Dipeptidyl peptidase-4 inhibitors administered in combination with metformin result in an additive increase in the plasma concentration of active GLP-1.

The aim of the study was to investigate the effects of a dipeptidyl peptidase-4 (DPP-4) inhibitor, of metformin, and of the combination of the two agents, on incretin hormone concentrations. Active and inactive (or total) incretin plasma concentrations, plasma DPP-4 activity, and preproglucagon (GCG) gene expression were determined after administration of each agent alone or in combination to mice with diet-induced obesity (DIO) and to healthy human subjects. In mice, metformin increased Gcg expression in the large intestine and elevated the plasma concentrations of inactive glucagon-like peptide 1 (GLP-1) (9-36) and glucagon. In healthy subjects, a DPP-4 inhibitor elevated both active GLP-1 and glucose dependent insulinotropic polypeptide (GIP), metformin increased total GLP-1 (but not GIP), and the combination resulted in additive increases in active GLP-1 plasma concentrations. Metformin did not inhibit plasma DPP-4 activity either in vitro or in vivo. The study results show that metformin is not a DPP-4 inhibitor but rather enhances precursor GCG expression in the large intestine, resulting in increased total GLP-1 concentrations. DPP-4 inhibitors and metformin have complementary mechanisms of action and additive effects with respect to increasing the concentrations of active GLP-1 in plasma.

Peroxisome proliferator-activated receptor-gamma ligands inhibit adipocyte 11beta -hydroxysteroid dehydrogenase type 1 expression and activity.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) has been shown to play an important role in the regulation of expression of a subclass of adipocyte genes and to serve as the molecular target of the thiazolidinedione (TZD) and certain non-TZD antidiabetic agents. Hypercorticosteroidism leads to insulin resistance, a variety of metabolic dysfunctions typically seen in diabetes, and hypertrophy of visceral adipose tissue. In adipocytes, the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) converts inactive cortisone into the active glucocorticoid cortisol and thereby plays an important role in regulating the actions of corticosteroids in adipose tissue. Here, we show that both TZD and non-TZD PPARgamma agonists markedly reduced 11beta-HSD-1 gene expression in 3T3-L1 adipocytes. This diminution correlated with a significant decrease in the ability of the adipocytes to convert cortisone to cortisol. The half-maximal inhibition of 11beta-HSD-1 mRNA expression by the TZD, rosiglitazone, occurred at a concentration that was similar to its K(d) for binding PPARgamma and EC(50) for inducing adipocyte differentiation thereby indicating that this action was PPARgamma-dependent. The time required for the inhibitory action of the TZD was markedly greater for 11beta-HSD-1 gene expression than for leptin, suggesting that these genes may be down-regulated by different molecular mechanisms. Furthermore, whereas regulation of PPARgamma-inducible genes such as phosphoenolpyruvate carboxykinase was maintained when cellular protein synthesis was abrogated, PPARgamma agonist inhibition of 11beta-HSD-1 and leptin gene expression was ablated, thereby supporting the conclusion that PPARgamma affects the down-regulation of 11beta-HSD-1 indirectly. Finally, treatment of diabetic db/db mice with rosiglitazone inhibited expression of 11beta-HSD-1 in adipose tissue. This decrease in enzyme expression correlated with a significant decline in plasma corticosterone levels. In sum, these data indicate that some of the beneficial effects of PPARgamma antidiabetic agents may result, at least in part, from the down-regulation of 11beta-HSD-1 expression in adipose tissue.

Novel peroxisome proliferator-activated receptor (PPAR) gamma and PPARdelta ligands produce distinct biological effects.

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARalpha, PPARdelta, and PPARgamma. PPARgamma has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPARgamma and PPARdelta that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPARgamma and PPARdelta directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARgamma agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabetic db/db mice all PPARgamma agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selective in vivo activation of PPARdelta did not significantly affect these parameters. In vivo PPARalpha activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPARgamma and PPARdelta; 2) ligand-dependent activation of PPARdelta involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARgamma activation (but not PPARdelta or PPARalpha activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARgamma agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARalpha activation is sufficient to affect triglyceride metabolism, PPARdelta activation does not appear to modulate glucose or triglyceride levels.

Analysis of the functional role of steroid receptor coactivator-1 in ligand-induced transactivation by thyroid hormone receptor.

The nuclear hormone receptors belonging to the steroid/thyroid/retinoid receptor superfamily are ligand-inducible transcription factors. These receptors modulate transcription of specific cellular genes, either positively or negatively, by interacting with specific hormone response elements located near the target promoters. Recent studies indicated that the hormone- occupied, DNA-bound receptor acts in concert with a cellular coregulatory factor, termed coactivator, and the basal transcription machinery to mediate gene activation. Consistent with this scenario, a number of nuclear proteins with potential coactivator function have been isolated. In the present study, we demonstrate that steroid receptor coactivator-1 (SRC-1), a recently isolated candidate coactivator, functions as a positive regulator of the thyroid hormone receptor (TR)-mediated transactivation pathway. In transient transfection experiments, coexpression of SRC-1 significantly enhanced ligand-dependent transactivation of a thyroid hormone response element (TRE)-linked promoter by human TRbeta. Our studies revealed that deletion of six amino acids (451-456) in the extreme COOH-terminal region of TRbeta resulted in a receptor that retained the ability to bind T3 but failed to be stimulated by SRC-1. These six amino acids are part of an amphipathic helix that is highly conserved among nuclear hormone receptors and contains the core domain of the ligand-dependent transactivation function, AF-2. In agreement with this observation, in vitro protein binding studies showed that SRC-1 interacted with a ligand binding domain peptide (145-456) of TRbeta in a T3-dependent manner, whereas it failed to interact with a mutant ligand binding domain lacking the amino acids (451-456). We demonstrated that a synthetic peptide containing the COOH-terminal amino acids (437-456) of TRbeta efficiently blocked the ligand-induced binding of SRC-1 to the receptor. These results suggest that the conserved amphipathic helix that constitutes the AF-2 core domain of TRbeta is critical for interaction with SRC-1 and thereby plays a central role in coactivator-mediated transactivation. We further observed that a heterodimer of TRbeta and retinoid X receptor-alpha (RXR alpha), either in solution or bound to a DR+4 TRE, recruited SRC-1 in a T3-dependent manner. The AF-2 of TR was clearly involved in this process because a TR-RXR heterodimer containing a mutant TRbeta (1-450) with impaired AF-2 failed to bind to SRC-1. Surprisingly, the RXR-specific ligand 9-cis-retinoic acid induced binding of SRC-1 to the RXR component of the TRE-bound heterodimer. This novel finding suggests that RXR, as a heterodimeric partner of TR, has the potential to play an active role in transcriptional regulation. Our results raise the interesting possibility that a RXR-specific ligand may modulate T3-mediated signaling by inducing additional interactions between TRE-bound TR-RXR heterodimer and the coactivator.

Transcriptional silencing by unliganded thyroid hormone receptor beta requires a soluble corepressor that interacts with the ligand-binding domain of the receptor.

Unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of genes bearing thyroid hormone response elements in their promoters. Binding of hormonal ligand to the receptor releases the transcriptional silencing and leads to gene activation. Previous studies showed that the silencing activity of TR is located within the C-terminal ligand-binding domain (LBD) of the receptor. To dissect the role of the LBD in receptor-mediated silencing, we used a cell-free transcription system containing HeLa nuclear extracts in which exogenously added unliganded TRbeta repressed the basal level of RNA polymerase II-driven transcription from a thyroid hormone response element-linked template. We designed competition experiments with a peptide fragment containing the entire LBD (positions 145 to 456) of TRbeta. This peptide, which lacks the DNA-binding domain, did not affect basal RNA synthesis from the thyroid hormone response element-linked promoter when added to a cell-free transcription reaction mixture. However, the addition of the LBD peptide to a reaction mixture containing TRbeta led to a complete reversal of receptor-mediated transcriptional silencing in the absence of thyroid hormone. An LBD peptide harboring point mutations, which severely impair receptor dimerization, also inhibited efficiently the silencing activity of TR, indicating that the relief of repression by the LBD was not due to the sequestration of TR or its heterodimeric partner retinoid X receptor into inactive homo- or heterodimers. We postulate that the LBD peptide competed with TR for a regulatory molecule, termed a corepressor, that exists in the HeLa nuclear extracts and is essential for efficient receptor-mediated gene repression. We have identified the region from positions 145 to 260 (the D domain) of the LBD as a potential binding site of the putative corepressor. We observed further that a peptide containing the LBD of retinoic acid receptor (RAR) competed for TR-mediated silencing, suggesting that the RAR LBD may bind to the same corepressor activity as the TR LBD. Interestingly, the RAR LBD complexed with its cognate ligand, all-trans retinoic acid, failed to compete for transcriptional silencing by TRbeta, indicating that the association of the LBD with the corepressor is ligand dependent. Finally, we provide strong biochemical evidence supporting the existence of the corepressor activity in the HeLa nuclear extracts. Our studies demonstrated that the silencing activity of TR was greatly reduced in the nuclear extracts preincubated with immobilized, hormone-free glutathione S-transferase-LBD fusion proteins, indicating that the corepressor activity was depleted from these extracts through protein-protein interactions with the LBD. Similar treatment with immobilized, hormone-bound glutathione S-transferase-LBD, on the other hand, failed to deplete the corepressor activity from the nuclear extracts, indicating that ligand binding to the LBD disrupts its interaction with the corepressor. From these results, we propose that a corepressor binds to the LBD of unliganded TR and critically influences the interaction of the receptor with the basal transcription machinery to promote silencing. Ligand binding to TR results in the release of the corepressor from the LBD and triggers the reversal of silencing by allowing the events leading to gene activation to proceed.

Ligand modulates the interaction of thyroid hormone receptor beta with the basal transcription machinery.

We investigated the molecular mechanisms underlying the transcriptional silencing and the hormone-induced activation of target genes by thyroid hormone receptor beta (TR-beta). We developed a cell-free transcription system containing HeLa cell nuclear extracts in which unliganded human TR-beta represses basal transcription from a promoter bearing thyroid hormone response elements. Binding of hormonal ligand to the receptor reverse this transcriptional silencing. Specific binding of TR-beta to the thyroid hormone response element at the target promoter is crucial for silencing. Studies employing TR-beta mutants indicate that the silencing activity is located within the C-terminal rather than the N-terminal domain of the receptor. Our studies reveal further that unliganded TR-beta inhibits the assembly of a functional transcription preinitiation complex (PIC) at the target promoter. We postulate that interaction with TR-beta impairs the function(s) of one or more assembling transcriptional complexes during the multistep assembly of a PIC. Consistent with this hypothesis, we observe that, in the absence of thyroid hormone, TR-beta or a heterodimer of TR-beta and retinoid-X-receptor undergoes direct protein-protein interactions with the transcription factor IIB-TATA binding protein complex, an early intermediate during PIC assembly. Binding of hormone to TR-beta dramatically reduces the interaction between the receptor and the transcription factor IIB-TATA binding protein complex. We propose that the role of ligand is to facilitate the assembly of functional PICs at the target promoter by reducing nonproductive interactions between TR-beta and the initiation factors.