Qiao-ng Li

Regulation of pancreatic PC1 and PC2 associated with increased glucagon-like peptide 1 in diabetic rats

The pancreatic processing enzymes, PC1 and PC2, convert proinsulin to insulin and convert proglucagon to glucagon and glucagon-like peptide 1 (GLP-1). We examined the effect of streptozotocin (STZ) treatment on the regulation of these enzymes and the production of insulin, glucagon, and GLP-1 in the rat. Pancreatic PC1 and PC2 mRNA increased >2-fold and >4-fold, respectively, in rats receiving intraperitoneal STZ (50 mg/kg) daily for 5 days. Immunocytochemistry revealed that, although pancreatic islet cells in the STZ-treated rats were sparse and atrophic PC1, PC2, glucagon, and GLP-1 immunoreactivity increased dramatically in the remaining islet cells. Heightened PC1 and PC2 expression was seen in cells expressing glucagon but not in insulin-expressing cells. Furthermore, in STZ-treated rats, bioactive GLP-1(7-36 amide) accumulated in pancreatic extracts and serum 3- and 2.5-fold, respectively, over control animals. This treatment also caused a 2-fold increase in the ratio of amidated forms of GLP-1 immunoreactivity to total glucagon immunoreactivity in the pancreas but did not affect the ratio of proinsulin to insulin. We conclude that hyperglycemic rats have an increased expression of prohormone converting enzymes in islet alpha cells, leading to an increase in amidated GLP-1, which can then exert an insulinotropic effect on the remaining beta cells.

Regulation of prohormone convertase 1 (PC1) by gp130-related cytokines

The processing of pro-opiomelanocortin (POMC) to generate bioactive ACTH in the anterior pituitary is mediated by prohormone convertase 1 (PC1). Leukemia inhibitory factor (LIF) and interleukin 6 (IL-6), two cytokines sharing the common gp130 receptor subunit and functioning through activation of the intracellular JAK/STAT pathway, induce POMC synthesis and ACTH release. We investigated the effects of LIF and IL-6 on PC1 expression and its subsequent processing of POMC. A significant time-dependent up-regulation of both PC1 protein and mRNA by LIF and IL-6 was seen in mouse corticotroph AtT-20 cells. IL-6 or LIF increased the synthesis of ACTH-related products with a concomitant increase in bioactive 5 and 13 kDa ACTH indicating coordinated regulation of substrate and processing enzyme. AtT-20 cells transiently transfected with a human PC1-promoter-luciferase reporter construct and treated with LIF or IL-6 showed significantly increased luciferase activity. Additionally, lipopolysaccharide (LPS) administration to rats resulted in an increase in both pituitary PC1 and POMC mRNA. These findings suggest that the ACTH increase induced by LIF and IL-6 is due to both increased POMC synthesis as well as increased POMC processing by up-regulation of PC1. These two coordinately regulated processing events probably exert central roles in the pathophysiological response to some stresses, such as inflammatory stress.

Leukemia inhibitory factor (LIF) modulates pro-opiomelanocortin (POMC) gene regulation in stably transfected AtT-20 cells overexpressing LIF

Leukemia inhibitory factor (LIF) levels are elevated in sepsis and correlate with shock and poor prognosis. We have previously shown that lipopolysaccharide (LPS) administration induces hypothalamic and pituitary LIF expression in vivo, which is associated with the acute rise in circulating adrenocorticotrophic hormone (ACTH) levels. As AtT-20 cells respond to LIF, we established murine LIF (mLIF) stably transfected AtT-20 cell lines to study LIF regulation of pro-opiomelanocortin (POMC) expression and ACTH secretion. Our results show that mLIF transfectants accumulated mLIF (up to 15.6±3.2 ng/mL after 24h) as well as increased ACTH secretion (up to 2.4-fold above control cells) in conditioned medium. The magnitude of ACTH induction correlated with mLIF concentrations in different transfectants (r=0.75–0.88,p<0.05). Moreover, mLIF transfectants showed a higher sensitivity to CRH stimulation with an increased ACTH production within 8 h (p<0.05), whereas control cells were responsive to CRH at 24h. Additionally, mLIF transfectants exhibited a maximum threefold ACTH induction, compared to 1.7-fold in control cells. Furthermore, mLIF transfectants have a blunted dexamethasone-mediated inhibition of ACTH (35% inhibition in control cells vs no inhibition in mLIF-transfected cells at 24h). These findings support and extend the previous observations of LIF acting at the pituitary level, and indicate that mLIF stably-transfected AtT-20 cells are a useful model for studying mLIF-mediated gene regulation in pituicytes.

Up-regulation of splenic prohormone convertases PC1 and PC2 in diabetic rats

Organisms respond to infection in a complex manner involving bidirectional interactions between the neuroendocrine and immune systems. Many of the bioactive endocrine/immune factors are synthesized in a precursor form and are expected to be activated by prohormone convertases (PCs). Since patients with both type 1 and type 2 diabetes have an increased incidence and severity of infections, we hypothesized that in a condition of hyperglycemia, these processing enzymes would be activated in an immune tissue, the spleen. To test this hypothesis, we treated rats with intraperitoneal streptozotocin (STZ) (50 mg/kg/day) daily for 5 days and measured splenic PC1 and PC2 mRNA by ribonuclease protection assay. We found that PC1 mRNA was increased 6.0+/-0.02-fold (P<0.05) and PC2 mRNA was increased 1.80+/-0.01-fold (P<0.005) in the spleen of rats that received STZ compared to rats that received vehicle. Western blot indicated that the 75-kDa form of PC1 was the only form of PC1 present in the spleen and that this form increased with STZ treatment. Immunohistochemistry revealed that PC1 was found in both the white pulp (T-lymphocytes) and red pulp (monocytes and macrophages) and that its increase in immunoreactivity occurred primarily in the white pulp. PC2 and pro-opiomelanocortin (POMC, a possible splenic substrate for PC1/PC2) immunoreactivity was found predominantly in the red pulp. STZ induced an increase in splenic PC1 and POMC, but not PC2 protein levels. We conclude that in the STZ model of diabetes, splenic PCs are induced, which could lead to an increased activation of many immune-derived hormones. We speculate that this up-regulation of prohormone converting enzymes may be related to the increased infections seen in patients with both type 1 and type 2 diabetes.

Prohormone convertase 2 enzymatic activity and its regulation in neuro-endocrine cells and tissues

We used the fluorometric substrate, pGlu-Arg-Thr-Lys-Arg-MCA and the C-terminal peptide of human 7B2(155-185), a specific inhibitor of prohormone convertase 2 (PC2), to specifically measure the enzymatic activity of the prohormone convertases, PC2. Using lysates from the pancreatic alpha cell line, alphaTC1-6 cells, which contain moderate levels of PC2 enzymatic activity, we determined that the PC2 assay was linear with respect to time of incubation and protein added and had a pH optimum of 5.5 and a calcium optimum of 2.5 mM. Rat pituitary contained high levels of PC2 enzymatic activity, while the hypothalamus and other brain regions contained moderate levels. This enzyme assay was used to document that both mice null for PC2 as well as mice null for the PC2 cofactor, 7B2, had only trace levels of PC2 activity in various brain regions, while mice heterozygous for these alleles had approximately half of the PC2 activity in most brain regions. PC2 enzymatic activity and PC2 mRNA levels were somewhat discordant suggesting that PC2 mRNA levels do not always reflect PC2 enzymatic activity.

Localization of Thyrotropin-Releasing Hormone mRNA Expression in the Rat Heart by in situ Hybridization Histochemistry

Our laboratory has recently detected mRNA of thyrotropin-releasing hormone (TRH) in the rat heart. The density of mRNA for TRH is five-fold higher in the atria than in the left and right ventricle. We also found TRH receptor mRNA and 3H-TRH-binding sites in both ventricles. Cardiac contractility was stimulated after intracoronary administration of TRH. This study was performed to investigate the localization of TRH in the heart. We utilized in situ hybridization histochemistry (ISHH) to localize TRH mRNA expression in the rat heart. ISHH was performed on fresh frozen heart tissue sections which were hybridized with a specific 35S-TRH oligo probe and subsequently processed by autoradiography. The autoradiographic signals corresponding to TRH mRNA were analyzed with an image program. For positive controls TRH mRNA was identified in the hypothalamic paraventricular nucleus. This test confirms the specificity of the TRH oligo probe. Cardiac hybridization signals were observed predominantly in the atria and localized preferentially in atrial connective tissues, vascular adventitia and atrial cardiomyocytes. No hybridization signals were found in ventricular cardiomyocytes. These observations suggest that TRH is synthesized in atrial myocytes and atrial vascular structures. Based on studies which show synthesis of the TRH receptors in ventricular cardiomyocytes, we hypothesize that atrial TRH is an endocrine source for the stimulation of ventricular contractility and that endothelial and adventitial TRH may play a role(s) in the regulation of the growth and/or vasomotor tome of the cardiac vascular system.