bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-Fiber Study of Contractile and Biochemical Properties of Skeletal Muscles in Streptozotocin-Induced Diabetic Rats
Authors: Stephenson Gabriela M M , O'Callaghan Alexia , Stephenson D George
Published: September 2013

Mechanically skinned, single muscle fibers, isometrically activated in pH and Ca2+ (Sr2+) buffered solutions were used to examine the function of the contractile apparatus in slow- and fast-twitch fibers from soleus (SOL, predominantly slow-twitch) and extensor digitorum longus (EDL, predominantly fast-twitch) muscles of streptozotocin (STZ)-induced diabetic rats and age-matched controls. Three and 14 days after STZ administration, the contractile properties of muscle fibers from diabetic rats did not differ significantly from those of controls with respect to several mechanical parameters, such as maximum Ca-activated tension, activation threshold, and sensitivity to Ca2+ and Sr2+. In contrast, 28 days after STZ administration, 37.5% of the fast-twitch EDL fibers developed maximum activated tensions (77.1 ± 10.4 kN/m2), which were significantly lower than those developed by controls (244.0 ± 14.3 kN/m2), and the slow-twitch SOL fibers displayed a significantly higher sensitivity to Ca2+ (and Sr2+) than the controls. All fibers from diabetic rats, including the low-tension EDL fibers and higher Ca sensitivity SOL fibers displayed control-like electrophoretic profiles of the major myofibrillar proteins. Taken together with data from earlier studies on the effects of long-term diabetes on whole skeletal muscle contractility, these results strongly suggest that 1) the decrease in tetanic tension output of EDL muscles induced by diabetes is caused mainly by direct effects of the diabetic condition on the contractile/regulatory system of a subpopulation of fast-twitch fibers, which develop little force, and 2) the diabetes-induced slowing of twitch times of SOL muscles is caused in part by the increased sensitivity to Ca2+ of the contractile apparatus in the slow-twitch fibers.
Immunohistochemical Characterization of Monocytes-Macrophages and Dendritic Cells Involved in the Initiation of the Insulitis and β-Cell Destruction in NOD Mice
Authors: Jansen Annemarie , Homo-Delarche Françoise , Hooijkaas Herbert , Leenen Pieter J , Dardenne Mireille , Drexhage Hemmo A
Published: September 2013

This immunohistochemical study describes the infiltration pattern of monocytes-macrophages and dendritic cells during the development of insulitis and diabetes in the NOD mouse. A panel of monoclonal antibodies (MoAbs) was used to analyze pancreases of nondiabetic (glucosuria negative) male and female NOD mice at 3, 7, 10, and 17 weeks of age. BALB/c female mice 17-weeks-old, diabetic NOD female mice 20- to 30-weeks-old, and nondiabetic NOD male mice 22-weeks-old were used as controls. Three MoAbs (viz., ER-MP23, MOMA1, and BM8) were special and appeared to identify macrophage/dendritic cell subsets that either had a characteristic infiltration pattern in the initial phases of the autoimmune reaction before T-cell infiltration or were typical for the later β-cell destructive insulitis process. 1) Raised numbers of ER-MP23+ and MOMA-1+ dendritic cells/macrophages were characteristic for the initial phases of the NOD insulitis in 3-week-old mice. The cells were found in and near swollen para-insular vessels. In 7-week-old mice, these ER-MP23+ and MOMA-1+ cells had accumulated around the islets and were the first hematopoietic cells detectable at these spots. 2) From 7 weeks of age onward, BM8+ macrophages could be found in the para- and peri-insulitis processes. However, only in females were these BM8+ macrophages found to infiltrate into the islets. In lymphoid tissues, ER-MP23 predominantly reacts with macrophages/dendritic cells present in the subcapsular and interfollicular sinuses of lymph nodes and the T-cell zones of these lymph nodes. ER-MP23 also reacts with tissue macrophages/dendritic cells. MOMA-1 reacts with the marginal metallophilic macrophages of the spleen and with sinus macrophages of the lymph node. Both populations of cells are likely to be involved in antigen presentation in lymphoid tissues as well as in the NOD peri-insulitis. BM8 in lymphoid tissues predominantly reacts with the phagocytosing macrophages present in the red pulp of the spleen. Because β-cell destruction and glucosuria almost exclusively take place in NOD females, our findings suggest that BM8+ macrophage infiltration into the female islets is linked to a β-cell destructive process, either as a destructive type of infiltration or as an infiltration meant to remove the β-cell debris caused by another immune assault.
Comparative Analysis of Vascular Endothelial Growth Factor Receptors on Retinal and Aortic Vascular Endothelial Cells
Authors: Thieme Hagen , Aiello Lloyd Paul , Takagi Hitoshi , Ferrara Napoleone , King George L
Published: September 2013

Ischemic eye disease often results in ocular neovascularization, presumably due to the elaboration of growth factors. Diabetic retinopathy is a classic example in which dramatic retinal neovascularization arises after ischemic retinal damage. The characterization of vascular endothelial growth factor (VEGF) as an angiogenic molecule whose expression is markedly induced by hypoxia makes it a promising candidate for mediating ischemic retinal neovascularization. Thus, we have characterized the structure, binding, and regulation of VEGF receptors in bovine retinal (BREC) and aortic endothelial cells (BAEC). VEGF stimulated a 2.1-fold increase in BREC number and DNA content at 0.6 nmol/l VEGF (P < 1 × 10−7). Scatchard binding analysis demonstrated specific high-affinity VEGF receptors on BREC with a Kd of 4.9 ± 0.6 × 10−11 mmol/l, similar to that observed for BAEC at 5.1 ± 0.4 × 10−11 mmol/l. BREC, however, possess 1.5 × 105 high-affinity receptors/cell, threefold more than BAEC (P < 0.003) and more than any cell type reported previously. 125I-VEGF affinity cross-linking revealed complexes at 220 and 170 kDa in BREC, but only a 220-kDa band of lesser intensity in BAEC. Cross-linking was displaceable in a dose-dependent manner by VEGF (P < 0.01) but not by other hormones. Hypoxia increased VEGF receptor number 50% in BREC without altering affinity. Antiphosphotyrosine immunoblotting showed VEGF-stimulated tyrosine autophosphorylation of VEGF receptor bands at 225 and 220 kDa and another band at 80 kDa within 1 min. These findings suggest that VEGF may mediate retinal vascular proliferation through large numbers of high-affinity receptors on retinal vascular endothelial cells and suggest that VEGF may be an important mediator of neovascularization induced by ischemic retinopathies such as diabetes.
Higher Proinsulin and Specific Insulin Are Both Associated With a Parental History of Diabetes in Nondiabetic Mexican-American Subjects
Authors: Haffner Steven M , Stern Michael P , Miettinen Heikki , Gingerich Ronald , Bowsher Ronald R
Published: September 2013

Both insulin resistance and decreased insulin secretion have been hypothesized to be precursors of non-insulin-dependent diabetes. An elevated proinsulin concentration reflects abnormal proinsulin processing and could indicate abnormal insulin secretion. We examined fasting insulin (measured by a radioimmunoassay that does not cross-react with proinsulin), as a marker of insulin resistance, and proinsulin and the fasting proinsulin-to-insulin ratio, as markers of impaired proinsulin processing, in 597 nondiabetic Mexican-Americans from the San Antonio Heart Study. Fasting insulin, proinsulin, and the fasting proinsulin-to-insulin ratio were higher in subjects with a parental history of diabetes than in subjects without such a history. These differences remained statistically significant after adjustment for obesity, body fat distribution, and glucose tolerance. A parental history of diabetes in nondiabetic Mexican-Americans is associated with an increase in fasting specific insulin and a disproportionate increase in proinsulin relative to insulin. These data suggest that both increased insulin resistance and abnormal processing of proinsulin are present in offspring of parents with diabetes.
Impaired Insulin-Induced Platelet Antiaggregating Effect in Obesity and In obese NIDDM Patients
Authors: Trovati Mariella , Mularoni Elena M , Burzacca Serenella , Ponziani Maria C , Massucco Paola , Mattiello Luigi , Piretto Valentina , Cavalot Franco , Anfossi Giovanni
Published: September 2013

To investigate the effects of insulin on platelets in obesity and in non-insulin-dependent diabetes mellitus (NIDDM)—classic insulin-resistant states—we determined ADP-induced platelet aggregation and platelet cGMP (guanosine 3',5'-cyclic monophosphate) content in platelet-rich plasma obtained from nine obese subjects and nine age-matched healthy volunteers and from eight NIDDM obese patients and nine age-matched healthy volunteers after a 3-min incubation with human recombinant insulin (0, 240, 480, 960, and 1,920 pmol/1). Platelet aggregation was evaluated using different ADP doses to measure the ADP concentration determined on the basis of a dose-response curve necessary to elicit a maximal aggregation of 50% (ED50). Insulin induced a dose-dependent decrease of platelet aggregation to ADP (P = 0.0001) in healthy subjects. A significant effect was evident starting from an insulin concentration of 240 pmol/1. On the contrary, in insulin-resistant subjects, insulin reduced platelet sensitivity to ADP only at a concentration of 1,920 pmol/1. When ADP ED50 values obtained in platelet-rich plasma incubated with insulin were expressed in percentage of the ADP ED50 values obtained in platelet-rich plasma without insulin, considered as 100%, we observed that ADP ED50 with 1,920 pmol/1 insulin was 153.6 ± 13.2% in the younger healthy subject group (P = 0.004), 150.0 ± 3.8% in the older healthy subject group (P = 0.0001), 116.1 ± 6.1% in obese subjects (P = 0.031), and 120.0 ± 8.6% in NIDDM patients (P = 0.05). In healthy subjects, insulin induced a dose-dependent increase of platelet cGMP (P = 0.0001). A significant effect was evident starting from an insulin concentration of 240 pmol/l; cGMP values rose from 7.9 ± 1.4 to 14.6 ± 2.5 pmol/109 platelets with 1,920 pmol/1 insulin in the younger healthy subject group (P = 0.0001) and from 7.5 ± 0.1 to 13.4 ± 1.4 pmol/109 platelets in the older healthy subject group (P = 0.003). In obese subjects and in NIDDM patients, insulin induced an increase of cGMP only at 1,920 pmol/l: from 7.6 ± 0.7 to 9.8 ± 1.4 pmol/109 platelets in obese subjects (P = 0.07) and from 7.5 ± 0.5 to 9.5 ± 0.9 pmol/109 platelets in NIDDM patients (P = 0.07). The platelet antiaggregating effect exerted by insulin, attributable to the increase of platelet cGMP content, is impaired in obesity and in obese NIDDM patients. These data indicate that human platelets are a site of insulin resistance.
Does Intra-Abdominal Adipose Tissue in Black Men Determine Whether NIDDM Is Insulin-Resistant or Insulin-Sensitive?
Authors: Banerji Mary Ann , Chaiken Rochelle L , Gordon David , Kral John G , Lebovitz Harold E
Published: September 2013

Insulin resistance in black Americans with non-insulin-dependent diabetes mellitus (NIDDM) is found in only 60% of those with a body mass index (BMI) of <30 kg/m2, suggesting that NIDDM can occur independent of peripheral insulin resistance. When insulin resistance is present, it is not necessarily correlated with obesity. Numerous studies have shown that increased amounts of intra-abdominal adipose tissue are associated with various metabolic abnormalities. We therefore investigated whether the occurrence of insulin resistance in black NIDDM men could be explained by the pattern of body adipose tissue distribution rather than total adiposity. Twenty-two near-normoglycemic black men (fasting plasma glucose [mean ± SD] = 104 ± 10 mg/dl, HbA1c = 4.6 ± 0.78%, age 48.9 ± 9.2 years, and BMI 26.5 ± 2.4 kg/m2) were studied. The euglycemic insulin clamp with 1 mU · kg−1 · min−1 insulin infusion and D-[3-3H]glucose was used to measure insulin action. Whole-body computed tomography with 22 scans was used to determine body composition. Total body adipose tissue was 19.6 ± 7.5 1, and the percentage of body fat was 27 ± 7. Glucose disposal ranged from 2.5 to 8.1 mg · kg−1 · min−1 (10 men were insulin-sensitive and 12 were insulin-resistant). There was a strong inverse correlation between glucose disposal and the proportion of total adipose tissue in the intra-abdominal region (r = −0.78, P < 0.001), while there was no correlation between glucose disposal and total muscle volume, BMI, total adipose tissue volume, or total subcutaneous adipose tissue volume. When insulin resistance is present, it is highly correlated with an increase in the proportion of intra-abdominal adipose tissue. The data raise the possibility that insulin resistance in black NIDDM men may be a consequence of increased intra-abdominal adipose tissue mass.
Effects of Acute, Subacute, and Chronic Diabetes on Carbohydrate and Energy Metabolism in Rat Sciatic Nerve: Relation to Mechanisms of Peripheral Neuropathy
Authors: Thurston Jean Holowach , McDougal David B , Hauhart Richard E , Schulz Demoy W
Published: September 2013

To address the problem of the pathogenesis of diabetic neuropathy, rats were made diabetic by alloxan administration, and sciatic nerves were sampled for electrolyte and water content and levels of selected carbohydrates and intermediates in energy metabolism at 3, 6, and 26 weeks. Significant increases were seen in the nerve content of glucose, sorbitol, and fructose. Decreases of myo-inositol were not statistically significant. Glucose-6-phosphate was increased at all times; fructose-1,6-bisphosphate was elevated at 6 and 26 weeks. Nerve ATP and phosphocreatine levels were both increased concomitantly, as was the energy charge. Nerve lactate levels increased only at 26 weeks when plasma lactate levels were also high. Plasma ketone bodies were elevated throughout the 26-week experimental interval. It is postulated that ketone bodies were being used as alternative metabolic fuels in diabetic nerve, thereby causing inhibition of pyruvate oxidation and increased aerobic production of lactate. Increased plasma ketone body levels could also inhibit hepatic lactate uptake. There was no other evidence for hypoxia/ischemia. Lactate:pyruvate ratios did not differ from control values at any time in these ketotic hypoinsulinemic animals. Five major hypotheses have been proposed to explain the pathogenesis of diabetic neuropathy: 1) hypoxia/ischemia, 2) hyperglycemie pseudohypoxia, 3) myo-inositol deficiency, 4) fructose and polyol accumulation and osmotic disequilibrium, and 5) nonenzymatic glycation of macromolecules by fructose and glucose. The data obtained in this study seem to fit best with hypotheses 4 and perhaps 5.
Human Hexokinase II mRNA and Gene Structure
Authors: Printz Richard L , Ardehali Hossein , Koch Stephen , Granner Daryl K
Published: September 2013

This study reports the isolation and characterization of the human hexokinase II (HKII) gene. This gene is ∼ 50 kilobases in length and contains 18 exons, ranging in size from 96 to 2,536 base pairs, that are exactly the same size as the corresponding exons in the rat HKII gene. A cDNA representing the entire open reading frame for HKII was synthesized using a series of polymerase chain reactions with human skeletal muscle RNA as the template, and this allowed us to deduce the complete structure of the HKII mRNA. The human HKII mRNA has 431 nucleotides (nt) of 5′ noncoding sequence, 2,751 nt of coding sequence, and 2,394 nt of 3′ noncoding sequence. The open reading frame encodes a protein of 917 amino acids with an estimated molecular mass of 102.4 kDa. There is a high degree of similarity in the amino acid and nt sequences of the rat and human glucokinase and HKII proteins and genes. This, coupled with the observation that the exon sizes are conserved, suggests a common evolutionary origin of the these two genes.
Morphological and Functional Characterization of βTC-6 Cells—an Insulin-Secreting Cell Line Derived From Transgenic Mice
Authors: Poitout Vincent , Stout Laurence E , Armstrong Michael B , Walseth Timothy F , Sorenson Robert L , Robertson R Paul
Published: September 2013

Morphological analysis of hormone content and functional assessment of hormone secretion were conducted in β TC-6 cells, an insulin-secreting cell line derived from transgenic mice expressing the large T-antigen of simian virus 40 (SV40) in pancreatic β-cells. We observed by immunohistochemistry and confocal microscopy that β TC-6 cells contain abundant insulin and small amounts of glucagon and somatostatin (SRIF). Glucagon usually co-localized with insulin, whereas cells containing SRIF did not contain insulin or glucagon. Static incubation and perifusion experiments demonstrated that β TC-6 cells at passage 30–45 secrete insulin in response to glucose. In static incubations, maximal stimulation was achieved for glucose concentrations > 2.8 mmol/l glucose, and the half-maximal effect was observed at 0.5 mmol/l. Maximal stimulation was four times > HIT-T15 cells at passage 72–81, although HIT cells had a greater response over their basal levels. The magnitude of the insulin response to glucose in perifusion was 1,734 ± 384 pmol.l−1. min and was 4.6-fold greater in the presence of 3-isobutyl-1-methylxanthine. Low amounts of glucagon were released in response to amino acids. Epinephrine (EPI), and to a lesser extent SRIF, inhibited phasic glucose-induced insulin secretion. A major portion of these inhibitory effects was mediated by pertussis toxin-sensitive substrates. Immunoblots detected the presence of the G-proteins Giα2, Giα3, and Goα2. These results indicate that βTC-6 cells are a glucose-responsive cell line in which insulin exocytosis is physiologically regulated by EPI and SRIF through Gi/Go-mediated mechanisms.
Identification of Four Amino Acid Substitutions in Hexokinase II and Studies of Relationships to NIDDM, Glucose Effectiveness, and Insulin Sensitivity
Authors: Echwald Søren M , Bjørbaek Christian , Hansen Torben , Clausen Jesper O , Vestergaard Henrik , Zierath Juleen R , Printz Richard L , Granner Daryl K , Pedersen Oluf
Published: September 2013

Human hexokinase (HK) II, a glucose phosphorylating enzyme in muscle tissue, plays a central role in glucose metabolism. Since reduced insulin-stimulated glucose uptake and reduced glucose-6-phosphate content in muscle have been demonstrated in pre-non-insulin-dependent diabetes mellitus (pre-NIDDM) and NIDDM subjects, we have examined the coding region of the HKII gene in NIDDM patients to determine whether these patients show genetic polymorphisms that are associated with or contribute to the disease. Single-strand conformational polymorphism analysis and nucleotide sequencing were initially performed on the entire coding region of the HKII gene of 38 insulin-resistant NIDDM patients and 5 healthy control subjects. This analysis revealed four missense mutations at codons 142 (Gln to His), 148 (Leu to Phe), 497 (Arg to Gln), and 844 (Arg to Lys) and an additional six exon polymorphisms that did not predict any change in amino acid composition of the protein. One homozygous and nine heterozygous carriers of the codon 142 mutation were found among the NIDDM patients. The mutations at codons 148, 497, and 844 were each found in one diabetic subject and only on one allele. There were no carriers of compound heterozygous mutations. A subsequent study of 301 patients with NIDDM and 151 healthy control subjects revealed no additional mutations at codons 148, 497, or 844. The total frequency of the mutated allele at codon 142 was 18.9% among the control subjects and 17.0% among the NIDDM patients (X2 = 0.56, P = 0.45). A population-based sample of 383 unrelated young healthy Caucasians was examined during a combined intravenous glucose and tolbutamide test to address whether the codon 142 amino acid polymorphism was associated with alterations in glucose effectiveness or insulin sensitivity. No evidence could, however, be provided for obvious relationships between the two estimates of whole-body insulin action and glucose turnover and the gene variant. In conclusion, four amino acid substitutions have been identified in the human HKII gene. Although the widespread mutation at codon 142 does not seem to be associated with an increased susceptibility to NIDDM or major abnormalities in whole-body glucose effectiveness or insulin sensitivity, further studies are needed to address the potential roles of the HKII mutations in intracellular energy metabolism.
Preventing Non-Insulin-Dependent Diabetes
Authors: Knowler William C , Narayan K M V , Hanson Robert L , Nelson Robert G , Bennett Peter H , Tuomilehto Jaakko , Scherstén Bengt , Pettitt David J
Published: September 2013

Many risk factors for non-insulin-dependent diabetes mellitus (NIDDM), such as obesity, physical inactivity, and high-fat diet, can potentially be modified. Furthermore, some of the metabolic abnormalities, such as insulin resistance and impaired glucose tolerance, that predict diabetes can be improved by behavior modification and drug treatment. Thus, at least to some extent, NIDDM may be preventable. Several small clinical trials have addressed the hypothesis that NIDDM can be prevented by dietary modification, physical activity, or drug treatment. Some studies suggest a preventive effect, but the conclusions are limited by considerations of sample size, randomization, or intensity of the interventions. Consequently, the hypothesis that NIDDM is preventable requires further testing.
Possible Role for TNF-α in Early Embryopathy Associated With Maternal Diabetes in the Rat
Authors: Pampfer Serge , Vanderheyden Ivo , Wuu Yan-Der , Baufays Laurence , Maillet Olivier , De Hertogh René
Published: September 2013

Tumor necrosis factor (TNF) bioactivity was assessed in culture media conditioned with uterine cells collected from control or diabetic rats on days 5 and 8 of pregnancy. On both days, diabetic uterine cells released significantly more biologically active TNF than did control cells, and this activity was significantly decreased by the addition of anti-TNF-α antibodies but not by the addition of normal IgG when WEHI 164 cells were used as a target. When uterine tissues from day 5 or day 8 pregnant diabetic rats were tested by Northern blot analysis, TNF-α mRNAs were twofold more abundant than in control samples, but the difference was not statistically significant (P = 0.086 and 0.100, respectively). Immunohistochemical analysis of diabetic day 5 uterine sections revealed that most of the TNF-α synthesis occurs in the epithelium lining the uterine lumen. Finally, the growth of day-5 embryos in culture medium conditioned with day-5 diabetic uterine cells was significantly reduced when compared with that of embryos in medium conditioned with control cells. Embryonic development was markedly improved when anti-TNF-α antibodies were added to the diabetic-cell conditioned medium. Our data support the hypothesis that TNF-α may be implicated in the developmental deficiencies observed in preimplantation embryos from pregnant diabetic rats.
A Prospective Study of the Role of Coxsackie B and Other Enterovirus Infections in the Pathogenesis of IDDM
Authors: Hyöty Heikki , Hiltunen Merja , Knip Mikael , Laakkonen Maria , Vähäsalo Paula , Karjalainen Jukka , Koskela Pentti , Roivainen Merja , Leinikki Pauli , Hovi Tapni , Åkerblom Hans K
Published: September 2013

Coxsackievirus B infections have been associated with clinical manifestation of insulin-dependent diabetes mellitus (IDDM) in several studies, but their initiating role in the slowly progressing β-cell damage is not known. This is the first prospective study designed to assess the role of coxsackie B and other enterovirus infections in the induction and acceleration of this process. Three separate series were studied: 1) an intrauterine exposure series comprising 96 pregnant mothers whose children subsequently manifested IDDM and 96 control mothers whose children remained nondiabetic; 2) a cohort of 22 initially unaffected siblings of diabetic children who were followed until they developed clinical IDDM (mean observation time, 29 months) and 110 control siblings who remained nondiabetic; 3) a case-control series comprising 90 children with newly diagnosed IDDM and 90 control subjects. Enterovirus infections were identified on the basis of significant increases in serum IgG, IgM, or IgA class antibodies against a panel of enterovirus antigens (capture radioimmunoassay). Enterovirus antibodies were significantly elevated in pregnant mothers whose children subsequently manifested IDDM, particularly in cases in which IDDM appeared at a very young age, before the age of 3 years (P < 0.005). Serologically verified enterovirus infections were almost two times more frequent in siblings who developed clinical IDDM than in siblings who remained nondiabetic (mean, 1.0 vs. 0.6 infections/follow-up year; P < 0.001). This difference was seen both close to the diagnosis of IDDM and several years before diagnosis. Up to 19% (10 of 52) of the infections in prediabetic siblings were associated with increases in islet cell antibody (ICA) levels, and 83% (10 of 12) of ICAs increase with enterovirus infections. The corresponding figures in control siblings were 3% (5 of 185, P < 0.001) and 38% (5 of 13, Ns). IgM class enterovirus antibodies were slightly elevated in young children (<3 years old)with newly diagnosed IDDM (P < 0.05), but not in older patients. These observations suggest that exposures to enterovirus infections, both in utero and in childhood, are able to induce β-cell damage and lead to clinical IDDM after a varying subclinical period.
Brainstem Dysfunction Is Provoked by a Less Pronounced Hypoglycemic Stimulus in Diabetic BB Rats
Authors: Jacob Ralph J , Weber Andrew B , Dziura James , Morgen Jennifer , Sherwin Robert S
Published: September 2013

Recent studies suggest that moderate hypoglycemia impairs brainstem function in normal humans and rats. To examine whether diabetes alters this response, simultaneous auditory-evoked potentials were recorded directly from the inferior colliculus (IC) and from the brainstem before and after controlled hypoglycemia (clamp) in awake insulin-dependent diabetic BB/Wor rats. Hyperglycemic diabetic animals were studied either during hyperinsulinemic euglycemia (5.6 mmol/l, n = 4) or mild hypoglycemia (3.5 mmol/l, n = 9). Nondiabetic controls were also studied at 3.5 mmol/l (n = 7) and at 2.8 mmol/l (n = 6). Brainstem function was not affected during euglycemia in diabetic rats. However, when plasma glucose was lowered to 3.5 mmol/l, the diabetic rats showed a 10% delay in IC evoked potential (ICEP) latency, whereas nondiabetic animals did not. This occurred despite similar counterregulatory hormones in both groups. The brainstem auditory-evoked potential (BAEP) localized the defect in the diabetic group to an area in or near the IC. When glucose levels were lowered to 2.8 mmol/l, however, brain function was impaired in nondiabetic rats as well. Again the defect was restricted to an area in or near the IC. We conclude that mild hypoglycemia causes a functional impairment in the IC region of the brainstem in both nondiabetic and diabetic rats. However, in the diabetic rats, this alteration occurs after a less pronounced hypoglycemic stimulus. Our findings suggest that chronic hyperglycemia leads to metabolic adaptions that render the diabetic brain more susceptible to mild hypoglycemia.
Hyperglycemia-Induced Thrombin Formation in Diabetes: The Possible Role of Oxidative Stress
Authors: Ceriello Antonio , Giacomello Roberta , Stel Giuliana , Motz Enrico , Taboga Claudio , Tonutti Laura , Pirisi Mario , Falleti Edmondo , Bartoli Ettore
Published: September 2013

Diabetes is characterized by the existence of a thrombosis-prone condition, possibly related to hyperglycemia. However, the mechanism linking hyperglycemia to the activation of the coagulation cascade is still unclear. It has been recently suggested that diabetes is accompanied by increased oxidative stress. In this work, the possibility that oxidative stress may be involved in the hyperglycemia-induced coagulation activation has been evaluated. Prothrombin fragment 1 + 2 (F1+2), which represents a reliable marker of the amount of thrombin released in the circulation, has been chosen for studying thrombin formation in vivo. In nine type II diabetic patients and in seven healthy control subjects, matched for age and body mass index, three different experiments were performed: oral glucose tolerance test (OGTT), intravenous antioxidant glutathione (GSH) administration for 2 h, and OGTT plus intravenous GSH administration. Samples were drawn at −15 min and every 30 min from 0 to 180 min. During the OGTT, F1+2 significantly increased in both diabetic and healthy subjects. GSH administration during OGTT normalized this phenomenon. GSH administered alone significantly decreased F1+2 in diabetic patients, while no effect was observed in the normal subjects. These data suggest that hyperglycemia may induce thrombin activation, possibly inducing an oxidative stress, and that antioxidant GSH may counterbalance this effect.
Elimination of Self-Peptide Major Histocompatibility Complex Class I Reactivity in NOD and β2-Microglobulin-Negative Mice
Authors: Huang Rong , Guo Jane , Li Xiangping , Faustman Denise L
Published: September 2013

Nonobese diabetic (NOD) mice and β2-microglobulin-gene–ablated mice (β2M –/–) show impaired presentation of major histocompatibility complex (MHC) class I and self-peptides, structures now recognized as critical for T-cell education to endogenous peptides. The naturally occurring NOD class I presentation abnormality appears to be attributable to, in part, a quantitative defect in the production of Tap-1 mRNA; Tap-1 with Tap-2 normally functions as a transporter for stable self-peptide and class I assembly. This study attempts to reverse NOD and β2M –/– mouse autoreactivity by introduced or reestablished syngeneic class I presentation. Introduction of MHC class I and self-peptides on syngeneic MHC class I-matched cells specifically prevented diabetes in NOD mice and eliminated in vitro class I–directed T-cell autoreactivity in NOD and β2M –/– mice. Reestablishment of endogenousclass I and self-peptide presentation in NOD mice was achieved with two well-described cures for the NOD mouse, complete Freund's adjuvant and mouse hepatitis virus. Both treatments induced Tap-1 mRNA, reestablished class I presentation of endogenous antigens, and eliminated in vitro and in vivo T-cell autoreactivity of self-peptides in the class I groove. These results substantiate a therapeutic role of self-peptide complexed with class I for T-cell education and suggest that some well-described NOD treatments may work, in part, through reestablishment of tolerance through class I and self-peptide.
Impaired Vasoconstriction to Endothelin 1 in Patients With NIDDM
Authors: Nugent Ailish G , McGurk Colm , Hayes J Randal , Johnston G Dennis
Published: September 2013

Microvascular disease is an important cause of morbidity in diabetes. There is evidence that impaired autoregulation of blood flow is involved in the pathogenesis of diabetic microangiopathy. The vascular endothelium plays a central role in the regulation of vascular tone. Endothelin (ET)-1 is a potent endothelium-derived vasoconstrictor substance that contributes to basal vascular tone. Impaired vasoconstriction in response to endogenous ET could result in hyperperfusion and subsequent microvascular damage. The purpose of our study was to determine whether vascular responses to locally administered ET-1 are impaired in NIDDM. Nine patients with NIDDM and 12 control subjects underwent cannulation of the nondominant brachial artery. Forearm blood flow (FBF) was measured at baseline and during the drug infusion using strain-gauge venous occlusion plethysmography. ET-1 (5 pmol/min) was infused for 60 min at a rate of 1 ml/min. FBF was measured during the first 5 min of the infusion and at 5-min intervals thereafter. Results were expressed as change in FBF from baseline (ml.100 ml−1 · min−1) and were analyzed using repeated measures analysis of variance and Dunnett's test of multiple comparisons. Control subjects showed a gradual onset of vasoconstriction in response to ET-1, which reached maximum at 35 min (1.1 ml.100 ml−1 · min−1; P < 0.01). There was no reduction in FBF in response to ET-1 in the diabetic group. The differences between the diabetic and control groups were significant (P < 0.03). In conclusion, ET-1 infused locally at 5 pmol/min does not cause vasoconstriction in patients with NIDDM.
Significance of Insulin for Glucose Metabolism in Skeletal Muscle During Contractions
Authors: Hespel P , Vergauwen L , Vandenberghe K , Richter E A
Published: September 2013

Glucose uptake rate in active skeletal muscles is markedly increased during exercise. This increase reflects a multifactorial process involving both local and systemic mechanisms that cooperate to stimulate glucose extraction and glucose delivery to the muscle cells. Increased glucose extraction is effected primarily via mechanisms exerted within the muscle cell related to the contractile activity per se. Yet contractions become a more potent stimulus of muscle glucose uptake as the plasma insulin level is increased. In addition, enhanced glucose delivery to muscle, which during exercise is essentially effected via increased blood flow, significantly contributes to stimulate glucose uptake. Again, however, increased glucose delivery appears to be a more potent stimulus of muscle glucose uptake as the circulating insulin level is increased. Furthermore, contractions and elevated flow prove to be additive stimuli of muscle glucose uptake at any plasma insulin level. In conclusion, the extent to which muscle glucose uptake is stimulated during exercise depends on various factors, including 1) the intensity of the contractile activity, 2) the magnitude of the exercise-associated increase in muscle blood flow, and 3) the circulating insulin level.
Dexamethasone-Induced Neuropeptide Y Expression in Rat Islet Endocrine Cells: Rapid Reversibility and Partial Prevention by Insulin
Authors: Myrsén Ulrika , Ahrén Bo , Sundler Frank
Published: September 2013

Neuropeptide Y (NPY) is a widely distributed neurotransmitter in the central and peripheral nervous system. In the normal rat pancreas, NPY is confined to neuronal elements, including fibers penetrating the islets. However, treatment of rats with the glucocorticoid dexamethasone (DEX) induces NPY expression also in islet cells. Previously performed double immunocytochemistry (ICC) and in situ hybridization (ISH) combined with ICC revealed that the majority of NPY-expressing islet cells are β-cells. The present study, using ICC, ISH, and Northern blot, addressed the question whether the islet cell expression of NPY induced by DEX is affected by concomitant insulin (60 U/kg body wt daily for 12 days) treatment. Further, the time course of NPY expression in the islet cells after DEX withdrawal was examined. Treatment with DEX (2 mg/kg body wt daily for 12 days) confirmed the induction of NPY expression in numerous cells, most of which were β-cells, dispersed within the islets. Northern blot analysis of RNA extracted from isolated islets of DEX-treated rats revealed a strong signal for NPY. Furthermore, DEX also induced NPY expression in isolated rat islets during a 5-day culture period in DEX (100 nmol/l). In vivo, the DEX-induced islet cell expression of NPY mRNA was rapidly reversed after cessation of DEX, being nondetectable 5 days post-treatment; NPY peptide was nondetectable 10 days post-treatment, indicating a slower turnover of the formed peptide. After combined treatment with DEX and insulin, the frequency of islet cells expressing NPY was markedly lower than after treatment with DEX alone. The vast majority of the NPY-expressing cells were β-cells. In conclusion, DEX-induced NPY expression in rat islet cells is dependent on continuous DEX treatment and is partly prevented by exogenous insulin. The results suggest that the DEX-induced islet NPY expression is regulated by insulin.
Effects of Glucose Ingestion Versus Infusion on Pulsatile Insulin Secretion: The Incretin Effect Is Achieved by Amplification of Insulin Secretory Burst Mass
Authors: Pørksen Niels , Munn Stephen , Steers Jeffery , Veldhuis Johannes D , Butler Peter C
Published: September 2013

In the present studies, we used a recently validated canine model to determine 1) if glucose ingestion stimulates insulin secretion by amplifying the pulsatile component of insulin release, and if so, 2) whether this effect is achieved preferentially through burst mass or frequency modulation, and 3) if the mechanism of incretin effect of insulin secretion is mediated via the pulsatile mode of secretion. We report that 30 g of glucose ingestion stimulates an ∼ 550% increase in the overall rate of insulin secretion (1.8 ± 0.2 to 11.6 ± 1.5 pmol.kg−1 · min−1), which is achieved via an ∼ 400% increase in the mass of insulin secreted per burst (202 ± 38 to 1,003 ± 147 pmol/pulse, P < 0.001) and a ∼ 40% increase in burst frequency (8.7 ± 0.5 to 12.3 ± 0.6 pulse/h, P < 0.001). Of the insulin secreted after glucose ingestion, 68% (±4) was released in discrete secretory bursts. Further analyses showed that the incretin effect of ingested (GPO) versus infused glucose (GIV) is achieved through regulation of pulsatile insulin secretion. Glucose ingestion led to an ∼ 70% greater rate of insulin secretion than intravenous glucose delivery (10.0 ± 1.6 vs. 5.9 ± 0.9 pmol.kg−1 · min−1, P < 0.005, GPO vs. GIV). This incretin effect was achieved by the specific mechanism of an ∼ 70% greater pulse mass (930 ± 196 vs. 558 ± 97 pmol/pulse, P < 0.02, GPO vs. GIV) but with a comparable pulse frequency (13.1 ± 0.9 vs. 12.0 ± 0.5 pulses/h, P = 0.14, n = 9 dogs, GPO vs. GIV). We conclude that in vivo glucose regulates overall insulin secretion almost exclusively by amplification of the pulsatile mode of insulin secretion, and that the incretin effect is achieved by preferential enhancement of insulin secretory burst mass.
Protein Tyrosine Phosphatase 1B Interacts With the Activated Insulin Receptor
Authors: Seely B Lynn , Staubs Patricia A , Reichart Donna R , Berhanu Paulos , Milarski Kim L , Saltiel Alan R , Kusari Jyotirmoy , Olefsky Jerrold M
Published: September 2013

Protein tyrosine phosphatase 1B (PTP1B) is a protein tyrosine phosphatase of unknown function, although increasing evidence supports a role for this phosphatase in insulin action. We have investigated the interaction of PTP1B with the insulin receptor using a PTP1B glutathione S-transferase (GST) fusion protein with a point mutation in the enzyme's catalytic domain. This fusion protein is catalytically inactive, but the phosphatase's phosphotyrosine binding site is maintained. The activated insulin receptor was precipitated from purified receptor preparations and whole-cell lysates by the inactive PTP1B-GST, demonstrating a direct association between the insulin receptor and PTP1B. A p120 of unknown identity was also precipitated from whole-cell lysates by the PTP1B fusion protein, but IRS-1 (pp185) was not. A catalytically inactive [35S]PTP1B-fusion protein bound directly to immobilized insulin receptor kinase domains and was displaced in a concentration-dependent manner. Finally, tyrosine-phosphorylated PTP1B was precipitated from whole-cell lysates by an anti-insulin receptor antibody after insulin stimulation. The site of interaction between PTP1B and the insulin receptor was studied using phosphopeptides modeled after the receptor's kinase domain, the NPXY domain, and the COOH-terminal. Each phosphopeptide inhibited the PTP1B-GST:insulin receptor interaction. Study of mutant insulin receptors demonstrated that activation of the kinase domain is necessary for the PTP1B:insulin receptor interaction, but receptors with deletion of the NPXY domain or of the COOH-terminal can still bind to the PTP1B-GST. We conclude that PTP1B can associate directly with the activated insulin receptor at multiple different phosphotyrosine sites and that dephosphorylation by PTP1B may play a significant role in insulin receptor signal transduction.
Responses of Leptin to Short-Term Fasting and Refeeding in Humans: A Link With Ketogenesis but Not Ketones Themselves
Authors: Kolaczynski Jerzy W , Considine Robert V , Ohannesian Joanna , Marco Cheryl , Opentanova Irina , Nyce Mark R , Myint Michael , Caro Jasé F
Published: September 2013

We investigated the response of leptin to short-term fasting and refeeding in humans. A mild decline in subcutaneous adipocyte ob gene mRNA and a marked fall in serum leptin were observed after 36 and 60 h of fasting. The dynamics of the leptin decline and rise were further substantiated in a 6-day study consisting of a 36-h baseline period, followed by 36-h fast, and a subsequent refeeding with normal diet. Leptin began a steady decline from the baseline values after 12 h of fasting, reaching a nadir at 36 h. The subsequent restoration of normal food intake was associated with a prompt leptin rise and a return to baseline values 24 h later. When responses of leptin to fasting and refeeding were compared with that of glucose, insulin, fatty acids, and ketones, a reverse relationship between leptin and β-OH-butyrate was found. Consequently, we tested whether the reciprocal responses represented a causal relationship between leptin and β-OH-butyrate. Small amounts of infused glucose equal to the estimated contribution of gluconeogenesis, which was sufficient to prevent rise in ketogenesis, also prevented a fall in leptin. The infusion of β-OH-butyrate to produce hyperketonemia of the same magnitude as after a 36-h fast had no effect on leptin. The study indicates that one of the adaptive physiological responses to fasting is a fall in serum leptin. Although the mediator that brings about this effect remains unknown, it appears to be neither insulin nor ketones.
Troglitazone Inhibits Fatty Acid Oxidation and Esterification, and Gluconeogenesis in Isolated Hepatocytes from Starved Rats
Authors: Fulgencio Jean-Pierre , Kohl Claude , Girard Jean , Pégorier Jean-Paul
Published: September 2013

The effects of troglitazone and pioglitazone on glucose and fatty acid metabolism were studied in hepatocytes isolated from 24-h-starved rats. These thiazolidinediones inhibited long-chain fatty acid (oleate) oxidation and produced a very oxidized mitochondrial redox state. By contrast, thiazolidinediones did not affect the rate of medium-chain fatty acid (octanoate) oxidation or the activity of mitochondrial carnitine palmitoyltransferase (CPT) I. Thiazolidinediones inhibited selectively triglyceride synthesis but not phospholipid synthesis. The combined inhibition of oleate oxidation and esterification by troglitazone was due to a noncompetitive inhibition of mitochondrial and microsomal long-chain acyl-CoA synthetase (ACS) activities. It was suggested that troglitazone must be metabolized into its sulfo-conjugate derivative in liver cells to inhibit mitochondrial and microsomal ACS activities. Thiazolidinediones inhibited glucose production from lactate/pyruvate or from alanine. Analysis of gluconeogenic metabolite concentrations suggested that troglitazone would inhibit gluconeogenesis at the level of pyruvate carboxylase and glyceraldehyde-3-phosphate dehydrogenase reactions. It was concluded that 1) at a similar concentration, troglitazone was more efficient than pioglitazone to inhibit fatty acid metabolism and gluconeogenesis and 2) the inhibition of gluconeogenesis by troglitazone could be the result of the inhibition of long-chain fatty acid oxidation (decrease in acetyl-CoA, NADH-to-NAD+, and ATP-to-ADP ratios).
Plasma Leptin Levels Correlate to Islet Function Independently of Body Fat in Postmenopausal Women
Authors: Larsson Hillevi , Elmståhl Sö , Ahrén Bo
Published: September 2013

It has previously been demonstrated that plasma leptin correlates to body fat content. Increased body fat content is accompanied by low insulin sensitivity, which is compensated with increased insulin secretion. We therefore studied whether plasma levels of leptin also correlate to insulin secretion and sensitivity in humans. Therefore, we examined insulin sensitivity by the euglycemic-hyperinsulinemic clamp technique and measured the insulin response to intravenous arginine (5 g) at fasting and 14 mmol/l glucose in postmenopausal women. Percent body fat content was determined with impedance measurements. Log plasma leptin significantly correlated to percent body fat (r = 0.84, P < 0.001). In women with normal glucose tolerance (n = 36), partial correlation studies controlling for body fat content revealed significant correlations between log plasma leptin and fasting insulin levels (r = 0.39, P = 0.029), the insulin response to arginine at both glucose levels (r = 0.38 and r = 0.37, P < 0.036 for both), and the glucose potentiation of arginine-stimulated insulin secretion (r = 0.40, P = 0.025). In contrast, in women with impaired glucose tolerance (n = 17), these correlations were not significant. Plasma leptin did not correlate with insulin sensitivity independently of body fat content. To study whether the correlation between leptin and insulin would be explained by insulin stimulating leptin secretion, we examined plasma leptin during hyperinsulinemic conditions (689 ± 41 pmol/l), under both euglycemia (5.0 mmol/l, n = 10) and hypoglycemia (2.5 mmol/l, n = 7). However, under both these conditions, plasma leptin was unaltered. In conclusion, plasma leptin 1) reflects body fat content and 2) correlates to insulin secretion independently of percent body fat in postmenopausal women with normal glucose tolerance.
Chronic Dosing With Aminoguanidine and Novel Advanced Glycosylation End Product-Formation Inhibitors Ameliorates Cross-Linking of Tail Tendon Collagen in STZ-Induced Diabetic Rats
Authors: Kochakian Maral , Manjula Belur N , Egan John J
Published: September 2013

Solubility of tail tendon collagen from normal, streptozotocin-induced diabetic Lewis rats, and diabetic animals treated with aminoguanidine and two novel advanced glycosylation end products (AGE)-formation inhibitors was investigated by limited pepsin digestion under acidic conditions. Assays were conducted using tail tendon collagen from Lewis rats obtained from two different vendors, Harlan and Charles River Laboratories. Collagen solubility was assessed by following the kinetics of pepsin digestion. The data revealed that the rate of digestion for diabetic animals is markedly slow relative to that of normals. More strikingly, the kinetics of the diabetic animals showed the feature of a lag in digestion regardless of the animal source. Experiments designed to optimize the difference in solubility between normal and diabetic animals demonstrated that Charles River animals exhibit a greater window of solubility than the Harlan animals. More importantly, a pronounced effect of aminoguanidine, an AGE-formation inhibitor, was observed in Charles River animals, but not in the Harlan animals, presumably because of the larger window of solubility between the normal and the diabetic animals in the former. These data indicated that the Charles River Lewis rats are an animal model that demonstrates greater efficacy in this assay. Analysis of in vivo screens designed to test efficacy of aminoguanidine and two novel AGE-formation inhibitors, ALT 462 and ALT 486, demonstrated that monitoring an in vivo dose response is highly dependent on the enzyme concentration as well as the time of digestion, and that 1.5 h of digestion and 10 microg/ml pepsin (5 μg pepsin/mg collagen) appeared optimal. Under these conditions, a 29% normalization of solubility was observed with aminoguanidine at 100 mg/kg body wt, whereas a similar normalization was observed at 10 mg/kg body wt for both ALT 462 and ALT 486. Thus, on a molar basis, ALT 462 and ALT 486 are at least 20 times more potent than aminoguanidine. This is the first demonstration of dose-dependent efficacy for AGE-formation inhibitors in animal models, and as such, this assay provides a method with which to assess the in vivo efficacy of other such inhibitors.