Reanalysis of study of pancreatic effects of incretin therapy: methodological deficiencies.

A recently published study by Butler et al. concluded that incretin treatment had adverse effects on the human type 2 diabetic pancreas including 'a marked expansion of the exocrine and endocrine pancreatic compartments, the former being accompanied by increased proliferation and dysplasia and the latter by α-cell hyperplasia with the potential for evolution into neuroendocrine tumours'. Incretin therapy has become widely used for type 2 diabetes, so these conclusions have instigated major concerns with regard to patient safety. We reassessed both the clinical case information and virtual microscopy images of the same 34 cases that were used in the Butler study as well as Network for Pancreatic Organ Donation (nPOD) cases that were not included. Whereas we would like to stress that it is important to investigate in depth any indication that incretin treatment may lead to inflammation or dysplasia in the pancreas, we find that the data presented in the Butler paper have serious methodological deficiencies that preclude any meaningful conclusions.

Rat neonatal beta cells lack the specialised metabolic phenotype of mature beta cells.

AIMS/HYPOTHESIS: Fetal and neonatal beta cells have poor glucose-induced insulin secretion and only gain robust glucose responsiveness several weeks after birth. We hypothesise that this unresponsiveness is due to a generalised immaturity of the metabolic pathways normally found in beta cells rather than to a specific defect. METHODS: Using laser-capture microdissection we excised beta cell-enriched cores of pancreatic islets from day 1 (P1) neonatal and young adult Sprague-Dawley rats in order to compare their gene-expression profiles using Affymetrix U34A microarrays (neonatal, n = 4; adult, n = 3). RESULTS: Using dChip software for analysis, 217 probe sets for genes/38 expressed sequence tags (ESTs) were significantly higher and 345 probe sets for genes/33 ESTs significantly lower in beta cell-enriched cores of neonatal islets compared with those of adult islets. Among the genes lower in the neonatal beta cells were key metabolic genes including mitochondrial shuttles (malate dehydrogenase, glycerol-3-phosphate dehydrogenase and glutamate oxalacetate transaminase), pyruvate carboxylase and carnitine palmitoyl transferase 2. Differential expression of these enzyme genes was confirmed by quantitative PCR on RNA from isolated neonatal (P2 until P28) and adult islets and with immunostaining of pancreas. Even by 28 days of age some of these genes were still expressed at lower levels than in adults. CONCLUSIONS/INTERPRETATION: The lack of glucose responsiveness in neonatal islets is likely to be due to a generalised immaturity of the metabolic specialisation of pancreatic beta cells.

Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells.

AIM/HYPOTHESIS: Neonatal beta cells lack glucose-stimulated insulin secretion and are thus functionally immature. We hypothesised that this lack of glucose responsiveness results from a generalised low expression of genes characteristic of mature functional beta cells. Important glucose-responsive transcription factors, Mafa and Pdx1, regulate genes involved in insulin synthesis and secretion, and have been implicated in late beta cell development. The aim of this study was to assess whether Mafa and/or Pdx1 regulates the postnatal functional maturation of beta cells. METHODS: By quantitative PCR we evaluated expression of these and other beta cell genes over the first month compared with adult. After infection with adenovirus expressing MAFA, Pdx1 or green fluorescent protein (Gfp), P2 rat islets were evaluated by RT-PCR and insulin secretion with static incubation and reverse haemolytic plaque assay (RHPA). RESULTS: At P2 most beta cell genes were expressed at about 10% of adult, but by P7 Pdx1 and Neurod1 no longer differ from adult; by contrast, Mafa expression remained significantly lower than adult through P21. Overexpression of Pdx1 increased Mafa, Neurod1, glucokinase (Gck) mRNA and insulin content but failed to enhance glucose responsiveness. Similar overexpression of MAFA resulted in increased Neurod1, Nkx6-1, Gck and Glp1r mRNAs and no change in insulin content but, importantly, acquisition of glucose-responsive insulin secretion. Both the percentage of secreting beta cells and the amount of insulin secreted per beta cell increased, approaching that of adult beta cells. CONCLUSIONS/INTERPRETATION: In the process of functional maturation acquiring glucose-responsive insulin secretion, neonatal beta cells undergo a coordinated gene expression programme in which Mafa plays a crucial role.

Why pancreatic islets should be regarded and regulated like organs.

There are strong reasons to say that pancreatic islets are organs before they are isolated and that they should be considered to be organs once transplanted. Thus, taking into account how much we have learned about the structure and function of islet micro-organs, it seems highly illogical to on one hand consider autologous islets be regulated as organ transplants and alloislets to be regulated with the very restrictive rules used for cell transplantation. It is particularly problematic that this policy has led to restrictions that have made it next to impossible for transplants of alloislets to be carried out in the US, which is a very sad situation for the country that made so many of the advances that brought islet transplantation to the clinic.

Rat islet cell aggregates are superior to islets for transplantation in microcapsules.

AIMS/HYPOTHESIS: Islet transplantation is a promising treatment for type 1 diabetes but is hampered by a shortage of donor human tissue and early failure. Research on islet cell transplantation includes finding new sources of cells and immunoisolation to protect from immune assault and tumourigenic potential. Small islet cell aggregates were studied to determine if their survival and function were superior to intact islets within microcapsules because of reduced oxygen transport limitation and inflammatory mediators. METHODS: Islet cell aggregates were generated by dispersing rat islets into single cells and allowing them to re-aggregate in culture. Rat islets and islet cell aggregates were encapsulated in barium alginate capsules and studied when cultured in low (0.5% or 2%) or normal (20%) oxygen, or transplanted into mice. RESULTS: Encapsulated islet cell aggregates were able to survive and function better than intact islets in terms of oxygen-consumption rate, nuclei counts, insulin-to-DNA ratio and glucose-stimulated insulin secretion. They also had reduced expression of pro-inflammatory genes. Islet cell aggregates showed reduced tissue necrosis in an immunodeficient transplant model and a much greater proportion of diabetic xenogeneic transplant recipients receiving islet cell aggregates (tissue volume of only 85 islet equivalents) had reversal of hyperglycaemia than recipients receiving intact islets. CONCLUSIONS/INTERPRETATION: These aggregates were superior to intact islets in terms of survival and function in low-oxygen culture and during transplantation and are likely to provide more efficient utilisation of islet tissue, a finding of importance for the future of cell therapy for diabetes.

OVO homologue-like 1 (Ovol1) transcription factor: a novel target of neurogenin-3 in rodent pancreas.

AIMS/HYPOTHESIS: The basic helix-loop-helix transcription factor neurogenin-3 (NGN3) commits the fates of pancreatic progenitors to endocrine cell types, but knowledge of the mechanisms regulating the choice between proliferation and differentiation of these progenitors is limited. METHODS: Using a chromatin immunoprecipitation cloning approach, we searched for direct targets of NGN3 and identified a zinc-finger transcription factor, OVO homologue-like 1 (OVOL1). Transactivation experiments were carried out to elucidate the functional role of NGN3 in Ovol1 gene expression. Embryonic and adult rodents pancreases were immunostained for OVOL1, Ki67 and NGN3. RESULTS: We showed that NGN3 negatively regulates transcription of Ovol1 in an E-box-dependent fashion. The presence of either NGN3 or NEUROD1, but not MYOD, reduced endogenous Ovol1 mRNA. OVOL1 was detected in pancreatic tissue around embryonic day 15.5, after which OVOL1 levels dramatically increased. In embryonic pancreas, OVOL1 protein levels were low in NGN3(+) or Ki67(+) cells, but high in quiescent differentiated cells. OVOL1 presence was maintained in adult pancreas, where it was detected in islets, pancreatic ducts and some acinar cells. Additionally OVOL1 presence was lacking in proliferating ductules in regenerating pancreas and induced in cells as they began to acquire their differentiated phenotype. CONCLUSIONS/INTERPRETATION: The timing of OVOL1 appearance in pancreas and its increased levels in differentiated cells suggest that OVOL1 promotes the transition of cells from a proliferating, less-differentiated state to a quiescent more-differentiated state. We conclude that OVOL1, a downstream target of NGN3, may play an important role in regulating the balance between proliferation and differentiation of pancreatic cells.

Single pancreatic beta cells co-express multiple islet hormone genes in mice.

AIMS/HYPOTHESIS: It is widely accepted that production of insulin, glucagon, somatostatin and pancreatic polypeptide in islet cells is specific to beta, alpha, delta and pancreatic polypeptide cells, respectively. We examined whether beta cells express other genes encoding islet hormones. METHODS: Nested RT-PCR was performed on single beta cells of transgenic mice with green fluorescent protein (GFP) driven by mouse insulin I promoter (MIP-GFP). RESULTS: Only 55% of adult beta cells expressed the insulin gene alone, while others expressed two or more islet hormone genes; 4% expressed all four hormone genes. In embryonic and neonatal cells, 60% to 80% of GFP(+) cells co-expressed pancreatic polypeptide and insulin genes in contrast to 29% in adult. To clarify cell fate, we conducted lineage tracing using rat insulin II promoter-cre mice crossed with reporter mice Gt(ROSA)26Sor-loxP-flanked STOP-cassette-GFP. All GFP(+) cells expressed insulin I and II genes, and showed similar heterogeneity of co-expression to that seen in MIP-GFP mice. Although we report expression of other hormone genes in a significant proportion of beta cells, our lineage tracing results demonstrate that after inducing InsII (also known as Ins2) expression, beta cell progenitors do not redifferentiate to non-beta cells. CONCLUSIONS/INTERPRETATION: This study shows co-expression of multiple hormone genes in beta cells of adult mice as well as in embryos and neonates. This finding could: (1) represent residual expression from beta cell precursors; (2) result from alternative developmental pathways for beta cells; or (3) denote the differentiation potential of these cells. It may be linked to functional heterogeneity. This heterogeneity in gene expression may provide a means to characterise the functional, cellular and developmental heterogeneity seen in beta cells.

Dimorphic histopathology of long-standing childhood-onset diabetes.

AIMS/HYPOTHESIS: Childhood diabetes is thought to usually result from autoimmune beta cell destruction (type 1A) with eventual total loss of beta cells. Analysis of C-peptide in children characterised at diabetes onset for autoantibodies shows heterogeneous preservation of insulin secretion in long-standing diabetes. The aim of this study was to characterise the pancreases of childhood-onset diabetes in order to define the pathological basis of this heterogeneity. METHODS: We evaluated 20 cadaveric organ donor pancreases of childhood-onset long-term patients for disease heterogeneity and obtained corresponding C-peptide measurements. RESULTS: Pancreases from the majority of cadaveric donors contained only insulin-deficient islets (14 of 20). The remaining six patients (30%) had numerous insulin-positive cells within at least some islets, with two different histological patterns. Pattern A (which we would associate with type 1A diabetes) had lobular retention of areas with 'abnormal' beta cells producing the apoptosis inhibitor survivin and HLA class I. In pattern B, 100% of all islets contained normal-appearing but quantitatively reduced beta cells without survivin or HLA class I. CONCLUSIONS/INTERPRETATION: Our data demonstrate that C-peptide secretion in long-standing diabetic patients can be explained by two different patterns of beta cell survival,possibly reflecting different subsets of type 1 diabetes.

Differentiation of COPAS-sorted non-endocrine pancreatic cells into insulin-positive cells in the mouse.

AIMS/HYPOTHESIS: The regenerative process in the pancreas is of particular interest, since insulin-producing beta cells are lost in diabetes. Differentiation of new beta cells from pancreatic non-endocrine cells has been reported in vivo and in vitro, a finding that implies the existence of pancreatic stem/progenitor cells. However, while tissue-specific stem cells are well documented in skin, intestine and testis, pancreatic stem cells have been elusive. We hypothesised that pancreatic stem/progenitor cells within the non-endocrine fraction could be a source of new islets in vitro. METHODS: To test if there were such cells within the pancreas, we generated pancreatic cell aggregates from tissue remaining after islet isolation from mouse insulin promoter 1-green fluorescent protein (MIP-GFP) mice. To eliminate any contamination of insulin-positive cells, we deleted all GFP-positive aggregates using COPAS Select and cultured with Matrigel. Immunohistochemistry, quantitative real-time PCR and single-cell nested RT-PCR were performed to confirm formation of insulin-producing cells. RESULTS: The GFP-negative cells were expanded as monolayers and then differentiated into three-dimensional cystic structures. After 1 week of culture, GFP-positive cells were found as clusters or single cells. By quantitative real-time PCR, no insulin mRNA was detected immediately after COPAS sorting, but after differentiation insulin mRNA of the whole preparation was 1.91 +/- 0.31% that of purified MIP-GFP beta cells. All GFP-positive cells expressed insulin 1; most expressed insulin 2, pancreas duodenum homeobox-1 and cytokeratin 19 by single cell nested RT-PCR. CONCLUSIONS/INTERPRETATION: Our data support the concept that within the exocrine (acinar and ductal) pancreas of the adult mouse there are cells that can give rise to insulin-positive cells in vitro.

Towards better understanding of the contributions of overwork and glucotoxicity to the beta-cell inadequacy of type 2 diabetes.

Type 2 diabetes (T2D) is characterized by reduction of beta-cell mass and dysfunctional insulin secretion. Understanding beta-cell phenotype changes as T2D progresses should help explain these abnormalities. The normal phenotype should differ from the state of overwork when beta-cells compensate for insulin resistance to keep glucose levels normal. When only mild hyperglycaemia develops, beta-cells are subjected to glucotoxicity. As hyperglycaemia becomes more severe, so does glucotoxicity. beta-Cells in all four of these situations should have separate phenotypes. When assessing phenotype with gene expression, isolated islets have artefacts resulting from the trauma of isolation and hypoxia of islet cores. An advantage comes from laser capture microdissection (LCM), which obtains beta-cell-rich tissue from pancreatic frozen sections. Valuable data can be obtained from animal models, but the real goal is human beta-cells. Our experience with LCM and gene arrays on frozen pancreatic sections from cadaver donors with T2D and controls is described. Although valuable data was obtained, we predict that the approach of taking fresh samples at the time of surgery is an even greater opportunity to markedly advance our understanding of how beta-cell phenotype evolves as T2D develops and progresses.

Minimal chronic hyperglycemia is a critical determinant of impaired insulin secretion after an incomplete pancreatectomy.

We now describe experiments that allow one to determine the consequences of B cell reduction alone vs. those that result from superimposed mild hyperglycemia. Male CD rats underwent a 60% pancreatectomy (Px); controls were sham operated. 1 wk later, either 10% sucrose (SUC) was substituted as fluid supply or tap water was continued (WAT). Plasma glucose and insulin values in Px-WAT remained equal to the sham groups; in Px-SUC the values were euglycemic for 25 d, but then nonfasting plasma glucose rose 15 mg/dl. After 6 wk, B cell mass in Px-WAT was reduced by 45% and non-B cell mass by 57%. In contrast, in Px-SUC both masses were comparable to the sham groups. The insulin response to 27.7 mM glucose was measured using the in vitro perfused pancreas. The responses were reduced in Px-WAT but in proportion to their reduced B cell mass; in contrast, it was 75% less than expected in Px-SUC. Also, the response to arginine given at 16.7 mM glucose was reduced only in Px-SUC. These results show that a lowering of B cell mass that does not result in hyperglycemia has no adverse effect on the remaining B cells. On the other hand, if even mild hyperglycemia develops, B cell function becomes impaired and results in inappropriately reduced insulin stores and insulin responses to marked stimuli.

Partial pancreatectomy in the rat and subsequent defect in glucose-induced insulin release.

To define the consequences of a known reduction of B cell mass in rats, 90% partial pancreatectomies were performed. For the 6 wk following surgery moderate hyperglycemia was maintained in the fed state but there were no differences in body weight nor plasma insulin concentrations compared with sham-pancreatectomized controls. 8-10 wk following surgery regeneration of the remnant was evident with remnant weight being 26%, B cell mass being 42%, and non-B cell mass being 47% of values found for control whole pancreas. There were comparable increases in the remnant content of insulin, glucagon, and somatostatin. Following meal challenges, intraperitoneal and intravenous glucose tolerance tests and intravenous arginine challenge given 6-7 wk after surgery, the insulin responses to glucose were blunted or absent but the responses following the meals or arginine were intact. Similarly, when the pancreatic remnant was perfused in vitro, insulin release after challenge with 300 mg/dl glucose was markedly reduced whereas intact responsiveness to 10 mM arginine was retained. These data suggest that the chronic stimulation of a reduced B cell mass can lead to a selective loss of glucose-induced insulin secretion.

Transplanted beta cell response to increased metabolic demand. Changes in beta cell replication and mass.

We determined the capacity of transplanted beta cells to modify their replication and mass when stimulated by changes in metabolic demand. Five groups of Lewis rats were studied: group 1 (Tx-Px) had a 95% pancreatectomy 14 d after transplantation of 500 islets; group 2 (Px-Tx) had a 95% pancreatectomy 14 d before transplantation of 500 islets; group 3 (Tx) was transplanted with 500 islets; group 4 (Px) had a 95% pancreatectomy; and group 5 (normal) was neither transplanted nor pancreatectomized. Blood glucose was normal in Tx-Px and Tx groups at all times. Px-Tx and Px groups developed severe hyperglycemia after pancreatectomy that was corrected in Px-Tx group in 83% of rats 28 d after transplantation. Replication of transplanted beta cells increased in Tx-Px (1.15 +/- 0.12%) and Px-Tx (0.85 +/- 0.12%) groups, but not in Tx group (0.64 +/- 0.07%) compared with normal pancreatic beta cells (0.38 +/- 0.05%) (P < 0.001). Mean beta cell size increased in Tx-Px (311 +/- 14 microns2) and Px-Tx (328 +/- 13 microns2) groups compared with Tx (252 +/- 12 microns2) and normal (239 +/- 9 microns2) groups (P < 0.001). Transplanted beta cell mass increased in Tx-Px (1.87 +/- 0.51 mg) and Px-Tx (1.55 +/- 0.21 mg) groups compared with Tx group (0.78 +/- 0.17 mg) (P < 0.05). In summary, changes in transplanted beta cells prevented the development of hyperglycemia in Tx-Px rats. Transplanted beta cells responded to increased metabolic demand increasing their beta cell mass.

Beta cell mass and growth after syngeneic islet cell transplantation in normal and streptozocin diabetic C57BL/6 mice.

In islet transplantation, nonimmunological factors such as limited growth capacity or increased death rate could reduce the beta cell mass in the graft and lead to failure of the transplant. We studied the evolution of beta cell replication and mass after transplantation of insufficient, minimally sufficient, or excessive islet tissue. Streptozocin diabetic C57BL/6 mice received 150 or 300 syngeneic islets under the kidney capsule and normal mice received 300 islets. In streptozocin diabetic mice 300 islets restored normoglycemia; beta cell replication in transplanted islets was similar to replication in normal pancreas and beta cell mass in the graft remained constant. In contrast, 150 islets were insufficient to achieve normoglycemia; beta cell replication was increased initially but not by 18 or 30 d despite persistent hyperglycemia, and beta cell mass fell progressively. When islets were transplanted into normal recipients, beta cell replication remained normal but beta cells underwent atrophy and mass in the graft was substantially reduced. Therefore, with a successful islet transplant, in diabetic mice beta cell replication and mass remain constant. In contrast, when insufficient islet tissue is transplanted an initial increase in beta cell replication can not compensate for a decline in beta cell mass. When excessive islet tissue is transplanted, beta cell mass is reduced despite normal beta cell replication.

Vanadate normalizes hyperglycemia in two mouse models of non-insulin-dependent diabetes mellitus.

We have studied the effects of oral administration of vanadate, an insulinometic agent and a potent inhibitor of phosphotyrosyl protein phosphatase (PTPase) in vitro, on blood glucose and PTPase action, in two hyperinsulinemic rodent models of non-insulin-dependent diabetes mellitus (NIDDM). Oral administration of vanadate (0.25 mg/ml in the drinking water) to ob/ob mice for 3 wk lowered blood glucose level from 236 +/- 4 to 143 +/- 2 mg/dl without effect on body weight. Administration of vanadate to db/db mice produced a similar effect. Electron microscopic examination revealed no signs of hepatotoxicity after 47 d of treatment. There was a slight reduction in insulin receptor autophosphorylation when tested by immunoblotting with antiphosphotyrosine antibody after in vivo stimulation, and the phosphorylation of the endogenous substrate of the insulin receptor, pp185, was markedly decreased in the ob/ob mice. Both cytosolic and particulate PTPase activities in liver of ob/ob mice measured by dephosphorylation of a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation were decreased by approximately 50% (P less than 0.01). In db/db diabetic mice, PTPase activity in the cytosolic fraction was decreased to 53% of control values (P less than 0.02) with no significant difference in the particulate PTPase activity. Treatment with vanadate did not alter hepatic PTPase activity as assayed in vitro, or receptor and substrate phosphorylation as assayed in vivo, in ob/ob mice despite its substantial effect on blood glucose. These data indicate that vanadate is an effective oral hypoglycemic treatment in NIDDM states and suggest that its major effects occurs distal to the insulin receptor tyrosine kinase.

An in vivo analysis of pancreatic protein and insulin biosynthesis in a rat model for non-insulin-dependent diabetes.

The purpose of these experiments was to estimate insulin biosynthesis in vivo in a rat model for non-insulin-dependent diabetes. Insulin biosynthesis rates were determined in 4-wk-old animals that had been injected with 90 mg/kg of streptozotocin 2 d postpartum. Control and diabetic animals did not differ in body weight or fasting plasma glucose. Fed plasma glucose was significantly elevated (186 +/- 13 micrograms/dl vs. 139 +/- 7 mg/dl, P less than 0.05) and pancreatic insulin content was reduced (41 +/- 2 micrograms/g vs. 63 +/- 8 micrograms/g, P less than 0.05) in the diabetic rats. Insulin biosynthesis was estimated in vivo by measuring and comparing [3H]leucine incorporation into proinsulin with that into total pancreatic protein 45 min after injection. Insulin biosynthesis was 0.391 +/- 0.07% of pancreas protein synthesized in control rats and 0.188 +/- 0.015% (P less than 0.05) in diabetic rats. In animals of the same age, the fractional and absolute rate of pancreatic protein synthesis were determined. Total pancreatic protein synthesis was not reduced in streptozotocin treated animals (185.5 +/- 14.1%/d vs. 158.6 +/- 14.9%/d, NS) but was markedly reduced in control rats after a 48-h fast (to 70.8 +/- 5.5%/d, P less than 0.01). Because total pancreatic protein synthesis was not decreased in the diabetic rats, the decrease in the fraction of radiolabel incorporated into insulin seems to represent an absolute decrease in the rate of insulin biosynthesis in this animal model for diabetes. Through RNA blot hybridization with 32P-labeled cloned rat insulin complementary DNA, proinsulin messenger RNA (mRNA) was estimated as the rate of insulin biosynthesis in control and diabetic animals. There was a 61% reduction in proinsulin mRNA at 4 wk and an 85% reduction at 7 wk (P less than 0.001) in the diabetic animals. After streptozotocin injection in neonatal rats, there is marked beta-cell damage and hyperglycemia. Beta-cell regeneration occurs with return to normoglycemia, but with age hyperglycemia develops. The reduction in insulin synthesis and proinsulin mRNA seemed disproportionate with the more modest reduction in beta-cell number. The importance of these observations is that, in this animal model, diabetes is associated with a limited ability to regenerate beta-cell mass and to synthesize insulin. The relationship between the defect in glucose-stimulated insulin release and impaired insulin biosynthesis has yet to be determined.

In situ glucose uptake and glucokinase activity of pancreatic islets in diabetic and obese rodents.

The present study evaluated the involvement of glucose transport and phosphorylation in glucose-stimulated insulin release from pancreatic islets. Using quantitative histochemical techniques, we investigated basal islet glucose content, islet glucose uptake in situ during acute extreme experimental hyperglycemia, and islet glucokinase activity in several animal models of diabetes and obesity. The basal islet glucose content in anaesthetized diabetic or obese rodents was either the same or higher than that in their relevant controls. The rate of glucose uptake of islet tissue in these animals after an i.v. glucose injection was different. The db+/db+ mouse and the obese Zucker rat exhibited significantly reduced islet glucose uptake rates. RIP-cHras transgenic mice, BHE/cdb rats and partially pancreatectomized rats showed normal islet glucose uptake rates. The activity of islet glucokinase was increased to a different degree related to the blood glucose level. All five animal models of diabetes or obesity exhibited either a delay or a reduction of insulin release in response to supra maximal glucose stimulation. Our results indicate that the impairment of glucose-induced insulin release in diabetes is not consistently associated with a reduction of islet glucose uptake nor a change of glucokinase activity.

The cholecystokinin-A receptor mediates inhibition of food intake yet is not essential for the maintenance of body weight.

Food intake and body weight are determined by a complex interaction of regulatory pathways. To elucidate the contribution of the endogenous peptide cholecystokinin, mice lacking functional cholecystokinin-A receptors were generated by targeted gene disruption. To explore the role of the cholecystokinin-A receptor in mediating satiety, food intake of cholecystokinin-A receptor-/- mice was compared with the corresponding intakes of wild-type animals and mice lacking the other known cholecystokinin receptor subtype, cholecystokinin-B/gastrin. Intraperitoneal administration of cholecystokinin failed to decrease food intake in mice lacking cholecystokinin-A receptors. In contrast, cholecystokinin diminished food intake by up to 90% in wild-type and cholecystokinin-B/gastrin receptor-/- mice. Together, these findings indicate that cholecystokinin-induced inhibition of food intake is mediated by the cholecystokinin-A receptor. To explore the long-term consequences of either cholecystokinin-A or cholecystokinin-B/gastrin receptor absence, body weight as a function of age was compared between freely fed wild-type and mutant animals. Both cholecystokinin-A and cholecystokinin-B/gastrin receptor-/- mice maintained normal body weight well into adult life. In addition, each of the two receptor-/- strains had normal pancreatic morphology and were normoglycemic. Our results suggest that although cholecystokinin plays a role in the short-term inhibition of food intake, this pathway is not essential for the long-term maintenance of body weight.

Pancreatic gastrin stimulates islet differentiation of transforming growth factor alpha-induced ductular precursor cells.

Gastrin is transiently expressed in fetal islets during a critical period of their development from protodifferentiated islet precursors in fetal pancreatic ducts. To examine the possible role of gastrin as an islet cell growth factor, postnatal islet growth was studied in transgenic mice which overexpress gastrin and TGF alpha in their pancreas. Overexpression of a TGF alpha transgene causes metaplastic ductules containing numerous insulin expressing cells that resemble protodifferentiated precursors of the fetal pancreas. However, islet mass of the TGF alpha transgenic mice was not increased. Pancreatic overexpression of gastrin from a chimeric insulin/gastrin transgene transcribed from the insulin promoter markedly decreased the TGF alpha-stimulated increase in pancreatic duct mass. Furthermore, pancreatic coexpression of both gastrin and TGF alpha significantly increased islet mass in mice expressing both transgenes. These findings indicate that TGF alpha and gastrin can act synergistically to stimulate islet growth, although neither peptide alone is sufficient. Islet growth may possibly be stimulated through gastrin promoting the differentiation of insulin-positive cells in the TGF alpha-induced metaplastic ducts. This transgenic study suggests that islet neogenesis can be reactivated in the ductular epithelium of the adult pancreas by local expression of two growth factors, gastrin and TGF alpha.

Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice.

Mutations in the gene for the transcription factor hepatocyte nuclear factor (HNF) 1alpha cause maturity-onset diabetes of the young (MODY) 3, a form of diabetes that results from defects in insulin secretion. Since the nature of these defects has not been defined, we compared insulin secretory function in heterozygous [HNF-1alpha (+/-)] or homozygous [HNF-1alpha (-/-)] mice with null mutations in the HNF-1alpha gene with their wild-type littermates [HNF-1alpha (+/+)]. Blood glucose concentrations were similar in HNF-1alpha (+/+) and (+/-) mice (7.8+/-0.2 and 7.9+/-0.3 mM), but were significantly higher in the HNF-1alpha (-/-) mice (13.1+/-0.7 mM, P < 0.001). Insulin secretory responses to glucose and arginine in the perfused pancreas and perifused islets from HNF-1alpha (-/-) mice were < 15% of the values in the other two groups and were associated with similar reductions in intracellular Ca2+ responses. These defects were not due to a decrease in glucokinase or insulin gene transcription. beta cell mass adjusted for body weight was not reduced in the (-/-) animals, although pancreatic insulin content adjusted for pancreas weight was slightly lower (0.06+/-0.01 vs. 0.10+/-0.01 microg/mg, P < 0.01) than in the (+/+) animals. In summary, a null mutation in the HNF-1alpha gene in homozygous mice leads to diabetes due to alterations in the pathways that regulate beta cell responses to secretagogues including glucose and arginine. These results provide further evidence in support of a key role for HNF-1alpha in the maintenance of normal beta cell function.