ABSTRACT
We previously reported children homozygous for two MC3R sequence variants (C17A+G241A) have greater fat mass than controls. Here we show, using homozygous knock-in mouse models in which we replace murine Mc3r with wild-type human (MC3R(hWT/hWT)) and double-mutant (C17A+G241A) human (MC3R(hDM/hDM)) MC3R, that MC3R(hDM/hDM) have greater weight and fat mass, increased energy intake and feeding efficiency, but reduced length and fat-free mass compared with MC3R(hWT/hWT). MC3R(hDM/hDM) mice do not have increased adipose tissue inflammatory cell infiltration or greater expression of inflammatory markers despite their greater fat mass. Serum adiponectin levels are increased in MC3R(hDM/hDM) mice and MC3R(hDM/hDM) human subjects. MC3R(hDM/hDM) bone- and adipose tissue-derived mesenchymal stem cells (MSCs) differentiate into adipocytes that accumulate more triglyceride than MC3R(hWT/hWT) MSCs. MC3R(hDM/hDM) impacts nutrient partitioning to generate increased adipose tissue that appears metabolically healthy. These data confirm the importance of MC3R signalling in human metabolism and suggest a previously-unrecognized role for the MC3R in adipose tissue development.
Subject(s)
Obesity/metabolism , Receptor, Melanocortin, Type 3/metabolism , Adipocytes/metabolism , Adiponectin/metabolism , Adipose Tissue/metabolism , Animals , Disease Models, Animal , Eating , Energy Metabolism , Fats/metabolism , Gene Knock-In Techniques , Humans , Leptin/metabolism , Mice , Obesity/genetics , Obesity/physiopathology , Receptor, Melanocortin, Type 3/geneticsABSTRACT
The melanocortin 3 receptor (MC3R) is involved in regulation of energy homeostasis. However, its transcript structure is not well understood. We therefore studied initiation and termination sites for hypothalamic murine Mc3r and human MC3R transcripts. Rapid Amplification of cDNA Ends (RACE) was performed for the 5' and 3' ends of murine and human hypothalamic RNA. 5' RACE experiments using hypothalamic murine RNA indicated mouse hypothalamus expresses two major Mc3r transcription start sites: one with a 5' UTR approximately 368 bases in length and another previously unknown transcript with a 5' UTR approximately 440 bases in length. 5' RACE experiments using human hypothalamic RNA identified a 5' UTR beginning 533 bases upstream of the start codon with a 248 base splice. 3' RACE experiments using hypothalamic murine RNA indicated the 3' UTR terminates approximately 1286 bases after the translational stop codon, with a previously unknown 787 base splice between consensus splice donor and acceptor sites. 3' RACE experiments using human MC3R transcript indicated the 3' UTR terminates approximately 115-160 bases after the translational stop codon. These data provide insight into melanocortin 3 receptor transcript structure.
Subject(s)
Hypothalamus/metabolism , Receptor, Melanocortin, Type 3/genetics , 3' Untranslated Regions , 5' Untranslated Regions , Animals , Base Sequence , DNA Primers/genetics , Humans , Mice , Nucleic Acid Amplification Techniques , Polymerase Chain Reaction , Transcription Initiation SiteABSTRACT
Extracellular inorganic pyrophosphate (PP(i)) is a potent suppressor of physiological calcification in bone and pathological calcification in blood vessels. Ectonucleotide pyrophosphatase/phosphodiesterases (eNPPs) generate PP(i) via the hydrolysis of ATP released into extracellular compartments by poorly understood mechanisms. Here we report that cultured vascular smooth muscle cells (VSMC) from rat aorta generate extracellular PP(i) via an autocrine mechanism that involves ATP release tightly coupled to eNPP activity. The nucleotide analog beta,gamma-methylene ATP (MeATP or AMPPCP) was used to selectively suppress ATP metabolism by eNPPs but not the CD39-type ecto-ATPases. In the absence of MeATP, VSMC generated extracellular PP(i) to accumulate >or=600 nM within 2 h while steadily maintaining extracellular ATP at 1 nM. Conversely, the presence of MeATP completely suppressed PP(i) accumulation while increasing ATP accumulation. Probenecid, which inhibits PP(i) efflux dependent on ANK, a putative PP(i) transporter or transport regulator, reduced extracellular PP(i) accumulation by approximately twofold. This indicates that autocrine ATP release coupled to eNPP activity comprises >or=50% of the extracellular PP(i)-generating capacity of VSMC. The accumulation of extracellular PP(i) and ATP was markedly attenuated by reduced temperature but was insensitive to brefeldin A, which suppresses constitutive exocytosis of Golgi-derived secretory vesicles. The magnitude of extracellular PP(i) accumulation in VSMC cultures increased with time postplating, suggesting that ATP release coupled to PP(i) generation is upregulated as cultured VSMC undergo contact-inhibition of proliferation or deposit extracellular matrix.
