Sirtuins, which are class III NAD-dependent histone deacetylases (HDACs) that regulate a number of physiological processes, play important roles in the regulation of metabolism, aging, oncogenesis and cancer progression. More recently, a role for the sirtuins in the regulation of steroid hormone receptor signaling is emerging. In this mini-review, we will summarize current research into the regulation of estrogen, androgen, progesterone, mineralocorticoid and glucocorticoid signaling by sirtuins in cancer. Sirtuins can regulate steroid hormone signaling through a variety of molecular mechanisms, including acting as co-regulatory transcription factors, deacetylating histones in the promoters of genes with nuclear receptor binding sites, directly deacetylating steroid hormone nuclear receptors, and regulating pathways which modify steroid hormone receptors through phosphorylation. Furthermore, disruption of sirtuin activity may be an important step in the development of steroid hormone-refractory cancers.
Sirtuin; steroid hormone receptor activity/regulation; SIRT1; endocrine cancer
During spermatogenesis, spermatids derived from meiosis simultaneously undergo extensive morphological transformation, to become highly specialized and metabolically quiescent cells, and transport across the seminiferous epithelium. Spermatids are also transported back-and-forth across the seminiferous epithelium during the epithelial cycle until they line up at the luminal edge of the tubule to prepare for spermiation at stage VIII of the cycle. Spermatid transport thus requires the intricate coordination of the cytoskeletons in Sertoli cells since spermatids are non-motile cells, lacking the ultrastructures of lamellipodia and filopodia, as well as the organized components of the cytoskeletons. In the course of preparing this brief review, we were surprised to see that, except for some earlier eminent morphological studies, little is known about the regulation of the microtubule (MT) cytoskeleton and the coordination of MT with the actin-based cytoskeleton to regulate spermatid transport during the epithelia cycle, illustrating that this is a largely neglected area of research required in the field. Herein, we summarize recent findings in the field regarding the significance of actin- and tubulin-based cytoskeletons in the Sertoli cell that support spermatid transport; we also highlight specific areas of research that deserve attentions in future studies.
Testis; MARKs; microtubule; spermatogenesis; seminiferous epithelial cycle; spermiogenesis; spermatid adhesion
In recent years, the roles of chronic stress and depression as an independent risk factor for decreased insulin sensitivity and the development of diabetes have been increasingly recognized. However, an understanding and the mechanisms linking insulin resistance and acute psychological stress are very limited. We hypothesized that acute psychological stress may cause the development of insulin resistance, which may be a risk factor in developing type 2 diabetes. We tested the hypothesis in a well-established mouse model using 180 episodes of inescapable foot shock (IES), followed by a behavioral escape test. In this study, mice that received IES treatment were tested for acute insulin resistance by measuring glucose metabolism and insulin signaling. When compared to normal and sham mice, mice that were exposed to IES resulting in escape failure (defined as IES with behavioral escape failure) displayed elevated blood glucose levels in both glucose tolerance and insulin tolerance tests. Furthermore, mice with IES exposure and behavioral escape failure exhibited impaired hepatic insulin signaling via the insulin-induced insulin receptor/insulin receptor substrate 1/Akt pathway, without affecting similar pathways in skeletal muscle, adipose tissue and brain. Additionally, a rise in murine growth-related oncogene KC/GRO was associated with impaired glucose metabolism in IES mice, suggesting a mechanism by which psychological stress by IES may influence glucose metabolism. The present results indicate that psychological stress induced by IES can acutely alter hepatic responsiveness to insulin and affect whole-body glucose metabolism.
Psychological stress; Inescapable foot shocks; Insulin signaling; Glucose metabolism; Acute insulin resistance
Aberrant redeployment of the ‘transient’ events responsible for bone development and postnatal longitudinal growth has been reported in some diseases in what is otherwise inherently ‘stable’ cartilage. Lessons may be learnt from the molecular mechanisms underpinning transient chondrocyte differentiation and function, and their application may better identify disease aetiology. Here, we review the current evidence supporting this possibility. We firstly outline endochondral ossification and the cellular and physiological mechanisms by which it is controlled in the postnatal growth plate. We then compare the biology of these transient cartilaginous structures to the inherently stable articular cartilage. Finally, we highlight specific scenarios in which the redeployment of these embryonic processes may contribute to disease development, with the foresight that deciphering those mechanisms regulating pathological changes and loss of cartilage stability will aid future research into effective disease-modifying therapies.
bone; cartilage; osteoarthritis; chondrocyte; endochondral ossification
Hypovitaminosis D is an important public health problem. Serum 25-hydroxyvitamin D (25-OHD) is now recognized as an independent predictor for cardiovascular and related diseases (CVD) as well as other chronic medical conditions. However, the biologic pathways through which these effects are mediated remain poorly understood. We hypothesized that exposing mesenchymal multipotent cells (MMCs) to the active form of vitamin D would increase the expression of selected antifibrotic factors that in turn would ameliorate the progression of chronic diseases. MMCs were primed with 5′-azacytidine to induce a fibrotic phenotype and then treated with active vitamin D (1,25D) or ethanol <0·1% as vehicle in a time course manner (30 min, 1, 5, and 24 h, and for 4 and 7 days). The addition of 1,25D to MMCs promotes: a) increased expression and nuclear translocation of the vitamin D receptor; b) decreased expression of TGFB1 and plasminogen activator inhibitor (SERPINE1), two well-known profibrotic factors; c) decreased expression of collagen I, III and other collagens isoforms; and d) increased expression of several antifibrotic factors such as BMP7 a TGFB1 antagonist, MMP8 a collagen breakdown inducer and follistatin, an inhibitor of the profibrotic factor myostatin. In conclusion, the addition of 1,25D to differentiated MMCs displays a decreased profibrotic signaling pathway and gene expression, leading to decrease in collagen deposition. This study highlights key mechanistic pathways through which vitamin D decreases fibrosis, and provides a rationale for studies to test vitamin D supplementation as a preventive and/or early treatment strategy for CVD and related fibrotic disorders.
As successful generation of insulin-producing cells could be used for diabetes treatment, a concerted effort is being made to understand the molecular programs underlying islet β-cell formation and function. The closely related MafA and MafB transcription factors are both key mammalian β-cell regulators. MafA and MafB are co-expressed in insulin+ β-cells during embryogenesis, while in the adult pancreas only MafA is produced in β-cells and MafB in glucagon+ α-cells. MafB−/− animals are also deficient in insulin+ and glucagon+ cell production during embryogenesis. However, only MafA over-expression selectively induced endogenous Insulin mRNA production in cell line-based assays, while MafB specifically promoted Glucagon expression. Here, we analyzed whether these factors were sufficient to induce insulin+ and/or glucagon+ cell formation within embryonic endoderm using the chick in ovo electroporation assay. Ectopic expression of MafA, but not MafB, promoted Insulin production; however, neither MafA nor MafB were capable of inducing Glucagon. Co-electroporation of MafA with the Ngn3 transcription factor resulted in the development of more organized cell clusters containing both insulin- and glucagon-producing cells. Analysis of chimeric proteins of MafA and MafB demonstrated that chick Insulin activation depended on sequences within the MafA C-terminal DNA-binding domain. MafA was also bound to Insulin and Glucagon transcriptional control sequences in mouse embryonic pancreas and β-cell lines. Collectively, these results demonstrate a unique ability for MafA to independently activate Insulin transcription.
Parathyroid hormone (PTH) is a major physiologic regulator of calcium, phosphorous and skeletal homeostasis. Cells of the osteoblastic lineage are key targets of PTH action in bone, and recent evidence suggests that osteocytes might be important in the anabolic effects of PTH. To understand the role of PTH signaling through the PTH/PTHrP receptors (PPR) in osteocytes and to determine the role(s) of these cells in mediating the effects of the hormone, we have generated mice in which PPR expression is specifically ablated in osteocytes. Transgenic mice in which the 10Kb-Dmp1 promoter drives a tamoxifen-inducible Cre –recombinase were mated with animals in which exon1 of PPR is flanked by Lox-P sites. In these animals, osteocyte-selective PPR knockout (Ocy-PPRcKO mice) could be induced by administration of tamoxifen. Histological analysis revealed a reduction in trabecular bone and mild osteopenia in Ocy-PPRcKO mice. Reduction of trabeculae number and thickness was also detected by μCT analysis whereas BV/TV% was unchanged. These findings were associated with an increase in Sost and sclerostin expression. When Ocy-PPRcKO mice were subjected to a low calcium diet, to induce secondary hyperparathyroidism, their blood calcium levels were significantly lower than littermate controls. Moreover, PTH was unable to suppress Sost and sclerostin expression in the Ocy-PPRcKO animals, suggesting an important role of PTH signaling in osteocytes for proper bone remodeling and calcium homeostasis.
osteocytes; parathyroid hormone; parathyroid hormone receptor
Parathyroid hormone (PTH) signaling via PTH 1 receptor (PTH1R) involves mitogen-activated protein kinase (MAPK) pathways. MAPK phosphatase 1 (MKP1) dephosphorylates and inactivates MAPKs in osteoblasts, the bone-forming cells. We previously showed that PTH1R activation in differentiated osteoblasts upregulates MKP1 and downregulates pERK1/2–MAPK and cyclin D1. In this study, we evaluated the skeletal phenotype of Mkp1 knockout (KO) mice and the effects of PTH in vivo and in vitro. Microcomputed tomography analysis of proximal tibiae and distal femora from 12-week-old Mkp1 KO female mice revealed osteopenic phenotype with significant reduction (8–46%) in bone parameters compared with wild-type (WT) controls. Histomorphometric analysis showed decreased trabecular bone area in KO females. Levels of serum osteocalcin (OCN) were lower and serum tartrate-resistant acid phosphatase 5b (TRAP5b) was higher in KO animals. Treatment of neonatal mice with hPTH (1–34) for 3 weeks showed attenuated anabolic responses in the distal femora of KO mice compared with WT mice. Primary osteoblasts derived from KO mice displayed delayed differentiation determined by alkaline phosphatase activity, and reduced expressions of Ocn and Runx2 genes associated with osteoblast maturation and function. Cells from KO females exhibited attenuated PTH response in mineralized nodule formation in vitro. Remarkably, this observation was correlated with decreased PTH response of matrix Gla protein expression. Expressions of pERK1/2 and cyclin D1 were inhibited dramatically by PTH in differentiated osteoblasts from WT mice but much less in osteoblasts from Mkp1 KO mice. In conclusion, MKP1 is important for bone homeostasis, osteoblast differentiation and skeletal responsiveness to PTH.
The calcium-sensing receptor (CaR) is a G-protein-coupled receptor (GPCR) that binds and signals in response to extracellular calcium and other polycations. It is highly expressed on parathyroid and kidney cells, where it participates in the regulation of systemic calcium homeostasis. It is also expressed on many other cell types and is involved in a wide array of biological functions such as cell growth and differentiation, ion transport and hormone secretion. It has been described to couple to several different G-proteins including Gαi/0, Gαq/11 and Gα12/13. Recently, it has also been shown to stimulate cAMP production by coupling to Gαs in immortalized or malignant breast cells. The CaR is expressed on cells in the anterior pituitary and had previously been described to stimulate cAMP production in these cells. In this report, we examined signaling from the CaR in murine pituitary corticotroph-derived, AtT-20 cells. We found that CaR activation led to the stimulation of cAMP production, and PTHrP and ACTH secretion from these cells. Furthermore, manipulation of cAMP levels was able to modulate PTHrP and ACTH secretion independent of changes in extracellular calcium. Finally, we demonstrated that the CaR couples to Gαs in AtT-20 cells. Therefore, in pituitary corticotroph-like cells, as in breast cancer cells, the CaR utilizes Gαs and activates cAMP production to stimulate hormone secretion.
G-protein coupled receptor; calcimimetic; intracellular signaling; cyclic-AMP
The adrenal glands are the primary source of mineralocorticoids, glucocorticoids and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by adrenocorticotrophic hormone (ACTH). Although it is well established that ACTH can stimulate steroidogenesis in the human adrenal gland, the effect of ACTH on overall production of different classes of steroid hormones has not been defined. In this study, we examined the effect of ACTH on the production of twenty-three steroid hormones in adult adrenal (AA) primary cultures and 20 steroids in the adrenal cell line, H295R. Liquid chromatography/tandem mass spectrometry analysis (LC-MS/MS) revealed that in primary cultures that cortisol and corticosterone were the two most abundant steroid hormones produced with or without ACTH treatment (48 h). Cortisol production responded the most to ACTH treatment, with a 64-fold increase. Interestingly, the production of two androgens, androstenedione and 11β-hydroxyandrostenedione, that were also produced in large amounts under basal conditions significantly increased after ACTH incubation. In H295R cells 11-deoxycortisol and androstenedione were the major products under basal conditions. Treatment with forskolin increased the percentage of 11β-hydroxylated products including cortisol and 11β-hydroxyandrostenedione. This study illustrates that normal adrenal cells respond to ACTH through the secretion of a variety of steroid hormones, thus supporting the role of adrenal cells as a source of both corticosteroids and androgens.
Adrenal cortex; ACTH; Steroidogenesis; LC-MS/MS
Phosphorylation, internalization, and desensitization of G protein-coupled receptors, such as the parathyroid hormone (PTH) and PTH-related peptide (PTHrP) receptor (PTH1R), are well characterized and known to regulate the cellular responsiveness in vitro. However, the role of PTH1R receptor phosphorylation in bone formation and osteoblast functions has not yet been elucidated. In previous studies, we demonstrated impaired internalization and sustained cAMP stimulation of a phosphorylation-deficient (pd) PTH1R in vitro, and exaggerated cAMP and calcemic responses to s.c. PTH infusion in pdPTH1R knock-in mouse model. In this study, we examined the impact of impaired PTH1R phosphorylation on the skeletal phenotype of mice maintained on normal, low, and high calcium diets. The low calcium diet moderately reduced (P<0.05) bone volume and trabecular number, and increased trabecular spacing in both wild-type (WT) and pd mice. The effects, however, seem to be less pronounced in the female pd compared to WT mice. In primary calvarial osteoblasts isolated from 2-week-old pd or WT mice, PTH and PTHrP decreased phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2), a member of mitogen-activated protein kinase, and cyclin D1, a G1/S phase cyclin, in vitro. In contrast to WT osteoblasts, down-regulation of cyclin D1 was sustained for longer periods of time in osteoblasts isolated from the pd mice. Our results suggest that adaptive responses of intracellular signaling pathways in the pd mice may be important for maintaining bone homeostasis.
In mammals, parathyroid hormone-related peptide (PTHrP, alias PTH-like hormone (Pthlh)) acts as a paracrine hormone that regulates the patterning of cartilage, bone, teeth, pancreas, and thymus. Beyond mammals, however, little is known about the molecular genetic mechanisms by which Pthlh regulates early development. To evaluate conserved pathways of craniofacial skeletogenesis, we isolated two Pthlh co-orthologs from the zebrafish (Danio rerio) and investigated their structural, phylogenetic, and syntenic relationships, expression, and function. Results showed that pthlh duplicates originated in the teleost genome duplication. Zebrafish pthlha and pthlhb were maternally expressed and showed overlapping and distinct zygotic expression patterns during skeletal development that mirrored mammalian expression domains. To explore the regulation of duplicated pthlh genes, we studied their expression patterns in mutants and found that both sox9a and sox9b are upstream of pthlha in arch and fin bud cartilages, but only sox9b is upstream of pthlha in the pancreas. Morpholino antisense knockdown showed that pthlha regulates both sox9a and sox9b in the pharyngeal arches but not in the brain or otic vesicles and that pthlhb does not regulate either sox9 gene, which is likely related to its highly degraded nuclear localization signal. Knockdown of pthlha but not pthlhb caused runx2b overexpression in craniofacial cartilages and premature bone mineralization. We conclude that in normal cartilage development, sox9 upregulates pthlh, which downregulates runx2, and that the duplicated nature of all three of these genes in zebrafish creates a network of regulation by different co-orthologs in different tissues.
Adiponectin is positively correlated with longevity and negatively correlated with many obesity-related diseases. While there are several circulating forms of adiponectin, the high molecular weight (HMW) version has been suggested to have the predominant bioactivity. Adiponectin gene expression and cognate serum protein levels are of particular interest in mice with altered growth hormone (GH) signaling as these mice exhibit extremes in obesity that are positively associated with insulin sensitivity and lifespan as opposed to the typical negative association of these factors. While a few studies have reported total adiponectin levels in young adult mice with altered GH signaling, much remains unresolved, including changes in adiponectin levels with advancing age, proportion of total adiponectin in the HMW form, adipose depot of origin, and differential effects of GH versus IGF1. Therefore, the purpose of this study was to address these issues using assorted mouse lines with altered GH signaling. Our results show that adiponectin is generally negatively associated with GH activity, regardless of age. Further, the amount of HMW adiponectin is consistently linked with the level of total adiponectin and not necessarily with previously reported lifespan or insulin sensitivity of these mice. Interestingly, circulating adiponectin levels correlated strongly with inguinal fat mass, implying the effects of GH on adiponectin are depot-specific. Interestingly rbGH, but not IGF1, decreased circulating total and HMW adiponectin levels. Taken together, these results fill important gaps in the literature related to GH and adiponectin and question the frequently reported associations of total and HMW adiponectin with insulin sensitivity and longevity.
adiponectin; high molecular weight adiponectin; growth hormone receptor; growth hormone; growth hormone deficiency; growth hormone antagonist
Leptin deficiency in mice results in chronic thymic atrophy, suppressed cell-mediated immunity, and decreased numbers of total lymphocytes, suggesting a key role for the metabolic hormone leptin in regulating thymopoiesis and overall immune homeostasis. The thymus unfortunately is highly susceptible to stress-induced acute involution. Prolonged thymus atrophy in stress situations can contribute to peripheral T cell deficiency or inhibit immune reconstitution. Little is known however about specific roles for leptin signaling in the thymus or the underlying mechanisms driving thymic involution or thymic recovery after acute stress. We report here that leptin receptor expression is restricted in thymus to medullary epithelial cells. Using a model of endotoxemia-induced acute thymic involution and recovery, we have demonstrated a role for supraphysiologic leptin in protection of thymic epithelial cells. We also present data in support of our hypothesis that leptin treatment decreases in vivo endotoxemia-induced apoptosis of double positive thymocytes and promotes proliferation of double negative thymocytes through a leptin receptor isoform b-specific mechanism. Furthermore, our studies have revealed that leptin treatment increases thymic expression of IL-7, an important soluble thymocyte growth factor produced by medullary thymic epithelial cells. Taken together, these studies support an intrathymic role for the metabolic hormone leptin in maintaining healthy thymic epithelium and promoting thymopoiesis, which is revealed when thymus homeostasis is perturbed by endotoxemia.
Thymopoiesis; Leptin; Leptin Receptor; Lipopolysaccharride
The steroid hormone aldosterone maintains sodium homeostasis and is therefore important in control of blood volume and pressure. Angiotensin II (AngII) and elevated extracellular potassium concentrations ([K+]e), the prime physiologic regulators of aldosterone secretion from adrenal glomerulosa cells, activate phospholipase D (PLD) in these cells. The role of Ca2+ in the activation by these agents is unknown, although nitrendipine, a voltage-dependent Ca2+ channel antagonist, does not inhibit AngII-elicited PLD activation, despite the fact that this compound blocked elevated [K+]e-stimulated PLD activity. PLD activation triggered by AngII was also unaffected by the T-type calcium channel inhibitor nickel. Nevertheless, Ca2+ influx was required for AngII-induced PLD activation in both primary cultures of bovine adrenal glomerulosa cells and a glomerulosa cell model, the NCI H295R adrenocortical carcinoma cell line. The involvement of store-operated Ca2+ (SOC) influx and Ca2+ release-activated Ca2+ (CRAC) influx pathways in PLD activation was investigated using thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor that empties the store to induce SOC influx, and the SOC inhibitor YM-58483 (BTP2), as well as a CRAC inhibitor, tyrphostin A9. In bovine glomerulosa cells tyrphostin A9 inhibited AngII-induced PLD activation without affecting elevated [K+]e-stimulated enzyme activity. On the other hand, differences were observed between the bovine adrenal glomerulosa and H295R cells in the involvement of Ca2+ influx pathways in PLD activation, with the involvement of the SOC pathway suggested in the H295R cells. In summary, our results indicate that Ca2+ entry only through certain Ca2+ influx pathways is linked to PLD activation.
NCI H295R cell line; protein kinase C; diacylglycerol; phosphatidic acid; thapsigargin; aldosterone; steroidogenesis
Physical inactivity can lead to obesity and fat accumulation in various tissues. Critical complications of obesity include type II diabetes and nonalcoholic fatty liver disease (NAFLD). Exercise has been reported to have ameliorating effects on obesity and NAFLD. However, the underlying mechanism is not fully understood. We showed that liver expression of macrophage migration inhibitory factor (MIF) was increased after 4 weeks of treadmill exercise. Phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase in human hepatocyte cell lines was enhanced after MIF treatment. These responses were accompanied by increases in lipid oxidation. Moreover, inhibition of either AMPK or cluster of differentiation 74 resulted in inhibition of MIF-induced lipid oxidation. Furthermore, the administration of MIF to a human hepatocyte cell line and mice liver reduced liver X receptor agonist-induced lipid accumulation. Taken together, these results indicate that MIF is highly expressed in the liver during physical exercise and may prevent hepatic steatosis by activating the AMPK pathway.
cytokines; lipid; molecular biology; liver; exercise
Pharmacological and genetic studies have suggested that melanocortin-4 receptor (MC4R) signaling in the paraventricular nucleus of hypothalamus (PVN) regulates appetite and energy balance. However, the specific role of MC4R signaling in PVN neurons in these processes remains to be further elucidated in normally developed animals. In the present study, we employed RNA interference to determine whether MC4R knockdown in the PVN modulates food intake and body weight in adult rats. Adeno-associated viral (AAV) vectors encoding short hairpin RNAs targeting MC4R (AAV-shRNA-MC4R) were generated to induce MC4R knockdown in the PVN. By in situ hybridization, we detected a high-level expression of Dicer, a key enzyme required for shRNA-mediated gene silencing, along the entire rostrocaudal extent of the PVN. Bilateral injection of AAV-shRNA-MC4R vectors into the PVN of the adult rat resulted in significant and specific reduction of MC4R mRNA expression. Animals with MC4R knockdown exhibited an increase in food intake and excessive body weight gain when exposed to a high-fat diet. Our results provide evidence that AAV-mediated silencing of MC4R on PVN neurons promotes hyperphagia and obesity in response to the dietary challenge in the adult animal.
The estrogen receptor α (ERα) is a transcription factor that mediates the biological effects of 17β-estradiol (E2). ERα transcriptional activity is also regulated by cytoplasmic signaling cascades. Here, several Gα protein subunits were tested for their ability to regulate ERα activity. Reporter assays revealed that overexpression of a constitutively active Gαo protein subunit potentiated ERα activity in the absence and presence of E2. Transient transfection of the human breast cancer cell line MCF-7 showed that Gαo augments the transcription of several ERα-regulated genes. Western blots of HEK293T cells transfected with ER±Gαo revealed that Gαo stimulated phosphorylation of ERK 1/2 and subsequently increased the phosphorylation of ERα on serine 118. In summary, our results show that Gαo, through activation of the MAPK pathway, plays a role in the regulation of ERα activity.
Previously it was shown that the type 1 deiodinase (D1) is subject to substrate dependent inactivation that is blocked by pretreatment with the inhibitor of D1 catalysis, propylthiouracil (PTU). Using HepG2 cells with endogenous D1 activity we found that while considerable D1-mediated catalysis of rT3 is observed in intact cells, there was a significant loss of D1 activity in sonicates assayed from the same cells in parallel. This rT3-mediated loss of D1 activity occurs despite no change in D1mRNA levels, and is blocked by PTU treatment, suggesting a requirement for catalysis. Endogenous D1 activity in sonicates was inactivated in a dose dependent manner in HepG2 cells, with a ~50% decrease after 10 nM rT3 treatment. Inactivation of D1 was rapid, occurring after only ½ hour of rT3 treatment. D1 expressed in HEK293 cells was inactivated by rT3 in a similar manner. 75Se labeling of the D1 selenoprotein indicated that after 4 hours rT3-mediated inactivation of D1 occurs without a corresponding decrease in D1 protein levels, though rT3 treatment causes a loss of D1 protein after 8-24 hours. Bioluminescence Resonance Energy Transfer (BRET) studies indicate that rT3 exposure increases energy transfer between the D1 homodimer subunits, and this was lost when the active site of D1 was mutated to alanine, suggesting that a post-catalytic structural change in the D1 homodimer could cause enzyme inactivation. Thus, both D1 and type 2 deiodinase (D2) are subject to catalysis-induced loss of activity although their inactivation occurs via very different mechanisms.
deiodinase; thyroid hormone action; thyroid hormone metabolism; selenoprotein
Sporadic primary hyperparathyroidism (PHPT), one of the most common endocrine disorders, is characterized by hypercalcemia and elevated PTH levels. The majority of cases are caused by a benign parathyroid adenoma, but somatic or de novo germ-line mutations that lead to adenoma formation have only been identified in few glands. GCMB is a parathyroid-specific transcription factor, which causes hypoparathyroidism when inactivated on both parental alleles or when a dominant-negative, heterozygous mutation is present. It is overexpressed in some parathyroid adenomas, and we therefore tested the hypothesis that GCMB mutations can be a cause of parathyroid adenomas. Nucleotide sequence analysis was performed on all coding exons and exon–intron borders of GCMB in 30 sporadic parathyroid adenomas and we identified several known polymorphisms that were either heterozygous or homozygous. In addition, one of the 30 investigated glands revealed a novel heterozygous missense mutation, c.1144G>A, which introduced methionine at position 382 for valine (V382M), a conserved amino acid residue. Western blot analysis using mutant GCMB (GCMB-V382M) from lysates of transiently transfected DF-1 fibroblasts, luciferase assays using extracts from these cells, and electrophoretic mobility assays failed to reveal differences between wild-type and mutant GCMB in expression level, transactivational capacity, and DNA-binding ability. Furthermore, pulse-chase experiments demonstrated no difference in half-life of wild-type and mutant protein. We conclude that mutations in the transcription factor GCMB do not seem to play a major role in the pathogenesis of PHPT.
Erratic regulation of glucose metabolism including hyperglycemia is a common condition of premature infants and is associated with increased morbidity and mortality.
To examine histological and ultra-structural differences in the endocrine pancreas in fetal (throughout gestation) and neonatal baboons.
Twelve fetal baboons were delivered at 125 days (d) gestational age (GA), 140dGA, or 175dGA. Eight animals were delivered at term (185dGA); half were fed for 5d. Seventy-three non-diabetic adult baboons were used for comparison. Pancreatic tissue was studied utilizing light microscopy, confocal imaging and electron microscopy.
The fetal and neonatal endocrine pancreas islet architecture became more organized as GA advanced. The percent areas of α-β-δ-cell type were similar within each fetal and newborn GA (NS), but were higher than the adults (P<0.05) regardless of GA. The ratio of β-cells within the islet (whole and core) increased with gestation (P<0.01). Neonatal baboons who survived for 5 days (feeding), had a 2.5-fold increase in pancreas weight compared to their counterparts euthanized at birth (P=0.01). Endocrine cells were found amongst exocrine ductal and acinar cells in 125,140 and 175dGA fetuses. Subpopulation of cells that co-expressed trypsin and glucagon/insulin show the presence of cells with mixed endo-exocrine lineage in fetuses.
The fetal endocrine pancreas has no prevalence of a of α-β-δ-cell type with larger endocrine cell percent areas than adults. Cells with mixed endocrine/exocrine phenotype occur during fetal development. Developmental differences may play a role in glucose homeostasis during the neonatal period and may have long term implications.
Insulin; glucagon; fetus; islet cells; primates
Fat infiltration within muscle is one of a number of features of vitamin D deficiency, which leads to a decline in muscle functionality. The origin of this fat is unclear, but one possibility is that it forms from myogenic precursor cells present in the muscle, which transdifferentiate into mature adipocytes. The current study examined the effect of the active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), on the capacity of the C2C12 muscle cell line to differentiate towards the myogenic and adipogenic lineages. Cells were cultured in myogenic or adipogenic differentiation media containing increasing concentrations (0, 10−13, 10−11, 10−9, 10−7 or 10−5 M) of 1,25(OH)2D3 for up to 6 days and markers of muscle and fat development were measured. Mature myofibres were formed in both adipogenic and myogenic media, but fat droplets were only observed in adipogenic media. Relative to controls, low physiological concentrations (10−13 and 10−11 M) of 1,25(OH)2D3 increased fat droplet accumulation, whereas high physiological (10−9 M) and supraphysiological concentrations (≥10−7 M) inhibited fat accumulation. This increased accumulation of fat with low physiological concentrations (10−13 and 10−11 M) was associated with a sequential up-regulation of Pparγ2 (Pparg) and Fabp4 mRNA, indicating formation of adipocytes, whereas higher concentrations (≥10−9 M) reduced all these effects, and the highest concentration (10−5 M) appeared to have toxic effects. This is the first study to demonstrate dose-dependent effects of 1,25(OH)2D3 on the transdifferentiation of muscle cells into adipose cells. Low physiological concentrations (possibly mimicking a deficient state) induced adipogenesis, whereas higher (physiological and supraphysiological) concentrations attenuated this effect.
myogenesis; adipogenesis; vitamin D; transdifferentiation
We have shown in rats that sodium salicylate (SS), which inhibits IkBa kinase B (IKKB), prevents hepatic and peripheral insulin resistance caused by short-term (7 h) i.v. administration of Intralipid and heparin (IH). We wished to further determine whether this beneficial effect of SS persisted after prolonged (48 h) IH infusion, which better mimics the chronic free fatty acid (FFA) elevation of obesity. Hence, we performed hyperinsulinemic euglycemic clamps with tritiated glucose methodology to determine hepatic and peripheral insulin sensitivity in rats infused with saline, IH, IH and SS, or SS alone. SS prevented peripheral insulin resistance (P<0.05) caused by prolonged plasma FFA elevation; however, it did not prevent hepatic insulin resistance. In skeletal muscle, protein levels of phospho-IkBa were augmented by prolonged IH administration and this was prevented by SS, suggesting that IH activates while SS prevents the activation of IKKB. Markers of IKKB activation, namely protein levels of phospho-IkBa and IkBa, indicated that IKKB is not activated in the liver after prolonged FFA elevation. Phosphorylation of serine 307 at insulin receptor substrate (IRS)-1, which is a marker of proximal insulin resistance, was not altered by IH administration in the liver, suggesting that this is not a site of hepatic insulin resistance in the prolonged lipid infusion model. Our results suggest that the role of IKKB in fat-induced insulin resistance is time and tissue dependent and that hepatic insulin resistance induced by prolonged lipid elevation is not due to an IRS-1 serine 307 kinase.
free fatty acids; insulin resistance; liver; glucose metabolism
Polycystic ovary syndrome (PCOS) is a common fertility disorder with metabolic sequelae. Our lab previously characterized reproductive phenotypes in a prenatally androgenized (PNA) mouse model for PCOS. PNA mice exhibited elevated testosterone and luteinising hormone (LH) levels, irregular oestrous cycles, and neuroendocrine abnormalities suggesting increased central drive to the reproductive system. In this study we examined metabolic characteristics of female PNA mice. PNA mice exhibited increased fasting glucose and impaired glucose tolerance (IGT) that were independent of age and were not associated with changes in body composition or peripheral insulin sensitivity. IGT was associated with defects in pancreatic islet function leading to an impaired response to high glucose, consistent with impaired insulin secretion. Exposure of isolated pancreatic islets to androgen in vitro demonstrated an impaired response to glucose stimulation similar to that in PNA mice, suggesting androgens may have activational in addition to organizational effects on pancreatic islet function. PNA mice also exhibited increased size of visceral adipocytes, suggesting androgens programmed differences in adipocyte differentiation and/or function. These studies demonstrate that in addition to causing reproductive axis abnormalities, in utero androgen exposure can induce long-term metabolic alterations in female mice.