The aim of the present study was to analyze the functional importance for the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) gene P2 promoter activity of the putative proximal Myc-associated zinc finger protein (MAZ) site localized at position bp -45 to -39 bp, taking advantage of a G/A mutation identified at position -40 in the human sequence. Wild-type 'full-length' (1285P2) and truncated (760P2) promoter sequences were inserted upstream to the luciferase basic (pLucB) and enhancer (pLucE) reporter gene expression vectors. Transient transfections in osteoblast-like SaOS-2 cells and renal cells (RC.SV3A2) showed that the -40 G/A mutation significantly impaired transcriptional activity of wild-type 1285P2-pLucB and 760P2-pLucE promoter constructs. Further truncation of the P2 sequence demonstrated that the sequence -109/-37 bp was essential for promoter activity. Co-transfection with a MAZ expression vector did not modify the wild-type 1285P2-pLucB construct reporter activity but significantly increased 2-fold the mutated construction activity (P<0.05). Electrophoretic mobility shift assays using SaOS-2 nuclear extracts and a double-stranded DNA fragment encompassing the -45 to -39 putative MAZ site (ds-MAZ-oligo) disclosed two specific DNA-protein complexes. Complex II (fast moving) had a lower affinity for the mutated MAZ motif than for the wild-type MAZ motif while complex I (slow moving) had the same affinity for both wild-type or mutated MAZ sequences. Competition studies with Sp1 consensus oligonucleotide (ds-Sp1-oligo) markedly reduced complex I intensity, with a concomitant increase in that of complex II. Finally, ribonuclease protection assays showed that P2-specific PTHR1 mRNA transcript expression was significantly decreased in SaOS-2 cells transfected with ds-MAZ-oligo as compared with that for control (P<0.001) and ds-Sp1-oligo (P<0.05). Taken together, our studies suggest that the putative -45 to -39 MAZ-binding site regulates the constitutive activity of human PTHR1 P2 promoter.
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- Abstract: Diabetes x
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- Abstract: Insulinoma x
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C Leroy, D Manen, R Rizzoli, M Lombes and C Silve
Michael A Gentile, Pascale V Nantermet, Robert L Vogel, Robert Phillips, Daniel Holder, Paul Hodor, Chun Cheng, Hongyue Dai, Leonard P Freedman and William J Ray
Androgens promote anabolism in the musculoskeletal system while generally repressing adiposity, leading to lean body composition. Circulating androgens decline with age, contributing to frailty, osteoporosis, and obesity; however, the mechanisms by which androgens modulate body composition are largely unknown. Here, we demonstrate that aged castrated rats develop increased fat mass, reduced muscle mass and strength, and lower bone mass. Treatment with testosterone or 5α-dihydrotestosterone (DHT) reverses the effects on muscle and adipose tissues while only aromatizable testosterone increased bone mass. During the first week, DHT transiently increased soleus muscle nuclear density and induced expression of IGF1 and its splice variant mechano growth factor (MGF) without early regulation of the myogenic factors MyoD, myogenin, monocyte nuclear factor, or myostatin. A genome-wide microarray screen was also performed to identify potential pro-myogenic genes that respond to androgen receptor activation in vivo within 24 h. Of 24 000 genes examined, 70 candidate genes were identified whose functions suggest initiation of remodeling and regeneration, including the type II muscle genes for myosin heavy chain type II and parvalbumin and the chemokine monocyte chemoattractant protein-1. Interestingly, Axin and Axin2, negative regulators of β-catenin, were repressed, indicating modulation of the β-catenin pathway. DHT increased total levels of β-catenin protein, which accumulated in nuclei in vivo. Likewise, treatment of C2C12 myoblasts with both IGF1Ea and MGF C-terminal peptide increased nuclear β-catenin in vitro. Thus, we propose that androgenic anabolism involves early downregulation of Axin and induction of IGF1, leading to nuclear accumulation of β-catenin, a pro-myogenic, anti-adipogenic stem cell regulatory factor.
Jan Christiansen, Astrid M Kolte, Thomas v O Hansen and Finn C Nielsen
Recent genome-wide association (GWA) studies of type 2 diabetes (T2D) have implicated IGF2 mRNA-binding protein 2 (IMP2/IGF2BP2) as one of the several factors in the etiology of late onset diabetes. IMP2 belongs to a family of oncofetal mRNA-binding proteins implicated in RNA localization, stability, and translation that are essential for normal embryonic growth and development. This review provides a background to the IMP protein family with an emphasis on human IMP2, followed by a closer look at the GWA studies to evaluate the significance, if any, of the proposed correlation between IMP2 and T2D.
B J Whitehouse and D R E Abayasekara
The role played by cyclic AMP (cAMP)-dependent protein kinases (PKAs) in rat adrenal steroidogenesis has been investigated using cAMP analogues which show partial selectivity for the type I and type II PKA isoenzymes. These were aminohexylamino-cAMP (AHA-cAMP; selective for site 1 on type I PKA), N 6-benzoyl-cAMP (BZ-cAMP; selective for site 2 on PKA types I and II) and 8-thiomethyl-cAMP (TM-cAMP; selective for site 1 on type II PKA). Positive cooperativity exists between the two nucleotide-binding sites, thus the presence of type I PKA was inferred when synergistic increases in corticosteroid production were obtained with AHA-cAMP plus BZ-cAMP and that of type II PKA when synergistic increases were obtained with TM-cAMP plus BZ-cAMP.
The effects of AHA-cAMP, TM-cAMP and BZ-cAMP (10–100 μmol/l) on aldosterone production by glomerulosa cell preparations and corticosterone production by fasciculata/reticularis cell preparations were compared. Dose-related stimulation of steroid production was obtained with each cAMP analogue in both types of cell preparation. Experiments were performed using the cAMP analogues in combination at doses which gave minimal stimulation individually. Cells were incubated with AHA-cAMP (66 and 100 μmol/l) or TM-cAMP (15, 30 and 45 μmol/l) in the presence and absence of 15μmol BZ-cAMP/l. Synergistic responses were obtained with both analogue pairs in both cell types. The synergism ratio in fasciculata/reticularis cell preparations for the type I PKA selective pair of analogues (100 μmol AHA-cAMP/l plus 15μmol BZ-cAMP/l) was significantly higher (P<0·01) than that for the type II selective pair (45μmol TM-cAMP/l plus 15μmol BZ-cAMP/l; 7·9±1·2 (mean±s.e.m.) and 2·6±0·3 respectively). In zona glomerulosa preparations the ratio was higher (P<0·05) for the type II selective pair (1·6±0·1 for AHA-cAMP plus BZ-cAMP and 2·8±0·4 for TM-cAMP plus BZ-cAMP).
The effects of 100μmol AHA-cAMP/l and 45μmol TM-cAMP/l on the response to ACTH (1 pmol/l–10 nmol/l) were examined. Synergistic responses were obtained in fasciculata/reticularis cells with both analogues in combination with low concentrations of ACTH (10 and 100 pmol/l). In zona glomerulosa cells only the addition of TM-cAMP (45 μmol/l) in combination with 10 pmol ACTH/1 gave rise to synergistic increases in aldosterone production, which suggests that there may be some compartmentalization of the cAMP-dependent pathway in these cells.
The results indicate that both isoenzymes of PKA are present in rat adrenocortical cells and can play a part in the control of steroidogenesis. Type I PKA activity appears dominant in the control of zona fasciculata/reticularis cell function whereas modulation of type II PKA activity plays a more significant role in the responses of zona glomerulosa cells.
Verónica Sancho, María V Trigo, Nieves González, Isabel Valverde, Willy J Malaisse and María L Villanueva-Peñacarrillo
Several kinases have been implicated in the metabolic response of human and rat myocytes to glucagon-like peptide-1 (GLP-1), exendin-4 (Ex-4) and exendin-9 (Ex-9). We have investigated, in isolated rat adipocytes, the changes caused by GLP-1, Ex-4 and Ex-9 compared with those provoked by insulin or glucagon, upon the activity of phosphatidylinositol-3-kinase (PI3K), protein kinase B (PKB), p42/44 MAP kinases (MAPKs) and p70s6 kinase (p70s6k), and the participation of these kinases and protein kinase C (PKC) in their action upon 2-deoxy-d-glucose uptake, lipolysis and lipogenesis. The study was conducted in normal rats, and extended to a streptozotocin-induced type-2 diabetic model (STZ-rats). The participation of distinct kinases was estimated by using potential kinase inhibitors, including wortmannin, PD98059, rapamycin, H-7 and RO31–8220. In normal rat adipocytes, GLP-1 and both exendins share with insulin an increasing action upon the activity of all kinases studied (except PKB), PI3K, p44 and p42 MAPKs and possibly PKC, all being required for their stimulating effect upon glucose uptake. Ex-4 and Ex-9, like GLP-1 and insulin, have lipogenic action, while only Ex-4 shares with GLP-1 its lipolytic effect which is antagonized by Ex-9. MAP kinases and PKC seem to have an essential role in the GLP-1 and Ex-4 lipolytic action, as does PI3K in that of Ex-4. An increase in PI3K and MAPKs activity for the lipogenic effect of Ex-4, Ex-9 and GLP-1 are required, and in the case of Ex-4 and Ex-9, a stimulation of p70s6k activity is also needed. In cells from STZ-rats the magnitude of the above parameters was, in general, comparable to that in normal animals, with some exceptions: basal PI3K activity and lipogenesis were higher, GLP-1, Ex-4 and Ex-9 failed to modify basal lipogenesis but increased PKB activity, insulin failed to affect the activity of MAPKs and the insulin-induced glucose uptake was impaired. The impaired insulin effects upon some of the variables in the STZ-rat, distinct from those of GLP-1 and exendins, adds knowledge to the mechanism of the beneficial action of GLP-1 and Ex-4 in diabetic states.
MS Wagner, R Morimoto, JM Dora, A Benneman, R Pavan and AL Maia
In the present study we show the expression profiles of both type 1 and type 2 iodothyronine deiodinases (D1 and D2) in a wide spectrum of mouse tIssues, and D2 regulation by thyroid status. A characteristic tIssue-specific expression for each isoform was observed. D2 transcripts were detected in most tIssues with variable levels of expression. The observed D2 mRNA tIssue distribution was similar to that described in rats and is in agreement with the view of different patterns of expression between rodents and humans. However, it is interesting to note that despite the low levels of D2 transcripts in mouse heart and testis in the euthyroid state, the induction of hypothyroidism caused a significant increase in D2 activity in these tIssues. Similar results were also obtained in adult rats. These results suggest a previously unrecognized role for type 2 deiodinase in controlling intracellular triiodothyronine levels in rodent heart and testis during states of thyroid hormone deficiency.
Bjarne Faurholm, Shaun Cochrane, Robert R Millar and Arieh A Katz
The marmoset type II GnRH receptor (GnRH-R) gene has the same structure and genomic organisation as the human and other type II GnRH-R genes. The gene consists of three exons and two introns and overlaps in the antisense orientation on its 5′ end with peroxisomal membrane protein 11β and on its 3′ end with the RNA-binding motif protein 8A. However, these genes occur only at one locus in the marmoset genome, while in the human at two loci. Employing 5′ rapid amplification of cDNA ends demonstrated that the marmoset type II GnRH-R gene has two transcriptional start sites at −341 and −567 nucleotides relative to the translational start codon and both start sites lack TATA and CAAT consensus sequences. A luciferase reporter construct with a 2.3 kb 5′ flanking region of the type II GnRH-R gene was active in a wide variety of cell lines tested, consistent with the wide tissue expression of the receptor. Progressive 5′ and 3′ deletions were employed to identify sequences required for basal expression of the type II GnRH-R gene. This analysis identified negative regulatory elements in the regions −2342/−1995, −1679/−1084 and −458/−1 and positive regulatory elements in the regions −1995/−1679, −1084/−458 and −458/−1 relative to the translational start site. The strongest of the positive regions located between −766/−665 has enhancer activity when cloned in front of a heterologous minimal promoter and is critical for basal expression of the type II GnRH-R.
Rene Cortese, Florian Eckhardt, Marianne Volleth, Manfred Wehnert, Uwe Koelsch, Peter Wieacker and Thomas Brune
Mutations in the LMNA gene cause various phenotypes including partial lipodystrophy, muscular dystrophies, and progeroid syndromes. The specific mutation position within the LMNA sequence can partially predict the phenotype, but the underlying mechanisms for the development of these different phenotypes are still unclear. To investigate whether different DNA methylation patterns contribute to the development of different phenotypes caused by LMNA mutations, we analyzed a panel of ten candidate genes related to fat metabolism, aging, and a tendency to different methylation patterns: CSPG2, ESR1, IGF1R, IGFR2, LMNA, MLH1, RANBP1, RARB, ZMPSTE24, and TGFBR1. We studied two independent families each comprising three individuals affected by familial partial lipodistrophy type 2 (FPLD2). Affected members in each family carried two different mutations of the LMNA gene (R482L and R471G respectively). In addition, we analyzed four progeria patients (2×LMNA/C G608G, 1×LMNA/C S143F, and 1×ZMPSTE24 IVS9-Ex10) and seven healthy adults. The gene encoding retinoic acid receptor B (RARB) showed a higher methylation in all six patients with FPLD2 when compared with the progeria patients with other LMNA mutations as well as the healthy controls (P<0.05). All other investigated genes showed no difference in the methylation patterns between the groups. A drug-induced inhibition of the retinol pathway is discussed as the key pathway for developing HAART-associated lipodystrophy and our data support a possible role of the retinol pathway in the development of lipodystrophy phenotypes.
K Gen, O Maruyama, T Kato, K Tomizawa, K Wakabayashi and Y Kato
Two types of cDNA (GTHα1 and -α2) encoding the α subunits of masu salmon (Oncorhynchus masou) gonadotrophin were cloned by the reverse transcription-polymerase chain reaction for pituitary mRNAs. The nucleotide sequences showed that the GTHα1 cDNA was 380 bp long, encoding 119 amino acids, and that GTHα2 cDNA was 365 bp long, encoding 114 amino acids. The masu salmon α subunit types had a few differences between the sequences, with homologies of 80% (nucleotide sequence) and 72% (amino acid sequence). The structural difference between the α1 and α2 subunits was predicted using hydropathic analysis. The evolutionary interval between masu and chum salmon was estimated to be 4·0 and 2·3 million years by comparing their GTHα1 and -α2 subunits respectively. These time values are roughly consistent with the evolutionary time interval (3·0 million years) estimated from fossil records and an isozyme study. Specific synthetic oligonucleotide probes were constructed and used for genomic Southern blot analyses. The restriction fragment sizes of the GTHα1 and -α2 genes were similar, and when their patterns were compared with those from four other teleosts, each species showed a different pattern from the others, but no difference between their respective α1 and α2 genes. Therefore, the structural features of the GTHα1 and -α2 genes may have diverged in a similar manner in these five teleosts.
SP Ip, TP Wong, SJ Tsai and PS Leung
Previous studies have shown that the expression of the major components from a local pancreatic renin-angiotensin system (RAS) was upregulated after chronic exposure to oxygen deprivation (10% oxygen). In the present study, the reversibility of expression for the pancreatic RAS affected by chronic hypoxia was investigated in the pancreas. Rats were first subject to hypoxia for one Month and they were then returned to normoxic conditions for a varying period of time (1, 2, 3 and 4 weeks). The degree of recovery in the expression of RAS components was analyzed with standard curve-quantitative competitive-reverse transcription-polymerase chain reaction (SC-QC-RT-PCR), Western blot analysis and a specific assay for angiotensin-converting enzyme (ACE) activity. Results from SC-QC-RT-PCR showed that the upregulated expression of angiotensin II type 1 (AT(1)) receptor mRNA following chronic hypoxia could be completely restored to the control level after the rats were returned to the normoxic condition for 3 weeks. The reversibility of mRNA expression for angiotensin II type 2 (AT(2)) receptor and angiotensinogen was observed after the return to normoxic conditions for 2 and 3 weeks respectively when compared with that of their respective controls. Results from Western blot analysis further confirmed that the expression of AT(1) receptor protein was also reversible after return to normoxic conditions for 4 weeks. In addition, the activation of ACE activity returned to its normal level in a time-dependent manner. These data indicate that the upregulation of a local pancreatic RAS affected by chronic hypoxia could be recoverable. The significance of its reversibility and adaptability following chronic hypoxia may be of physiological relevance to the pancreas.