Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells.

The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5(+)) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and to allow for a response to tissue injury. Here, we identify a subpopulation of slowly cycling ISCs marked by mouse telomerase reverse transcriptase (mTert) expression that can give rise to Lgr5(+) cells. mTert-expressing cells distribute in a pattern along the crypt-villus axis similar to long-term label-retaining cells (LRCs) and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert(+) cells give rise to all differentiated intestinal cell types, persist long term, and contribute to the regenerative response following injury. Consistent with other highly regenerative tissues, our results demonstrate that a slowly cycling stem cell population exists within the intestine.

Generation of mTert-GFP mice as a model to identify and study tissue progenitor cells.

Stem cells hold great promise for regenerative medicine, but remain elusive in many tissues in part because universal markers of "stemness" have not been identified. The ribonucleoprotein complex telomerase catalyzes the extension of chromosome ends, and its expression is associated with failure of cells to undergo cellular senescence. Because such resistance to senescence is a common characteristic of many stem cells, we hypothesized that telomerase expression may provide a selective biomarker for stem cells in multiple tissues. In fact, telomerase expression has been demonstrated within hematopoietic stem cells. We therefore generated mouse telomerase reverse transcriptase (mTert)-GFP-transgenic mice and assayed the ability of mTert-driven GFP to mark tissue stem cells in testis, bone marrow (BM), and intestine. mTert-GFP mice were generated by using a two-step embryonic stem cell-based strategy, which enabled primary and secondary screening of stably transfected clones before blastocyst injection, greatly increasing the probability of obtaining mTert reporter mice with physiologically appropriate regulation of GFP expression. Analysis of adult mice showed that GFP is expressed in differentiating male germ cells, is enriched among BM-derived hematopoietic stem cells, and specifically marks long-term BrdU-retaining intestinal crypt cells. In addition, telomerase-expressing GFP(+) BM cells showed long-term, serial, multilineage BM reconstitution, fulfilling the functional definition of hematopoietic stem cells. Together, these data provide direct evidence that mTert-GFP expression marks progenitor cells in blood and small intestine, validating these mice as a useful tool for the prospective identification, isolation, and functional characterization of progenitor/stem cells from multiple tissues.

Fatty acid translocase (FAT/CD36) is localized on insulin-containing granules in human pancreatic beta-cells and mediates fatty acid effects on insulin secretion.

The membrane receptor FAT/CD36 facilitates the major fraction of long-chain fatty acid (FA) uptake by muscle and adipose tissues. In line with the well-known effects of FA metabolism on carbohydrate utilization and insulin responsiveness, altered expression of CD36 has been linked to phenotypic features of the metabolic syndrome including insulin resistance and dyslipidemia. FA metabolism is also known to significantly affect insulin secretion. However, the role of CD36 in this process remains unknown, since its expression levels and function in the pancreas have not been explored. In the present study, freshly isolated human islets and a mouse-derived beta-cell line (MIN6) were shown positive for CD36 expression by RT-PCR, Western blot, and immunofluorescence. The identity of the PCR product was confirmed by microsequencing. The identified transcript was translated and the protein was expressed and subjected to the known posttranslational glycosylation. Fluorescence resonance energy transfer analysis and subcellular protein fractionation indicated that insulin and CD36 are colocalized in the secretory granules of beta-cells. Islet CD36 functioned in FA uptake because this process was blocked by the irreversible CD36 inhibitor sulfosuccinimidyl-oleate. More importantly, sulfosuccinimidyl-oleate reversed enhancing and inhibiting effects, respectively, of acute and long-term palmitate incubations on glucose-dependent insulin secretion. In conclusion, our study demonstrates that human islets express CD36 in the plasma membrane as well as in the insulin secretory granules. CD36 activity appears important for uptake of FA into beta-cells as well as for mediating their modulatory effects on insulin secretion.

GLP-1 inhibition of pancreatic islet cell apoptosis.

Apoptosis plays an important role in the normal physiology of the pancreas, the pathogenesis of diabetes mellitus (DM) and the success rate of islet transplantation. Glucagon-like peptide-1 (GLP-1), an incretin hormone with multiple effects on glucose metabolism and pancreatic gene expression, has recently been found to have antiapoptotic properties. This new property of GLP-1 has clinical relevance for the treatment of patients with overt DM, possible prevention of DM during the stage of impaired glucose tolerance and improvement in the outcome of islet transplantation. The pleiotropic effects of GLP-1 have fostered considerable interest in evaluating the efficacy of GLP-1, and might lead in the near future to its use in the prevention and/or treatment of DM.

Glucagon-like peptide-1 promotes islet cell growth and inhibits apoptosis in Zucker diabetic rats.

A constant remodeling of islet cell mass mediated by proliferative and apoptotic stimuli ensures a dynamic response to a changing demand for insulin. In this study, we investigated the effect of glucagon-like peptide-1 (GLP-1) in Zucker diabetic rats, an animal model in which the onset of diabetes occurs when the proliferative potential and the rate of beta-cell apoptosis no longer compensate for the increased demand for insulin. We subjected diabetic rats to a 2-d infusion of GLP-1 and tested their response to an ip glucose tolerance test. GLP-1 produced a significant increase of insulin secretion, which was paralleled by a decrease in plasma glucose levels (P < 0.001 and P < 0.01, respectively). Four days after the removal of the infusion pumps, rats were killed and the pancreas harvested to study the mechanism by which GLP-1 ameliorated glucose tolerance. Ex vivo immunostaining with the marker of cell proliferation, Ki-67, showed that the metabolic changes observed in rats treated with GLP-1 were associated with an increase in cell proliferation of the endocrine and exocrine component of the pancreas. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining, a marker of cellular apoptosis, indicated a reduction of apoptotic cells within the islet as well in the exocrine pancreas in GLP-1-treated rats. Double immunostaining for the apoptotic marker caspase-3 and for insulin showed a significant reduction of caspase-3 expression and an increase in insulin content in GLP-1-treated animals. Finally, staining of pancreatic sections with the nuclear dye 4,6-Diaminidino-2-phenyl-dihydrochloride demonstrated a marked reduction of fragmented nuclei in the islet cells of rats treated with GLP-1. Our findings provide evidence that the beneficial effects of GLP-1 in Zucker diabetic rats is mediated by an increase in islet cell proliferation and a decrease of cellular apoptosis.

Glucagon-like peptide 1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets.

The peptide hormone, glucagon-like peptide 1 (GLP-1), has been shown to increase glucose-dependent insulin secretion, enhance insulin gene transcription, expand islet cell mass, and inhibit beta-cell apoptosis in animal models of diabetes. The aim of the present study was to evaluate whether GLP-1 could improve function and inhibit apoptosis in freshly isolated human islets. Human islets were cultured for 5 d in the presence, or absence, of GLP-1 (10 nm, added every 12 h) and studied for viability and expression of proapoptotic (caspase-3) and antiapoptotic factors (bcl-2) as well as glucose-dependent insulin production. We observed better-preserved three-dimensional islet morphology in the GLP-1-treated islets, compared with controls. Nuclear condensation, a feature of cell apoptosis, was inhibited by GLP-1. The reduction in the number of apoptotic cells in GLP-1-treated islets was particularly evident at d 3 (6.1% apoptotic nuclei in treated cultures vs. 15.5% in controls; P < 0.01) and at d 5 (8.9 vs. 18.9%; P < 0.01). The antiapoptotic effect of GLP-1 was associated with the down-regulation of active caspase-3 (P < 0.001) and the up-regulation of bcl-2 (P < 0.01). The effect of GLP-1 on the intracellular levels of bcl-2 and caspase-3 was observed at the mRNA and protein levels. Intracellular insulin content was markedly enhanced in islets cultured with GLP-1 vs. control (P < 0.001, at d 5), and there was a parallel GLP-1-dependent potentiation of glucose-dependent insulin secretion (P < 0.01 at d 3; P < 0.05 at d 5). Our findings provide evidence that GLP-1 added to freshly isolated human islets preserves morphology and function and inhibits cell apoptosis.

The short half-life of glucagon-like peptide-1 in plasma does not reflect its long-lasting beneficial effects.

The incretin hormone glucagon-like peptide-1 (GLP-1) is capable of ameliorating glucose-dependent insulin secretion in subjects with diabetes. However, its very short half-life (1.5-5 min) in plasma represents a major limitation for its use in the clinical setting. The present study was designed to characterize the duration of the effect of GLP-1 in the Zucker diabetic fatty (ZDF) rat. ZDF rats were subjected to a 48 h infusion of human GLP-1 (30 pmol/kg per min), followed by an i.p. glucose tolerance test (IPGTT) (1 g/kg body weight), 2 h after removing the infusion pump. At 15 min from the beginning of the test, GLP-1-treated animals had lower plasma glucose levels (442+/-38 mg/dl) than saline-infused controls (583+/-63 mg/dl, P<0.01). This was reflected in the higher insulin levels attained in the GLP-1-treated animals (1999+/-163 vs 1250+/-51 pmol/l, GLP-1 vs saline respectively, P<0.01). Repetition of the IPGTT on day 3, 9 and 16 from the removal of the infusion pump revealed a surprising lasting 'memory' of the exposure to GLP-1. Indeed, the best insulin secretory response was observed approximately 1 week after discontinuation of the GLP-1 infusion, and lasted up to 3 weeks from the early exposure to GLP-1. Detection of fasting plasma levels of GLP-1 during the 3 weeks of the experiment showed a very rapid decline, consistent with the data reported by others. Our findings provide evidence for a long-lasting beneficial effect of GLP-1 that persists for weeks even when the circulating levels of GLP-1 are back to normal.

Skeletal muscle mitochondrial function is associated with longitudinal growth velocity in children and adolescents.

CONTEXT: Periods of rapid growth require an increase in energy use and substrate formation. Mitochondrial function contributes to each of these and therefore may play a role in longitudinal growth. METHODS: Twenty-nine children and adolescents of ages 8-15 yr were enrolled in a comprehensive longitudinal assessment of glucose homeostasis and mitochondrial function. Fasting laboratory studies and an estimate of mitochondrial function (as assessed by the time to recovery of phosphocreatine (PCr) concentration after submaximal quadriceps extension/flexion exercise using (31)P magnetic resonance spectroscopy) were obtained at baseline and annually for 2 yr. RESULTS: Data were complete for 23 subjects. Subjects were 11.3 ± 1.9 (sd) yr old at the beginning of the study; 61% were male. Average annualized growth velocity at 1 yr for boys was 7.1 ± 1.5 cm/yr and for girls 6.5 ± 1.7 cm/yr. More rapid recovery of PCr concentration, suggestive of greater skeletal muscle oxidative phosphorylation capacity at baseline, was associated with faster growth velocity in the subsequent year (r(2) = 0.29; P = 0.008). In multivariate modeling, baseline mitochondrial function remained significantly and independently associated with growth (R(2) for model = 0.51; P = 0.05 for effect of phosphocreatine recovery time constant), controlling for age, gender, Tanner stage, body mass index Z-score, and height Z-score. CONCLUSIONS: We report a novel association between time to recovery of PCr concentration after submaximal exercise and faster annual linear growth in healthy children. Future studies are needed to determine the physiological mechanisms and clinical consequences of this observation.

The role of GLP-1 in the regulation of islet cell mass.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone capable of restoring euglycemia in glucose-intolerant subjects and improving glucose control in individuals with type 2 diabetes. Whether the antidiabetic properties of GLP-1 are exclusively the result of its acute postprandial action is being investigated. A GLP-1-dependent differentiation of pancreatic precursor cells into mature beta-cells has been proposed. In addition, GLP-1 has been shown to have antiapoptotic activity in cultured insulin-secreting cells and in an animal model in which diabetes occurs as a consequence of an excessive rate of beta-cell apoptosis. Studies from our laboratory, and others, lead us to propose that GLP-1 is a growth factor for pancreatic cells and it is a regulator of islet cell mass. The aim of this article is to review those reports that have emphasized the role of GLP-1 as a regulator of islet cell mass as well as its insulin secretory action.

Application of phage display peptide library to autoimmune diabetes: identification of IA-2/ICA512bdc dominant autoantigenic epitopes.

Autoantigenic epitope mapping represents a critical issue in autoimmune diseases. The islet tyrosine phosphatase-like protein IA-2/ICA512bdc is a major autoantigen in type 1 diabetes (IDDM), but the epitopes responsible for autoantibody binding have been only partially defined. The aim of our study was to identify ICA512bdc epitopes, and in particular mini-epitopes, utilizing a novel strategy for autoimmune diseases. The study was performed in three sequential steps: (1) construction of a lambda-phage surface-displayed ICA512bdc cDNA library with the methodology of tagged random priming with peptides displayed as a fusion to the C terminus of the capsid protein D; (2) affinity selection of the resulting library, followed by immunoscreening, enzyme-linked immunosorbent assay and sequence analysis of positive clones, and (3) radioimmunoprecipitation to detect autoantibodies to the selected clones. This strategy resulted in the identification of two epitopes (IA-2 residues 761 - 964 and 929 - 979), which were recognized by 100 % and 62.9 % ICA512bdc-positive IDDM patients, respectively. Interestingly, the larger clone was detected also by a proportion (16.7 %) of new onset ICA512bdc-negative patients, thus suggesting that this region contains not only the main autoantigenic repertoire of ICA512bdc molecule, but is able to detect IA-2 autoantibodies in even higher percentages of patients. In addition, this study showed the existence of multiple epitopes located in the C-terminal domain of the IA-2 protein, one of which is formed by the 50 C-terminal amino acids, and provided evidence that the strategy used represents a valid tool for identification of epitopes within autoantigenic molecules.