ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus-pituitary-adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MC1R-MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD.adrenocorticotropic hormone (ACTH) ͉ familial glucocorticoid deficiency (FGD) ͉ hypothalamus-pituitary-adrenal ͉ zona fasciculata T he adrenal gland regulates a number of essential physiological functions in adult organisms through the production of steroids and catecholamines. Maintenance of adrenal structure and function is regulated through the integration of extra-and intracellular signals. The pituitary hormone ACTH (i.e., adrenocorticotropic hormone), which is derived from the proopiomelanocortin (POMC) polypeptide precursor, is the principal regulator that stimulates adrenal glucocorticoid (GC) biosynthesis and secretion via the membrane-bound specific receptor for ACTH, ACTH receptor/melanocortin 2 receptor (MC2R) (1).It was previously demonstrated that, although POMC knockout (KO) mice are born at the expected Mendelian frequency, three-quarters of POMC KO mice undergo neonatal death. Furthermore, those mice surviving to adulthood exhibit obesity, pigmentation defects, and adrenal insufficiency (2-4). POMC KO mice possess macroscopically detectable adrenal glands that lack normal architecture (2, 4, 5). These results demonstrate the importance of POMC-derived peptides in regulating the hypothalamus-pituitary-adrenal axis and adrenal development.Familial glucocorticoid deficiency (FGD), or hereditary unresponsiveness to ACTH [Online Mendelian Inheritance in Man (OMIM) no. 202200; www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?idϭ202200], is an autosomal recessive disorder ...
BackgroundPerilipin A (PeriA) exclusively locates on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Previously, we reported that adipocyte specific overexpression of PeriA caused resistance to diet-induced obesity and resulted in improved insulin sensitivity. In order to better understand the biological basis for this observed phenotype, we performed additional studies in this transgenic mouse model.Methodology and Principal FindingsWhen compared to control animals, whole body energy expenditure was increased in the transgenic mice. Subsequently, we performed DNA microarray analysis and real-time PCR on white adipose tissue. Consistent with the metabolic chamber data, we observed increased expression of genes associated with fatty acid β-oxidation and heat production, and a decrease in the genes associated with lipid synthesis. Gene expression of Pgc1a, a regulator of fatty acid oxidation and Ucp1, a brown adipocyte specific protein, was increased in the white adipose tissue of the transgenic mice. This observation was subsequently verified by both Western blotting and histological examination. Expression of RIP140, a regulator of white adipocyte differentiation, and the lipid droplet protein FSP27 was decreased in the transgenic mice. Importantly, FSP27 has been shown to control gene expression of these crucial metabolic regulators. Overexpression of PeriA in 3T3-L1 adipocytes also reduced FSP27 expression and diminished lipid droplet size.ConclusionsThese findings demonstrate that overexpression of PeriA in white adipocytes reduces lipid droplet size by decreasing FSP27 expression and thereby inducing a brown adipose tissue-like phenotype. Our data suggest that modulation of lipid droplet proteins in white adipocytes is a potential therapeutic strategy for the treatment of obesity and its related disorders.
Journal of Lipid ResearchObesity is associated with metabolic dysfunction and increased risk for diabetes, cardiovascular disease, cancer, and early mortality ( 1, 2 ). In both lean and obese states, triacylglycerol is predominately stored within lipid droplets of adipocytes. Perilipin A (PeriA) is the most abundant phosphoprotein on adipocyte lipid droplets and is an important regulator of lipid storage and release (lipolysis) ( 3, 4 ). In the absence of hormonal stimulation (basal state), PeriA functions to inhibit the actions of lipases on stored neutral lipids, thereby maintaining a low rate of constitutive lipolysis (promotes lipid storage) ( 5-10 ). Catecholamines enhance adipocyte lipolysis by stimulating adenylate cyclase activity and consequently activating cAMP-dependent protein kinase A (PKA), which simultaneously phosphorylates perilipin and hormone-sensitive lipase (HSL) ( 3,11,12 ). PKA-dependent phosphorylation of multiple serine residues on perilipin releases CGI-58 from the lipid droplet protein, resulting in enhanced activity of adipose triglyceride lipase (ATGL) ( 13, 14 ) and enhanced HSL accessibility to lipid stores ( 4,5,10,(15)(16)(17). As a result of these combined actions, lipolysis is dramatically increased.Abstract Perilipin A is the most abundant phosphoprotein on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Perilipin null mice exhibit diminished adipose tissue, elevated basal lipolysis, reduced catecholamine-stimulated lipolysis, and increased insulin resistance. To understand the physiological consequences of increased perilipin expression in vivo, we generated transgenic mice that overexpressed either human or mouse perilipin using the adipocyte-specifi c aP2 promoter/enhancer. Phenotypes of female transgenic and wild-type mice were characterized on chow and high-fat diets (HFDs). When challenged with an HFD, transgenic mice exhibited lower body weight, fat mass, and adipocyte size than wild-type mice. Expression of oxidative genes was increased and lipogenic genes decreased in brown adipose tissue of transgenic mice. Basal and catecholaminestimulated lipolysis was decreased and glucose tolerance signifi cantly improved in transgenic mice fed a HFD. Perilipin overexpression in adipose tissue protects against HFD-induced adipocyte hypertrophy, obesity, and glucose intolerance. Alterations in brown adipose tissue metabolism may mediate the effects of perilipin overexpression on body fat, although the mechanisms by which perilipin overexpression alters brown adipose tissue metabolism remain to be determined. Our fi ndings demonstrate a novel role for perilipin expression in adipose tissue metabolism and regulation of obesity and its metabolic complications. -Miyoshi,
Perilipin, a family of phosphoproteins located around lipid droplets in adipocytes, is essential for enlargement of lipid droplets and lipolytic reaction by hormone-sensitive lipase. Thiazolidinediones, peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been shown to increase perilipin expression in fully differentiated adipocytes. However, the precise mechanism of transcriptional regulation of murine perilipin gene heretofore remains unclear. We determined the transcription start site of murine perilipin gene by RNA ligase-mediated rapid amplification of the cDNA ends method. We generated luciferase reporter gene constructs containing various lengths of the 5'-flanking region of the murine perilipin gene and assayed promoter/enhancer activities using differentiated 3T3-L1 adipocytes. We identified a functional PPAR-responsive element (PPRE) in the murine perilipin promoter, and this was confirmed by gel EMSAs using nuclear extracts from differentiated 3T3-L1 adipocytes. Furthermore, point mutations of the identified functional PPRE markedly reduced both the reporter gene activity in differentiated 3T3-L1 adipocytes and PPARgamma/thiazolidinedione-induced transactivation in NIH-3T3 fibroblasts. Real-time RT-PCR revealed that thiazolidinedione up-regulates endogenous perilipin mRNA levels. We propose that PPARgamma plays a significant role in the transcriptional regulation of murine perilipin gene via the PPRE in its promoter.
The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) in cells of the renal proximal tubule mediates the reduction in membrane expression of the sodiumdependent P i co-transporters, NPT2a and NPT2c, and thus suppresses the re-uptake of P i from the filtrate. In most cell types, the liganded PTHR1 activates G␣ S /adenylyl cyclase/cAMP/PKA (cAMP/PKA) and G␣ q/11 /phospholipase C/phosphatidylinositol 1,4,5-trisphosphate (IP 3 )/Ca 2؉ /PKC (IP 3 /PKC) signaling pathways, but the relative roles of each pathway in mediating renal regulation P i transport remain uncertain. We therefore explored the signaling mechanisms involved in PTH-dependent regulation of NPT2a function using potent, long-acting PTH analogs, M-PTH(1-28) (where M ؍ Ala P uptake, and these responses persisted for more than 24 h after ligand wash-out, whereas that of PTH(1-34) was terminated by 4 h. When injected into wild-type mice, both M-modified PTH analogs induced prolonged reductions in blood P i levels and commensurate reductions in NPT2a expression in the renal brush border membrane. Our findings suggest that the acute down-regulation of NPT2a expression by PTH ligands involves mainly the cAMP/PKA signaling pathway and are thus consistent with the elevated blood P i levels seen in pseudohypoparathyroid patients, in whom G␣ smediated signaling in renal proximal tubule cells is defective.Parathyroid hormone (PTH), 2 the most important peptide hormone regulator of calcium homeostasis, also contributes importantly to the regulation blood phosphorus levels. These physiologically vital actions of PTH are mediated through the PTH/PTH-related peptide (PTHrP) receptor (PTHR1), which is abundantly expressed in the renal proximal tubules (1, 2). Activation of this G protein-coupled receptor by PTH suppresses the reabsorption of P i by diminishing protein levels of the sodium-dependent phosphate co-transporter NPT2a, the major phosphate transporter in the renal proximal tubules (3). In addition, PTH regulates the renal type 2c sodium-dependent phosphate co-transporter (NPT2c), as shown by studies in rats rendered hyperphosphatemic by thyroparathyroidectomy, in which exogenous PTH administration causes a marked reduction in the level of NPT2c in renal brush border membrane vesicles (4). This more recently discovered transporter plays a critical role in P i homeostasis in humans because homozygous or compound heterozygous loss-of-function mutations in NPT2c cause hereditary hypophosphatemic rickets with hypercalciuria (5-7).The OK cell line, which is derived from opossum kidneyproximal tubular cells and thus provides endogenous expression of the PTHR1 and NPT2a, is currently the only genetically non-manipulated cell line for studying PTH-dependent inhibition of phosphate transport (8 -10). As in most other cells expressing the PTHR1, stimulation of OK cells with PTH agonists results in the activation of two G protein-dependent signaling pathways, the G␣ s /adenylyl cyclase/cAMP/PKA (cAMP/PKA) pathway and the G␣ q/11 /PLC/IP 3 /Ca 2ϩ /P...
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