Rat brain myo-inositol 3-phosphate synthase is a phosphoprotein.

The therapeutic effects of lithium in bipolar disorder are poorly understood. Lithium decreases free inositol levels by inhibiting inositol monophosphatase 1 and myo-inositol 3-phosphate synthase (IPS). In this study, we demonstrate for the first time that IPS can be phosphorylated. This was evident when purified rat IPS was dephosphorylated by lambda protein phosphatase and analyzed by phospho-specific ProQ-Diamond staining and Western blot analysis. These techniques demonstrated a mobility shift consistent with IPS being phosphorylated. Mass spectral analysis revealed that Serine-524 (S524), which resides in the hinge region derived from exon 11 of the gene, is the site for phosphorylation. Further, an antibody generated against a synthetic peptide of IPS containing monophosphorylated-S524, was able to discriminate the phosphorylated and non-phosphorylated forms of IPS. The phosphoprotein is found in the brain and testis, but not in the intestine. The intestinal IPS isoform lacks the peptide bearing S524, and hence, cannot be phosphorylated. Evidences suggest that IPS is monophosphorylated at S524 and that the removal of this phosphate does not alter its enzymatic activity. These observations suggest a novel function for IPS in brain and other tissues. Future studies should resolve the functional role of phospho-IPS in brain inositol signaling.

Reconstructing the pancreas: restoration of normoglycemia, exocrine function, and islet innervation by islet transplantation to the pancreas.

Impaired function in transplanted islets may be ascribed in part to disturbed reinnervation. The objectives of this study were to determine whether islet transplantation to the pancreas in the presence of nerve growth factor (NGF) would restore islet innervation and endocrine and exocrine pancreatic function. Streptozotocin-diabetic Lewis rats received 800 syngeneic islets beneath the pancreatic capsule in the presence or absence of NGF (20 ng/d for 14 days). Fasting blood glucose was measured for 3 months. The pancreata were isolated and perfused in situ. Pancreatic juice was collected for amylase determination. The sympathetic trunks were isolated and stimulated electrically. The tissues were immunostained for nerve markers. All islet recipients remained euglycemic (4.2 +/- 0.6 mmol/L glucose). Ductal amylase concentrations were restored to near normal levels in contrast to diabetic controls (normal rat 98 +/- 8 U/L, islet transplant 78.4 +/- 9 U/L, diabetic control 14.5 +/- 8 U/L). NGF enhanced the innervation of transplanted islets in contrast to control islet transplants. Sympathetic adrenergic innervation was significantly increased by NGF (tyrosine hydroxylase [P < .001] and neuropeptide Y [P < .05]). No differences in parasympathetic innervation were observed (vesicular acetylcholine transporter). Electrical stimulation of the sympathetic trunks in the presence of 4 micromol/L phentolamine and 5 micromol/L atropine resulted in increased insulin secretion in NGF-treated islet transplants (164%) compared with control transplants (30%). The combination of growth factors and the pancreatic site may allow the use of fewer islets than conventional islet transplant sites and promote more normal transplanted islet function by the enhancement of islet reinnervation.

Protection from ischemic cell death by the induction of cytoglobin.

Methods to reduce beta-cell loss after islet isolation and transplantation must be developed if islet transplantation is to become a preferred treatment for diabetes. Most recent research has focused on the reduction of toxicity from immunosuppressants and the enhancement of revascularization by growth factors such as vascular endothelial growth factor. Cytoglobin is an intracellular oxygen-binding protein found in islet beta-cells, inducible by hypoxia. It is our hypothesis that cytoglobin induction and overexpression may improve survival and function of transplanted islets by preventing ischemic cell death. Lewis rat islets and MIN6 cells were transfected with the cytoglobin gene. Control and transfected cells and islets were held for 4 hours at 20% oxygen before glucose challenge. Another group of islets and cells was held for 4 hours at 20% and then 1% oxygen prior to glucose challenge. Untreated or transfected Lewis rat islets (n = 800) were transplanted beneath the renal capsule of streptozotocin diabetic Lewis rats. In another study, Sprague-Dawley islets were transfected and transplanted into streptozotocin diabetic Lewis rats. Fasting blood glucose was used as an indicator of islet function and survival. Cytoglobin transfected islets and cells retained the ability to secrete insulin at low oxygen concentrations in contrast to controls. Cytoglobin over expression reduced the development of central islet necrosis after 5 days in tissue culture. Cytoglobin inhibited the onset of immunorejection (14 +/- 2 days) as compared with controls islets (5 +/- 2 days). Cytoglobin induction may be a useful adjunct to islet transplantation.

Beta-cell sparing in transplanted islets by vascular endothelial growth factor.

We have reported that vascular endothelial growth factor (VEGF) promotes the revascularization of transplanted islets, thereby reducing the initial number required to prevent diabetes. The present study was undertaken to assess other mechanisms of beta-cell sparing by VEGF. For in vitro studies, islets were cultured for 14 days with versus without 20 ng/mL VEGF. Viability, necrosis, and apoptosis were examined by specific staining (Alcein AM, propidium iodide, and annexin/phosphatidylserine). The effects of VEGF on islets were also examined in a proteomic study. In vivo streptozotocin-treated diabetic Lewis rats received 1000 Lewis or Sprague-Dawley islets beneath the renal capsule. Oxygen levels at the transplant site were monitored by a Clark-type oxygen electrode. Fasting blood glucose served as an indicator of islet survival and function. VEGF enhanced oxygen levels at the transplant site. Syngeneic recipients were euglycemic for over 6 months, whereas control islets failed within 30 to 60 days. VEGF prevented allograft rejection for over 14 days, whereas controls were rejected within 6 to 7 days. Immunostaining suggested that VEGF inhibited the presentation of MHC II antigen and promoted islet survival by the inhibition of necrosis and apoptosis. Our proteomic study suggested VEGF preserved systems required for cellular preservation (heat shock proteins) and insulin secretion. VEGF promotes the preservation of isolated and transplanted islets by a variety of mechanisms, including enhanced oxygenation and inhibition of immune rejection, necrosis, and apoptosis. The provision of exogenous VEGF may be a useful adjunct to islet transplantation.

Interstitial insulin during euglycemic-hyperinsulinemic clamp in obese and lean individuals.

Transcapillary insulin transport has been considered a rate-limiting step of insulin action. However, direct measurement of interstitial insulin levels during physiologic levels of insulinemia have not been performed. We determined changes in interstitial insulin in eight healthy non-obese men and seven healthy obese men by microdialysis during a euglycemic-hyperinsulinemic clamp. Interstitial insulin was determined in the subcutaneous tissue of the abdomen and thigh. Steady-state insulin concentrations were reached approximately 10 minutes after the start of insulin infusion in the subcutaneous tissue of the abdomen and thigh and returned to basal levels approximately 10 minutes after the infusion was discontinued. There was no difference in the rapidity of change in interstitial insulin between obese and lean individuals at either site studied, irrespective of the pattern of fat distribution. The relative change in dialysate insulin concentration during the euglycemic clamp did not differ between obese and lean individuals at either site studied. It was also unaffected by the waist to hip ratio. The rapid change in interstitial insulin concentration could be of physiologic significance in determining the effects of changes in circulating insulin concentration. We conclude that transcapillary insulin transport in adipose tissue is unaffected by obesity and the pattern of fat distribution in healthy men. It is also concluded that when interstitial insulin is determined directly, transcapillary insulin transport is rapid and does not demonstrate a significant lag phase.

In situ binding of islet hormones in the isolated perfused rat pancreas: evidence for local high concentrations of islet hormones via the islet-acinar axis.

Insulin and somatostatin reportedly affect pancreatic acinar cell function via specific receptor binding. Theoretically peri-insular levels depend on the islet-acinar portal system, but the actual hormone levels have never been demonstrated. Rat pancreata were perfused anterogradely or retrogradely with 125I-insulin, -somatostatin, or -glucagon (each, approximately equal to 10(-11) mol/l). Tracer binding was determined from differences between influx and efflux radioactivity. Saturable binding was observed for insulin and somatostatin, but not for glucagon. Binding in the absence of unlabelled peptides was significantly higher during retrograde perfusion than during anterograde perfusion for insulin (25.9 +/- 2.6 vs 16.0 +/- 2.1%, mean +/- SD; each, n = 4; p < 0.001) and somatostatin (18.4 +/- 2.0 vs 13.6 +/- 1.2%; each, n = 3; p < 0.05). Non-specific binding was similar in both directions. These findings are attributable to endogenous hormones acting as unlabelled ligands competing with the tracers during anterograde perfusion. This conclusion was supported by the demonstration that endogenous insulin stimulation by D-glucose, but not by L-glucose, caused a decrease in labelled insulin binding only during anterograde perfusion. Displacement curves obtained during retrograde perfusion showed that interstitial concentrations of insulin and somatostatin were 7.5 x 10(-9) and 1.1 x 10(-9) mol/l, respectively. Thus, the exocrine pancreas is indeed exposed to locally high concentrations of islet hormones.

Lack of delay in hepatic insulin transit as indicated by concordance of insulin secretory and peripheral insulin pulses.

The liver is an important site of insulin metabolism and action. It has often been assumed that the liver may diminish the amplitude of the insulin secretory waveform without altering hepatic insulin transit time. However, the significant extraction and metabolism of hepatic insulin has the potential to delay hepatic insulin transit. To examine hepatic insulin transit, we studied the concordance of calculated insulin secretory peaks with peripheral insulin peaks in 12 healthy men of varying body weight and fat distribution. Adiposity was determined by percent body fat, and fat distribution by the waist to hip ratio. Arterialized peripheral venous samples for insulin and C-peptide assays were obtained every 2 minutes for 90 minutes. Pancreatic insulin secretion rates were estimated with individual C-peptide kinetics using a two-compartment model. Concordance between insulin secretory peaks and peripheral insulin peaks was assessed by the hypergeometric probability model. A significant concordance between secretory and peripheral insulin pulses was demonstrated in seven of 12 subjects (P < .00001). The mean pulse intervals for insulin secretion were similar to the mean pulse intervals for peripheral insulin. The degree of concordance between the insulin secretory peaks and peripheral insulin pulses was unrelated to adiposity or body fat distribution. Significant synchronicity exists between insulin secretory peaks and peripheral insulin peaks in healthy men. We conclude that despite significant hepatic insulin extraction and metabolism, hepatic insulin transit may not be delayed in healthy men.

Suppressive role of the islet-acinar axis in the perfused rat pancreas.

BACKGROUND: The stimulating effects of insulin on the exocrine pancreas are well known. The effects of other islet hormones, however, are controversial. The aim of the present study was to determine whether the islet-acinar axis, as a whole, is stimulatory or inhibitory. Because we have shown that retrograde perfusion reverses the islet-acinar directed microcirculation, retrograde perfusion was expected to remove the overall effects of islet hormones from the acinar tissue. METHODS: Rat pancreata were perfused (7 mmol/L glucose plus 3 mmol/L mixed amino acids) either anterogradely or retrogradely. Pancreatic juice flow, protein output, and amylase output were measured. RESULTS: When perfusion was switched from anterograde to retrograde, juice flow increased threefold without changes in protein and amylase output. When cerulein (10(-10) mol/L) was infused, retrograde protein and amylase responses were larger than anterograde responses (each, n = 7; 2.71 +/- 0.23 vs. 1.71 +/- 0.11 mg/40 minutes; 173 +/- 17 vs. 98 +/- 8 U/40 minutes; mean +/- SE; both, P < 0.01). Somatostatin-14 and rat pancreatic polypeptide (each, 10(-9) mol/L) reduced the retrograde protein and amylase responses, but not juice flow, to the anterograde response levels. Conversely, these peptides did not affect exocrine function during anterograde perfusion. CONCLUSIONS: A suppressive role of the islet-acinar axis via endogenous somatostatin and/or pancreatic polypeptide is suggested.

Exocrine interstitial insulin and somatostatin in the perfused dog pancreas.

To determine whether the islet-acinar relationship is mediated microvascularly or by diffusion from islets, isolated dog pancreata were perfused anterogradely (n = 4) or retrogradely (n = 5). Interstitial fluid (ISF) was sampled utilizing a microdialysis technique. Three to five microdialysis probes were placed in the dorsal lobe of each pancreas. Insulin and somatostatin were measured in dialysate, as well as in perfusate, under both resting (5.6 mM glucose) and stimulated (12.2 mM glucose plus 20 mM arginine) conditions. During the stimulative period, retrograde ISF responses were markedly less than anterograde responses, whereas perfusate (intravascular) anterograde and retrograde responses were not different; ratios of the sum of increments above basal levels of ISF concentrations to the sum of increments of perfusate concentrations (sigma delta ISF/sigma delta perfusate) of insulin and somatostatin during anterograde vs. retrograde perfusion were 35.1 +/- 8.8 (SE; n = 9) vs. 13.8 +/- 2.8% (n = 10) and 37.1 +/- 14.2 (n = 7) vs. 8.5 +/- 4.2% (n = 9), respectively (both, P < 0.05). These data strongly suggest that these islet hormones may be delivered to the exocrine interstitial space via a directed microcirculation from the islet to the acinar tissue.

Lack of insulin feedback inhibition in non-obese and obese men.

The existence of insulin feedback inhibition is a controversial issue. The present study adopted a novel approach to determine whether insulin feedback inhibition exists in vivo during physiologic hyperinsulinemia and if it could contribute to enhanced insulin secretion in obesity. Serial plasma insulin and C-peptide levels were determined during a basal state and a hyperinsulinemic clamp (287 pmol/min/m2) and following discontinuation of the insulin infusion under euglycemic conditions. Insulin secretion rates were derived from plasma C-peptide levels and individual C-peptide kinetics using a two-compartment model. Eight non-obese and nine obese men were recruited for the studies, which were performed in random order. Men with significant variations in glucose levels during hyperinsulinemia were excluded from the analysis. Plasma glucose levels were similar between the non-obese and obese groups during all phases of the study, and similar plasma insulin levels were achieved in both groups during euglycemic hyperinsulinemia. In obese men, C-peptide levels were significantly greater compared with non-obese men during euglycemic hyperinsulinemia (P < .05). However, neither the non-obese nor the obese group demonstrated significant suppression of insulin secretion rates during euglycemic hyperinsulinemia. Expressing the data in absolute terms or as a percent of basal did not alter the results. Moreover, there was no significant change between the non-obese and the obese group during the rapid onset and cessation of hyperinsulinemia. Under euglycemic conditions, physiologic hyperinsulinemia does not induce suppression of endogenous insulin secretion in non-obese or obese men.

Relationship of insulin secretory pulses to sex hormone-binding globulin in normal men.

Insulin has emerged as a regulator of sex hormone-binding globulin (SHBG) production in vitro and in vivo. A role for insulin in regulating SHBG exists in insulin resistant states such as obesity and polycystic ovary syndrome. The relationship of in vivo insulin secretion rates to SHBG levels in healthy normal men is less well documented. Hepatic synthesis of SHBG may be influenced by quantitative insulin exposure as well as qualitative characteristics such as frequency and amplitude of insulin secretory pulses. The present study was undertaken to assess these relationships in 10 normal men. Adiposity was determined by the body mass index and fat distribution by the waist hip ratio. Peripheral insulin sensitivity was determined by the euglycemic clamp technique at an insulin infusion rate of 287 pmol/min.m2. SHBG levels were determined in the fasting state by RIA. Arterialized venous samples for C-peptide were obtained every 2 min for 90 min in the basal state. Individual C-peptide kinetics were derived after a bolus injection of biosynthetic human C-peptide and a previously validated two compartmental model. Insulin secretion rates at each time point were calculated using the plasma C-peptide values and the C-peptide kinetics. Insulin secretion rates were unrelated to SHBG concentrations (r = -0.29, P > 0.05). The insulin secretory pulse interval had a significant positive association with SHBG levels (r = 0.86, P < 0.05). Insulin secretory pulse amplitude, body mass index, waist hip ratio, and peripheral insulin sensitivity were not associated with SHBG concentrations in a regression analysis. We postulate that insulin secretory pulse frequency may be an important determinant of SHBG synthesis in normal man.

Body fat distribution and peripheral insulin sensitivity in healthy men: role of insulin pulsatility.

Abdominal fat distribution is associated with insulin resistance in healthy young men. Factors modulating this phenomenon remain unclear. Pulsatile insulin release has been implicated as a potential regulator of insulin action. The relationship of pulsatility of peripheral insulin levels to fat distribution and peripheral insulin sensitivity was examined in 10 healthy men. Fat distribution was determined by the waist to hip ratio. Peripheral insulin sensitivity was assessed by the euglycemic clamp at an insulin infusion rate of 287 pmol/min.m2. Pulsatility of insulin was assessed by sampling every 2 min for 90 min in the basal state. The characteristics of insulin pulses were assessed by the computer program Pulsar. The waist to hip ratio was negatively associated with insulin sensitivity (r = -0.70, P less than 0.05) and insulin pulse interval (r = -0.66, P less than 0.05). The insulin pulse interval was positively correlated with peripheral insulin sensitivity (r = 0.73, P less than 0.05). The insulin interpulse interval was the primary determinant of insulin sensitivity. The increased frequency of insulin pulses may play a role in inducing insulin resistance in individuals with abdominal fat distribution.

Endothelin does not affect experimentally induced corneal neovascularization.

Endothelin, a potent vasoconstrictor, was found to be ineffective in the treatment of experimentally induced corneal neovascularization. Endothelin was administered topically, subconjunctivally, and intraluminally in serial concentrations ranging from 0.0005 to 5.0 micrograms/mL in New Zealand white rabbits without effect. Electron microscopy of the neovascular cornea revealed the vessels consisted only of endothelin and pericytes. Hence, the vessels were not responsive to endothelin because they lacked contractile smooth muscle.

Induction of angiogenesis by growth factors: relevance to pancreatic islet transplantation.

Biodegradable pellets releasing 20 ng/day of endothelial cell growth factor alpha (alpha ECGF) or a- or b-fibroblast growth factor (FGF) and 90 micrograms/day of heparin were implanted beneath the renal capsule in rats and dogs and the muscularis/serosal border of the pyloric stomach in dogs to test for angiogenesis in a potential pancreatic islet transplant site. These factors were also tested in vitro to determine whether the capillary bed of the isolated islet could be preserved. alpha ECGF was superior to a- or bFGF in promoting endothelial cell growth and capillary formation in isolated islets. Both a- or bFGF and alpha ECGF induced the development of a dense capillary bed in the dog stomach, whereas in the kidney site alpha ECGF was more effective in the rat than was a- or bFGF. Priming the isolated islet as well as the transplant site prior to islet transplantation resulted in islet blood flow being established within 3 days in contrast to 7-14 days in controls.

Perfusion with anti-insulin gamma globulin indicates a B to A to D cellular perfusion sequence in the pancreas of the rhesus monkey, Macaca mulatta.

The cellular sequence of intraislet vascular perfusion has been shown to be important in the regulation of islet hormone secretion in the rat and dog islet. In order to test whether a B to A to D sequence of islet cellular perfusion is also present in a nonhuman primate, pancreata from the rhesus monkey, Macaca mulatta, were isolated and perfused in vitro in the presence and absence of anti-insulin gamma globulin. In the presence of the insulin antibody, efflux concentration of insulin decreased rapidly (-95 +/- 1.8%), whereas glucagon and somatostatin concentrations increased (111 +/- 28% and 239 +/- 38%, respectively). These results suggest the presence of a B-A-D cellular sequence of vascular perfusion within the monkey islet. The present results also strongly support the hypothesis that a B-A-D sequence of islet perfusion is important in the regulation of islet hormone secretion and further emphasize the central role of the B-cell in intraislet cellular interactions. The results also suggest that, despite differences in islet anatomy, a B-A-D order of islet cellular perfusion may be the preferred functional sequence among mammalian species.