Perifused Rat Islets Research Articles

Comparative effects of LN 5330 and diazoxide on insulin release and 86Rb + permeability in perifused rat islets of Langerhans

The benzothiadiazine derivative LN 5330 (chloro-7 trifluoromethyl-6 benzothiadiazine-1,2,4 dioxide-1,1) has been shown to inhibit insulin secretion and calcium uptake. The present study was carried out to investigate the effects of LN 5330 on insulin release and 86Rb + efflux from perifused rat pancreatic islets; a comparison was made with the structural analogue diazoxide. In the presence of 8.3 mM glucose, LN 5330 (100 μM) accelerated 86Rb + efflux while reducing insulin output from the islets. LN 5330 induced a dose-dependent acceleration of 86Rb + efflux and appeared to be a more potent activator of 86Rb + efflux than diazoxide. The stimulatory effect of LN 5330 on 86Rb + efflux persisted in the absence of extracellular calcium. In the absence of glucose, 86Rb + permeability also increased, LN 5330 being again significantly more efficient than diazoxide at an equimolar concentration. These data indicate that the benzothiadiazine derivative LN 5330 inhibits insulin secretion by increasing the potassium permeability of the plasma membrane. It is suggested that, like diazoxide, this drug could activate the ATP-sensitive K + channel.

Oscillations in cytosolic free Ca2+, oxygen consumption, and insulin secretion in glucose-stimulated rat pancreatic islets

Insulin secretion in the intact organism, and by the perfused pancreas and groups of isolated perifused islets, is pulsatile. We have proposed a metabolic model of glucose-induced insulin secretion in which oscillations in the ATP/ADP ratio drive alterations in metabolic and electrical events that lead to insulin release. A key prediction of our model is that metabolically driven Ca2+ oscillations will also occur. Using the fluorescent Ca2+ probe, fura 2, digital image analysis, and sensitive O2 electrodes, we investigated cytosolic free Ca2+ responses and O2 consumption in perifused rat islets that had been maintained in culture for 1-4 days. We found that elevated ambient glucose increased the average cytosolic free Ca2+ level, the ATP/ADP ratio, and oxygen consumption, as previously found in freshly isolated islets. Oscillatory patterns were obtained for Ca2+, O2 consumption, and insulin secretion in the presence of 10 and 20 mM glucose. Very low amplitude oscillations in cytosolic free Ca2+ were observed at 3 mM nonstimulatory glucose levels. Evaluation of the Ca2+ responses of a large series of individual islets, monitored by digital image analysis and perifused at both 3 and 10 mM glucose, indicated that the rise in glucose concentration caused more than a doubling of the average cytosolic free Ca2+ value and a 4-fold increase in the amplitude of the oscillations with little change in period. The pattern of Ca2+ change within the islets was consistent with recruitment of responding cells. The coexistence of oscillations with similar periods in insulin secretion, oxygen consumption, and cytosolic free Ca2+ is consistent with the model of metabolically driven pulsatile insulin secretion.

Content of CoA-esters in perifused rat islets stimulated by glucose and other fuels

Content of CoA-esters in perifused rat islets stimulated by glucose and other fuels

Content of CoA-Esters in Perifused Rat Islets Stimulated by Glucose and Other Fuels

Fluorometry and high-performance liquid chromatography were used to measure the content of free CoA and the esters of acetate, malonate, succinate, and long-chain fatty acids in isolated perifused rat pancreatic islets exposed to 25 mM glucose or a mixture of fuels (25 mM glucose plus 10 mM glutamine, 10 mM lactate, and 1 mM pyruvate) to assess the role of intermediates of lipid metabolism as candidate metabolic coupling factors in the mechanism of fuel-induced insulin secretion. Insulin secretion was stimulated in a biphasic manner with the fuel mixture, showing twice the potency compared with high glucose alone. Islets perifused for 3 min with high glucose alone or the fuel mixture compared with 2.5 mM glucose showed a significant increase in malonyl-CoA and succinyl-CoA and a decrease in acetyl-CoA. Free CoA and long-chain acyl-CoA levels were unaltered. Perifused islets stimulated with 25 mM glucose for 30 min showed a significant increase in succinyl-CoA and long-chain acyl-CoA and decrease in acetyl-CoA, whereas malonyl-CoA was not affected. However, when islets were stimulated by the fuel mixture for 30 min, malonyl-CoA was maintained at a high level, and the change in succinyl-CoA and long-chain acyl-CoA was similar to that observed in islets stimulated with 25 mM glucose alone. The acetyl-CoA concentration in the islets stimulated with the fuel mixture decreased slightly. These results confirm the viability of the hypothesis that malonyl-CoA and long-chain acyl-CoA serve as metabolic coupling factors in signal transduction when islets are stimulated by high glucose or glucose combined with other fuels.

Comparative effects of linogliride fumarate and tolbutamide sodium on insulin secretion and glucose utilization in rat pancreatic islets

AbstractThe effects of linogliride fumarate (0.1 mmol/liter) and tolbutamide sodium (0.5 mmol/liter) on insulin secretion and glucose utilzation were directly compared in isolated perifused rat islets. Both compounds potentiated glucose‐primed (5.5 mmol/liter) insulin release. The insulin secretory response at the concentration chosen was qualitatively different for the two agents. In the presence of glucose, the insulin secretory effect of tolbutamide was biphasic, whereas linoglirde predominantly increased second‐phase insulin secretion. In the absence of exogenous glucos, tolbutamide stimulated first‐phase insulin release wherease linogliride was ineffective without glucose in the perifusion medium. Neither linogliride (0.1 mmol/liter) nor tolbutamide (0.5 mmol/liter) stimulated islet cell glucose usage as measured by conversion of [5−3H]‐glucose to 3H2O. The insulin secretagogue effect of linogliride was completely abolished when islet cell glucose usage was partially blocked (22% reduction) by mannoheptulose (MH) (10 mmol/liter). When glucose usage was significantly inhibited (55%) by 2‐deoxyglucose (2DG) (10 mmol/liter), linogliride‐stimulated insulin secretion was not significantly reduced. In contrast, tolbutamide‐stimulated first‐phase insulin release was not inhibited when glucose usage was reduced by either MH or 2‐DG, wherease secondphase insulin release was significantly inhibited. In summary, linogliride potentiates insulin secretion in isolated islets by a glucose‐dependent process, but with a kinetic profile and response to metabolic inhibitors different from that of tolbutamide.

Chronicin VivoHyperglycemia 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.

Effect of Galanin on Arginine-Stimulated Pancreatic Hormone Release from Isolated Perifused Rat Islets

The effect of galanin on pancreatic hormone release was studied using isolated perifused rat pancreatic islets. In the presence of 100 mg/dl glucose, 10(-8) mol/L galanin significantly inhibited the basal somatostatin release compared with the perifusion without galanin, whereas there was no significant change in the basal insulin and glucagon release. However, under stimulation of 20 mmol/L arginine, 10(-8) mol/L galanin significantly enhanced glucagon release and suppressed insulin and somatostatin release. These effects disappeared immediately after cessation of galanin infusion. Additionally, 10(-8) mol/L galanin significantly enhanced the first and second phase of glucagon release stimulated by arginine, whereas arginine-stimulated insulin and somatostatin releases were significantly inhibited in both phases. In the cysteamine-treated rat islets, neither enhancement of glucagon release nor suppression of insulin release by galanin was reproducible. These findings indicate two possible explanations. First, it is suggested that the effects of galanin on insulin and glucagon release may be direct and reversed by non-specific effect of cycteamine. Secondly, it seems likely that galanin-enhanced glucagon release may be indirect and in part due to the concomitant somatostatin suppression. Galanin may have an important regulatory function on endocrine pancreas.

Interactions between lithium, inositol and mono-oleoylglycerol in the regulation of insulin secretion from isolated perifused rat islets

In response to stimulation by 20 mM-glucose, 15 mM-4-methyl-2-oxopentanoate or 10 mM-glyceraldehyde, isolated perifused rat islets respond with brisk biphasic insulin-secretory responses. The inclusion of 10 mM-LiCl significantly decreased second-phase insulin release in response to all agonists. Inositol, at a concentration (10 mM) which has no effect on secretion in the presence of 2.75 mM-glucose, restored significantly glucose-, 4-methyl-2-oxopentanoate- or glyceraldehyde-induced second-phase release from Li+-treated islets. The addition of the diacylglycerol kinase inhibitor mono-oleoylglycerol, at a concentration (25 microM) which has no stimulatory effect on insulin secretion in the presence of 2.75 mM-glucose, significantly amplified both the first- and second-phase insulin responses to 20 mM-glucose. This amplifying effect of mono-oleoylglycerol was readily reversible and dependent on Ca2+ influx into the beta-cell. Li+ decreased the amplified insulin response to 20 mM-glucose plus mono-oleoylglycerol. Inositol restored release under this condition. These findings suggest that Li+ inhibits release by sequestering inositol into biosynthetically ineffective inositol phosphates. By limiting phosphoinositide resynthesis, the continued hydrolysis of phosphoinositides is diminished. Our results with mono-oleoylglycerol suggest further that diacylglycerol content may play a critically important role in the regulation of both the first and second phases of insulin secretion.

Interactions between cholinergic agonists and enteric factors in the regulation of insulin secretion from isolated perifused rat islets.

The ability of the cholinergic agonist carbachol to sensitize islets to the action of combined glucose, cholecystokinin and gastric inhibitory polypeptide was determined in isolated rat islets. In response to this combination, peak first phase insulin secretion from control islets averages 85 +/- 5 pg.islet-1.min-1 (mean +/- SEM) and the insulin secretory rates measured 35-40 min after the onset of stimulation averages 127 +/- 34 pg.islet-1.min-1. A prior 20 min exposure to 1 mmol/l carbachol potentiates the modest insulin stimulatory response to this combination of stimulants: peak first phase release is 354 +/- 61 pg.islet-1.min-1, and release measured 35-40 min after the onset of stimulation is 179 +/- 34 pg.islet-1.min-1. This sensitizing effect of carbachol lasts for at least 40 min and can be duplicated by the natural in vivo agonist acetylcholine. These results demonstrate that cholinergic stimulation of isolated islets primes them to the subsequent stimulatory effect of a moderate increase in the circulating glucose level and to several postulated incretin factors. If operative in vivo, this communications network between cephalic and enteric factors represents a remarkable control system to ensure the release of insulin in amounts commensurate to meet the anticipated and actual insulin requirements for insulin-mediated fuel disposition.

The effect of monooleoylglycerol on insulin secretion from isolated perifused rat islets.

The effect of monooleoylglycerol on cholecystokinin- and tolbutamide-induced insulin secretion was examined in isolated perifused rat islets. In the presence of 5.5 mmol/l glucose, addition of 10 nmol/l cholecystokinin or 50 mumol/l tolbutamide had practically no effect on insulin secretion. Combined tolbutamide and cholecystokinin led to a biphasic insulin secretory response which was significantly enhanced by addition of 50 mumol/l monooleoylglycerol, an inhibitor of diacylglycerol kinase. Monooleoylglycerol (50 mumol/l) alone had a minimal stimulatory effect on insulin release in the presence of 5.5 mmol/l glucose. Perifusion of islets with 1 mumol/l forskolin had no significant effect on basal insulin secretion in the presence of 5.5 mmol/l glucose, but markedly enhanced the responses to both cholecystokinin plus tolbutamide, and to the combination of cholecystokinin, tolbutamide and monooleoylglycerol. Lowering the glucose level to 2.75 mmol/l abolished the profound stimulatory effect to these agonist combinations on insulin release. Finally, monooleoylglycerol also enhanced the first and second phase insulin secretory responses induced by 20 mmol/l glucose. These results are discussed in relationship to the possible role of protein kinase C in mediating insulin secretion.

Interleukin 1 is a potent stimulator of islet insulin secretion and phosphoinositide hydrolysis

The insulin stimulatory effect of 7 mM glucose on isolated perifused rat islets is dramatically potentiated by the monokine interleukin 1 (IL-1). At levels (10(-10) -10(-8) M) noted in vivo during sepsis, it reversibly amplifies peak second phase insulin release to the hexose. At 2.75 mM glucose, however, IL-1 has no effect on insulin secretion. IL-1 also potentiates glyceraldehyde (2 mM)- and alpha-ketoisocaproate (5 mM)-induced insulin secretion. In islets whose phosphoinositides were prelabeled with myo-[2-3H]inositol, 2.0-5.0 nM IL-1 increases the efflux of [3H]inositol from subsequently perifused islets, the parallel accumulation of labeled inositol phosphates, and insulin secretion in the simultaneous presence of 7 mM glucose but not 2.75 mM glucose. In support of these in vitro observations, the in vivo infusion of IL-1 (40 micrograms/kg body wt) elevated circulating plasma insulin levels two-to fourfold. These results establish IL-1 as a potent, readily reversible, glucose-dependent modulator of stimulated insulin secretion and further suggest that its positive impact on insulin release is mediated, at least in part, by phosphoinositide-derived second messenger molecules. IL-1-induced insulin secretion may participate in the multiple metabolic and immunologic adaptations occurring during sepsis.

Phosphoinositide hydrolysis and insulin release from isolated perifused rat islets. Studies with glucose

Phosphoinositide hydrolysis and insulin release from isolated perifused rat islets. Studies with glucose

Phosphoinositide hydrolysis and insulin release from isolated perifused rat islets. Studies with glucose.

The ability of glucose to promote the hydrolysis of prelabeled [2-3H]inositol-containing phosphoinositides (PI) was assessed by measuring the efflux of 3H in response to glucose and the accumulation of labeled inositol phosphates. The inclusion of nonradioactive inositol (1 mM) in the perifusion medium dramatically improved our ability to monitor glucose-induced increases in 3H efflux. Efflux studies with this method revealed the following. 1) 3H efflux is significantly greater at 7 than at 2.75 mM glucose, and this parallels a small but significant increase in insulin secretion. 2) D-manno-Heptulose reduces 3H efflux with 7 mM glucose to a level approximating that seen in the presence of 2.75 mM glucose and has no effect on 3H efflux with 2.75 mM glucose. 3) In the presence of 20 mM glucose plus 1 mM inositol, 3H efflux is rapid and biphasic, a response that parallels the timing and amplitude of the biphasic pattern of insulin secretion. Direct measurements of labeled inositol and inositol phosphate levels in islets revealed the following. 4) After 50 min of perifusion with 2.75 or 7 mM glucose, labeled inositol phosphates were significantly greater with 7 mM glucose. 5) In response to 20 mM glucose alone, islet levels of free inositol, inositol monophosphate (IP1), and inositol bisphosphate (IP2) increased. 6) In response to 20 mM glucose plus 1 mM cold inositol, islet levels of free inositol increased, whereas islet levels of IP1, IP2, and inositol trisphosphate (IP3) were reduced compared with values obtained with 20 mM glucose alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic impact of cholecystokinin and gastric inhibitory polypeptide on the regulation of insulin secretion

The modulation of insulin output from isolated perifused rat islets by the intestinal peptides cholecystokinin (CCK) and gastric inhibitory polypeptide (GIP) was assessed. In the presence of 7 mmol/L (126 mg/dL) glucose, but not 2.75 mmol/L (50 mg/dL) glucose, CCK (5 nmol/L) or GIP (50 ng/mL) alone evoke small insulin secretory responses. However, the combination of GIP (50 ng/mL) plus CCK (5 nmol/L) together with 7 mmol/L glucose results in a markedly amplified insulin secretory response. CCK (50 nmol/L) alone increases phosphoinositide (PI) hydrolysis in islets, an event reflected by an increase in 3H efflux from myo[2- 3H]inositol prelabeled islets and parallel accumulations of labeled inositol phosphates. GIP (50 ng/mL) alone has no effect on PI hydrolysis. However, GIP reduces the quantitative impact of CCK on PI turnover, an effect attributable to the capacity of GIP to elevate islet cAMP levels. CCK has no significant effect on islet cAMP levels. The results support the concept that the synergistic action of these peptides on insulin output is mediated by their ability to generate separate beta cell second messenger molecules. Nutrient-regulated intestinal release of various peptides represents a remarkable control system to ensure the release of insulin from the beta cell in amounts commensurate with both the quantity and quality of nutrient intake.

Antagonistic effect of dibutyryl cyclic GMP on caerulein induced insulin secretion in isolated perifused rat islets.

The effects of caerulein and dibutyryl cyclic GMP on isolated rat islets were studied using the perifusion system. One, 20, 50 and 100 nM caerulein stimulated insulin secretion in the perifused rat islets, in a dose dependent manner. Insulin secretion reached a maximal level in the presence of 50 nM caerulein. Caerulein-induced insulin secretion showed a predominant first and minimal second phase. After cessation of stimulation with 50 nM caerulein, the insulin secretion level did not return to the basal value, but enhanced hormone secretion was sustained to a significant extent (p less than 0.01). The simultaneous administration of 10(-3) M dibutyryl cyclic GMP, a specific membrane antagonist against cholecystokinin (CCK), inhibited the first and second phases of caerulein-induced insulin secretion. Moreover, with this nucleotide, "persistent" insulin secretion induced by 50 nM caerulein was inhibited. These data indicate that caerulein directly stimulates pancreatic B cells and that this peptide causes "persistent" insulin secretion. Dibutyryl cyclic GMP counteracts the effects of caerulein. Thus, both pancreatic B cells and pancreatic acinar cells possibly possess a CCK receptor.

Antagonistic effect of dibutyryl cyclic GMP on caerulein induced insulin secretion in isolated perifused rat islets

Antagonistic effect of dibutyryl cyclic GMP on caerulein induced insulin secretion in isolated perifused rat islets

Role of phosphoinositide metabolism in induction of memory in isolated perifused rat islets.

Prior exposure of isolated perifused rat islets to 20 mM glucose or 10 mM glyceraldehyde amplifies their subsequent insulin secretory response to 10 mM glucose. The involvement of phosphoinositide turnover in the induction of this "time-dependent potentiation" (TDP) was investigated. In islets in which inositol-containing phospholipids were prelabeled with myo-[2-3H]inositol, the addition of 20 mM glucose augments the efflux of 3H. This effect persists for approximately 50 min after the cessation of stimulation. Direct measurements of labeled inositol phosphate accumulation confirmed that this increase in 3H efflux is primarily the result of a persistent increase in phosphoinositide (PI) hydrolysis and not due to the slow efflux and/or degradation of performed [3H]inositol phosphates. The duration of the increase in 3H efflux parallels the duration of TDP. Mannoheptulose abolishes both the increase in 3H efflux evoked by 20 mM glucose and TDP. The omission of extracellular calcium plus 0.5 mM ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid also abolishes both of these effects of high glucose. D-Glyceraldehyde (10 mM) addition to 3H-inositol-prelabeled islets results in an acute efflux of 3H, a persistent efflux after removal of the D-glyceraldehyde from the perifusion medium, and the induction of TDP. Similar to the results obtained with high glucose, the return of 3H efflux rates to prestimulatory values is accompanied by the abolition of TDP. These results suggest that events associated with persistent stimulant-induced increases in phosphoinositide hydrolysis may participate in the induction and maintenance of TDP.

Insulinotropic effects of vanadate.

Vanadium compounds are known to affect multiple membrane and cytosolic phosphoenzymes from various tissues; the most characterized effect is the inhibition of Na+-K+-ATPase. Since we previously reported that immunoreactive insulin (IRI) secretagogues tend to inhibit rat islet cation-dependent ATPases, we examined the effects of sodium vanadate on rat IRI secretion from incubated and perifused rat islets. In the presence of 2.4 mM Ca2+, vanadate (10(-3) M) induced biphasic IRI secretion with a background glucose of 100 mg/dl. In the absence of extracellular Ca2+, IRI released from incubated islets by vanadate at 100 and 300 mg/dl glucose was doubled and tripled, respectively. Furthermore, this stimulatory effect was completely abolished by known inhibitors of IRI release such as somatostatin, epinephrine, and diphenylhydantoin. Although we found the expected dose-dependent inhibition by vanadate of islet membrane Na+-K+-ATPase activity, the mechanism of action of vanadate on IRI secretion remains unknown. Vanadate probably interacts in a complex fashion with different islet phosphoenzymes and may prove to be a useful probe to further unravel the mechanisms leading to insulin secretion.

Influence of the oral hypoglycemic agent linogliride (McN-3935) on insulin secretion from isolated rat islets of Langerhans.

The influence of a new orally effective hypoglycemic compound, linogliride (McN-3935), on insulin release from isolated perifused rat islets was investigated. At a concentration of 100 microM, linogliride was without effect on insulin secretion in the absence of glucose. While 5.5 mM glucose alone produced a weak secretagogue effect, the secretory response was dramatically (5- to 6-fold) increased by the addition of 100 microM linogliride. This concentration of linogliride did not affect the conversion of [5-3H]glucose to 3H2O, a measure of the rate of glycolysis by the islet. Insulin secretion in response to the combination of 100 microM linogliride and 5.5 mM glucose was abolished by the omission of extracellular calcium. Mannoheptulose (10 mM), an inhibitor of glucose phosphorylation and glucose-stimulated insulin secretion, markedly attenuated the insulinotropic effect of linogliride in parallel with reduced glucose usage. Paradoxically, in the presence of another metabolic inhibitor, 2-deoxyglucose (10 mM), the insulinotropic effect of linogliride (100 microM) in the presence of 5.5 mM glucose was not diminished despite the reduced glucose usage. Linogliride significantly increased the secretory response to other secretagogues, including 2.5 mM D-glyceraldehyde, 15 mM N-acetylglucosamine, 10 mM leucine, and 100 nM of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. These data suggest that linogliride amplifies a cellular signal generated during beta-cell activation by various stimulants.

HPLC analysis of nucleotide profiles in glucose-stimulated perifused rat islets

Nucleotide concentrations were measured in isolated pancreatic islets from rats using HPLC. This method was used to study the role of nucleotides in islets, which were perifused for several hours with different substrates and exhibited signs of exhaustion. Using islet tissue extracts, it was possible to determine 11 nucleotides. It was hoped that alterations in islet nucleotide concentrations would provide clues for the biochemical basis for the exhaustion phenomenon; however, changes in nucleotide concentrations that were observed in exhausted islet tissue did not correlate with corresponding changes in insulin release. Therefore, they are unlikely to be directly related to islet exhaustion.