demonstrated in vivo in subjects with NIDDM, resides in part at the level of the glucose transport system.
Nonenzymatic Glucosylation of High-Density Lipoprotein Accelerates Its Catabolism in Guinea pigs
Authors: Witztum Joseph L , Fisher Milton , Pietro Tiziana , Steinbrecher Urs P , Elam Richard L
Published: September 2013

Nonenzymatic glucosylation of lysine residues of high-density lipoprotein (HDL) was shown to occur in vitro. Most of the incorporated glucose was localized to apoprotein A-l, but other apoproteins were glucosylated as well. Glucosylated high-density lipoproteins (glcHDL) had enhanced electrophoretic mobility on agarose. With increasing amounts of glucose incorporated there was a proportionate increase in the rate of clearance of glcHDL when injected intravenously into guinea pigs. When 60% of lysines were derivatized, clearance of glcHDL was 60% faster than that of control HDL. When as few as 2% of lysines were glucosylated, there was still an 8% increase in the rate of clearance. Uptake of glcHDL by macrophages was not increased. The accelerated clearance of glcHDL from plasma may be relevant to the decreased HDL levels observed in diabetic subjects.
The Relative Importance of First- and Second-Phase Insulin Secretion in Countering the Action of Glucagon on Glucose Turnover in the Conscious Dog
Authors: Steiner Kurt E , Mouton Stephanie M , Bowles Charles R , Williams Phillip E , Cherrington Alan D
Published: September 2013

While it is known that glucagon induces a biphasic release of insulin when infused into a normal animal, it is not known whether the resultant pattern of insulin secretion has important metabolic consequences. To ascertain whether such is the case, glucagon was elevated fourfold in the presence of first-, second-, or combined first- and second-phase insulin release to determine ability of the latter hormone to antagonize the effect of glucagon on glucose turnover in the conscious dog. To separate the effects of the different phases of insulin release the “pancreatic clamp” technique, in which somatostatin is given to inhibit the endocrine pancreas and replacement amounts of insulin and glucagon are given intraportally, was used. In this way, a rise in glucagon (∼220 pg/ml) was brought about in the presence of simulated first-phase (peak IRI 25 μU/ml at 5 min; basal by 30 min), secondphase (peak IRI 19 μU/ml at 30 min and sustained elevation thereafter), or first- plus second-phase (peak IRI 33 μU/ml at 5 min; 17 μU/ml at 30 min and sustained elevation thereafter) insulin release.

Optimal glycemic control required both first- and second-phase insulin release. A selective deficiency of first-phase release resulted in a transient (2 h) worsening of the glucagon-induced hyperglycemia (twofold the normal increment). This defect was attributable to a larger initial rise in glucose production (3.6 ± 0.6 mg/kg · min) than that observed when both phases of insulin release were present (0.9 ± 0.4 mg/kg · min). First-phase insulin release had no significant effect on glucose clearance. A selective deficiency of second-phase release resulted in marked (sixfold) and prolonged worsening of the glucagon-induced hyperglycemia. In this case, however, the hyperglycemia was primarily the result of a defect in glucose clearance. Glucose clearance fell by 29 ± 7% instead of rising by 30 ± 4% as it did when both firstand second-phase release were present. Glucose production was mildly elevated between 15 and 75 min when second-phase insulin release was deficient relative to that apparent when both the first- and secondphases of release were present; this also contributed to the abnormal hyperglycemia.

We conclude that both phases of insulin release are vital to full counterregulation of the action of glucagon on glucose metabolism. First-phase insulin release is important to counter the quick effect of glucagon on glucose production, while second-phase insulin release is important to sustain that inhibition and to augment glucose utilization. Absence of first-phase release results in a transient (2 h) and moderate (20–30 mg/dl) glycemic defect while an absence of second-phase release results in a prolonged and dramatic (70–100 mg/dl) defect.
Ultrastructural Aspects of the Liver Perisinusoidal Space in Diabetic Patients With and Without Microangiopathy
Authors: Bernuau Dominique , Guillot Raymonde , Durand Anne-Marie , Raoux Nadine , Gabreau Thierry , Passa Philippe , Feldmann Gérard
Published: September 2013

To determine whether abnormalities of the perisinusoidal space of Disse are present in the liver of diabetic patients with microangiopathy, an ultrastructural stereologic study of the space of Disse was performed in six insulin-treated diabetics with severe pro-liferative retinopathy and six insulin-treated diabetics with normal fluorescein angiography, six patients with familial unconjugated hyperbilirubinemia were studied as controls. No patient had clinical and/or biochemical hepatic abnormalities and none suffered from any of the pathologic conditions known to be associated with collagenization of the perisinusoidal space. In control patients, the space of Disse of liver sinusoids contained occasional small deposits of collagen fibers. The relative volume of these fibers per unit of sinusoid represented 2.63 ± 0.82%. In all diabetic patients with retinopathy, marked deposition of collagen fibers within the perisinusoidal space was constantly observed, a finding confirmed by ultrastructural stereo-logic analysis which showed that the relative volume of collagen fibers per unit of sinusoid represented 7.33 ± 1.44% and differed significantly from control patient values (P < 0.001). On the contrary, the relative volume of collagen fibers within the space of Disse in diabetic patients without retinopathy (3.95 ± 2.96%) did not differ significantly from control patient values. These findings demonstrate that collagenization of the space of Disse is positively correlated with the presence of diabetic microangiopathy. Ultrastructural examination of the liver sinusoids might constitute a sensitive and useful approach for detecting the early changes of the microcirculation in diabetic patients.
Photoreactive Insulin Derivatives: Comparison of Biologic Activity and Labeling Properties of Three Analogues in Isolated Rat Adipocytes
Authors: Wang Chih-Chen , Hedo Jose A , Ronald Kahn C , Saunders Derek T , Thamm Peter , Brandenburg Dietrich
Published: September 2013

The biologic and chemical properties of three photoreactive insulin derivatives, NαA1-(2-nitro-4-azido-phenyl) insulin (A1-Nap-Ins), NαB1-(2-nitro-4-azidophenyl acetyl) insulin (B1-Napa-Ins), and NεB29-(2-nitro-4-azidophenyl acetyl) insulin (B29-Napa-Ins), were characterized in isolated rat adipocytes. In the absence of photolysis, the relative affinities of these three analogues for the insulin receptor were 0.07, 1.0, and 0.55 when compared with native beef insulin, and these correlated well with the ability of these analogues to stimulate glucose oxidation in a fat cell bioassay. After photolysis and extensive washing to remove noncovalently bound insulin, the Bland B29-photoreactive insulins produced a persistent biologic effect in the isolated adipocytes, with up to 90% of the maximal stimulatory level of insulin. By contrast, photolysis did not produce a persistent biologic effect of the A1-Nap-Ins, even when its concentration was increased 10-fold to account for the difference in receptor affinity.

All three analogues were iodinated and used, for affinity labeling of the insulin receptor in isolated adipocytes. The structure of the insulin receptor was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis and autoradiography. When gels were run under reducing conditions, all three analogues labeled predominantly a protein with an apparent mol wt (Mr) of 138,000, corresponding to the a subunit of the insulin receptor. Minor components with apparent Mr = 90-120K were also identified using both the B1 and B29 analogues, and a minor band was observed at 45K with the B29-Napa-Ins only. The A1-Nap-Ins also strongly labeled a component of Mr = 85,000; this was, however, only partially inhibited by insulin.

Under honreducing conditions, the photoinsulins were linked to receptor oligomers of Mr = 330-350K, 220K, and 185K, as well as to a small amount of free a subunits. With increasing reductant concentrations, these oligomers decreased and the intensity of the a subunit band increased.

These data suggest that although all three photoinsulins bind predominantly to the a subunit, only those linked via the B-chain produce a persistent biologic effect. These studies also suggest that the insulin receptor of isolated fat cells in the native state exists in various oligomeric forms, as well as free subunits, in the plasma membrane.