Plasma B-cell tropin (ACTH22-39) concentration in lean and obese (ob/ob) mice and lean and obese (fa/fa) Zucker rats

A method has been developed for the measurement of plasma concentrations of Beta-cell tropin (BCT), which is a potent insulinotropic and lipogenic peptide secreted by the pituitary. The method was employed to compare plasma Beta-cell tropin concentrations between lean and genetically obese (ob/ob) mice and between lean and genetically obese (fa/fa) Zucker rats. The plasma concentration in lean mice was 0.17 +/- 0.02 (5)nmole/l (mean +/- SEM, n = 5), while that in obese (ob/ob) mice was significantly higher, being 2.88 +/- 1.13 (5)nmole/l. The plasma BCT concentration in Zucker rats was 0.14 +/- 0.02 (15)nmole/l, while that in obese Zucker (fa/fa) rats was significantly higher, being 1.69 +/- 0.72 (16)nmole/l. These results explain previously observed differences in the Beta-cell tropin-like biological activity in plasma from lean and obese animals, and support the hypothesis that the peptide has a role in the development of hyperinsulinaemia and obesity.     

RX871024 reduces NO production but does not protect against pancreatic beta-cell death induced by proinflammatory cytokines

The imidazoline compound RX871024 reduces IL-1beta-induced NO production thereby protecting against IL-1beta-induced beta-cell apoptosis. The aim of this study was to evaluate whether imidazolines RX871024 and efaroxan protect beta-cells against death in the presence of a combination of the cytokines IL-1beta, IFNgamma, and TNFalpha. To address this issue, experiments involving different methods for detection of cell death, different concentrations of the cytokines, and a variety of conditions of preparation and culturing of ob/ob mouse islets and beta-cells have been carried out. Thoroughly performed experiments have not been able to demonstrate a protective effect of RX871024 and efaroxan on beta-cell death induced by the combination of cytokines. However, the inhibitory effect of RX871024 on NO production in ob/ob mouse islets and beta-cells was still observed in the presence of all three cytokines and correlated with the decrease in p38 MAPK phosphorylation. Conversely, efaroxan did not affect cytokine-induced NO production. Our data indicate that a combination of pro-inflammatory cytokines IL-1beta, IFNgamma, and TNFalpha, conditions modelling those that take place in type 1 diabetes, induces pancreatic beta-cell death that does not directly correlate with NO production and cannot be counteracted with imidazoline compounds.     

Carbon monoxide stimulates insulin release and propagates Ca2+ signals between pancreatic beta-cells

A key question for understanding the mechanisms of pulsatile insulin release is how the underlying beta-cell oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) are synchronized within and among the islets in the pancreas. Nitric oxide has been proposed to coordinate the activity of the beta-cells by precipitating transients of [Ca2+]i. Comparing ob/ob mice and lean controls, we have now studied the action of carbon monoxide (CO), another neurotransmitter with stimulatory effects on cGMP production. A strong immunoreactivity for the CO-producing constitutive heme oxygenase (HO-2) was found in ganglionic cells located in the periphery of the islets and in almost all islet endocrine cells. Islets from ob/ob mice had sixfold higher generation of CO (1 nmol.min-1.mg protein-1) than the lean controls. This is 100-fold the rate for their constitutive production of NO. Moreover, islets from ob/ob mice showed a threefold increase in HO-2 expression and expressed inducible HO (HO-1). The presence of an excessive islet production of CO in the ob/ob mouse had its counterpart in a pronounced suppression of the glucose-stimulated insulin release from islets exposed to the HO inhibitor Zn-protoporhyrin (10 microM) and in a 16 times higher frequency of [Ca2+]i transients in their beta-cells. Hemin (0.1 and 1.0 microM), the natural substrate for HO, promoted the appearance of [Ca2+]i transients, and 10 microM of the HO inhibitors Zn-protoporphyrin and Cr-mesoporphyrin had a suppressive action both on the firing of transients and their synchronization. It is concluded that the increased islet production of CO contributes to the hyperinsulinemia in ob/ob mice. In addition to serving as a positive modulator of glucose-stimulated insulin release, CO acts as a messenger propagating Ca2+ signals with coordinating effects on the beta-cell rhythmicity.     

The long-acting GLP-1 derivative NN2211 ameliorates glycemia and increases beta-cell mass in diabetic mice

NN2211 is a long-acting, metabolically stable glucagon-like peptide-1 (GLP-1) derivative designed for once daily administration in humans. NN2211 dose dependently reduced the glycemic levels in ob/ob mice, with antihyperglycemic activity still evident 24 h postdose. Apart from an initial reduction in food intake, there were no significant differences between NN2211 and vehicle treatment, and body weight was not affected. Histological examination revealed that beta-cell proliferation and mass were not increased significantly in ob/ob mice with NN2211, although there was a strong tendency for increased proliferation. In db/db mice, exendin-4 and NN2211 decreased blood glucose compared with vehicle, but NN2211 had a longer duration of action. Food intake was lowered only on day 1 with both compounds, and body weight was unaffected. beta-Cell proliferation rate and mass were significantly increased with NN2211, but with exendin-4, only the beta-cell proliferation rate was significantly increased. In conclusion, NN2211 reduced blood glucose after acute and chronic treatment in ob/ob and db/db mice and was associated with increased beta-cell mass and proliferation in db/db mice. NN2211 is currently in phase 2 clinical development.     

Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis

The adipocyte-derived hormone adiponectin has been shown to play important roles in the regulation of energy homeostasis and insulin sensitivity. In this study, we analyzed globular domain adiponectin (gAd) transgenic (Tg) mice crossed with leptin-deficient ob/ob or apoE-deficient mice. Interestingly, despite an unexpected similar body weight, gAd Tg ob/ob mice showed amelioration of insulin resistance and beta-cell degranulation as well as diabetes, indicating that globular adiponectin and leptin appeared to have both distinct and overlapping functions. Amelioration of diabetes and insulin resistance was associated with increased expression of molecules involved in fatty acid oxidation such as acyl-CoA oxidase, and molecules involved in energy dissipation such as uncoupling proteins 2 and 3 and increased fatty acid oxidation in skeletal muscle of gAd Tg ob/ob mice. Moreover, despite similar plasma glucose and lipid levels on an apoE-deficient background, gAd Tg apoE-deficient mice showed amelioration of atherosclerosis, which was associated with decreased expression of class A scavenger receptor and tumor necrosis factor alpha. This is the first demonstration that globular adiponectin can protect against atherosclerosis in vivo. In conclusion, replenishment of globular adiponectin may provide a novel treatment modality for both type 2 diabetes and atherosclerosis.     

Cell-specific Ca(2+) responses in glucose-stimulated single and aggregated beta-cells

A rise in the cytoplasmic calcium concentration ([Ca(2+)](i)) is a key event for insulin exocytosis. We have recently found that the 'early [Ca(2+)](i) response' in single ob/ob mouse beta-cells is reproduced during consecutive glucose stimulations. It, therefore, appears that the response pattern is a characteristic of the individual beta-cell. We have now investigated if a cell-specific [Ca(2+)](i) response is a general phenomenon in rodent beta-cells, and if it can be observed when cells are functionally coupled. With the use of the fura-2 technique, we have studied the 'early [Ca(2+)](i) response' in single dispersed beta-cells, in beta-cell clusters of different size and in intact islets from the ob/ob mouse during repeated glucose stimulation (20mM). beta-Cells from lean mouse and rat, and intact islets from lean mouse were also investigated. Significant correlations between the first and second stimulation were found for the parameters lag-time for Ca(2+) rise (calculated as the time from start of stimulation of the cell until the first value above an extrapolated baseline), nadir of initial lowering (difference between the baseline and lowest [Ca(2+)](i) value), and peak height (difference between baseline and the highest [Ca(2+)](i) value of the first calcium peak) in single dispersed beta-cells, in 'single beta-cell within a small cluster', in clusters of medium and large size, and in single dispersed beta-cells from lean mouse and rat. The lag-times for Ca(2+) rise and peak heights were correlated within the pairs of stimulation also in intact ob/ob islets. In summary, despite a large heterogeneity of the 'early [Ca(2+)](i) response' among individual cells, the lag-time for [Ca(2+)](i) rise, the nadir of initial lowering and the height of the first peak response can be identified as cell-specific markers in beta-cells.     

Positional cloning of "Lisch-Like", a candidate modifier of susceptibility to type 2 diabetes in mice

In 404 Lep(ob/ob) F2 progeny of a C57BL/6J (B6) x DBA/2J (DBA) intercross, we mapped a DBA-related quantitative trait locus (QTL) to distal Chr1 at 169.6 Mb, centered about D1Mit110, for diabetes-related phenotypes that included blood glucose, HbA1c, and pancreatic islet histology. The interval was refined to 1.8 Mb in a series of B6.DBA congenic/subcongenic lines also segregating for Lep(ob). The phenotypes of B6.DBA congenic mice include reduced beta-cell replication rates accompanied by reduced beta-cell mass, reduced insulin/glucose ratio in blood, reduced glucose tolerance, and persistent mild hypoinsulinemic hyperglycemia. Nucleotide sequence and expression analysis of 14 genes in this interval identified a predicted gene that we have designated "Lisch-like" (Ll) as the most likely candidate. The gene spans 62.7 kb on Chr1qH2.3, encoding a 10-exon, 646-amino acid polypeptide, homologous to Lsr on Chr7qB1 and to Ildr1 on Chr16qB3. The largest isoform of Ll is predicted to be a transmembrane molecule with an immunoglobulin-like extracellular domain and a serine/threonine-rich intracellular domain that contains a 14-3-3 binding domain. Morpholino knockdown of the zebrafish paralog of Ll resulted in a generalized delay in endodermal development in the gut region and dispersion of insulin-positive cells. Mice segregating for an ENU-induced null allele of Ll have phenotypes comparable to the B.D congenic lines. The human ortholog, C1orf32, is in the middle of a 30-Mb region of Chr1q23-25 that has been repeatedly associated with type 2 diabetes.     

Increased insulin translation from an insulin splice-variant overexpressed in diabetes, obesity, and insulin resistance

Type 2 diabetes occurs when pancreatic beta-cells become unable to compensate for the underlying insulin resistance. Insulin secretion requires beta-cell insulin stores to be replenished by insulin biosynthesis, which is mainly regulated at the translational level. Such translational regulation often involves the 5'-untranslated region. Recently, we identified a human insulin splice-variant (SPV) altering only the 5'-untranslated region and conferring increased translation efficiency. We now describe a mouse SPV (mSPV) that is found in the cytoplasm and exhibits increased translation efficiency resulting in more normal (prepro)insulin protein per RNA. The RNA stability of mSPV is not increased, but the predicted secondary RNA structure is altered, which may facilitate translation. To determine the role of mSPV in insulin resistance and diabetes, mSPV expression was measured by quantitative real-time RT-PCR in islets from three diabetic and/or insulin-resistant, obese and nonobese, mouse models (BTBRob/ob, C57BL/6ob/ob, and C57BL/6azip). Interestingly, mSPV expression was significantly higher in all diabetic/insulin-resistant mice compared with wild-type littermates and was dramatically induced in primary mouse islets incubated at high glucose. This raises the possibility that the mSPV may represent a compensatory beta-cell mechanism to enhance insulin biosynthesis when insulin requirements are elevated by hyperglycemia/insulin resistance.     

Pioglitazone preserves pancreatic islet structure and insulin secretory function in three murine models of type 2 diabetes

Thiazolidinediones may slow the progression of type 2 diabetes by preserving pancreatic beta-cells. The effects of pioglitazone (PIO) on structure and function of beta-cells in KKA(y), C57BL/6J ob/ob, and C57BL/KsJ db/db mice (genetic models of type 2 diabetes) were examined. ob/ob (n = 7) and db/db (n = 9) mice were randomly assigned to 50-125 mg.kg body wt-1.day-1 of PIO in chow beginning at 6-10 wk of age. Control ob/ob (n = 7) and db/db mice (n = 9) were fed chow without PIO. KKA(y) mice (n = 15) were fed PIO daily at doses of 62-144 mg.kg body wt-1.day-1. Control KKA(y) mice (n = 10) received chow without PIO. Treatment continued until euthanasia at 14-26 wk of age. Blood was collected at baseline (before treatment) and just before euthanasia and was analyzed for glucose, glycosylated hemoglobin, and plasma insulin. Some of the splenic pancreas of each animal was resected and partially sectioned for light or electron microscopy. The remainder of the pancreas was assayed for insulin content. Compared with baseline and control groups, PIO treatment significantly reduced blood glucose and glycosylated hemoglobin levels. Plasma insulin levels decreased significantly in ob/ob mice treated with PIO. All groups treated with PIO exhibited significantly greater beta-cell granulation, evidence of reduced beta-cell stress, and 1.5- to 15-fold higher levels of pancreatic insulin. The data from these studies suggest that comparable effects would be expected to slow the progression of type 2 diabetes, either delaying or possibly preventing progression to an insulin-dependent state.     

Effect of perchlorate on calcium uptake and insulin secretion in mouse pancreatic islets.

Microdissected beta-cell-rich pancreatic islets of non-inbred ob/ob mice were used in studies of how perchlorate (CIO4-) affects stimulus-secretion coupling in beta-cells. CIO4- at 16 mM potentiated D-glucose-induced insulin release, without inducing secretion at non-stimulatory glucose concentrations. The potentiation mainly applied to the first phase of stimulated insulin release. In the presence of 20 mM-glucose, the half-maximum effect of CIO4- was reached at 5.5 mM and maximum effect at 12 mM of the anion. The potentiation was reversible and inhibitable by D-mannoheptulose (20 mM) or Ca2+ deficiency. CIO4- at 1-8 mM did not affect glucose oxidation. The effects on secretion were paralleled by a potentiation of glucose-induced 45Ca2+ influx during 3 min. K+-induced insulin secretion and 45Ca2+ uptake were potentiated by 8-16 mM-CIO4-. The spontaneous inactivation of K+-induced (20.9 mM-K+) insulin release was delayed by 8 mM-CIO4-. The anion potentiated the 45Ca2+ uptake induced by glibenclamide, which is known to depolarize the beta-cell. Insulin release was not affected by 1-10 mM-trichloroacetate. It is suggested that CIO4- stimulates the beta-cell by affecting the gating of voltage-controlled Ca2+ channels.     

Glucose inhibits 45Ca efflux from pancreatic beta-cells also in the absence of Na+-Ca2+ countertransport

During perifusion with medium deprived of Ca2+, addition of glucose or omission of Na+ resulted in prompt and quantitatively similar inhibitions of 45Ca efflux from beta-cell rich pancreatic islets microdissected from ob/ob mice. Glucose had no additional inhibitory effect when Na+ was isoosmotically replaced by sucrose or choline+. When K+ was used as a substitute for Na+, the inhibitory effect of Na+ removal on 45Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting 45Ca efflux in the presence or absence of Na+ is difficult to reconcile with the postulate that the Na+-Ca2+ countertransport mechanism is a primary site of action for glucose.     

Glucose-induced increase of potassium in pancreatic beta-cells associated with reduced mobilization of the ion

The potassium contents of beta-cell-rich pancreatic islets from ob/ob-mice were measured with an integrating flame photometer. After exposure to 5 mM glucose islet potassium increased by 17 +/- 2%, no additional effect being seen with increase of the sugar to 20 mM. Glucose counteracted the loss of islet potassium obtained on removal of the ion from the incubation medium, halving the initial disappearance rate. Whereas the effect of glucose in suppressing the mobilisation of potassium was mimicked by tolbutamide and quinine, it was antagonized by diazoxide. It is concluded that the glucose interference with the outward transport of K+ is sufficient to raise the beta-cell content of the ion.     

Cytosolic O-GlcNAc accumulation is not involved in beta-cell death in HIT-T15 or Min6

O-linked N-acetylglucosamine (O-GlcNAc) is attached to and detached from proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. It has been proposed that streptozotocin induces pancreatic beta-cell death by blocking O-GlcNAcase and increasing O-GlcNAc. To elucidate the relationship between cytosolic O-GlcNAc accumulation and beta-cell death, we treated beta-cell lines HIT-T15 and Min6 with glucosamine. Glucosamine markedly reduced cell viability in both cell lines only at 10 mM. The measurement of cytosolic O-GlcNAc under glucosamine treatment revealed that O-GlcNAc accumulation was observed even at 2 mM glucosamine and maximized at 5 mM, but did not occur very well at 10 mM. Furthermore, 100 microM PUGNAc, an inhibitor of O-GlcNAcase, increased cytosolic O-GlcNAc but did not induce cell death in these cells. Therefore, no correlation between accumulation of cytosolic O-GlcNAc and beta-cell death was suggested. Alternatively, inosine partially rescued cell death induced by glucosamine in Min6 cells, suggesting that energy depletion partly contributes to beta-cell death by glucosamine.     

FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice

The metabolic syndrome is a worldwide problem mainly caused by obesity. FTO was found to be a obesity-risk gene in humans and FTO deficiency in mice led to reduction in adipose tissue. Thus, FTO is an important factor for the development of obesity. Leptin-deficient mice are a well characterized model for analysing the metabolic syndrome. To determine the relevance of FTO for the development of the metabolic syndrome we analysed different parameters in combined homozygous deficient mice (Lep^ob/ob;Fto^−/−). Lep^ob/ob;Fto^−/− mice showed an improvement in analysed hallmarks of the metabolic syndrome in comparison to leptin-deficient mice wild type or heterozygous for Fto. Lep^ob/ob;Fto^−/− mice did not develop hyperglycaemia and showed an improved glucose tolerance. Furthermore, extension of beta-cell mass was prevented in Lep^ob/ob;Fto^−/−mice and accumulation of ectopic fat in the liver was reduced. In conclusion this study demonstrates that FTO deficiency has a protective effect not only on the development of obesity but also on the metabolic syndrome. Thus, FTO plays an important role in the development of metabolic disorders and is an interesting target for therapeutic agents.     

Roles for bone-marrow-derived cells in beta-cell maintenance

With more than 177 million people suffering from diabetes worldwide, and the number expected to double by 2030, finding new ways to treat this disease is a high priority. Intensive effort is being directed towards developing mechanisms for increasing beta-cell expansion as a diabetic therapeutic. Recent studies, in which adult bone marrow has been used to induce beta-cell expansion in mice, have shown both exciting and controversial results. In these reports, marrow-derived cells can contribute towards beta-cell maintenance both by promoting endogenous beta-cell expansion and possibly by transdifferentiation into beta-cells. These studies reveal mechanisms for potential therapeutic intervention.     

Isolation of corticotropin-β-lipotropin common precursor from ovine pituitary glands

Ovine corticotropin-β-lipotropin common precursor was purified to homogeneity from commercial frozen ovine pituitary glands. A crude preparation was obtained following a procedure published elsewhere (Lee, T.H. and Lee, M.S. (1977) Biochemistry 16, 2824–2829) and was purified by gel filtration on Sephadex G-200 in the presence of 0.5% SDS and 0.1% 2-mercaptoethanol, and under an atmosphere of nitrogen. The gel filtration was repeated once. The partially purified preparation obtained from the second Sephadex G-200 gel filtration was further fractionated by preparative SDS-acrylamide gel eletrophoresis, using immunoprecipitated and electrophoretically purified [¹²⁵I]corticotropin-β-lipotropin common precursor as a marker. The preparation was judged homogenous by the appearance of a single protein band in analytical SDS-acrylamide gel electrophoresis, which exhibited both corticotropin and β-lipotropin immunoreactivities, and a single symmetrical peak in high-pressure liquid chromatography on a reverse phase C18 column. The isolated ovine corticotropin-β-lipotropin common precursor possessed specific activities of 116 μg of immunoreactive corticotropin and 210 μg of immunoreactive β-lipotropin per mg of protein, equivalent to 89 and 62% of theoretical values, respectively. The amino acid composition of the homogeneous preparation was determined.     

Whole Organism High Content Screening Identifies Stimulators of Pancreatic Beta-Cell Proliferation

Inducing beta-cell mass expansion in diabetic patients with the aim to restore glucose homeostasis is a promising therapeutic strategy. Although several in vitro studies have been carried out to identify modulators of beta-cell mass expansion, restoring endogenous beta-cell mass in vivo has yet to be achieved. To identify potential stimulators of beta-cell replication in vivo, we established transgenic zebrafish lines that monitor and allow the quantification of cell proliferation by using the fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. Using these new reagents, we performed an unbiased chemical screen, and identified 20 small molecules that markedly increased beta-cell proliferation in vivo. Importantly, these structurally distinct molecules, which include clinically-approved drugs, modulate three specific signaling pathways: serotonin, retinoic acid and glucocorticoids, showing the high sensitivity and robustness of our screen. Notably, two drug classes, retinoic acid and glucocorticoids, also promoted beta-cell regeneration after beta-cell ablation. Thus, this study establishes a proof of principle for a high-throughput small molecule-screen for beta-cell proliferation in vivo, and identified compounds that stimulate beta-cell proliferation and regeneration.     

Unraveling the pathogenesis of type 1 diabetes with proteomics: present and future directions

Type 1 diabetes (T1D) is the result of selective destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. T1D is due to a complex interplay between the beta-cell, the immune system, and the environment in genetically susceptible individuals. The initiating mechanism(s) behind the development of T1D are largely unknown, and no genes or proteins are specific for most T1D cases. Different pro-apoptotic cytokines, IL-1 beta in particular, are present in the islets during beta-cell destruction and are able to modulate beta-cell function and induce beta-cell death. In beta-cells exposed to IL-1 beta, a race between destructive and protective events are initiated and in susceptible individuals the deleterious events prevail. Proteins are involved in most cellular processes, and it is thus expected that their cumulative expression profile reflects the specific activity of cells. Proteomics may be useful in describing the protein expression profile and thus the diabetic phenotype. Relatively few studies using proteomics technologies to investigate the T1D pathogenesis have been published to date despite the defined target organ, the beta-cell. Proteomics has been applied in studies of differentiating beta-cells, cytokine exposed islets, dietary manipulated islets, and in transplanted islets. Although that the studies have revealed a complex and detailed picture of the protein expression profiles many functional implications remain to be answered. In conclusion, a rather detailed picture of protein expression in beta-cell lines, islets, and transplanted islets both in vitro and in vivo have been described. The data indicate that the beta-cell is an active participant in its own destruction during diabetes development. No single protein alone seems to be responsible for the development of diabetes. Rather the cumulative pattern of changes seems to be what favors a transition from dynamic stability in the unperturbed beta-cell to dynamic instability and eventually to beta-cell destruction.     

糖脂毒性对胰腺β细胞的功能损伤作用及机制

现在关于高糖高脂对胰腺β细胞的毒性机制已经有了明显的进展,但还不完全清楚。实际上,β细胞响应过量营养物质的过程是一个连续的过程,包括β细胞补偿和β细胞功能失调。在早期,β细胞应对高糖高脂的反应是一个积极主动的过程;而到后期,过量的糖脂会导致胰岛素分泌下降,削弱胰岛素基因表达量,并促进胰岛β细胞凋亡。最终对2型糖尿病的发展有促进作用。综述了近年来细胞水平和分子水平,在葡萄糖存在的条件下,脂肪酸对胰腺β细胞的损伤作用及其机制的研究进展,重在说明葡萄糖和脂肪酸在2型糖尿病发展中的共同作用。