In this study, an insulinoma-associated antigen-1 (INSM1)-binding site in the proximal promoter sequence of the insulin gene was identified. The co-transfection of INSM1 with rat insulin I/II promoter-driven reporter genes exhibited a 40–50% inhibitory effect on the reporter activity. Mutational experiments were performed by introducing a substitution, GG to AT, into the INSM1 core binding site of the rat insulin I/II promoters. The mutated insulin promoter exhibited a three- to 20-fold increase in the promoter activity over the wild-type promoter in several insulinoma cell lines. Moreover, INSM1 overexpression exhibited no inhibitory effect on the mutated insulin promoter. Chromatin immunoprecipitation assays using βTC-1, mouse fetal pancreas, and Ad-INSM1-transduced human islets demonstrated that INSM1 occupies the endogenous insulin promoter sequence containing the INSM1-binding site in vivo. The binding of the INSM1 to the insulin promoter could suppress ∼50% of insulin message in human islets. The mechanism for transcriptional repression of the insulin gene by INSM1 is mediated through the recruitment of cyclin D1 and histone deacetylase-3 to the insulin promoter. Anti-INSM1 or anti-cyclin D1 morpholino treatment of fetal mouse pancreas enhances the insulin promoter activity. These data strongly support the view that INSM1 is a new zinc-finger transcription factor that modulates insulin gene transcription during early pancreas development.
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Hong-Wei Wang, Michelle Muguira, Wei-Dong Liu, Tao Zhang, Chiachen Chen, Rebecca Aucoin, Mary B Breslin, and Michael S Lan
K Cheng, L Wei, L-Y Chaung, W W-S Chan, B Butler, and R G Smith
H2N,d-Arg,Pro,Lys,Pro,d-Phe,Gln,d-Trp,Phe,d-Trp,Leu, Leu,NH2 (L-756,867), a weak substance P antagonist, inhibited L-692,429-stimulated GH release from rat primary pituitary cells in a dose-dependent manner. At a concentration of 50 nm, L-756,867 shifted the dose–response curve of L-692,429-induced GH release to the right by about tenfold. It also impaired the ability of L-692,429 to potentiate the effect of growth hormone-releasing factor (GRF) on GH release. Substance P (1 μm) had no effect on basal or L-692,429-stimulated GH release. When tested in anesthetized rats, L-756,867 inhibited L-692,429- and growth hormone-releasing hexapeptide- (GHRP-6)-stimulated GH secretion in a dose-dependent manner. Complete inhibition was observed at an i.v. dose of 100 μg/kg of L-756,867. However, at the same concentration, it had no effect on GRF-induced GH secretion. d-Lys3-GHRP-6, a GHRP-6 antagonist, had no effect on GHRP-6 or L-692,429-induced GH secretion even at an i.v. dose of 2 mg/kg. These results indicate that L-692,429 and GHRP-6 stimulate GH release both in vitro and in vivo via a common receptor and signaling pathway which is different from that of substance P in spite of the fact that their effects are inhibited by a weak substance P antagonist.
Journal of Endocrinology (1997) 152, 155–158
Y Yang, J Cao, W Xiong, J Zhang, Q Zhou, H Wei, C Liang, J Deng, T Li, S Yang, and L Xu
It has been documented that stress or glucocorticoids have conflicting effects on memory under different conditions. However, it is not fully understood why stress can either impair or enhance memory. Here, we have examined the performance of six age groups of Wistar rats in a water maze spatial task to evaluate the effects of stress under different conditions. We found that the impairment or enhancement effect of an 'elevated platform' (EP) stress on memory was dependent on previous stress experience and on age. EP stress impaired memory retrieval in water maze naive animals, but enhanced rather than impaired memory retrieval in young water maze stress-experienced animals. Furthermore, exogenously applied corticosterone or foot shock stress before water maze training prevented the impairment of memory retrieval that should be induced by treatment with corticosterone or foot shock before the 'probe trial'. Again, memory retrieval was enhanced in young animals under these conditions, and this enhancement can be prevented by the glucocorticoid receptor antagonist RU 38486. Thus, glucocorticoid receptor activation not only induced impairment of memory but also increased the capacity of young animals to overcome a later stress. The present findings suggest that the effect of stress on memory can be switched from impairment to enhancement dependent on both stress experience and age.
S Wei, Y Feng, FY Che, H Pan, N Mzhavia, LA Devi, AA McKinzie, N Levin, WG Richards, and LD Fricker
ProSAAS is a neuroendocrine peptide precursor that potently inhibits prohormone convertase 1 in vitro. To explore the function of proSAAS and its derived peptides, transgenic mice were created which express proSAAS using the beta-actin promoter. The body weight of transgenic mice was normal until approximately 10-12 weeks, and then increased 30-50% over wild-type littermates. Adult transgenic mice had a fat mass approximately twice that of wild-type mice, and fasting blood glucose levels were slightly elevated. In the pituitary, the levels of several fully processed peptides in transgenic mice were not reduced compared with wild-type mice, indicating that the proSAAS transgene did not affect prohormone convertase 1 activity in this tissue. Because the inhibitory potency of proSAAS-derived peptides towards prohormone convertase 1 is much greater in the absence of carboxypeptidase E activity, the proSAAS transgene was also expressed in carboxypeptidase E-deficient Cpe (fat/fat) mice. Although the transgenic mice were born in the expected frequency, 21 of 22 proSAAS transgenic Cpe (fat/fat) mice died between 11 and 26 weeks of age, presumably due to greatly elevated blood glucose. The levels of several pituitary peptides were significantly reduced in the proSAAS transgenic Cpe (fat/fat) mice relative to non-transgenic Cpe (fat/fat) mice, suggesting that the transgene inhibited prohormone convertase 1 in these mice. Taken together, these results are consistent with a role for proSAAS-derived peptides as neuropeptides that influence body weight independently of their function as inhibitors of prohormone convertase 1.
Tusty-Jiuan Hsieh, Pierre Fustier, Chih-Chang Wei, Shao-Ling Zhang, Janos G Filep, Shiow-Shiu Tang, Julie R Ingelfinger, I George Fantus, Pavel Hamet, and John S D Chan
We reported previously that insulin inhibits the stimulatory effect of high glucose on the expression of angiotensinogen (ANG) gene in both rat immortalized renal proximal tubular cells (IRPTCs) and non-diabetic rat renal proximal tubular cells (RPTCs), but has no effect in diabetic rat RPTCs. In the present study we investigated whether hyperglycaemia-induced resistance to the insulin-induced inhibition of expression of the ANG gene is mediated via the generation of reactive oxygen species (ROS) in RPTCs. Rat IRPTCs were cultured for 2 weeks in high-glucose (25 mM) or normal-glucose (5 mM) medium plus angiotensin II (Ang II) with or without a superoxide scavenger (tiron), or inhibitors of: NADPH oxidase (diphenylene iodinium, DPI), Ang II type 1 and 2 receptors (losartan and PD123319), angiotensin-converting enzyme (perindopril), protein kinase C (GF 109203X), or glutamine:fructose-6-phosphate amino-transferase (azaserine). Cellular generation of ROS, and ANG and renin mRNA levels were assessed by lucigenin assay and specific reverse transcriptase-PCR respectively. Phosphorylation of p44/42 mitogen-activated protein kinase (p44/42 MAPK) was evaluated by western blotting. Prolonged exposure of IRPTCs to high concentrations of glucose or Ang II evoked generation of ROS and resistance to the insulin-induced inhibition of expression of the ANG gene and of p44/42 MAPK phosphorylation. Co-incubation of IRPTCs with tiron, DPI, losartan, PD123319, perindopril, GF 109203X or azaserine prevented ROS generation, restoring the inhibitory action of insulin on ANG gene expression and on p44/42 MAPK phosphorylation. In conclusion, our studies demonstrate that blockade of both ROS generation and activation of the intrarenal renin–angiotensin system improves the inhibitory action of insulin on ANG gene expression in IRPTCs in conditions of high glucose.