Molecular endocrinology最新文献

The human somatostatin receptor 3 (sst3) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst3 receptor is unique among ssts in that it exhibits a very long intracellular C-terminal tail containing a huge number of potential phosphate acceptor sites. Consequently, our knowledge about the functional role of the C-terminal tail in sst3 receptor regulation is very limited. Here, we have generated a series of phosphorylation-deficient mutants that enabled us to determine crucial sites for its agonist-induced β-arrestin mobilization, internalization, and down-regulation. Based on this information, we generated phosphosite-specific antibodies for C-terminal Ser(337)/Thr(341), Thr(348), and Ser(361) that enabled us to investigate the temporal patterns of sst3 phosphorylation and dephosphorylation. We found that the endogenous ligand somatostatin induced a rapid and robust phosphorylation that was completely blocked by the sst3 antagonist NVP-ACQ090. The stable somatostatin analogs pasireotide and octreotide promoted clearly less phosphorylation compared with somatostatin. We also show that sst3 phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst3 receptor occurred at a considerable slower rate. In addition, we also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1α and 1β as key regulators of sst3 phosphorylation and dephosphorylation, respectively. Thus, we here define the C-terminal phosphorylation motif of the human sst3 receptor that regulates its agonist-promoted phosphorylation, β-arrestin recruitment, and internalization of this clinically relevant receptor.

The pathophysiology of metabolic diseases such as coronary artery disease, diabetes, and obesity is complex and multifactorial. Developing new strategies to prevent or treat these diseases requires in vitro models with which researchers can extensively study the molecular mechanisms that lead to disease. Human pluripotent stem cells and their differentiated derivatives have the potential to provide an unlimited source of disease-relevant cell types and, when combined with recent advances in genome editing, make the goal of generating functional metabolic disease models, for the first time, consistently attainable. However, this approach still has certain limitations including lack of robust differentiation methods and potential off-target effects. This review describes the current progress in human pluripotent stem cell-based metabolic disease research using genome-editing technology.

Skeletal muscle remodels metabolic capacity, contractile and exercise phenotype in response to physiological demands. This adaptive remodeling response to physical activity can ameliorate/prevent diseases associated with poor diet and lifestyle. Our previous work demonstrated that skeletal muscle-specific transgenic expression of the neuron-derived orphan nuclear receptor, Nor-1 drives muscle reprogramming, improves exercise endurance, and oxidative metabolism. The current manuscript investigates the association between exercise, Nor-1 expression and the role of Nor-1 in adaptive remodeling. We demonstrate that Nor-1 expression is induced by exercise and is dependent on calcium/calcineurin signaling (in vitro and in vivo). Analysis of fatigue-resistant transgenic mice that express Nor-1 in skeletal muscle revealed increased hypertrophy and vascularization of muscle tissue. Moreover, we demonstrate that transgenic Nor-1 expression is associated with increased intracellular recycling, ie, autophagy, involving 1) increased expression of light chain 3A or LC3A-II, autophagy protein 5, and autophagy protein 12 in quadriceps femoris muscle extracts from Tg-Nor-1 (relative to Wild-type (WT) littermates); 2) decreased p62 expression indicative of increased autophagolysosome assembly; and 3) decreased mammalian target of rapamycin complex 1 activity. Transfection of LC3A-GFP-RFP chimeric plasmid demonstrated that autophagolysosome formation was significantly increased by Nor-1 expression. Furthermore, we demonstrated a single bout of exercise induced LC3A-II expression in skeletal muscle from C57BL/6 WT mice. This study, when combined with our previous studies, demonstrates that Nor-1 expression drives multiple physiological changes/pathways that are critical to the beneficial responses of muscle to exercise and provides insights into potential pharmacological manipulation of muscle reprogramming for the treatment of lifestyle induced chronic diseases.

Many studies have provided evidence to demonstrate the beneficial renal effects of resveratrol (RESV) due to its antioxidant character and its capacity for activation of surtuin 1. However, the molecular mechanisms underlying the protective role of RESV against kidney injury are still incompletely understood. The present study used Lepr db/db (db/db) and Lepr db/m (db/m) mice as models to evaluate the effect of RESV on diabetic nephropathy (DN). RESV reduced proteinuria and attenuated the progress of renal fibrosis in db/db mice. Treatment with RESV markedly attenuated the diabetes-induced changes in renal superoxide dismutase copper/zinc, superoxide dismutase manganese, catalase, and malonydialdehyde as well as the renal expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), α-smooth muscle actin (α-SMA), and E-cadherin in db/db mice. The kidney expression of the IGF-1 receptor (IGF-1R) was increased in db/db mice, but the expression of 3-hydroxy-3-methylglutaryl reductase degradation (HRD1), a ubiquitin E3 ligase, was significantly decreased in the DN model. RESV treatment dramatically decreased IGF-1R and increased HRD1 expressions, consistent with data obtained with HKC-8 cells. HRD1 physically interacted with IGF-1R in HKC-8 cells and liquid chromatography and tandem mass spectrometry (LC-MS/MS) data supported the concept that IGF-1R is one of the HRD1 substrates. HRD1 promoted the IGF-1R ubiquitination for degradation in HKC-8 cells, and the down-regulation of HRD1 reversed the protective effects of RESV in HKC-8 cells. In summary, we have demonstrated that RESV reduces proteinuria and attenuates the progression of renal fibrosis in db/db mice. These protective effects of RESV on DN were associated with the up-regulation of HRD1, induced by RESV, and the promotion of IGF-1R ubiquitination and degradation.

GPR119 is a G protein-coupled receptor expressed on intestinal L cells that synthesize and secrete the blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1). GPR119 agonists stimulate the release of GLP-1 from L cells, and for this reason there is interest in their potential use as a new treatment for type 2 diabetes mellitus. AS1269574 is one such GPR119 agonist, and it is the prototype of a series of 2,4,6 trisubstituted pyrimidines that exert positive glucoregulatory actions in mice. Here we report the unexpected finding that AS1269574 stimulates GLP-1 release from the STC-1 intestinal cell line by directly promoting Ca(2+) influx through transient receptor potential ankyrin 1 (TRPA1) cation channels. These GPR119-independent actions of AS1269574 are inhibited by TRPA1 channel blockers (AP-18, A967079, HC030031) and are not secondary to intracellular Ca(2+) release or cAMP production. Patch clamp studies reveal that AS1269574 activates an outwardly rectifying membrane current with properties expected of TRPA1 channels. However, the TRPA1 channel-mediated action of AS1269574 to increase intracellular free calcium concentration is not replicated by GPR119 agonists (AR231453, oleoylethanolamide) unrelated in structure to AS1269574. Using human embryonic kidney-293 cells expressing recombinant rat TRPA1 channels but not GPR119, direct TRPA1 channel activating properties of AS1269574 are validated. Because we find that AS1269574 also acts in a conventional GPR119-mediated manner to stimulate proglucagon gene promoter activity in the GLUTag intestinal L cell line, new findings reported here reveal the surprising capacity of AS1269574 to act as a dual agonist at two molecular targets (GPR119/TRPA1) important to the control of L-cell function and type 2 diabetes mellitus drug discovery research.

Better understanding the mechanisms underlying adipogenesis may provide novel therapeutic targets in the treatment of obesity. Most studies investigating the mechanisms underlying adipogenesis focus on highly regulated transcriptional pathways; little is known about the epigenetic mechanisms in this process. Here, we determined the role of DNA methylation in regulating 3T3-L1 adipogenesis in early and late stage of differentiation. We found that inhibiting DNA methylation pharmacologically by 5-aza-2'-deoxycytidine (5-aza-dC) at early stage of 3T3-L1 differentiation markedly suppressed adipogenesis. This inhibition of adipogenesis by 5-aza-dC was associated with up-regulation of Wnt10a, an antiadipogenic factor, and down-regulation of Wnt10a promoter methylation. In contrast, inhibiting DNA methylation by 5-aza-dC at late stage of differentiation enhanced the lipogenic program. The differential effects of 5-aza-dC on adipogenesis were confirmed by gain or loss of function of DNA methyltransferase 1 using genetic approaches. We further explored the molecular mechanism underlying the enhanced lipogenesis by inhibition of DNA methylation at late stage of differentiation. The Srebp1c promoter is enriched with CpG sites. Chromatin immunoprecipitation assays showed that DNA methyltransferase 1 bound to the methylation region at the Srebp1c promoter. Pyrosequencing analysis revealed that the DNA methylation at the key cis-elements of the Srebp1c promoter was down-regulated in adipogenesis. Further, luciferase reporter assays showed that the Srebp1c promoter activity was dramatically up-regulated by the unmethylated promoter compared with the fully methylated promoter. Thus DNA methylation appears to exert a biphasic regulatory role in adipogenesis, promoting differentiation at early stage while inhibiting lipogenesis at late stage of 3T3-L1 preadipocyte differentiation.

GH receptor (GHR) binds GH at the cell surface via its extracellular domain and initiates intracellular signal transduction, resulting in important anabolic and metabolic actions. GH signaling is subject to dynamic regulation, which in part is exerted by modulation of cell surface GHR levels. Constitutive and inducible metalloprotease-mediated cleavage of GHR regulate GHR abundance and thereby modulate GH action. We previously demonstrated that GHR proteolysis is catalyzed by the TNF-α converting enzyme (TACE; ADAM17). Tissue inhibitors of metalloproteases-3 (TIMP3) is a natural specific inhibitor of TACE, although mechanisms underlying this inhibition are not yet fully understood. In the current study, we use two model cell lines to examine the relationships between cellular TACE, TIMP3 expression, GHR metalloproteolysis, and GH sensitivity. These two cell lines exhibited markedly different sensitivity to inducible GHR proteolysis, which correlated directly to their relative levels of mature TACE vs unprocessed TACE precursor and indirectly to their levels of cellular TIMP3. Our results implicate TIMP3 as a modulator of cell surface GHR abundance and the ability of GH to promote cellular signaling; these modulatory effects may be conferred by endogenous TIMP3 expression as well as exogenous TIMP3 exposure. Furthermore, our analysis suggests that TIMP3, in addition to regulating the activity of TACE, may also modulate the maturation of TACE, thereby affecting the abundance of the active form of the enzyme.