Increasing uncoupling protein-2 in pancreatic beta cells does not alter glucose-induced insulin secretion but decreases production of reactive oxygen species.

AIMS/HYPOTHESIS: Levels of uncoupling protein-2 (UCP2) are regulated in the pancreatic beta cells and an increase in the protein level has been associated with mitochondrial uncoupling and alteration in glucose-stimulated insulin secretion. However, it is not clear whether an increase in uncoupling protein-2 per se induces mitochondrial uncoupling and affects ATP generation and insulin secretion. MATERIALS AND METHODS: Transgenic mice with beta cell-specific overexpression of the human UCP2 gene and INS-1 cells with doxycycline-inducible overproduction of the protein were generated and the consequences of increased levels of UCP2 on glucose-induced insulin secretion and on parameters reflecting mitochondrial uncoupling were determined. RESULTS: In transgenic mice, an increase in beta cell UCP2 protein concentration did not significantly modify plasma glucose and insulin levels. Glucose-induced insulin secretion and elevation in the ATP/ADP ratio were unaltered by an increase in UCP2 level. In INS-1 cells, a similar increase in UCP2 level did not modify glucose-induced insulin secretion, cytosolic ATP and ATP/ADP ratio, or glucose oxidation. Increased levels of UCP2 did not modify the mitochondrial membrane potential and oxygen consumption. Increased UCP2 levels decreased cytokine-induced production of reactive oxygen species. CONCLUSION/INTERPRETATION: The results obtained in transgenic mice and in the beta cell line do not support the hypothesis that an increase in UCP2 protein per se uncouples the mitochondria and decreases glucose-induced insulin secretion. In contrast, the observation that increased UCP2 levels decrease cytokine-induced production of reactive oxygen species indicates a potential protective effect of the protein on beta cells, as observed in other cell types.

Characterization of two distinct liver progenitor cell subpopulations of hematopoietic and hepatic origins.

Despite extensive studies, the hematopoietic versus hepatic origin of liver progenitor oval cells remains controversial. The aim of this study was to determine the origin of such cells after liver injury and to establish an oval cell line. Rat liver injury was induced by subcutaneous insertion of 2-AAF pellets for 7 days with subsequent injection of CCl(4). Livers were removed 9 to 13 days post-CCl(4) treatment. Immunohistochemistry was performed using anti-c-kit, OV6, Thy1, CK19, AFP, vWF and Rab3b. Isolated non-parenchymal cells were grown on mouse embryonic fibroblast, and their gene expression profile was characterized by RT-PCR. We identified a subpopulation of OV6/CK19/Rab3b-expressing cells that was activated in the periportal region of traumatized livers. We also characterized a second subpopulation that expressed the HSCs marker c-kit but not Thy1. Although we successfully isolated both cell types, OV6/CK19/Rab3b(+) cells fail to propagate while c-kit(+)-HSCs appeared to proliferate for up to 7 weeks. Cells formed clusters which expressed c-kit, Thy1 and albumin. Our results indicate that a bona fide oval progenitor cell population resides within the liver and is distinct from c-kit(+)-HSCs. Oval cells require the hepatic niche to proliferate, while cells mobilized from the circulation proliferate and transdifferentiate into hepatocytes without evidence of cell fusion.

Beta-cell-targeted expression of a dominant-negative hepatocyte nuclear factor-1 alpha induces a maturity-onset diabetes of the young (MODY)3-like phenotype in transgenic mice.

Mutations in the transcription factor hepatocyte nuclear factor-1 alpha (HNF-1 alpha) cause maturity-onset diabetes of the young 3, a severe form of diabetes characterized by pancreatic beta-cell dysfunction. We have used targeted expression of a dominant-negative mutant of HNF-1 alpha to specifically suppress HNF-1 alpha function in beta-cells of transgenic mice. We show that males expressing the mutant protein became overtly diabetic within 6 wk of age, whereas females displayed glucose intolerance. Transgenic males exhibited impaired glucose-stimulated insulin secretion, detected both in vivo and in the perfused pancreas. Pancreatic insulin content was markedly decreased in diabetic animals, whereas the glucagon content was increased. Postnatal islet development was altered, with an increased alpha-cell to beta-cell ratio. beta-Cell ultrastructure showed signs of severe beta-cell damage, including mitochondrial swelling. This animal model of maturity-onset diabetes of the young 3 should be useful for the further elucidation of the mechanism by which HNF-1 alpha deficiency causes beta-cell dysfunction in this disease.

Activation of protein kinase B/cAkt in hepatocytes is sufficient for the induction of expression of the gene encoding glucokinase.

Inhibitors of signalling pathways were used to dissect the mechanism of insulin action on expression of the gene encoding glucokinase in cultured rat hepatocytes. Wortmannin and LY 294002 completely prevented the insulin-induced increase in glucokinase mRNA seen in unhibited cells, indicating that the phosphoinositide 3-kinase module has a key role. A ligand inducible protein kinase B (PKB, also termed cAkt) fusion protein was expressed by using adenoviral transduction of hepatocytes in primary culture. The PKB activity of this protein was shown to be activated in transduced hepatocytes within 30 min of the addition of 4-hydroxytamoxifen and to stay high for 8 h, as a result of serine phosphorylation at position 473 of PKB. The increase in PKB activity was reflected in the hyperphosphorylation of phosphorylated, heat and acid stable regulated by insulin protein (PHAS-I; also termed 4E-BP1, for eukaryotic initiation factor 4E-binding protein 1), a protein involved in the regulation of translation initiation. These effects were comparable to the insulin-induced activation of endogenous PKB and phosphorylation of PHAS-I in non-transduced hepatocytes. The addition of tamoxifen to transduced hepatocytes resulted in an induction of glucokinase mRNA with kinetics and magnitude similar to those of insulin-induced mRNA accumulation. The effect of tamoxifen depended on stimulated PKB activity because it did not occur in hepatocytes that were transduced with a mutant PKB fusion protein that was refractory to activation with tamoxifen. These results establish that acute activation of PKB is sufficient to produce an insulin-like induction of glucokinase in isolated hepatocytes. Together with the inhibition by phosphoinositide 3-kinase inhibitors, they suggest that the activation of PKB might be critical in mediating the induction of glucokinase by insulin. In addition, experiments showed that PD98059 decreased by half the increase in glucokinase mRNA brought about by insulin, suggesting a contributory role of the mitogen-activated protein kinase cascade.

Junctional communication of pancreatic beta cells contributes to the control of insulin secretion and glucose tolerance.

Proper insulin secretion requires the coordinated functioning of the numerous beta cells that form pancreatic islets. This coordination depends on a network of communication mechanisms whereby beta cells interact with extracellular signals and adjacent cells via connexin channels. To assess whether connexin-dependent communication plays a role in vivo, we have developed transgenic mice in which connexin 32 (Cx32), one of the vertebrate connexins found in the pancreas, is expressed in beta cells. We show that the altered beta-cell coupling that results from this expression causes reduced insulin secretion in response to physiologically relevant concentrations of glucose and abnormal tolerance to the sugar. These alterations were observed in spite of normal numbers of islets, increased insulin content, and preserved secretory response to glucose by individual beta cells. Moreover, glucose-stimulated islets showed improved electrical synchronization of these cells and increased cytosolic levels of Ca(2+). The results show that connexins contribute to the control of beta cells in vivo and that their excess is detrimental for insulin secretion.

Short-term insulin-induced glycogen formation in primary hepatocytes as a screening bioassay for insulin action.

We describe a novel bioassay to measure specific insulin-like activity in primary cultures of rat hepatocytes by determination of [3H]glycogen from d-[6-3H]glucose. The dose-response curve of insulin in this assay exhibited an EC50 of 0.42 (+/-0.04) nM, which is comparable to the dissociation constant of insulin from its receptor in hepatocytes. We used this assay to examine possible residual insulin-like activity of the four major fragments formed upon insulin degradation by insulin protease. Fragments A1-13B1-9, A1-14B1-9,and A14-21B14-30 showed no measurable activity. Although preparations of fragment A14-21B10-30 displayed dose-dependent agonist activity with an EC50 of 380 (+/-40) nM, we conclude that this was due to an insulin-like impurity since the chemically synthesized fragment showed no such activity. In summary, this bioassay demonstrates the action of insulin on glycogen formation in hepatocytes and provides a rapid and sensitive measurement of insulin-like activity which could facilitate screening studies.

Liver-specific enhancer of the glucokinase gene.

Glucokinase gene regions that are important for liver specific expression of the enzyme have been functionally identified using transient transfection of rat hepatocytes. Maximal luciferase activity was elicited by a reporter plasmid with 3.4 kilobase pairs of genomic DNA flanking the liver glucokinase promoter. Deletion of a gene fragment between -1000 and -600 with respect to the start of transcription resulted in a 60% decrease in luciferase activity. Further reduction, close to background level, occurred upon deletion of a 90-base pair sequence between -123 and -34. Reporter plasmids with the liver glucokinase promoter and any length of flanking sequence were minimally active in INS-1 insulinoma cells, and conversely reporters with the beta-cell-specific promoter were ineffective in primary hepatocytes. In FTO-2B hepatoma cells, a differentiated line expressing many liver-specific traits but not the endogenous glucokinase gene, the promoter proximal region between -123 and -34 markedly stimulated the expression of transfected plasmids above background. However, addition of the flanking region up to -1000 inhibited luciferase expression. The gene fragment from -1003 to -707 was shown to be a bona fide, hepatocyte-specific enhancer by the following criteria: 1) it stimulated reporter expression by more than 10- and 5-fold when inserted directly upstream of the glucokinase TATA box or complete promoter, respectively, regardless of orientation; 2) it stimulated gene expression from the heterologous SV 40 promoter 4-fold; 3) it was also effective from a downstream position; and 4) in contrast to the enhancer effect in primary hepatocytes, the sequence acted as a silencer in FTO-2B cells and was neutral in INS-1 cells. Both the promoter proximal and the enhancer regions were marked by DNase I hypersensitive sites in the chromatin of primary hepatocytes but not hepatoma or insulinoma cells. Seven footprinted elements termed A through G were mapped in the enhancer by the in vitro DNase I protection assay. Elements A-C may bind liver enriched factors, because they were not protected by spleen nuclear extract. In hepatocyte transfection, the downstream half of the enhancer containing elements A-C was about half as effective as the complete enhancer in stimulating glucokinase promoter activity. Site-directed mutagenesis of element A virtually abrogated the activity of the half-enhancer, whereas mutation of element C had a more moderate effect. The sequence between -732 and -578 upstream of the liver start of transcription in the human glucokinase gene displays 79% sequence identity with the downstream half of the rat enhancer. The human gene fragment ligated to the minimal rat liver glucokinase promoter was shown to work as an enhancer in the hepatocyte transfection system.

Glucokinase and cytosolic phosphoenolpyruvate carboxykinase (GTP) in the human liver. Regulation of gene expression in cultured hepatocytes.

Glucokinase and phosphoenolpyruvate carboxykinase are key enzymes of glucose metabolism in the rat liver. The former is considered to be instrumental in regulating glucose hepatic release/uptake according to the glycaemia level, and cytosolic phosphoenolpyruvate carboxykinase is a major flux-generating enzyme for gluconeogenesis. The level of expression of both enzymes and the regulation of their mRNAs in the human liver cell were investigated. Surgical biopsies of liver from patients undergoing partial hepatectomies and parenchymal hepatocytes derived from the biopsies were used to assay glucokinase, hexokinase and phosphoenolpyruvate carboxykinase activities. Hepatocytes were placed in culture and the actions of insulin, glucagon and cAMP on glucokinase and phosphoenolpyruvate carboxykinase mRNAs were studied. The main results are: (a) glucokinase accounts for 95% of the glucose phosphorylation activity of human hepatocytes, although this fact is masked in assays of total liver tissue; (b) glucokinase activity is set at a lower level in human hepatocytes than in rat hepatocytes, and vice-versa for the gluconeogenic enzyme phosphoenolpyruvate carboxykinase; and (c) as previously shown in rat liver, glucokinase and phosphoenolpyruvate carboxykinase mRNAs are regulated in a reciprocal fashion in human hepatocytes, insulin inducing the first enzyme and repressing the latter, whereas glucagon has opposite effects. These data have interesting implications with respect to metabolic regulation and intracellular hormone signaling in the human liver.

In vivo phosphorylation of the Na,K-ATPase alpha subunit in sciatic nerves of control and diabetic rats: effects of protein kinase modulators.

The phosphorylation state of the Na,K-ATPase alpha subunit has been examined in 32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit 32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on alpha-subunit 32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca(2+)-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the 32P labeling of the alpha subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca(2+)-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased 32P labeling of the alpha subunit. In contrast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significant in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such nerves. In conclusion, the altered effect of PK activators on Na,K-ATPase phosphorylation state is consistent with the view that a defect in PKC activation exists in diabetic nerves.

Alpha, beta I, beta II, delta, and epsilon protein kinase C isoforms and compound activity in the sciatic nerve of normal and diabetic rats.

Defective protein kinase C (PKC) has been implicated in impaired Na+,K(+)-ATPase activity in the sciatic nerve of streptozotocin-induced diabetic rats. In the present study, alpha, beta I, beta II, gamma, delta, and epsilon isoform-specific antibodies were used in parallel to the measurement of compound PKC activity for the characterization of PKC distribution and isoform expression in sciatic nerves of normal and diabetic rats. To distinguish isoform expression between the axonal and glial compartments, PKC isoforms were evaluated in nerves subjected to Wallerian degeneration and in a pure primary Schwann cell culture. alpha, beta I, beta II, delta, and epsilon but no gamma isoforms were detected in sciatic nerve. Similar immunoreactivity was observed in degenerated nerves 3-4 days after transection except for diminished beta I and epsilon species; in Schwann cell cultures, only alpha, beta II, delta, and epsilon were detected. In normal nerves, two-thirds of PKC compound activity was found in the cytosol and 50% of total enzyme activity translocated to the Na+,K(+)-ATPase-enriched membrane fraction with phorbol myristate acetate. Similar redistribution patterns were observed for the immunoreactivity of all isoforms with the exception of delta, which did not translocate to the membrane with phorbol myristate acetate. No abnormality in compound PKC activity, in the immunoreactive intensity, or in the distribution of PKC isoforms could be detected in rat sciatic nerve after 6-12 weeks of diabetes. Thus, defective activation rather than decreased intrinsic PKC activity may occur in diabetic neuropathy.

Glucagon-like peptide-I-(7-37) suppresses hyperglycemia in rats.

Glucagon-like peptide-(GLP) I-(7-37) is an endogenous hormone that has recently been demonstrated to be a potent insulin secretagogue. In these studies, GLP was administered during oral and intravenous (IV) glucose tolerance tests (OGTT and IVGTT, respectively) to determine whether this peptide could enhance postprandial insulin levels and thus reduce glycemic excursions. Surprisingly, during OGTT, GLP administration did not augment insulin secretion; however, GLP administration resulted in significantly lower glycemic excursions. In fasted rats, glycemic excursions were significantly reduced 10 and 20 minutes after receiving GLP (P < .001). Fed rats that received GLP had virtually no initial increase in plasma glucose level after administration of oral glucose. During IVGTT, glucose alone increased insulin levels eightfold, while administration of both glucose and GLP resulted in a 15-fold increase (P < .001). These IVGTT data support previous studies that show GLP to be a potent and glucose-dependent insulin secretagogue. Furthermore, all of these studies suggest that GLP reduces postprandial glycemic excursion and thus may be useful in the treatment of non-insulin-dependent diabetes mellitus.

Cyclic adenosine monophosphate prevents the glucocorticoid-mediated inhibition of insulin gene expression in rodent islet cells.

Dexamethasone negatively regulates insulin gene expression in HIT-15 cells. In vivo, however, an excess of glucocorticoids results in an increase in insulin biosynthesis and peripheral hyperinsulinemia. To resolve this contradiction, we have studied the effects of dexamethasone in primary rat islet cells. We show here that dexamethasone decreases insulin mRNA levels in single islet cells, as in HIT-15 cells, but does not affect these levels in reaggregated islet cells and increases them in intact islets of Langerhans. Because cAMP is an important regulator of insulin gene expression and intracellular cAMP content may be decreased in single beta cells, we investigated whether cAMP could prevent the inhibitory effect of dexamethasone on insulin mRNA levels. In the presence of cAMP analogues, the inhibitory action of dexamethasone was not only prevented, but insulin mRNA increased to levels comparable to those observed when cAMP analogues were used alone. We conclude that the insulin gene is negatively regulated by dexamethasone in single islet cells, but that other factors such as cAMP prevent this effect when the native environment of islet cells is preserved. Our results indicate that insulin gene regulation is influenced by cell to cell contacts within the islet, and that intracellular cAMP levels might be influential in this regulation.

Unimpaired effect of insulin on glucokinase gene expression in hepatocytes challenged with amylin.

Amylin appears to interfere with the action of insulin in muscle and possibly in liver. We have attempted to detect a direct antagonism between amylin and insulin in cultured rat hepatocytes. The stimulation of glucokinase gene expression was used as a marker of insulin action. Amylin proved ineffective in suppressing subsequent accumulation of glucokinase mRNA in response to maximal or submaximal doses of insulin. When applied to cells already induced by prior incubation with insulin alone, amylin failed to reverse induction, in contrast to the effectiveness of glucagon under the same conditions. Thus, amylin is not a physiological antagonist of insulin in the control of hepatic glucokinase gene expression.

Inverse relationship between glucose metabolism and glucose-induced insulin secretion in rat insulinoma cells.

Slowly growing X-ray-induced rat insulinomas and derived cell lines have been used as a model system for glucose-induced insulin release. During perfusions of tumors transplanted under the kidney capsule, the carbohydrates glucose and D-glyceraldehyde increased insulin secretion. These stimuli and the amino acids leucine and alanine also provoked insulin release in freshly isolated tumor cells. Under these conditions, glucose utilization had a Km of 4.6 mM and maximal velocity of 0.9 nmol/min/10(6) cells. A continuous cell line was established from such a preparation. In culture, glucose-induced insulin secretion was no longer detectable while responses to D-glyceraldehyde and amino acids were retained. Glucose metabolism in the cell line showed a decrease in Km to 0.7 mM glucose and an increased maximal velocity of 1.4 nmol/min/10(6) cells. Attempts to revert these alterations were undertaken using glucose-deficient culture medium to diminish glycolytic flux. Basal insulin release was lowered, while the growth pattern of the cells remained unchanged. Another approach involved the use of sodium butyrate which has been demonstrated to promote differentiation in other cell systems. Whereas sodium butyrate markedly increased cellular insulin content, the secretory responses were not improved. These results provide evidence that the loss of glucose-induced insulin secretion is paralleled by alterations in glucose metabolism.

Stimulation by insulin of glucokinase gene transcription in liver of diabetic rats.

The purpose of this work was to investigate the molecular mechanism responsible for the induction of hepatic glucokinase in diabetic rats acutely treated with insulin. Experimental diabetes was provoked by injection of streptozotocin 8-10 days before the experiments. Regular insulin was given by three intraperitoneal injections at 8-h intervals, and the time course of glucokinase induction was followed over a time period of 24 h. The amount of glucokinase in liver was estimated by Western blotting of total cytosol protein with affinity-purified antibodies, as well as by conventional enzyme activity assay. Both measurements showed that glucokinase was reduced by more than 90% in the livers of diabetic rats as compared to normal controls. Following insulin administration, the amount (and activity) of glucokinase increased in a time-dependent fashion, after an initial lag of 4 h, to reach 65% of the nondiabetic control level 24 h after the initial dose of insulin. Northern blot analysis with a cloned cDNA probe was used to quantitate glucokinase mRNA. In contrast with the slow onset of enzyme accumulation, the amount of glucokinase mRNA was shown to be increased dramatically as early as 1 h after insulin administration. The abundance of specific mRNA increased until 8 h after the initial dose of insulin. Subsequently, the level of the mRNA decayed rapidly so that little message was left after 16 h and virtually none after 24 h. Run-on transcription experiments with isolated nuclei showed that the rate of transcription of the glucokinase gene was increased about 20-fold within 45 min of insulin administration and returned to the prestimulation level after 8 h. From these data, it was concluded that the induction of glucokinase resulted primarily from a burst in the transcriptional activity of the gene, leading to a short-term accumulation of glucokinase mRNA. The more sustained elevation of the enzyme level can be accounted for by the long half-life of the enzyme (greater than 30 h). The virtually immediate activation of glucokinase gene transcription suggests a direct effect of insulin on the liver cell.

Direct stimulation of enzyme secretion from rat exocrine pancreas by neurotensin and its naturally occurring fragments.

Neurotensin stimulates amylase release from dispersed pancreatic acini at concentrations as low as 10(-15) M. The naturally occurring fragments of neurotensin (NT), NT 1-8 and NT 1-11, also stimulate amylase secretion at concentrations that occur in peripheral plasma (10(-11) M). The analogue D-Phe11 neurotensin was as potent as neurotensin itself with respect to stimulation of amylase secretion. Basal plasma neurotensin levels were approximately 10 pmol/L, a concentration that stimulates the exocrine pancreas in vitro. Increases in plasma neurotensin levels induced by infusion of neurotensin caused greater increases in secretion of pancreatic amylase than did similar changes of neurotensin concentration in vitro. These results suggest that in addition to its direct action, other factors are involved in modulation of the effects of neurotensin on pancreatic exocrine function in vivo.

Regulation of pancreatic exocrine function: a role for cell-to-cell communication?

The effect of heptanol, an alkanol which decreases gap junctional permeability, was investigated in the perfused rat pancreas. Under basal conditions, heptanol (3.5 mM) caused a three- to fourfold increase of pancreatic juice, protein, and amylase outputs. The effect on enzyme secretion was fully reversible upon removal of the alkanol and was not secondary to the release of acetylcholine from nerve endings, since it was not prevented by addition of atropine (10(-6) M) to the perfusate. By contrast, another alkanol, hexanol (3 mM), which does not decrease coupling between acinar cells in spite of anesthetic properties analogous to those of heptanol, did not alter pancreatic secretion. The effect of heptanol was not mediated by a significant stimulation of cyclic AMP, nor did the alkanol increase the secretion of lactic dehydrogenase, a cytosolic marker. Analysis of the numerical density of freeze-fractured and immunolabeled gap junctions between acinar cells did not show differences between heptanol-perfused and control pancreases. In addition, heptanol did not alter carbachol (10(-6) M and 10(-5) M)-evoked amylase release. Since heptanol blocks cell coupling, apparently without interfering with the main intracellular pathways triggering enzyme release, we suggest that downregulation of direct cell-to-cell communications increases pancreatic exocrine secretion. Thus, cell coupling is probably involved in the regulation of the secretory activity of acinar cells.

Differences in pancreatic enzyme release from ventral and dorsal areas of the rat pancreas.

The exocrine secretory function of the ventral and dorsal areas of the rat pancreas was investigated separately. In the isolated perfused rat pancreas, caerulein (10(-12), 10(-11), and 10(-10) M) and carbachol (10(-7) and 10(-6) M) caused a greaterfold increase in pancreatic secretion from the dorsal than the ventral area. Caerulein (10(-9) M) and carbachol (10(-5) M) were supramaximal concentrations with respect to secretion from the dorsal area, but this was not the case in the ventral area. Similar findings were also observed when ventral and dorsal secretions were studied in vivo in anesthetized animals. By contrast, in dispersed acini the sensitivity and responsiveness of tissue from the dorsal and ventral areas were similar to both secretagogues. The possible influence of the endocrine pancreas via the insuloacinar portal system was studied in the perfused pancreas of diabetic rats treated with insulin in vivo. Carbachol (10(-6) M)-stimulated secretion from the ventral area was similar (except for amylase) to that of controls, while a significantly reduced (P less than 0.001) output was measured from the dorsal pancreas. These results demonstrate that in the intact pancreas differences of exocrine secretion exist between the ventral and dorsal areas and suggest that insulin may contribute, at least in part, to the expression of such differences.

Activity of the insulo-acinar axis in the isolated perfused rat pancreas.

The object of the present investigation was to determine whether insulin secreted by the endocrine pancreas and carried in the insulo-acinar portal system has a direct effect on pancreatic enzyme secretion. For this purpose, the isolated rat pancreas was perfused in a nonrecirculating system. The perfusate contained 3 mM glucose, and either caerulein or vaso-active intestinal polypeptide was used to stimulate exocrine secretion. The amount of insulin reaching the exocrine pancreas was reduced by two different experimental procedures. In the first, use was made of streptozotocin-diabetic rats treated with insulin in vivo. Treatment was such that the contents of amylase and lipase, vastly altered in the untreated diabetic state, were normalized before the perfusion studies. In the second procedure, insulin reaching the exocrine pancreas was reduced by antiinsulin serum in the perfusate. In these procedures, the reduced insulin bioavailability was associated with a reduction in caerulein- and vasoactive intestinal polypeptide-stimulated enzyme release, which was shown as a reduction of maximum responsiveness to caerulein without alteration of sensitivity. By contrast, in dispersed pancreatic acini where the insulo-acinar axis was completely disrupted, amylase secretion from diabetic and nondiabetic tissue was identical over a wide range of caerulein concentrations, showing that the secretory defect seen in the perfusion studies was not inherent to the exocrine tissue. The results show that basal insulin secretion has a direct effect on pancreatic enzyme output and that the insulo-acinar axis may play an important role in the regulation of acinar cell function.

A new, rapid, method for preparation of dispersed pancreatic acini.

A new method for the preparation of pancreatic acini is described. The method is simple and much more rapid than previously described techniques, the time required for preparation of pancreatic acini being 20 min from removal of the pancreas. Acini prepared with this method perform in a superior manner when stimulated by either caerulein or secretin. Thus this new technique would be ideal for use in binding and secretion studies.