bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasminogen activator inhibitor 1, transforming growth factor-beta1, and BMI are closely associated in human adipose tissue during morbid obesity.
Authors: Alessi M C , Bastelica D , Morange P , Berthet B , Leduc I , Verdier M , Geel O , Juhan-Vague I
Published: March 2007

In adipose tissue from both obese mice and humans, plasminogen activator inhibitor 1 (PAI-1) expression has been reported to be upregulated to levels of increased plasma PAI-1. This elevated expression has been shown to be partly controlled by tumor necrosis factor (TNF)-alpha in mice. In humans, increased PAI-1 expression is associated with insulin resistance characterized by visceral fat accumulation. Therefore, the aim of this study was to investigate the expression pattern of PAI-1 and TNF-alpha (antigen and mRNA) in visceral human adipose fat in comparison with subcutaneous (SC) fat. Because transforming growth factor (TGF)-beta1 is a potent inducer of PAI-1 synthesis and has been shown to influence adipocyte metabolism, this work was extended to TGF-beta1 quantification. A total of 32 obese individuals (BMI 42 +/- 6.8 kg/m2) were investigated. Freshly collected visceral adipose tissue did not exhibit a higher content of PAI-1 or TGF-beta1 than did SC tissue. Although most of the TNF-alpha values were at the detection limit of the methods, TNF-alpha antigen was 3-fold higher and TNF-alpha mRNA was 1.2-fold higher in visceral fat. The levels of tissue TGF-beta1 antigen correlated well with those of PAI-1 antigen, regardless of the fat depot studied (SC tissue: n = 21, r = 0.72, P = 0.0006; visceral tissue: n = 20, r = 0.49, P < 0.03), and they were both significantly associated with BMI. Conversely, no relationship was observed between the levels of TNF-alpha and PAI-1 or TNF-alpha and BMI. Tissue PAI-1 levels were also significantly correlated with those of circulating PAI-1. These results describe, in severe obesity, a proportional increase in tissue PAI-1 and TGF-beta1 in visceral and SC tissues. This increased PAI-1 expression could be the result of tissue cytokine disturbances, such as elevated TGF-beta1 expression.
Combined antioxidant and COMT inhibitor treatment reverses renal abnormalities in diabetic rats.
Authors: Lal M A , Körner A , Matsuo Y , Zelenin S , Cheng S X , Jaremko G , DiBona G F , Eklöf A C , Aperia A
Published: March 2007

The development and progression of diabetic nephropathy is dependent on glucose homeostasis and many other contributing factors. In the present study, we examined the effect of nitecapone, an inhibitor of the dopamine-metabolizing enzyme catechol-O-methyl transferase (COMT) and a potent antioxidant, on functional and cellular determinants of renal function in rats with streptozotocin-induced diabetes. Administration of nitecapone to diabetic rats normalized urinary sodium excretion in a manner consistent with the dopamine-dependent inhibition of proximal tubule Na,K-ATPase activity. Hyperfiltration, focal glomerulosclerosis, and albuminuria were also reversed by nitecapone, but in a manner that is more readily attributed to the antioxidant potential of the agent. A pattern of elevated oxidative stress, measured as CuZn superoxide dismutase gene expression and thiobarbituric acid-reactive substance content, was noted in diabetic rats, and both parameters were normalized by nitecapone treatment. In diabetic rats, activation of glomerular protein kinase C (PKC) was confirmed by isoform-specific translocation and Ser23 phosphorylation of the PKC substrate Na,K-ATPase. PKC-dependent changes in Na,K-ATPase phosphorylation were associated with decreased glomerular Na,K-ATPase activity. Nitecapone-treated diabetic rats were protected from these intracellular modifications. The combined results suggest that the COMT-inhibitory and antioxidant properties of nitecapone provide a protective therapy against the development of diabetic nephropathy.
Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice.
Authors: Zykova S N , Jenssen T G , Berdal M , Olsen R , Myklebust R , Seljelid R
Published: March 2007

Macrophage dysfunction is a likely mechanism underlying common diabetic complications such as increased susceptibility to infection, accelerated atherosclerosis, and disturbed wound healing. There are no available studies on the function of tissue macrophages in diabetes in humans. We have therefore studied peritoneal macrophages from diabetic type 2-like db/db mice. We found that the release of tumor necrosis factor-alpha and interleukin-1beta from lipopolysaccharide plus interferon-gamma-stimulated macrophages and vascular endothelial growth factor from both stimulated and nonstimulated macrophages was significantly reduced in diabetic animals compared with nondiabetic controls. Nitric oxide production from the stimulated db/db macrophages was significantly higher than that in the db/+ cultures, whereas there was no difference in their ability to generate reactive oxygen species. When studied both at light and electron microscopic levels, macrophages in diabetic animals had an altered morphological appearance compared with those of normal controls. We conclude that the function and morphology of the macrophages are disturbed in db/db mice and that this disturbance is related to the mechanisms underlying common inflammatory and degenerative manifestations in diabetes.
Oxygen consumption oscillates in single clonal pancreatic beta-cells (HIT).
Authors: Porterfield D M , Corkey R F , Sanger R H , Tornheim K , Smith P J , Corkey B E
Published: March 2007

Based on population studies, we have hypothesized that changes in metabolism in pancreatic beta-cells precede changes in Ca2+. It is well known from single-cell Ca2+ studies that variable oscillatory patterns in Ca2+ occur in response to glucose stimulation. The present studies, using the clonal beta-cell line HIT-T-15, were undertaken to evaluate the relationship between glucose concentration, insulin secretion, and O2 consumption and to determine the Ca2+ dependency of glucose-induced changes in O2 consumption. In population studies, an excellent correlation was found between respiration and insulin secretion, with half-maximal values at approximately 1 mmol/l glucose for both respiration and secretion. In the absence of Ca2+, glucose stimulated O2 consumption but not insulin secretion. In single clonal beta-cells, a self-referencing O2 electrode was used to assess O2 consumption. Large-amplitude oscillations were found to occur in response to stimulation by glucose and were blocked by uncoupling respiration with carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). They were also blocked and respiration totally inhibited by antimycin A, an inhibitor of complex III of the respiratory chain. Half of the cells sampled (approximately 100 total) exhibited increased oscillatory O2 consumption in response to glucose. Oscillations in O2 occurred in response to glucose even in the absence of Ca2+, and their amplitude increased further on restoration of a normal extracellular Ca2+ level. These studies indicated that oscillatory O2 consumption was not dependent on Ca2+ but that the amplitude of the O2 oscillations increased in the presence of Ca2+, possibly reflecting the additional work involved in insulin secretion and Ca2+ pumping. These studies demonstrated, for the first time, a direct correlation between O2 consumption and insulin secretion, the oscillatory nature of O2 consumption in single cells, and the feasibility of using a highly sensitive noninvasive on-line self-referencing O2 electrode to monitor single beta-cell respiration.
Coronary calcium in adults with type 1 diabetes: a stronger correlate of clinical coronary artery disease in men than in women.
Authors: Olson J C , Edmundowicz D , Becker D J , Kuller L H , Orchard T J
Published: March 2007

We studied the relationship of coronary artery calcification (CAC), a marker of coronary atherosclerosis, with prevalent clinical coronary artery disease (CAD) and established cardiovascular disease (CVD) risk factors in a type 1 diabetic population. At the 10-year follow-up examination of the Pittsburgh Epidemiology of Diabetes Complications (EDC) Study cohort, 302 adults (mean age 38.1 +/- 7.8 years) received electron beam tomography (EBT) scanning of the heart and a clinical examination. Clinical CAD was defined as a confirmed history of myocardial infarction (MI), angiographic stenosis > or =50%, Pittsburgh EDC Study physician-diagnosed angina, or ischemic electrocardiogram (ECG). CAC correlated with most CVD risk factors. CAC had 84 and 71% sensitivity for clinical CAD in men and women, respectively, and 100% sensitivity for MI or obstructive CAD. A CACS cut point of 400 was the most efficient coronary calcium correlate of CAD. In subjects with angina only, CAC sensitivity was 83% in men and 46% in women. In logistic regression, CAC, ECG R-R variation, peripheral vascular disease, and Beck Depression Inventory independently correlated with prevalent CAD in men and overall. Except for CAC, the same variables independently correlated with CAD in women, and age also entered the model. CAC was an independent correlate of MI or obstructive CAD in both sexes and was the strongest independent correlate in men, but CAC was not independently associated with angina and ischemic ECG in either sex. It is concluded that EBT-detected CAC is strongly correlated with CAD in type 1 diabetes-particularly in men.
Albuminuria in patients with type 1 diabetes is directly linked to changes in the lysosome-mediated degradation of albumin during renal passage.
Authors: Osicka T M , Houlihan C A , Chan J G , Jerums G , Comper W D
Published: March 2007

Previous studies by our group have shown that albumin is metabolized in rodents during renal passage and excreted in the urine as a mixture of intact protein and albumin-derived fragments. The aim of this study was to examine whether albumin is metabolized during renal passage in nondiabetic volunteers and in type 1 diabetic patients with varying levels of albuminuria. Nine nondiabetic normoalbuminuric volunteers and 11 type 1 diabetic patients with albumin excretion rates varying from normoalbuminuria to macroalbuminuria were studied. Each subject received an intravenous injection of tritium-labeled albumin ([3H]-albumin). Urine was collected at 4 h and 24 h after injection and analyzed by size exclusion chromatography. The amount of intact and fragmented albumin was quantified, and each fraction was analyzed by radioimmunoassay (RIA) for albumin. [3H]-albumin in nondiabetic volunteers was metabolized during renal passage to small peptide fragments not detectable by conventional RIA (only 0.05-3.8% of the total urinary radioactivity was associated with intact albumin). The process responsible for albumin fragmentation was similar in diabetic patients with normoalbuminuria (intact albumin represented 0.01-4.0% of total urinary radioactivity). However, there was a reduction in the fragmentation ratio (fragmented:intact) in diabetic patients with micro- or macroalbuminuria (intact albumin represented 2.7-55.5%, P = 0.048). This change in the fragmentation ratio was directly related to the degree of albuminuria. These results have important implications for understanding the mechanisms underlying albuminuria in nondiabetic volunteers and type 1 diabetic patients. In nondiabetic volunteers, the renal processing of albumin involves a relatively rapid and comprehensive degradation of albumin to small fragments (range 1-15 kDa). The degradation process is inhibited in diabetic nephropathy in proportion to the level of albuminuria detected by RIA.
Type 2 diabetes-like hyperglycemia in a backcross model of NZO and SJL mice: characterization of a susceptibility locus on chromosome 4 and its relation with obesity.
Authors: Plum L , Kluge R , Giesen K , Altmüller J , Ortlepp J R , Joost H G
Published: March 2007

A backcross model of New Zealand obese mice (NZO) with the lean, atherosclerosis-resistant SJL strain was established to locate genes responsible for obesity, insulin resistance, and type 2 diabetes-like hyperglycemia. In male NZO x F1 backcross mice, a major susceptibility locus for the development of hyperglycemia and hypoinsulinemia (Nidd/SJL) was identified on chromosome 4 between the markers D4Mit278 and D4Mit232, 10-28 cM distal of the previously described Nidd1 locus. The diabetogenic allele has presumably been contributed by the SJL genome, and it appeared to be responsible for approximately 60% of the total prevalence of hyperglycemia. The presence of Nidd/SJL did not alter body weight or weight gain by week 12. Thereafter, it was associated with reduced weight gain or weight loss, presumably as a consequence of decompensated hyperglycemia. In all male backcross mice, the prevalence of hyperglycemia at week 22 increased with the body weight at week 12, suggesting that the development of hyperglycemia was dependent on the degree of obesity. In the absence of Nidd/SJL, mice weighing <50 g at week 12 did not develop hyperglycemia by week 22. In contrast, in animals carrying the diabetogenic allele, the prevalence of hyperglycemia was 20 and 64% when the 12-week weight was <45 and 45-50 g, respectively. These data are consistent with the conclusion that Nidd/SJL represents a diabetes gene that lowers the obesity threshold for the development of hyperglycemia and hypoinsulinemia.
Nonsense mutation of islet-1 gene (Q310X) found in a type 2 diabetic patient with a strong family history.
Authors: Shimomura H , Sanke T , Hanabusa T , Tsunoda K , Furuta H , Nanjo K
Published: March 2007

Islet-1 (Isl-1) is one of the transcription factors that play an important role for the formation of the islet cells. We scanned the Isl-1 gene in 77 Japanese type 2 diabetic patients with a family history and found a heterozygous nonsense mutation (Q310X) in 1 diabetic patient. The mutation was not found in 180 nondiabetic subjects. This mutation is located in the putative transactivation domain and deletes 40 amino acids of the COOH-terminal lesion. The Q310X mutant exhibited a 50% reduction in activity compared with the wild-type when tested for stimulation of transcription of a human amylin promoter-linked luciferase reporter gene in betaTC3 cells. The patient was a 49-year-old nonobese man who was diagnosed as having type 2 diabetes at 32 years of age and has been treated with sulfonylureas. The mutation was found in his mother, who has type 2 diabetes, and in his 14-year-old daughter, who has normal glucose tolerance but a relatively low insulin response. This is the first reported finding of Isl-1 gene mutation in type 2 diabetes. Although Isl-1 is not a common predisposing gene for Japanese type 2 diabetes, the mutation in this gene may be a rare cause of diabetes in isolated families.
Heterozygous expansion of the GAA tract of the X25/frataxin gene is associated with insulin resistance in humans.
Authors: Hebinck J , Hardt C , Schöls L , Vorgerd M , Briedigkeit L , Kahn C R , Ristow M
Published: March 2007

Friedreich's ataxia (FA) is an autosomal recessive disease that has been attributed to a GAA triplet repeat expansion in the first intron of the X25/frataxin gene. Impaired glucose tolerance is present in up to 39% of FA patients, and clinically apparent diabetes is seen in approximately 18% of the affected individuals. Subjects carrying the X25/frataxin GAA repeat in a heterozygous state do not develop FA and, therefore, represent an ideal model to study the underlying metabolic defects that contribute to the diabetes associated with this disorder. In the present study, we have compared 11 first-degree relatives of FA patients (i.e., parents or heterozygous siblings of FA patients) with matched normal control subjects to study the parameters of glucose metabolism. An oral glucose tolerance test revealed diabetes in one of the heterozygous subjects who was excluded from further analyses. Using an octreotide-based quantification of insulin sensitivity, 8 of the remaining 10 study subjects showed pronounced insulin resistance, reflecting a significant difference from the control group (P = 0.001). In conclusion, a heterozygous expansion of the X25/frataxin GAA repeat in healthy individuals is associated with insulin resistance and might be considered a genetic co-factor in the pathogenesis of mitochondrial subtypes of diabetes.
The K121Q variant of the human PC-1 gene is not associated with insulin resistance or type 2 diabetes among Danish Caucasians.
Authors: Rasmussen S K , Urhammer S A , Pizzuti A , Echwald S M , Ekstrøm C T , Hansen L , Hansen T , Borch-Johnsen K , Frittitta L , Trischitta V , Pedersen O
Published: March 2007

The human plasma-cell membrane differentiation antigen-1 (PC-1) has been shown to inhibit insulin receptor tyrosine kinase activity. Recently, a K121Q polymorphism in the human PC-1 gene was found in a Sicilian population and was shown to be strongly associated with insulin resistance. The objectives of the present investigation were to examine in the Danish Caucasian population whether the K121Q variant was associated with type 2 diabetes or, in glucose-tolerant subjects, with impaired whole-body insulin sensitivity. We genotyped 404 Danish type 2 diabetic patients and found that the allele frequency of the variant was 0.14 (95% CI 0.12-0.16), whereas the allele frequency was 0.16 (95% CI 0.13-0.19) among 237 matched glucose-tolerant control subjects (P = 0.6). In the control subjects, there were no significant differences among wild-type, heterozygous, or homozygous subjects in regard to 1) serum insulin and plasma glucose levels at fasting, 60 min, or 120 min during an oral glucose tolerance test (OGTT) or 2) the estimates of insulin resistance obtained from the homeostasis model assessment (HOMA). Furthermore, we investigated the impact of the variant in 2 other Danish population samples that comprised 356 young healthy subjects and 226 glucose-tolerant offspring of type 2 diabetic probands, respectively. In all of the study populations, the polymorphism was not associated with an altered insulin sensitivity index as estimated from an intravenous glucose tolerance test in combination with an intravenous injection of tolbutamide. In addition, among the 226 offspring, the variations in serum insulin and serum C-peptide responses measured during an OGTT were not related to the PC-1 genotype. In conclusion, the K121Q polymorphism of the human PC-1 gene is not associated with type 2 diabetes or insulin resistance among Danish Caucasians.
Glucose Sensing in Pancreatic β-Cells
Authors: Schuit Frans C. , Huypens Peter , Heimberg Harry , Pipeleers Daniel G.
Published: March 2007

Nutrient homeostasis is known to be regulated by pancreatic islet tissue. The function of islet β-cells is controlled by a glucose sensor that operates at physiological glucose concentrations and acts in synergy with signals that integrate messages originating from hypothalamic neurons and endocrine cells in gut and pancreas. Evidence exists that the extrapancreatic cells producing and secreting these (neuro)endocrine signals also exhibit a glucose sensor and an ability to integrate nutrient and (neuro)hormonal messages. Similarities in these cellular and molecular pathways provide a basis for a network of coordinated functions between distant cell groups, which is necessary for an appropriate control of nutrient homeostasis. The glucose sensor seems to be a fundamental component of these control mechanisms. Its molecular characterization is most advanced in pancreatic β-cells, with important roles for glucokinase and mitochondrial oxidative fluxes in the regulation of ATP-sensitive K+ channels. Other glucose-sensitive cells in the endocrine pancreas, hypothalamus, and gut were found to share some of these molecular characteristics. We propose that similar metabolic signaling pathways influence the function of pancreatic α-cells, hypothalamic neurons, and gastrointestinal endocrine and neural cells.
Effects of Tungstate, a New Potential Oral Antidiabetic Agent, in Zucker Diabetic Fatty Rats
Authors: Muñoz María Carmen , Barberà Albert , Domínguez Jorge , Fernàndez-Alvarez Josefa , Gomis Ramon , Guinovart Joan J.
Published: March 2007

Tungstate was orally administered to 7.5-week-old male Zucker diabetic fatty (ZDF) rats that already showed moderate hyperglycemia (180 ± 16 mg/dl). The animals became normoglycemic for ∼10 days. Then, glycemia started to rise again, although it did not reach the initial values until day 24, when levels stabilized at ∼200 mg/dl for the duration of the experiment. Untreated ZDF rats showed steadily increased blood glucose levels between 7.5 and 10 weeks of age, when they reached a maximum value of 450 ± 19 mg/dl, which was maintained throughout the experiment. In addition, tolerance to intraperitoneal glucose load improved in treated diabetic rats. Serum levels of triglycerides were elevated in untreated diabetic rats compared with their lean counterparts (ZLC). In the liver of diabetic animals, glucokinase (GK), glycogen phosphorylase a (GPa), liverpyruvate kinase (L-PK), and fatty acid synthase (FAS) activities decreased by 81, 30, 54, and 35%, respectively, whereas phosphoenolpyruvate carboxykinase (PEPCK) levels increased by 240%. Intracellular glucose-6-phosphate (G6P) decreased by 40%, whereas glycogen levels remained unaffected. Tungstate treatment of these rats induced a 42% decrease in serum levels of triglycerides and normalized hepatic G6P concentrations, GPa activity, and PEPCK levels. GK activity in treated diabetic rats increased to 50% of the values of untreated ZLC rats. L-PK and FAS activity increased to higher values than those in untreated lean rats (1.7-fold L-PK and 2.4-fold FAS). Hepatic glycogen levels were 55% higher than those in untreated diabetic and healthy rats. Tungstate treatment did not significantly change the phosphotyrosine protein profile of primary cultured hepatocytes from diabetic animals. These data suggest that tungstate administration to ZDF rats causes a considerable reduction of glycemia, mainly through a partial restoration of hepatic glucose metabolism and a decrease in lipotoxicity.
Expression and Role of Bcl-2 in Rat Blastocysts Exposed to High D-Glucose
Authors: Pampfer Serge , Cordi Sabine , Vanderheyden Ivo , Van Der Smissen Patrick , Courtoy Pierre J. , Van Cauwenberge Anne , Alexandre Henri , Donnay Isabelle , De Hertogh René
Published: March 2007

Bcl-2 mRNA expression was detected in rat blastocysts by in situ hybridization. The distribution of mRNA expression was rather heterogenous, with ∼2% of high-expressing cells. In vitro exposure to 28 mmol/l D-glucose for 24 h resulted in a significant increase in the proportion of these cells compared with control embryos in either 6 mmol/l D-glucose or 28 mmol/l D+L-glucose. Heterogeneity in the expression of Bcl-2 was also observed at the protein level by immunocytochemistry. Exposure to 28 mmol/l D-glucose significantly increased the incidence of chromatin degradation (karyolysis) and nuclear fragmentation (karyorhexis), two nuclear markers of apoptosis in rat blastocysts. When two different antisense oligodeoxynucleotides designed to block Bcl-2 expression were added to 28 mmol/l D-glucose, the incidence of karyolysis (but not karyorhexis) was increased compared with embryos in 28 mmol/l D-glucose alone. These data suggest that Bcl-2 is involved in the protective response against the induction of karyolysis in blastocysts on their exposure to high concentrations of D-glucose in vitro, whereas karyorhexis appears to result from the activation of an intracellular pathway that is independent of Bcl-2.
IGF-I mRNA and Signaling in the Diabetic Retina
Authors: Gerhardinger Chiara , McClure Kimberly D. , Romeo Giulio , Podestà Francesca , Lorenzi Mara
Published: March 2007

IGF-I promotes the survival of multiple cell types by activating the IGF-I receptor (IGF-IR), which signals downstream to a serine/threonine kinase termed Akt. Because in diabetes vascular and neural cells of the retina undergo accelerated apoptosis, we examined IGF-I synthesis and signaling in the human and rat diabetic retina. In retinas obtained postmortem from six donors aged 64 ± 8 years with a diabetes duration of 7 ± 5 years, IGF-I mRNA levels were threefold lower than in the retinas of six age-matched nondiabetic donors (P = 0.005). In the retinas of rats with 2 months' duration of streptozotocin-induced diabetes, IGF-I mRNA levels were similar to those of control rats, but after 5 months of diabetes they failed to increase to the levels recorded in age-matched controls (P < 0.02). Retinal IGF-I expression was not altered by hypophysectomy, proving to be growth-hormone independent. IGF-IR levels were modestly increased in the human diabetic retinas (P = 0.02 vs. nondiabetic retinas) and were unchanged in the diabetic rats. Phosphorylation of the IGF-IR could be measured only in the rat retina, and was not decreased in the diabetic rats (94 ± 18% of control values). In the same diabetic rats, phosphorylation of Akt was 123 ± 21% of control values. There was not yet evidence of increased apoptosis of retinal microvascular cells after 5 months of streptozotocin-induced diabetes. Hence, in the retina of diabetic rats, as in the retina of diabetic human donors, IGF-I mRNA levels are substantially lower than in age-matched nondiabetic controls, whereas IGF-IR activation and signaling are not affected, at least for some time. This finding suggests that in the diabetic retina, the activation of the IGF-IR is modulated by influences that compensate for, or are compensated by, decreased IGF-I synthesis.
Effect of Oral Creatine Supplementation on Human Muscle GLUT4 Protein Content After Immobilization
Authors: Eijnde B. Op 't , Ursø B. , Richter E.A. , Greenhaff P.L. , Hespel P.
Published: March 2007

The purpose of this study was to investigate the effect of oral creatine supplementation on muscle GLUT4 protein content and total creatine and glycogen content during muscle disuse and subsequent training. A double-blind placebo-controlled trial was performed with 22 young healthy volunteers. The right leg of each subject was immobilized using a cast for 2 weeks, after which subjects participated in a 10-week heavy resistance training program involving the knee-extensor muscles (three sessions per week). Half of the subjects received creatine monohydrate supplements (20 g daily during the immobilization period and 15 and 5 g daily during the first 3 and the last 7 weeks of rehabilitation training, respectively), whereas the other 11 subjects ingested placebo (maltodextrine). Muscle GLUT4 protein content and glycogen and total creatine concentrations were assayed in needle biopsy samples from the vastus lateralis muscle before and after immobilization and after 3 and 10 weeks of training. Immobilization decreased GLUT4 in the placebo group (-20%, P < 0.05), but not in the creatine group (+9% NS). Glycogen and total creatine were unchanged in both groups during the immobilization period. In the placebo group, during training, GLUT4 was normalized, and glycogen and total creatine were stable. Conversely, in the creatine group, GLUT4 increased by ∼40% (P < 0.05) during rehabilitation. Muscle glycogen and total creatine levels were higher in the creatine group after 3 weeks of rehabilitation (P < 0.05), but not after 10 weeks of rehabilitation. We concluded that 1) oral creatine supplementation offsets the decline in muscle GLUT4 protein content that occurs during immobilization, and 2) oral creatine supplementation increases GLUT4 protein content during subsequent rehabilitation training in healthy subjects.
Amplitude Modulation of Pulsatile Insulin Secretion by Intrapancreatic Ganglion Neurons
Authors: Sha Lei , Westerlund Johanna , Szurszewski Joseph H. , Bergsten Peter
Published: March 2007

Neuron activity and insulin release were measured simultaneously from 33 preparations of intrapancreatic canine ganglia and pancreatic parenchyma adjacent to the ganglia. The electrical activity of single neurons of the ganglia was recorded with intracellular microelectrodes, and insulin release from the attached islets was determined with an enzyme-linked immunosorbent assay. Insulin release was 62 ± 18 fmol preparation/min in the presence of 10 mmol/l glucose and pulsatile (3.7 ± 0.4 min/pulse). Corresponding measurements of neuronal electrical activity showed a stable membrane potential of -53.5 ± 0.6 mV. Short, high-frequency (20 Hz) preganglionic nerve stimulation evoked action potentials and, in 46% of the preparations, a threefold rise in the insulin secretory rate associated with increased amplitude of the insulin pulses. The effects were blocked by 10 μmol/l tetrodotoxin (TTX). In other preparations, continuous low-frequency (0.05-0.5 Hz) preganglionic nerve stimulation evoked action potentials and, in 50% of the preparations, a gradual increase of insulin release associated with augmentation of insulin pulse amplitude without alteration of the duration. The effects were blocked by 50 μmol/l hexamethonium (HEX). In the remaining preparations, no change in insulin release was observed during nerve stimulation. In the absence of stimulation, neither TTX nor HEX affected the membrane potential or insulin secretion. These first simultaneous measurements of intrapancreatic ganglion activity and insulin secretion are consistent with amplitude modulation of pulsatile insulin secretion induced by changes in electrical activity in a population of intrapancreatic ganglion neurons.
Distinct Effects of Saturated and Monounsaturated Fatty Acids on β-Cell Turnover and Function
Authors: Maedler K. , Spinas G.A. , Dyntar D. , Moritz W. , Kaiser N. , Donath Marc Y.
Published: March 2007

Glucotoxicity and lipotoxicity contribute to the impaired β-cell function observed in type 2 diabetes. Here we examine the effect of saturated and unsaturated fatty acids at different glucose concentrations on β-cell proliferation and apoptosis. Adult rat pancreatic islets were cultured onto plates coated with extracellular matrix derived from bovine corneal endothelial cells. Exposure of islets to saturated fatty acid (0.5 mmol/l palmitic acid) in medium containing 5.5, 11.1, or 33.3 mmol/l glucose for 4 days resulted in a five- to ninefold increase of β-cell DNA fragmentation. In contrast, monounsaturated palmitoleic acid alone (0.5 mmol/l) or in combination with palmitic acid (0.25 or 0.5 mmol/l each) did not affect DNA fragmentation. Increasing concentrations of glucose promoted β-cell proliferation that was dramatically reduced by palmitic acid. Palmitoleic acid enhanced the proliferation activity in medium containing 5.5 mmol/l glucose but had no additional effect at higher glucose concentrations (11.1 and 33.3 mmol/l). The cell-permeable ceramide analog C2-ceramide mimicked both the palmitic acid—induced β-cell apoptosis and decrease in proliferation. Moreover, the ceramide synthetase inhibitor fumonisin B1 blocked the deleterious effects of palmitic acid on β-cell viability. Additionally, palmitic acid but not palmitoleic acid decreased the expression of the mitochondrial adenine nucleotide translocator and induced release of cytochrome c from the mitochondria into the cytosol. Finally, palmitoleic acid improved β-cell—secretory function that was reduced by palmitic acid. Taken together, these results suggest that the lipotoxic effect of the saturated palmitic acid involves an increased apoptosis rate coupled with reduced proliferation capacity of β-cells and impaired insulin secretion. The deleterious effect of palmitate on β-cell turnover is mediated via formation of ceramide and activation of the apoptotic mitochondrial pathway. In contrast, the monounsaturated palmitoleic acid does not affect β-cell apoptosis, yet it promotes β-cell proliferation at low glucose concentrations, counteracting the negative effects of palmitic acid as well as improving β-cell function.
Decreased In Situ Insulin Receptor Dephosphorylation in Hyperglycemia-Induced Insulin Resistance in Rat Adipocytes
Authors: Tang Shangguo , Le-Tien Hoang , Goldstein Barry J. , Shin Phillip , Lai Robert , Fantus I. George
Published: March 2007

The regulation of insulin receptor (IR) tyrosine (tyr) phosphorylation is a key step in the control of insulin signaling. Augmented IR tyr dephosphorylation by protein tyrosine phosphatases (PTPs) may contribute to insulin resistance. To investigate this possibility in hyperglycemia-induced insulin resistance, primary cultured rat adipocytes were rendered insulin-resistant by chronic exposure (18 h) to 15 mmol/l glucose combined with 10-7 mol/l insulin. Insulin-resistant adipocytes showed a decrease in insulin sensitivity and a maximum response of 2-deoxyglucose uptake, which was associated with a decrease in maximum insulin-stimulated IR tyr phosphorylation in situ. To assess tyr dephosphorylation, IRs of insulin-stimulated permeabilized adipocytes were labeled with [γ-32P]ATP and chased for 2 min with unlabeled ATP in the presence of EDTA. In a nonradioactive protocol, insulin-stimulated adipocytes were permeabilized and exposed to EDTA and erbstatin for 2 min, and IRs were immunoblotted with anti-phosphotyrosine (pY) antibodies. Both methods showed a similar diminished extent of IR tyr dephosphorylation in resistant cells. Immunoblotting of four candidate IR-PTPs demonstrated no change in PTP1B or the SH2 domain containing phosphatase-2 (SHP-2), whereas a significant decrease in leukocyte antigen-related phosphatase (LAR) (51 ± 3% of control) and an increase in PTP-α (165 ± 16%) were found. Activity of immunoprecipitated PTPs toward a triple tyr phosphorylated IR peptide revealed a correlation with protein content for PTP1B, SHP-2, and LAR but a decrease in apparent specific activity of PTP-α. The data indicate that decreased IR tyr phosphorylation in hyperglycemia-induced insulin resistance is not due to enhanced dephosphorylation. The diminished IR tyr dephosphorylation observed in this model is associated with decreased LAR protein content and activity.
The Protein-Retaining Effects of Growth Hormone During Fasting Involve Inhibition of Muscle-Protein Breakdown
Authors: Nørrelund Helene , Nair K. Sreekumaran , Jørgensen Jens Otto Lunde , Christiansen Jens Sandahl , Møller Niels
Published: March 2007

The metabolic response to fasting involves a series of hormonal and metabolic adaptations leading to protein conservation. An increase in the serum level of growth hormone (GH) during fasting has been well substantiated. The present study was designed to test the hypothesis that GH may be a principal mediator of protein conservation during fasting and to assess the underlying mechanisms. Eight normal subjects were examined on four occasions: 1) in the basal postabsorptive state (basal), 2) after 40 h of fasting (fast), 3) after 40 h of fasting with somatostatin suppression of GH (fast-GH), and 4) after 40 h of fasting with suppression of GH and exogenous GH replacement (fast+GH). The two somatostatin experiments were identical in terms of hormone replacement (except for GH), meaning that somatostatin, insulin, glucagon and GH were administered for 28 h; during the last 4 h, substrate metabolism was investigated. Compared with the GH administration protocol, IGF-I and free IGF-I decreased 35 and 70%, respectively, during fasting without GH. Urinary urea excretion and serum urea increased when participants fasted without GH (urea excretion: basal 392 ± 44, fast 440 ± 32, fast-GH 609 ± 76, and fast+GH 408 ± 36 mmol/24 h, P < 0.05; serum urea: basal 4.6 ± 0.1, fast 6.2 ± 0.1, fast-GH 7.0 ± 0.2, and fast+GH 4.3 ± 0.2 mmol/l, P < 0.01). There was a net release of phenylalanine across the forearm, and the negative phenylalanine balance was higher during fasting with GH suppression (balance: basal 9 ± 3, fast 15 ± 6, fast-GH 17 ± 4, and fast+GH 11 ± 5 nmol/min, P < 0.05). Muscle-protein breakdown was increased among participants who fasted without GH (phenylalanine rate of appearance: basal 17 ± 4, fast 26 ± 9, fast-GH 33 ± 7, fast+GH 25 ± 6 nmol/min, P < 0.05). Levels of free fatty acids and oxidation of lipid decreased during fasting without GH (P < 0.01). In summary, we find that suppression of GH during fasting leads to a 50% increase in urea-nitrogen excretion, together with an increased net release and appearance rate of phenylalanine across the forearm. These results demonstrate that GH—possibly by maintenance of circulating concentrations of free IGF-I—is a decisive component of protein conservation during fasting and provide evidence that the underlying mechanism involves a decrease in muscle protein breakdown.
Inhibition of Cytokine-Induced NF-κB Activation by Adenovirus-Mediated Expression of a NF-κB Super-Repressor Prevents β-Cell Apoptosis
Authors: Heimberg Harry , Heremans Yves , Jobin Christian , Leemans Ruth , Cardozo Alessandra K. , Darville Martine , Eizirik Décio L.
Published: March 2007

Cytokine-induced β-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-κB (NF-κB) is activated by interleukin-1β (IL-1β), and its activity promotes the expression of several β-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1β + γ-interferon [IFN-γ])-induced expression of NF-κB in β-cell apoptosis, rat β-cells were infected with the recombinant adenovirus AdIκB(SA)2, which contained a nondegradable mutant form of inhibitory κB (IκB(SA)2, with S32A and S36A) that locks NF-κB in a cytosolic protein complex, preventing its nuclear action. Expression of IκB(SA)2 inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-κB. Cytokine-induced gene expression of several NF-κB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIκB(SA)2, as established by reverse transcriptase–polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, β-cell survival after IL-1β + IFN-γ treatment was significantly improved by IκB(SA)2 expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-κB activation, under in vitro conditions, has primarily a pro-apoptotic function in β-cells.
The Expression of Hormone-Sensitive Lipase in Clonal β-Cells and Rat Islets Is Induced by Long-Term Exposure to High Glucose
Authors: Winzell Maria Sörhede , Svensson Håkan , Arner Peter , Ahrén Bo , Holm Cecilia
Published: March 2007

Hormone-sensitive lipase (HSL) is expressed and enzymatically active in β-cells and has been proposed to be involved in the generation of the lipid-derived signal that seems to be necessary for glucose-stimulated insulin secretion. In this study, we investigated whether the expression of HSL in INS-1 cells and in rat islets is affected by exposure to high glucose concentrations. Incubation of INS-1 cells in 25 mmol/l glucose for 16 and 32 h induced HSL protein expression twofold, whereas no effect was observed after 4 and 8 h of incubation. The HSL activity, defined as the diglyceride lipase activity inhibited by anti-rat HSL antibodies, constituted ∼25% of total diglyceride lipase activity and was induced to a similar extent as HSL protein levels. The glucose effect at 16 h on HSL protein expression level was confirmed in freshly isolated rat islets. Exposure of INS-1 cells to different glucose concentrations for 16 h showed that the inductive effect on HSL protein levels was maximum at 20 mmol/l glucose (2- to 2.5-fold). Northern blot analysis demonstrated a more than threefold elevation of HSL mRNA levels. The induction was blocked by actinomycin D, and the half-life of the transcript seemed to be unchanged by high glucose, suggesting a transcriptional nature of the glucose effect on HSL gene expression. The nonmetabolizable glucose analog 2-deoxyglucose, which has no mitogenic effect, induced HSL ∼1.3-fold, whereas mannose was similar to glucose, stimulating HSL expression 1.7- to 2-fold. The results suggest that HSL is involved in the β-cell responses to hyperglycemia and also in generating the lipid signal that is needed in stimulus-secretion coupling.
Noninvasive In Vivo Measurement of β-Cell Mass in Mouse Model of Diabetes
Authors: Moore Anna , Bonner-Weir Susan , Weissleder Ralph
Published: March 2007

Pancreatic β-cell mass (BCM) is a major determinant of the quantity of insulin that can be secreted. BCM is markedly reduced in type 1 diabetes because of selective autoimmune destruction of β-cells. Accurate assessment of BCM in human diabetes is limited to autopsy studies, which usually suffer from inadequate clinical information; thus, the development of noninvasive means of BCM measurement could be important in intervention therapy. The goal of this study was to develop such noninvasive methods for measuring BCM featuring target-specific imaging probes and to investigate whether this technique is feasible, accurate, and predictive of BCM in normal and diabetic states. Using a β-cell–specific monoclonal antibody IC2, modified with a radioisotope chelator for nuclear imaging, we showed that highly specific binding and accumulation to β-cells occurs after intravenous administration of the probe, with virtually no binding to exocrine pancreas or stromal tissues. Furthermore, we observed a direct correlation between accumulation of the probe with BCM in diabetic and normal animals. Nuclear imaging of the animals that received an injection of the radioactive probe showed major difference in signal intensity between normal and diabetic pancreases. The results from this study set the route for further development of imaging probes for measuring BCM that would aid in diagnosis and treatment of diabetic patients in the clinic.
Protein Kinase Cζ Activation Mediates Glucagon-Like Peptide-1–Induced Pancreatic β-Cell Proliferation
Authors: Buteau Jean , Foisy Sylvain , Rhodes Christopher J. , Carpenter Lee , Biden Trevor J. , Prentki Marc
Published: March 2007

Glucagon-like peptide-1 (GLP-1), an insulinotropic and glucoincretin hormone, is a potentially important therapeutic agent in the treatment of diabetes. We previously provided evidence that GLP-1 induces pancreatic β-cell growth nonadditively with glucose in a phosphatidylinositol-3 kinase (PI-3K)–dependent manner. In the present study, we investigated the downstream effectors of PI-3K to determine the precise signal transduction pathways that mediate the action of GLP-1 on β-cell proliferation. GLP-1 increased extracellular signal-related kinase 1/2, p38 mitogen-activated protein kinase (MAPK), and protein kinase B activities nonadditively with glucose in pancreatic β(INS 832/13) cells. GLP-1 also caused nuclear translocation of the atypical protein kinase C (aPKC) ζ isoform in INS as well as in dissociated normal rat β-cells as shown by immunolocalization and Western immunoblotting analysis. Tritiated thymidine incorporation measurements showed that the p38 MAPK inhibitor SB203580 suppressed GLP-1–induced β-cell proliferation. Further investigation was performed using isoform-specific pseudosubstrates of classical (α, β, and γ) or ζ aPKC isoforms. The PKCζ pseudosubstrate suppressed the proliferative action of GLP-1, whereas the inhibitor of classical PKC isoforms had no effect. Overexpression of a kinase-dead PKCζ acting as a dominant negative protein suppressed GLP-1–induced proliferation. In addition, ectopic expression of a constitutively active PKCζ mutant stimulated tritiated thymidine incorporation to the same extent as GLP-1, and the glucoincretin had no growth-promoting action under this condition. The data indicate that GLP-1–induced activation of PKCζ is implicated in the β-cell proliferative signal of the insulinotropic hormone. The results are consistent with a model in which GLP-1–induced PI-3K activation results in PKCζ translocation to the nucleus, which may play a role in the pleiotropic effects (DNA synthesis, metabolic enzymes, and insulin gene expression) of the glucoincretin.
Phosphorylation-Dependent Nucleocytoplasmic Shuttling of Pancreatic Duodenal Homeobox-1
Authors: Elrick Lucy J. , Docherty Kevin
Published: March 2007

Pancreatic duodenal homeobox-1 (PDX-1) is a homeodomain protein that plays an important role in the development of the pancreas and in maintaining the identity and function of the islets of Langerhans. It also regulates the expression of the insulin gene in response to changes in glucose and insulin concentrations. Glucose and insulin regulate PDX-1 by way of a signaling pathway involving phosphatidylinositol 3-kinase (PI 3-kinase) and SAPK2/p38. Activation of this pathway leads to phosphorylation of PDX-1 and its movement into the nucleus. To investigate the intracellular trafficking of PDX-1, immunocytochemistry was used to localize PDX-1 in the human β-cell line NesPDX-1, in which PDX-1 is overexpressed, and in MIN6 β-cells. In low-glucose conditions, PDX-1 localized predominantly to the nuclear periphery, with some staining in the cytoplasm. After stimulation with glucose, PDX-1 was present in the nucleoplasm. The translocation of PDX-1 to the nucleoplasm was complete within 15 min and occurred in 5−10 mmol/l glucose. Insulin and sodium arsenite, an activator of the stress-activated pathway, also stimulated PDX-1 movement from the nuclear periphery to the nucleoplasm. When cells were transferred between high glucose− and low glucose−containing medium, PDX-1 rapidly shuttled between the nuclear periphery and the nucleoplasm. Glucose- and insulin-stimulated translocation of PDX-1 to the nucleoplasm was inhibited by wortmannin and SB 203580, indicating that a pathway involving PI 3-kinase and SAPK2/p38 was involved; translocation was unaffected by PD 098959 and rapamycin, suggesting that neither mitogen-activated protein kinase nor p70s6k were involved. Arsenite-stimulated import of PDX-1 into the nucleus was inhibited by SB 203580 but not by wortmannin. Export from the nucleoplasm to the nuclear periphery was inhibited by calyculin A and okadaic acid, suggesting that dephosphorylation of PDX-1 was involved. These results demonstrated that PDX-1 shuttles between the nuclear periphery and nucleoplasm in response to changes in glucose and insulin concentrations and that these events are dependent on PI 3-kinase, SAPK2/p38, and a nuclear phosphatase(s).
Inducible and Reversible β-Cell Autoimmunity and Hyperplasia in Transgenic Mice Expressing a Conditional Oncogene
Authors: Berkovich Irina , Efrat Shimon
Published: March 2007

Expression of the SV40 T antigen (Tag) in pancreatic β-cells in transgenic mice has been shown to induce β-cell tumorigenesis. We generated transgenic mice in which Tag expression is inducible and reversible by the tet-on gene regulation system. These mice develop β-cell tumors only when treated with the inducer doxycycline (dox). Tag expression in vivo is reversible upon dox withdrawal. As a result, β-cell proliferation is greatly reduced, indicating that genetic changes, which may occur in the transformed cells, do not allow Tag-independent proliferation. Induction of Tag expression after immune recognition of self-antigens has been established triggers an autoimmune response against β-cells, as evidenced by insulitis. Shut-off of Tag expression results in elimination of insulitis, suggesting that this process depends on continuous expression of the target antigen. In addition, the reversibility of autoimmunity suggests that β-cell damage caused by the anti-Tag immune response does not elicit secondary responses to other newly exposed β-cell antigens, which would have persisted after Tag elimination. β-Cell proliferation in this model is accompanied by cell apoptosis. Apoptosis persisted for several weeks in the islets after dox removal. In close to 40% of the mice analyzed, this process reduced the islet size back to normal, suggesting the existence of a homeostatic mechanism that maintains β-cell mass within the normal range.