GLP-1(28-36) improves ß-cell mass and glucose disposal in streptozotocin-induced diabetic mice and activates cAMP/PKA/ß-catenin signaling in ß-cells in vitro.

Recent studies have demonstrated that the COOH-terminal fragment of the incretin hormone glucagon-like peptide-1 (GLP-1), a nonapeptide GLP-1(28-36)amide, attenuates diabetes and hepatic steatosis in diet-induced obese mice. However, the effect of this nonapeptide in pancreatic ß-cells remains largely unknown. Here, we show that in a streptozotocin-induced mouse diabetes model, GLP-1(28-36)amide improved glucose disposal and increased pancreatic ß-cell mass and ß-cell proliferation. An in vitro investigation revealed that GLP-1(28-36)amide stimulates ß-catenin (ß-cat) Ser(675) phosphorylation in both the clonal INS-1 cell line and rat primary pancreatic islet cells. In INS-1 cells, the stimulation was accompanied by increased nuclear ß-cat content. GLP-1(28-36)amide was also shown to increase cellular cAMP levels, PKA enzymatic activity, and cAMP response element-binding protein (CREB) and cyclic AMP-dependent transcription factor-1 (ATF-1) phosphorylation. Furthermore, GLP-1(28-36)amide treatment enhanced islet insulin secretion and increased the growth of INS-1 cells, which was associated with increased cyclin D1 expression. Finally, PKA inhibition attenuated the effect of GLP-1(28-36)amide on ß-cat Ser(675) phosphorylation and cyclin D1 expression in the INS-1 cell line. We have thus revealed the beneficial effect of GLP-1(28-36)amide in pancreatic ß-cells in vitro and in vivo. Our observations suggest that GLP-1(28-36)amide may exert its effect through the PKA/ß-catenin signaling pathway.

Pancreatic islet-specific overexpression of Reg3ß protein induced the expression of pro-islet genes and protected the mice against streptozotocin-induced diabetes mellitus.

Reg family proteins have been implicated in islet ß-cell proliferation, survival, and regeneration. The expression of Reg3ß (pancreatitis-associated protein) is highly induced in experimental diabetes and acute pancreatitis, but its precise role has not been established. Through knockout studies, this protein was shown to be mitogenic, antiapoptotic, and anti-inflammatory in the liver and pancreatic acinars. To test whether it can promote islet cell growth or survival against experimental damage, we developed ß-cell-specific overexpression using rat insulin I promoter, evaluated the changes in normal islet function, gene expression profile, and the response to streptozotocin-induced diabetes. Significant and specific overexpression of Reg3ß was achieved in the pancreatic islets of RIP-I/Reg3ß mice, which exhibited normal islet histology, ß-cell mass, and in vivo and in vitro insulin secretion in response to high glucose yet were slightly hyperglycemic and low in islet GLUT2 level. Upon streptozotocin treatment, in contrast to wild-type littermates that became hyperglycemic in 3 days and lost 15% of their weight, RIP-I/Reg3ß mice were significantly protected from hyperglycemia and weight loss. To identify specific targets affected by Reg3ß overexpression, a whole genome DNA microarray on islet RNA isolated from the transgenic mice revealed more than 45 genes significantly either up- or downregulated. Among them, islet-protective osteopontin/SPP1 and acute responsive nuclear protein p8/NUPR1 were significantly induced, a result further confirmed by real-time PCR, Western blots, and immunohistochemistry. Our results suggest that Reg3ß is unlikely an islet growth factor but a putative protector that prevents streptozotocin-induced damage by inducing the expression of specific genes.

Coordinated age-dependent and pancreatic-specific expression of mouse Reg2Reg3α, and Reg3ß genes.

Reg family proteins such as Reg1 and islet neogenesis-associated protein (INGAP) have long been implicated in the growth and/or neogenesis of pancreatic islet cells. Recent reports further suggest similar roles to be played by new members such as Reg2, Reg3α, and Reg3ß. We have studied their age-, isoform-, and tissue-specific expressions. RNA and protein were isolated from C57BL/6 mice aged 7, 30, and 90 days. Using real-time polymerase chain reaction, the levels of Reg gene expression in the pancreas were 20-600-fold higher than that in other tissues (≫duodenum>stomach>liver); gene expression of Reg2, Reg3α, and Reg3ß was age dependent as it was hardly detectable at day 7, increased drastically at day 30, and significantly decreased at day 90; the levels of pancreatic proteins displayed similar age-dependent variations. Using dual-labeled immunofluorescence, Reg2, Reg3α, and Reg3ß were abundantly expressed in most acinar cells of the pancreas, in contrast to INGAP which exhibited stepwise increases from day 7 to day 90 and colocalized with the α-cells. These new Reg genes were mainly expressed in the pancreas, with clear age-dependent and isoform-specific patterns.

[Association of L-selectin gene polymorphism with susceptibility to coronary heart disease].

OBJECTIVE: To investigate the possible association between L-selectin gene P213S polymorphism and coronary heart disease (CHD) in Chinese population. METHODS: In total 212 CHD patients diagnosed by angiography and 230 healthy controls were studied. The genotype and allele frequencies of L-selectin gene polymorphism were assayed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The frequency of the L-selectin gene 213P allele in CHD patients was significantly higher than that in the control group (77.59% vs 69.35%, P=0.006). Compared with the SS genotype, PP homozygote had a significantly increased CHD risk (OR=2.70 and OR=2.15 using unadjusted and adjusted Logistic regression models, respectively). No association was found between the severity of CHD and the Lselectin gene P213S polymorphism CONCLUSION: Our findings suggest that L-selectin gene 213P mutant allele might contribute to susceptibility of Chinese individuals to contract CHD.

Deteriorated high-fat diet-induced diabetes caused by pancreatic ß-cell-specific overexpression of Reg3ß gene in mice.

Reg family proteins have long been implicated in islet ß-cell proliferation, survival, and regeneration. In our previous study, we reported that Reg3ß overexpression did not increase islet growth but prevented streptozotocin-induced islet damage by inducing specific genes. In order to explore its role in type 2 diabetes (T2D), we established high-fat diet (HFD)-induced obesity and diabetes in RIP-I/Reg3ß mice. Glucose and insulin tolerance tests, immunofluorescence for insulin, eIF2α, and GLUT2 in islets, Western blots on phosphorylated AMPKα and hepatic histology were performed. Both RIP-I/Reg3ß and wild-type mice gained weight rapidly and became hyperglycemic after 10 weeks on the HFD. However, the transgenic mice exhibited more significant acceleration in blood glucose levels, further deterioration of glucose intolerance and insulin resistance, and a lower intensity of insulin staining. Immunofluorescence revealed similar magnitude of islet compensation to a wild-type HFD. The normal GLUT2 distribution in the transgenic ß-cells was disrupted and the staining was obviously diminished on the cell membrane. HFD feeding also caused a further decrease in the level of AMPKα phosphorylation in the transgenic islets. Our results suggest that unlike its protective effect against T1D, overexpressed Reg3ß was unable to protect the ß-cells against HFD-induced damage.

[Association of C-159T polymorphism in promoter region of CD14 and coronary heart disease].

OBJECTIVE: To investigate the distribution of CD14 promoter gene -159(C>T) polymorphism in Hubei Han population of China and analyze the association of CD14 polymorphisms with coronary heart disease (CHD). METHODS: Genotypes of CD14 were typed in 162 CHD patients and 196 controls by polymerase chain reaction-restriction fragment length polymorphism. Selected coronary angiography was performed in 162 CHD patients. RESULTS: CD14 promoter -159 genotype frequencies of CC, CT and TT were 27.4%, 45.6%, 27.0% and 14.8%, 46.5%, 38.7% in normal control group and CHD group respectively. Genotype distribution was in accordance with Hardy-Weinberg equilibrium. There existed statistically significant difference in frequencies of allele and genotype in CD14 C-159T polymorphism between CHD group and control group (Genotype: Chi2=0.654, P < 0.05, CT vs CC, OR=1.245, 95%CI: 1.001-1.473, TT vs CC, OR=2.374, 95%CI 2.012-2.649; Allele: Chi2=0.547, P < 0.05, T vs C, Chi2=0.547, P < 0.05, OR=3.105, 95%CI: 2.493-3.539). The distributions of allele and genotype in CD14 -159(C>T) were of statistically significant difference between non-myocardial infarction subgroup and myocardial infarction subgroup (Genotype: Chi2=0.782, P < 0.05, CT vs CC, OR=2.375, 95%CI: 2.017-2.689, TT vs CC, OR=3.459, 95%CI: 3.003-3.846. Allele: Chi2=2.374, P < 0.05, T vs C, Chi2=2.374, P < 0.05, OR=4.011, 95%CI: 3.814-4.279). However, no statistically significant difference was found among the subgroups of oneìtwo and three stenosed vessels. CONCLUSION: The T allele of the C-159T polymorphism of CD14 gene may be a risk factor for myocardial infarction.

The expression of dominant negative TCF7L2 in pancreatic beta cells during the embryonic stage causes impaired glucose homeostasis.

OBJECTIVE: Disruption of TCF7L2 in mouse pancreatic ß-cells has generated different outcomes in several investigations. Here we aim to clarify role of ß-cell TCF7L2 and Wnt signaling using a functional-knockdown approach. METHODS: Adenovirus-mediated dominant negative TCF7L2 (TCF7L2DN) expression was conducted in Ins-1 cells. The fusion gene in which TCF7L2DN expression is driven by P TRE3G was utilized to generate the transgenic mouse line TCF7L2DN Tet . The double transgenic line was created by mating TCF7L2DN Tet with Ins2-rtTA, designated as ßTCFDN. ß-cell specific TCF7L2DN expression was induced in ßTCFDN by doxycycline feeding. RESULTS: TCF7L2DN expression in Ins-1 cells reduced GSIS, cell proliferation and expression of a battery of genes including incretin receptors and ß-cell transcription factors. Inducing TCF7L2DN expression in ßTCFDN during adulthood or immediately after weaning generated no or very modest metabolic defect, while its expression during embryonic development by doxycycline feeding in pregnant mothers resulted in significant glucose intolerance associated with altered ß-cell gene expression and reduced ß-cell mass. CONCLUSIONS: Our observations support a cell autonomous role for TCF7L2 in pancreatic ß-cells suggested by most, though not all, investigations. ßTCFDN is a novel model for further exploring the role of TCF7L2 in ß-cell genesis and metabolic homeostasis.

New insight into the mechanisms underlying the function of the incretin hormone glucagon-like peptide-1 in pancreatic ß-cells: the involvement of the Wnt signaling pathway effector ß-catenin.

During the past two decades, the exploration of function of two incretin hormones, namely glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP), has led to the development of two categories of novel therapeutic agents for diabetes and its complications, known as GLP-1 receptor (GLP-1R) agonists and DPP-IV inhibitors. Mechanisms underlying the function of GLP-1, however, still need to be further explored. GLP-1 not only functions as an incretin hormone in stimulating insulin secretion in response to nutritional, hormonal and neuronal stimulations, but also acts as an "insulin-like" factor in ß-cell and extra-pancreatic organs. In addition to these insulinotropic and insulinomimetic effects, GLP-1 was shown to exert its protective effect in ß-cell by repressing the expression of TxNIP, a mediator of glucolipotoxicity. A number of recent studies have shown that the Wnt signaling pathway effector, the bipartite transcription factor ß-catenin/TCF, controls not only the production of GLP-1, but also the function of GLP-1. Furthermore, previously assumed "degradation" products of GLP-1(7-36)amide, including GLP-1(9-36)amide and GLP-1(28-36)amide, have been shown to exert beneficial effect in pancreas and extra-pancreatic tissues or cell lineages. Here we summarized our current knowledge on the metabolic, proliferative and protective effects of GLP-1(7-36)amide and its cleavage fragments, mainly focusing on pancreatic ß-cells and the involvement of the Wnt signaling pathway effector ß-catenin.