Effects of 5-hydroxytryptamine on rubidium ion fluxes and insulin release in cultured pancreatic islets

We have incubated pancreatic islets isolated from noninbred ob/ob mice and NMRI mice for 3 days with or without 5-hydroxytryptamine (5-HT) in the medium and tested the effect of such long term treatment on subsequent insulin release and 86Rb+ accumulation and efflux. Two tenths millimolars of 5-HT abolished insulin release in response to 20 mM glucose. Two tenths millimolars of 5-HT also diminished the ability of islets to accumulate 86Rb+ and the effect of 10 mM glucose on 86Rb+ efflux. One one-hundredth millimolars of 5-HT had no effect on insulin release or 86Rb+ fluxes. Clearly, islets subjected to 5-HT for 3 days at concentrations that do not elicit demonstrable effects in short term incubations show a reduced secretory response. However, the physiological role of the high affinity uptake system for 5-HT in islet cells [Michaelis-Menten constant (Km) = 1.6 microM] remains unknown.     

Decreased cyclic AMP and Insulin responses to glucose in pancreatic islets of diabetic chinese hamsters

Summary The dose as well as the time kinetics of insulin and adenosine-3,5-monophosphate (cyclic AMP) responses to glucose were compared in pancreatic islets isolated from normal and diabetic Chinese hamsters. The insulin content in diabetic islets was about one-half that in normal islets. Insulin release in diabetic islets incubated for 10 min with glucose 60–1000 mg/l00 ml was from one-third to one-half that in normal islets. Glucose 1000 mg/l00 ml stimulated three-fold increases in insulin release without increasing the accumulation of [³H] cyclic AMP in either normal or diabetic islets prelabelled with [³H] adenine. However, in the presence of 1.0 mM of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), glucose 150 mg/l00 ml elicited significant increases of insulin release (+ 134%) and [³H] cyclic AMP accumulation in islets (+ 44%) and incubation medium (+ 48%) of islets of normal but not diabetic hamsters. Also, in perifusion experiments with 0.1 mM IBMX, glucose 500 mg/l00 ml produced threefold greater increases in insulin release and two-fold greater increases in efflux of cyclic AMP in normal than diabetic islets. By contrast with the lesser effects of glucose in diabetic islets, 1.0 mM IBMX increased islet and medium cyclic AMP, as well as insulin release, similarly in normal and diabetic islets. It is suggested that the impairment of glucose induced insulin release in islets of the diabetic Chinese hamster may be due to a defective interaction of glucose with the adenylate cyclase-cyclic AMP system in the pancreatic B cell.This work was presented in part at a meeting of the European Association for the Study of Diabetes, Sept. 1975, Munich, Germanyon leave from the Department of Endocrinology, Karolinska Hospital, Stockholm     

Glucose utilization in relation to insulin secretion in NZO and C57Bl mouse islets

Further studies of insulin secretion from isolated pancreatic islets of the NZO strain of obese hyperglycemic mouse have shown markedly impaired insulin secretory responses to D-glucose in islets from fasted or fed mice. NZO islets at a low glucose concentration (3.3 mM) showed a significant insulin secretory response to 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, but in the presence of this agent showed no significant additional response to increased glucose concentration. This contrasted with the situation in islets from an arbitrarily chosen control strain of mouse (C57Bl) which showed a small or insignificant response to 0.5 mM IBMX at a low glucose concentration, but a greatly enhanced response to glucose in the presence of IBMX. In contrast to the relative refractoriness of the NZO islets to glucose, they showed a large response to D,L-glyceraldehyde, at least equal to that found in the control islets. Glucose utilization was studied by measuring the conversion of D-[5-3H]glucose to [3H]H2O. In islets from both fasted and fed NZO mice, glucose utilization, when calculated on the basis of islet DNA content, was markedly reduced at high glucose concentrations compared to that in islets from the control strain. It is concluded that the relative unresponsiveness of NZO islets to glucose is associated with, and perhaps due to, a decreased rate of glucose utilization. The preserved responsiveness to glyceraldehyde suggests that the reduced glucose utilization may be due to a partial metabolic block before the triose phosphate step in the islet glycolytic pathway.     

Phosphate flush and glucose metabolism in pancreatic islets of young and old diabetic mice (C57BL/KsJ-db/db)

Pancreatic islets from normal C57BL/KsJ-+/+-mice and diabetic C57BL/KsJ-db/db-mice were collagenase-isolated and incubated with 33P-labelled inorganic phosphate. No significant difference was observed in phosphate uptake between normal and diabetic mouse islets whether the animals were young (6-8 weeks) or old (27 +/- 9 weeks). When 33P-labelled islets were perifused with non-radioactive medium, all types of islets exhibited a brisk and transient peak of phosphate release in response to a change of glucose concentration from 2.8 to 16.7 mmol/l. Expressed in absolute terms, both the basal and peak efflux of phosphate appeared to be diminished in the diabetic mice. In relative terms (peak over basal), the glucose-stimulated phosphate efflux was not lower in diabetic than in normal mice. At both a low (3 mmol/l) and a high (20 mmol/l) glucose concentration, the production of 3H2O from D-[5-3H]glucose was reduced in old but not in young diabetic mouse islets. The percentage increase in glucose metabolism in response to a rise in glucose concentration from 3 to 20 mmol/l was about the same in all types of islets. The results add to previous observations of disturbed ionic fluxes in the pancreatic islets of diabetic KsJ-db/db-mice. These effects are probably not due to gross alterations in glycolytic metabolism but more probably reflect alterations in the function of the beta-cell plasma membrane.     

Demonstration of glucose inhibition of insulin release in the presence of diazoxide

beta-Cell-rich pancreatic islets from ob/ob mice were taken for measurements of insulin release in response to glucose after culture in RPMI 1640 medium. The stimulatory effect of 20 mmol/l glucose was converted into an inhibition when the medium was supplemented with 400 mumol/l diazoxide. Glucose inhibition of insulin release was observed when the islets had been cultured in the presence of 1 or 20 mmol/l glucose in media either containing or lacking Ca2+. The data provide further evidence for an inhibitory component in the action of glucose on insulin release, suggesting that glucose stimulation of the Ca2+ efflux is essential for the appearance of this inhibition.     

Forskolin-induced desensitization of pancreatic beta-cell insulin secretory responsiveness: possible involvement of impaired information flow in the inositol-lipid cycle.

Isolated rat islets of Langerhans were incubated for 2 h in a myo-[2-3H]inositol-containing solution to label their phosphoinositides. Also included during this labeling period was forskolin (0.1-5 microM), a compound established to elevate islet cAMP levels. These islets were subsequently perifused, and their insulin secretory responses to 20 mM glucose or 1 microM of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) were assessed. Determined in parallel with secretion were [3H] inositol efflux patterns and, at the termination of the perifusion, labeled inositol phosphate accumulation. The following major observations were made. 1) Forskolin had no deleterious effect on the total amount of [3H]inositol incorporated by the islets during the labeling period. 2) However, labeling in forskolin resulted in subsequent dose-dependent decreases in 20 mM glucose-induced insulin secretion, [3H]inositol efflux and inositol phosphate accumulation. 3) Inclusion of the diacylglycerol (DAG) kinase inhibitor monooleoylglycerol (50 microM) restored to a significant degree glucose-induced release from forskolin-desensitized islets. 4) Pretreatment with 5 microM forskolin had no deleterious effect on TPA-induced insulin release. 5) Prior exposure to forskolin also impaired phosphoinositide hydrolysis in response to cholecystokinin stimulation. 6) Similar to forskolin, labeling in isobutylmethylxanthine (1 mM) reduced in a parallel fashion islet [3H]inositol efflux and insulin secretion in response to 20 mM glucose stimulation. These findings demonstrate that prior chronic elevation of islet cAMP levels suppresses the activation of phospholipase-C in response to subsequent stimulation. Defective insulin secretory responsiveness of these islets appears to be the result of impaired generation of phosphoinositide-derived second messenger molecules, particularly DAG. By substituting for DAG, however, TPA circumvents this biochemical lesion and evokes a normal insulin secretory response from forskolin-pretreated islets.     

Effects of guanosine 3',5'-monophosphate on glucose utilization in isolated islets of Langerhans

Dynamic changes in total glucose utilization in isolated islets of Langerhans of the rat were determined by quantitation of the formation of 3H2O from D-[5-3H]glucose. The addition of 8-bromo-cGMP (8-Br-cGMP) or monobutyryl cGMP to the islets during a linear phase of glucose utilization resulted in concentration- and time-dependent increases in glucose utilization. Effects of the analogs of cGMP on glucose utilization were noted as early as 5 min after the onset of stimulation in the presence of 10 mM glucose. 8-Br-cGMP also increased the utilization of 1 mM glucose within 20 min. Stimulatory effects of 8-Br-cGMP were observed in the presence of cycloheximide or N-acetylglucosamine. Neither 8-bromo-cAMP (8-Br-cAMP) nor monobutyryl cAMP induced significant changes in glucose utilization at 1 or 10 mM glucose. In the presence of 3-isobutyl-1-methylxanthine (IBMX), 8-Br-cGMP, but not 8-Br-cAMP, induced a rapid change in glucose utilization. N-Methyl-N'-nitro-N-nitrosoguanidine, which activates guanylate cyclase, also stimulated glucose utilization in the presence of IBMX by 3-fold. IBMX alone did not change glucose utilization. In contrast, 8-Br-5'-GMP reduced glucose utilization, whereas 8-bromoinosine 3',5'-monophosphate and 8-bromoguanosine did not change glucose utilization. Sodium bromide did not affect glucose utilization. Glucose-stimulated insulin release was potentiated by 8-Br-cGMP, whereas insulin release from islets incubated in the absence of glucose or the presence of glyceraldehyde or 2-ketoisocaproic acid was not altered by 8-Br-cGMP. Thus, glucose utilization in pancreatic islets is modulated by cGMP, and the secretory response to 8-Br-cGMP is glucose dependent.     

Phosphoinositide hydrolysis and insulin secretion in response to glucose stimulation are impaired in isolated rat islets by prolonged exposure to the sulfonylurea tolbutamide

Isolated rat islets of Langerhans were incubated for 2 h in a [3H]inositol-containing medium supplemented with 7 mM glucose and the sulfonylurea tolbutamide (50-200 microM). After labeling, the ability of these islets to respond during a subsequent perifusion to 20 mM glucose or 15 mM alpha-ketoisocaproate (KIC) was assessed. The following major observations were made. Prior exposure to tolbutamide inhibited [3H]inositol efflux, inositol phosphate accumulation, and the insulin secretory responses of subsequently perifused islets to 20 mM glucose stimulation. When present during the 2-h labeling period, the calcium channel blocker nitrendipine (500 nM), a compound that abolishes tolbutamide-induced increases in PI hydrolysis, blocked these inhibitory effects of tolbutamide. In addition, the diacylglycerol kinase inhibitor monooleoylglycerol (50 microM) restored the impaired second phase insulin secretory response noted after a 2-h tolbutamide exposure. Prior exposure to tolbutamide (200 microM) also desensitized the islet, in terms of [3H] inositol phosphate accumulation, [3H]inositol efflux, and insulin secretory responses, to 15 mM KIC. The inclusion of monooleoylglycerol during the stimulatory period with KIC restored second phase insulin secretion. The results support the conclusion that chronic tolbutamide-induced increases in PI hydrolysis render the beta-cell insensitive to a subsequent 20-mM glucose or 15-mM KIC stimulus. Blocking tolbutamide-induced increases in PI hydrolysis during the labeling period eliminates the adverse effects of the sulfonylurea. The ineffectiveness of glucose and KIC to maintain insulin secretory responses from prior tolbutamide-exposed islets appears to be the result of the inability of these agonists to appropriately activate PI hydrolysis.     

Interleukin-1 alpha exerts glucose-dependent stimulatory and inhibitory effects on islet cell phosphoinositide hydrolysis and insulin secretion

Isolated rat islets were incubated with myo-[2-3H]inositol to label their phosphoinositides (PI). Labeling was carried out in the presence of various glucose levels (2.75-10 mM) with or without human recombinant interleukin-1 alpha (IL-1). After the labeling period, insulin release, [3H]inositol efflux, and the accumulation of labeled inositol phosphates in perfused islets were assessed under various conditions. The following major observations were made. 1) In islets labeled for 2 h with [3H]inositol in the presence of 2.75 mM glucose, subsequent perifusion with 5.0 nM IL-1 increased insulin output, [3H]inositol efflux, and [3H]inositol phosphate accumulation in the simultaneous presence of 7 mM, but not 2.75 mM, glucose. 2) Mannoheptulose, a competitive inhibitor of islet glucokinase, blocked the stimulatory effects of IL-1 noted in the presence of 7 mM glucose. In other experiments, the conditions used during the 2-h labeling period with myo-[2-3H]inositol were varied. The following major observations were made in islets subsequently stimulated during the perifusion with 20 mM glucose. 3) Islets labeled with [3H]inositol in the presence of 2.75 mM glucose with or without 5.0 nM IL-1 responded with similar increases in PI hydrolysis and insulin output. 4) Compared to that with 2.75 mM glucose alone, labeling in the presence of 7 mM glucose alone was without any adverse effect on the subsequent PI and insulin responses of perifused islets to 20 mM glucose. 5) Labeling in the presence of 7 mM glucose plus 5.0 nM IL-1 resulted in a significant reduction in the subsequent PI and insulin responses. 6) These inhibitory effects of IL-1 were abolished if mannoheptulose was included during the 2-h incubation with 7 mM glucose plus 5.0 nM IL-1. 7) The diacylglycerol kinase inhibitor 1-monooleoylglycerol (100 microM) significantly restored insulin output after IL-1 exposure (with 7 mM glucose). 8) Similar to the results obtained with 7 mM glucose plus IL-1, incubation of islets with 8-10 mM glucose alone produced dose-dependent impairments of [3H]inositol efflux patterns and inositol phosphate accumulation. Insulin secretion was also impaired. These results demonstrate that IL-1 has glucose-dependent stimulatory and inhibitory effects on beta-cell function. Both effects appear to involve alterations in islet PI hydrolysis.     

Interleukin-1 induces time-dependent potentiation in isolated rat islets: possible involvement of phosphoinositide hydrolysis

Prior exposure of isolated perfused rat islets to the monokine interleukin-1 (IL-1) amplifies their subsequent insulin secretory response to 10 mM glucose. This potentiating effect of the monokine is dose dependent, lasts for at least 45 min after IL-1 removal from the medium, and is not confined to glucose; IL-1 also potentiates the insulin secretory responses to tolbutamide and glyceraldehyde. IL-1 exposure of islets incubated with myo-[2-3H]inositol to label their phosphoinositides (PI) results in an increase in [3H]inositol efflux, an event that persists long after removal of IL-1 from the medium. Direct measurements of labeled inositol phosphate accumulation substantiate the concept that this sustained [3H]inositol efflux response is the direct result of a sustained increase in PI hydrolysis. These results expand the list of compounds that induce time-dependent potentiation in islets to include IL-1. This action of the monokine, mediated at least in part by PI-derived second messenger molecules, may contribute to its postulated effects on insulin and glucose homeostasis.     

An autoradiographic study of [(18)F]FDG uptake to islets of Langerhans in NOD mouse

To evaluate the potential of in vivo imaging of accumulation of lymphocytes to islets of Langerhans (insulitis), we compared 2-[¹⁸F]fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) uptake in the pancreas and pancreatic islets of healthy BALB/c mice, phenotypically healthy NOD mice with insulitis and diabetic NOD mice. [¹⁸F]FDG was injected i.v. to 14 female BALB/c mice (age 13 ± 3 weeks, plasma glucose 8 ± 2 mmol/l) and 21 age-matched female NOD mice (plasma glucose 8 ± 4 mmol/l, p = 0.06). The mice were killed 90-min post injection and distribution of radioactivity was analysed using digital autoradiography. There was no correlation of plasma glucose concentration with the [¹⁸F]FDG uptake values. Uptake of radioactivity in NOD mice to the islets affected by insulitis was up to 2.3 times higher (p = 0.001) than that to unaffected islets in the same pancreas. Uptake to NOD islets with insulitis was also clearly enhanced (1.0–2.3 times higher) compared to the islets in the BALB/c mice.

In conclusion, NOD mouse islets with insulitis accumulate [¹⁸F]FDG markedly more than islets without insulitis or BALB/c islets. However, the relatively small difference in the [¹⁸F]FDG intensity between healthy and diseased islets, combined with the limited resolution ability of the positron emission tomography (PET), probably prevent the use of [¹⁸F]FDG in PET studies aiming at in vivo documentation of onset and progression of insulitis and prediabetes in mouse and man.     

Biochemical mechanisms involved in monomethyl succinate-induced insulin secretion.

Esters of succinic acid stimulate insulin secretion from pancreatic beta-cells. Using collagenase-isolated rat islets, the transduction mechanisms involved were investigated. In freshly isolated perifused islets, monomethyl succinate (MMSucc), in the presence of basal (2.75 mM) glucose, stimulated insulin release in a biphasic pattern. This secretory response was dependent on extracellular calcium movement into the beta-cell, since the calcium channel blocker nitrendipine (5 microM) abolished it. The glucokinase inhibitor mannoheptulose (20 mM) had no effect on its secretory action, while the protein kinase-C inhibitor staurosporine (20 nM) reduced secretion to MMSucc. In addition, while ineffective alone, the diacylglycerol kinase inhibitor monooleoylglycerol (25 microM) potentiated MMSucc-induced insulin release. A similarly amplified response occurred in the presence of forskolin (0.25 microM), a compound that elevates islet cAMP levels. The sodium salt of succinic acid (20 mM) had no effect on insulin release in the presence or absence of forskolin. Prior treatment with MMSucc in the presence of 2.75 mM glucose sensitized islets to the usually weak insulin secretory effect of 7.5 mM glucose. Other groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide pools. These islets were subsequently stimulated, and the kinetics of [3H]inositol efflux and insulin secretion were measured. MMSucc induced a rapid and sustained dose-dependent increase in [3H]inositol efflux rates. In batch-incubated islets, MMSucc increased inositol phosphate levels. Finally, MMSucc (20 mM), in the presence of 8 mM glucose, did not influence the detritiation of [5-3H]glucose, but reduced the oxidation of [U-14C] glucose. These results support the following conclusions. First, MMSucc is a potent activator of islet phosphoinositide hydrolysis. Second, the activation of protein kinase-C appears to contribute to the acute insulin secretory effect of MMSucc. Third, MMSucc-induced increases in phosphoinositide hydrolysis contribute at least in part to its ability to acutely stimulate insulin release and prime the beta-cell to subsequent stimulation. Finally, mitochondrial events associated with the oxidative metabolism of MMSucc may underlie its insulinotropic action.     

Noradrenergic stimulation of serotonin release from rat pineal glands in vitro

The pharmacodynamics of serotonin (5-hydroxytryptamine; 5-HT) uptake and release were studied in rat pineal glands. Initially, uptake was tested by incubating pineals with several concentrations of [3H]5-HT. The incubation media also contained [14C]mannitol to which cells are impermeable. Since [14C]mannitol accumulates only in extracellular spaces, the radio-labelled sugar was used to determine the differential distribution of [3H]5-HT in pineal compartments. Intracellular accumulation of 3H in pineal glands increased linearly as a function of time for [3H]5-HT concentrations ranging from 1 to 10 mumol/l. The ratio of 3H to 14C also increased for the same time-interval, indicating that the glands accumulated [3H]5-HT preferentially in non-extracellular spaces. [3H]5-HT accumulated in pineal glands which were denervated for more than 7 days before testing, suggesting that uptake is not restricted to adrenergic terminals but also occurs in pinealocytes. In addition to uptake, spontaneous and noradrenaline-stimulated release of [3H]5-HT was tested in perifusion and/or step-transfer systems. Spontaneous release of [3H]5-HT was biphasic consisting of rapid and slower efflux phases. In contrast, release of [14C]mannitol was monophasic, characterized exclusively by rapid efflux. Since [14C]mannitol does not enter cells, the rapid and slower phases of [3H]5-HT efflux may represent release from pineal extracellular and intracellular compartments respectively. The identity of [3H]5-HT in pineal glands and perifusion media was confirmed by thin-layer chromatography. When L-noradrenaline was added to the perifusion media, [3H]5-HT efflux during the slower phase of release was significantly increased above the non-stimulated state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic in vivo hyperglycemia impairs phosphoinositide hydrolysis and insulin release in isolated perifused rat islets

We examined the effect of chronic hyperglycemia on phosphoinositide hydrolysis and insulin secretion in isolated perifused rat islets. Rats were infused for 44 h with 40% dextrose in order to raise and maintain the plasma glucose concentration at 350 mg/dl. Control animals were infused with equiosmolar amounts of mannitol. In vivo insulin secretion and rats of glucose disposal were monitored throughout the study. At the end of the infusion, islets were collagenase isolated, and phosphoinositide (PI) hydrolysis (assessed by measuring the increment in [3H]inositol efflux as well as labeled inositol phosphates) and insulin output in response to a 20-mM glucose challenge were quantitated. Plasma insulin concentration and in vivo glucose disposal rates decreased significantly, by 47% and 35% respectively, after 6-8 h of hyperglycemia. In islets perifused immediately after isolation, prior in vivo hyperglycemia markedly altered the pattern of insulin output in response to 20-mM glucose challenge. Compared to mannitol infusion, 20 mM glucose stimulation resulted in an exaggerated first phase insulin secretory response (1121 +/- 88 vs. 467 +/- 75 pg/islets.min) and a blunted second phase insulin secretory response (392 +/- 90 vs. 1249 +/- 205 pg/islet.min). In islets prelabeled with myo-[2-3H]inositol for 2 h, PI hydrolysis, particularly [3H]inositol efflux in response to glucose stimulation was also reduced (0.28 +/- 0.03%/min) compared to that in mannitol-infused animals (0.53 +/- 0.08%/min). Two hours of preincubation in a low glucose medium (2.75 mM) were able to completely reverse the islet defect in both PI hydrolysis and insulin secretion. Our results demonstrate that chronic in vivo hyperglycemia impairs PI hydrolysis in perifused rat islets and suggest that this defect accounts in part for the abnormal pattern of glucose-induced insulin secretion.     

Long-term leptin treatment of ob/ob mice improves glucose-induced insulin secretion.

OBJECTIVE: Previous studies have demonstrated that leptin inhibits glucose-stimulated insulin secretion from isolated islets, although a lack of leptin effect on insulin secretion has also been reported. The effect of long term in vivo leptin treatment of insulin secretion has, however, not been established. Therefore, in the present study, we have evaluated the effect of long term in vivo treatment of leptin on glucose-induced insulin secretion in ob/ob mice. METHODS: After 7 days' treatment of leptin (100 microg daily s.c.), insulin release was measured in isolated islets by batch incubation followed by radioimmunoassay. Glucose utilization and oxidation were measured by measuring the formation of (3)H(2)O and (14)CO(2) from [5-(3)H] and [U-(14)C] glucose, respectively. Glucose-6-phosphatase activity was measured by measuring the conversion of (14)C-glucose-6-P to (14)C-glucose. In addition, immunohistochemistry of pancreatic specimens was undertaken for study of expression of insulin, GLUT-2 and hormone-sensitive lipase (HSL). RESULTS: Leptin treatment significantly improved insulin secretion both at 5.5 mM (by 15%; P<0.05) and 16.7 mM (by 85%; P<0.001) glucose, compared to vehicle-treated controls. Furthermore, whereas leptin treatment did not affect islet insulin or DNA contents, a significant decrease in islet triglyceride content and glucose-6-phosphatase activity was observed. Moreover, the immunocytochemical data revealed an increased immunostaining for insulin, GLUT-2 and hormone-sensitive lipase (HSL) in islets from leptin-treated ob/ob mice. CONCLUSION: The results suggest that long-term leptin treatment of ob/ob mice improves glucose-stimulated insulin secretion in parallel with reduced glucose-6-phosphatase activity, increased HSL and decreased triglyceride levels in islets. These perturbations may explain the improvement of glucose-stimulated insulin secretion induced by leptin.     

Effect of ciglitazone on glucose uptake and insulin sensitivity in skeletal muscle of the obese (ob/ob) mouse: distinct insulin and glucocorticoid effects

The oral hypoglycemic agent, ciglitazone, (5-[4-(1-methylcyclohexylmethoxy)benzyl]-thiazolidine-2,4-dione), was fed for nine days to genetically obese (ob/ob) mice aged 5 to 6 weeks. This treatment resulted in a lowering of plasma glucose and circulating insulin levels, but did not cause a fall in plasma corticosterone levels. Basal 2-deoxy-D-glucose uptake by the perfused hindquarters of ob/ob mice was unchanged by ciglitazone feeding. In the presence of 0.1 mU/mL insulin in the perfusion medium, there was a significant increase in the uptake rate of 2-deoxy-D-glucose by the skeletal muscle of ciglitazone-treated ob/ob mice, while there was no insulin effect in untreated ob/ob mice. Insulin at a concentration of 1 mU/mL caused a further stimulation of 2-deoxy-D-glucose transport. However, this response was significantly lower than the maximal stimulation in lean mice. Ciglitazone feeding did not have any effect on [5-3H]-glucose metabolism by the perfused muscle which remained subnormal, suggesting that the posttransport metabolism of glucose was limited by substrate availability. In the perfused mouse liver, net [14C]-glucose production from [14C]-lactate was unchanged by ciglitazone treatment while gluconeogenesis from [14C]-alanine was reduced. These findings show that ciglitazone produces its hypoglycemic effect by improving the insulin sensitivity in skeletal muscle, as others have reported in the adipose tissue. The presence of elevated plasma levels of corticosterone and lower levels of insulin in ciglitazone-treated ob/ob mice suggests that the adrenal glucocorticoids are responsible for the basal defects in glucose transport and the hyperinsulinemia is responsible for the insulin insensitivity.     

Release of immunoreactive and radioactively prelabelled endogenous (pro)-insulin from isolated islets of rat pancreas in the presence of exogenous insulin.

To study the influence of insulin on its own secretion, collagenase-isolated islets of rat pancreas were prelabelled with [3H]leucine for 2 h. After washing the islets, (pro-)insulin release was stimulated by glucose in the presence or absence of exogenous insulin (up to 2-5 mu./ml). Hormone release was unchanged by the presence of exogenous insulin as judged by determination of both immunoreactive insulin and radioactivity incorporated into the proinsulin and insulin fractions of the medium. No direct feedback mechanism for insulin secretion was apparent from this study.     

Constitutive expression of a megakaryocytic functional property by murine erythroleukemia (Friend) cells: the incorporation of serotonin.

Murine Friend-derived erythroleukemia cells (MEL) are generally believed to be unipotential progenitors inducible to terminal erythroid differentiation. However, we found that MEL can constitutively incorporate significant amounts of radiolabeled serotonin ([3H]5-HT). Because this process is typical of cells belonging to the megakaryocytic lineage, we investigated the significance and mechanisms of 5-HT incorporation in the MEL system. We observed that: 1) normal murine erythroid cells and erythroid progenitors do not incorporate [3H]5-HT, as well as normal murine myeloid cells and the human myeloid cell line HL-60; on the other hand, the human erythroleukemia cell lines K562 and HEL, which have been shown to constitutively express megakaryocytic features, were able to incorporate [3H]5-HT; 2) MEL incorporated 5-HT by an active and saturable mechanism, dependent on temperature and sodium concentration in the medium; and 3) 5-HT uptake was very rapid. Moreover, because about 65% of cell-associated radioactivity was no longer displaced by the cold substrate, we assumed it to represent "true" cytoplasmic internalization. Finally, 5-HT incorporation by MEL was inhibited by clomipramine, ouabain, and reserpine, which are known inhibitors of 5-HT uptake in platelets. The commitment of MEL to terminal erythroid differentiation by hexamethylene bisacetamide or dimethyl sulfoxide greatly reduced the capacity to incorporate [3H]5-HT. These results seem to suggest that the MEL system, although mainly erythroid as regards its differentiation capability, constitutively expresses features of the megakaryocytic lineage, possibly disclosed by the ability to incorporate 5-HT. This hypothesis was further supported by the findings that 30%-40% of uninduced MEL were labeled by a polyclonal antibody raised against murine platelets that selectively recognized megakaryocytes in murine bone marrow smears.     

Stimulation by D-glucose of 36Cl- efflux from prelabeled rat pancreatic islets

D-glucose was previously reported to cause a concentration-related decrease in the 36Cl- content of prelabeled islets prepared from ob/ob mice, a current animal model of inherited obesity. From these findings, it was inferred that the hexose stimulates Cl- efflux from islet cells and that such an increase in Cl- permeability may partly mediate glucose-induced depolarization of insulin-producing cells. The aim of the present study was to investigate the possible extension of these findings to islets prepared from normal rats by measuring the changes evoked by increasing concentrations of D-glucose in 36Cl- outflow itself from prelabeled isolated islets. After 60 min preincubation at 37 degrees C in the presence of 3 mM D-glucose and 36Cl- (75 microCi/mL), the islets were incubated for 8-10 min at 37 degrees C in the presence of increasing concentrations of the hexose (3-20 mM). The changes in 36Cl- outflow during incubation indicated that D-glucose, in excess of a threshold concentration close to 5 mM, indeed increases effluent radioactivity from the prelabeled islets. It is proposed, therefore, that a gating of volume-sensitive anion channels in glucose-stimulated insulin-producing islet cells participates in the depolarization of the plasma membrane recorded in the range of insulinotropic concentrations of the hexose.     

Endogenous islet uncoupling protein-2 expression and loss of glucose homeostasis in ob/ob mice

We hypothesized that the loss of glucose homeostasis in ob/ob mice is associated with upregulation of islet uncoupling protein-2 (UCP2) expression, leading to impaired glucose-stimulated insulin secretion (GSIS). Changes in glucose homeostasis in lean and ob/ob mice from 5 to 16 weeks were assessed by fasting blood glucose, plasma insulin, oral glucose tolerance, and tissue insulin sensitivity. In vitro GSIS and ATP content were assayed in isolated islets, while UCP2 expression was determined by quantitative real-time PCR and immunoblotting. Short-term reduction of UCP2 expression was achieved through transfection of islets with specific small interfering RNA. Insulin resistance was detected in 5-week-old ob/ob mice, but GSIS and blood glucose levels remained normal. By 8 weeks of age, ob/ob mice displayed fasting hyperglycemia, hyperinsulinemia and glucose intolerance, and also had elevated non-esterified fatty acid concentration in plasma. In vitro, GSIS and ATP generation were impaired in ob/ob islets. Islet UCP2 expression was elevated at 5 and 8 weeks of age. Short-term knockdown of islet UCP2 increased GSIS in islets of lean mice, but had no effect in islets from ob/ob mice. Loss of glucose homeostasis and impairment of insulin secretion from isolated islets at 8 weeks in ob/ob mice is preceded by an increase in UCP2 expression in islets. Moreover, the glucolipotoxic conditions observed are predicted to increase UCP2 activity, contributing to lower islet ATP and GSIS.