bluuebunny/crossref_metadata_2025 huggingface dataset:
Decreased Activity of Acyl-CoA:Cholesterol Acyltransferase by Insulin in Human Intestinal Cell Line Caco-2
Authors: Jiao Sheng , Moberly James B , Cole Thomas G , Schonfeld Gustav
Published: September 2013

Diabetes mellitus is accompanied by increased intestinal cholesterol synthesis and cholesterol esterification. Both are reversed by insulin therapy. To assess whether the action of insulin on cholesterol esterification by intestinal cells is direct or mediated by other effectors associated with diabetes, we investigated the effect of insulin on the activity of microsomal acyl-CoA:cholesterol acyltransferase (ACAT) and on the incorporation rate of [14C]oleic acid into cholesteryl oleate in the Caco-2 human intestinal cell line. Microsomal ACAT activity of cells that were incubated with insulin for 3 h at a concentration of 1, 10, and 100 μU/ml was decreased by 48, 58, and 74%, respectively, compared with cells cultured in insulinfree medium. This effect was evident as soon as 10 min after the addition of 10 μU/ml insulin. The inhibition by insulin was reversible. After incubation for 24 h, intracellular esterified-cholesterol content and the ratio of esterified to nonesterified cholesterol were significantly lower in the cells treated with insulin (100 μU/ml) than in those not treated with insulin (esterified cholesterol 48.6 ± 2.0 vs. 74.2 ± 4.3 nmol/mg protein, respectively, P < .005; esterified to nonesterified ratio 0.280 ± 0.008 vs. 0.359 ± 0.059, respectively, P < .05). Cells cultured on filters manifested physiologic polarity; >90% of [14C]oleic acid-labeled cholesterol ester secreted by cells was secreted into the basolateral chambers. Incorporation of [14C]oleic acid into cholesteryl oleate over 24 h in the cells and in the medium of the basolateral chamber was suppressed by 100 μU/ml insulin by 23 and 40%, respectively. These findings indicate that insulin acts directly on the enterocytes to suppress intestinal cholesterol ester synthesis and secretion.
In Vitro Relationship of CD4 Cells From Type I Diabetic Patients and Xenogeneic β-Cell Membranes
Authors: Segain Jean-Pierre , Valentin Alexis , Bardet Stephane , Feve Bruno , Sevestre Helene , Houssaint Elisabeth , Charbonnel Bernard , Sai Pierre
Published: September 2013

In a rosette assay, 63 patients with recent-onset type I (insulin-dependent) diabetes mellitus had a higher (P < .001) number of lymphocytes adhering to rat insulinoma RINm5F cells (diabetic rosettes) than 153 healthy control (background rosettes) or 20 nondiabetic subjects with other organ-specific autoimmune diseases. Furthermore, lymphocytes from diabetic patients displayed a highly correlated (r = .97, P < .001) binding on two different xenogeneic β-cell lines (RIN and hamster insulinoma HIT cells). This phenomenon was not found on a panel of seven non β-cell lines (e.g., exocrine pancreatic cells, endocrine cells). By increasing lymphocyte-to-RIN ratios (0.25:1 to 30:1), the supernumerary RIN-adherent lymphocytes from diabetic patients, expressed as the percentage of lymphocytes involved in conjugates, were only detectable at lower ratios (0.25:1 to 4:1), and their binding efficiency was two times higher than that of control lymphocytes. This efficiency fell at higher ratios (>4:1) to the level of background rosettes that remained constant through the ratio scale. This specific RIN-rosette formation was abrogated when lymphocytes from diabetic patients were preabsorbed on β-cells (either HIT or RIN) but not on non-β-cells, whereas preabsorption of control lymphocytes did not modify the number of background rosettes. In addition, diabetic rosettes, but not background rosettes, were inhibited by competition with RIN membrane extracts but not by non-β-cell extracts. Moreover, diabetic rosettes were inhibited during blocking experiments with anti-CD3 monoclonal antibody (MoAb) but not with unrelated MoAbs. Finally, diabetic rosettes were abolished after depletion of CD3+ or CD4+ cells but remained after elimination of CD8+ or CD16+ cells. Enrichment experiments confirmed the implication of CD3+/CD4+ cells but not of B-lymphocytes. Thus, CD3+/CD4+ cells from diabetic patients specifically recognize xenogeneic β-cell membrane antigens. This reinforces our hypothesis that diabetic rosettes are markers of cellular immunity in type I diabetes.
Comparison of Insulin Secretory Patterns in Obese Nondiabetic LA/N-cp and Obese Diabetic SHR/N-cp Rats: Role of Hyperglycemia
Authors: Recant Lillian , Voyles Nancy R , Timmers Kim I , Bhathena Sam J , Solomon Darley , Wilkins Sam , Michaelis Otho E
Published: September 2013

Obese diabetic SHR/N-(cp/cp) rats are a genetic model for non-insulin-dependent diabetes mellitus. When SHR/N-cp rats are overtly diabetic, they are hyperinsulinemic and hyperglycemic in the fed state when consuming commercial chow or semipurified high-carbohydrate diets. Obese SHR/N-cp rats were hyperinsulinemic by 4 wk of age, although hyperglycemia did not appear until 3–4 wk later and was exacerbated by a high-sucrose diet (mean ± SE 1488 ± 238 (μ/ml insulin and 425 ± 51 mg/dl glucose). The control SHR/N-cp rats (+ /?) on the sucrose diet remained lean and normoglycemic. The obese diabetic SHR/N-cp rats showed three alterations in pancreas perfusion data (not present in control rats): 7) paradoxically high insulin secretion at low glucose levels (2.5 mM), 2) secretion of insulin in response to arginine (10 mM) in the absence of glucose, and 3) impaired response of insulin secretion to high glucose (16.7 mM). To determine whether hyperglycemia was responsible for the abnormalities of insulin secretion, perfusion studies were conducted in obese nondiabetic LA/N-cp rats and compared with the SHR/N-cp rats. The obese LA/N-cp rats resembled the corpulent SHR/N-cp rats in every way, except that they were normoglycemic on the sucrose diet. The obese LA/N-cp rats had two of the three alterations in insulin secretion shown by obese SHR/N-cp rats, lacking only the impaired response to high glucose, suggesting that hyperglycemia was required for that defect to occur. This finding is supported by the partial reversal of this defect in the obese SHR/N-cp rats after fasting for 48–72 h, when plasma glucose levels had been close to normal for >24–48 h. Additional evidence was obtained in younger, prediabetic obese SHR/N-cp rats that also showed the first two of the three defects and showed only a slightly abnormal response to high glucose. Paradoxically, high insulin secretion at low glucose levels and hypersecretion of insulin in response to arginine without glucose are early abnormalities associated with the development of obesity in these models, whereas decreased maximal response to high glucose develops later and is associated with hyperglycemia.
Molecular and Clinical Characterization of an Insertional Polymorphism of the Insulin-Receptor Gene
Authors: Elbein Steven C
Published: September 2013

A restriction-fragment–length polymorphism (RFLP) detected with the human insulin-receptor cDNA and the enzyme Sac I has been reported to be associated with non-insulin-dependent diabetes mellitus (NIDDM) in White and Black populations and segregated with diabetes in two small pedigrees with maturity-onset diabetes of the young. A size difference of ∼500 base pairs (bp) was demonstrated between the alleles of this and several other RFLPs that mapped to the same 300-bp region near the transmembrane coding region of the cDNA (β-chain, thus suggesting the presence of an insertion in this region that could affect insulin-receptor function. Genomic DNA fragments containing this RFLP were cloned from an individual heterozygous for the putative insertion, and the differing fragments of the two alleles were sequenced. The presence of a 400-bp insertion was thus confirmed and was demonstrated to be entirely within an intron. No significant coding-region differences from published cDNA sequences were detected in four exons sequenced from the region of the insertional allele. The sequenced regions included multiple Alu repeat sequences. The RFLP was unusual in that the larger allele consisted of an additional Alu repeat sequence that included a new Pst I site. Because the nature and location of the insertion did not suggest a role in insulin-receptor function, the association of this RFLP with NIDDM and hyperinsulinemia was reexamined in a small sample of Whites. No association could be demonstrated, and the insertion also failed to segregate with NIDDM in five White pedigrees. The lack of evidence for a role of this insertion by molecular, genetic, and physiological analyses suggests that this RFLP should be used cautiously as a marker for NIDDM, although the sequence data suggested that this region might be predisposed to recombination and mutation.
Mechanism for Underestimation of Isotopically Determined Glucose Disposal
Authors: Yki-Järvinen Hannele , Consoli Agostino , Nurjhan Nurjahan , Young Andrew A , Gerich John E
Published: September 2013

Use of [3H]glucose and a one-compartment model to determine glucose kinetics frequently underestimates the rate of glucose production (Ra). To assess to what extent an isotope effect, a tracer contaminant, or inadequacy of the model was responsible, we measured glucose Ra and forearm clearance of tracer and unlabeled glucose at various concentrations of plasma insulin (∼50, ∼160, and ∼1800 μU/ml) and plasma glucose (∼90, ∼160, ∼250, and ∼400 mg/dl) under steady-state and non-steady-state conditions. Under isotopic steady-state conditions, the clearances of tracer and unlabeled glucose across the forearm were identical, and exogenous glucose infusion rates did not differ significantly from the isotopically determined glucose Ra (10.0 ±1.3 vs. 10.5 ± 1.0 mg ⋅ kg−1 fat-free mass ⋅ min−1, respectively). However, under isotopic non-steady-state conditions, the isotopically determined Ra was significantly lower than the glucose infusion rate (11.5 ± 1.3 vs. 13.7 ± 1.5 mg ⋅ kg−1 fat-free mass ⋅ min−1, respectively, P < .001), and the underestimation was related to the deviation from the isotopic steady state. When [3H]glucose specific activity of plasma samples from experiments with the greatest underestimation of Ra was determined by high-performance liquid chromatography, <7% of the underestimation could be accounted for by a contaminant. These results indicate that inadequacy of the one-compartment model is responsible for underestimation of glucose Ra under non-steady-state conditions and that there is no detectable isotopic effect or appreciable contaminant of [3-3H]glucose. We conclude that under isotopic steady-state conditions, [3-3H]glucose is a reliable tracer for glucose kinetic studies in humans.
Tissue-Specific Alterations in Somatostatin mRNA Accumulation in Streptozocin-Induced Diabetes
Authors: Papachristou Dimitrios N , Pham Khan , Zingg Hans H , Patel Yogesh C
Published: September 2013

In streptozocin-induced diabetes in rats, there is a marked increase in the content and release of immunoreactive somatostatin (SLI) from the pancreas and upper gut. To elucidate whether these SLI changes are associated with alterations in somatostatin gene transcription, we measured somatostatin mRNA (SmRNA) accumulation in these and other SLI-producing tissues. Pancreas, stomach, jejunum, hypothalamus, and cerebral cortex were removed from control rats, 6-wk-diabetic rats, and diabetic rats treated with insulin for 6 wk. Total RNA was isolated by centrifugation through CsCI and fractionated on agarose gels. A sensitive radiodensitometric hybridization assay was used to determine SmRNA levels in absolute amounts by in vitro synthesized sense-strand RNA as a quantitative standard and antisense cRNA as a specific probe. SLI was determined by radioimmunoassay. SmRNA exhibited size heterogeneity between the different control and diabetic tissues. A 2- to 3-fold increase in total SmRNA was found in pancreas and stomach of the diabetic rats that suppressed toward normal with insulin treatment. These two tissues also exhibited significant 1.6- to 2.6-fold increases in SLI, respectively. The remaining tissues showed no diabetes-related changes in SLI or SmRNA. We conclude that in insulinopenic diabetes, tissue SLI and SmRNA accumulation undergo parallel changes; are increased in pancreas and upper gut, reflecting augmented somatostatin synthesis; are reciprocally related to insulin acting directly or indirectly on somatostatin-producing cells; and are unchanged in the lower gut and brain, suggesting tissue-specific regulation of somatostatin gene transcription in diabetes.
Development of a Method for Isolation of Islets From Human Fetal Pancreas
Authors: Kover Karen , Moore Wayne V
Published: September 2013

Three methods for the preparation of islets from human fetal pancreas (17.4 ± 1.2 wk gestational age) were compared. In each method, the pancreases were minced and followed by 1) no collagenase digestion, 2) 5 min of collagenase digestion, or 3) 14 min of collagenase digestion. The culture conditions prevented adherence of the fragments. Culture for 6–7 wk of minced fetal pancreas without collagenase digestion resulted in fragments that were a mixture of cells positive for insulin or glucagon, ducts, necrotic debris, and other unidentified cells with complete degeneration of the acinar cells. Culture of minced pancreas digested for 5 min with collagenase resulted in fragments that superficially appeared to be islets but did not have the size characteristics of human fetal islets and contained fibrous and duct elements not seen in islets. Culture of minced pancreas digested for 14 min with collagenase resulted in islets that were released into the medium and harvested by picking. These islets were morphologically similar to islets of the intact human fetal pancreas and isolated islets from rat neonatal pancreas. These islets and fragments were viable for at least 7–8 wk in culture.
Primary Association of HLA-DQw8 With Type I Diabetes in DR4 Patients
Authors: Owerbach David , Gunn Sheila , Gabbay Kenneth H
Published: September 2013

DNA from 164 Caucasian type I (insulin-dependent) diabetic patients and 200 Caucasian nondiabetic control blood donors were analyzed by the polymerase chain reaction technique for HLA-DR4 and the associated Dw and DQB subtypes of DR4. The DQw8 subtype of HLA-DR4 was associated with type I diabetes in all DR4 subgroups (DR4/3 and DR4/non-3). Dw subtypes of DR4 other than Dw10 did not confer additional association with type I diabetes. Thus, the DQ region appears to provide the primary major histocompatibility association with type I diabetes in most DR4 patients.
Ganglioside Treatment and Improved Axonal Regeneration Capacity in Experimental Diabetic Neuropathy
Authors: Triban Chiara , Guidolin Diego , Fabris Michele , Marini Paolo , Schiavinato Antonella , Donà Massimo , Bortolami Maria Cristina , Giamberardino Luigi Di , Fiori Mario G
Published: September 2013

The efficacy of gangliosides in enhancing axonal regeneration and maturation in the early stages of diabetic neuropathy was assessed by quantitative analysis of immunostained serial sections of the sciatic nerve. Sprague-Dawley rats were made diabetic with a single injection of alloxan (100 mg/kg). One week later they were injected daily intraperitoneally with either a highly purified ganglioside mixture (10 mg/kg) or sterile saline for 4 wk. At the end of the treatment, sciatic nerves were crushed and allowed to regenerate for 1 wk without ganglioside treatment. The animals were then killed, and the nerves were frozen and processed for immunohistochemistry and electron microscopy. The number of regrowing axons was counted with a computerized image-analysis system on cross sections taken at predefined distances along the regenerating stump and stained with monoclonal antibody iC8 specific for the 145,000-M subunit of the neurofilaments. In untreated diabetic animals the number of axons able to regenerate and sustain elongation for ≥13 mm from the crush point was reduced by 40% with respect to control rats. Ganglioside treatment was effective in compensating almost completely for this dramatic reduction. Electron microscopy confirmed that the immunofluorescence counts corresponded to regenerating axons containing neurofilaments. These results suggest that gangliosides are able to compensate for the derangements of axonal transport of cytoskeletal proteins reported in experimental diabetic neuropathy.
Autonomic Neuropathy in BB Rats and Alterations in Bladder Function
Authors: Paro Maurizio , Prosdocimi Marco , Zhang Wei-Xian , Sutherland Garnette , Sima Anders A F
Published: September 2013

In vivo urinary bladder function was examined in BB rats after 4 and 6 mo of diabetes, and the data were correlated with morphometric changes in the pelvic and hypogastric nerves, which constitute the micturition reflex arc. After controlled bladder distension, diabetic animals revealed irregular bladder contractions at frequencies that were reduced to 33% of normal values and with significantly increased amplitudes. The abnormal micturition in diabetic animals was elicited at moderately elevated threshold volumes. These functional abnormalities of the diabetic bladder were associated with a progressive axonopathy of afferent myelinated sensory fibers and later-occurring axonal atrophy of unmyelinated efferent preganglionic fibers. These data suggest that diabetic urinary bladder dysfunction is initiated by a visceral sensory neuropathy involving the afferent limb of the micturition reflex arc.
Increased β-Cell Secretory Responsiveness to Ceruletide and TPA in Streptozocin-Induced Mildly Diabetic Rats
Authors: Okabayashi Yoshinori , Otsuki Makoto , Ohki Atsushi , Tani Satoshi , Baba Shigeaki
Published: September 2013

We examined the effects of various stimuli on immunoreactive insulin (IRI) and glucagon (IRG) release from perfused pancreases isolated from control and streptozocin-induced diabetic (STZ-D) rats. Diabetes was induced by injecting 30 mg/kg STZ into rats fasted for 16–18 h 12–17 days before our experiments. Glucose (11.1 mM) caused a distinct biphasic pattern of IRI release from the control pancreas, whereas the first phase was marginal and the second phase was absent in the diabetic pancreas. Arginine (20 mM)-induced IRI release was similar in both groups, whereas IRG release was greater in the control rats than in the diabetic rats. Thus, this model of STZ-D simulates a certain class of non-insulin-dependent diabetes mellitus (NIDDM). In these diabetic animals, the cholecystokinin (CCK) analogue ceruletide (620 pM) caused a significantly greater increase in IRI release in the presence of 5.6 mM glucose than in the control rats, but ceruletide caused a similar IRG release in both groups. Because CCK and ceruletide stimulate phosphoinositide turnover in pancreatic islets, we examined the effects of carbachol and phorbol ester TPA on IRI release in the presence of 5.6 mM glucose. Carbachol (10 μM), which is thought to generate similar second messengers as ceruletide, induced greater IRI release in diabetic than in control rats. TPA (100 nM) caused a significantly greater increase in IRI release from the diabetic than the control pancreas. Our results demonstrate that the insulin-releasing mechanism involved in protein kinase C activation is enhanced in this model of NIDDM. Considering the diminished glucose sensitivity of islets from STZ-D rats, the augmented insulinotropic effect of CCK may facilitate the maintenance of glucose homeostasis in these animals.
Functional Pancreatic β-Cell Line From SV40 T-Antigen Transgenic Mouse
Authors: Gilligan Ann , Jewett Lorraine , Simon Daniela , Damjanov Ivan , Matschinsky Franz M , Weik Heather , Pinkert Carl , Knowles Barbara B
Published: September 2013

Cell line lgSV195, derived from a pancreatic tumor that arose in an SV40 T-antigen transgenic mouse, retains certain morphological and physiological characteristics of pancreatic β-cells throughout in vitro and in vivo passage. Insulin secretion is stimulated by exposure of these cells to fetal bovine serum and a combination of 3-isobutyl-1-methylxanthine and glutamine but not by concentrations of glucose in the physiological range. Insulin processing appears to be intact. Neither class I nor class II major histocompatibility complex (MHC) antigens are routinely expressed at the cell surface; however, MHC class I– but not class II–encoded gene products are detected after treatment with recombinant interferon-γ (IFN-γ) alone or in combination with tumor necrosis factor. Cytolysis of lgSV195 cells by SV40 T-antigen-specific H-2b-restricted lymphocytes is similarly dependent on IFN-γ pretreatment. These results emphasize that SV40 T-antigen transgenic mice are likely sources of cell lines that retain their differentiated function in vitro. The lgSV195 cell line provides an accessible model in which to investigate the control of gene expression and function of pancreatic β-cells.
Tyrosine Hydroxylase Activity in Sympathetic Nervous System of Rats With Streptozocin-Induced Diabetes
Authors: Schmidt Robert E , Cogswell Barbara E
Published: September 2013

The activity of tyrosine hydroxylase (TOH), the rate-limiting enzyme in norepinephrine biosynthesis, was measured in selected sympathetic ganglia to develop a quantitative measure of sympathetic autonomic neuropathy in streptozocin-induced diabetic rats. Surprisingly, TOH activity was elevated twofold in diabetic prevertebral ganglia innervating the alimentary tract (i.e., superior mesenteric, celiac, and inferior mesenteric), which has terminal processes that develop neuroaxonal dystrophy in this model system. TOH activity of paravertebral ganglia (i.e., superior cervical and stellate) with nonalimentary targets was not increased in the same animals. Increased TOH activity in the prevertebral ganglia 1) developed within the 1st wk of diabetes and persisted for 10 mo, 2) did not represent a change in TOH affinity for d-1,6-methyl-5,6,7,8-tetrahydropterine cofactor, 3) was prevented by both nicotinamide pretreatment and early institution of insulin therapy, and 4) did not develop as a result of classical transsynaptic induction. Pair-feeding experiments confirmed that the most likely cause of increased TOH activity in this system was the marked hypertrophy and hyperplasia of the diabetic bowel resulting from compensatory hyperphagia. We conclude that TOH activity does not represent a suitable marker for sympathetic autonomic neuropathy in this experimental system. Rather, the increase appears to be an example of a selective increase in the synthesis of neurotransmitter enzymes, possibly in response to increased trophic support provided by the expanded target, i.e., the hypertrophic gut. The additional synthetic stress imposed on prevertebral neurons by the expansion of the innervation of the alimentary target coupled with the complex diabetic metabolic milieu may contribute to the development and selective distribution of dystrophic axonopathy to the innervation of the alimentary tract.
Decreased Platelet Phosphoinositide Turnover and Enhanced Platelet Activation in IDDM
Authors: Bastyr Edward J , Kadrofske Mark M , Dershimer R Charles , Vinik Aaron I
Published: September 2013

Individuals with diabetes mellitus may have increased in vivo platelet activity. Abnormal platelet function could contribute to the increased incidence of vascular disease in diabetes mellitus. The biochemical mechanism(s) for platelet hyperactivation is unknown. We examined the hypothesis that platelet phosphoinositide turnover, a key signal-transducing mechanism involved in platelet activation, was abnormal in diabetic subjects. Platelets were harvested from 16 subjects with insulin-dependent diabetes mellitus (IDDM) and 19 healthy, nondiabetic control subjects of comparable age. Plasma β-thromboglobulin (β-TBG), a specific marker of platelet activity in vivo, was increased in IDDM (67.1 ± 7.3 ng/ ml) compared with control (41.0 ± 6.0 ng/ml) subjects (P < .005). [32P]orthophosphate (32P1) incorporation into the individual phosphoinositides and phosphatidic acid (PA) reached isotopie equilibrium by 120 min for IDDM and control subjects. Specific activity (dpm 32P/μg phosphorus) of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was not different between IDDM and control subjects. Under these conditions, basal 32P, incorporation into PIP2 and PIP but not phosphatidylinositol (PI) or PA was significantly lower in IDDM subjects. There was significantly decreased [32P]PIP2 and [32P]PIP hydrolysis and decreased [32P]PA formation in IDDM after platelet stimulation with 4 U/ml human thrombin. There were no differences in [32P]PI hydrolysis between the two groups. The mass of PIP2 was reduced (P < .005) in the platelets from IDDM (0.71 ± 0.23 nmol/109 platelets) compared with control (1.65 ± 0.53 nmol/109 platelets) subjects. Similarly, PIP was lower (P < .001) in IDDM (0.66 ± 0.09 nmol/109 platelets) than in control (2.92 ± 0.43 nmol/109 platelets) subjects. There were no differences in PI and PA mass between the two groups. We conclude that increased platelet activation in IDDM is associated with a decrease in platelet polyphosphoinositide content and hydrolysis of PIP and PIP2. These data suggest that altered phosphoinositide turnover may play a role in abnormal platelet function in IDDM.
Differing Attachment of Diabetogenic and Nondiabetogenic Variants of Encephalomyocarditis Virus to β-Cells
Authors: Kaptur Paulina E , Thomas Donald C , Giron David J
Published: September 2013

The D variant of encephalomyocarditis (EMC-D) virus does not induce the production of interferon (IFN) and produces an insulin-dependent diabetes mellitus (IDDM)-like syndrome in certain mouse strains. In contrast, the B variant (EMC-B) virus, which is serologically identical to EMC-D virus, is a good inducer of IFN and is nondiabetogenic. It has been postulated that IFN may play a major role in determining the ability of these two viruses to infect pancreatic β-cells. However, recent studies have shown that ICR Swiss and BALB/cByJ male mice are not protected by IFN against EMC-D virus-induced IDDM. Furthermore, treatment of these two strains of mice with anti-IFN γ-globulin before infection with EMC-B virus does not result in diabetes. These observations suggest that mechanisms other than the IFN system are involved in determining the ability of the viruses to infect and destroy β-cells. Studies were initiated to identify other mechanisms of action. In this communication, we show that up to six times more EMC-D than EMC-B virus attaches to primary β-cells extracted from male ICR Swiss mice. This difference in ability to attach to β-cells may account for the difference in the diabetic potential of this mouse strain.
Autoregulation of Renal Blood Flow in Streptozocin-Induced Diabetic Rats
Authors: Hashimoto Yasuki , Ideura Terukuni , Yoshimura Ashio , Koshikawa Shozo
Published: September 2013

Autoregulation of renal blood flow (RBF) was studied in male Wistar rats. We studied 11 control rats, 11 rats with severe streptozocin (STZ)-induced hyperglycemia (diabetic group), and 10 moderately hyperglycemie rats made diabetic by injection of STZ but given 2–8 U s.c. insulin daily (insulin-treated group). RBF was measured by an electromagnetic flowmeter during stepwise reduction of renal perfusion pressure 4–8 wk after injection of STZ (older group). RBF autoregulation of the diabetic group was impaired compared with the control group. In the insulin-treated group, autoregulatory capability was less attenuated than in the diabetic group. The average autoregulatory index (ARI) of the diabetic group (0.61 ± 0.05) was greater than that of the control (0.24 ± 0.02, P < .01) and the insulin-treated (0.33 ± 0.07, P < .05) groups. To study the relationship between autoregulation and the duration of diabetes, an autoregulatory study was also made in a group of 22 rats (11 diabetic and 11 control) that were tested 2–3 wk after injection of STZ (younger group). The ARI in the younger diabetic group was smaller than that in the older diabetic group (P < .05). The results suggest that in uncontrolled diabetes RBF fluctuates with blood pressure change, and protection against hypertensive injury of glomerular capillaries may be diminished. Autoregulatory disability develops with time, and insulin treatment diminishes impairment of autoregulation. These findings may also explain the adverse consequences of hypertension on the progression of diabetic nephropathy in poorly controlled diabetes.
Cooperation of Adipocytes and Endothelial Cells Required for Catecholamine Stimulation of PGI2 Production by Rat Adipose Tissue
Authors: Parker Judy , Lane John , Axelrod Lloyd
Published: September 2013

Disturbances of prostaglandin I2 (PGI2, prostacyclin) production by adipose tissue contribute to the pathogenesis of diabetic ketoacidosis and may contribute to the pathogenesis of hypertension and vascular disease. We studied the cellular basis of PGI2 production in adipose tissue, measured as release of 6-keto-PGF1α in response to epinephrine. Adipocytes did not produce PGI2 when nonfat cells were removed by repeated washing. The nonadipocyte cellular constituents of adipose tissue (nonfat cells) did not produce PGI2 in the absence of adipocytes. Both adipocytes and nonfat cells were required for PGI2 production in response to epinephrine. Adipocytes pretreated with 0.2 mM aspirin to inhibit PGH synthase nevertheless promoted PGI2 production when mixed with nonfat cells. Nonfat cells preincubated with aspirin did not produce PGI2 when mixed with adipocytes. The nonfat cells converted arachidonic acid to PGI2 but adipocytes did not. Epinephrine stimulated lipolysis and PGI2 production in a dose-dependent parallel manner, but the responses were distinct above 10−6 M. Characterization of the nonfat cells by fractionation on a Percoli density gradient followed by measurement of angiotensincon vert ing enzyme activity and 6-keto-PGF1α production indicated that the nonfat cells were predominantly vascular endothelial cells. We conclude that catecholamine-stimulated PGI2 production in adipose tissue results from the cooperation of adipocytes and vascular endothelial cells. The adipocytes provide arachidonic acid, which is converted to PGI2 by the vascular endothelial cells. Because adipose tissue is located near blood vessels throughout the body, adipocytes may be an important source of arachidonic acid for vascular endothelial cells in various circumstances in health and disease. Our findings raise the possibility that adipocytes may, under some circumstances, release arachidonic acid into the systemic circulation where it is used by vascular endothelial cells throughout the body to produce PGI2 and other eicosanoids.
Revelation of Specificity of 64K Autoantibodies in IDDM Serums by High-Resolution 2-D Gel Electrophoresis: Unambiguous Identification of 64K Target Antigen
Authors: Bækkeskov Steinunn , Warnock Garth , Christie Michael , Rajotte Ray V , Larsen Peter Mose , Fey Stephen
Published: September 2013

Antibodies in serums from newly diagnosed insulin-dependent (type I) diabetes mellitus (IDDM) patients and individuals experiencing early phases of β-cell destruction specifically immunoprecipitate a minor pancreatic islet cell membrane protein of 64,000 Mr (64K). In this report, we demonstrate the use of two-dimensional (2-D) gel electrophoresis to unambiguously identify the 64K antigen. By nonequilibrium pH-gradient gel electrophoresis in the first dimension and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second dimension, the 64K protein separates into two components, designated α and β, that differ in size but display identical charge heterogeneity. The high resolution of the 2-D method efficiently separates the 64K components from background proteins in immunoprecipitates from crude detergent lysates of islets. The background proteins were identified as major cellular proteins carried nonspecifically through the immunoprecipitation procedure. The high affinity and specificity of the 64K autoantibodies were demonstrated by the exclusive and > 1000-fold purification of this minor protein by immunoprecipitation with IDDM serums. The 2-D analyses did not reveal additional proteins specifically immunoprecipitated by IDDM serums, suggesting that the 64K protein is the only protein antigen specifically and consistently recognized by IDDM autoantibodies in the relatively stringent conditions of immunoprecipitation. Moreover, the 2-D analyses demonstrate that purification of membrane protein fractions from both human and rat islets before the immunoprecipitation efficiently removes background proteins and substantially increases the specificity of 64K autoantibody measurements by traditional methods.
Production of Endothelin 1 by Cultured Bovine Retinal Endothelial Cells and Presence of Endothelin Receptors on Associated Pericytes
Authors: Takahashi Kazuhiro , Brooks Roger A , Kanse Sandip M , Ghatei Mohammad A , Kohner Eva M , Bloom Stephen R
Published: September 2013

Endothelinlike immunoreactivity was detected by radioimmunoassay in medium conditioned by cultured endothelial cells obtained from bovine retinal microvessels (9.2 ± 6.5 pM, n = 4). Sephadex G-25 column chromatography and fast-protein liquid chromatography revealed that most of the endothelinlike immunoreactivity was eluted in an identical position to synthetic endothelin 1. Retinal capillary pericyte-conditioned medium contained 2.9 pM endothelinlike immunoreactivity. In contrast to endothelial cells, retinal pericytes were found to bind endothelin. The dissociation constant and binding capacity were 0.14 nM and 1.5 × 105 sites/cell (n = 3), respectively. These findings suggest that endothelin produced by the retinal endothelial cells binds to the pericytes, adding support to the suggestion that pericytes in the retina may have a musclelike function.
Fasting and Postprandial Concentrations of Somatostatin-28 and Somatostatin-14 in Type II Diabetes in Men
Authors: D'Alessio David A , Ensinck John W
Published: September 2013

Recent evidence suggests that somatostatin-28 (SRIF-28), cleaved from prosomatostatin by cells of the upper intestine, acts as a nutrient-stimulated inhibitor of insulin secretion in healthy men. A role for SRIF-28 in the pathophysiology of diabetes has not been previously explored, although several groups have measured circulating somatostatinlike immunoreactivity (SLI) in diabetic subjects. To investigate the possible mediation of abnormal insulin secretion in diabetes by SRIF-28, plasma levels were measured in 10 non-insulin-dependent diabetic men and 9 age- and weight-matched control subjects. Concentrations of SRIF-14 and SLI were also obtained. Subjects were admitted for study after an overnight fast, blood was collected before and at 30-min intervals for 4 h after a fat meal, and plasma samples were analyzed for SRIF-28 and SRIF-14 by specific methods. Basal glucose levels in the diabetic men were significantly higher than in control subjects (10.2 ± 1 vs. 5.8 ± 0.2 mM), but insulin levels were similar (79 ± 14.2 vs. 93.3 ± 14.2 pM). The diabetic men had significantly lower basal SRIF-28 levels than the control subjects (11.4 ± 0.6 vs. 14.6 ± 1.0 pM, P = 0.017). After fat intake, SRIF-28 levels throughout the 4 h of study were indistinguishable in the two groups (270 vs. 292% of basal). Basal SRIF-14 and SLI levels were not significantly different in the two groups, and SRIF-14 and SLI concentrations rose similarly after the meal. There were no correlations between basal SRIF-28 and glucose or insulin levels. The lower concentrations of the β-cell inhibitor SRIF-28 in diabetic men may represent an adaptation to relative insulin deficiency.
Lack of Growth Hormone Effect on Insulin-Associated Suppression of Insulinlike Growth Factor Binding Protein 1 in Humans
Authors: Conover Cheryl A , Butler Peter C , Wang Michael , Rizza Robert A , Lee Phillip D K
Published: September 2013

Insulinlike growth factor binding protein 1 (IGFBP-1) has been shown to modulate the metabolic and mitogenic actions of the growth hormone (GH)- dependent peptide insulinlike growth factor I. Previous studies showed that levels of IGFBP-1 are regulated by insulin. The relative role of GH in the regulation of IGFBP-1 levels is less well defined and was examined in our study with a contiguous two-part protocol. Overnight (part A) and pre- and post-morning meal (part B) blood samples were obtained from eight healthy adults during a constant infusion of saline (SAL) or 4 μg · kg−1 · min−1 GH. Five of eight subjects were restudied with glucose (GLUC) infused during part B (SAL + GLUC) to match glucose and insulin to levels observed during GH infusion. During SAL infusion, IGFBP-1 levels measured by specific radioimmunoassay showed a marked immediate decline after the evening meal in part A, with a subsequent nocturnal rise of 2.4- to 17.3-fold. GH infusion resulted in a similar meal-induced fall in IGFBP-1 levels but led to a delayed nocturnal rise in IGFBP-1, which was associated with elevated postprandial insulin concentrations. During part B, changes in plasma IGFBP-1 levels showed a similar pattern, with a delayed postprandial increase observed during both GH and SAL + GLUC infusions. The half-life of IGFBP-1 disappearance was calculated at ∼2 h for all three infusion groups. Comparison of venous and arterialized blood samples showed no consistent pattern of difference, arguing against peripheral tissue clearance or compartmentalization as the mechanism for the rapid rise and fall in IGFBP-1 levels. Our studies, the first to measure GH, insulin, and glucose in concurrent sampling and under controlled physiological conditions, support previous investigations suggesting that insulin (not GH or glucose) is the primary regulator of plasma IGFBP-1 levels. Furthermore, we postulate that the observed fluctuations in plasma levels are caused by a direct insulin effect on hepatic IGFBP-1 production.
Biochemical Studies of RT6 Alloantigens in BB/Wor and Normal Rats: Evidence for Intact Unexpressed RT6a Structural Gene in Diabetes-Prone BB Rats
Authors: Crisá Laura , Greiner Dale L , Mordes John P , MacDonald Richard G , Handler Eugene S , Czech Michael P , Rossini Aldo A
Published: September 2013

Lymphocytes bearing the T-lymphocyte differentiation antigen RT6 play an important immunoregulatory role in the development of autoimmune diabetes in BB rats. Immunofluorescence studies suggest that diabetesprone (DP)- but not diabetes-resistant (DR)-BB rat lymphocytes fail to express RT6 antigen during ontogeny. Two alloantigenic forms of the molecule exist, i.e., RT6.1 and RT6.2; both are linked to cell membranes by a phosphatidylinositol (PI) linkage. In these studies, Pl-phospholipase C (PLC) treatment of lymphocytes from BB and normal rats followed by immunoabsorption and sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of released proteins with anti-RT6 allotype-specific monoclonal antibodies was performed. RT6.1 in several nondiabetic rat strains was found to consist of a family of nonglycosylated and variably glycosylated molecules: an N-Glycanase–resistant 24,000- to 26,000- Mr peptide and four N-Glycanase–sensitive peptides of 29,000, 31,000, 33,000, and 34,000 Mr In contrast, RT6.2 was found to be a 24,000- to 26,000-Mr nonglycosylated polypeptide. The electrophoretic pattern of RT6.1 was observed to be the same when the antigen was extracted from W3/25+ (CD4+) versus W3/25- T lymphocytes or from resting versus mitogen-activated cells. A pattern of bands characteristic of the RT6.1 antigen found in normal rat strains was detected after PLC treatment or detergent solubilization of lymphocytes obtained from DR rats. In contrast, no evidence of either RT6 species was found after PLC or detergent treatment of comparable numbers of T lymphocytes from DP-BB rats. Interestingly, T lymphocytes from Wistar-Furth (RT6.2+) × DP (RT6−) F1 crosses were observed to coexpress both RT6.2 and RT6.1 molecules, with the electrophoretic pattern of RT6.1 being similar to that obtained in DR and other rat strains. This study provides biochemical evidence that DP rats may have an intact RT6a structural gene.
Abnormal Redox Status Without Increased Lipid Peroxidation in Sugar Cataract
Authors: Woollard Alison C S , Bascal Zainab A , Armstrong Graeme R , Wolff Simon P
Published: September 2013

In conflict with a previous report, we find that phenolic inhibitors of lipid peroxidation (butylated hydroxytoluene [BHT] and butylated hydroxyanisole [BHA]) do not have significant inhibitory effect on galactosemic cataract formation. This is consistent with the lack of enhancement of stable products of lipid peroxidation (measured by the thiobarbituric acid assay) in the lenses of galactosemic rats. This does not imply that oxidative stress plays no role in galactosemic cataract formation (indeed, we find that galactosemic lens homogenates contain increased amounts of an Fe2+ oxidant, possibly a peroxide), but rather that BHT- and BHA-inhibitable lipid peroxidation specifically has no role to play. In instances where drugs appear to inhibit galactosemic cataract formation, other effects caused by the drugs, e.g., inhibition of feeding or induction of general detoxification pathways, must be considered.
Na+-K+-ATPase and Changes in ATP Hydrolysis, Monovalent Cation Affinity, and K+ Occlusion in Diabetic and Galactosemic Rats
Authors: Tehrani Shahram T , Yamamoto Judi J , Garner Margaret H
Published: September 2013

This study showed that steady-state kinetics of ATP hydrolysis by Na+-K+-ATPase are altered in the BB Wistar diabetic rat and experimental galactosemia. Four days after onset, this change was not evident if NaCNBH3 was omitted during enzyme preparations (indicating reversibility). Ninety days after onset, NaCNBH3 reduction was not necessary to see the change in ATP hydrolysis kinetics (indicating nonreversibility). The change in steady-state ATP hydrolysis was similar to that reported earlier for Na+-K+-ATPase of the lens epithelium and kidney medulla of diabetic individuals and for two in vitro glycosylation models. Our study also showed that the affinities of Na+-K+-ATPase for K+ are altered, and Na+-K+-ATPase-dependent K+ occlusion is inhibited in diabetic and galactosemic animals. Because K+ occlusion is required for efficient K+ transport, this finding supports previous in vitro studies that indicated that glycosylation inhibits pump-dependent K+ transport. Furthermore, our study suggested an irreversible impairment of Na+-K+-ATPase function in the diabetic BB Wistar rat as early as 15 days after onset, even when blood glucose was maintained at 6.7 mM by daily insulin injection.
Is Insulinlike Growth Factor I Associated With Diabetic Retinopathy?
Authors: Dills Diana G , Moss Scot E , Klein Ronald , Klein Barbara E K , Davis Mark
Published: September 2013

Insulinlike growth factor I (IGF-I) is the mediator of the growth-promoting effects of growth hormone and has been suspected of playing a role in the pathogenesis of proliferative diabetic retinopathy (PDR). However, previous attempts to correlate IGF-I levels with PDR have yielded conflicting results. We determined IGF-I levels in a large population-based study of 682 earlyonset (diagnosed before 30 yr of age) adult (≥18 yr old) insulin-taking diabetic subjects. PDR was found in 25% of the population. IGF-I levels were measured by radioimmunoassay. The mean serum level of IGF-I was 277 ± 108 μg/L (mean ± SD). Spearman rank correlations showed statistically significant negative correlations between IGF-I levels and age (r = −0.51, P < 0.0001), duration of disease (r = −0.36, P < 0.0001), and glycosylated hemoglobin (r = −0.09, P < 0.05). There was a significant trend (P < 0.001) toward decreasing risk of PDR with increasing IGF-I. However, after controlling for duration of diabetes, glycosylated hemoglobin, diastolic blood pressure, and the presence of proteinuria and/or creatinine >265 μM in a multiple logistic regression model, IGF-I was not significantly associated with PDR. These data suggest that IGF-I may not be a risk factor for the development of PDR.
Contribution of Impaired Muscle Glucose Clearance to Reduced Postabsorptive Systemic Glucose Clearance in NIDDM
Authors: Gerich John E , Mitrakou Asimina , Kelley David , Mandarino Lawrence , Nurjhan Nurjahan , Reilly James , Jenssen Trond , Veneman Thiemo , Consoli Agostino
Published: September 2013

The reduced postabsorptive rates of systemic glucose clearance in non-insulin-dependent diabetes mellitus (NIDDM) are thought to be the consequence of insulin resistance in peripheral tissues. Although the peripheral tissues involved have not been identified, it is generally assumed to be primarily muscle, the major site of insulin-mediated glucose disposal. To test this hypothesis, we measured postabsorptive systemic and forearm glucose utilization and clearance in 15 volunteers with NIDDM and 15 age- and weightmatched nondiabetic volunteers. Although systemic glucose utilization was increased in NIDDM subjects (14.5 ± 0.5 vs. 11.2 ± 0.2 μmol · kg−1 · min−1, P < 0.001), systemic glucose clearance was reduced 1.40 ± 0.06 vs. 2.13 ± 0.05 ml · kg−1 · min−1, P < 0.01). Although forearm glucose utilization was increased in NIDDM subjects (0.663 ± 0.058 vs. 0.411 ± 0.019 μmol · dl−1 · min−1, P < 0.001), forearm glucose dl−1 clearance was reduced (0.628 ± 0.044 vs. 0.774 ± 0.037 ml · L−1 · min−1, P < 0.01). However, extrapolation of forearm data to total-body muscle indicated that impaired clearance reduced muscle glucose disposal by only 61 ± 21 μmol<min, whereas impaired systemic clearance reduced systemic glucose disposal by 662 ± 82 μmol<min. Thus, impaired muscle glucose clearance accounted for <10% of the reduced systemic glucose clearance in NIDDM subjects. Therefore, we conclude that muscle insulin resistance plays only a minor role in the reduced systemic glucose clearance found in NIDDM in the postabsorptive state and propose that reduced brain glucose clearance is largely responsible.
Hepatic and Extrahepatic Responses to Insulin in NIDDM and Nondiabetic Humans: Assessment in Absence of Artifact Introduced by Tritiated Nonglucose Contaminants
Authors: Butler Peter C , Kryshak Edward J , Schwenk W Frederick , Haymond Morey W , Rizza Robert A
Published: September 2013

It is well established that patients with non-insulindependent diabetes mellitus (NIDDM) are resistant to insulin. However, the contribution of hepatic and extrahepatic tissues to insulin resistance remains controversial. The uncertainty may be at least in part due to errors introduced by the unknowing use in previous studies of impure isotopes to measure glucose turnover. To determine hepatic and extrahepatic responses to insulin in the absence of these errors, steady-state glucose turnover was measured simultaneously with [6-3H]- and [6-14C]glucose during sequential 5- and 4-h infusions of insulin at rates of 0.4 and 10 mil · kg−1 · min−1 in diabetic and nondiabetic subjects. At low insulin concentrations, [6-3H]- and [6-14C]glucose gave similar estimates of glucose turnover. Hepatic glucose release was equal to but not below zero in the nondiabetic subjects, but persistent glucose release (P < 0.001) and decreased glucose uptake (P < 0.001) was observed in the diabetic patients. At high insulin concentrations, both isotopes underestimated glucose turnover during the 1st h after initiation of the highdose insulin infusion. More time (P < 0.05) was required to reachieve steady state in NIDDM than nondiabetic subjects. At steady state, [6-3H] but not [6-14C]glucose systematically underestimated (P < 0.05) glucose turnover in both groups due to the presence of a tritiated nonglucose contaminant. The percentage of radioactivity in plasma due to tritiated contaminants was linearly related to turnover. When plasma [6-3H]glucose specific activity was corrected for the presence of the contaminant in each subject, both [6-3H]- and [6-14C]glucose indicated that diabetic patients had hepatic and extrahepatic insulin resistance. We conclude that in these experiments, although the presence of a low percentage of tritiated nonglucose contaminant in the tracer may alter estimates of the severity of hepatic and extrahepatic insulin resistance in patients with NIDDM, it does not obscure their presence