bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Sorbinil, Dietary myo-Inositol Supplementation, and Insulin on Resolution of Neuroaxonal Dystrophy in Mesenteric Nerves of Streptozocin-Induced Diabetic Rats
Authors: Schmidt Robert E , Plurad Santiago B , Coleman Bill D , Williamson Joseph R , Tilton Ronald G
Published: September 2013

Previous studies indicate that experimental diabetic autonomic neuropathy can be largely prevented by initiating therapy at the onset of diabetes. More clinically relevant, however, is the ability of therapy to reverse established neuropathy produced by long-standing diabetes. We have examined the effect of selected therapies on established neuroaxonal dystrophy (NAD) in ileal mesenteric nerves, a rat model of diabetic autonomic neuropathy. Groups of 3-mo-old rats were made diabetic with streptozocin (STZ-D) and allowed to survive untreated for 5 mo, at which time they were begun on sorbinil, dietary myo-inositol, and daily insulin therapies or left untreated for an additional 2 or 4 mo. Ultrastructural evidence of NAD was demonstrated in ileal mesenteric nerves of rats with untreated 5-mo STZ-D and increased with the duration of diabetes. No lesions were demonstrated in control rats of any age. myo-inositol or sorbinil administration failed to alter the severity of diabetes as measured by its metabolic indices. Institution of sorbinil or insulin treatment at 5 mo of diabetes prevented the increase in, but did not normalize, NAD at 7 or 9 mo. Dietary myo-inositol failed to significantly reverse established NAD or prevent its initial development. Morphometric examination of ileal mesenteric nerves demonstrated a decrease in the number of axons comprising each diabetic Schwann cell unit, suggestive of chronic cycles of axonal degeneration and regeneration. This parameter, clearly abnormal by 5 mo of diabetes, was not normalized by 2 or 4 mo of insulin, sorbinil, or myo-inositol treatment. These observations indicate that treatment with insulin or an aldose reductase inhibitor, but not myo-inositol, initiated after the development of structural axonopathy significantly inhibited progression of NAD for the duration of treatment. (ABSTRACT TRUNCATED AT 250 WORDS)
Endocrine-Metabolic Function in Remission-Phase IDDM During Administration of Cyclosporine
Authors: Dupre John , Jenner Morris R , Mahon Jeffrey L , Purdon Cameron , Rodger N Wilson , Stiller Calvin R
Published: September 2013

We have studied the endocrine-metabolic status of patients in non–insulin-receiving (NIR) remission of insulin-dependent diabetes mellitus (IDDM) within 6–60 mo of diagnosis during administration of cyclosporine, in comparison with nondiabetic subjects. IDDM patients in NIR remission were recognized when target glycemic control (plasma glucose and mean capillary blood glucose levels <7.8 mM before meals) was maintained without administration of insulin for at least 2 wk. In so-called isoglycemic tests, 50 g glucose was administered orally, and the glycemic curve was simulated in a subsequent study by programmed intravenous infusion of glucose. Under these conditions, the subjects with diabetes exhibited obvious glucose intolerance: acute β-cell responses to intravenous glucose were virtually absent but significant, although subnormal responses were present after oral glucose. The responses of plasma immunoreactive gastric inhibitory polypeptide to oral glucose were normal. After bolus intravenous injections of glucose, the patients with diabetes again exhibited glucose intolerance; acute responses of immunoreactive insulin (IRI) and C-peptide were present, although grossly obtunded. On intravenous infusion of arginine (30 g in 30 min), the patients with diabetes showed substantial but subnormal increases in plasma IRI and C-peptide. Intravenous infusion of arginine elicited increments of plasma immunoreactive glucagon (IRGI) in both groups, and this response was slightly exaggerated in the patients with diabetes. On ingestion of a standard mixed meal (Sustacal) delivering 600 cal, there was a modest but significantly greater increase in plasma glucose levels in the diabetic subjects. This was associated with substantial increases in plasma IRI and C-peptide in both groups, whereas the responses of both hormones were significantly reduced in the patients with diabetes. Plasma IRGI did not change in response to Sustacal in nondiabetic or NIR subjects. Thus, patients in NIR remission of diabetes as defined exhibited grossly subnormal responses of the μ-cells to intravenous glucose, with relatively well-preserved responses to oral glucose, intravenous arginine, and a mixed meal. Evidence is cited that cyclosporine does not affect the endocrine-metabolic responses examined in this study. It appears that patients in this condition revert to a state similar to that in the interval shortly preceding manifestation of overt hyperglycemia.
Dioctanoylglycerol Regulation of Cytosolic Ca2+ by Protein Kinase C-Independent Mechanism in HIT T-15 Islet Cells
Authors: Thomas Thomas P , Martin Dennis B , Pek Sumer Belbez
Published: September 2013

The effect of activators of protein kinase C (PKC) on cytosolic concentration of free Ca2+ ([Ca2+]i) was assessed in insulin-secreting islet cell line HIT T-15. Dioctanoylglycerol (DiC8) and 12-O-tetradecanoylphorbol-13-acetate (TPA) evoked activation of PKC. Basal [Ca2+]i was 65–160 nM. DiC8 induced triphasic increases in [Ca2+]i; phase 2 was the most prominent and consistent one. With 25–150 μM DiC8, [Ca2+], increased in a dose-dependent manner during phase 2; half-maximal stimulatory dose was 53 μM. TPA did not evoke any increase in [Ca2+]i. Staurosporine, sphingosine, and H7, which are inhibitors of PKC, did not block DiC8-induced rise in [Ca2+]i. DiC8-induced rise in [Ca2+]i was also seen in cells that had been depleted of PKC by prior exposure to TPA. DiC8-induced rise in [Ca2+]i still occurred in the presence of the Ca2+-channel blocker verapamil or when the extracellular Ca2+ had been reduced from 2.5 mM to 30 nM by EGTA. Three immediate metabolites of DiC8, monooctanoylglycerol, octanoate, and glycerol, did not evoke any change in [Ca2+]i. Monooleoylglycerol and R59022, which induce increases in endogenous diacylglycerol (DAG) by inhibiting DAG kinase, evoked increases in [Ca2+]i. DiC8 did not cause any change in inositol 1,4,5- trisphosphate levels. DiC8 evoked biphasic increases in insulin release; the second-phase increase in [Ca2+]i preceded the late phase of insulin secretion. Exogenous DAGs should be used with caution in assessing PKC function. Changes in the generation of DAGs must be included among the mechanisms by which Ca2+ homeostasis is regulated in islet cells. Endogenous DAGs may amplify insulin secretion not only via activation of PKC but also via increases in [Ca2+]i.
Scintigraphic Studies in Rats: Kinetics of Insulin Analogues Covering Wide Range of Receptor Affinities
Authors: Jensen Ivan , Kruse Viggo , Larsen Ulla Dahl
Published: September 2013

Whole-body kinetics of 123I-labeled human insulin and five insulin analogues were investigated by scintigraphic studies in rats. The amino acid substitutions and the relative receptor affinities (RAs), determined by binding to HepG2 cells, were: GluB12, des-B30 insulin, RA 0.15%; AspB9,GluB27 insulin, RA 18%; AspB28 insulin, RA 80%; AspB10 insulin, RA 327%; and HisA8,HisB4,GluB10,HisB27 insulin, RA 687%. All analogues were compared with human insulin (RA 100%). The analogue with RA 0.15% showed a significantly slower disappearance in the heart window, no liver uptake, and the greatest kidney radioactivity, the latter probably caused by high plasma concentrations. The low-affinity analogue (RA 18%) reached a surprisingly high hepatic peak value, although significantly lower than insulin. Kidney radioactivity was higher than for insulin. The analogue with RA 80% showed liver and kidney radioactivities that were not significantly different from those of insulin. The two high-affinity analogues (RAs 327 and 687%) showed peak liver radioactivities not significantly different from insulin. However, liver radioactivity after the peaks declined significantly more slowly. Compared with insulin, the kidney radioactivity curves were not significantly different. We conclude that high-affinity insulin analogues will bind to any available receptor that, because of the large number of receptors in the periphery and the distribution of cardiac output, favors extrahepatic elimination. In contrast, low-affinity analogues bind to receptors several times before they are eliminated. This leads to recirculation and, thus, hepatic elimination due to the high receptor density there. It follows that hepatopreferential binding cannot be expected solely by use of an insulin analogue with a particular receptor affinity.
Pathobiology of Endothelial and Other Vascular Cells in Diabetes Mellitus: Call for Data
Authors: Lorenzi Mara , Cagliero Enrico
Published: September 2013

Because the pathogenetic understanding of diabetic vascular complications remains fragmentary, and even the best available interventions may prove insufficient to arrest the progression of certain lesions, new avenuses of investigation should be pursued. One of these should be the early in vivo investigation of the cells that endure the pathological process (pericytes and endothelial and mesangial cells), preferably in humans. The abnormal vascular architecture (i.e., capillary acellularity, microaneurysms, thickened basement membranes, and mesangial expansion) and the hemostatic and hemodynamic alterations observed in diabetes point to an adaptive/maladaptive replicative and biosynthetic program triggered by the metabolic perturbation, but positive documentation of cellular changes in vivo remains grossly insufficient. Critical review of current knowledge of microangiopathy permits elaboration of specific questions that, with the tools provided by the new molecular technology, may be posed about vascular cells in situ. Knowing whether and how the cell types involved in the vascular complications of diabetes modify their differentiated functions may offer novel targets for intervention and, most important, should provide a much needed “sounding board” against which to test the viability and refine the focus of pathogenetic hypotheses.
Comparison of Effects of Neuropeptide Y and Norepinephrine on Insulin Secretion and Vascular Resistance in Perfused Rat Pancreas
Authors: Skoglund Gunnar , Gross René A , Bertrand Gyslaine R , Ahrén Bo , Loubatières-Mariani Marie-Madeleine
Published: September 2013

Neuropeptide Y (NPY) and norepinephrine (NE) behave like cotransmitters in intrapancreatic adrenergic nerves. Therefore, in the isolated rat pancreas, we 1) studied and compared the effect of increasing concentrations of NE and NPY given alone on insulin secretion induced by 8.3 mM glucose and on pancreatic vascular flow rate and 2) investigated the effects of combinations of NPY and NE at low concentrations. NE induced a dose-dependent inhibition of insulin release between 1 and 50 nM (max 80%); β-cells appeared sensitive to NPY at 0.1 nM, but the maximal reduction of insulin release was comparatively weak (25–30%) at 10 nM. The study of the effect of combinations of NE and NPY at different concentrations suggests that the two neurotransmitters act in an additive way to inhibit insulin secretion. NPY (0.1–10 nM) induced a marked dose-dependent reduction of pancreatic outflow rate with a biphasic pattern between 1 and 10 nM. On the other hand, at low concentrations (1 and 2 nM), NE induced a progressive increase in pancreatic outflow rate; a clear but transient decrease could only be observed at 50 nM before a secondary increase. A combined treatment with two effective concentrations of NPY (0.1 nM) and NE (1 nM) resulted in a progressive reversal by NE of NPY's vasoconstrictive effect. We conclude that β-cell function and pancreatic vascular resistance were not affected in the same way by NPY and NE: NPY appeared to be a strong vasoconstrictor but only a slight inhibitor of insulin release, whereas NE strongly inhibited β-cells and was a comparatively poor vasoconstrictor, even able to increase vascular output at low concentration. When combined at the concentrations used, they showedinhibitory additive effects on insulin secretion but exerted opposite effects and were antagonistic on pancreatic flow rate.
Multifactorial Origin of Hypoglycemic Symptom Unawareness in IDDM: Association With Defective Glucose Counterregulation and Better Glycemic Control
Authors: Clarke William L , Gonder-Frederick Linda A , Richards Fredrick E , Cryer Philip E
Published: September 2013

To assess potential relationships between unawareness of hypoglycemic symptoms and both defective glucose counterregulation and therapy-associated altered glycemic thresholds, symptoms and hormonal responses to hypoglycemia were quantitated during standardized insulin infusion tests in 41 patients with insulin-dependent diabetes mellitus (IDDM). The glycemic thresholds for both neurogenic and neuroglycopenic symptoms (and those for both epinephrine and pancreatic polypeptide release) were at lower plasma glucose concentrations in both patients with defective (n = 9, 22%) and those with adequate glucose counterregulation and, among the latter, in patients with lower compared with higher glycosylated hemoglobin levels. The data are consistent with the concept that both defective glucose counterregulation and improved glycemic control contribute to excessive hypoglycemia in IDDM by reducing awareness of symptoms of developing hypoglycemia and by impairing physiological defenses against hypoglycemia. Thus, hypoglycemic symptom unawareness is multifactorial in origin and may be partly reversible.
Effects of Lipid on Basal Carbohydrate Metabolism in Normal Men
Authors: Boden Guenther , Jadali Fayegh
Published: September 2013

We investigated the effects of infusion of a 20% triglyceride emulsion plus heparin (LH) on carbohydrate (CHO) metabolism during basal insulin and glucose turnover conditions in normal male subjects. In study 1, LH or saline was infused at 0.5 and 1.5 ml/min for 2 h each. Plasma free fatty acids rose from ∼0.4 to 0.8 mM with the low rate and to between 1.6 and 2.1 mM with the high rate. Similar increases occurred in plasma concentrations of glycerol, acetoacetate, and β-hydroxybutyrate. LH infusions resulted in significant increases in C-peptide concentrations but had no effects on any of the other measured parameters of CHO metabolism. In study 2, LH or saline was infused as in study 1, but the compensatory insulin release was prevented by intravenous infusion of somatostatin and replacement of basal insulin and glucagon concentrations. This resulted in significant increases in plasma glucose (from 4.5 ± 0.2 to 7.1 ± 0.6 mM, P < 0.001) and hepatic glucose output (from 9.0 ± 1.5 to 11.3 ± 1.4 μmol · kg−1 · min−1, P < 0.05) and a decrease in glucose clearance (from 2.32 ± 0.13 to 1.44 ± 0.11 ml · kg−1 · min−1, P < 0.05). We conclude that lipids can have adverse effects on CHO metabolism under basal conditions and that healthy individuals can compensate for these effects with additional secretion of insulin.
Uncoupling of Attenuated myo-[3H]Inositol Uptake and Dysfunction in Na+-K+-ATPase Pumping Activity in Hypergalactosemic Cultured Bovine Lens Epithelial Cells
Authors: Cammarata Patrick R , Tse Daniel , Yorio Thomas
Published: September 2013

Attenuation of both the active transport of myo-inositol and Na+-K+-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na+-K+-ATPase and myo-inositol uptake. The Na+-K+-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-[3H]inositol was lowered after 20 h of incubation in galactose and remained significantly below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-[3H]inositol uptake. No significant difference in the rates of passive efflux of myo-[3H]inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil. These observations support the contention that the aldose reductase reaction or formation of the product of the aldose reductase reaction disrupted lens cell myo-inositol uptake. Although the coadministration of sorbinil to the galactose medium protected against the attendant decrease in transport activity, the cessation of exposure of BLECs to galactose was sufficient stimulus to normalize myo-inositol uptake. Moreover, these observations suggested that the deficit in myoinositol uptake elicited by exposure to galactose was reversible and occurred independently of changes in Na+-K+-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.
Conversion of Oral Glucose to Lactate in Dogs: Primary Site and Relative Contribution to Blood Lactate
Authors: Youn Jang H , Bergman Richard N
Published: September 2013

We evaluated the relative contribution of oral glucose to arterial lactate and the relative role of the splanchnic bed in converting glucose to lactate in normal healthy dogs. After an oral glucose load (1.2 g/kg) spiked with [U-14C]glucose (16.9 μCi/kg; protocol 1, n = 7), arterial blood lactate increased from 0.43 ± 0.03 mM at basal to a peak of 1.04 ± 0.07 mM at 45 min and then slowly decreased to 0.47 ± 0.07 mM at 240 min. Arterial blood [14C]lactate peaked at 60 min and then decreased slowly to ∼35% of the peak at 4 h. When arterial blood lactate peaked at 45 min, the proportion of arterial lactate that was derived from oral glucose was 34 ± 3%. The integrated area under the curve of lactate derived from exogenous glucose was 40 ± 2% of that of total lactate. The splanchnic bed released lactate and [14C]lactate during the initial 2 h after oral [14C]glucose. Thus, the splanchnic bed apparently contributed to the conversion of exogenous glucose to lactate. In the matched experiments (protocol 2, n = 5), dogs were given the same amount of oral glucose but no [14C]glucose, and [U-14C]lactate was infused into the right atrium to match the splanchnic [14C]lactate release from the first experiment. Despite a well-matched splanchnic [14C]lactate contribution, arterial concentrations of [14C]lactate were markedly lower in protocol 2 compared with protocol 1. The integrated area under the [14C]lactate profile in protocol 2 was only 11 ± 1% of that in protocol 1. These results indicate that the splanchnic bed is responsible for only 11% of arterial blood lactate that was derived from oral glucose. We concluded that 1) after oral glucose loading, a major portion of circulating lactate has its origin not in exogenous glucose but in endogenous sources, and 2) the splanchnic bed is not the major site of oral glucose conversion to lactate after glucose ingestion.
Initiation of Rejection of Established Islet Allografts by Third-Party Thyroid Allografts and Splenic Dendritic Cells
Authors: Kover Karen , Moore Wayne V
Published: September 2013

Due to concerns of cross-reactivity between renal and islet allografts in initiation of rejection, we determined the ability of donor-specific and third-party splenic dendritic cells (DCs) and thyroids (whole-organ transplant) to initiate rejection of established islet allografts. Purified islets from neonatal F-344 (RT1Lv1) rats were transplanted bilaterally under the kidney capsule of Wistar-Furth (W/F, RT1u) rats without immunosuppression. The islet allografts were not rejected by 21 days posttransplantation. On day 22, freshly isolated or cultured DCs were injected intraperitoneally into the host. Both freshly isolated and cultured donor-specific (F-344) and some third-party (Buffalo, RT1b; ACI, RT1a) DCs initiated rejection of the islets as indicated by lymphocyte infiltration and destruction of the allograft. DCs, whether freshly isolated or cultured for 8 days from the recipient strain (W/F) and one third-party rat (Brown Norway, RT1n), did not initiate rejection. Splenic DCs from the Lewis (RT1l) rat, which has the same class I and II antigen haplotype as F-344 islet donor rats, also initiated rejection. Only 103–104 DCs isolated from the spleen of donor rats were required to initiate rejection of the allograft. In a parallel series of W/F rats with islet allografts, a thyroid (half lobe) from the islet donor strain (F-344), recipient strain (W/F), or third-party rat (Buffalo, Brown Norway, or ACI) was inserted under the kidney capsule at 22 days post–islet transplantation. At 35 days, all thyroids and most islet allografts exhibited active or complete rejection after thyroid transplant from Buffalo, F-344, or ACI rats. Islet allografts challenged by thyroids from recipient or Brown Norway rats did not show signs of rejection. Intraperitoneal injection of donor-specific buffy coat cells from donor blood or heat-killed cells did not initiate rejection. The cross-reactivity of splenic DCs and thyroid allografts from donor-specific and third-party strains in initiation of islet allograft rejection suggests that a subsequent transplantation may initiate an immune response in an established allograft. This may be important in selection of islets and timing of transplantation into individuals with established renal or islet allografts.
NIT-1, a Pancreatic β-Cell Line Established From a Transgenic NOD/Lt Mouse
Authors: Hamaguchi Kazuyuki , Gaskins H Rex , Leiter Edward H
Published: September 2013

NOD/Lt mice harboring a hybrid rat insulin-promoter/SV40 large T-antigen gene spontaneously develop β-cell adenomas. NIT-1 is a pancreatic β-cell line established from one of these transgenic mice. Immunocytochemical staining of passage 18 cells showed most contained insulin, with <5% containing glucagon, and none containing pancreatic polypeptide or somatostatin. Glucagon content radioimmunoassayed in cell extracts was only 0.27% of the insulin content. Two-hour insulin secretion at 16.5 mM glucose was 638 ng/106 cells (41% of intracellular content) compared to only 1.3 ng glucagon (32% of intracellular content). Stimulated insulin secretion was consistently observed in response to 11 and 16.5 mM glucose between passages 11 and 19. At passage 19, both theophylline and tolbutamide stimulated insulin secretion at 5.5 mM glucose. Northern-blot analysis confirmed high levels of insulin mRNA but only trace glucagon mRNA and undetectable somatostatin mRNA. Interferon-γ (IFN-β)–induced MHC class I RNA expression was correlated with markedly increased antigen expression at the cell surface. Similarly, a MHC-linked “occult” class I–like antigen detected by Cr release assay only after exposure of standard NOD/Lt islet cells to IFN-γ was strongly induced by IFN-γ in NIT-1 cells. Cell surface MHC class II antigen was not constitutively expressed on NIT-1 cells and could not be detected after IFN-γ incubation, despite demonstration of IFN-β-induced Aa, Ab, and li invariant-chain RNA transcripts. Similarly IFN-γ induction of intercellular adhesion molecule 1 (Icam-1) transcripts was not accompanied by demonstrable cell surface expression of ICAM-1 antigen. NIT-1 cells exhibited ultrastructural features typical of differentiated mouse β-cells, including numerous p-granules. An unusual finding was that NIT-1 cells were shedding mature type C retrovirus from their surfaces. Because this cell line represents a source of immortalized NOD β-cell genome, it will be useful for a wide variety of studies dealing with β-cell antigens.
Validation and Evaluation of Test for Sympathetic Cholinergic Function in Diabetes Mellitus
Authors: Pestell Richard G , Kirsner Richard L G , Best James D
Published: September 2013

Skin potential response (SPR), an electrodermal measure of sudomotor nerve function, was shown in this study to examine specifically sympathetic cholinergic fibers by abolition of the response during atropine infusion but not during propranolol infusion. The difference between responses in the left and right arms (SPR-D) was used to assess autonomic nerve function in 136 patients with diabetes and 52 control subjects. In 82% of the diabetic population (112 of 136), SPR-D was >2SD above the mean control response compared with 42% (57 of 137) >2SD below the mean control age-related value for a standard autonomic test predominantly of parasympathetic function, the R-R interval variation with breathing. Of 15 patients with clinical diabetic autonomic neuropathy, SPR wave forms were bizarre or absent in 5 patients compared with 18 of 121 patients′ without clinical autonomic neuropathy (χ 2 = 3.5, P = 0.062). Measurement of SPR-D provides an easily determined measure of sympathetic cholinergic nerve function and may be a useful component of a group of tests for autonomic nerve function in diabetes.
Atrial Natriuretic Peptide and Response to Changing Plasma Volume in Diabetic Nephropathy
Authors: Lieberman Jonathan S , Parra Lilia , Newton Lynne , Scandling John D , Loon Nicholas , Myers Bryan D
Published: September 2013

We evaluated the renal and hormonal responses to volume expansion induced by water immersion in subjects with diabetic nephropathy (n = 12) and in healthy control subjects (n = 9). Immersion induced similar average increments in sodium excretion (±223 vs. 176 μmol/min) and comparable decrements in renovascular resistance (RVR; –15 vs. – 16 U). However, whereas the control subjects responded uniformly, the response among diabetic subjects was highly variable, with a subset of patients exhibiting paradoxical antinatriuresis and vasoconstriction. Immersion was associated with marked elevation of atrial natriuretic peptide (ANP) in plasma of diabetic versus control subjects (61 ± 9 vs. 19 ± 2 pM, respectively; P < 0.001). Yet for each picomolar increment in plasma ANP during immersion, the corresponding increases in urinary excretion of cyclic guanosine monophosphate (26 vs. 279 pmol/min) and sodium (9 vs. 47 μmol/min) and the reciprocal lowering of RVR (0.7 vs. 1.9 U) were blunted in the diabetic versus control group. Volume contraction in the postimmersion period was associated with disproportionate antinatriuresis and renal vasoconstriction in the diabetic group, despite a persistent elevation of ANP (29 ± 2 vs. 16 ± 2 pM, P < 0.01). We propose that renal insensitivity to ANP in diabetic nephropathy could contribute to altered vasoreactivity and abnormal excretory responsiveness to changing plasma volume. Blunted natriuresis in response to ANP release and enhanced sodium retention during volume contraction could account for the expanded extracellular fluid volume that has consistently been reported to accompany the development of diabetic nephropathy.
Transplantation of Purified Islet Cells in Diabetic Rats: II. Immunogenicity of Allografted Islet β-Cells
Authors: Pipeleers Daniel G , Pipeleers-Marichal Miriam , Vanbrabandt Bart , Duys Sykvie
Published: September 2013

This study examines whether the survival of allografted rat islet β-cells is influenced by the presence of other pancreatic donor cells. Grafts (RT1u/l) of different cellular composition were intraportally transplanted in streptozocin-induced diabetic rats (RT1n/n). All grafts corrected the diabetic state within 3 days. Implants of freshly isolated islets contained various endocrine and nonendocrine cell types; they became diffusely infiltrated within 1 wk and were completely destroyed within 2 wk. A 4-day culture period did not lead to major changes in the cellular composition of the islets or in their survival as allograft. Islet cell aggregates prepared after islet dissociation and cell purification were less acutely infiltrated and less rapidly rejected. Aggregates composed of sorted MHC class II–negative cells maintained basal normoglycemia in 3 of 5 recipients for 5 wk but only in 1 of 5 for 20 wk. Aggregates of purified islet β-cells remained relatively free of diffuse infiltrations during the 1st wk and preserved the normalized state in 7 of 13 recipients for 5 wk; after 20 wk, 6 of 13 were still aglucosuric, but 40% of the implants were diffusely infiltrated and depleted of insulin. Reaggregation of purified islet β-cells with purified islet endocrine non–²-cells promoted their long-term survival as allograft: 11 of 13 recipients of mixed islet endocrine cells maintained normal basal glycemia over 20 wk; their implants contained relatively constant insulin reserves and remained virtually devoid of diffuse infiltrations. These results demonstrate that techniques aiming at the elimination of surface MHC class II–positive cells are less successful in preparing rat islet allografts of low immunogenicity than methods of positive cell selection. Pure islet β-cells are immunogenic as an allograft but illicit a milder and less-acute immune attack than undissociated islet tissue. Nonendocrine and damaged islet cells are suspected of enhancing the rapidity and intensity of the cytotoxic reaction. Survival of allografted β-cells is markedly prolonged by the presence of islet endocrine non–²-cells within the graft. The mechanisms underlying this effect have not yet been elucidated; they may involve immune and metabolic interactions of the endocrine non–²-cells. We conclude that purification of islet endocrine cells represents a new and powerful method for preparing insulin-producing allografts that can survive in hosts without pharmacological immunosuppression.
Trans-Hydroxyl Group Configuration on Carbons 2 and 3 of Glucose: Responsible for Acute Inhibition of myo-Inositol Transport?
Authors: Yorek Mark A , Stefani Mark R , Dunlap Joyce A , Ro Kee S , Davidson Eric P
Published: September 2013

Cultured neuroblastoma, cerebral μvessel endothelial, and retinoblastoma cells were used to examine the mechanism of acute inhibition by D-glucose of myo-inositol uptake. Acute exposure of the cells to 30 mM D-glucose caused a significant decrease in Na+-dependent myo-inositol uptake in all three cell types. The effect of D-glucose to acutely inhibit myo-inositol uptake was dependent on the extracellular glucose concentration and was not reversed by sorbinil. 2-Deoxy-D-glucose (30 mM), 3-O-methyl-D-glucose (30 mM), and cytochalasin B (100 μM) did not acutely inhibit myo-inositol uptake. These data suggest that the hydroxyl groups on carbons 2 and 3 of D-glucose, which in a Haworth projection appear trans to each other, are important for inhibitory activity. Other monosaccharides (30 mM) having a similar 2,3-trans–diol configuration, L-glucose, D- and L-fucose, D- and L-galactose, D- and L-xylose, and D-arabinose, all to varying degrees significantly inhibited myo-inositol uptake. In all cases, the L-isomers were more potent inhibitors of myo-inositol uptake than the corresponding D-isomers. Monosaccharides (30 mM) having hydroxyl groups on carbons 2 and 3 in a cis configuration, D-mannose, L-rhamnose, D-allose, and D-ribose, did not acutely inhibit myo-inositol uptake. Replacing the hydroxyl group with a fluorine on carbons 2 or 3 of D-glucose negated its inhibitory activity of myo-inositol uptake. In contrast, replacing the hydroxyl group with a fluorine on carbon 6 of D-glucose did not block its inhibition of myo-inositol uptake. 4-Fluoro-4-deoxy-D-glucose did not block myo-inositol uptake by neuroblastoma cells but was almost as effective as D-glucose in inhibiting myo-inositol uptake by cerebral microvessel endothelial and retinoblastoma cells. These data suggest that the trans diol on carbons 2 and 3 of D-glucose is necessary for 4-Fluoro-4-deoxy-D-glucose did not block myo-inositol uptake by neuroblastoma cells but was almost as effective as D-glucose in inhibiting myo-inositol uptake by cerebral microvessel endothelial and retinoblastoma cells. These data suggest that the trans diol on carbons 2 and 3 of D-glucose is necessary for this site.
Evidence for Dual Control Mechanism Regulating Hepatic Glucose Output in Nondiabetic Men
Authors: Clore John N , Glickman Penny S , Helm Shirley T , Nestler John E , Blackard William G
Published: September 2013

We previously reported a fall in hepatic glucose output (HGO) during sleep accompanied by reductions in glucose utilization (Rd) and free fatty acids (FFAs). This study was undertaken to determine the potential role of changes in Rd and FFA on HGO in nondiabetic men. To determine if the fall in HGO during sleep could be reversed by FFA elevation, seven nondiabetic men underwent [3-3H]glucose infusions from 2200 to 0800, with heparin (90 mU · kg−1 · min−1) added at 0200. Glucose appearance (Ra) fell from 11.7 ± 1.1 at 2430 to 8.9 ± 0.8 μmol · kg−1 · min−1 (P < 0.05) at 0200. The fall in Ra was associated with decreases in FFA (0.57 ± 0.10 to 0.48 ± 0.07 mM) and glycerol (0.08 ± 0.01 to 0.06 ± 0.01 mM). Infusion of heparin significantly increased FFA and glycerol (1.09 ± 0.21 and 0.11 ± 0.01 mM, respectively, P < 0.01) and resulted in a significant fall in plasma alanine, suggesting that gluconeogenesis had been increased. However, rates of glucose turnover were indistinguishable from overnight studies without heparin. In additional studies (n = 6), intralipid and heparin-induced FFA elevation (from 0.61 ± 0.07 to 0.95 ± 0.05 mM, P < 0.01) stimulated gluconeogenesis ([U-14C]alanine to glucose) twofold (188 ± 22% increase compared to 114 ± 6% in saline control studies, P < 0.01). However, despite increasing gluconeogenesis, overall HGO did not change (10.6 ± 0.5 vs. 10.7 ± 0.6 μmol · kg−1 · min−1) during lipid infusion. On the other hand, when the increase in glucose recirculated to the liver as a result of decreased overnight Rd was mimicked by an incremental infusion of glucose after an overnight fast, the fall in HGO observed during sleep was reproduced independent of changes in plasma glucose and insulin values. This study suggests that peripheral events such as Rd and lipolysis may regulate HGO in nondiabetic men by a dual-control mechanism independent of insulin secretion. The relative contributions of gluconeogenesis and glycogenolysis to HGO may vary according to the levels of gluconeogenic promoters (e.g., FFA) and substrates without altering overall HGO. However, HGO may be regulated quantitatively by changes in Rd, which alter the amount of glucose recirculated to the liver.
Induction of Islet Cytodifferentiation by Fetal Mesenchyme in Adult Pancreatic Ductal Epithelium
Authors: Dudek Ronald W , Lawrence Irvin E , Hill Ronald S , Johnson Robert C
Published: September 2013

Recombinant tissue consisting of adult ductal epithelium isolated from pancreas and fetal mesenchyme was transplanted subcutaneously in the inguinal region of nude mice or epididymal fat pads of rats with a tissue chamber device for short-term (8-day) or long-term (6- to 12-wk) duration. We found that recombinant tissue underwent morphogenesis and cytodifferentiation, thereby forming islets that contained cells immunocytochemically positive for insulin and glucagon. Islet cytodifferentiation occurred in ∼20% of the recombinants. In recombinants that developed into islets, the tissue was always in close association with an extracellular matrix, nerves, and blood vessels. Controls consisting of mesenchyme alone or duct epithelium alone showed no evidence of morphogenesis of cytodifferentiation. Pancreatic rudiments were also implanted to serve as positive controls. This is the first demonstration of islet cytodifferentiation from adult duct epithelium.
Ca2+-Calmodulin–Dependent Phosphorylation of Islet Secretory Granule Proteins
Authors: Watkins Dudley T
Published: September 2013

The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing [32P]ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10−4 M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 μM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 μM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca2+-calmodulin–dependent protein kinases and endogenous substrates are present in islet secretory granules.
Ion Channels of Glucose-Responsive and -Unresponsive β-cells
Authors: Soria Bernat , Chanson Marc , Giordano Emanuele , Bosco Domenico , Meda Paolo
Published: September 2013

To assess whether different electrophysiological characteristics could account for the heterogeneous secretion of individual β-cells in vitro, we used patch-clamp configurations to study currents in plaque-forming (insulin-secreting) and non–plaque-forming rat pancreatic β-cells that were distinguished in a reverse hemolytic plaque assay (RHPA) after a 30-min stimulation by 16.7 mM glucose. RHPA showed that the population of single β-cells under study was stimulated (P < 0.01–0.001) to secrete insulin by 16.7 mM glucose, 100 μM tolbutamide, 20 μM glyburide, or 30 mM KCl but, under these conditions, also comprised β-cells that did not secrete detectable amounts of insulin. Under current clamp conditions, secreting and nonsecreting β-cells showed analogous resting membrane potentials (∼60 mV) and were similarly depolarized by 30 mm KCl and 100 μM tolbutamide. Under voltage-clamp conditions, total membrane conductance (∼6 nS) was also similar in the glucose-responsive and -unresponsive β-cells, which, when monitored in the whole-cell configuration after RHPA, showed the following currents: a voltage-dependent Na+ current, a voltage-activated Ba2+ current, a voltage-dependent K+ delayed-rectifier current, a voltage-dependent Ca2+-activated K+ current, and a voltage-independent and tolbutamide-sensitive K+ current. In the cell-attached configuration and the presence of 2.8 mM glucose, secreting and nonsecreting β-cells displayed a similar single-channel activity that was abolished when glucose concentration was raised to 16.7 mM. We conclude that β-cells studied after RHPA have an electrically normal membrane whether they release insulin in response to 16.7 mM glucose or not.
Von Willebrand Factor and Development of Diabetic Nephropathy in IDDM
Authors: Stehouwer Coen D A , Stroes Erik S G , Hackeng Wil H L , Mulder Paul G H , Den Ottolander Gerard J H
Published: September 2013

We tested the hypothesis that dysfunction of vascular endothelium, indicated by an increase in plasma level of von Willebrand factor (vWF), is present in patients with insulin-dependent diabetes mellitus (IDDM) who develop diabetic nephropathy (DN). DN was classified as absent (urinary albumin excretion [UAE] rate <15 microgram/min), incipient (UAE rate 15–200 μgrams/min), or clinical (UAE rate > 200 μg/min). We followed a cohort of 59 patients for a median of 3 yr. At baseline, 52 patients had no DN, 6 had incipient DN, and 1 had clinical DN. At follow-up, 38 patients had no DN (group 1). Incipient DN had developed in 14 patients and worsened in 3 patients. Clinical DN had worsened in 1 patient. Together, these 18 patients comprised group 2. A decrease in UAE was observed in the remaining three patients with incipient DN at baseline (group 3). In group 1, vWF—measured by immunoelectrophoresis and expressed as a percentage of normal—increased slightly (median 10%, range –43 to 145, P = 0.009). In group 2, vWF increased in all patients (median 80%, range –14 to –206 [corrected], P = 0.0002 vs. baseline and group 1). In group 3, vWF decreased (median –19%, range –44 to –18). After correction for possible confounders, i.e., age, varying duration of follow-up, and initial level of vWF, the difference in vWF change between groups 1 and 2 remained significant (P = 0.009). Poor glycemic control at baseline, estimated by glycosylated hemoglobin, was a significant predictor of increases in vWF in both group 1 and groups 1 and 2 combined. We conclude that dysfunction of vascular endothelium is present in patients who develop DN. Poor glycemic control may be related to endothelial dysfunction in patients with and without DN. Our datasuggest that changes in vWF may serve as a useful marker of the state of the vascular endothelium in diabetes.
Lack of Effect of Islet Amyloid Polypeptide in Causing Insulin Resistance in Conscious Dogs During Euglycemic Clamp Studies
Authors: Kassir A A , Upadhyay A K , Lim T J , Moossa A R , Olefsky J M
Published: September 2013

In this study, we administered constant intravenous infusions of human islet amyloid polypeptide (hIAPP) to conscious dogs during euglycemic glucose-clamp studies. The doses of hIAPP used (5 and 50 pmol · kg−1 · min−1) raised the circulating IAPP levels ∼12- and 50-fold above basal levels, respectively. Studies were conducted at two different insulin infusion rates, resulting in steady-state plasma insulin levels of ∼600 and 2800 pM. According to our results, the hIAPP infusions did not lead to any measurable change in the insulin-stimulated glucose disposal rate at either insulin infusion rate. Additionally, we observed no effect of IAPP on hepatic glucose production. Although we did not observe any effect of hIAPP on any of the aspects of glucose or insulin metabolism measured, we did find a consistent hypocalcemic effect of this peptide at the 50-pmol · kg−1 · min−1 infusion rate. Shortly after the onset of hIAPP infusion, serum calcium levels fell by 10–15% and remained at these levels throughout the course of the hIAPP infusion. In summary, 1) infusion of hIAPP at doses of 5 or 50 pmol · kg−1 · min−1 in conscious dogs raised the circulating IAPP level 12- to 50-fold above basal; 2) during these infusion studies, no effect of hIAPP was observed on any of the aspects of glucose or insulin homeostasis measured; 3) 50 pmol · kg−1 · min−1 hIAPP lead to a prompt reduction in plasma calcium concentrations with intravenous administration.
Pancreas Transplantation in Humans With Diabetes Mellitus
Authors: Robertson R Paul
Published: September 2013

Pancreas transplantation, when successful, is a reproducibly effective method to normalize glycemia without the use of exogenous insulin treatment in patients with diabetes mellitus. Success rates for combined pancreas and kidney transplantation are ∼70%, and patient survival rates are ∼90% 1 yr postoperatively. Metabolic benefits of this procedure include normalization of levels of fasting plasma glucose and HbA1C. Glucose-induced insulin secretion and intravenous glucose tolerance are normalized. Improvements are also observed in glucose recovery after insulin-induced hypoglycemia and in glucagon secretion during hypoglycemia. Pancreas transplantation is also associated with normalization of kidney structure and both motor and sensory nerve function. However, no benefits have been observed with regard to pancreatic polypeptide secretion, kidney function, and the retinal pathology of diabetes mellitus. Pancreas transplantation has reached a point in its history where the operative technique and its ancillary medical therapy have been optimized. Improvement in the rates of success, morbidity, and mortality will probably depend on improvement in immunosuppressive drugs and the physical condition of the recipients themselves. The time is at hand when we need to carefully consider whether it is ethical and advisable to make pancreas transplantation available to individuals who have fewer chronic complications of diabetes mellitus. Future studies of pancreas transplantation must incorporate more rigid experimental controls than have been used in the past to better assess the relative merits of this procedure.
Identification of G Protein α-Subunits in RINm5F Cells and Their Selective Interaction with Galanin Receptor
Authors: Cormont Mireille , Le Marchand-Brustel Yannick , Van Obberghen Emmanuel , Spiegel Allen M , Sharp Geoffrey W G
Published: September 2013

Galanin, an inhibitor of insulin secretion in pancreatic β-cells, exerts its multiple effects through mechanisms that are sensitive to pertussis toxin (PTX). G proteins have been characterized in RINm5F cells. By ADP ribosylation and immunoblotting, the α-subunits of Gi1, Gi2, Gi3, and two forms of Go were identified, Gi12 being predominant. As expected from a G protein–linked receptor, GTP and its nonhydrolyzable analogue GTP-γ-S decreased tracer galanin binding to cell membranes. This resulted from a change in receptor affinity without any modification in the number of sites. Selective antibodies against the COOH-terminal decapeptide of the α-subunits of the Gi and Go proteins were used to block G protein interaction before we studied galanin binding. Antibody AS, which selectively recognizes Giα1 and Giα2, decreased tracer galanin binding to membranes at concentrations where there were no effects of other antibodies specifically directed against Giα3 or Gαo. These data suggest that Gi1 and/or Gi2 interact with the galanin receptor and probably mediate the effects of galanin in pancreatic β-cells.