bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of T Lymphocytes Infiltrating Human Pancreas Allograft Affected by Isletitis and Recurrent Diabetes
Authors: Santamaria Pere , Nakhleh Raouf E , Sutherland David E , Barbosa Jose J
Published: September 2013

We studied a human leukocyte antigen—identical pancreas graft transplanted into an insulin-dependent (type l) diabetic patient shortly after onset of recurrent diabetes to characterize the putative autoreactive T lymphocytes mediating the lesion. The immunohistopathological analysis revealed the presence of isletitis and a selective loss of β-cells. The isletitis was mostly constituted by CD8+/T-lymphocyte receptor α,β (TCR α,β+) T lymphocytes surrounding and infiltrating the affected islets. CD4−/CD8−/TCRγ,δ+ T lymphocytes were observed within the islets. Incubation of the tissue in 15% interleukin 2 induced the migration and initial expansion of the infiltrating cells (66% CD3+lymphocytes) for up to 2 wk; most T lymphocytes in this initial isolate were CD4+ (92% CD4+ and 7% CD8+). Long-term anti-CD3 stimulation of this T-lymphocyte population induced the selectivegrowth of CD8+/TCRαβ+ (75%) and CD4−/CD8−/TCRγδ+ v(all V1δ+) (17%) T lymphocytes. Therefore, this strategy selectively expanded the T lymphocytes, found to be the predominantly islet-infiltrating cells, rather than the lymphocytes predominating in the initial isolate. Anti-CD3 did not stimulate growth of T lymphocytes in cultures of three isletitis-free pancreas graft biopsies. In a control experiment with a CD4+-rich T-lymphocyte population, long-term anti-CD3 stimulation and cloning of cytomegalovirus (CMV)-primed peripheral blood mononuclear cells from a CMV+ subject selectively induced the growth of CD4+ T-lymphocyte clones, all CMVspecific.Application of the same strategy to a CD8+ and CD3+/CD4−/CD8−/TCRγδ+ T-lymphocyterich population from a Graves' disease thyroid resulted in expansion of CD8+ but not CD4−/CD8− lymphocytes. We conclude that 1) the selective T-lymphocyte expansionproperties of anti-CD3 monoclonal antibody in long-term cultures is not dependent on thephenotype of the T lymphocytes but most likely on their activation state and 2) application of this strategy to T lymphocytes isolated from pancreas grafts transplanted into diabetic patients undergoing recurrent diabetes may allow the characterization of the putative CD8+ cells mediating type l diabetes.
The Vascular Order of Islet Cellular Perfusion in the Human Pancreas
Authors: Stagner John I , Samols Ellis
Published: September 2013

The vascular order of pancreatic islet cellular perfusion is important in the intraislet regulation of hormone secretion. Establishment of the sequence of interaction is fundamental to understanding the physiology and pathophysiology of the human islet. Intraislet insulin from the β-cell regulates both net hormone secretion and pulsatile secretion from α- and δ-cells. In terms of vascular perfusion, the δ-cell is perfused last and does not directly affect α- or β-cells in humans.
Electrophysiology of Stimulus-Secretion Coupling in Human β-Cells
Authors: Misler Stanley , Barnett David W , Gillis Kevin D , Pressel David M
Published: September 2013

Herein, we review the applicability to human β-cells of an electrophysiologically based hypothesis of the coupling of glucose metabolism to insulin secretion. According to this hypothesis, glucose metabolism leads to the generation of intracellular intermediates (including ATP), which leads to closure of ATP-sensitive K+ channels. Channel closure results in membrane depolarization, the onset of electrical activity, and voltage-dependent Ca2+ entry. The resultant rise in cytosolic Ca2+ leads to Ca2+-dependent exocytosis of insulin granules. We found that most of the published experimental evidence for human β-cells supports this hypothesis. In addition, we present three other emerging lines of evidence in support of this hypothesis for human islet β-cells: 1) the effects of pH1-altering maneuvers on insulin secretion and electrical activity; 2) preliminary identification of LVA and HVA single Ca2+ channel currents; and 3) validation of the feasibility of Cm measurements to track insulin granule exocytosis. On the basis of this last new line of evidence, we suggest that combinations of Cm measurements and electrical activity/membrane current measurements may help define the roles of diverse electrical activity patterns, displayed by human β-cells, in stimulus-induced insulin secretion.
Impact of Insulin Deficiency on Glucose Fluxes and Muscle Glucose Metabolism During Exercise
Authors: Wasserman David H , Mohr Thomas , Kelly Paul , Lacy D Brooks , Bracy Deanna
Published: September 2013

Exercise in the insulin-deficient diabetic state is characterized by a further increase in elevated circulating glucose and NEFA levels and by excessive counterregulatory hormone levels. The aim of this study was to distinguish the direct glucoregulatory effects of insulinopenia during exercise from the indirect effects that result from the metabolic and hormonal environment that accompanies insulin deficiency. For this purpose, dogs underwent 90 min of treadmill exercise during SRIF infusion with (SRIF+INS, n = 8) or without (SRIF-INS, n = 6) intraportal insulin replacement. Glucagon was not replaced, thus allowing assessment of the direct effect of insulinopenia at the liver independent of the potentiation of glucagon action. Glucose was infused to maintain euglycemia. Hepatic glucose production (Ra); glucose utilization (Rd); and LGIcU, LGIcE, and LGIcO were assessed with tracers ([3H]glucose, [14C]glucose) and arteriovenous differences. With exercise, insulin fell from 66 ± 6 to 42 ± 6 pM in the SRIF+INS group, and was undetectable in the SRIF-INS group. Plasma glucose was 6.33 ± 0.38 and 6.26 ± 0.30 mM at rest in the SRIF+INS and SRIF-INS groups, respectively, and was unchanged with exercise. Ra rose from 7.5 ± 2.3 to 16.5 ± 2.2 μmol · kg−1 · min−1 and 9.1 ± 2.0 to 31.4 ± 3.9 μmol · kg−1 · min−1 with exercise in the SRIF+INS and SRIF-INS groups, whereas Rd rose from 19.5 ± 2.0 to 46.8 ± 3.9 μmol · kg−1 · min−1 and 15.1 ± 1.8 to 29.9 ± 3.3 μmol · kg−1 · min−1. LGIcU rose from 36 ± 9 to 112 ± 25 μmol/min and 1 5 ± 4 t o 59 ± 13 μmol/min and LGIcO rose from 5 ± 2 to 61 ± 12 μmol/min and 5 ± 3 to 32 ± 9 μmol/min with exercise in the SRIF+INS and SRIF-INS groups, respectively. Arterial levels and limb balances of NEFAs and glycerol were similar in the two groups. In summary, during exercise: 1) marked insulinopenia attenuates the increases in muscle glucose uptake and oxidation by ∼50%, independent of changes in circulating metabolic substrate levels; 2) substantial increases in muscle glucose uptake and oxidation are, however, still present even in the absence of detectable insulin levels; and 3) insulinopenia facilitates the increase in Ra, independent of the potentiation of basal glucagon action. In conclusion, marked insulinopenia contributes directly to the exacerbation of glucoregulation during exercise in the diabetic state by limiting the rises in glucose uptake and metabolism and by enhancing hepatic glucose production.
Human GLUT4/Muscle-Fat Glucose-Transporter Gene: Characterization and Genetic Variation
Authors: Buse John B , Yasuda Kazuki , Lay Tracy P , Seo Tracy S , Olson Ann Louise , Pessin Jeffrey E , Karam John H , Seino Susumu , Bell Graeme I
Published: September 2013

Four overlapping DNA fragments spanning 32 kb containing the human GLUT4 facilitative glucose-transporter gene were isolated and characterized. The sequence of the GLUT4 gene (∼6.3 kb) and 2.0 kb of the promoter region was determined. The sequence of the promoter revealed potential binding sites for transcription factors known to regulate gene expression in muscle cells and adipocytes. However, transfection of constructs including 2 kb of the GLUT4 promoter fused to the bacterial CAT gene into 3T3-L1 adipocytes displayed only weak promoter activity. Because insulin resistance plays a prominent role in the development of NIDDM, genetic variation in the sequence of GLUT4 also was evaluated. Oligonucleotide primer pairs were selected that allowed the protein-coding region of the human GLUT4 gene to be amplified by PCR. The sequence of the protein-coding region of the GLUT4 gene and all intron-exon junctions was determined for a single diabetic Pima Indian and was identical to that of the cloned gene and cDNA. SSCP analysis was used to screen patients with diabetes mellitus and normal, healthy nondiabetic individuals for mutations at the GLUT4 locus. In addition to the silent substitution in the codon for Asn130 (AAC or AAT) and a Val383(GTC)→IIe(ATC) replacement described previously, two new variants were identified. One was a T→A substitution in intron 1 that was found in 1 of 36 NIDDM patients who were typed for this variant. The second was a lle385(ATT)→Thr(ACT) replacement that occurred in 1 normal individual and was not found in any of 676 other normal and diabetic subjects. A large and racially diverse group of normal and diabetic individuals also was screened for the lle383 polymorphism. It occurred in both diabetic and nondiabetic subjects. There is no indication from our data that these polymorphisms are associated with NIDDM.
Lilly Lecture: Molecular Mechanisms of Insulin Resistance: Lessons From Patients With Mutations in the Insulin-Receptor Gene
Authors: Taylor Simeon I
Published: September 2013

Insulin resistance contributes to the pathogenesis of NIDDM. We have investigated the molecular mechanisms of insulin resistance in patients with genetic syndromes caused by mutations in the insulin-receptor gene. In general, patients with two mutant alleles of the insulin-receptor gene are more severly insulinresistant than are patients who are heterozygous for a single mutant allele. These mutations can be put into five classes, depending upon the mechanisms by which they impair receptor function. Some mutations lead to a decrease in the number of insulin receptors on the cell surface. For example, some mutations decrease the level of insulin receptor mRNA or impair receptor biosynthesis by introducing a premature chain termination codon (class 1). Class 2 mutations impair the transport of receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane. Mutations that accelerate the rate of receptor degradation (class 5) also decrease the number of receptors on the cell surface. Other mutations cause insulin resistance by impairing receptor function—either by decreasing the affinity to bind insulin (class 3) or by impairing receptor tyrosine kinase activity (class 4). The prevalence of mutations in the insulin receptor gene is not known. However, theoretical calculations suggest that ∼0.1–1% of the general population are heterozygous for a mutation in the insulinreceptor gene; the prevalence is likely to be higher among people with NIDDM. Accordingly, it is likely that mutations in the insulin-receptor gene may be a con-tributory cause of insulin resistance in a subpopulation with NIDDM.
Islet Transplantation With Immunoisolation
Authors: Lanza Robert P , Sullivan Susan J , Chick William L
Published: September 2013

Immunoisolation is a potentially important approach to transplanting islets without need for immunosuppressive drugs. Immunoisolation systems have been conceived in which the transplanted tissue Is separated from the immune system of the host by an artificial barrier. These systems offer a solution to the problem of human islet procurement by permitting use of Islets Isolated from animal pancreases. The devices used are referred to as biohybrid artificial organs because they combine synthetic, selectively permeable membranes that block immune rejection with living transplants. Three major types of biohybrid pancreas devices have been studied. These include devices anastomosed to the vascular system as AV shunts, diffusion chambers, and microcapsules. Results In diabetic rodents and dogs indicate that biohybrid pancreas devices significantly improve glucose homeostasis and can function for more than a year. Recent progress made with this approach is discussed, and some of the remaining problems that must be resolved to bring this technology to clinical reality are addressed.
Experimental Transplantation With Principal Islets of Teleost Fish (Brockmann Bodies): Long-Term Function of Tilapia Islet Tissue in Diabetic Nude Mice
Authors: Wright James R , Polvi Sherilyn , Maclean Heather
Published: September 2013

Certain teleost fish have macroscopically visible islets called BBs that are anatomically discrete. BBs were harvested from Oreochromis nilotica (tilapia) with microscissors, divided, and cultured overnight at 37°C before transplantation into STZ-induced diabetic nude mice. Each mouse received BB fragments from 3-5 fish weighing in aggregate ∼1.7 kg. Non-FPGs were monitored 5 days/wk. Recipients remained normoglycemic (plasma glucose <11.1 mM) for 50 days posttransplantation. Mice bearing 50-day-old grafts had essentially normal GTTs. Left nephrectomies then were performed to remove the grafts, and plasma glucose levels in recipient mice rose to >22.2 mM. Histological examination of graft-bearing kidneys showed viable, vascularized islet tissue containing numerous well-granulated β-cells; examination of recipient native pancreases revealed small islets composed predominately of non-β-cells.
Insulin Induces the Translocation of GLUT4 From a Unique Intracellular Organelle to Transverse Tubules in Rat Skeletal Muscle
Authors: Marette André , Burdett Elena , Douen André , Vranic Mladen , Klip Amira
Published: September 2013

Skeletal muscle surface membrane is constituted by the PM domain and its specialized deep invaginations known as TTs. We have shown previously that insulin induces a rapid translocation of GLUT4s from an IM pool to the PM in rat skeletal muscle (6). In this study, we have investigated the possibility that insulin also stimulates the translocation of GLUT4 proteins to TTs, which constitute the largest area of the cell surface envelope. PM, TTs, and IM components of control and insulinized skeletal muscle were isolated by subcellular fractionation. The TTs then were purified further by removing vesicles of SR origin by using a Ca-loading procedure. Ca-loading resulted in a five- to sevenfold increase in the purification of TTs in the unloaded fraction relative to the loaded fraction, assessed by immunoblotting with an anti-OHP-receptor monoclonal antibody. In contrast, estimation of the content of Ca2+-ATPase protein (a marker of SR) with a specific polyclonal antibody revealed that most, if not all, SR vesicles were recovered in the Ca-loaded fraction. Western blotting with an anti-COOH-terminal GLUT4 protein polyclonal antibody revealed that acute insulin injection in vivo (30 min) increased the content of GLUT4 (by 90%) in isolated PMs and markedly enhanced (by 180%) GLUT4 content in purified TTs. Importantly, these insulin-dependent changes in GLUT4 content of PM and purified TTs were seen in the absence of changes in the α1-subunit of the Na+-K+-ATPase, a surface membrane marker. Isolated IM components such as LSR, HSR, and triads (terminal cisternae plus junctional TT) contained low or barely detectable amounts of GLUT4; furthermore, insulin treatment did not change the distribution of the transporter protein within these fractions. In contrast, a unique IM fraction that was not associated with either SR or triad markers, contained significant amounts of GLUT4 and showed an insulin-dependent decrease (40%) in GLUT4 protein content. These results show that acute insulin treatment induces the translocation of GLUT4s to both the PM and TTs from a unique intracellular organelle not associated with the SR.
Improved Specificity of ICA Assays in the Fourth International Immunology of Diabetes Serum Exchange Workshop
Authors: Greenbaum Carla J , Palmer Jerry P , Nagataki S , Yamaguchi Y , Molenaar J L , Van Beers W A M , Maclaren Noel K , Lernmark Åke ,   Participating Laboratories
Published: September 2013

The goal of the Fourth International Workshop for Standardization of ICA Measurements was to determine the specificity of ICA assays and their ability to distinguish between control sera (n = 57) and sera from IDDM-related individuals—representing relatives of IDDM patients (n = 21), healthy individuals who later developed IDDM (n = 8), or newly diagnosed IDDM patients (n = 23). Results from 28 laboratories were analyzed. The mean specificity (percentage of control sera reported as negative) among 27 laboratories was 91%, including 6 laboratories with 100% specificity. Nevertheless, 78% of laboratories found at least one control sample >0 JDF U. Among samples from first-degree relatives, the mean concordance was 86%, including three sera found negative (0 JDF U) by all laboratories. Among individuals who later developed diabetes, the mean concordance was 93%, with two sera found positive by 100% of laboratories. In sera from newly diagnosed IDDM patients, the mean concordance was 82%. Three sera were found positive and one serum negative by all laboratories. The JDF U of the sera considered to be positive were significantly greater than each laboratory's average for the controls. In conclusion, the results from laboratories participating in the Fourth International ICA Workshop demonstrated excellent specificity, good concordance, and an ability to separate control sera from defined, IDDM-related subjects.
Polygenic Nature of Spontaneous Diabetes in the Rat: Permissive MHC Haplotype and Presence of the Lymphopenic Trait of the BB Rat Are Not Sufficient to Produce Susceptibility
Authors: Colle Eleanor , Fuks Abraham , Poussier Philippe , Edouard Philippe , Guttmann Ronald D
Published: September 2013

We describe the phenotypic characteristics of animals in the fifth backcross-intercross generation of a breeding program in which the RT1 u haplotype and the phenotypic trait responsible for the T-lymphopenia of BB rats have been transferred to the ACI background. In this generation of animals, 24% were lymphopenic with decreased numbers of PBL expressing CD5, TCRα, and RT6. The PBL of the lymphopenic animals had a decreased mitogenic response to ConA. All of the nonlymphopenic animals were homozygous for RT6.2. Phenotypic analysis of intestinal IEL revealed that this was also the case for the lymphopenic animals. Moreover, IEL of the lymphopenic animals exhibited a pattern of staining (increased numbers of TCRαβ+CD4+CD8+ and decreased numbers of TCRαβ+CD4−CD8+) similar to that of BB DP animals. The ACI.1U(BB)-lymphopenic animals, although having two of the genetic traits associated with the expression of spontaneous diabetes mellitus, uniformly fail to develop diabetes. Breeding studies in which these animals were crossed with BB and hBB rats suggest that other genes are necessary for development of overt diabetes.
PGE2 Prevents Anomalies Induced by Hyperglycemia or Diabetic Serum in Mouse Embryos
Authors: Goto Motoko P , Goldman Allen S , Uhing Michael R
Published: September 2013

Both a high level of D-glucose in the medium and serum from a diabetic rat can induce neural-tube fusion defects and growth retardation in cultured mouse and rat embryos. To test our hypothesis that a deficiency of PGs may be involved in the mechanism of hyperglycemia- and diabetic serum-induced teratogenesis and growth retardation, we added PGE2 to the medium of a whole mouse embryo culture containing either normal rat serum and 52.7 mM D-glucose (hyperglycemic) or diabetic rat serum and 22.2 mM D-glucose (diabetic). After a 24-h culture, 94% of hyperglycemic embryos and 81% of diabetic embryos had neural-tube fusion defects; in addition, the number of somites, the morphological score, and the protein content of the embryos were significantly lower than those of controls. Supplementing the medium with PGE2 at concentrations of 0.028–28.4 nM (hyperglycemic) or 28.4 nM (diabetic) significantly reduced the incidence of neural-tube defects and increased the number of somites, the morphological score, and the protein content. These results strongly support the hypothesis that the teratogenicity of diabetic serum, as well as the teratogenic action of hyperglycemic culture, are mediated through a deficiency of PGs.
Longitudinal Study of Plasma Lipoproteins and Hormones During Pregnancy in Normal and Diabetic Women
Authors: Montelongo Adela , Lasunción Miguel Angel , Pallardo Luis Felipe , Herrera Emilio
Published: September 2013

Plasma lipoproteins were studied longitudinally at the 1st, 2nd, and 3rd trimester of gestation and at postpartum and postlactation in 12 age-matched PGDM women, 9 GDM women, and 12 healthy control subjects. FPG and HbA1c were higher in every case in PGDM women than in control subjects, whereas in GDM patients, glucose was augmented only after parturition. FFA and β-hydroxybutyrate levels were higher in both PGDM and GDM patients than in control subjects during gestation but not after parturition. Total TGs and VLDL, LDL, and HDL TGs increased with gestational time in the three groups and declined at postpartum, and although total cholesterol and VLDL, LDL, and HDL cholesterol followed a similar trend, their rise was less pronounced, and the decline after parturition was slower than that of the TGs in the three groups, with no difference among them. The VLDL TG/cholesterol ratio declined in the three groups at the 3rd gestational trimester, whereas in both LDL and HDL, the TG/cholesterol ratio, but not the cholesterol/phospholipid ratio, increased during gestation in the three groups, indicating a specific enrichment of TGs in these particles. The increase in apoA-I and apoB with gestation was parallel to the respective changes in HDL and LDL cholesterol and, again, no difference was observed between the three groups. Plasma levels of β-estradiol, progesterone, and prolactin increased sharply with gestation and declined at postpartum in the three groups, but absolute values of β-estradiol and prolactin, at the three trimesters of gestation, were lower in PGDM patients, but progesterone levels were lower than controls in GDM women only at the 3rd trimester. The logarithm for each of these hormones correlated linearly with VLDL, LDL, and HDL TGs, and the highest correlation coefficient value corresponded to the regression between β-estradiol and HDL TGs. Because estrogens are known to increase VLDL production, decrease hepatic lipase activity, and increase HDL TG levels, we propose that the decreased estradiol levels in our diabetic patients impede an exaggerated rise of circulating lipoproteins above the normal range. We also propose that the development or lack of development of a dyslipidemic condition in diabetic pregnancy depends on the balance between the metabolic control and the level of sex hormones.
Chromatographic Quantitation of Plasma and Erythrocyte Pentosidine in Diabetic and Uremic Subjects
Authors: Odetti Patrizio , Fogarty Jack , Sell David R , Monnier Vincent M
Published: September 2013

Pentosidine is a fluorescent advanced Maillard/glycosylation product and protein cross-link present in elevated amounts in skin from diabetic and uremic subjects. A high-performance liquid chromatographic (HPLC) assay was developed to quantitate pentosidine in plasma and erythrocytes and other tissue proteins with low levels of pentosidine. High protein content and presence of basic amino acids and O2 during acid hydrolysis led to the formation of fluorescent artifacts that could be separated from true pentosidine through combined reverse-phase ion-exchange HPLC. No true pentosidine was formed during acid hydrolysis of ribated protein, suggesting that Amadori products do not generate artifactual pentosidine during hydrolysis. With the combined reverse-phase ion-exchange chromatographic assay, we found a 2.5-fold (P < 0.001) and a 23-fold (P < 0.001) elevation of mean ± SD plasma protein pentosidine in diabetic (2.4 ± 1.2 pmol/mg) and uremic (21.5 ± 10.8 pmol/mg) subjects compared with healthy (0.95 ± 0.33 pmol/mg) subjects. Pentosidine in hemolysate was normal in diabetes but dramatically elevated in uremia (0.6 ± 0.4 pmol/mg hemoglobin, P < 0.001). Although the precise nature of the pentosidine precursor sugar is unknown, plasma pentosidine may be a useful marker for monitoring the biochemical efficacy of trials with aminoguanidine or other treatment modalities. Furthermore, pentosidine in plasma proteins may act as a signal for advanced glycosylation end product-mediated receptor uptake by macrophages and other cells and contribute to accelerated atherosclerosis in diabetes and uremia.
Effect of Acute Hyperglycemia on Glucose Metabolism in Skeletal Muscles in IDDM Patients
Authors: Vaag Allan , Hother-Nielsen Ole , Skött Peter , Andersen Pernille , Richter Erik A , Beck-Nielsen Henning
Published: September 2013

The effect of acute hyperglycemia on glucose metabolism in skeletal muscles was assessed during replacement insulin infusion in 11 patients with insulin-dependent diabetes mellitus (IDDM). With a primed continuous [3-3H]glucose infusion and indirect calorimetry, glucose metabolism was assessed during a basal period (plasma glucose [PG] 5 mM) and during a hyperglycemic period (4-h i.v. glucose infusion, PG 12.1 mM). Biopsies were taken from the vastus lateralis muscle during both periods. On a control day, glucose metabolism was assessed in 10 patients during a basal period (PG 5.2 mM) and after 4 h with no glucose infusion (PG 4.2 mM). Nonoxidative glucose disposal increased during hyperglycemia (32 ± 7 vs. 51 ± 9 mg · m−2 · min−1, P < 0.05), whereas glucose oxidation remained constant. On the control day, nonoxidative glucose disposal decreased from the basal to the second (control) period (33 ± 7 vs. 22 ± 6 mg · m−2 · min−1, P < 0.05), and glucose oxidation remained constant. The activity of glycogen synthase in muscle biopsies (fractional velocities [0.1 and 10 mM glucose 6-phosphate (G6P)]) decreased slightly during hyperglycemia (18 ± 2 vs. 12 ± 2%, P < 0.05) and on the control day (26 ± 4 vs. 20 ± 3%, P < 0.05). Hyperglycemia increased the intracellular concentration of free glucose, corrected for estimated extracellular glucose (0.56 ± 0.11 vs. 1.43 ± 0.19 mM, P < 0.01), G6P (0.14 ± 0.04 vs. 0.23 ± 0.08 mM, P < 0.02), and lactate (2.88 ± 0.33 vs. 4.46 ± 0.61 mM, P < 0.05), whereas these substrate concentrations remained constant on the control day. Muscle glycogen content failed to increase during hyperglycemia but decreased from the basal to the second (euglycemic) period on the control day (398 ± 16 vs. 369 ± 15 mmol glucose/kg dry wt, P < 0.05). In conclusion, hyperglycemia predominantly stimulates nonoxidative glucose disposal during low physiological insulin concentrations. The mechanism may partly be an allosteric activation of glycogen synthase in skeletal muscles in vivo due to an accumulation of G6P. The accumulation of free glucose and G6P indicates that glucose metabolism in skeletal muscles is not regulated solely by the membrane glucose transport system.
Dose-Response Relationship Between Lymph Insulin and Glucose Uptake Reveals Enhanced Insulin Sensitivity of Peripheral Tissues
Authors: Ader Marilyn , Poulin Renee A , Yang Yeon J , Bergman Richard N
Published: September 2013

To examine the role of transcapillary insulin transport to peripheral insulin sensitivity in vivo, we performed dose-response experiments in which both plasma and thoracic duct lymph insulin and glucose utilization (Rd) were measured in conscious dogs. Euglycemic clamps (n = 22) consisted of a 3-h activation period in which insulin was infused (rates: “physiological” 3.6, 5.4, 7.2 pmol · min−1 · kg−1; “pharmacological” 108 pmol · min−1 · kg−1), followed by a 3-h deactivation period. [14C]inulin was also infused as a diffusionary marker. Insulin sensitivity was estimated as the ED50. When based on plasma insulin, ED50 was 480 pM. However, when calculated from lymph (i.e., interstitial) insulin measurements, ED50 was 240 pM. Thus, interstitial insulin measurements reveal that insulin sensitivity of peripheral tissues is approximately twice that estimated from plasma insulin and is similar to sensitivity reported for suppression of hepatic glucose production. Furthermore, although [14C]inulin achieved equilibrium between plasma and lymph within 180 min, within the physiological range, steady state plasma insulin was higher than insulin in lymph (306 ± 18, 474 ± 42, and 780 ± 60 pM vs. 180 ± 18, 318 ± 12, and 504 ± 36 pM; P < 0.0001); plasma insulin achieved steady state faster than lymph insulin (6 ± 1, 6 ± 2, and 11 ± 3 min vs. 29 ± 4, 16 ± 6, and 44 ± 8 min; P < 0.01) and disappeared faster (5 ± 2, 7 ± 2, and 15 ± 6 min vs. 37 ± 8, 32 ± 4, and 43 ± 9 min; P < 0.01). The time course of lymph insulin at each dose was similar to that of Rd, and at each dose, unlike plasma insulin, lymph insulin was strongly correlated with Rd (r = 0.93 or better). At pharmacological hyperinsulinemia (plasma 35232 ± 5250 pM, lymph 27366 ± 4380 pM), Rd rose faster than lymph insulin and disappeared more slowly than insulin. Thus, lymph insulin data indicate that the periphery is more sensitive to insulin than previously realized from estimates based solely on plasma hormone. Furthermore, lymph insulin is proportional to Rd within the physiological but not pharmacological range of insulin, indicating that transcapillary insulin transport is rate limiting for insulin action in this range. Finally, based on in vivo lymph (i.e., interstitial) insulin measurements, peripheral tissue is almost twice as sensitive to insulin than previously realized.
Lipid-Lowering Therapy and Macrovascular Disease in Diabetes Mellitus
Authors: Garg Abhimanyu
Published: September 2013

Patients with diabetes mellitus are at increased risk of morbidity and mortality from macrovascular disease manifesting as coronary heart disease, cerebrovascular accidents, and peripheral vascular disease. Increased frequency of dyslipidemia, hyperglycemia, obesity, hypertension, and associated nephropathy may contribute to accelerated atherogenesis in diabetic patients. Therefore, besides intensive control of hyperglycemia, management of dyslipidemia, hypertension, and obesity should also be emphasized in diabetic patients. Those who smoke should be strongly encouraged to quit smoking. Besides attempts to achieve normal levels of plasma lipoproteins, consideration also should be given to normalization of compositional abnormalities of various lipoproteins in patients with diabetes mellitus. The therapeutic goals for cholesterol reduction should be lower in diabetic patients than nondiabetic subjects. The first step is to achieve good metabolic control of diabetes mellitus by diet, exercise, and weight reduction and, if needed, with sulfonylureas or insulin therapy. Because most of the patients with insulin-dependent diabetes mellitus achieve normal levels of plasma lipoproteins with intensive insulin therapy, lipid-lowering medications are rarely needed. In patients with non-insulin-dependent diabetes mellitus, however, dyslipidemia often persists despite good glycemic control. Lipid-lowering medications should be considered in such patients. Because nicotinic acid can cause marked deterioration in glycemic control, and bile acid-binding resins may accentuate hypertriglyceridemia, these agents are less desirable for use by diabetic patients. Inhibitors of hydroxymethylglutaryl coenzyme A reductase may be preferred in patients with elevated LDL cholesterol and mild hypertriglyceridemia. For diabetic patients with marked hypertriglyceridemia, however, fibric acid derivatives should be the drug of choice.
Quantitative and Qualitative Lipoprotein Abnormalities in Diabetes Mellitus
Authors: Taskinen Marja-Riitta
Published: September 2013

In people with diabetes, the concentration of an individual lipoprotein or apolipoprotein can be highly variable and is totally different in the two major forms of the disease. Alterations in the concentrations of major lipids and lipoproteins are well characterized in both IDDM and NIDDM. In general, the lipoprotein pattern is antiatherogenic in individuals with IDDM who are treated and have optimal glycemic control. In contrast, NIDDM is associated with atherogenic changes of serum lipids and lipoproteins regardless of the mode of treatment. In people with both types of diabetes, the distribution of apoE phenotype seems to be similar to that in nondiabetic populations. IDDM patients with microalbuminuria show atherogenic changes of lipoproteins and have elevated levels of Lp(a), which is a risk factor of coronary artery disease. Whether glycemic control influences the concentration of Lp(a) is still an open question. An important issue is that the concentration of a lipoprotein can be normal without excluding compositional abnormalities that are potentially atherogenic. Such alterations are present in people with both IDDM and NIDDM. Consequently, it has been questioned whether the target values to start treatment should be lower in diabetic than in nondiabetic populations.
Receptor-Mediated Interactions of Advanced Glycosylation End Products With Cellular Components Within Diabetic Tissues
Authors: Vlassara Helen
Published: September 2013

AGEs are nonenzymatically glycosylated adducts of proteins that accumulate in vascular tissues with aging and at an accelerated rate in people with diabetes; AGEs are closely linked to tissue damage due to their high reactivity in protein cross-linking. A macrophage-monocyte receptor system for AGE moieties is shown to mediate the uptake of AGE-modified proteins by a process that also induces cachectin-TNF, IL-1, IGF-I, and PDGF secretion. Thus, in addition to removing senescent glucose-modified proteins and cells, AGE-mediated release of growth-promoting factors may represent a mechanism by which macrophages signal mesenchymal cells the need for replacement of senescent proteins. The age of the macrophage correlates inversely with the binding and removal capacity of the AGE receptor, possibly preventing the clearance of cross-linked proteins and the compounding aging-related tissue damage. In addition to monocyte and macrophages, other cells express similar receptors for AGE-proteins, including endothelial cells, fibroblasts, and mesangial cells. Endothelial cell AGE-receptors mediate transcytosis of AGEs to the subendothelium, induce increased permeability, and enhance endothelium-dependent procoagulant activity. Renal mesangial AGE receptors mediate PDGF-dependent extracellular matrix protein production. Fibroblast AGE receptors may influence cellular proliferation by EGF and EGF-receptor regulation. These findings, in connection with the known abundance of AGEs in aged and diabetic tissues, indicate that AGE-ligand-receptor interactions are crucial for the development of age- and diabetes-related vascular tissue and renal pathology.
Apolipoprotein A-I-Containing Particles and Reverse Cholesterol Transport in IDDM
Authors: Fievet Catherine , Theret Nathalie , Shojaee Negin , Duchateau Philippe , Castro Graciela , Ailhaud Gerard , Drouin Pierre , Fruchart Jean-Charles
Published: September 2013

Lipoprotein particles containing apoA-l but not apoA-II are, among high-density lipoproteins, effective protectors against atherosclerosis that act by promoting the efflux of cellular cholesterol and the reverse cholesterol transport process. Because previous studies showed that in vitro nonenzymatic glycosylation of HDL impairs HDL receptor-mediated cholesterol efflux, we isolated Lp A-I from two poorly controlled insulin-dependent diabetic patients and compared the chemical composition and ability to promote cholesterol efflux with the same particles purified from two matched nondiabetic control subjects. No differences in lipid composition or in the ability to promote cholesterol efflux from cultured adipose cells were noted between the two types of Lp A-I preparations. However, when we separated Lp A-I from diabetic subjects by degree of glycosylation, the specifically glycosylated subfractions were about 50% less effective in producing cholesterol efflux than the nonglycosylated particles.
Metformin Normalizes Nonoxidative Glucose Metabolism in Insulin-Resistant Normoglycemic First-Degree Relatives of Patients With NIDDM
Authors: Widén Elisabeth I M , Eriksson Johan G , Groop Leif C
Published: September 2013

Many first-degree relatives of patients with non-insulin-dependent diabetes mellitus (NIDDM) are characterized by insulin resistance. Because metformin improves peripheral insulin sensitivity, we examined the acute effect of metformin and placebo on glucose and lipid metabolism in nine insulin-resistant first-degree relatives of NIDDM patients with the euglycemic insulin-clamp technique combined with indirect calorimetry and infusion of [3-3H]glucose. Either placebo or 500 mg metformin was taken in random order twice the day before and once 1 h before the clamp. Nine healthy individuals without family history of diabetes served as control subjects. Basal plasma glucose was normal and did not differ between the metformin and the placebo study (4.8 ± 0.2 vs. 5.0 ± 0.2 mM) and neither did basal hepatic glucose production (10.59 ± 0.54 vs. 10.21 ± 0.80 μmol · kg−1 · min−1). Insulin-stimulated glucose disposal was significantly increased by 25% after metformin compared with placebo (26.67 ± 2.87 vs. 21.31 ± 1.73 μmol kg−1 min−1 P < 0.05). The enhancement in glucose utilization was primarily due to normalization of nonoxidative glucose disposal (from 8.02 ± 1.35 to 15.07 ± 2.69 μmol kg−1 min−1 P < 0.01, vs. 15.65 ± 2.72 μmol kg−1 min−1 in control subjects). In contrast, glucose oxidation during the clamp was slightly lower after metformin compared with both placebo (11.59 ± 0.83 vs. 13.30 ± 1.00 μmol kg−1 min−1 P = 0.06) and healthy control subjects (15.68 ± 1.38 μmol kg−1 min−1 P < 0.05). We conclude that acutely administered metformin improves peripheral insulin sensitivity in insulin-resistant normoglycemic individuals primarily by stimulating the nonoxidative pathway of glucose metabolism.
Detection of Mutations in Insulin Receptor Gene by Denaturing Gradient Gel Electrophoresis
Authors: Barbetti Fabrizio , Gejman Pablo V , Taylor Simeon I , Raben Nina , Cama Alessandro , Bonora Enzo , Pizzo Paolo , Moghetti Paolo , Muggeo Michele , Roth Jesse
Published: September 2013

Denaturing gradient gel electrophoresis (DGGE) has been used to screen for mutations in the insulin receptor gene. Each of the 22 exons was amplified by the polymerase chain reaction (PCR). For each exon, one of the two PCR primers contained a guanine-cytosine (GC) clamp at its 5′ end. The DNA was analyzed by electrophoresis through a polyacrylamide gel containing a gradient of denaturants. Two geometries for the gels were compared; the gradient of denaturants was oriented either parallel or perpendicular to the electric field. The sensitivity of the technique was evaluated by determining whether DGGE succeeded in detecting known mutations and polymorphisms in the insulin receptor gene. With parallel gels, 12 of 16 sequence variants were detected. The use of perpendicular gels increased the sensitivity of detection so that all 16 sequence variants were successfully detected when DNA was analyzed by a combination of perpendicular and parallel gels. Furthermore, DGGE was used to investigate a patient with leprechaunism whose insulin receptor genes had not previously been studied. Two mutant alleles were identified in this patient. The allele inherited from the father had a mutation substituting alanine for Val-28; in the allele inherited from the mother, arginine was substituted for Gly-366.
Dual Functional Role of Membrane Depolarization/Ca2+ Influx in Rat Pancreatic B-Cell
Authors: Sato Yoshihiko , Aizawa Toru , Komatsu Mitsuhisa , Okada Naomi , Yamada Takashi
Published: September 2013

Transient exposure of rat pancreatic B-cell to 50 mM K+ ([K+50]) makes exocytosis unresponsive to further depolarization, i.e., stimulation with 100 mM K+ or 1 uM glyburide, which closes the ATP-sensitive K+ (K+ATP) channel, simultaneously with [K+50] does not produce any greater insulin secretion compared with [K+50] alone. In sharp contrast, 16.7 mM glucose ([G16.7]) applied simultaneously with [K+50] elicits an insulin response markedly greater than that produced by [K+50] alone, which is not attenuated by 100 uM diazoxide, an inhibitor of K+ATP channel closure. [G16.7]-Induced Insulin secretion at the basal K+ concn of 4.7 mM was greatly (93%) suppressed by 100 uM diazoxide. Insulin secretion induced by [K+50] plus [G16.7] ([K+50 + G16.7]) was markedly suppressed (70%) by 1 uM nifedipine, a Ca2+-channel blocker and was completely abolished by 2 mM 2-cyclohexen-1-one, which reportedly decreases reduced glutathione level and blocks glucokinase. This finding indicates that insulin release induced by [K+50 + G16.7] is not due to leakage produced by toxic stimuli but to activation of exocytosis. When graded concentrations (25 and 50 mM) of K+ were applied simultaneously with [G16.7] in the presence of 100 uM diazoxide, insulin response was clearly dependent on K+ concentration, indicating that the physiological range of membrane depolarization also activates the glucose-responsive effector. Membrane depolarization/Ca2+ influx directly stimulates hormone exocytosis on one hand and activates the K+ATP channel-independent glucose-responsive effector or effectors on the other in the B-cell. The nature of the glucose-responsive effector or effectors remains to be established.
Mechanisms Involved in Degradation of Human Insulin by Cytosolic Fractions of Human, Monkey, and Rat Liver
Authors: Wroblewski Victor J , Masnyk Michael , Khambatta Sunita S , Becker Gerald W
Published: September 2013

The degradation of native and 125I-labeled human insulin (HI) was examined in the cytosolic fraction of human, monkey, and rat liver. The purpose of these studies was to provide a species comparison of the interaction of insulin-degrading enzyme (IDE) and protein disulfide isomerase (PDI) in the degradation of HI. Western-blot analysis with monoclonal antibodies indicated the presence of both IDE and PDI in the cytosolic fraction of human and monkey liver. In contrast, rat liver cytosol contained, detectable levels of IDE only. A species comparison of metabolic profiles was performed by fractionating peptide products with reversed-phase high-performance liquid chromatography. After a 60-min incubation, human liver cytosol degraded unlabeled HI into three major products. Two of these peptides coeluted with the products of the incubation of HI with purified rat liver PDI. The three peptides were isolated and determined by NH2-terminal sequence analysis to be intact A chain, B chain, and des(Phe1)–B chain. Human liver cytosol also formed 125I–A chain and 125I–B chain as major products when specifically labeled 125I–HI isomers were used as substrate. Significant proteolytic degradation was observed only when reactions with human liver cytosol were supplemented with Mn2+. In contrast, monkey and rat liver cytosol proteolytically degraded 125I–HI isomers to small peptide fragments. The rat and monkey metabolic profiles were similar to each other and to that observed with Mn2+-supplemented human liver cytosol. Proteolysis in monkey and rat was sensitive to inhibition by EDTA. When proteolysis was inhibited in monkey cytosol, the formation of B chain, reflecting the activity of PDI, was evident. Immunoinhibition of PDI with ascites containing monoclonal antibodies to PDI prevented the metabolism of 125I-(B26)HI in monkey and Mn2+ supplemented human liver cytosol. PDI antibodies did not inhibit the proteolysis of 125I-(B26)-HI in rat cytosol or the proteolysis of 125I-(B26)–B chain by monkey or Mn2+-supplemented human liver cytosol. These results are consistent with the involvement of both PDI and IDE in the degradation of HI by nonrodent and human liver cytosol.
Aminoguanidine, a Novel Inhibitor of Nitric Oxide Formation, Prevents Diabetic Vascular Dysfunction
Authors: Corbett John A , Tilton Ronald G , Chang Kathy , Hasan Khalid S , Ido Yasuo , Wang Jin Lin , Sweetland Michael A , Lancaster Jack R , Williamson Joseph R , McDaniel Michael L
Published: September 2013

Increased blood flow and vascular leakage of proteins preferentially affect tissues that are sites of diabetic complications in humans and animals. These vascular changes in diabetic rats are largely prevented by aminoguanidine. Glucose-induced vascular changes in nondiabetic rats are also prevented by aminoguanidine and by NG-monomethyl-L-arginine (NMMA), an established inhibitor of nitric oxide (NO·) formation from L-arginine. Aminoguanidine and NMMA are equipotent inhibitors of interleukin-1 β-induced 1) nitrite formation (an oxidation product of NO·) and cGMP accumulation by the rat β-cell insulinoma cell line RINm5F, and 2) inhibition of glucose-stimulated insulin secretion and formation of iron-nitrosyl complexes by islets of Langerhans. In contrast, NMMA is ∼ 40 times more potent than aminoquanidine in elevating blood pressure in nondiabetic rats. These results demonstrate that aminoguanidine inhibits NO. production and suggest a role for NO· in the pathogenesis of diabetic vascular complications.
Glucose as Regulator of Glucose Transport Activity and Glucose-Transporter mRNA in Hamster β-Cell Line
Authors: Inagaki Nobuya , Yasuda Koichiro , Inoue Gen , Okamoto Yoshimasa , Yano Hideki , Someya Yoshimichi , Ohmoto Yasuka , Deguchi Kyohei , Imagawa Ken-Ichi , Imura Hiroo , Seino Yutaka
Published: September 2013

To investigate the role of glucose in regulating glucose transporters in pancreatic β-cells, we studied the hamster clonal β-cell line HIT-T15, which retains responsiveness to glucose. Northern blot analysis demonstrates that GLUT2 and GLUT1 mRNA are abundant in HIT cells. After a 24-h culture with various concentrations of glucose (0–22.2 mM [0–400 mg/dl]), the GLUT2 mRNA level in HIT cells increased by 40% at 22.2 mM (400 mg/dl) glucose compared with 11.1 mM (200 mg/dl) without a change in mRNA stability. It also decreased proportionally to the reduction of glucose concentration. Glucose deprivation resulted in a decrease of GLUT2 mRNA to an almost undetectable level, with a marked increase in the degradation rate of mRNA. In contrast, the GLUT1 mRNA was not affected by glucose. We show that glucose uptake is highest in HIT cells incubated at 2.8–5.5 mM (50–99 mg/dl) glucose for 24 h, and that levels in cells cultured at 0 mM (0 mg/dl) and 22.2 mM (400 mg/dl) glucose decrease to ∼ 20% of the maximum level. This decrease is consistent with the effects of glucose on glucose-stimulated insulin secretion in HIT cells. Our results indicate that glucose is involved in regulating GLUT2 mRNA and glucose uptake activity and that the glucose responsiveness of the insulin secretion correlates with the glucose-induced change in glucose uptake activity in HIT cells.
Cell and Matrix Components of the Glomerular Mesangium in Type I Diabetes
Authors: Steffes Michael W , Bilous Rudolf W , Sutherland David E R , Mauer S Michael
Published: September 2013

In a cross-sectional study, glomerular basement membrane (GBM) width, the volume fractions of the mesangium (VvMes), its cell (VvCell) and matrix (VvMatx) components, and surface density of the peripheral capillary surface (SvPGBM) were measured in renal biopsies from 187 nondiabetic living related and cadaveric donors of kidneys for transplantation and from 150 patients with insulin-dependent (type I) diabetes mellitus of 1–41 yr duration. In the diabetic patients, the matrix was the major factor in the expansion of the mesangium. However, both VvCell (0.11 ± 0.04) and VvMatx (0.20 ± 10) in diabetic patients exceeded the same measurements in nondiabetic subjects (0.07 ± 0.02 for each component) (P < 0.001 in each case). Linear regression analysis demonstrated significant correlations (P < 0.001 for all) between GBM width, VvMes, VvCell, VvMatx, or SvPGBM and either urinary albumin excretion and creatinine clearance, with the higher correlation coefficients in all cases with albuminuria. Of the structural parameters, VvMatx correlated best with either functional measure by stepwise regression, with GBM as an added factor only with albuminuria. Therefore, although models of diabetic glomerulopathy must consider enlargement of both mesangial cells and matrix, the predominant factor in the progression of structural and functional renal disease is mesangial matrix expansion.