bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and Functional Characterization of Recombinant GAD65 Autoantibodies Derived by IgG Repertoire Cloning From Patients With Type 1 Diabetes
Authors: Jury Karin , Söhnlein Petra , Vogel Monique , Richter Wiltrud
Published: March 2007

The generation of human monoclonal autoantibodies is critical for understanding humoral immune responses in autoimmunity. In this study, we isolated the first human recombinant antibodies to glutamate decarboxylase (rGAD65ab) by IgG repertoire cloning, phage display of Fab fragments, and biopanning from two patients at onset of type 1 diabetes. We demonstrate that natural Ig heavy- and light-chain pairings of autoantibodies can be isolated by the recombinant approach and have a major selection advantage over other rGAD65ab. Among eight rGAD65ab, three (rGAD65ab A–C) displayed all functional and structural properties of known disease-related GAD65ab, including reactivity in the enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), islet cell antibody (ICA) test, and variable gene usage. Dominant epitope recognition was directed to the previously defined epitope EP-1 in the middle of GAD65, corroborating its immunodominance in the molecule. New features, such as assay-dependent GAD65 reactivity and new epitope recognition, were observed in two rGAD65ab (D and E). These antibodies were positive in the GAD65 ELISA and ICA test but not in the GAD65 RIA, providing the first examples for ICA with incongruent results in solid-phase and fluid-phase assays. In conclusion, phage display–derived antibodies reflected well the natural autoantibody response in type 1 diabetes and may allow further characterization of assay-dependent features of GAD65ab and the recognition of epitopes in solid- but not fluid-phase assays.
Decrease in β-Cell Mass Leads to Impaired Pulsatile Insulin Secretion, Reduced Postprandial Hepatic Insulin Clearance, and Relative Hyperglucagonemia in the Minipig
Authors: Kjems Lise L. , Kirby Barbara M. , Welsh Elizabeth M. , Veldhuis Johannes D. , Straume Marty , McIntyre Susan S. , Yang Dongchang , Lefèbvre Pierre , Butler Peter C.
Published: March 2007

Most insulin is secreted in discrete pulses at an interval of ∼6 min. Increased insulin secretion after meal ingestion is achieved through the mechanism of amplification of the burst mass. Conversely, in type 2 diabetes, insulin secretion is impaired as a consequence of decreased insulin pulse mass. β-cell mass is reported to be deficient in type 2 diabetes. We tested the hypothesis that decreased β-cell mass leads to decreased insulin pulse mass. Insulin secretion was examined before and after an ∼60% decrease in β-cell mass achieved by a single injection of alloxan in a porcine model. Alloxan injection resulted in stable diabetes (fasting plasma glucose 7.4 ± 1.1 vs. 4.4 ± 0.1 mmol/l; P < 0.01) with impaired insulin secretion in the fasting and fed states and during a hyperglycemic clamp (decreased by 54, 80, and 90%, respectively). Deconvolution analysis revealed a selective decrease in insulin pulse mass (by 54, 60, and 90%) with no change in pulse frequency. Rhythm analysis revealed no change in the periodicity of regular oscillations after alloxan administration in the fasting state but was unable to detect stable rhythms reliably after enteric or intravenous glucose stimulation. After alloxan administration, insulin secretion and insulin pulse mass (but not insulin pulse interval) decreased in relation to β-cell mass. However, the decreased pulse mass (and pulse amplitude delivered to the liver) was associated with a decrease in hepatic insulin clearance, which partially offset the decreased insulin secretion. Despite hyperglycemia, postprandial glucagon concentrations were increased after alloxan administration (103.4 ± 6.3 vs. 92.2 ± 2.5 pg/ml; P < 0.01). We conclude that an alloxan-induced selective decrease in β-cell mass leads to deficient insulin secretion by attenuating insulin pulse mass, and that the latter is associated with decreased hepatic insulin clearance and relative hyperglucagonemia, thereby emulating the pattern of islet dysfunction observed in type 2 diabetes.
Calpains Play a Role in Insulin Secretion and Action
Authors: Sreenan Seamus K. , Zhou Yun-Ping , Otani Kenichi , Hansen Polly A. , Currie Kevin P.M. , Pan Chien-Yuan , Lee Jean-Pyo , Ostrega Diane M. , Pugh William , Horikawa Yukio , Cox Nancy J. , Hanis Craig L. , Burant Charles F. , Fox Aaron P. , Bell Graeme I. , Polonsky Kenneth S.
Published: March 2007

Studies of the genetic basis of type 2 diabetes suggest that variation in the calpain-10 gene affects susceptibility to this common disorder, raising the possibility that calpain-sensitive pathways may play a role in regulating insulin secretion and/or action. Calpains are ubiquitously expressed cysteine proteases that are thought to regulate a variety of normal cellular functions. Here, we report that short-term (4-h) exposure to the cell-permeable calpain inhibitors calpain inhibitor II and E-64-d increases the insulin secretory response to glucose in mouse pancreatic islets. This dose-dependent effect is observed at glucose concentrations above 8 mmol/l. This effect was also seen with other calpain inhibitors with different mechanisms of action but not with cathepsin inhibitors or other protease inhibitors. Enhancement of insulin secretion with short-term exposure to calpain inhibitors is not mediated by increased responses in intracellular Ca2+ or increased glucose metabolism in islets but by accelerated exocytosis of insulin granules. In muscle strips and adipocytes, exposure to both calpain inhibitor II and E-64-d reduced insulin-mediated glucose transport. Incorporation of glucose into glycogen in muscle also was reduced. These results are consistent with a role for calpains in the regulation of insulin secretion and insulin action.
Combined Treatment With Benzylamine and Low Dosages of Vanadate Enhances Glucose Tolerance and Reduces Hyperglycemia in Streptozotocin-Induced Diabetic Rats
Authors: Marti Luc , Abella Anna , Carpéné Christian , Palacín Manuel , Testar Xavier , Zorzano Antonio
Published: March 2007

Semicarbazide-sensitive amine oxidase (SSAO) is highly expressed in adipose cells, and substrates of SSAO, such as benzylamine, in combination with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 recruitment in 3T3-L1 and rat adipocytes. Here we examined whether acute and chronic administration of benzylamine and vanadate in vivo enhances glucose tolerance and reduces hyperglycemia in diabetic rats. Acute intravenous administration of these drugs enhanced glucose tolerance in nondiabetic rats and in streptozotocin (STZ)-induced diabetic rats. This occurred in the absence of changes in plasma insulin concentrations. However, the administration of benzylamine or vanadate alone did not improve glucose tolerance. The improvement caused by benzylamine plus vanadate was abolished when rats were pretreated with the SSAO-inhibitor semicarbazide. Chronic administration of benzylamine and vanadate exerted potent antidiabetic effects in STZ-induced diabetic rats. Although daily administration of vanadate alone (50 and 25 μmol · kg−1 · day−1 i.p.) for 2 weeks had little or no effect on glycemia, vanadate plus benzylamine reduced hyperglycemia in diabetic rats, enhanced basal and insulin-stimulated glucose transport, and upregulated GLUT4 expression in isolated adipocytes. In all, our results substantiated that acute and chronic administration of benzylamine with low dosages of vanadate have potent antidiabetic effects in rats.
Adipocyte Produces Matrix Metalloproteinases 2 and 9
Authors: Bouloumié Anne , Sengenès Coralie , Portolan Ghyslaine , Galitzky Jean , Lafontan Max
Published: March 2007

Adipocyte hypertrophy and hyperplasia together with angiogenesis contribute to the growth of the fat mass. Because changes in the extracellular matrix (ECM) components are often associated with such cellular remodeling, we studied the adipocyte expression of the matrix metalloproteinases (MMPs) 2 and 9, two key enzymes involved in the modulation of ECM. The present study provides the first evidence that human adipose tissue produces and secretes MMP-2 and -9 as shown by gelatin zymography analysis performed on media conditioned by human subcutaneous adipose tissue and human preadipocytes in primary cultures and by reverse transcriptase–polymerase chain reaction (RT-PCR) analysis on transcripts from mature human adipocytes. The further characterization performed on the murine 3T3F442A preadipocyte cell line demonstrates that MMP expression, assessed by RT-PCR and Western blot analysis, as well as activity, assessed by gelatin zymography analysis, increased during the adipocyte differentiation, whereas the expression of tissue inhibitor metalloproteinases 1 and 2 were abolished or not affected, respectively. Finally, preadipocyte treatment with MMP inhibitors such as batimastat and captopril, as well as neutralizing antibodies, markedly decreased adipocyte differentiation as demonstrated by the inhibition in the appearance of lipogenic (triglycerides) and lipolytic (glycerol release and hormone-sensitive lipase expression) markers. These data suggest that MMP-2 and -9 could be important key regulators of adipocyte differentiation. Thus, the adipocyte-derived MMPs might represent a new target for the inhibition of adipose tissue growth.
Abnormal Glucose Handling by the Kidney in Response to Hypoglycemia in Type 1 Diabetes
Authors: Cersosimo Eugenio , Garlick Peter , Ferretti John
Published: March 2007

The frequent occurrence of hypoglycemia in people with type 1 diabetes is attributed to abnormalities in the blood glucose counterregulatory response. In view of recent findings indicating that the kidney contributes to prevent and correct hypoglycemia in healthy subjects, we decided to investigate the role of renal glucose handling in hypoglycemia in type 1 diabetes. Twelve type 1 diabetic patients and 14 age-matched normal individuals were randomized to hyperinsulinemic-euglycemic (n = 6 diabetic subjects and n = 8 control subjects) or hypoglycemic (n = 6 each) clamps with blood glucose maintained either stable near 100 mg/dl (5.6 mmol/l) or reduced to 54 mg/dl (3.0 mmol/l). All study subjects had their renal vein catheterized under fluoroscopy, and net renal glucose balance and renal glucose production and utilization rates were measured using a combination of arteriovenous concentration difference with stable isotope dilution technique. Blood glucose and insulin were comparable in both groups in all studies. In patients with diabetes, elevations in plasma glucagon, epinephrine, and norepinephrine were blunted, and both the compensatory rise in endogenous glucose production and in the net glucose output by the kidney seen in normal subjects with equivalent hypoglycemia were absent. Renal glucose balance switched from a mean ± SE baseline net uptake of 0.6 ± 0.4 to a net output of 4.5 ± 1.3 μmol · kg−1 · min−1 in normal subjects, but in patients with diabetes there was no net renal contribution to blood glucose during similar hypoglycemia (mean ± SE net glucose uptake [baseline 0.7 ± 0.4] remained at 0.4 ± 0.3 μmol · kg−1 · min−1 in the final 40 min of hypoglycemia; P < 0.01 between groups). We conclude that adrenergic stimulation of glucose output by the kidney, which represents an additional defense mechanism against hypoglycemia in normal subjects, is impaired in patients with type 1 diabetes and contributes to defective glucose counterregulation.
PPARγ Ligands Increase Expression and Plasma Concentrations of Adiponectin, an Adipose-Derived Protein
Authors: Maeda Norikazu , Takahashi Masahiko , Funahashi Tohru , Kihara Shinji , Nishizawa Hitoshi , Kishida Ken , Nagaretani Hiroyuki , Matsuda Morihiro , Komuro Ryutaro , Ouchi Noriyuki , Kuriyama Hiroshi , Hotta Kikuko , Nakamura Tadashi , Shimomura Iichiro , Matsuzawa Yuji
Published: March 2007

Insulin resistance and its dreaded consequence, type 2 diabetes, are major causes of atherosclerosis. Adiponectin is an adipose-specific plasma protein that possesses anti-atherogenic properties, such as the suppression of adhesion molecule expression in vascular endothelial cells and cytokine production from macrophages. Plasma adiponectin concentrations are decreased in obese and type 2 diabetic subjects with insulin resistance. A regimen that normalizes or increases the plasma adiponectin might prevent atherosclerosis in patients with insulin resistance. In this study, we demonstrate the inducing effects of thiazolidinediones (TZDs), which are synthetic PPARγ ligands, on the expression and secretion of adiponectin in humans and rodents in vivo and in vitro. The administration of TZDs significantly increased the plasma adiponectin concentrations in insulin resistant humans and rodents without affecting their body weight. Adiponectin mRNA expression was normalized or increased by TZDs in the adipose tissues of obese mice. In cultured 3T3-L1 adipocytes, TZD derivatives enhanced the mRNA expression and secretion of adiponectin in a dose- and time-dependent manner. Furthermore, these effects were mediated through the activation of the promoter by the TZDs. On the other hand, TNF-α, which is produced more in an insulin-resistant condition, dose-dependently reduced the expression of adiponectin in adipocytes by suppressing its promoter activity. TZDs restored this inhibitory effect by TNF-α. TZDs might prevent atherosclerotic vascular disease in insulin-resistant patients by inducing the production of adiponectin through direct effect on its promoter and antagonizing the effect of TNF-α on the adiponectin promoter.
Glucose and Palmitic Acid Induce Degeneration of Myofibrils and Modulate Apoptosis in Rat Adult Cardiomyocytes
Authors: Dyntar Daniela , Eppenberger-Eberhardt Monika , Maedler Kathrin , Pruschy Martin , Eppenberger Hans M. , Spinas Giatgen A. , Donath Marc Y.
Published: March 2007

Several studies support the concept of a diabetic cardiomyopathy in the absence of discernible coronary artery disease, although its mechanism remains poorly understood. We investigated the role of glucose and palmitic acid on cardiomyocyte apoptosis and on the organization of the contractile apparatus. Exposure of adult rat cardiomyocytes for 18 h to palmitic acid (0.25 and 0.5 mmol/l) resulted in a significant increase of apoptotic cells, whereas increasing glucose concentration to 33.3 mmol/l for up to 8 days had no influence on the apoptosis rate. However, both palmitic acid and elevated glucose concentration alone or in combination had a dramatic destructive effect on the myofibrillar apparatus. The membrane-permeable C2-ceramide but not the metabolically inactive C2-dihydroceramide enhanced apoptosis of cardiomyocytes by 50%, accompanied by detrimental effects on the myofibrils. The palmitic acid–induced effects were impaired by fumonisin B1, an inhibitor of ceramide synthase. Sphingomyelinase, which activates the catabolic pathway of ceramide by metabolizing sphingomyeline to ceramide, did not adversely affect cardiomyocytes. Palmitic acid–induced apoptosis was accompanied by release of cytochrome c from the mitochondria. Aminoguanidine did not prevent glucose-induced myofibrillar degeneration, suggesting that formation of nitric oxide and/or advanced glycation end products play no major role. Taken together, these results suggest that in adult rat cardiac cells, palmitic acid induces apoptosis via de novo ceramide formation and activation of the apoptotic mitochondrial pathway. Conversely, glucose has no influence on adult cardiomyocyte apoptosis. However, both cell nutrients promote degeneration of myofibrils. Thus, gluco- and lipotoxicity may play a central role in the development of diabetic cardiomyopathy.
Overexpression of Cu2+/Zn2+ Superoxide Dismutase Protects Against Early Diabetic Glomerular Injury in Transgenic Mice
Authors: Craven Patricia A. , Melhem Mona F. , Phillips Stephen L. , DeRubertis Frederick R.
Published: March 2007

Ex vivo and in vitro observations implicate superoxide as a mediator of cell injury in diabetes, but in vivo evidence is lacking. In the current studies, parameters of glomerular injury were examined in hemizygous nondiabetic transgenic mice (SOD) and streptozotocin-diabetic (D) transgenic mice (D-SOD), which overexpress human cytoplasmic Cu2+/Zn2+ superoxide dismutase (SOD-1), and in corresponding wild-type littermates (WT, D-WT) after 4 months of diabetes. In both SOD and D-SOD mice, renal cortical SOD-1 activity was twofold higher than values in the WT mice; blood glucose and glycosylated hemoglobin (GHb) levels did not differ in the two diabetic groups. Urinary albumin excretion, fractional albumin clearance, urinary transforming growth factor-β (TGF-β) excretion, glomerular volume, glomerular content of immunoreactive TGF-β, and collagen α1 (IV) and renal cortical malondialdehyde (MDA) levels were significantly higher in D-WT mice compared with corresponding values in D-SOD mice. Glomerular volume, glomerular content of TGF-β and collagen IV, renal cortical MDA, and urinary excretion of TGF-β in D-SOD mice did not differ significantly from corresponding values in either the nondiabetic SOD or WT mice. In separate groups of mice studied after 8 months of diabetes, mesangial matrix area, calculated as a fraction of total glomerular tuft area, and plasma creatinine were significantly higher in D-WT but not in D-SOD mice, compared with corresponding values in the nondiabetic mice. In vitro infection of mesangial cells (MC) with a recombinant adenovirus encoding human SOD-1 increased SOD-1 activity threefold over control cells and prevented the reduction of aconitase activity, an index of cellular superoxide, and the increase in collagen synthesis that otherwise occurred in control MC in response to culture with 300 or 500 mg/dl glucose. Thus, increases in cellular SOD-1 activity attenuate diabetic renal injury in vivo and also prevent stimulation of MC matrix protein synthesis induced in vitro by high glucose.
Changes in Matrix Proteoglycans Induced by Insulin and Fatty Acids in Hepatic Cells May Contribute to Dyslipidemia of Insulin Resistance
Authors: Olsson Urban , Egnell Ann-Charlotte , Lee Mariam Rodríguez , Lundén Gunnel Östergren , Lorentzon Malin , Salmivirta Markku , Bondjers Göran , Camejo Germán
Published: March 2007

Insulin resistance and type 2 diabetes are associated with elevated circulating levels of insulin, nonesterified fatty acids (NEFAs), and lipoprotein remnants. Extracellular matrix proteoglycan (PG) alterations are also common in macro- and microvascular complications of type 2 diabetes. In liver, extracellular heparan sulfate (HS) PGs contribute to the uptake of triglyceride-rich lipoprotein remnants. We found that HepG2 cells cultured with 10 or 50 nmol/l insulin or 300 μmol/l albumin-bound linoleic acid changed their PG secretion. The glycosaminoglycans (GAGs) of the secreted PGs from insulin-treated HepG2 cells were enriched in chondroitin sulfate (CS) PGs. In contrast, cells exposed to linoleic acid secreted PGs with decreased content of CS. Insulin caused a moderate increase in mRNA for versican (secreted CS PG), whereas linoleic acid markedly decreased mRNA for versican in HepG2 cells, as did the peroxisomal proliferator-activated receptor-α agonist bezafibrate. The effects of insulin or linoleic acid on syndecan 1, a cell surface HS PG, were similar to those on versican, but less pronounced. The livers of obese Zucker fa/fa rats, which are insulin-resistant and have high levels of insulin, NEFAs, and triglyceride-rich remnants, showed increased expression of CS PGs when compared with lean littermates. These changes in PG composition decreased the affinity of remnant β-VLDL particles to PGs isolated from insulin-treated HepG2 cells and obese rat livers. The results indicated that insulin and NEFAs modulate the expression of PGs in hepatic cells. We speculate that in vivo this exchange of CS for HS may reduce the clearance of remnant β-VLDLs and contribute to the dyslipidemia of insulin resistance.
Association of a Novel Point Mutation (C159G) of theCTLA4Gene With Type 1 Diabetes in West Africans but not in Chinese
Authors: Osei-Hyiaman Douglas , Hou Lifang , Zhiyin Ren , Zhiming Zhang , Yu Haiquin , Amankwah Abena Agyeiwaa , Harada Shoji
Published: March 2007

Here, we report on the detection of a novel point mutation of the CTLA4 gene at nucleotide position 159 (C→G) leading to amino acid substitution at position 53 (I→M), as well as its association with type 1 diabetes in two ethnically distinct populations. Subjects included 182 unrelated type 1 diabetes children and 201 control subjects from Ghana, West Africa. The Chinese study population consisted of 350 type 1 diabetic children and 420 healthy control subjects from central China. Polymerase chain reaction–single-strand conformation polymorphism and sequence analysis were used to screen for polymorphisms in the CTLA4 gene. CTLA4 49 (A→G) mutation conferred a risk of type 1 diabetes in the Chinese children (odds ratio 1.78, 95% CI 1.58–2.0), but not in the West African children (1.17, 0.84–1.64). On the other hand, the novel CTLA4 159 (C→G) mutation conferred a risk of type 1 diabetes in the West African children (2.1, 1.54–2.86), but not in the Chinese type 1 diabetic children. The novel CTLA4 gene polymorphism at nucleotide position 159 significantly associated with type 1 diabetes in West Africans, but not in Chinese. On the other hand, the CTLA4 gene polymorphism at nucleotide position 49 significantly associated with type 1 diabetes in Chinese, but not in West Africans.
Overexpression of Dominant-Negative Mutant Hepatocyte Nuclear Factor-1α in Pancreatic β-Cells Causes Abnormal Islet Architecture With Decreased Expression of E-Cadherin, Reduced β-cell Proliferation, and Diabetes
Authors: Yamagata Kazuya , Nammo Takao , Moriwaki Makoto , Ihara Arisa , Iizuka Katsumi , Yang Qin , Satoh Tomomi , Li Ming , Uenaka Rikako , Okita Kohei , Iwahashi Hiromi , Zhu Qian , Cao Yang , Imagawa Akihisa , Tochino Yoshihiro , Hanafusa Toshiaki , Miyagawa Jun-ichiro , Matsuzawa Yuji
Published: March 2007

One subtype of maturity-onset diabetes of the young (MODY)-3 results from mutations in the gene encoding hepatocyte nuclear factor (HNF)-1α. We generated transgenic mice expressing a naturally occurring dominant-negative form of human HNF-1α (P291fsinsC) in pancreatic β-cells. A progressive hyperglycemia with age was seen in these transgenic mice, and the mice developed diabetes with impaired glucose-stimulated insulin secretion. The pancreatic islets exhibited abnormal architecture with reduced expression of glucose transporter (GLUT2) and E-cadherin. Blockade of E-cadherin–mediated cell adhesion in pancreatic islets abolished the glucose-stimulated increases in intracellular Ca2+ levels and insulin secretion, suggesting that loss of E-cadherin in β-cells is associated with impaired insulin secretion. There was also a reduction in β-cell number (50%), proliferation rate (15%), and pancreatic insulin content (45%) in 2-day-old transgenic mice and a further reduction in 4-week-old animals. Our findings suggest various roles for HNF-1α in normal glucose metabolism, including the regulation of glucose transport, β-cell growth, and β-cell–to–β-cell communication.
Ghrelin Is Present in Pancreatic α-Cells of Humans and Rats and Stimulates Insulin Secretion
Authors: Date Yukari , Nakazato Masamitsu , Hashiguchi Suzuko , Dezaki Katsuya , Mondal Muhtashan S. , Hosoda Hiroshi , Kojima Masayasu , Kangawa Kenji , Arima Terukatsu , Matsuo Hisayuki , Yada Toshihiko , Matsukura Shigeru
Published: March 2007

Ghrelin, a novel growth hormone–releasing peptide isolated from human and rat stomach, is a 28–amino acid peptide with a posttranslational acylation modification that is indispensable for stimulating growth hormone secretion by increasing intracellular Ca2+ concentration. It also functions in the regulation of feeding behavior, energy metabolism, and gastric acid secretion and motility. Using two different antibodies against the NH2- and COOH-terminal regions of ghrelin, we studied its localization in human and rat pancreas by immunohistochemistry. Ghrelin-immunoreactive cells were identified at the periphery of pancreatic islets in both species. Ghrelin co-localized exclusively with glucagon in rat islets, indicating that it is produced in α-cells. We identified ghrelin and des-acyl ghrelin in the rat pancreas using reverse-phase high-performance liquid chromatography combined with two radioimmunoassays. We also detected mRNA encoding ghrelin and its receptor in the rat pancreatic islets. Ghrelin increased the cytosolic free Ca2+ concentration in β-cells and stimulated insulin secretion when it was added to isolated rat pancreatic islets. These findings indicate that ghrelin may regulate islet function in an endocrine and/or paracrine manner.
High Glucose via Peroxynitrite Causes Tyrosine Nitration and Inactivation of Prostacyclin Synthase That Is Associated With Thromboxane/Prostaglandin H2 Receptor–Mediated Apoptosis and Adhesion Molecule Expression in Cultured Human Aortic Endothelial Cells
Authors: Zou Ming-Hui , Shi Chaomei , Cohen Richard A.
Published: March 2007

Loss of the modulatory role of the endothelium may be a critical initial factor in the development of diabetic vascular diseases. Exposure of human aortic endothelial cells (HAECs) to high glucose (30 or 44 mmol/l) for 7–10 days significantly increased the release of superoxide anion in response to the calcium ionophore A23187. Nitrate, a breakdown product of peroxynitrite (ONOO−), was substantially increased in parallel with a decline in cyclic guanosine monophosphate (GMP). Using immunochemical techniques and high-performance liquid chromatography, an increase in tyrosine nitration of prostacyclin (PGI2) synthase (PGIS) associated with a decrease in its activity was found in cells exposed to high glucose. Both the increase in tyrosine nitration and the decrease in PGIS activity were lessened by decreasing either nitric oxide or superoxide anion, suggesting that ONOO− was responsible. Furthermore, SQ29548, a thromboxane/prostaglandin (PG) H2 (TP) receptor antagonist, significantly reduced the increased endothelial cell apoptosis and the expression of soluble intercellular adhesion molecule-1 that occurred in cells exposed to high glucose, without affecting the decrease in PGIS activity. Thus, exposure of HAECs to high glucose increases formation of ONOO−, which causes tyrosine nitration and inhibition of PGIS. The shunting of arachidonic acid to the PGI2 precursor PGH2 or other eicosanoids likely results in TP receptor stimulation. These observations can explain several abnormalities in diabetes, including 1) increased free radicals, 2) decreased bioactivity of NO, 3) PGI2 deficiency, and 4) increased vasoconstriction, endothelial apoptosis, and inflammation via TP receptor stimulation.
Chemokine Receptor Genotype Is Associated With Diabetic Nephropathy in Japanese With Type 2 Diabetes
Authors: Nakajima Kunihiro , Tanaka Yasushi , Nomiyama Takashi , Ogihara Takeshi , Piao Lianshan , Sakai Ken , Onuma Tomio , Kawamori Ryuzo
Published: March 2007

Glomerular infiltration of monocytes/macrophages occurs in diabetic patients with nephropathy, and chemokine receptor signals are thought to play a key role in the development of nephropathy. Recently, polymorphism of the chemokine receptor (CCR)2 coding region V64I and the CCR5 promoter region 59029 (G/A) have been identified. Accordingly, we evaluated the effects of these genotypes on diabetic nephropathy. CCR2 V64I and CCR5 59029 (G/A) were detected by polymerase chain reaction–restriction fragment–length polymorphism in 401 patients with type 2 diabetes who had a serum creatinine <2.0 mg/dl. Although the CCR2 V64I genotype showed no association with nephropathy, the frequency of the CCR5 59029 A-positive genotype (G/A or A/A) was significantly higher in patients with microalbuminuria (urinary albumin-to-creatinine ratio [ACR] ≥30 and <300 mg/gCre, 86%) and patients with macroalbuminuria (ACR ≥300 mg/gCre, 87%) than in patients with normoalbuminuria (ACR <30 mg/gCre, 75%; P = 0.0095). Polytomic logistic regression analysis showed that the CCR5 59029 A-positive genotype was associated with nephropathy (odds ratio 2.243, P = 0.0074). These results suggest that the CCR5 promoter 59029 A genotype may be an independent risk factor for diabetic nephropathy in patients with type 2 diabetes.
The Angiotensin-Converting Enzyme DD Genotype Is Associated With Glomerulopathy Lesions in Type 2 Diabetes
Authors: Solini Anna , Dalla Vestra Michele , Saller Alois , Nosadini Romano , Crepaldi Gaetano , Fioretto Paola
Published: March 2007

Genetic factors are important in conferring diabetic nephropathy (DN) risk. The insertion/deletion (I/D) polymorphism of the ACE gene has been described to be associated with DN risk and progression. The renal lesions underlying DN in type 2 diabetes are heterogeneous; only a subset of patients, characterized by a faster decline of renal function, have diabetic glomerulopathy. This study explored the relations between diabetic glomerulopathy and the ACE genotype distribution in 77 type 2 diabetic patients with an albumin excretion rate ≥20 μg/min. Using morphometric analysis of kidney biopsies, mesangial and mesangial matrix fractional volumes [Vv(mes/glom) and Vv(MM/glom)] and glomerular basement membrane (GBM) width were estimated. We found that 13 patients were II, 30 were ID, and 34 were DD. Clinical features and renal function were similar in the three groups; in contrast, the DD patients had the highest Vv(MM/glom) and GBM width. Subdividing patients in tertiles of GBM width and Vv(MM/glom), from the lowest (I) to the highest (III) values, the DD carriers had an odds ratio of 6.11 (95% CI 1.84–20.3) and 10.67 (2.51–45.36), respectively, for the likelihood of being in tertile III than I for GBM width and Vv(MM/glom). Multiple regression analysis revealed the I/D polymorphism as an independent determinant of GBM thickening in addition to diabetes duration and HbA1c. In conclusion, the ACE DD genotype is associated with diabetic glomerulopathy lesions, making the study of this polymorphism helpful in identifying those type 2 diabetic patients at higher risk of fast DN progression.
Regulation of Glucose Transport and Insulin Signaling by Troglitazone or Metformin in Adipose Tissue of Type 2 Diabetic Subjects
Authors: Ciaraldi Theodore P. , Kong Alice P.S. , Chu Neelima V. , Kim Dennis D. , Baxi Sunita , Loviscach Mattias , Plodkowski Ray , Reitz Richard , Caulfield Michael , Mudaliar Sunder , Henry Robert R.
Published: March 2007

Type 2 diabetic subjects failing glyburide therapy were randomized to receive additional therapy with either metformin (2,550 mg/day) or troglitazone (600 mg/day) for 3–4 months. Biopsies of subcutaneous abdominal adipose tissue were obtained before and after therapy. Glycemic control was similar with both treatments. Metformin treatment increased insulin-stimulated whole-body glucose disposal rates by 20% (P < 0.05); the response to troglitazone was greater (44% increase, P < 0.01 vs. baseline, P < 0.05 vs. metformin). Troglitazone-treated subjects displayed a tendency toward weight gain (5 ± 2 kg, P < 0.05), increased adipocyte size, and increased serum leptin levels. Metformin-treated subjects were weight-stable, with unchanged leptin levels and reduced adipocyte size (to 84 ± 4% of control, P < 0.005). Glucose transport in isolated adipocytes from metformin-treated subjects was unaltered from pretreatment. Glucose transport in both the absence (321 ± 134% of pre-Rx, P < 0.05) and presence of insulin (418 ± 161%, P < 0.05) was elevated after troglitazone treatment. Metformin treatment had no effect on adipocyte content of GLUT1 or GLUT4 proteins. After troglitazone treatment, GLUT4 protein expression was increased twofold (202 ± 42%, P < 0.05). Insulin-stimulated serine phosphorylation of Akt was augmented after troglitazone (170 ± 34% of pre-Rx response, P < 0.05) treatment and unchanged by metformin. We conclude that the ability of troglitazone to upregulate adipocyte glucose transport, GLUT4 expression, and insulin signaling can contribute to its greater effect on whole-body glucose disposal.
A Distinct Th1 Immune Response Precedes the Described Th2 Response in Islet Xenograft Rejection
Authors: Krook Henrik , Hagberg Anette , Song Zhenshun , Landegren Ulf , Wennberg Lars , Korsgren Olle
Published: March 2007

Previous studies using semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) have demonstrated that islet xenograft rejection in mice is dominated by Th2-associated cytokines, i.e., interleukin (IL)-4 and IL-10. However, immunohistochemical stainings show that the morphological pattern in this model is more reminiscent of a delayed-type hypersensitivity (DTH) reaction, which is associated with a Th1 response. This study was designed to resolve the mechanisms of acute cellular xenograft rejection in rats transplanted with fetal porcine islet-like cell clusters (ICCs). Real-time quantitative RT-PCR was used to quantify the mRNA expression of cytokines in the grafts and lymph nodes, and the findings were related to the immunopathology of the rejecting grafts. By day 1, mRNA expression levels of IL-1β, IL-2, IL-12p40, interferon-γ, and tumor necrosis factor-α were already induced in the lymph nodes. From days 3 to 12, an increasing amount of activated macrophages was seen in the grafts, whereas T- and NK-cells were fewer and mainly accumulated in the periphery of the grafts. Most of the ICCs were rejected by day 5. Transcripts of Th1-associated cytokines were dominant in both regional lymph nodes and in the grafts, with peak levels on days 3 and 5, respectively. The mRNA expression of IL-4 was increased on day 12, and it correlated with the infiltration of eosinophils and an increased level of xenoreactive IgG. The data presented indicate that an islet xenograft triggers a sequential activation of 1) a Th1-associated response characterized by graft destruction in a DTH-like reaction and then 2) a subsequent Th2-associated response characterized by increased levels of xenoreactive antibodies.
Ghrelin Is Not Necessary for Adequate Hormonal Counterregulation of Insulin-Induced Hypoglycemia
Authors: Lucidi Paola , Murdolo Giuseppe , Di Loreto Chiara , De Cicco Arianna , Parlanti Natascia , Fanelli Carmine , Santeusanio Fausto , Bolli Geremia B. , De Feo Pierpaolo
Published: March 2007

Ghrelin is a novel enteric hormone that stimulates growth hormone (GH), ACTH, and epinephrine; augments plasma glucose; and increases food intake by inducing the feeling of hunger. These characteristics make ghrelin a potential counterregulatory hormone. At present, it is not known whether ghrelin increases in response to insulin-induced hypoglycemia. To answer this question, we compared plasma ghrelin concentrations after a short-term insulin infusion that was allowed or not (euglycemic clamp) to cause hypoglycemia (2.7 ± 0.2 mmol/l at 30 min) in five healthy volunteers. In both studies, plasma ghrelin concentrations decreased (P < 0.01) after insulin infusion (hypoglycemia by 14%, euglycemia by 22%), reached a nadir at 30 min, and returned to baseline at 60 min, without differences between the hypoglycemia and the euglycemia studies. Glucagon, cortisol, and GH increased in response to hypoglycemia despite the decreased ghrelin. There was a strong correlation (R2 = 0.91, P < 0.002) between the insulin sensitivity of the subjects and the percentage suppression of ghrelin from baseline. These data demonstrate that ghrelin is not required for the hormonal defenses against insulin-induced hypoglycemia and that insulin can suppress ghrelin levels in healthy humans. These results raise the possibility that postprandial hyperinsulinemia is responsible for the reduction of plasma ghrelin that occurs during meal intake.
The Effect of Thiazolidinediones on Plasma Adiponectin Levels in Normal, Obese, and Type 2 Diabetic Subjects
Authors: Yu Joseph G. , Javorschi Sandrine , Hevener Andrea L. , Kruszynska Yolanta T. , Norman Rodney A. , Sinha Madhur , Olefsky Jerrold M.
Published: March 2007

The insulin-sensitizing effects of thiazolidinediones are thought to be mediated through peroxisome proliferator-activated receptor-γ, a nuclear receptor that is highly abundant in adipose tissue. It has been reported that adipocytes secrete a variety of proteins, including tumor necrosis factor-α, resistin, plasminogen activator inhibitor-1, and adiponectin. Adiponectin is a fat cell-secreted protein that has been reported to increase fat oxidation and improve insulin sensitivity. Our aim was to study the effects of troglitazone on adiponectin levels in lean, obese, and diabetic subjects. Ten diabetic and 17 nondiabetic subjects (8 lean, BMI <27 kg/m2 and 9 obese, BMI >27 kg/m2) participated in the study. All subjects underwent an 80 mU · m−2 · min−1 hyperinsulinemic-euglycemic glucose clamp before and after 3 months’ treatment with the thiazolidinedione (TZD) troglitazone (600 mg/day). Fasting plasma glucose significantly decreased in the diabetic group after 12 weeks of treatment compared with baseline (9.1 ± 0.9 vs. 11.1 ± 0.9 mmol/l, P < 0.005) but was unchanged in the lean and obese subjects. Fasting insulin for the entire group was significantly lower than baseline (P = 0.02) after treatment. At baseline, glucose disposal rate (Rd) was lower in the diabetic subjects (3.4 ± 0.5 mg · kg−1 · min−1) than in the lean (12.3 ± 0.4) or obese subjects (6.7 ± 0.7) (P < 0.001 for both) and was significantly improved in the diabetic and obese groups (P < 0.05) after treatment, and it remained unchanged in the lean subjects. Baseline adiponectin levels were significantly lower in the diabetic than the lean subjects (9.0 ± 1.7 vs. 16.7 ± 2.7 μg/ml, P = 0.03) and rose uniformly in all subjects (12.2 ± 2.3 vs. 25.7 ± 2.6 μg/ml, P < 10–4) after treatment, with no significant difference detected among the three groups. During the glucose clamps, adiponectin levels were suppressed below basal levels in all groups (10.2 ± 2.3 vs. 12.2 ± 2.3 μg/ml, P < 0.01). Adiponectin levels correlated with Rd (r = 0.46, P = 0.016) and HDL cholesterol levels (r = 0.59, P < 0.001) and negatively correlated with fasting insulin (r = −0.39, P = 0.042) and plasma triglyceride (r = −0.61, P < 0.001). Our findings show that TZD treatment increased adiponectin levels in all subjects, including normal subjects in which no other effects of TZDs are observed. Insulin also appears to suppress adiponectin levels. We have confirmed these results in normal rats. These findings suggest that adiponectin can be regulated by obesity, diabetes, TZDs, and insulin, and it may play a physiologic role in enhancing insulin sensitivity.
Hyperinsulinemia in African-American Children
Authors: Arslanian Silva A. , Saad Rola , Lewy Vered , Danadian Kapriel , Janosky Janine
Published: March 2007

African-American (AA) children are hyperinsulinemic and insulin resistant compared with American White (AW) children. This study investigated 1) whether AA/AW differences in insulinemia are associated with differences in insulin clearance; 2) whether dietary patterns, mainly carbohydrate and fat intake, play a role; and 3) whether the quantitative relationship between insulin sensitivity and secretion is similar between AA and AW children. Forty-four prepubertal children (22 AA and 22 AW) with comparable body composition and visceral adiposity were studied. All underwent a 3-h hyperinsulinemic (40 mU · m−2 · min−1)-euglycemic clamp to calculate insulin sensitivity and insulin clearance and a 2-h hyperglycemic clamp (12.5 mmol/l) to assess first- and second-phase insulin responses. Twenty-four-hour food recalls were analyzed for macronutrient intake. Insulin clearance (19.5 ± 0.7 vs. 22.9 ± 1.1 ml · min−1 · kg−1 fat-free mass [FFM]; P = 0.011) and insulin sensitivity were lower in AA versus AW children (14.8 ± 1.0 vs. 18.9 ± 1.4 μmol · min−1 · kg−1 FFM; P = 0.021). Both insulin clearance and insulin sensitivity correlated inversely with dietary fat/carbohydrate ratio, which was higher in AA than in white children. Fasting C-peptide and insulin were higher in AA children with no difference in proinsulin levels. First- and second-phase insulin concentrations and glucose disposition index (insulin sensitivity × first-phase insulin) were higher in AA than in white children (12.8 ± 2.1 vs. 7.2 ± 0.6 μmol · min−1 · kg−1 FFM; P = 0.019). In conclusion, the hyperinsulinemia observed in AA children is due to both lower insulin clearance and higher insulin secretion compared with their white peers. The quantitative relationship between insulin secretion and sensitivity is upregulated in AA children. This suggests that increased insulin secretion in AA children is not merely a compensatory response to lower insulin sensitivity. Dietary factors may have a role. Additional studies are needed to determine whether metabolic/nutritional factors, possibly mediated through free fatty acids, may play a role in the hyperinsulinism observed in AA children.
Effects of Insulin Treatment in Type 2 Diabetic Patients on Intracellular Lipid Content in Liver and Skeletal Muscle
Authors: Anderwald Christian , Bernroider Elisabeth , Krs̆s̆ák Martin , Stingl Harald , Brehm Attila , Bischof Martin G. , Nowotny Peter , Roden Michael , Waldhäusl Werner
Published: March 2007

Insulin resistance is frequently associated with increased lipid content in muscle and liver. Insulin excess stimulates tissue lipid accumulation. To examine the effects of insulin and improved glycemia on insulin sensitivity and intracellular lipids, we performed stepped (1, 2, and 4 mU · min−1 · kg−1) hyperinsulinemic-euglycemic clamps in eight type 2 diabetic and six nondiabetic control subjects at baseline and after 12 and 67 h of insulin-mediated near-normoglycemia (118 ± 7 mg/dl). Intrahepatocellular lipids (IHCLs) and intramyocellular lipids (IMCLs) of soleus (IMCL-S) and tibialis anterior muscle (IMCL-TA) were measured with 1H nuclear magnetic resonance spectroscopy. At baseline, nondiabetic subjects had an approximate twofold higher insulin sensitivity (P < 0.02) and lower IHCLs than diabetic patients (5.8 ± 1.2 vs. 18.3 ± 4.2%, P < 0.03), in whom IMCL-TA negatively correlated with insulin sensitivity (r = −0.969, P < 0.001). After a 67-h insulin infusion in diabetic patients, IMCL-S and IHCLs were increased (P < 0.05) by ∼36 and ∼18%, respectively, and correlated positively with insulin sensitivity (IMCL-S: r = 0.982, P < 0.0005; IHCL: r = 0.865, P < 0.03), whereas fasting glucose production, measured with d-[6,6-2H2]glucose, decreased by ∼10% (P < 0.04). In conclusion, these results indicate that IMCLs relate to insulin resistance in type 2 diabetic patients at baseline and that insulin-mediated near-normoglycemia for ∼3 days reduces fasting glucose production but stimulates lipid accumulation in liver and muscle without affecting insulin sensitivity.
Insulin Sensitively Controls the Glucagon Response to Mild Hypoglycemia in the Dog
Authors: Igawa Kayano , Mugavero Mike , Shiota Masakazu , Neal Doss W. , Cherrington Alan D.
Published: March 2007

In the present study, we examined how the arterial insulin level alters the α-cell response to a fall in plasma glucose in the conscious overnight fasted dog. Each study consisted of an equilibration (−140 to −40 min), a control (−40 to 0 min), and a test period (0 to 180 min), during which BAY R 3401 (10 mg/kg), a glycogen phosphorylase inhibitor, was administered orally to decrease glucose output in each of four groups (n = 5). In group 1, saline was infused. In group 2, insulin was infused peripherally (3.6 pmol · kg− 1 · min−1), and the arterial plasma glucose level was clamped to the level seen in group 1. In group 3, saline was infused, and euglycemia was maintained. In group 4, insulin (3.6 pmol · kg−1 · min−1) was given, and euglycemia was maintained by glucose infusion. In group 1, drug administration decreased the arterial plasma glucose level (mmol/l) from 5.8 ± 0.2 (basal) to 5.2 ± 0.3 and 4.4 ± 0.3 by 30 and 90 min, respectively (P < 0.01). Arterial plasma insulin levels (pmol/l) and the hepatic portal-arterial difference in plasma insulin (pmol/l) decreased (P < 0.01) from 78 ± 18 and 90 ± 24 to 24 ± 6 and 12 ± 6 over the first 30 min of the test period. The arterial glucagon levels (ng/l) and the hepatic portal-arterial difference in plasma glucagon (ng/l) rose from 43 ± 5 and 5 ± 2 to 51 ± 5 and 10 ± 5 by 30 min (P < 0.05) and to 79 ± 16 and 31 ± 15 (P < 0.05) by 90 min, respectively. In group 2, in response to insulin infusion, arterial insulin (pmol/l) was elevated from 48 ± 6 to 132 ± 6 to an average of 156 ± 6. The hepatic portal-arterial difference in plasma insulin was eliminated, indicating a complete inhibition of endogenous insulin release. The arterial glucagon level (ng/l) and the hepatic portal-arterial difference in plasma glucagon (ng/l) did not rise significantly (40 ± 5 and 7 ± 4 at basal, 44 ± 4 and 9 ± 4 at 90 min, and 44 ± 8 and 15 ± 7 at 180 min). In group 3, when euglycemia was maintained, the insulin and glucagon levels and the hepatic portal-arterial difference remained constant. In group 4, the arterial plasma glucose level remained basal (5.9 ± 1.1 mmol/l) throughout, whereas insulin infusion increased the arterial insulin level to an average of 138 ± 6 pmol/l. The hepatic portal-arterial difference in plasma insulin was again eliminated. Arterial glucagon level (ng/l) and the hepatic portal-arterial difference in plasma glucagon (ng/l) did not change significantly (43 ± 2 and 9 ± 2 at basal, 39 ± 3 and 9 ± 2 at 90 min, and 37 ± 3 and 7 ± 2 at 180 min). Thus, a difference of ∼120 pmol/l in arterial insulin completely abolished the response of the α-cell to mild hypoglycemia.
Naltrexone, an Opioid Antagonist, Facilitates Reepithelialization of the Cornea in Diabetic Rat
Authors: Zagon Ian S. , Jenkins Joe B. , Sassani Joseph W. , Wylie James D. , Ruth Torre B. , Fry Jamie L. , Lang C. Max , McLaughlin Patricia J.
Published: March 2007

Ulcers and erosions of the corneal epithelium, as well as delays in resurfacing of the cornea after wounding, are major causes of ocular morbidity and visual loss in diabetes. To study whether intervention by the opioid antagonist naltrexone (NTX; 30 mg/kg, twice daily) can restore reepithelialization in diabetic cornea, we induced diabetes in rats by intravenous injection of 65 mg/kg streptozotocin. After confirmation of diabetes, 5-mm-diameter epithelial defects that did not include the limbus were created by mechanical scraping of the cornea. At 4 and 8 weeks, corneal reepithelialization was markedly subnormal, with delays ranging from 11% to 17-fold in the diabetic animals compared with control counterparts. Rats that were diabetic for 8 weeks also had a significant decrease in the incidence of complete wound closure. At 4 and 8 weeks, diabetic animals that were receiving NTX had an acceleration in reepithelialization compared with diabetic animals that were receiving vehicle and even surpassed controls. DNA synthesis in the corneal epithelium of diabetic rats was decreased up to 90% of control levels, and NTX exposure of diabetic subjects elevated the labeling index by up to eightfold from diabetic animals that were receiving vehicle. Opioid growth factor and opioid growth factor receptor distribution were comparable in diabetic and control animals. These results indicate a delay in reepithelialization that is dependent on the duration of diabetes and that intervention of endogenous opioid-receptor interfacing with an opioid antagonist can facilitate the process of wound healing.