bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin Increases Guanosine-3′ ,5′-Cyclic Monophosphate in Human Platelets: A Mechanism Involved in the Insulin Anti-Aggregating Effect
Authors: Trovati Mariella , Massucco Paola , Mattiello Luigi , Mularoni Elena , Cavalot Franco , Anfossi Giovanni
Published: September 2013

To investigate whether insulin reduces platelet aggregability through a modulation of the guanosine-3′,5′-cyclic monophosphate (cGMP) concentrations, we determined by a radioimmunoassay the cGMP values in the platelet-rich plasma (PRP) obtained from 17 healthy volunteers and incubated for 3 min with different concentrations of human recombinant insulin (0, 240, 480, 720, 960, and 1,920 pM). Insulin induced a dose-dependent cGMP increase, from 18.5 ± 3.3 to 42.0 ± 6.4 pmol/109 platelets (P = 0.0001). This increase was completely blunted when PRP was preincubated for 20 min with the tyrosine kinase inhibitor genistein (10 μM) or with the guanylate cyclase inhibitor methylene blue (10 μM), but the increase remained highly significant (P = 0.003 and 0.009) when PRP was preincubated for 20 min with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX, 500 μM) or with the nitric oxide synthase inhibitor NG-mono-methyl-L-arginine (L-NMMA, 30 μM). Finally, the insulin-induced decrease of platelet aggregability to collagen and ADP was completely blunted when PRP was preincubated with 10 μM of the guanylate cyclase inhibitor methylene blue. This study demonstrates that the platelet anti-aggregatory effect exerted by insulin is attributable to the insulin-induced increase of cGMP that is due to a direct receptor-mediated platelet guanylate cyclase activation.
Impact of Lisinopril and Atenolol on Kidney Function in Hypertensive NIDDM Subjects With Diabetic Nephropathy
Authors: Nielsen Flemming S , Rossing Peter , Gall Mari-Anne , Skøtt Peter , Smidt Ulla M , Parving Hans-Henrik
Published: September 2013

Diabetic nephropathy is characterized by hypertension and a relentless decline in kidney function. Angiotensin-converting enzyme inhibitors have been claimed to preserve kidney function better than an equal blood pressure (BP) reduction with conventional antihypertensive treatment (renoprotection). We compared the effect on kidney function of lisinopril (10–20 mg/day) and atenolol (50–100 mg/day) in hypertensive NIDDM patients (mean age 60 ± 8 years) with diabetic nephropathy. Forty-three (21 lisinopril and 22 atenolol) patients were enrolled in a 1-year randomized double-blind parallel study. Eight patients dropped out, and the results for the remaining 35 patients (16 lisinopril and 19 atenolol) are presented. Diuretics were required in 10 of 16 lisinopril patients and 12 of 19 atenolol patients. The following variables were measured: 24-hour ambulatory BP (Takeda TM2420), albuminuria (enzyme-linked immunosorbent assay), fractional albumin clearance, and glomerular filtration rate (GFR) ([51Cr]EDTA technique). The average reduction in mean arterial BP during the 12 months was identical in the two groups 12 ± 2 vs. 11 ± 1 mmHg in the lisinopril and atenolol group, respectively. Albuminuria was on average reduced 45% in the lisinopril group vs. 12% in the atenolol group (P < 0.01), and fractional albumin clearance was on average reduced 49% in the lisinopril group vs. 1% in the atenolol group (P < 0.05). GFR declined identically in the two groups 11.7 ± 2.3 vs. 11.6 ± 2.3 ml.min−1.year−1 in the lisinopril and atenolol groups, respectively. In conclusion, both drugs arrested the progressive rise in albuminuria characteristically found in diabetic nephropathy, but lisinopril reduced albuminuria more than an equally effective antihypertensive treatment with atenolol. Longer follow-up is required to clarify if this difference is of importance for the progression in kidney function.
High Prevalence of Autoantibodies to Glutamic Acid Decarboxylase in Long-Standing IDDM Is Not a Marker of Symptomatic Autonomic Neuropathy
Authors: Zanone Maria M , Petersen Jacob S , Peakman Mark , Mathias Christopher J , Watkins Peter J , Dyrberg Thomas , Vergani Diego
Published: September 2013

Immune reactivity to the enzyme glutamic acid decarboxylase (GAD), a pancreatic islet autoantigen, is present at the diagnosis of insulin-dependent diabetes mellitus (IDDM). Because GAD is also highly expressed in the nervous system, we investigated the presence of autoantibodies to the isoform GAD65 in patients with diabetic neuropathy, which is a debilitating complication of the disease. We studied 39 patients with autonomic and somatic neuropathy, 28 patients matched for age and IDDM duration, and 13 patients with a shorter duration of IDDM, all with no diabetic complications, as well as 50 recently diagnosed diabetic patients, 23 neurologic patients with idiopathic autonomic failure unrelated to IDDM, and 72 healthy subjects. An immunoprecipitation radioligand assay was used to detect anti-GAD65 autoantibodies with in vitro transcribed and translated human islet GAD65 as antigen. Autoantibodies to GAD65 were present in 56% of the diabetic patients with neuropathy, 57% of the long-duration and 69% of the short-duration diabetic control subjects, 78% of the recently diagnosed patients, and 13% of the nondiabetic neuropathic patients. Among the diabetic patients with neuropathy, there was no correlation between the presence of anti-GAD65 antibodies and the presence of autoantibodies to sympathetic ganglia, vagus nerve, or adrenal medulla structures identified by immunofluorescence. Our study shows that anti-GAD65 antibodies are present in a high proportion of patients with diabetic neuropathy but are not exclusively associated with it, rendering it unlikely that they have a role as a disease marker or that they are pathogenetic. Our findings also show that humoral autoimmunity to GAD65 is a major feature of diabetes of long duration (up to 52 years), although the source of the persistent stimulus for this reaction and its significance remain to be established.
Opposite Effects of β-cell Differentiation and Growth on Reg Expression in Human Fetal Pancreatic Cells
Authors: Otonkoski Timo , Mally Martin I , Hayek Alberto
Published: September 2013

Reg is a gene associated with regeneration of pancreatic islets. We have previously shown that nicotinamide induces differentiation of human fetal β-cells in tissue culture and that hepatocyte growth factor/scatter factor (HGF/SF) is mitogenic for the fetal β-cells. We now tested whether these conditions, supporting either differentiation or growth, are associated with changes in reg gene expression in human fetal pancreatic cells. Culture for 7 days with 10 mM nicotinamide led to a fourfold decrease in reg mRNA levels (23 ± 12% of control, n = 5, P < 0.001). In contrast, HGF/SF increased reg expression threefold (302 ± 68% of control, n = 4, P < 0.05). Nicotinamide, which does not alter the differentiation level of adult β-cells, did not significantly affect reg expression in adult human islets (84 ± 4% of control, n = 2, NS). Thus, a higher level of endocrine differentiation is associated with a lower level of reg expression, and a higher rate of β-cell replication results in increased reg transcription. These results provide the first evidence of a molecular marker, the reg gene, to distinguish between proliferation and differentiation of human β-cells.
Protection of Encapsulated Human Islets Implanted Without Immunosuppression in Patients With Type I or Type II Diabetes and in Nondiabetic Control Subjects
Authors: Scharp David W , Swanson Carol J , Olack Barbara J , Latta Paul P , Hegre Orion D , Doherty Edward J , Gentile Frank T , Flavin Karen S , Ansara Maha F , Lacy Paul E
Published: September 2013

Human islets were macroencapsulated in permselective hollow fiber membrane devices and successfully allotransplanted subcutaneously with > 90% viability after 2 weeks in situ. Recipients were patients with type I or type II diabetes and normal control subjects; none was immunosuppressed. Between 150 and 200 islet equivalents were implanted in each of the nine patients. No adverse patient complications were observed. Biocompatibility of devices was excellent. Insulin-positive β-cells were confirmed in encapsulated islets recovered from the implanted devices in all patient populations including the type I diabetic patients. Glucose-stimulated insulin release could be demonstrated in vitro from recovered islets. These data demonstrate that macroencapsulated human islets can survive at the subcutaneous site and that permselective membranes can be designed to protect against both allogeneic immune responses as well as the autoimmune component of type I diabetes.
Reduced Levels of mRNA Encoding Endoskeletal and Growth-Associated Proteins in Sensory Ganglia in Experimental Diabetes
Authors: Mohiuddin Liza , Fernyhough Paul , Tomlinson David R
Published: September 2013

This study investigated changes in levels of mRNAs encoding the three neurofilament (NF) proteins NF-L (low), NF-M (medium), and NF-H (high) and two growth-associated proteins, GAP-43 and Tα1 α-tubulin, in lumbar dorsal root ganglia of control and streptozocin-induced diabetic rats. After 8 weeks of diabetes the animals were killed, and total RNA was isolated from the L4 and L5 dorsal root ganglia and subjected to Northern blotting, with constant amounts of total RNA loaded onto each lane. A truncated sense RNA for GAP-43 was included as an internal standard during RNA isolation to enable accurate quantification of mRNA levels. The filters were probed sequentially with 32P-labeled cDNAs encoding NF-L, NF-M, NF-H, GAP-43, T α 1 α-tubulin, and citrate synthase. Hybridizing RNAs were detected by autoradiography and quantified by image analysis. Hybridization signals were normalized to those of the internal standard. In diabetes, NF-L mRNA levels (2.5- and 4-kilobase [kb] transcripts) were decreased by 35 (P = 0.002) and 34% (P < 0.001), respectively, the NF-H mRNA level was decreased by 65% (P < 0.001), but the NF-M mRNA remained unchanged. T α 1 α-tubulin and GAP-43 mRNA levels were reduced by 56 (P < 0.001) and 30% (P < 0.05), respectively. Levels of citrate synthase mRNA were unchanged. These data indicate a selective defect of expression of growth-associated and endoskeletal proteins in experimentally induced diabetes.
Autoantibodies Against Oxidatively Modified Low-Density Lipoproteins in NIDDM
Authors: Bellomo Giorgio , Maggi Elena , Poli Maurizio , Agosta Fulvia G , Bollati Paola , Finardi Giorgio
Published: September 2013

Diabetes is an independent risk factor in the development of atherosclerosis, although the pathophysiological processes underlying this association are poorly understood. The oxidation of low-density lipoprotein (LDL) is considered a key event in the development and progression of atherosclerosis because it generates molecular epitopes that are more atherogenic than parent LDL. A total of 138 patients suffering from non-insulin-dependent diabetes mellitus (NIDDM) and 80 matched control subjects were investigated. LDL oxidation was evaluated as the presence of autoantibodies against oxidatively modified LDL, since they mirror the in vivo occurrence of oxidative processes. NIDDM patients had an antibody ratio (calculated as the ratio of antibodies against modified versus native LDL) significantly higher than control subjects for Cu2+-oxidized LDL (1.88 ± 0.6 vs. 1.05 ± 0.3, P < 0.01, for IgG), malondialdehyde-modified LDL (2.54 ± 0.73 vs. 2.04 ± 0.11, P < 0.01, for IgG and 3.96 ± 1.51 vs. 2.90 ± 0.15, P < 0.01, for IgM), and malondialdehyde-modified human serum albumin (1.79 ± 0.54 vs. 1.46 ± 0.1, P < 0.05 for IgG). The possible role played by glycation in sensitizing LDL to oxidation was investigated by measuring autoantibodies against both glycated LDL (glycLDL) and glycoxydated LDL (glycoxLDL). NIDDM patients had an antibody ratio significantly higher than control subjects for anti-glycLDL and anti-glycoxLDL IgG (1.79 ± 0.38 vs. 1.12 ± 0.23, P < 0.01 and 2.55 ± 1.03 vs. 1.39 ± 0.44, P < 0.01, respectively) but not anti-glycLDL and anti-glycox-LDL IgM. These results demonstrate that in NIDDM patients enhanced LDL oxidation occurs in vivo and that LDL glycation may represent a predisposing event that facilitates subsequent oxidative modifications.
Tissue Factor Pathway Inhibitor Activity in Patients With IDDM
Authors: Leurs Paul B , Oerle Rene van , Hamulyak Karly , Wolffenbuttel Bruce H R
Published: September 2013

Until now, several endothelium-dependent hemostatic parameters have been proposed as markers of vascular endothelial dysfunction in diabetes. We studied tissue factor pathway inhibitor (TFPI) activity in insulin-dependent diabetes mellitus (IDDM) patients without macro-or microvascular complications, before and after intravenous administration of heparin, in comparison with age-matched control subjects. We also examined the effect of acute hyperglycemia on TFPI activity in healthy men. A clotting and a chromogenic assay were used for determining TFPI activity. In the clotting assay, the COOH-terminus of TFPI is essential, but in the chromogenic assay, it is of minor importance. When the chromogenic assay was used, TFPI activity before heparin injection was significantly higher in the IDDM patients (92 ± 24 vs. 112 ± 23%, P < 0.01). The postheparin increase in TFPI activity, measured with both assays, was significantly higher in the diabetic subjects (area under the curve: clotting assay 64 ± 14 vs. 81 ± 24, P < 0.05; chromogenic assay 82 ± 26 vs. 121 ± 35, P < 0.0001). A positive correlation between TFPI activity and glycated hemoglobin was demonstrated. Acute hyperglycemia did not alter TFPI activity. It can be concluded that TFPI activity, especially after stimulation with heparin, is affected by chronic hyperglycemia in diabetic subjects without vascular complications. Alterations in TFPI activity may therefore reflect early endothelial dysfunction.
Somatostatin Selectively Couples to Goα in HIT-T15 Cells
Authors: Seaquist Elizabeth R , Armstrong Michael B , Gettys Thomas W , Walseth Timothy F
Published: September 2013

Previously, we have demonstrated that somatostatin mediates all of its inhibitory effects on glucose-induced insulin secretion from the HIT-T15 cell through pertussis toxin-sensitive G-proteins and that the membrane fraction of this clonal line of pancreatic β-cells contains six such proteins: Giα1, Giα2, Giα3, and three forms of Goα. To determine the specificity of somatostatin receptor–G-protein coupling in HIT-T15 cells, we examined the ability of antisera specific for the COOH-terminus of G α subtypes to inhibit somatostatin-induced augmentation of membrane GTPase activity. GTPase activity increased in membranes as a function of GTP. At all concentrations of GTP studied, 1 μmol/l somatostatin stimulated GTPase activity. Pertussis-toxin pretreatment prevented the effects of somatostatin. Antisera selective for Goα subtypes reduced the effects of somatostatin on GTPase activity (GTPase activity in absence of antisera, 125 ± 3% of control; in the presence of antisera 976, 110 ± 2% of control; n = 13, P < 0.001), whereas antisera directed against Giα1, Giα2, Giα3, and Gsα were without effect. Somatostatin also significantly prevented cyclic AMP accumulation during perifusion with 11.1 mmol/l glucose through a pertussis toxin–sensitive mechanism. These data indicate that the somatostatin receptor couples to Goα in the HIT-T15 cell and suggest that Goα may link somatostatin to cyclic AMP metabolism in pancreatic β-cells.
Treatment of IFN-γ Transgenic Mice With Anti–IFN-γ Reveals the Remodeling Capacity of the Adult Pancreas
Authors: Gu Danling , Molony Leslie , Krahl Troy , Sarvetnick Nora
Published: September 2013

Pancreatic expression of γ-interferon (IFN-γ) initiates a cascade of pathogenic changes that include pancreatic inflammation, islet cell destruction, hyperglycemia, and islet regeneration. In this study, we explore the developmental plasticity of the adult pancreas and particularly its ability to return to normoglycemia and to remodel itself from an advanced pathogenic state. This was approached by treating adult transgenic mice with a pulse of anti–IFN-γ antibody and determining the functional and morphological status of the pancreas. We demonstrated that anti–IFN-γ antibody administration led to the reduction of hyperglycemic blood glucose levels in transgenic mice. We also observed that the pancreas returned from a profoundly perturbed state toward normality. Analysis of the mitotic index indicated that cell proliferation previously associated with islet cell regeneration was greatly reduced after anti–IFN-γ administration. Our results highlight the ability of the adult pancreas to remodel itself and return from a complex pathological state to normalcy once the trophic signal inducing this pathology is removed. These data also suggest that anti–IFN-γ administration may have important clinical implications for treatment of chronic pancreatitis in humans.
Crosses of NOD Mice With the Related NON Strain: A Polygenic Model for IDDM
Authors: McAleer Marcia A , Reifsnyder Peter , Palmer Sheila M , Prochazka Michal , Love Jennifer M , Copeman James B , Powell Elizabeth E , Rodrigues Nanda R , Prins Jan-Bas , Serreze David V , DeLarato Nicole H , Wicker Linda S , Peterson Laurence B , Schork Nicholas J , Todd John A , Leiter Edward H
Published: September 2013

Chromosome locations of non–major histocompatibility complex (MHC) genes contributing to insulin-dependent diabetes mellitus (IDDM) in mice have been determined by outcrossing NOD mice to other inbred strains congenic for the NOD MHC haplotype (H2g7). At least nine non-MHC IDDM susceptibility genes (Idd) were previously identified at first backcross (BC1) after outcross of NOD to C57BL/10.H2g7 congenic mice (B10.H2g7). We investigated whether the same set of Idd loci segregated with IDDM susceptibility after outcross of NOD to NON.H2g7 congenic mice. Since the outcrosses to NON.H2g7 and B10.H2g7 were performed in the same vivarium, direct comparisons were made of the chromosomal locations and relative strengths of Idd alleles in diabetic progeny from the two different outcrosses. In comparison with the NOD × B10.H2g7 outcross, the NOD × NON.H2g7 outcross produced significantly higher IDDM frequencies in F1, F2, and BC1 generations. The high F2 diabetes frequency allowed evaluation of the effects of homozygous expression of both the susceptibility and the resistance allele at Idd loci. This analysis demonstrated that no single non-MHC Idd locus was essential for the onset of diabetes in this cross. After outcross to NON.H2g7, Idd4 (chromosome [Chr] 11), Idd5 (Chr 1), and Idd8 (Chr 14) did not segregate with IddM in either the BC1 or the F2 generation. Diabetogenic NOD-derived alleles at Idd2 (Chr 9), Idd3 (Chr 3), and Idd10 (Chr 3) were segregating in the BC1. An NON-derived allele contributing to susceptibility on Chr 7 (Idd7) was also detected. Dominant traits, detectable only in the F2 cross, were encoded by Chr 4 (Idd9) and two newly mapped loci on Chr 13 (Idd14) and 5 (Idd15). A third dominant trait was encoded by Chr 6 (possibly Idd6), but here, in contrast to Idd9, Idd14, and Idd15, the NON allele was diabetogenic. Stepwise logistic regression analysis of the BC1 and F2 data confirmed that the ability to identify certainty of the non-MHC Idd loci was contingent on the extent of homozygosity for NOD background genes. This study shows that the diabetogenic phenotype can be achieved through the actions of variable combinations of MHC-unlinked genes and a diabetogenic MHC haplotype.
Regulation of Insulin Receptor mRNA Splicing in Rat Tissues: Effect of Fasting, Aging, and Diabetes
Authors: Vidal Hubert , Auboeuf Didier , Beylot Michel , Riou Jean Paul
Published: September 2013

Recent findings suggested that alterations in insulin receptor isoform expression might be involved in the molecular mechanism of insulin resistance. Using reverse transcription reaction followed by competitive polymerase chain reaction, we measured the level of the receptor mRNA variants in rat insulin-sensitive tissues, under conditions of decreased insulin effectiveness (fasting, aging, and diabetes). The liver expressed the mRNA variant with exon 11 predominantly, and the hind limb skeletal muscles expressed the mRNA without exon 11. The heart and epididymal adipose tissue expressed both variants. Fasting and streptozocin-induced diabetes increased the level of receptor mRNAs in the liver but did not modify the repartition between the two variants. The modification of the expression ratio, in favor of the form with exon 11, found by some authors in the skeletal muscle of insulin-resistant patients was not observed in rat muscles that expressed >99% of the form without exon 11 under all the conditions tested. In adipose tissue, the proportion of both mRNA variants was never altered (45% of exon 11-positive [Ex11+]), while the total receptor mRNA concentration changed markedly during fasting or aging. The only modification observed in the isoform distribution was a significant decrease in Ex11+ mRNA concentration in the liver, muscle, and heart of old rats. We conclude that alternative splicing of insulin receptor mRNA is not involved in the impairment of insulin action during fasting or diabetes. Its potential role in the insulin resistance of old animals remains to be defined.
Diabetes, Diabetes, and the American Diabetes Association
Authors: Cryer Philip E
Published: September 2013

Now at the end of my tenure as editor of Diabetes, I wish to offer some personal thoughts about our journal, the disease we oppose, and our Association. I am pleased, but not satisfied, with our journal. I am disappointed, but not surprised, that the disease remains recalcitrant. I am concerned, but optimistic, about our Association. The challenges are great, but so are the opportunities.
Decreased Insulin Secretion and Increased Insulin Resistance Are Independently Related to the 7-Year Risk of NIDDM in Mexican-Americans
Authors: Haffner Steven M , Miettinen Heikki , Gaskill Sharon P , Stern Michael P
Published: September 2013

The relative importance of insulin resistance and abnormal insulin secretion as risk factors for the development of non-insulin-dependent diabetes mellitus (NIDDM) is still controversial. Few data are available on insulin secretion as a risk factor for the development of NIDDM, especially in subjects with normal glucose tolerance. We examined the relation of fasting insulin (as a markerof insulin resistance) and the ratio of change in insulin to change in glucose during the first 30min after glucose ingestion (ΔI30/ΔG30) (as a marker ofinsulin secretion) as predictors of the 7-year development of NIDDM in 714 initially nondiabetic Mexican-Americans. NIDDM developed in 99 subjects. The relative risk of NIDDM increased with higherquartiles of fasting insulin (quartile 1 [low], 1.0; quartile 2, 1.5; quartile 3, 2.0; and quartile 4 [high], 3.7; P < 0.0001) and lower ΔI30/ΔG30 (quartile 1 [low], 6.9; quartile 2, 1.9; quartile 3, 1.1; quartile 4 [high], 1.0; P < 0.001). Subjects with both increased fasting insulin and decreased ΔI30/ΔG30 had independent increases in NIDDM incidence (P < 0.001). Further, when we stratified subjects by baseline glucose tolerance, both increased fasting insulin and decreased ΔI30/ΔG30 significantly predicted NIDDM in subjects with both impaired and normal glucose tolerance at baseline. We conclude that both decreasedinsulin secretion (as assessed by low ΔI30/ΔG30) and increased insulin resistance (as assessed by fasting insulin) predict the development of NIDDM in Mexican-Americans, a group previously characterized as having hyperinsulinemia and insulin resistance. This study provides the first evidence that decreased insulin secretion predicts the development of NIDDM in subjects with normal glucose tolerance, suggesting that deficient insulin secretion and insulin resistance occur early as a precursor of NIDDM.
Growth Factor/Matrix-Induced Proliferation of Human Adult β-Cells
Authors: Hayek Alberto , Beattie Gillian M , Cirulli Vicenzo , Lopez Ana D , Ricordi Camillo , Rubin Jeffrey S
Published: September 2013

Proliferation of human β-cells in vitro is desirable for both transplantation and biological studies. In this study, human pancreatic islets obtained from cadavers were kept in tissue culture plates that favored cell attachment. When the cells attached to the matrix produced by the rat-bladder carcinoma cell line 804G, 5′-bromo-2′-deoxyuridine (BrdU) labeling increased from 4.7 ± 2.5 to 13.2 ± 2.2%, while cells simultaneously labeled for insulin and BrdU increased from 0 to 32%. Addition of the growth factor hepatocyte growth factor/scatter (HGF/SF) increased BrdU labeling to 17.5 ± 1.8 and the percentage of double positive (BrdU + insulin) cells to 69%. This is the first in vitro demonstration that human β-cells grown in monolayer culture are able to replicate when exposed to selected matrices and growth factors. These experiments add further evidence that HGF/SF is an important mitogenic agent for human β-cells.
Effect of Intracerebroventricular Insulin Infusion on Diabetic Hyperphagia and Hypothalamic Neuropeptide Gene Expression
Authors: Sipols Alfred J , Baskin Denis G , Schwartz Michael W
Published: September 2013

To test the hypothesis that diabetic hyperphagia results from insulin deficiency in the brain, diabetic rats (streptozotocin-induced) were given an intracerebroventricular (ICV) infusion of saline or insulin (at a dose that did not affect plasma glucose levels) for 6 days. Food and water intake were significantly increased in diabetic rats, but only food intake was affected by ICV insulin. Diabetic hyperphagia was reduced 58% by ICV insulin compared with ICV saline (P < 0.05) and was accompanied by a 69% increase in diabetes-induced weight loss (P < 0.05). To evaluate whether central nervous system (CNS) insulin deficiency affects expression of neuropeptides involved in food intake, in situ hybridization was done for neuropeptide Y (NPY), which stimulates feeding, in the hypothalamic arcuate nucleus and for cholecystokinin (CCK) and corticotropin-releasing hormone (CRH), which inhibit feeding, in the hypothalamic paraventricular nucleus. In diabetic rats, NPY mRNA hybridization increased 280% (P < 0.05), an effect reduced 40% by ICV insulin (P < 0.05). CCK mRNA hybridization increased 50% in diabetic rats (P < 0.05), a response reduced slightly by ICV insulin (P < 0.05), whereas CRH mRNA hybridization decreased 33% in diabetic rats (P < 0.05) and was unchanged by ICV insulin. The results demonstrate that CNS infusion of insulin to diabetic rats reduces both hyperphagia and overexpression of hypothalamic NPY mRNA. This observation supports the hypothesis that a deficiency of insulin in the brain is an important cause of diabetic hyperphagia and that increased hypothalamic NPY gene expression contributes to this phenomenon.
Kinin, a Mediator of Diabetes-Induced Glomerular Hyperfiltration
Authors: Jaffa Ayad A , Rust Philip F , Mayfield Ronald K
Published: September 2013

Renal kallikrein is increased in diabetic patients and streptozotocin (STZ)-induced diabetic rats with hyperflltration. Chronic inhibition of renal kallikrein reduces glomerular filtration rate (GFR) and renal plasma flow (RPF) in hyperfiltering STZ-induced diabetic rats. To investigate whether these actions of kallikrein and its inhibition are kinin-mediated, we used a B2-kinin receptor antagonist (BKA). In STZ-induced diabetic rats with hyperflltration, renal kallikrein excretion rate was significantly increased (P < 0.01), and kinin excretion rate was increased 57%, as compared with control rats. Left kidney GFR and RPF were measured before and during a 40-min infusion of BKA (0.5 μg · kg−1 · min−1) or vehicle. Infusion of the kinin receptor antagonist reduced the GFR and RPF significantly. GFR was reduced by 18%, from an average baseline value of 2.07 ± 0.11 to 1.70 ± 0.06 ml/min, P < 0.001 (means ± SE). RPF was reduced by 25%, from 6.74 ± 0.38 to 5.06 ± 0.17 ml/min, P < 0.001. Total renal vascular resistance was significantly increased during BKA infusion, P < 0.001. Vehicle infusion for the same period had no significant effect on GFR, RPF, or renal vascular resistance. These findings further support the hypothesis that increased renal production of kinins contributes to the renal vasodilation of diabetes.
Does Insulin-Like Growth Factor I Predict Incidence and Progression of Diabetic Retinopathy?
Authors: Wang Qin , Dills Diana G , Klein Ronald , Klein Barbara E K , Moss Scot E
Published: September 2013

We evaluated the relationship of insulin-like growth factor (IGF)-I to incidence and progression of diabetic retinopathy over a 6-year interval in a large population-based study of diabetes in southern Wisconsin. Participants included people with younger-onset diabetes (n = 66 adolescents, n = 661 adults 2:18 years of age) and older-onset diabetes (n = 285 for those using insulin, n = 248 for those not using insulin). Fundus photographs were graded in a masked fashion using standardized protocols to determine the severity of retinopathy in each eye. Serum IGF-I levels were measured during 1984–1986 using a double-antibody radioimmunoassay. Mean IGF-I was highest in adolescents (499.1 μg/l), lower in younger-onset adults (280.1 μg/l), and lowest in the older-onset group (205.7 and 221.2 μg/l for older-onset group using insulin and not using insulin, respectively). The incidence of retinopathy was not significantly higher in people with higher IGF-I levels in any group. The odds of developing diabetic retinopathy in 6 years for each 10 μg/l increase in IGF-I after controlling for age, glycosylated hemoglobin, and duration of diabetes at baseline was 1.21 (95% confidence interval [CI] 0.95–1.54) for adolescents; 1.00 (95% CI 0.93–1.08) for younger-onset adults; 0.93 (95% CI 0.85–1.02) for the older-onset group using insulin; and 0.99 (95% CI 0.95–1.04) for the older-onset group not using insulin. In summary, IGF-I was not associated with 6-year incidence or progression of diabetic retinopathy in any of the groups.
Regulation of Postprandial Whole-Body Proteolysis in Insulin-Deprived IDDM
Authors: Biolo Gianni , Inchiostro Sandro , Tiengo Antonio , Tessari Paolo
Published: September 2013

Suppression of tissue proteolysis is an important mechanism of postprandial protein anabolism, and it may be mediated by insulin, hyperaminoacidemia, or both. To evaluate whether insulin is essential in the regulation of this process, we have investigated the effect of mixed-meal ingestion on whole-body protein breakdown in insulin-deprived insulin-dependent diabetes mellitus (IDDM) patients and normal control subjects. Endogenous phe-nylalanine and leucine rate of appearance (Ra) from proteolysis were measured at steady-state conditions using a multiple stable isotope technique before and after the constant administration of a synthetic mixed meal. In the postabsorptive state, the IDDM patients exhibited accelerated intracellular leucine Ra (IDDM, 2.64 ± 0.19 μmol · min−1 · kg−1; control, 2.02 ± 0.08 μmol · min−1 · kg−1; P < 0.05) and plasma phenylalanine Ra (IDDM, 0.73 ± 0.03 μmol · min−1 · kg−1; control, 0.61 ± 0.04 μmol · min−1 · kg−1; P < 0.05). During meal ingestion, endogenous phenylalanine and leucine Ra values were suppressed in both the insulin-deficient IDDM (P < 0.05) and control subjects (P < 0.05). Although postmeal endogenous leucine and phenylalanine Ra values remained greater (P < 0.05) in IDDM, the Δ changes from the basal endogenous leucine Ra (IDDM, −0.56 ± 0.11 μmol · min−1 · kg−1; control, −0.56 ± 0.09 μmol · min−1 · kg−1) and phenylalanine Ra (IDDM, −0.13 ± 0.01 μmol · min−1 · kg−1; control, −0.14 ± 0.02 μmol · min−1 · kg−1) were similar in both groups. In the IDDM patients, the postmeal increases from the basal leucine concentration were onefold greater (P < 0.05) than in the control subjects. In conclusion, in IDDM patients, whole-body proteolysis was suppressed during meal ingestion despite insulin withdrawal, which was possibly mediated by excessive hyperaminoacidemia.
Two Distinct Glutamic Acid Decarboxylase Auto-Antibody Specificities in IDDM Target Different Epitopes
Authors: Daw Kendra , Powers Alvin C
Published: September 2013

Although most individuals with insulin-dependent diabetes mellitus (IDDM) have autoantibodies to glutamic acid decarboxylase (GAD), antibodies to GAD are also present in some individuals with a low risk of developing diabetes. The GAD autoantibodies of IDDM are specific for the GAD65 isoform, do not bind denatured GAD protein, and target epitope(s) dependent on conformation of the protein. However, the IDDM epitopes have been difficult to further define because the antibodies do not bind GAD protein fragments or synthetic peptides. Since the GAD67 isoform is highly homologous to GAD65 but is usually not a target of the GAD autoantibodies in IDDM sera, we created six GAD65/GAD67 chimeric proteins to maintain the overall GAD protein conformation and used these chimeric proteins to map conformation-dependent epitopes of GAD65 targeted by IDDM sera. We find that the GAD binding present in most IDDM sera (n = 11 of 12) is composed of two distinct GAD antibody specificities that target different conformation-dependent regions of the GAD65 protein, one that is located between amino acids 240 and 435 (termed IDDM-E1) and one that is located between amino acids 451 and 570 (termed IDDM-E2). One DDDM serum (it = 1 of 12) bound only the IDDM-E1 region. Identification of epitopes targeted by IDDM sera may allow one to distinguish between GAD antibody-positive individuals at high and low risk of developing IDDM and to determine if differences in the autoimmune repertoire directed at GAD are present. The chimeric GAD65/GAD67 proteins may also be useful in designing GAD assays specific for IDDM.
Insulin Inhibits Liver Expression of the CCAAT/Enhancer-Binding Protein β
Authors: Bosch Fatima , Sabater Jordi , Valera Alfons
Published: September 2013

The CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that is abundant in the liver. The concentration of C/EBPβ mRNA in the liver of mice and rats fed a high-carbohydrate diet, which causes a rise in blood insulin levels, was lower (80 and 65%, respectively) than that detected in animals fed a standard diet. Similarly, the expression of the human insulin gene in the liver of transgenic mice led to a decrease in the concentration of C/EBPβ mRNA. However, no change was detected in the mRNA levels of C/EBα or cAMP regulatory element-binding protein transcription factors in the livers of these mice. Furthermore, the expression of the C/EBPβ gene increased in the liver of diabetic rats and decreased in the liver of diabetic animals treated with vanadate, an insulin mimetic agent. In addition, a decrease in C/EBPβ protein was observed in liver nuclei from mice after insulin injections, in mice fed a high-carbohydrate diet, and in transgenic mice expressing the insulin gene in the liver. These results suggest that insulin might control gene expression in vivo, at least in part, by a mechanism involving a decrease in the transcription factor C/EBPβ.
Regulation of Glycogen Synthase by Glucose, Glucosamine, and Glutamine:Fructose-6-Phosphate Amidotransferase
Authors: Crook Errol D , Zhou Jianxin , Daniels Marc , Neidigh John L , McClain Donald A
Published: September 2013

The hexosamine biosynthesis pathway has been hypothesized to mediate some of the regulatory as well as the deleterious effects of glucose. We have stably overexpressed the cDNA for human glutamine:fructose-6-phosphate amidotransferase (GFA), the rate-limiting enzyme in the hexosamine biosynthesis pathway, in rat-1 fibroblasts. Two cell lines expressing the human RNA were selected by Northern analysis, and they exhibited 51-95% increases in GFA activity. Insulin-stimulated glycogen synthase (GS) activity and net glycogen synthesis were assayed, and GFA cells revealed decreased insulin sensitivity for both GS and net glycogen synthesis. The ED50 for insulin stimulation of GS was 2.45 ± 0.4 nmol/l insulin in controls and 5.29 ± 1.01 nmol/l in GFA cells (P < 0.005). For insulin-stimulated glycogen synthesis, the ED50 was 3.43 ± 0.88 nmol/l in controls and 5.54 ± 0.98 nmol/l in GFA cells (P < 0.005). There were no significant differences in maximally insulin-stimulated or total GS activities, insulin binding or receptor number, or glucose uptake between GFA and control cells. We also examined the effects of glucose on GS activity. GFA cells had a twofold increase in GS activity at low glucose (0.5 mmol/l) when compared with controls (P < 0.025). Both GFA and control cells had an ∼75–80% decrease in GS activity as glucose concentration was increased from 0.5 to 20 mmol/l. This change in GS activity was not observed until after 12 h in culture. GFA cells were more sensitive to the effects of glucose. The ID50 values for glucose were 1.72 ± 0.08 mmol/1 in GFA cells and 5.60 ± 2.05 mmol/l in control cells (P ? 0.05). Glucosamine mimicked the effects of glucose but at a much higher potency with an ED50 of 0.667 ± 0.15 mmol/1 in control cells cultured in low glucose. The changes in activity seen with glucose are not due to the osmotic effects of glucose or the carryover of intracellular effector molecules into the in vitro GS assay. In sum, the stable overexpression of the cDNA for human GFA leads to decreased insulin sensitivity of GS and glycogen synthesis and is accompanied by an increased sensitivity to inhibition of GS by high glucose. We conclude that glucose regulates glycogen synthesis through the metabolism of glucose to hexosamines.
Expression of the Co-stimulator Molecule B7–1 in Pancreatic β-Cells Accelerates Diabetes in the NOD Mouse
Authors: Wong Susan , Guerder Sylvie , Visintin Irene , Reich Eva-Pia , Swenson Karl E , Flavell Richard A , Janeway Charles A
Published: September 2013

B7-1 is a co-stimulatory molecule that signals T-cells that recognize antigen to proliferate and differentiate into effector T-cells. The same cell must present antigen and express co-stimulatory molecules, such as B7-1, to activate naive T-cells. Thus, tissues that do not express co-stimulatory molecules would not be expected to induce immune responses, while expression of a co-stimulator on tissue cells may convert them into effective antigen-presenting cells and induce autoimmunity. To test this, transgenic mice have been generated that express B7-1 on the β-cells of the pancreatic islets of Langerhans. On a B6 genetic background, B7-1 expression on β-cells does not predispose to diabetes. B6 mice are resistant to diabetes. However, when B7-1 is expressed on the β-cells of B6 mice backcrossed once to the genetically susceptible NOD strain, the onset of diabetes is accelerated and the autoimmune attack intensified. This illustrates that B7-1 is a very potent co-stimulatory molecule in vivo and that its presence on the surface of tissue cells can potentiate the autoimmune process.
Correction of Hypertriglyceridemia with Low High-Density Lipoprotein Cholesterol by the Novel Compound NO-1886, a Lipoprotein Lipase-Promoting Agent, in STZ-Induced Diabetic Rats
Authors: Tsutsumi Kazuhiko , Inoue Yasuhide , Shima Atsushi , Murase Toshio
Published: September 2013

We have previously reported that the novel compound NO-1886 increased lipoprotein lipase (LPL) activity, with resulting elevation of high-density lipoprotein (HDL) cholesterol in normal rats (J Clin Invest 92:411–417, 1993). The aim of this study was to ascertain whether the compound has the same action in diabetes, because hypertriglyceridemia with low HDL cholesterol is an extremely common concomitant condition in diabetes. Streptozotocin-induced diabetic rats showed marked elevation of plasma triglyceride and reduction of HDL cholesterol. Both single and repeated administration of NO-1886 increased postheparin plasma LPL activity, with resulting reduction of plasma triglyceride and elevation of HDL cholesterol. Repeated administration increased the amount of LPL mRNA in adipose tissue and myocardium. The compound had no effects on plasma glucose and insulin levels. Our study indicates that the compound is potentially beneficial for the treatment of hypertriglyceridemia with low HDL cholesterol in diabetes.
Counterregulation of Hypoglycemia: Skeletal Muscle Glycogen Metabolism During Three Hours of Physiological Hyperinsulinemia in Humans
Authors: Cohen Neil , Rossetti Luciano , Shlimovich Pavel , Halberstam Meyer , Hu Meizhu , Shamoon Harry
Published: September 2013

We examined the role of skeletal muscle in counterregulation of hypoglycemia (3.4 ± 0.1 mmol/l) in 12 nondiabetic individuals (age 26 ± 1 years, body mass index 24.2 ± 0.7 kg/m2) during physiological hyperinsulinemia (280 ± 25 pmol/l) compared with euglycemia (4.8 ± 0.1 mmol/l). During hypoglycemia, hepatic glucose output (3-[3H]-glucose) was greater (7.72 ± 2.72 mumol.kg−1 · min−1, P < 0.01), glucose uptake was ∼ 49% lower (21.20 ± 3.55 mumol.kg−1 · min−1, P < 0.005), and glucose clearance was reduced (P < 0.002) compared with euglycemia. Rates of flux of plasma-derived glucosyl units through glycolysis were similar in the two experiments, while glycogen synthetic rates were significantly reduced during hypoglycemia (P < 0.01) and accounted entirely for the reduction in glucose disposal. The insulin-induced activation of skeletal muscle glycogen synthase (reflected by Km decline by ∼ 50% from 0.408 ± 0.056 mmol/l and fractional velocity increase by ∼ twofold from 21.8 ± 2.7%) was completely abolished in hypoglycemia. In concert, glycogen phosphorylase activity increased during hypoglycemia by ∼ 40% (P = 0.0001). Hypoglycemia resulted in seven- to eightfold increments in plasma epinephrine (P < 0.0001) and growth hormone (P < 0.001) and 40-60% increments in plasma glucagon (P < 0.005) and cortisol (P < 0.05). We conclude that, in this model of mild hypoglycemia of moderate duration, the majority of the glucose made available during the counterregulatory process (∼ 60-70%) is due to the limitation of glucose disposal, mostly via decreased glycogen synthetic activity in skeletal muscle.
Measurement of Proinsulin and Intermediates: Validation of Immunoassay Methods by High-Performance Liquid Chromatography
Authors: Ostrega Diane , Polonsky Kenneth , Nagi Dinesh , Yudkin John , Cox Lorna J , Clark Penelope M S , Hales C Nicholas
Published: September 2013

Human proinsulin and 32–33 split proinsulin have been measured in the peripheral circulation by immunoradiometric assays (IRMAs) and have been shown to be elevated in impaired glucose tolerance and non-insulin-dependent diabetes mellitus (NIDDM). The IRMA for 32–33 split proinsulin did not discriminate between this molecule and des-32 or des-31,32 split proinsulin. We describe the comparison of IRMA for human plasma proinsulin and 32–33 split proinsulins with assays combined with high-performance liquid chromatography (HPLC), which can discriminate between 32–33 split, des-32 split, and des-31,32 split proinsulin. Subjects were those with normal glucose tolerance (n = 8) and those with NIDDM (n = 17), who were studied while fasting and 30 min after a glucose load. After collection, blood was centrifuged promptly, and the serum/plasma was stored frozen until assay. Both IRMA and HPLC methods were calibrated against synthetic peptides. Interassay coefficients of variation for the IRMA for proinsulin and 32-33 split proinsulin were < 13% over the ranges 3.8–65 pmol/l and 6.4–65 pmol/l, respectively. The following regression lines were obtained: proinsulin IRMA ½ −0.143 + 1.066 HPLC, r = 0.860; 32–33 split proinsulin IRMA ½ 0.048 + 1.051 HPLC; and des-31,32 split proinsulin, r = 0.814. For both analytes, there was no significant difference in the relationship of IRMA to HPLC results between the various subject groups and various time points. Thus, the IRMA for proinsulin has been validated by an independent method. The 32–33 split proinsulin IRMA predominately measures des-31,32 split proinsulin, which is the major partially processed proinsulin present in human plasma of both normal and NIDDM subjects.
Hyperglycemia-Induced Changes in Na+/myo-Inositol Transport, Na+-K+-ATPase, and Protein Kinase C Activity in Proximal Tubule Cells
Authors: Cole Judith A , Walker Ruth E W , Yordy Michael R
Published: September 2013

In many tissues, hyperglycemia alters the activities of the Na+-dependent myo-inositol (Na/MI) transporter, Na+-K+-ATPase, and protein kinase C (PKC). However, little is known concerning adaptive changes in renal proximal tubular function after acute or chronic hyperglycemia. We examined hyperglycemia-induced changes in Na/MI transport, Na+-K+-ATPase activity, and PKC activity using three proximal tubule–like cell lines (JTC12, LLC-PK1, and OK/E cells) and primary cultures of human proximal tubular epithelium (HK cells) cultured for varying periods in low- or high-glucose media, myo-Inositol (MI) transport was mediated by a high-affinity (Km ∼50 mumol/l) Na+-dependent saturable process in the four cell lines. Hyperglycemia produced a time-dependent and persistent increase in Na/MI transport in all cell lines. Chronic hyperglycemia increased the Km for MI transport in LLC-PK1 cells and increased the Vmax in both LLC-PK1 and JTC12 cells. Glucose competitively inhibited Na/MI transport in all low-glucose cells and in high-glucose HK, JTC12, and OK/E cells but had no effect on transport in high-glucose LLC-PK1 cells. Acute hyperglycemia also produced time-dependent increases in Na+-K+-ATPase activity in all cell lines, a change that persisted only in HK cells. A 24-h exposure to high glucose had no effect on PKC activity in any of the cell lines but increased Ca/phospholipid-dependent PKC activity in membrane fractions from chronically high-glucose LLC-PK1 and OK/E cells. These data suggest that hyperglycemia causes acute changes in proximal tubule function and long-lived adaptive responses in Na/MI transport and the PKC signaling pathway.
Evidence for Linkage Between a Region on Chromosome 1p and the Acute Insulin Response in Pima Indians
Authors: Thompson D Bruce , Janssen Rachel C , Ossowski Victoria M , Prochazka Michal , Knowler William C , Bogardus Clifton
Published: September 2013

A low acute insulin response (AIR) is a predictor of non-insulin-dependent diabetes mellitus (NIDDM) in insulin-resistant Pima Indians. We have initiated a search for regions of the genome linked with the AIR using sib-pair linkage analysis as a first step in identifying genes that are determinants of this phenotype. Eighteen short tandem-repeat polymorphisms from chromosome 1 were genotyped in over 900 Pima Indians and tested for linkage with NIDDM and in a subset of Pima Indians for linkage with AIR. The anonymous DNA marker D1S198 on chromosome 1p was linked with AIR (P = 0.000056) in 175 sib pairs from 60 families, all with normal glucose tolerance, but no linkage was observed between D1S198 and NIDDM (P = 0.44, 996 sib pairs). Additional markers genotyped on chromosome 1 did not show linkage with AIR or NIDDM. This study indicates that a locus on chromosome 1p may be a determinant of the phenotypic variation seen in the AIR.