Making ß(-like)-cells from exocrine pancreas.

Creating an abundant source of ß(-like)-cells has been a major goal in diabetes research for many decades. The concept of cell plasticity has inspired many strategies towards regenerative medicine, but its successes have been limited until very recently. Today, most cell types in the pancreas are considered candidates for the generation of ß(-like)-cells through transdifferentiation. While ß(-like)-cells that are in vitro differentiated from human embryonic stem cells are already being grafted in patients, ß(-like)-cells generated by transdifferentiation are not yet ready for clinical application. These cells would however offer several advantages over the current ß(-like)-cells generated by directed differentiation, especially concerning safety issues. In addition, perfect control of the transdifferentiation efficiency would through targeted drug delivery support a non-invasive cell therapy for diabetes. Lastly, focusing on the exocrine pancreas as prime candidate makes sense in view of their abundance and high plasticity. Keeping these hopeful perspectives in mind, it is worth to continue focused research on the mechanisms that control transdifferentiation from pancreas exocrine to ß-cells.

Accessory cells for ß-cell transplantation.

Despite recent advances, insulin therapy remains a treatment, not a cure, for diabetes mellitus with persistent risk of glycaemic alterations and life-threatening complications. Restoration of the endogenous ß-cell mass through regeneration or transplantation offers an attractive alternative. Unfortunately, signals that drive ß-cell regeneration remain enigmatic and ß-cell replacement therapy still faces major hurdles that prevent its widespread application. Co-transplantation of accessory non-islet cells with islet cells has been shown to improve the outcome of experimental islet transplantation. This review will highlight current travails in ß-cell therapy and focuses on the potential benefits of accessory cells for islet transplantation in diabetes.

Differentiating neural crest stem cells induce proliferation of cultured rodent islet beta cells.

AIMS/HYPOTHESIS: Efficient stimulation of cycling activity in cultured beta cells would allow the design of new strategies for cell therapy in diabetes. Neural crest stem cells (NCSCs) play a role in beta cell development and maturation and increase the beta cell number in co-transplants. The mechanism behind NCSC-induced beta cell proliferation and the functional capacity of the new beta cells is not known. METHODS: We developed a new in vitro co-culture system that enables the dissection of the elements that control the cellular interactions that lead to NCSC-dependent increase in islet beta cells. RESULTS: Mouse NCSCs were cultured in vitro, first in medium that stimulated their proliferation, then under conditions that supported their differentiation. When mouse islet cells were cultured together with the NCSCs, more than 35% of the beta cells showed cycle activity. This labelling index is more than tenfold higher than control islets cultured without NCSCs. Beta cells that proliferated under these culture conditions were fully glucose responsive in terms of insulin secretion. NCSCs also induced beta cell proliferation in islets isolated from 1-year-old mice, but not in dissociated islet cells isolated from human donor pancreas tissue. To stimulate beta cell proliferation, NCSCs need to be in intimate contact with the beta cells. CONCLUSIONS/INTERPRETATION: Culture of islet cells in contact with NCSCs induces highly efficient beta cell proliferation. The reported culture system is an excellent platform for further dissection of the minimal set of factors needed to drive this process and explore its potential for translation to diabetes therapy.

Glucose promotes survival of rat pancreatic beta cells by activating synthesis of proteins which suppress a constitutive apoptotic program.

This study demonstrates that rat islet beta cells constitutively express an apoptotic program which is activated when mRNA or protein synthesis is blocked. Apoptotic beta cells were detectable by electron microscopy after treatment with actinomycin D or cycloheximide. With a fluorescence microscopic assay both agents were found to increase the number of apoptotic beta cells dose- and time-dependently, up to 70% after 1 wk of culture; virtually no apoptotic beta cells occurred in control preparations or in conditions leading to primary necrosis. Thus, survival of beta cells seems dependent on synthesis of proteins which suppress an endogenous suicide program. This mechanism explains earlier observed effects of glucose on survival of cultured beta cells. Glucose is known to dose-dependently increase the percentage of beta cells in active biosynthesis and the percentage that survives during culture. It is now demonstrated that the glucose-induced survival of beta cells cultured for 1 wk results from a dose-dependent reduction in the percentage of beta cells dying in apoptosis (49% at 3 mM glucose, 40% at 6 mM, 9% at 10 mM). Thus, intercellular differences in glucose sensitivity appear responsible for the heterogeneity in beta cell sensitivity to apoptotic conditions. These data indicate that glucose promotes survival of beta cells by activating synthesis of proteins which suppress apoptosis. The present model allows for further investigation of the regulation of apoptosis in beta cells and the identification of agents which induce or prevent beta cell death.

Methyl succinate antagonises biguanide-induced AMPK-activation and death of pancreatic beta-cells through restoration of mitochondrial electron transfer.

BACKGROUND AND PURPOSE: Two mechanisms have been proposed to explain the insulin-sensitising properties of metformin in peripheral tissues: (a) inhibition of electron transport chain complex I, and (b) activation of the AMP activated protein kinase (AMPK). However the relationship between these mechanisms and their contribution to beta-cell death and dysfunction in vitro, are currently unclear. EXPERIMENTAL APPROACH: The effects of biguanides (metformin and phenformin) were tested on MIN6 beta-cells and primary FACS-purified rat beta-cells. Cell metabolism was assessed biochemically and by FACS analysis, and correlated with AMPK phosphorylation state and cell viability, with or without fuel substrates. KEY RESULTS: In MIN6 cells, metformin reduced mitochondrial complex I activity by up to 44% and a 25% net reduction in mitochondrial reducing potential. In rat beta-cells, metformin caused NAD(P)H accumulation above maximal glucose-inducible levels, mimicking the effect of rotenone. Drug exposure caused phosphorylation of AMPK on Thr(172) in MIN6 cell extracts, indicative of kinase activation. Methyl succinate, a complex II substrate, appeared to bypass metformin blockade of complex I. This resulted in reduced phosphorylation of AMPK, establishing a link between biguanide-induced mitochondrial inhibition and AMPK activation. Corresponding assessment of cell death indicated that methyl succinate decreased biguanide toxicity to beta-cells in vitro. CONCLUSIONS AND IMPLICATIONS: AMPK activation can partly be attributed to metformin's inhibitory action on mitochondrial complex I. Anaplerotic fuel metabolism via complex II rescued beta-cells from metformin-associated toxicity. We propose that utilisation of anaplerotic nutrients may reconcile in vitro and in vivo effects of metformin on the pancreatic beta-cell.

STAT3 modulates ß-cell cycling in injured mouse pancreas and protects against DNA damage.

Partial pancreatic duct ligation (PDL) of mouse pancreas induces a doubling of the ß-cell mass mainly through proliferation of pre-existing and newly formed ß-cells. The molecular mechanism governing this process is still largely unknown. Given the inflammatory nature of PDL and inflammation-induced signaling via the signal transducer and activator of transcription 3 (STAT3), the activation and the role of STAT3 in PDL-induced ß-cell proliferation were investigated. Duct ligation stimulates the expression of several cytokines that can act as ligands inducing STAT3 signaling and phosphorylation in ß-cells. ß-Cell cycling increased by conditional ß-cell-specific Stat3 knockout and decreased by STAT3 activation through administration of interleukin-6. In addition, the level of DNA damage in ß-cells of PDL pancreas increased after deletion of Stat3. These data indicate a role for STAT3 in maintaining a steady state in the ß-cell, by modulating its cell cycle and protection from DNA damage.

Interleukin-1beta-induced alteration in a beta-cell phenotype can reduce cellular sensitivity to conditions that cause necrosis but not to cytokine-induced apoptosis.

Previous work has shown that interleukin-1beta (IL-1beta) alters protein expression in beta-cells. This alteration is associated with cell death in isolated rat islets but not in isolated rat beta-cells. We examined whether IL-1beta pretreatment of isolated beta-cells influences their sensitivity to toxic agents. After a 24-h culture with IL-1beta (30 U/ml), beta-cells exhibited a lower expression of the beta-cell-specific protein transcription factor pancreatic and duodenal homeobox gene (PDX)-1, glucose transporter GLUT2, and proinsulin convertase PC2, with a marked reduction (60-70%) in glucose-induced insulin production and selective sensitivity to the toxins alloxan (ALX) and streptozotocin (STZ). On the other hand, the cells presented an increased expression of Mn-superoxide dismutase, heat shock protein 70, inducible heme oxygenase, and inducible nitrite oxide synthase. This IL-1beta-induced alteration in beta-cell phenotype resulted in a reduced cellular sensitivity to the beta-cell-specific toxins ALX and STZ; the production of nontoxic conditions of nitric oxide (NO) also rendered the cells less susceptible to radical-induced damage. Exposure to IL-1beta can thus protect beta-cells against conditions that cause necrosis; however, it did not protect against apoptosis induced by the additional presence of interferon-gamma or tumor necrosis factor-alpha. Release of IL-1beta in the endocrine pancreas is thus not necessarily the cause of massive NO-dependent beta-cell destruction. On the contrary, IL-1beta may protect these cells against necrosis, though with a loss of their characteristic phenotype and homeostatic functions.

Adult human pancreatic duct and islet cells exhibit similarities in expression and differences in phosphorylation and complex formation of the homeodomain protein Ipf-1.

The homeodomain transcription factor encoded by the pancreatic and duodenal homeobox gene-1 (Ipf-1) is essential for pancreatic ontogenesis. Whether Ipf-1 is also involved in the neogenesis of beta-cells in the adult pancreas is unknown. We examined whether Ipf-1 is expressed in adult human pancreatic ducts, which are thought to generate new beta-cells. In tissue sections, virtually all duct cells were immunopositive for Ipf-1, as were the islet beta-cells but not the acinar cells. After isolation and culture, both duct and islet cell preparations contained the Ipf-1 immunoreactive proteins p42 and p45 (42 and 45 kDa, respectively) in similar proportions, but the expression levels were twofold lower in duct cells. After 4 h of labeling, the endocrine cells exhibited a sevenfold higher phosphorylation of p42 than the duct cells, whereas p45 was phosphorylated only in endocrine cells. Homeobox binding transcription factor complexes with Ipf-1 in duct cells differed from those in endocrine cells in terms of gel mobility, sequence specificity, and affinity. The observed similarities in Ipf-1 expression by adult human pancreatic duct cells and endocrine cells may reflect their common ontogenic origin, whereas the differences in Ipf-1 phosphorylation and complex formation may correlate with their divergent differentiation.

Role of pancreatic beta-cells in the process of beta-cell death.

Studies on the pathogenesis of type 1 diabetes have mainly focused on the role of the immune system in the destruction of pancreatic beta-cells. Lack of data on the cellular and molecular events at the beta-cell level is caused by the inaccessibility of these cells during development of the disease. Indirect information has been collected from isolated rodent and human islet cell preparations that were exposed to cytotoxic conditions. This article reviews in vitro experiments that investigated the role of beta-cells in the process of beta-cell death. beta-Cells rapidly die in necrosis because of toxic levels of oxidizing radicals or of nitric oxide; they progressively become apoptotic after prolonged culture at low glucose or with proinflammatory cytokines. Their susceptibility to necrosis or apoptosis varies with their functional state and thus with the environmental conditions. A change in cellular phenotype can alter its recognition of potentially cytotoxic agents and its defense mechanisms against cell death. These observations support the view that beta-cells are not necessarily passive victims of a cytotoxic process but can actively participate in a process of beta-cell death. Their role will be influenced by neighboring non-beta-cells, which can make the islet internal milieu more protective or toxic for the beta-cells. We consider duct cells as potentially important contributors to this local process.

Characterisation of portal hypertension models by microspheres in anaesthetised rats: a comparison of liver flow.

Several portal hypertensive animal models are available and frequently used for haemodynamic studies. The portal venous inflows, measured with microspheres in pentobarbital anaesthetised rats, are compared here. The partial portal vein ligation model is characterised by a high portal venous inflow, together with extensive portal systemic shunting, at the cost of portal sinusoidal flow. In carbon tetrachloride-induced micronodular cirrhosis, portal sinusoidal flow, which reaches liver parenchyma, is high, and this is more pronounced in the presence of ascites. In bile duct ligation and excision-induced cirrhosis, an increase in liver weight was not equally followed by an increase in portal sinusoidal flow, pointing to a relatively underperfused liver.

The dihydrofolate reductase-encoding gene dyrA of the hyperthermophilic bacterium Thermotoga maritima.

The structural gene (dyrA) encoding dihydrofolate reductase (DHFR) of Thermotoga maritima has been cloned, sequenced and expressed in Escherichia coli. The dyrA gene, located immediately upstream from the gene encoding aspartate carbamoyltransferase (pyrB), encodes a highly thermostable enzyme with a distinct thermophilic activity profile. Important structural features are conserved among all bacterial DHFR, yet the DHFR of T. maritima appears unique in a number of insertions and deletions, some of which are reminiscent of eukaryotic DHFR.

AMP-activated protein kinase can induce apoptosis of insulin-producing MIN6 cells through stimulation of c-Jun-N-terminal kinase.

We have recently shown that conditions known to activate AMP-activated protein kinase (AMPK) in primary beta-cells can trigger their apoptosis. The present study demonstrates that this is also the case in the MIN6 beta-cell line, which was used to investigate the underlying mechanism. Sustained activation of AMPK was induced by culture with the adenosine analogue AICA-riboside or at low glucose concentrations. Both conditions induced a sequential activation of AMPK, c-Jun-N-terminal kinase (JNK) and caspase-3. The effects of AMPK on JNK activation and apoptosis were demonstrated by adenoviral expression of constitutively active AMPK, a condition which reproduced the earlier-described AMPK-dependent effects on pyruvate kinase and acetyl-coA-carboxylase. The effects of JNK activation on apoptosis were demonstrated by the observations that (i). its inhibition by dicumarol prevented caspase-3 activation and apoptosis, (ii). adenoviral expression of the JNK-interacting scaffold protein JIP-1/IB-1 increased AICA-riboside-induced JNK activation and apoptosis. In primary beta-cells, AMPK activation was also found to activate JNK, involving primarily the JNK 2 (p54) isoform. It is concluded that prolonged stimulation of AMPK can induce apoptosis of insulin-producing cells through an activation pathway that involves JNK, and subsequently, caspase-3.

The role of the codon first letter in the relationship between genomic GC content and protein amino acid composition.

Analysis of the statistical distribution of amino acid compositions within 22 protein families shows that a GC bias generally affects proteins with a variety of functions from the extreme thermophile Thermus. This results in evident enrichment in amino acids of the group L, V, A, P, R and G and underrepresentation of amino acids of the group I, M, F, S, T, C and W. The strong amino acid composition biases noted in Thermus proteins are not related to thermoadaptation; they were also found in mesophilic homologues encoded by GC-rich genes. The results of a comparative analysis on large samples of translated sequences from 30 organisms, representing the three major kingdoms of life and including extremophiles, indicate a universal correlation between the usage of particular amino acids and the genomic GC content. It is concluded that the codon first letter plays a dominant role in translating the genomic GC signature into protein amino acid composition and sequences.

Blood antioxidant levels in patients with alcoholic liver disease correlate with the degree of liver impairment and are not specific to alcoholic liver injury itself.

BACKGROUND: Enhanced production of reactive oxygen species may play a pathogenic role in alcoholic liver injury. AIMS: To investigate whether various antioxidant parameters in blood are affected in different stages of alcoholic liver disease and how specific the changes are relative to non-alcoholic cirrhosis. METHODS: Patients with alcohol abuse without cirrhosis (n=14), with alcoholic cirrhosis [Child-Pugh scores A (n=9), B (n=5) and C (n=18)] and with non-alcoholic cirrhosis [Child-Pugh score C (n=6)] and healthy controls (n=13) were studied. Levels of reduced glutathione and glutathione peroxidase activity in blood, erythrocytic superoxide dismutase activity and carotenoids, alpha-tocopherol and malondialdehyde in plasma were measured. RESULTS: Levels of reduced glutathione were significantly decreased in Child-Pugh score C cirrhotics, alcoholic or not in origin, whereas oxidized glutathione and glutathione peroxidase activity were not affected. Superoxide dismutase activity and alpha-tocopherol levels were not significantly different in the various groups. Carotenoid levels were significantly lower in alcoholic cirrhotics (Child-Pugh score C) vs. controls. Malondialdehyde levels were elevated only in cirrhotics Child-Pugh score C, alcoholic or non-alcoholic. CONCLUSIONS: Levels of reduced glutathione and malondialdehyde reflect the degree of liver impairment, more than the relation with alcohol intake. Decreases in several antioxidant levels are not specific to alcoholic liver injury.

Oxidative breakdown of octanoic acid is maintained in patients with cirrhosis despite advanced disease.

As an octanoic acid 13CO2 breath test is frequently used to test gastric emptying of solid food, the purpose of the present study was to study whether oxidative breakdown of octanoic acid is affected by severe liver disease. The design of our study was twofold. First, cirrhotic patients (n = 82) of varying severity were compared with healthy controls (n = 17). Values of half-time, time point of maximal expiration and cumulative recovery of octanoic acid breath tests (OBT) were not significantly different between them. Secondly, cirrhotic patients (n = 10) were studied before placement of transjugular intrahepatic portosystemic shunt, 4-7 days later and 1-2 months later. Values of half-time, time point of maximal expiration and cumulative recovery of consecutive OBTs did not change significantly. The OBT may therefore be a suitable test in the future to detect delayed gastric emptying of solids in cirrhotic patients with reduced liver function and portal hypertension.

Neurogenin 3+ cells contribute to ß-cell neogenesis and proliferation in injured adult mouse pancreas.

We previously showed that injury by partial duct ligation (PDL) in adult mouse pancreas activates Neurogenin 3 (Ngn3)(+) progenitor cells that can differentiate to ß cells ex vivo. Here we evaluate the role of Ngn3(+) cells in ß cell expansion in situ. PDL not only induced doubling of the ß cell volume but also increased the total number of islets. ß cells proliferated without extended delay (the so-called 'refractory' period), their proliferation potential was highest in small islets, and 86% of the ß cell expansion was attributable to proliferation of pre-existing ß cells. At sufficiently high Ngn3 expression level, upto 14% of all ß cells and 40% of small islet ß cells derived from non-ß cells. Moreover, ß cell proliferation was blunted by a selective ablation of Ngn3(+) cells but not by conditional knockout of Ngn3 in pre-existing ß cells supporting a key role for Ngn3(+) insulin(-) cells in ß cell proliferation and expansion. We conclude that Ngn3(+) cell-dependent proliferation of pre-existing and newly-formed ß cells as well as reprogramming of non-ß cells contribute to in vivo ß cell expansion in the injured pancreas of adult mice.

Nutrient sensing in pancreatic beta cells suppresses mitochondrial superoxide generation and its contribution to apoptosis.

Excessively high glucose concentrations have been shown to damage tissues through stimulation of mitochondrial superoxide generation. This effect has therefore been considered as a potential cause for dysfunction and death of pancreatic beta cells in diabetes. We have examined whether the rate of glucose metabolism in isolated rat beta cells is correlated with their formation of oxygen radicals. It was found that high rates of glucose metabolism did not stimulate the formation of superoxide and H(2)O(2) but suppressed it. The higher rates of superoxide production in beta cells with lower mitochondrial metabolic activity contributed to the susceptibility of these cells to apoptosis.

Effects of varying doses of spironolactone without and with nitrates on portal vein pressure and kidney function in partial portal vein ligated rats.

The optimal dose of spironolactone to reduce portal vein pressure, alone or in combination with isosorbide-5-mononitrate (IsMn), has not been identified. We studied the effects of 8 days oral treatment with spironolactone, IsMn or both on portal pressure, plasma volume and renal sodium handling in rats with partial portal vein ligation. At daily doses of 0.33; 0.50; 1, and 1.50 mg/kg, spironolactone reduced portal pressure (all P < .05) as compared with placebo. Only the highest dose significantly lowered plasma volume (10.1 +/- 0.7 vs. 13.0 +/- 0.3 mL; P < .03) and enhanced urinary fractional sodium excretion (0.73 +/- 0.04 vs. 0.58 +/- 0.03%; P < .03) and the (Na+)/(K+) ratio in urine (0.43 +/- 0.04 vs. 0.30 +/- 0.03; P < .02). IsMn at doses of 0.25; 0.50, and 1 mg/kg decreased portal pressure (all P < .05) without a change in plasma volume but with a tendency (not significant) to lower fractional sodium excretion. IsMn impaired free water clearance at doses of 0.5 and 1 mg/kg (P < .05). Combinations of spironolactone 1 mg/kg with IsMn 0.5 or 1 mg/kg have no additive effect on portal pressure compared with spironolactone or IsMn alone. The higher the dose of IsMn in the combination, the more the natriuretic effect of spironolactone is opposed. Low doses of spironolactone are as effective as a higher dose to reduce portal pressure. This reduced portal pressure was independent of changes in plasma volume and diuretic effect, which suggests that spironolactone might have a direct vasoactive effect on the splanchnic circulation. To counteract sodium retention of nitrovasodilators, combination with high doses of spironolactone seems advantageous.

Hypercalcemia, monoclonal protein, and osteolytic bone lesions in chronic lymphocytic leukemia.

We describe a patient with a long history of typical chronic lymphocytic leukemia (CLL) who developed hypercalcemia, osteolytic bone lesions, and a monoclonal protein, all features of a secretory plasma cell disorder. These features in CLL have been reported in only four previous cases. The hypercalcemia in our patient is felt to result from an increase in the osteoclastic process.

Structure and expression of a pyrimidine gene cluster from the extreme thermophile Thermus strain ZO5.

On a 4.7-kbp HindIII clone of Thermus strain ZO5 DNA, complementing an aspartate carbamoyltransferase mutation in Escherichia coli, we identified a cluster of four potential open reading frames corresponding to genes pyrR, and pyrB, an unidentified open reading frame named bbc, and gene pyrC. The transcription initiation site was mapped at about 115 nucleotides upstream of the pyrR translation start codon. The cognate Thermus pyr promoter also functions in heterologous expression of Thermus pyr genes in E. coli. In Thermus strain ZO5, pyrB and pyrC gene expression is repressed three- to fourfold by uracil and increased twofold by arginine. Based on the occurrence of several transcription signals in the Thermus pyr promoter region and strong amino acid sequence identities (about 60%) between Thermus PyrR and the PyrR attenuation proteins of two Bacillus sp., we propose a regulatory mechanism involving transcriptional attenuation to control pyr gene expression in Thermus. In contrast to pyr attenuation in Bacillus spp., however, control of the Thermus pyr gene cluster would not involve an antiterminator structure but would involve a translating ribosome for preventing formation of the terminator RNA hairpin. The deduced amino acid sequence of Thermus strain ZO5 aspartate carbamoyltransferase (ATCase; encoded by pyrB) exhibits the highest similarities (about 50% identical amino acids) with ATCases from Pseudomonas sp. For Thermus strain ZO5 dihydroorotase (DHOase; encoded by pyrC), the highest similarity scores (about 40% identity) were obtained with DHOases from B. caldolyticus and Bacillus subtilis. The enzyme properties of ATCase expressed from truncated versions of the Thermus pyr gene cluster in E. coli suggest that Thermus ATCase is stabilized by DHOase and that the translation product of bbc plays a role in feedback inhibition of the ATCase-DHOase complex.