Pancreatic Secretion In Rats Research Articles

Assessment of exocrine pancreatic function by secretin‐stimulated magnetic resonance cholangiopancreaticography and diffusion‐weighted imaging in healthy controls

To characterize and quantify exocrine pancreatic function by secretin-stimulated magnetic resonance cholangiopancreaticography (s-MRCP) and diffusion-weighted imaging (DWI) in healthy subjects and compare these findings to morphological features, ie, pancreatic volume and secretin-stimulated peak bicarbonate concentration measured in pancreatic juice. Pancreatic magnetic resonance imaging (MRI) (1.5 T) was performed in 20 healthy volunteers among which 10 underwent gastroscopy with duodenal intubation. MRI included T2-weighted imaging and DWI acquired before and 1, 5, 9, and 13 minutes after secretin administration. Secreted pancreatic juice volumes were calculated based on the sequential T2-weighted images and pancreatic volumes and apparent diffusion coefficient (ADC) values were estimated. The mean pancreatic secretion rate declined from 9.5 mL/min at 1-5 minutes (postsecretin) to 2.9 mL/min at 9-13 minutes. Pancreatic head ADC values significantly increased from baseline (1.29 × 10(-3) mm(2) /s) to 1 minute postsecretin (1.48 × 10(-3) mm(2) /s) (P = 0.003). Secreted pancreatic juice volume at 1 minute after secretin correlated positively with peak bicarbonate concentration (n = 10, P = 0.05). Secretin-stimulated MRCP and DWI can characterize and quantify exocrine pancreatic function in healthy subjects. These imaging methods may prove relevant for patients with exocrine pancreatic dysfunction.

TLQP-21, a VGF-derived peptide, stimulates exocrine pancreatic secretion in the rat

The aims of this paper were to study: (1) the effects of TLQP-21 (non-acronic name), the C-terminal region of the VGF (non-acronic name), polypeptide (from residue 557 to 576 of VGF), on in vitro amylase release from rat isolated pancreatic lobules and acinar cells; (2) the mechanism through which TLQP-21 regulates exocrine pancreatic secretion, by using the muscarinic receptor antagonist atropine (10−6M) and the cyclo-oxygenase inhibitor, indomethacin (10−6M). On pancreatic lobules of rats, concentrations of TLQP-21 from 10−7 to 10−5M significantly (p<0.05) induced a 2–3-fold increase of baseline pancreatic amylase release, measured at the end of 60min incubation period. Co-incubation with atropine 10−6M did not antagonise the enzyme outflow induced by the peptide. On the contrary, co-incubation of TLQP-21 (10−7 and 10−6M) with indomethacin, at concentration of 10−6M, which alone did not modify enzyme secretion, completely suppressed the increase of amylase evoked by TLQP-21 on pancreatic lobules. On rat pancreatic acinar cells, TLQP-21, at all the concentrations tested, was unable to affect exocrine pancreatic secretion, indicating an indirect mechanism of action on acinar cells. These results put in evidence, for the first time, that TLQP-21, a VGF-derived peptide, modulates exocrine pancreatic secretion in rats through a stimulatory mechanism involving prostaglandin release. In conclusion, TLQP-21 could be included among the neurohumoral signals regulating pancreatic exocrine secretion, and increases the knowledge concerning the systems controlling this function.

Arginine-induced pancreatitis: involvement of the autonomic nervous system?

to the editor: Dawra et al. ([1][1]) were able to trigger acute pancreatitis in mice after the administration of l-arginine. In addition, acute inflammation in the pancreas was associated with lung injury. With respect to this, we would like to offer some insight into the possible physiological,

Vagally mediated, nonparacrine effects of cholecystokinin-8s on rat pancreatic exocrine secretion

Cholecystokinin (CCK) has been proposed to act in a vagally dependent manner to increase pancreatic exocrine secretion via actions exclusively at peripheral vagal afferent fibers. Recent evidence, however, suggests the CCK-8s may also affect brain stem structures directly. We used an in vivo preparation with the aims of 1) investigating whether the actions of intraduodenal casein perfusion to increase pancreatic protein secretion also involved direct actions of CCK at the level of the brain stem and, if so, 2) determining whether, in the absence of vagal afferent inputs, CCK-8s applied to the dorsal vagal complex (DVC) can also modulate pancreatic exocrine secretion (PES). Sprague-Dawley rats (250-400 g) were anesthetized and the common bile-pancreatic duct was cannulated to collect PES. Both vagal deafferentation and pretreatment with the CCK-A antagonist lorglumide on the floor of the fourth ventricle decreased the casein-induced increase in PES output. CCK-8s microinjection (450 pmol) in the DVC significantly increased PES; the increase was larger when CCK-8s was injected in the left side of the DVC. Protein secretion returned to baseline levels within 30 min. Microinjection of CCK-8s increased PES (although to a lower extent) also in rats that underwent complete vagal deafferentation. These data indicate that, as well as activating peripheral vagal afferents, CCK-8s increases pancreatic exocrine secretion via an action in the DVC. Our data suggest that the CCK-8s-induced increases in PES are due mainly to a paracrine effect of CCK; however, a relevant portion of the effects of CCK is due also to an effect of the peptide on brain stem vagal circuits.

Variation in exocrine pancreatic secretion in rats due to different commercial diets

The diet fed to laboratory animals is one of many variables that can confound research results. The authors investigated the effect of the composition of commercial standard rodent diets on exocrine pancreatic function in rats. They compared two widely used commercial animal diets and found that diet composition greatly influences pancreatic secretion. Their results indicate that commercial diets should conform to the recommended composition requirements to avoid alterations in physiological functions that would eventually affect the results of biomedical research and that investigators should be keenly aware of the composition of the diets being fed to their animals.

Serotonin-3 receptors in gastric mechanisms of cholecystokinin-induced satiety

our understanding of the controls of food intake has increased substantially during the last 30 years. Important in this has been the recognition of the meal as a controlled, physiologically relevant unit of energy intake. During a meal, ingested nutrients accumulate in the stomach and gradually

Autocrine regulation of single pancreatic beta-cell survival.

Function and survival of cells depend in part on the presence of growth factors. We explored the autocrine regulation of insulin and nerve growth factor (NGF) on single adult rat pancreatic beta-cell survival and hormone secretion. When NGF or insulin signaling were blocked in culture media, cell survival decreased compared with control cells, with apoptosis being the main mechanism of cell death. To further explore the role of glucose in beta-cell survival, we cultured the cells for 16 h in 2.6 mmol/l glucose and observed that nearly 17% of the cells developed apoptosis; this effect was partially prevented by NGF and almost completely inhibited by insulin treatment. A high K+ concentration had the same effect, suggesting that insulin and NGF secretion by the cells was responsible for the survival effects and not glucose per se. Blocking NGF signaling with an NGF antibody or with K252a reduced insulin biosynthesis and secretion in the cells that survived the treatment. Moreover, the functional beta-cell subpopulation with a higher insulin secretion rate is more susceptible to K252a. These results further indicate that NGF and insulin play important autoregulatory roles in pancreatic beta-cell survival and function and strongly suggest the need to explore new focuses in diabetes treatment.

Stimulation of rat pancreatic exocrine secretion by urocortin and corticotropin releasing factor

Neural and hormonal mechanisms control pancreatic secretion. The effects of the corticotropin releasing factor (CRF) related neuropeptide urocortin (UCN) on pancreatic exocrine secretion were examined. In anesthetized male rats, pancreatic secretion volume and total protein were assayed. UCN increased pancreatic secretory volume and protein secretion and potentiated cholecytokinin-stimulated protein secretion. Astressin, a non-specific CRF receptor antagonist, inhibited UCN-stimulated protein output while CRF 2 receptor antagonist, antisauvagine-30, was without effect. Atropine, but not subdiaphragmatic vagotomy, inhibited UCN-mediated secretion. In acinar cells, UCN did not stimulate release of amylase nor intracellular cAMP. UCN is a pancreatic exocrine secreatagogue with effects mediated through cholinergic intrapancreatic neurons.

Effect of C-domain N-glycosylation and deletion on rat pancreatic alpha-amylase secretion and activity.

Even though all animal alpha-amylases include glycosylation sequons (Asn-Xaa-Thr/Ser) in their sequences, amylases purified from natural sources are not quantitatively glycosylated. When wild-type rat pancreatic alpha-amylase, which contains two glycosylation sequons, was expressed in animal cell lines the protein displayed a very low rate of glycosylation (approx. 2%), even after Brefeldin A treatment to increase the contact with the glycosylation machinery. Site-directed mutagenesis of the first glycosylation sequon (Asn(410)-->Gln) resulted in 90% of the protein being glycosylated at the second glycosylation sequon (Asn(459)). Mutation of the second sequon completely inhibited glycosylation. In order to ascertain if the interference in the glycosylation of Asn(459) that was eliminated by the Asn(410)-->Gln mutation could be due to the position of the asparagine residue in the Cys(448)-Cys(460) disulphide bridge, these cysteine residues were mutated to serine residues. The resulting mutant was found to be 100% glycosylated. All mutants with mutations in the C-domain had specific activities identical to that of the wild-type enzyme, indicating that enzymic activity is independent of the structure and modification of the C-terminal domain. To further test the independence of the C-domain with respect to the two N-terminal domains of the protein, which harbour the catalytic site, the last seven of the ten beta\beta-strands that make up the beta-sandwich configuration of the domain were deleted. The truncated protein was not secreted from cells and all enzyme activity was destroyed. These observations show that Asn(459) is the only site that can be glycosylated in wild-type amylase, and confirm the relative independence of the C-terminal domain of alpha-amylase with respect to enzyme activity. In addition, they also establish that the C-terminal domain is absolutely essential for the correct post-translational folding of the enzyme that is responsible for its activity and allows for its secretion.

Roles of 5-HT receptors in the release and action of secretin on pancreatic secretion in rats.

5-Hydroxytryptamine (serotonin, 5-HT) is a hormone and neurotransmitter regulating gastrointestinal functions. 5-HT receptors are widely distributed in gastrointestinal mucosa and the enteric nervous system. Duodenal acidification stimulates not only the release of both 5-HT and secretin but also pancreatic exocrine secretion. We investigated the effect of 5-HT receptor antagonists on the release of secretin and pancreatic secretion of water and bicarbonate induced by duodenal acidification in anesthetized rats. Both the 5-HT(2) receptor antagonist ketanserin and the 5-HT(3) receptor antagonist ondansetron at 1-100 microg/kg dose-dependently inhibited acid-induced increases in plasma secretin concentration and pancreatic exocrine secretion. Neither the 5-HT(1) receptor antagonists pindolol and 5-HTP-DP nor the 5-HT(4) receptor antagonist SDZ-205,557 affected acid-evoked release of secretin or pancreatic secretion. None of the 5-HT receptor antagonists affected basal pancreatic secretion or plasma secretin concentration. Ketanserin or ondansetron at 10 microg/kg or a combination of both suppressed the pancreatic secretion in response to intravenous secretin at 2.5 and 5 pmol x kg(-1) x h(-1) by 55-75%, but not at 10 pmol x kg(-1) x h(-1). Atropine (50 microg/kg) significantly attenuated the inhibitory effect of ketanserin on pancreatic secretion but not on the release of secretin. These observations suggest that 5-HT(2) and 5-HT(3) receptors mediate duodenal acidification-induced release of secretin and pancreatic secretion of fluid and bicarbonate. Also, regulation of pancreatic exocrine secretion through 5-HT(2) receptors may involve a cholinergic pathway in the rat.

Stimulation of rat pancreatic exocrine secretion by cocaine- and amphetamine-regulated transcript peptide

Cocaine- and amphetamine-regulated transcript (CART) peptide is a recently described neuropeptide that has been localized to areas of the central and peripheral nervous systems. CART has been shown to be involved in feeding behavior when injected centrally, however, its effects upon peripheral tissues have not been studied. This report describes the effects of CART peptide on rat pancreatic exocrine secretion. Infusion of CART peptide caused four-fold increases in amylase secretion from anesthetized rats that had been fashioned with a bile–pancreatic duct cannula. CART peptide-induced increases in pancreatic secretion appear to involve pathways that are sensitive to both acetylcholine (ACh) and cholecystokinin (CCK) since pre-treatment with atropine (ACh receptor antagonist) or L-364,718 (CCK-A receptor antagonist) inhibited the effects of CART peptide on amylase secretion. Pre-treatment with a combination of atropine and L-364,718 abolished the effects of CART peptide. When isolated rat pancreatic acini were exposed to varying doses of CART peptide, no increase in amylase secretion was observed. The results of the present study suggest that CART peptide has stimulatory effects upon pancreatic exocrine secretion. CART peptide-induced increases in pancreatic secretion appear to be indirectly mediated as no direct effect upon pancreatic acini was shown. CART peptide likely acts upon either peripheral or central regulators of pancreatic secretory function that are distant from the acinar unit.

Kinesin is involved in regulation of rat pancreatic amylase secretion

Kinesin has recently been localized to zymogen granules of pancreatic acini and is suggested to participate in exocytosis of exocrine pancreas. We examined the function of kinesin in regulated exocytosis of pancreatic acini in this study. Kinesin function in exocytosis was examined by introducing hexahistidine-tagged recombinant kinesin protein and antikinesin monoclonal antibody into streptolysin-O-permeabilized acini. Intracellular localization of introduced recombinant kinesin was investigated by immunohistochemistry. Interaction between recombinant kinesin and the microtubule network was confirmed by nocodazole pretreatment of acini. Kinesin regulation by secretagogues was investigated by examining their effect on adenosine triphosphatase (ATPase) activity of endogenous kinesin. Recombinant kinesin enhanced calcium-stimulated amylase release from streptolysin-O-permeabilized acini. Introduced recombinant kinesin was localized to both the microtubule network and zymogen granule. Nocodazole pretreatment of acini abolished the enhancing effect of recombinant kinesin on calcium-stimulated amylase release. Antikinesin antibody inhibited amylase release stimulated by the combination of calcium and cyclic adenosine monophosphate (cAMP) but not that stimulated by calcium alone. Secretin and 8-bromo-cAMP increased ATPase activity of endogenous kinesin. Kinesin plays a stimulatory role in regulated exocytosis of pancreatic acini and is involved in stimulus-secretion coupling through a cAMP-dependent pathway.

Serotonin released from intestinal enterochromaffin cells mediates luminal non–cholecystokinin-stimulated pancreatic secretion in rats

Background & Aims: Similar to cholecystokinin (CCK), non–CCK-dependent duodenal factors stimulate vagal mucosal afferent fibers to mediate pancreatic enzyme secretion via a common cholinergic pathway. We tested the hypothesis that 5-hydroxytryptamine (5-HT) released from enterochromaffin (EC) cells plays an important role in the transduction of luminal information to the central nervous system via vagal afferent fibers to mediate pancreatic secretion. Methods: Pancreatic secretions were examined in conscious rats after intragastric administration of chopped rodent chow in the presence and absence of CCK or 5-HT 3 and 5-HT 2 antagonists. Pancreatic responses to intraduodenal administration of maltose, hyperosmolar NaCl, and light mucosal stroking were examined in rats pretreated with various pharmacological antagonists or after surgical or chemical ablation of vagal and 5-HT neural pathways. Results: Administration of L364,718 inhibited 54% of pancreatic protein secretion evoked by intragastric administration of rodent chow. L364,714 and ICS 205-930, a 5-HT 3 antagonist, combined produced a 94% inhibition. Vagal afferent rootlet section eliminated pancreatic secretions evoked by intraduodenal stimuli. p-Chlorophenylalanine, a 5-HT synthesis inhibitor, but not 5,7-hydroxytryptamine, a 5-HT neurotoxin, also eliminated the pancreatic response to these luminal stimuli. The 5-HT 3 antagonist markedly inhibited pancreatic secretion induced by maltose and hyperosmolar NaCl. 5-HT 2 and 5-HT 3 antagonists combined inhibited the pancreatic response to light stroking of the mucosa. Conclusions: Luminal factors such as osmolality, disaccharides, and mechanical stimulation stimulated pancreatic secretion via intestinal vagal mucosal afferent fibers. It is likely that 5-HT originating from intestinal EC cells activated 5-HT 3 and 5-HT 2 receptors on vagal afferent fibers to mediate luminal factor–stimulated pancreatic secretion. GASTROENTEROLOGY 2000;118:1197-1207

Effect of exogenous insulin and glucagon on exocrine pancreatic secretion in rats in vivo.

The physiological roles of the islet hormones insulin and glucagon in the control of exocrine pancreatic secretion is not clear. It is still unknown whether these hormones have a stimulatory or an inhibitory effect on the basal exocrine pancreatic secretion. Thirty anesthetized rats were stimulated with doses of insulin and glucagon administered by continuous intravenous infusion. Doses varying from physiological to supraphysiological were used. Different groups of 5 rats were given each of these doses. The volume of pancreatic juice and amylase, lipase and trypsin activity, as well as enzyme output, were measured 0, 20, 40, and 60 min after starting infusion. The insulin, glucagon, and glucose levels were determined in serum at 0, 10, 30, and 60 min. In the insulin group, the secreted volume of pancreatic juice increases with the maximum dose. All insulin doses results in amylase and lipase decreased activity. When submaximum and maximum insulin doses are administered, the trypsin activity also decreases. In the glucagon group, the activity of lipase and trypsin decreases regardless the dose, whereas the amylase activity decreases with submaximum and supramaximum doses. Both insulin and glucagon affect the basal exocrine pancreatic secretion in vivo when physiological doses are administered.

Canine pancreatic juice stimulates the release of secretin and pancreatic secretion in the dog.

A secretin-releasing factor (SRF) was found in canine pancreatic juice that increases plasma secretin and stimulates pancreatic secretion in rats, suggesting that a positive feedback mechanism may be involved in the regulation of pancreatic secretion. In the present study, we investigated to determine whether or not SRF releases endogenous secretin and stimulates exocrine pancreatic secretion in conscious dogs. Fresh pancreatic juice was collected from four dogs by intravenous administration of secretin at 0.5 microg. kg(-1). h(-1) and CCK at 0.2 microg. kg(-1). h. The juice was boiled for 10 min at 100 degrees C. Experiments were carried out in phase I of spontaneous cycle of interdigestive pancreatic secretion. The testing solutions were infused intraduodenally in separate experiments: NaHCO3 solution (0.1 M, 4.5 ml/min, 60 min), a corn oil (Lipomul, 2 ml/min, 10 min), boiled pancreatic juice (BPJ, 4.5 ml/min, 60 min), and mixture of BPJ and Lipomul. Pancreatic secretion of fluid and bicarbonate was significantly increased by either BPJ or a mixture of BPJ and Lipomul (34- and 31-fold or 41- and 38-fold, respectively). Plasma secretin level also significantly increased by 164.7 +/- 13.4% and 223.1 +/- 35.0%, respectively, from basal concentration of 1.7 +/- 0.5 pM. In contrast, neither bicarbonate solution nor Lipomul influenced the plasma secretin level or pancreatic secretion. In addition, when Lipomul was incubated with BPJ, no fatty acid was produced. Thus the increased pancreatic secretion in the dog infused with a combination of BPJ and Lipomul was caused by SRF in BPJ, which released endogenous secretin. Moreover, the increases by BPJ of both plasma secretin level and bicarbonate secretion were completely blocked by intravenous administration of an antisecretin antibody in these dogs. The observations suggest that SRF in pancreatic juice exerts a positive feedback effect on exocrine pancreatic secretion that is mediated by the release of secretin in the interdigestive state in dogs.

Diazepam-binding inhibitor33-50 elicits Ca2+ oscillation and CCK secretion in STC-1 cells via L-type Ca2+ channels.

We recently isolated and characterized 86-amino acid CCK-releasing peptide from porcine intestinal mucosa. The sequence of this peptide is identical to that of porcine diazepam-binding inhibitor (DBI). Intraduodenal administration of DBI stimulates the CCK release and elicits pancreatic secretion in rats. In this study we utilized a murine tumor cell line (STC-1 cells) that contains CCK to examine if DBI directly acts on these cells to stimulate CCK release. We investigated the cellular mechanisms responsible for this action. We showed that DBI33-50, a biologically active fragment of DBI1-86, significantly stimulated CCK secretion in STC-1 cells. This action was abolished by Ca2+-free medium. The mean basal intracellular Ca2+ concentration ([Ca2+]i) was 52 nM in fura 2-loaded STC-1 cells. DBI33-50 (1-1,000 nM) elicited Ca2+ oscillations; DBI33-50 (10 nM) increased the oscillation frequency to 5 cycles/10 min and elicited a net [Ca2+]i increase (peak - basal) to 157 nM. In contrast, bombesin and forskolin caused an initial transient [Ca2+]i followed by a small sustained [Ca2+]i plateau. Withdrawal of extracellular Ca2+ abolished Ca2+ oscillations stimulated by DBI33-50. L-type Ca2+ channel blockers nifedipine and diltiazem (3-10 microM) markedly attenuated DBI-stimulated Ca2+ oscillations. In other cell types L-type Ca2+ channels are activated by cAMP-protein kinase A. DBI33-50 failed to stimulate cAMP formation in STC-1 cells. Similarly, DBI33-50 had no effect on myo-inositol 1,4, 5-trisphosphate concentration ([IP3]), whereas bombesin caused an eightfold increase in [IP3] over basal. In addition, inhibitors of phospholipase C (U-73122), phospholipase A2 (ONO-RS-082), and protein tyrosine kinase (genistein) did not alter the Ca2+ oscillations elicited by DBI33-50. It appears that DBI33-50 acts directly on STC-1 cells to elicit Ca2+ oscillations via the voltage-dependent L-type Ca2+ channels, resulting in the secretion of CCK. Mediation of this action is by intracellular mechanisms independent of the traditional signal transduction pathways, including phospholipase C, phospholipase A2, protein tyrosine kinase, and cAMP systems.

P–350 - Effect of Z-203, a CCK-A receptor antagonist, on rat pancreatic exocrine secretion and experimental acute pancreatitis models

P–350 - Effect of Z-203, a CCK-A receptor antagonist, on rat pancreatic exocrine secretion and experimental acute pancreatitis models

Central CGRP inhibits pancreatic enzyme secretion by modulation of vagal parasympathetic outflow.

Calcitonin gene-related peptide (CGRP) is a potent inhibitor of pancreatic enzyme secretion in vivo. Recent studies have shown that CGRP exerts its inhibitory action at a central vagal site. The present study investigates the mechanism responsible for the central action of CGRP. Rats were fitted with lateral cerebroventricular cannulas, using stereotaxic instruments, 4 days before pancreatic secretion studies. In anesthetized rats, administration of 2-deoxy-D-glucose (2-DG) (75 mg/kg iv) or CCK-8 (40 pmol. kg-1. h-1) produced a 100 and 75% increase in protein secretion, respectively, which was completely blocked by atropine. Intracerebroventricular (ICV) administration of CGRP (0.03-0.6 nmol/h) resulted in a dose-related inhibition of pancreatic protein secretion evoked by 2-DG or CCK-8. CGRP administered by the ICV route was 10-40 times more potent than CGRP given by the intravenous route. In contrast, ICV administration of CGRP had no significant effect on pancreatic protein secretion evoked by electrical vagal stimulation or bethanechol, which directly activates the pancreatic muscarinic receptor. Chemical sympathectomy induced by pretreatment with guanethedine (20 mg/kg ip, 2 days) or alpha-adrenergic receptor blockade with phentolamine did not alter the inhibitory effects of CGRP. We recently demonstrated that CCK stimulated the enteropancreatic neural pathways to mediate pancreatic secretion in rats with a chronic vagotomy. ICV-administered CGRP did not affect CCK-stimulated pancreatic secretion in rats with a chronic vagotomy. In conclusion, CGRP in the central nervous system inhibits pancreatic enzyme secretion stimulated by 2-DG and CCK-8, which act through vagal pathways. The inhibitory action of CGRP is not mediated by the sympathetic nervous system but appears to depend on intact vagus nerves.

Contrasting effects of circulating nitric oxide and nitrergic transmission on exocrine pancreatic secretion in rats

Background—Nitric oxide (NO) blockade byl-nitroarginine methyl ester (l-NAME) inhibits pancreatic secretion in vivo and aggravates caerulein induced pancreatitis. Nitric oxide synthase (NOS) is present in pancreatic islets, endothelium, and nerve...

Desensitization of Mitochondrial Ca2+ and Insulin Secretion Responses in the Beta Cell

The role of mitochondria in the desensitization of insulin secretion was investigated. In rat pancreatic beta cells, both insulin secretion and mitochondrial [Ca2+] increases were desensitized following two challenges with the mitochondrial substrate methyl succinate. In the beta cell line INS-1, similar results were observed when a 5-min interval separated two 5-min pulses. In contrast, ATP generation monitored in luciferase-expressing INS-1 cells was stimulated to the same extent during both exposures to methyl succinate. Succinate, like alpha-glycerophosphate, activates the electron transport chain at complex II. As a consequence, the mitochondrial membrane hyperpolarizes, promoting ATP synthesis and Ca2+ influx into the mitochondria through the uniporter. The mitochondrial desensitization was further studied in permeabilized INS-1 cells. Increasing extramitochondrial [Ca2+] from 100 to 500 nM enhanced succinate oxidation 4-fold. At 500 nM Ca2+, 1 mM succinate caused a blunted mitochondrial [Ca2+] increase upon the second, compared with the first, stimulation. These effects were mimicked by alpha-glycerophosphate, and there was cross-desensitization between the two compounds. Succinate hyperpolarized the mitochondrial membrane during both the first and second applications. This suggests that the uniporter itself, rather than the respiratory chain, is desensitized. These results emphasize the key role of the mitochondria not only in the stimulation of insulin secretion, but also in its desensitization.