Cell culture The mouse MA-10 Leydig cell line ( Ascoli 1981 ) was provided by Dr Mario Ascoli (University of Iowa, Iowa City, Iowa) and maintained in DMEM-F12 supplemented with penicillin and streptomycin, 15% horse serum, and incubated at 37°C in 5
Houssein S Abdou, Nicholas M Robert and Jacques J Tremblay
M. G. Castro, J. Brooke, A. Bullman, M. Hannah, B. P. Glynn and P. J. Lowry
The mouse corticotrophic tumour cell line AtT-20 naturally synthesizes pro-opiomelanocortin (POMC) which is proteolytically processed to N-POMC(1–76), ACTH, β-lipotrophin and β-endorphin. The processed products are stored in secretory vesicles and released upon stimulation with specific secretagogues. ArT-20 cells which have been stably transfected with the human corticotrophin-releasing hormone (CRH) gene store and secrete immunoreactive CRH. The present results demonstrate that the CRH precursor is proteolytically processed in the transfected cells to yield the 41 amino acid neuropeptide CRH(1–41). On stimulation with the secretagogue noradrenaline, CRH(1–41) was released into the medium, while the precursor was not. Whilst treatment of wild-type ArT-20 cells with exogenous CRH(1–41) (1 nm) caused a fourfold stimulation of ACTH release above basal levels, the peptide had no effect on ACTH release from the stably transfected cells R1 and R4. These results suggest that the endogenous CRH produced by the transfected R1 and R4 cells may cause down-regulation of their CRH receptors, and thus exogenous CRH cannot cause further stimulation of ACTH release in these cells. We propose that the CRH precursor is correctly processed in the transfected AtT-20 cells (R1 and R4) and that the foreign prohormone is sorted into the secretory pathway.
W Becker, R Kluge, T Kantner, K Linnartz, M Korn, G Tschank, L Plum, K Giesen and HG Joost
New Zealand obese (NZO) mice exhibit severe insulin resistance of hepatic glucose metabolism. In order to define its biochemical basis, we studied the differential expression of genes involved in hepatic glucose and lipid metabolism by microarray analysis. NZOxF1 (SJLxNZO) backcross mice were generated in order to obtain populations with heterogeneous metabolism but comparable genetic background. In these backcross mice, groups of controls (normoglycemic/normoinsulinemic), insulin-resistant (normoglycemic/hyperinsulinemic) and diabetic (hyperglycemic/hypoinsulinemic) mice were identified. At 22 weeks, mRNA was isolated from liver, converted to cDNA, and used for screening of two types of cDNA arrays (high-density filter arrays and Affymetrix oligonucleotide microarrays). Differential gene expression was ascertained and assessed by Northern blotting. The data indicate that hyperinsulinemia in the NZO mouse is associated with: (i) increased mRNA levels of enzymes involved in lipid synthesis (fatty acid synthase, malic enzyme, stearoyl-CoA desaturase) or fatty acid oxidation (cytochrome P450 4A14, ketoacyl-CoA thiolase, acyl-CoA oxidase), (ii) induction of the key glycolytic enzyme pyruvate kinase, and (iii) increased mRNA levels of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase. These effects were enhanced by a high-fat diet. In conclusion, the pattern of gene expression in insulin-resistant NZO mice appears to reflect a dissociation of the effects of insulin on genes involved in glucose and lipid metabolism. The data are consistent with a hypothetical scenario in which an insulin-resistant hepatic glucose production produces hyperinsulinemia, and an enhanced insulin- and substrate-driven lipogenesis further aggravates the deleterious insulin resistance of glucose metabolism.
F. M. Ng, N. A. Adamafio and J. E. Graystone
The effects of two preparations of highly purified human GH (hGH) on lipid metabolism were studied in the GH-deficient little mouse (50–60 days old). Marked decreases in incorporation of [14C]glucose into fatty acid and in the activity of acetyl-CoA carboxylase in the epididymal fat pads were observed after i.p. injection of hGH at a dose of 1·0μg/g body weight or after continuous infusion of hGH by osmotic minipump. The rate of glucose incorporation into fatty acid decreased from 107·0 ± 27·6 (s.e.m.) to 38·1 ± 19·6 μmol/g tissue per h after a single injection of hGH and from 174·1±28·5 to 56·3±20·3 μmol/g tissue per h after continuous infusion of hGH for 2 days. Activity of the lipogenic enzyme acetyl-CoA carboxylase was also reduced by more than 50% in the epididymal fat pad from hGH-treated mice in comparison with the corresponding control animals. Incubation of isolated fat pads with hGH (0·1 μg/ml) revealed similar inhibitory effects of the hormone on fatty acid synthesis and acetyl-CoA carboxylase activity. No lipolytic effect of hGH was found as determined by the rate of glycerol release from epididymal fat pads of little mice following hormone treatment in vivo or in vitro. The results lend strong support to the conclusion that GH inhibits lipogenesis but has no effect on lipolysis in adipose tissues, and indicate that the physiological role of GH in lipid metabolism is concerned mainly with the regulation of anabolic rather than catabolic processes.
F Varga, S Spitzer, M Rumpler and K Klaushofer
Thyroid hormones are important regulators of bone development and metabolism. We have demonstrated that tri-iodothyronine (T3) increased and 1,25-dihydroxyvitamin D3 (1,25D3) attenuated the T3-stimulated expression of osteocalcin (OCN) in the osteoblast-like cell line MC3T3-E1. By means of transfection of promoter-reporter gene constructs we investigated the basal and the regulated transcription of this gene by both hormones. We found that a 0.67 kbp and a 1.3 kbp fragment of the mouse OCN OG2 promoter containing two Runx2 binding sites were significantly more active than a smaller fragment containing only one Runx2 binding site. The longer promoter fragments showed a higher reporter gene expression when the transfected cells were treated with 10(-7) M T3. This expression was attenuated by 1,25D3 dose-dependently. These fragments contain a sequence homologue to the recently identified binding site for the 1,25D3 receptor (VDR) in the rat OCN promoter. Deletion of a part of the promoter containing this VDR response element-like sequence (VDRE) resulted in a higher basal expression but abrogated the regulation by T3 and 1,25D3. Electrophoretic mobility shift assays revealed that the deleted sequence was able to bind both in vitro-translated chicken thyroid hormone receptor (TR) and proteins from nuclear extracts that reacted with an antiserum against TR. From these data we conclude that the VDRE-like sequence of the OG2 promoter contains a thyroid hormone response element.
I Chakraborty, S K Das, J Wang and S K Dey
Cyclo-oxygenase (COX) is a rate-limiting enzyme that converts arachidonic acid to prostaglandins (PGs) and exists in two isoforms, COX-1 and COX-2. In the rodent, increased uterine vascular permeability at sites of blastocyst apposition is one of the earliest prerequisite events in the implantation process. This event is preceded by generalized uterine edema and luminal closure, and coincides with the initial attachment reaction between the trophectoderm and luminal epithelium. Vasoactive PGs are implicated in these processes. Here we demonstrate that COX genes are differentially regulated in the peri-implantation mouse uterus. During the preimplantation period (days 1–4), the COX-1 gene was expressed in the uterine epithelium mainly on day 4 until the initiation of attachment reaction in the evening after which the expression was downregulated. This COX-1 expression coincides with the generalized uterine edema required for luminal closure. In contrast, the COX-2 gene was expressed in the luminal epithelium and subepithelial stromal cells at the anti-mesometrial pole exclusively surrounding the blastocyst at the time of attachment reaction on day 4 and persisted through the morning of day 5. This uterine gene was not expressed at the sites of blastocyst apposition during progesterone (P4) treated delayed implantation, but was readily induced in the uterus surrounding the activated blastocysts after termination of the delay by estradiol-17β (E2). The results suggest that PG synthesis catalyzed by COX-2 is important for localized increased uterine vascular permeability and attachment reaction. The COX-1 gene that was downregulated from the time of attachment reaction on day 4 was again expressed in the mesometrial and anti-mesometrial secondary decidual beds on days 7 and 8. These results suggest that PGs generated by COX-1 are involved in decidualization and/or continued localized endometrial vascular permeability observed during this period. In contrast, the COX-2 gene, expressed at the anti-mesometrial pole on days 4 and 5, switched its expression to the mesometrial pole from day 6 onward. These results suggest that PGs produced at this site by COX-2 are involved in angiogenesis for the establishment of placenta. In the ovariectomized mice, the COX-1 gene was induced in the epithelium by a combined treatment with P4 and E2. However, P4 and/or E2 treatments failed to influence the uterine COX-2 gene. Overall, the results suggest that the uterine COX-1 gene is influenced by ovarian steroids, while the COX-2 gene is regulated by the implanting blastocyst during early pregnancy.
K Phillips, MA Park, LH Quarrie, M Boutinaud, JD Lochrie, DJ Flint, GJ Allan and J Beattie
The mouse mammary epithelial cell line HC11 upregulates the synthesis of beta-casein (a differentiation marker) following treatment with the lactogenic hormone mix dexamethasone, insulin and prolactin (DIP). We demonstrate that the basal levels of IGF-binding protein (IGFBP)-5 secreted by undifferentiated HC11 cells are upregulated 10-fold during DIP-induced cellular differentiation whereas the level of the other IGFBP species secreted by HC11 cells (IGFBP-2) is downregulated during this process. As previously reported, the combination of all three of these hormones is required for synthesis of the differentiation marker beta-casein, whereas basal IGFBP-5 secretion is evident in the absence of any hormonal treatment and, unlike beta-casein, secretion of this protein can be stimulated by binary combinations of the hormones (although maximal levels of IGFBP-5 are achieved in the presence of all three lactogenic hormones). Additionally, levels of IGFBP-5 can be increased by DIP treatment under conditions (non-competency of HC11 cultures or presence of epidermal growth factor) where DIP treatment does not increase synthesis of beta-casein. For IGFBP-2, dexamethasone is a potent inhibitor of secretion whilst prolactin stimulated the secretion of this binding protein into the medium. For the IGFBP axis in HC11 cells we conclude that, although the levels of IGFBP-5 and -2 are influenced by the state of cellular differentiation, the hormonal regulation of the levels of these IGFBP species can be dissociated from the regulation of beta-casein synthesis. In a further series of experiments we demonstrate that IGF-I is able to replace insulin in the DIP lactogenic hormone mix and by the use of a specific IGF-I receptor blocking antibody indicate that the action of IGF-I is mediated through the cell surface IGF-I receptor and not by cross-reaction of IGF-I ligand at the insulin receptor. We discuss our data in the context of the potential role of the IGF axis in the process of cell differentiation and illustrate the significance of our findings in the context of the physiology and life cycle of the mammary epithelial cell.
Y Y Liu, W Jia, I E Wanke, D A Muruve, H P Xiao and N C W Wong
′; antisense: 5′-GCGGCGGCCGCCTAGTTGCAGTAGTTCTCCAG-3′. Primers for GAPDH were used as previously reported ( Zerbini et al . 2003 ). Immunoblotting HepG2 cells or mouse liver lysates were extracted in buffer (50 mmol/l Tris–HCl, pH 7.4, 150 mmol/l NaCl, 1% NP-40
E Takahashi, N Miyamoto and T Nagasu
The omega-agatoxin-IVA-sensitive P/Q-type Ca(2+) channel plays a role in insulin release from the pancreatic islets of beta cells. To dissect the molecular mechanisms underlying beta cell expression of the P/Q-type channel, we characterized the 5'-upstream region of the mouse alpha(1A) subunit gene using transgenic mice and HIT insulinoma cells. The E. coli lacZ reporter gene was expressed in pancreatic acini and islets in transgenic mice carrying the 6.3 kb or 3.0 kb of the 5'-upstream region, although those with 1.5 kb or 0. 5 kb of the 5'-upstream region failed to show reporter expression on histological examination. As the expression of alpha(1A)subunit gene could not be detected in acini using RT-PCR analysis, the reporter expression in acini might have been ectopic expression. When linked to the placental alkaline phosphatase reporter gene to examine promoter activity for beta cell expression, the 6.3 kb and 3.0 kb fragment of the 5'-upstream region, but not the smaller 1.5 kb fragment, were able to drive reporter gene expression in HIT cells. The sequence between 3.0 and 1.5 kb upstream of the start codon enhanced thymidine kinase promoter activity in HIT cells, but not in fibroblast NIH3T3 cells. These results suggested that the beta cell-specific elements of the alpha(1A) subunit gene are likely to be located in the distal upstream region (-3021 to-1563) of the 5'-upstream sequence and that the 6.3 kb fragment of the 5'-upstream region alone might be a lack of a negative cis-regulatory element(s) to suppress the alpha(1A) subunit gene expression in acini.
D J Flint, M Boutinaud, C B A Whitelaw, G J Allan and A F Kolb
-like growth factor I (IGF-I) interaction with its receptor on the epithelial cells, resulting in cell death. More recently we have shown that IGFBP-5 mRNA levels are also significantly increased during involution in the mouse mammary gland ( Boutinaud et al