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.

TRH is negatively regulated by T3 both in the hypothalamic paraventricular nucleus and transient transfection models. Mutations in hTR beta 1 genes are associated with the syndrome of generalized resistance to thyroid hormone. To investigate potential effects of mutant TRs on T3 regulation of the hTRH gene, transient gene expression assays were performed in human neuroblastoma (HTB-11) cells with an hTRH promoter-luciferase construct, wild type (WT) hTR beta 1, and three qualitatively distinct hTR beta 1 mutant forms (ED, OK and PV). In the presence of T3 (10(-9) M), liganded WT-hTR beta 1 inhibited hTRH promoter activity significantly (40%). Cotransfection of each of the two mutants (ED and OK) achieved similar levels of inhibition only at 10 to 100 fold increased T3 concentrations. Of interest, a 10x excess of mutant ED or OK could also exert dominant negative effects upon WT hTR beta 1-T3 mediated inhibitory actions on the hTRH promoter. In contrast, mutant TR-PV exerted neither inhibitory nor dominant negative effects at even higher concentrations of T3. Moreover, all three unliganded mutant forms stimulated TRH promoter activity significantly in the absence of T3, despite their different mutations in the ligand-binding domain (LBD). These data demonstrate that thyroid hormone resistance at the level of TRH gene regulation, due to reduced inhibitory actions of mutant TR-T3 complexes, as well as dominant negative effects upon WT hTR beta 1 mediated inhibition, likely contribute to elevated TSH values observed in the syndrome of thyroid hormone resistance.

Thyrotropin-releasing hormone (TRH) is the key regulator of the synthesis and secretion of TSH in animals and humans (Wilber and Yamada 1990). The biological implications of this peptide, the first releasing hormone to be characterized, has generated a large literature regarding both pituitary TSH and extrapituitary roles of TRH as a neurotransmitter and/or neuro-modulator in the central nervous system (O'Leary and O'Connor 1995, Morley 1981). In this review, new areas of TRH biology are explored, focused on the differential regulation of the TRH gene by triiodothyronine (T3) and other substances in the hypothalamus and two unexpected extrahypothalamic loci, the heart and testis. These new directions should enlarge our understanding concerning how hormones like T3 regulate genes negatively and selectively with the identical receptors and DNA elements required for positive gene stimulation. In addition, regulatory studies of the TRH gene by T3 should be relevant to other hormone receptor interactions with DNA sequences in general, as glucocorticoids, mineral-ocorticoids, sex steroids, vitamin D, and retinoic acid are ligands for homologous receptor proteins in the nuclear receptor superfamily.

The thyrotropin-releasing hormone (TRH) gene is regulated negatively at the transcriptional level by thyroid hormone (T3). T3 positive regulatory effects on other target genes, such as the growth hormone gene, are mediated through heterodimerization of thyroid hormone receptors (TRs) with RXR or other auxiliary nuclear protein(s). To explore whether an accessory co-suppressor protein(s) may be involved in T3 inhibitory regulation of human TRH gene transcription, transient gene expression studies have been carried out using a hTRH-luciferase (TRH-Luc) chimetric reporter construct, an hTR beta 1 expression construct, and pABgal-hTR beta 1 ligand-binding domain (LBD) fusion constructs, cotransfected into a human neuroblastoma cell line (HTB-11,ATCC). Results herein indicate that T3-dependent inhibitory regulation (48-60% of control) of the hTRH gene promoter by hTR beta 1-T3 complexes could be abrogated completely by cotransfection of a 10 x excess of hTR beta 1-LBD (TR 168-456 aa) in a pABgal94 vector. In striking contrast, cotransfection of a 10 x excess of highly truncated hTR beta 1-LBD (TR 452-456 aa) failed to reverse T3-mediated TRH promoter inhibition. This squelching effect by excessive intact TR-LBD, moreover, could not be reversed by raising T3 concentration 100-fold (from 10(-8) to 10(-6) M), thus excluding a squelching effect of T3 itself by excess LBD. These results suggest that negative regulation of the hTRH gene promoter activity by TR beta 1-T3 complexes involves interactions with an accessory co-suppressor protein, which may bridge DNA-bound TR beta 1-T3 complexes to the transcriptional initiation complex.

Thyrotropin releasing hormone (TRH) gene is regulated negatively at the transcriptional level by thyroid hormone (T3) in rat anterior hypothalamus. The actions of T3 upon other target genes are known to be mediated through the thyroid hormone receptors (TR), TRα and TRβ. To explore whether the inhibitory regulation of human (h) TRH gene transcription by T3 is TR isoform specific and whether TRH gene transcription can be modulated as well by unliganded TR isoforms, transient gene expression studies have been carried out using hTRH-luciferase (TRH-Luc) chimeric constructs and TR expression constructs, co-transfected into a human neuroblastoma cell line (HTB-11). Data herein demonstrate T3-dependent inhibitory regulation of the hTRH gene promoter by TR-T3 complexes. Moreover, significant inhibition (39%-60%) could be achieved by T3 bound to either hTRα1, hTRβ1, or rTRβ1, β2 and was comparable quantitatively, indicating an absence of TR isoform specificity for T3 inhibition. Conversely, basal promoter activity of the hTRH gene could be activated significantly by unliganded hTRα1, β1, rTRβ1, and β2 (150% to 334%), but not by hTRα2. Thus, TRs appear to exert opposite effects on hTRH gene transcription, depending on the presence or absence of ligand T3. These dual effects of TR suggest that the addition of the T3 ligand effects conformational changes that can abrogate the initiation of transcription.