Tacrolimus suppresses glucose-induced insulin release from pancreatic islets by reducing glucokinase activity

Tacrolimus is widely used for immunosuppressant therapy, including various organ transplantations. One of its main side effects is hyperglycemia due to reduced insulin secretion, but the mechanism remains unknown. We have investigated the metabolic effects of tacrolimus on insulin secretion at a concentration that does not influence insulin content. Twenty-four-hour exposure to 3 nM tacrolimus reduced high glucose (16.7 mM)-induced insulin secretion (control 2.14 +/- 0.08 vs. tacrolimus 1.75 +/- 0.02 ng.islet(-1).30 min(-1), P < 0.01) without affecting insulin content. In dynamic experiments, insulin secretion and NAD(P)H fluorescence during a 20-min period after 10 min of high-glucose exposure were reduced in tacrolimus-treated islets. ATP content and glucose utilization of tacrolimus-treated islets in the presence of 16.7 mM glucose were less than in control (ATP content: control 9.69 +/- 0.99 vs. tacrolimus 6.52 +/- 0.40 pmol/islet, P < 0.01; glucose utilization: control 103.8 +/- 6.9 vs. tacrolimus 74.4 +/- 5.1 pmol.islet(-1).90 min(-1), P < 0.01). However, insulin release from tacrolimus-treated islets was similar to that from control islets in the presence of 16.7 mM alpha-ketoisocaproate, a mitochondrial fuel. Glucokinase activity, which determines glycolytic velocity, was reduced by tacrolimus treatment (control 65.3 +/- 3.4 vs. tacrolimus 49.9 +/- 2.8 pmol.islet(-1).60 min(-1), P < 0.01), whereas hexokinase activity was not affected. These results indicate that glucose-stimulated insulin release is decreased by chronic exposure to tacrolimus due to reduced ATP production and glycolysis derived from reduced glucokinase activity.     

Reduced sensitivity to dexamethasone of pancreatic islets from obese (fa/fa) rats.

The direct effects of dexamethasone exposure on insulin secretion from islets of fa/fa rats and their lean littermates (Fa/?) were compared. After 72 h culture in 1 nM dexamethasone, glucose (27.5 mM)-stimulated insulin secretion over 90 min from islets of lean rats was significantly decreased compared with islets cultured without dexamethasone (12.9 +/- 1.4 vs. 5.7 +/- 1.0% of total islet content, p < 0.05). Higher doses of dexamethasone for 24-48 h culture produced similar effects. For islets of fa/fa rats, the minimum inhibitory concentration of dexamethasone was 10-fold higher, and islets required at least 48 h exposure for inhibitory effects to be observed. Dexamethasone also decreased the insulin response by islets to glybenclamide, indicating that dexamethasone effects were not specific to glucose transport or metabolism. The results suggest that islets of fa/fa rats may be less sensitive to direct inhibitory effects of glucocorticoids on glucose-stimulated insulin release than islets of lean animals.     

In vivo insulin responsiveness for glucose uptake and production at eu- and hyperglycemic levels in normal and diabetic rats.

Whole body glucose uptake (BGU) and hepatic glucose production (HGP) at maximal plasma insulin concentrations (+/- 5000 microU/ml) were determined by eu- (EC) (6 mM) and hyperglycemic (HC) (20 mM) clamps (120 min), combined with [3-3H]glucose infusion, in normal and streptozotocin-treated (65 mg/kg) 3-day diabetic, conscious rats. In normal rats, during EC, BGU was 12.4 +/- 0.4 mg/min and during HC, when urinary glucose loss was 0.54 +/- 0.09 mg/min, BGU was 25.5 +/- 1.6 mg/min. However, throughout the final 60 min of HC, glucose infusion rate (GIR) was not constant but a linear decline in time (r = -0.99) of 17%, P less than 0.0001, was observed indicating a hyperglycemia-induced desensitization process. In diabetic rats, during EC, BGU was 7.7 +/- 0.3 mg/min and during HC, BGU was 15.5 +/- 1.4 mg/min. Throughout the final 60 min of HC, GIR was constant, suggesting that the hyperglycemia-induced desensitization process was already completed. In normal and diabetic rats, HGP was similar: during EC 0.2 +/- 0.5 mg/min and 0.1 +/- 0.5 mg/min, and during HC 0.4 +/- 0.4 mg/min and 0.5 +/- 0.6 mg/min, respectively. In vitro adipocyte and muscle insulin receptor studies showed normal to increased receptor number and increased receptor autophosphorylation in diabetic compared to normal rats. In conclusion: (i) 3-day diabetic rats show, at maximal plasma insulin concentrations, insulin resistance to BGU, but not to HGP. The resistance to BGU is equally present (reduction of 38%) at eu- and hyperglycemic levels as compared to normal rats. (ii) 3-day diabetic rats reveal no defect in adipocyte and muscle insulin receptor function. These data indicate that the diabetes induced insulin resistance for BGU is at the post-receptor level and due to a decreased maximal capacity (Vmax) for glucose uptake, with no change in affinity, or Km.     

Hexosamines regulate sensitivity of glucose-stimulated insulin secretion in beta-cells

Hexosamines serve a nutrient-sensing function through enzymatic O-glycosylation of proteins. We previously characterized transgenic (Tg) mice with overexpression of the rate-limiting enzyme in hexosamine production, glutamine:fructose-6-phosphate amidotransferase, in beta-cells. Animals were hyperinsulinemic, resulting in peripheral insulin resistance. Glucose tolerance deteriorated with age, and males developed diabetes. We therefore examined islet function in these mice by perifusion in vitro. Young (2-mo-old) Tg animals had enhanced sensitivity to glucose of insulin secretion. Insulin secretion was maximal at 20 mM and half maximal at 9.9 +/- 0.5 mM glucose in Tg islets compared with maximal at 30 mM and half maximal at 13.5 +/- 0.7 mM glucose in wild type (WT; P < 0.005). Young Tg animals secreted more insulin in response to 20 mM glucose (Tg, 1,254 +/- 311; WT, 425 +/- 231 pg x islet(-1) x 35 min(-1); P < 0.01). Islets from older (8-mo-old) Tg mice became desensitized to glucose, with half-maximal secretion at 16.1 +/- 0.8 mM glucose, compared with 11.8 +/- 0.7 mM in WT (P < 0.05). Older Tg mice secreted less insulin in response to 20 mM glucose (Tg, 2,256 +/- 342; WT, 3,493 +/- 367 pg x islet(-1) x 35 min(-1); P < 0.05). Secretion in response to carbachol was similar in WT and Tg at both ages. Glucose oxidation was blunted in older Tg islets. At 5 mM glucose, islet CO2 production was comparable between Tg and WT. However, WT mice increased islet CO2 production 2.7 +/- 0.4-fold in 20 mM glucose, compared with only 1.4 +/- 0.1-fold in Tg (P < 0.02). Results demonstrate that hexosamines are involved in nutrient sensing for insulin secretion, acting at least in part by modulating glucose oxidation pathways. Prolonged excess hexosamine flux results in glucose desensitization and mimics glucose toxicity.     

Characteristics of insulin and glucagon release from the perfused pancreas, intact isolated islets, and dispersed islet cells

There are a variety of different tissue preparations which have been used to study secretion from the endocrine pancreas and there are considerable differences in the results obtained from these. The purpose of this study was to compare several preparations in one laboratory using the same rats, buffers, and radioimmunoassays. The preparations included the isolated perfused rat pancreas, fresh isolated intact islets and dispersed cells, and cultured islets and cells. Insulin release from the perfused rat pancreas at 2.8 mM glucose was so low that it could not be measured, such that over a 90-min time period the amount of insulin released was less than 0.004% of pancreatic insulin content. In contrast, islets in static incubation appear to release 2.0% of their stored content and dispersed cells appear to release 2.6% of their content. Samples were taken at early time points during incubations of fresh islets and dispersed cells, and it was found that almost all of the insulin found at the end of a 90-min incubation period was present during the first 5 min. It is therefore suspected that the true secretory rate of insulin at a low glucose concentration is far lower than had been generally appreciated. Glucagon release patterns showed similarities in that with isolated islets and dispersed cells a disproportionate amount of glucagon release was found during a 0- to 30-min incubation period when compared with the 30- to 90-min period. In summary, artifacts have been identified in some of the in vitro systems used for the study of endocrine pancreatic secretion and these deserve greater recognition.     

Kinetic characteristics and regulation of hexose transport in a galactokinase-negative Chinese hamster fibroblast cell line: a good model for studies on sugar transport in cultured mammalian cells

We report the kinetic characteristics for D-galactose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose transport in a galactokinase null-allele mutant of a Chinese hamster V79 cell line. GalKl cells exhibited a Km and Vmax for D-galactose, 2-deoxy-D-glucose, and 3-O-methyl-D-glucose transport of 8.6 +/- 2.6 mM and 26.1 +/- 7.2 nmol/mg p/min, 4.1 +/- 1.2 mM and 40.3 +/- 9.5 nmol/mg p/min, and 7.01 +/- .85 mM and 11.6 +/- 4.8 nmol/mg p/30 s, respectively. Nonsaturable hexose uptake was determined using cytochalasin B inhibition of galactose uptake (89.6 +/- 3.7% of galactose uptake was cytochalasin B inhibitable) and L-glucose uptake (7.5% of the galactose uptake). D-Galactose was not metabolized and effluxed rapidly from preloaded cells. The Kls for the inhibition of D-galactose transport were 4.5 +/- 2.5 mM for D-glucose, 7.0 +/- 2.0 mM for 2-deoxy-D-glucose, 6 mM for 2-deoxy-D-galactose and 6.0 +/- 0.6 mM for 3-O-methyl-D-glucose. This indicates the operation of a single common carrier. The hexose transport rate decreased 50-60% after 24 h serum deprivation. Addition of insulin was shown to increase hexose transport (more than twofold) in serum-deprived cells. Hexose transport rates increased substantially in glucose-deprived, D-fructose- or D-galactose-fed cells as compared to glucose-fed cells. Since GalKl does not metabolize galactose, the hexose transport increases induced by feeding cells galactose suggest that carrier interaction with ligand is not a significant factor in transport regulation in GalKl. The kinetic and regulatory characteristics of D-galactose transport in the GalKl cell line indicate that this system is a good model to study sugar transport from a mechanistic and regulatory point of view.     

Effect of sucrose diet on insulin secretion in vivo and in vitro and on triglyceride storage and mobilisation of the heart of rats

Basal heart triacylglycerol (TG) (mumole triacylglycerol/g of dry weight) (- before "in vitro" Langendorff perfusion -) was significantly higher in animals rendered chronically hypertriglyceridaemic (H) by a 63% sucrose-rich diet than in controls (C, standard diet); 28 +/- 2.6 means + SEM vs. 19.3 +/- 1.2; respectively (p less than 0.01). After 40' perfusion with Krebs-Henseleit buffer + 5.5 mM glucose, 2.5 mM Ca++, TG content fell to 14.2 +/- 0.6 in C and 14.9 +/- 1.9 in H (n.S.). Administration of 1 n mol x min-1 of glucagon (Gn) from min 20 to 40 reduced TG to 9.0 +/- 0.5 in C (p less than 0.05). In contrast no effect of Gn was observed in H (TG at min 40: 16.7 +/- 2.5). Glycogen (Gly) content (mumol/g of dry weight) after Gn perfusion fell from 30 +/- 1.9 to 17 +/- 2.1 (p less than 0.01) in C, while again no effect was recorded in H. "In vivo" plasma glucose fractional coefficient disappearance rate was lower (p less than 0.001) in H: 1.01 x 10(-2) +/- 0.09 x 10(-2) vs 2.61 x 10(-2) +/- 0.14 x 10(-2) in C, in spite of H showing hyperinsulin secretion. Hyperinsulinism was further documented by "in vitro" Iri release studies from incubated pancreas pieces. In the absence of glucose (G) from the incubation medium H produced 541 +/- 19.8 mU/mg weight Tissue/20', while C produced 91.2 +/- 12.7 (p less than 0.001). With 100 mg% G, H released 1058 +/- 259 and C 377 +/- 82.5 (p less than 0.001). It is suggested that hyperinsulin secretion plus insulin resistance may account for the above findings.     

Respiratory depression produced by centrally administered taurine in the cat

The effects of taurine (0.8-64.8 mumol) were studied on respiratory activity following intracisternal (cisterna magna) and intracerebroventricular (lateral ventricle) injections in cats anesthetized with alpha-chloralose. Respiratory activity was measured by using a Fleisch pneumotachograph and monitoring tracheal airflow. The flow signal was integrated to obtain tidal volume (VT) and respiratory rate (f) was obtained by counting the number of VT excursions over one minute. Inspiratory (TI), expiratory (TE) and total (TTOT) cycle durations were also determined during this time period. In addition, end-tidal CO2 was continuously monitored. Associated changes in arterial pressure (femoral artery cannula) and heart rate were also determined. After injections into the cisterna magna, taurine caused dose-related decreases in minute ventilation (VE). The maximal decrease in VE was from 495 +/- 59 to 64 +/- 14 ml/min (p less than 0.05), and was due to both decreases in VT (from 27 +/- 3 to 5 +/- 1 ml; p less than 0.05) and f (from 18 +/- 1 to 12 +/- 2 breaths/min; p less than 0.05). TE and TTOT were increased from 2.4 +/- 0.4 to 4.5 +/- 0.6 sec (p less than 0.05) and from 3.7 +/- 0.4 to 6.4 +/- 0.8 sec (p less than 0.05), respectively. Mean inspiratory flow (VT/TI), a measure of inspiratory drive, was decreased from 21 +/- 4 to 4 +/- 2 ml/sec (p less than 0.05). Apnea occurred in 5 of 6 animals after the 64.8 mumol dose. This respiratory depression occurred without any significant change in arterial pressure. After lateral ventricle injections, taurine also caused dose-related, but not as pronounced, decreases in respiratory activity. In addition, taurine caused significant decreases (p less than 0.05) in arterial pressure in doses that decreased VE. Taurine administered intravenously had no significant cardiorespiratory depressant effects. These data indicate that centrally administered taurine produces respiratory depression and, depending on the route of CNS administration, also produces hypotension.     

Effects of porcine diazepam-binding inhibitor on insulin and glucagon secretion in vitro from the rat endocrine pancreas.

Porcine diazepam-binding inhibitor (pDBI) is a novel peptide that has been isolated from the small bowel of the pig, and that occurs also in the islet D-cells. We have studied its effects on hormone release in vitro from the endocrine pancreas of the rat. In isolated islets, pDBI (10(-9)-10(-6)M) did not affect basal insulin release at 3.3 mM glucose, whereas stimulated release at 8.3 mM glucose was dose-dependently suppressed by 32-69% (P less than 0.01). Furthermore, insulin secretion stimulated by either 16.7 mM glucose or 1 mM IBMX (3-isobutyl-1-methylxanthine) or 1 micrograms/ml glibenclamide was suppressed by pDBI at 10(-8) M (by 28-30%, P less than 0.05) and 10(-7) M (by 43-47%, P less than 0.01). In contrast, islet insulin secretion induced by 20 mM arginine was unaffected by these concentrations of pDBI. In the perfused rat pancreas, pDBI (10(-8) M) enhanced by 30% (P less than 0.05) the first phase (0-5 min) of arginine-stimulated insulin release, whereas the second phase (5-20 min) was unchanged. Moreover, pDBI suppressed by 28% (P less than 0.05) the second phase of arginine-induced glucagon release. Arginine-induced somatostatin release was not significantly affected by the peptide. Since pDBI immunoreactivity has been localized also to islet D-cells, the present results suggest that pDBI may act as a local modulator of islet hormone release.     

Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.     

Insulin secretion and acetylcholinesterase activity in monosodium l-glutamate-induced obese mice.

OBJECTIVE: Pancreatic islets isolated from mice treated neonatally with monosodium L-glutamate (MSG) were used to study insulin secretion. MATERIALS AND METHODS: Total acetylcholinesterase (AchE) activity of tissue extract was measured as a cholinergic activity marker. Obesity recorded in 90-day-old MSG mice (OM) by Lee index reached 366.40 +/- 1.70, compared to control mice (CM) 324.40 +/- 1.10 (p < 0.0001). Glucose 5.6 mM induced insulin secretion of 36 +/- 5 pg/15 min from islets of CM and 86 +/- 13 from OM (p < 0.001). When glucose was raised to 16.7 mM, islets from OM secreted 1,271 +/- 215 and 1,017 +/- 112 pg/30 min to CM. AchE activity of pancreas from OM was 0.64 +/- 0.02 nmol of substrate hydrolyzed/min/mg of tissue and 0.52 +/- 0.01 to CM (p < 0.0001). Liver of obese animals also presented increase of AchE activity. RESULTS: These indicate that OM insulin oversecretion in low glucose may be attributed, at least in part, to an enhancement of parasympathetic tonus.     

Subsensitivity to insulin in adipocytes from rats submitted to foot-shock stress

We examined the effect of three daily foot-shock stress sessions on glucose homeostasis, insulin secretion by isolated pancreatic islets, insulin sensitivity of white adipocytes, and glycogen stores in the liver and soleus muscle of rats. Stressed rats had plasma glucose (128.3 +/- 22.9 mg/dL) and insulin (1.09 +/- 0.33 ng/mL) levels higher than the controls (glucose, 73.8 +/- 3.5 mg/dL; insulin, 0.53 +/- 0.11 ng/mL, ANOVA plus Fisher's test; p < 0.05). After a glucose overload, the plasma glucose, but not insulin, levels remained higher (area under the curve 8.19 +/- 1.03 vs. 4.84 +/- 1.33 g/dL 30 min and 102.7 +/- 12.2 vs. 93.2 +/- 16.1 ng/mL 30 min, respectively). Although, the area under the insulin curve was higher in stressed (72.8 +/- 9.8 ng/mL) rats than in control rats (34.9 +/- 6.9 ng/mL) in the initial 10 min after glucose overload. The insulin release stimulated by glucose in pancreatic islets was not modified after stress. Adipocytes basal lipolysis was higher (stressed, 1.03 +/- 0.14; control, 0.69 +/- 0.11 micromol of glycerol in 60 min/100 mg of total lipids) but maximal lipolysis stimulated by norepinephrine was not different (stressed, 1.82 +/- 0.35; control, 1.46 +/- 0.09 micromol of glycerol in 60 min/100 mg of total lipids) after stress. Insulin dose-dependently inhibited the lipolytic response to norepinephrine by up to 35% in adipocytes from control rats but had no effect on adipocytes from stressed rats. The liver glycogen content was unaltered by stress, but was lower in soleus muscle from stressed rats than in control rats (0.45 +/- 0.04 vs. 0.35 +/- 0.04 mg/100 mg of wet tissue). These results suggest that rats submitted to foot-shock stress develop hyperglycemia along with hyperinsulinemia as a consequence of insulin subsensitivity in adipose tissue, with no alteration in the pancreatic sensitivity to glucose. Foot-shock stress may therefore provide a useful short-term model of insulin subsensitivity.     

Insulin release from collagenase-isolated islets of rat pancreas in the presence of cyclic AMP and somatostatin.

In order to study the role of cyclic AMP in the inhibition by somatostatin of glucose-induced insulin release, the effect of somatostatin on the potentiation by dibutyryl-cyclic AMP (db-cAMP) of insulin release from isolated pancreatic islets of rats was examined. Isolated islets were obtained from the rat pancreas by the collagenase method. Ten islets were incubated for periods of 30 min in Krebs-Ringer bicarbonate buffer containg albumin and glucose 2.0 mg/ml in the presence or absence of somatostatin (1 microgram/ml or 100 ng/ml) and/or db-cAMP 1 mM. Glucose-induced insulin release was reduced by somatostatin in concentrations of 1 microgram/ml. Somatostatin in a concentration of 100 ng/ml significantly abolished the potentiation by db-cAMP of insulin release (p less than 0;01), in spite of exerting no inhibition of glucose-induced insulin release. However, in the presence of theophylline 5 mM, somatostatin 100 ng/ml did not show that inhibitory effect on the potentiated insulin release.     

Insulin release and eflux of [32P]Phosphate from islets of rat Langerhans treated with iodoacetic acid and the anomers of D-glucose.

To clarify the insulin-releasing mechanism, we studied insulin release and the efflux of [32P]phosphate by glucose at 0.1 mM/min of gradient level or at 16.7 mM, and other metabolism in islets of rat Langerhans. When treated with 1 mM iodoacetic acid (IAA) plus the anomers of D-glucose at 2.8 mM for 6 min at 37 degrees C, islets elicited insulin at half the control rate under the step-wise stimulation by glucose and at the same rate as the control under the slow-rise stimulation by glucose. Using islets treated with IAA plus the alpha anomer at 16.7 mM, the step-wise stimulation secreted insulin at half a rate of the control and the slow-rise stimulation at the rate lower than the control, which was not significantly different from the control rate. Treatment with IAA plus the beta anomer at 16.7 mM inhibited insulin release under both types of stimulations by glucose. The step-wise stimulation caused the same rapid efflux of [32P]phosphate from IAA-treated islets as from the control islets, except for islets treated with IAA plus the beta anomer at 16.7 mM. The rate of glucose utilization in islets was inhibited by all IAA-treatments to the same extent, being merely half the control rate. Treatments with IAA plus the anomers at 16.7 mM significantly reduced the formation of [3H]-cAMP and the activity of protein phosphokinase in islets, while in the presence of the anomers at 2.8 mM IAA produced no significant effect. Neither IAA-treatments altered the uptake of 45Ca and the ATP content in islets. The uptake of [14C]IAA was significantly enhanced by the presence of the beta anomer at 16.7 mM to two times the control level. On the basis of these results, we suggested that the B cell might contain both glucoreceptors and rate-sensors of glucose controlling insulin release and the former might be less sensitive to IAA as compared with the latter.     

Fetal rat islet insulin deficiency following maternal administration of streptozotocin

Pancreatic islets were isolated from the fetuses of normal rats and rats made diabetic by the iv administration of streptozotocin (STZ) on either Day 3 or 5 of pregnancy. Of the rats made diabetic on Day 3, one group also received insulin injections at the appearance of glucosuria. Maternal blood glucose on Day 20 of gestation was significantly different in the diabetic rats (405 +/- 27 mg/dl) from the normal (97 +/- 1 mg/dl) and insulin-treated diabetic rats (69 +/- 9 mg/dl). While fetal weight was significantly decreased in the STZ-treated rats (2.64 +/- 0.13 g vs 3.52 +/- 0.05 g for the control group, P less than 0.005), fetal glucose was significantly higher in the STZ-treated than in normal pups (342 +/- 11 vs 35 +/- 1 mg/dl, P less than 0.005). Both fetal weight and glucose were normalized by insulin treatment: 3.16 +/- 0.18 g and 31 +/- 7 mg/dl, respectively. Insulin release from fetal islets of diabetic dams was blunted after a week in culture both in basal and stimulated conditions. After 2 weeks in culture, there was partial recovery in the insulin response to glucose but it did not equal to that measured in fetal islets from the normal and insulin-treated diabetic rats. These data suggest maternal hyperglycemia severely impairs fetal weight and insulin release from fetal rat islets in vitro, and correction of the hyperglycemia by insulin treatment not only improves fetal weight and glucose concentrations, but it also normalizes insulin release from fetal rat islets in vitro.     

Effect of beta-adrenoceptor blockade on H+ and K+ flux in exercising humans

The effect of beta-adrenoceptor blockade (beta B) on muscle release and uptake of H+ and K+ in humans during maximal exercise has been investigated. Eight volunteers cycled intermittently at power outputs corresponding to 100% of maximal O2 uptake. Prior to exercise either propranolol (beta B) or saline (control) was infused into the femoral vein. Arterial and femoral venous blood samples were drawn at rest, during exercise, and during 30-min recovery. Peak arterial blood values for K+, lactic acid (LA), and base deficit (BD) (mean +/- SE) were respectively 5.5 +/- 0.1, 9.5 +/- 0.6, and 11.7 +/- 0.9 mmol/l during beta B and 5.1 +/- 0.1, 8.3 +/- 0.6, and 10.3 +/- 1.0 for control (P less than 0.05). The release of K+ from the working leg did not differ between treatments during exercise, but K+ uptake during late recovery (5-30 min) was slightly lower during beta B. Thus the higher arterial K+ levels during exercise (beta B) cannot be attributed to greater release by active muscle but are likely due to decreased K+ uptake by noncontracting muscle. Arterial-femoral venous differences for LA and BD did not differ significantly between treatments. Additionally LA exchange across the leg was similar to H+ exchange (arterial-femoral venous differences for BD) under all conditions. During early recovery (1-5 min), regardless of experimental treatment, BD levels iin arterial blood were higher than LA (P less than 0.05). These elevated BD levels may be due to unequal removal rates between LA and H+ equivalents by nonexercised tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pancreatic hormone response to neuropeptide Y (NPY) perifusion in vitro

Available data on the effect of neuropeptide Y (NPY) on insulin release are conflicting and little data exist regarding the effect of NPY on glucagon secretion. The purpose of the present study, therefore, was to characterize the direct effect of NPY on the release of these pancreatic hormones and to examine the role of glucose on these interactions. Using a perifused mouse islet system, we found that NPY suppressed both basal and glucose-stimulated insulin secretion. Thus, basal insulin release assessed as mean integrated area under the curve/20 min (AUC/20 min) decreased from 1446 +/- 143 pg to 651 +/- 112 pg (P less than 0.05) with the addition of 2 x 10(-8) M NPY and the AUC/20 min for glucose stimulated insulin output decreased from 1973 +/- 248 pg to 1426 +/- 199 pg (P less than 0.05). In both cases, this inhibitory effect was followed after removing NPY by a stimulation of insulin secretion which was typical of a 'rebound off-response'. In contrast, NPY exerted a stimulatory effect on basal glucagon release and significantly reversed the suppressive effect of high glucose on glucagon output. The basal glucagon AUC/20 min increased from 212 +/- 103 pg to 579 +/- 316 pg (P less than 0.05), while glucagon secretion in the presence of 27.7 mM glucose increased from 75 +/- 26 pg to 255 +/- 28 pg (P less than 0.01). In conclusion, we have shown that the direct effect of NPY on the endocrine pancreas is to suppress insulin but stimulate glucagon secretion. These data are compatible with a role for NPY in the regulation of pancreatic hormone output.     

Glucoregulation and hormonal responses to maximal exercise in non-insulin-dependent diabetes

Maximal dynamic exercise results in a postexercise hyperglycemia in healthy young subjects. We investigated the influence of maximal exercise on glucoregulation in non-insulin-dependent diabetic subjects (NIDDM). Seven NIDDM and seven healthy control males bicycled 7 min at 60% of their maximal O2 consumption (VO2max), 3 min at 100% VO2max, and 2 min at 110% VO2max. In both groups, glucose production (Ra) increased more with exercise than did glucose uptake (Rd) and, accordingly, plasma glucose increased. However, in NIDDM subjects the increase in Ra was hastened and Rd inhibited compared with controls, so the increase in glucose occurred earlier and was greater [147 +/- 21 to 169 +/- 19 (30 min postexercise) vs. 90 +/- 4 to 100 +/- 5 (SE) mg/dl (10 min postexercise), P less than 0.05]. Glucose levels remained elevated for greater than 60 min postexercise in both groups. Glucose clearance increased during exercise but decreased postexercise to or below (NIDDM, P less than 0.05) basal levels, despite increased insulin levels (P less than 0.05). Plasma epinephrine and glucagon responses to exercise were higher in NIDDM than in control subjects (P less than 0.05). By use of the insulin clamp technique at 40 microU.m-2.min-1 of insulin with plasma glucose maintained at basal levels, glucose disposal in NIDDM subjects, but not in controls, was enhanced 24 h after exercise. It is concluded that, because of exaggerated counter-regulatory hormonal responses, maximal dynamic exercise results in a 60-min period of postexercise hyperglycemia and hyperinsulinemia in NIDDM. However, this event is followed by a period of increased insulin effect on Rd that is present 24 h after exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnesium uptake by intestinal brush-border membranes of spontaneously hypertensive rats.

Magnesium uptake by intestinal brush-border membranes (BBM) was studied in duodenal and jejunal vesicles of the spontaneously hypertensive rat (SHR) and normotensive control, the Wistar-Kyoto (WKY) rat. In the duodenum, no statistical difference was evidenced between the two types of rats. By contrast, initial rates of magnesium uptake in jejunal vesicles were lower in SHR (5.4 +/- 2.1 nmol/mg protein x 10 sec) in comparison to WKY rats (11.0 +/- 2.5 nmol/mg protein x 10 sec) at a magnesium concentration of 1 mM (P less than 0.01). In jejunal BBM, kinetic analysis of magnesium uptake showed three components in WKY rats, with one being diffusional. In SHR, only two components were seen, with the diffusional one being absent. The two saturable components showed Vmax of 6.5 +/- 1.3 and 26.2 +/- 6.0 nmol/mg protein x 10 sec and apparent Km of 0.22 +/- 0.12 mM and 1.9 +/- 0.4 mM in WKY rats, and Vmax of 10.9 +/- 3.5 and 14.8 +/- 5.9 nmol/mg protein x 10 sec and apparent Km of 0.43 +/- 0.23 mM and 1.3 +/- 0.2 mM in SHR. Only the component with the lowest apparent affinity appeared statistically different in SHR as compared with WKY rats for both Vmax and apparent Km (P less than 0.05). Time course evolution of magnesium uptake in jejunal BBM indicated, by extrapolation at zero time, that 2.5 and 5.1 nmol magnesium/mg protein in SHR and WKY rats, respectively, would be in the bound state. The study of the influence of medium osmolarity on 60-min magnesium uptakes was also indicative of a smaller binding compartment in jejunal BBM of SHR (3.70 and 8.26 nmol/mg protein in SHR and WKY rats, respectively); at the four osmolarities assayed, the 60-min uptakes were significantly lower in SHR as compared with WKY rats (P less than 0.01). From 60-min glucose uptakes, a smaller volume of jejunal BBM vesicles was determined for SHR as compared with WKY rats (0.34 +/- 0.06 and 0.63 +/- 0.17 microliter/mg of protein in SHR and WKY rats respectively, P less than 0.05), this volume being significantly augmented by the presence of 1 mM MgCl2 (0.48 +/- 0.05 and 1.27 +/- 0.02 microliter/mg of protein in SHR and WKY rats respectively, P less than 0.01). These results suggest that magnesium uptake and binding by jejunal BBM are altered in SHR in comparison to WKY rats, implying a possible role of the small intestine in the abnormalities of magnesium metabolism in genetic hypertension.     

An inhibitor of sweet taste response modulates glucose-induced phosphoinositide breakdown in rat pancreatic islets.

We studied the effect of a specific-competitive inhibitor of the sucrose taste response, p-nitrophenyl-D-glucopyranoside (PNP-Glu) on insulin release and phosphoinositide metabolism in rat pancreatic islets. The alpha-anomer, but not the beta-anomer, of PNP-Glu at a concentration of 5 mM inhibited insulin release induced by 10 mM glucose. Islets were labeled by exposure for 2 h to 10 uCi of myo-[2-3H] inositol solution supplemented with 2.8 mM glucose. Forty islets were then incubated in the presence of 10 mM LiCl, 1 mM inositol and 10 mM glucose with or without the anomers of PNP-Glu. [3H] radioactivity in the incubation medium remained significantly greater in the presence of the alpha-anomer of PNP-Glu than in the presence of glucose alone after 5- and 20-min incubation. The inositol monophosphate levels in the islets incubated with glucose alone were increased more than in the islets with alpha-anomer. The beta-anomer of PNP-Glu did not change either glucose-induced insulin release or phosphoinositide breakdown. A patch-clamp study revealed that neither anomer affected the glucose-dependent ATP-sensitive K(+)-channels. These results indicate that the anomeric preference for glucose in insulin release in the pancreatic islets is closely associated with phosphoinositide breakdown.