Gs? deficiency in the dorsomedial hypothalamus underlies obesity associated with Gs? mutations.
ABSTRACT: Gs?, encoded by Gnas, mediates hormone and neurotransmitter receptor-stimulated cAMP generation. Heterozygous Gs?-inactivating mutations lead to obesity in Albright hereditary osteodystrophy (AHO) patients, but only when the mutations occur on the maternal allele. This parent-of-origin effect is due to Gs? imprinting in the CNS, although the relevant CNS regions are unknown. We have now shown that mice with a Gnas gene deletion disrupting Gs? expression on the maternal allele, but not the paternal allele, in the dorsomedial nucleus of the hypothalamus (DMH) developed obesity and reduced energy expenditure without hyperphagia. Although maternal Gnas deletion impaired activation of brown adipose tissue (BAT) in mice, their responses to cold environment remained intact. Similar findings were observed in mice with DMH-specific deficiency of melanocortin MC4R receptors, which are known to activate Gs?. Our results show that Gs? imprinting in the DMH underlies the parent-of-origin metabolic phenotype that results from Gs? mutations and that DMH MC4R/Gs? signaling is important for regulation of energy expenditure and BAT activation, but not the metabolic response to cold.
Project description:Central melanocortin 4 receptors (MC4Rs) stimulate energy expenditure and inhibit food intake. MC4Rs activate the G protein Gs?, but whether Gs? mediates all MC4R actions has not been established. Individuals with Albright hereditary osteodystrophy (AHO), who have heterozygous Gs?-inactivating mutations, only develop obesity when the Gs? mutation is present on the maternal allele because of tissue-specific genomic imprinting. Furthermore, evidence in mice implicates Gs? imprinting within the central nervous system (CNS) in this disorder. In this study, we examined the effects of Gs? in MC4R-expressing cells on metabolic regulation. Mice with homozygous Gs? deficiency in MC4R-expressing cells (MC4RGsKO) developed significant obesity with increased food intake and decreased energy expenditure, along with impaired insulin sensitivity and cold-induced thermogenesis. Moreover, the ability of the MC4R agonist melanotan-II (MTII) to stimulate energy expenditure and to inhibit food intake was impaired in MC4RGsKO mice. MTII failed to stimulate the secretion of the anorexigenic hormone peptide YY (PYY) from enteroendocrine L cells, a physiological response mediated by MC4R-Gs? signaling, even though baseline PYY levels were elevated in these mice. In Gs? heterozygotes, mild obesity and reduced energy expenditure were present only in mice with a Gs? deletion on the maternal allele in MC4R-expressing cells, whereas food intake was unaffected. These results demonstrate that Gs? signaling in MC4R-expressing cells is required for controlling energy balance, thermogenesis, and peripheral glucose metabolism. They further indicate that Gs? imprinting in MC4R-expressing cells contributes to obesity in Gs? knockout mice and probably in individuals with Albright hereditary osteodystrophy as well.
Project description:BACKGROUND:The stimulatory G-protein ?-subunit encoded by GNAS exons 1-13 (GNAS-Gs?) mediates signal transduction of multiple G protein-coupled receptors, including arginine vasopressin receptor 2 (AVPR2). Various germline-derived loss-of-function GNAS-Gs? variants of maternal and paternal origin have been found in pseudohypoparathyroidism type Ia and pseudopseudohypoparathyroidism, respectively. Specific somatic gain-of-function GNAS-Gs? variants have been detected in McCune-Albright syndrome and may result in phosphate wasting. However, no germline-derived gain-of-function variant has been identified, implying that such a variant causes embryonic lethality. METHODS:We performed whole-exome sequencing in two families with dominantly inherited nephrogenic syndrome of inappropriate antidiuresis (NSIAD) as a salient phenotype after excluding a gain-of-function variant of AVPR2 and functional studies for identified variants. RESULTS:Whole-exome sequencing revealed two GNAS-Gs? candidate variants for NSIAD: GNAS-Gs? p.(F68_G70del) in one family and GNAS-Gs? p.(M255V) in one family. Both variants were absent from public and in-house databases. Of genes with rare variants, GNAS-Gs? alone was involved in AVPR2 signaling and shared by the families. Protein structural analyses revealed a gain-of-function-compatible conformational property for p.M255V-Gs?, although such assessment was not possible for p.F68_G70del-Gs?. Both variants had gain-of-function effects that were significantly milder than those of McCune-Albright syndrome-specific somatic Gs? variants. Model mice for p.F68_G70del-Gs? showed normal survivability and NSIAD-compatible phenotype, whereas those for p.M255V-Gs? exhibited severe failure to thrive. CONCLUSIONS:This study shows that germline-derived gain-of-function rare variants of GNAS-Gs? exist and cause NSIAD as a novel Gs?-mediated genetic disease. It is likely that AVPR2 signaling is most sensitive to GNAS-Gs?'s gain-of-function effects.
Project description:The murine Gnas (human GNAS) locus gives rise to G?s and different splice variants thereof. The G?s promoter is not methylated thus allowing biallelic expression in most tissues. In contrast, the alternative first Gnas/GNAS exons and their promoters undergo parent specific methylation, which limits transcription to the non-methylated allele. Pseudohypoparathyroidism type Ia (PHP1A) or type Ib (PHP1B) are caused by heterozygous maternal GNAS mutations suggesting that little or no G?s is derived in some tissues from the non-mutated paternal GNAS thereby causing hormonal resistance. Previous data had indicated that G?s is mainly derived from the maternal Gnas allele in brown adipose tissue (BAT) of newborn mice, yet it is biallelically expressed in adult BAT. This suggested that paternal G?s expression is regulated by an unknown factor(s) that varies considerably with age. To extend these findings, we now used a strain-specific SNP in Gnas exon 11 (rs13460569) for evaluation of parent-specific G?s expression through the densitometric quantification of BanII-digested RT-PCR products and digital droplet PCR (ddPCR). At all investigated ages, G?s transcripts were derived in BAT predominantly from the maternal Gnas allele, while kidney and liver showed largely biallelic G?s expression. Only low or undetectable levels of other paternally Gnas-derived transcripts were observed, making it unlikely that these are involved in regulating paternal G?s expression. Our findings suggest that a cis-acting factor could be implicated in reducing paternal G?s expression in BAT and presumably in proximal renal tubules, thereby causing PTH-resistance if the maternal GNAS/Gnas allele is mutated.
Project description:Approximately 100 genes undergo genomic imprinting. Mutations in fewer than 10 imprinted genetic loci, including GNAS, are associated with complex human diseases that differ phenotypically based on the parent transmitting the mutation. Besides the ubiquitously expressed Gsalpha, which is of broad biological importance, GNAS gives rise to an antisense transcript and to several Gsalpha variants that are transcribed from the nonmethylated parental allele. We previously identified two almost identical GNAS microdeletions extending from exon NESP55 to antisense (AS) exon 3 (delNESP55/delAS3-4). When inherited maternally, both deletions are associated with erasure of all maternal GNAS methylation imprints and autosomal-dominant pseudohypoparathyroidism type Ib, a disorder characterized by parathyroid hormone-resistant hypocalcemia and hyperphosphatemia. As for other imprinting disorders, the mechanisms resulting in abnormal GNAS methylation are largely unknown, in part because of a paucity of suitable animal models. We now showed in mice that deletion of the region equivalent to delNESP55/delAS3-4 on the paternal allele (DeltaNesp55(p)) leads to healthy animals without Gnas methylation changes. In contrast, mice carrying the deletion on the maternal allele (DeltaNesp55(m)) showed loss of all maternal Gnas methylation imprints, leading in kidney to increased 1A transcription and decreased Gsalpha mRNA levels, and to associated hypocalcemia, hyperphosphatemia, and secondary hyperparathyroidism. Besides representing a murine autosomal-dominant pseudohypoparathyroidism type Ib model and one of only few animal models for imprinted human disorders, our findings suggest that the Nesp55 differentially methylated region is an additional principal imprinting control region, which directs Gnas methylation and thereby affects expression of all maternal Gnas-derived transcripts.
Project description:Skeletal bone formation and maintenance requires coordinate functions of several cell types, including bone forming osteoblasts and bone resorbing osteoclasts. Gs?, the stimulatory subunit of heterotrimeric G proteins, activates downstream signaling through cAMP and plays important roles in skeletal development by regulating osteoblast differentiation. Here, we demonstrate that Gs? signaling also regulates osteoclast differentiation during bone modeling and remodeling. Gnas, the gene encoding Gs?, is imprinted. Mice with paternal allele deletion of Gnas (Gnas<sup>+/p-</sup>) have defects in cortical bone quality and strength during early development (bone modeling) that persist during adult bone remodeling. Reduced bone quality in Gnas<sup>+/p-</sup> mice was associated with increased endosteal osteoclast numbers, with no significant effects on osteoblast number and function. Osteoclast differentiation and resorption activity was enhanced in Gnas<sup>+/p-</sup> cells. During differentiation, Gnas<sup>+/p-</sup> cells showed diminished pCREB, ?-catenin and cyclin D1, and enhanced Nfatc1 levels, conditions favoring osteoclastogenesis. Forskolin treatment increased pCREB and rescued osteoclast differentiation in Gnas<sup>+/p-</sup> by reducing Nfatc1 levels. Cortical bone of Gnas<sup>+/p-</sup> mice showed elevated expression of Wnt inhibitors sclerostin and Sfrp4 consistent with reduced Wnt/?-catenin signaling. Our data identify a new role for Gs? signaling in maintaining bone quality by regulating osteoclast differentiation and function through cAMP/PKA and Wnt/?-catenin pathways.
Project description:XL?s, a variant of the stimulatory G protein ?-subunit (Gs?), can mediate receptor-activated cAMP generation and, thus, mimic the actions of Gs? in transfected cells. However, it remains unknown whether XL?s can act in a similar manner in vivo. We have now generated mice with ectopic transgenic expression of rat XL?s in the renal proximal tubule (rptXL?s mice), where Gs? mediates most actions of PTH. Western blots and quantitative RT-PCR showed that, while Gs? and type-1 PTH receptor levels were unaltered, protein kinase A activity and 25-hydroxyvitamin D 1-?-hydroxylase (Cyp27b1) mRNA levels were significantly higher in renal proximal tubules of rptXL?s mice than wild-type littermates. Immunohistochemical analysis of kidney sections showed that the sodium-phosphate cotransporter type 2a was modestly reduced in brush border membranes of male rptXL?s mice compared to gender-matched controls. Serum calcium, phosphorus, and 1,25 dihydroxyvitamin D were within the normal range, but serum PTH was ?30% lower in rptXL?s mice than in controls (152 ± 16 vs. 222 ± 41 pg/ml; P < 0.05). After crossing the rptXL?s mice to mice with ablation of maternal Gnas exon 1 (E1(m-/+)), male offspring carrying both the XL?s transgene and maternal Gnas exon 1 ablation (rptXL?s/E1(m-/+)) were significantly less hypocalcemic than gender-matched E1(m-/+) littermates. Both E1(m-/+) and rptXL?s/E1(m-/+) offspring had higher serum PTH than wild-type littermates, but the degree of secondary hyperparathyroidism tended to be lower in rptXL?s/E1(m-/+) mice. Hence, transgenic XL?s expression in the proximal tubule enhanced Gs?-mediated responses, indicating that XL?s can mimic Gs? in vivo.
Project description:Pseudohypoparathyroidism (PHP) is caused by reduced expression of genes within the GNAS cluster, resulting in parathormone resistance. The cluster contains multiple imprinted transcripts, including the stimulatory G protein α subunit (Gs-α) and NESP55 transcript preferentially expressed from the maternal allele, and the paternally expressed XLas, A/B and antisense transcripts. PHP1b can be caused by loss of imprinting affecting GNAS A/B alone (associated with STX16 deletion), or the entire GNAS cluster (associated with deletions of NESP55 in a minority of cases). We performed targeted genomic next-generation sequencing (NGS) of the GNAS cluster to seek variants and indels underlying PHP1b. Seven patients were sequenced by hybridisation-based capture and fourteen more by long-range PCR and transposon-mediated insertion and sequencing. A bioinformatic pipeline was developed for variant and indel detection. In one family with two affected siblings, and in a second family with a single affected individual, we detected maternally inherited deletions of 40 and 33 bp, respectively, within the deletion previously reported in rare families with PHP1b. All three affected individuals presented with atypically severe PHP1b; interestingly, the unaffected mother in one family had the detected deletion on her maternally inherited allele. Targeted NGS can reveal sequence changes undetectable by current diagnostic methods. Identification of genetic mutations underlying epigenetic changes can facilitate accurate diagnosis and counselling, and potentially highlight genetic elements critical for normal imprint setting.
Project description:The imprinted Gnas cluster is involved in obesity, energy metabolism, feeding behavior, and viability. Relative contribution of paternally expressed proteins XL?s, XLN1, and ALEX or a double dose of maternally expressed Gs? to phenotype has not been established. In this study, we have generated two new mutants (Ex1A-T-CON and Ex1A-T) at the Gnas cluster. Paternal inheritance of Ex1A-T-CON leads to loss of imprinting of Gs?, resulting in preweaning growth retardation followed by catch-up growth. Paternal inheritance of Ex1A-T leads to loss of imprinting of Gs? and loss of expression of XL?s and XLN1. These mice have severe preweaning growth retardation and incomplete catch-up growth. They are fully viable probably because suckling is unimpaired, unlike mutants in which the expression of all the known paternally expressed Gnasxl proteins (XL?s, XLN1 and ALEX) is compromised. We suggest that loss of ALEX is most likely responsible for the suckling defects previously observed. In adults, paternal inheritance of Ex1A-T results in an increased metabolic rate and reductions in fat mass, leptin, and bone mineral density attributable to loss of XL?s. This is, to our knowledge, the first report describing a role for XL?s in bone metabolism. We propose that XL?s is involved in the regulation of bone and adipocyte metabolism.
Project description:The Gnas locus in the mouse is imprinted with a complex arrangement of alternative transcripts defined by promoters with different patterns of monoallelic expression. The Gnas transcript is subject to tissue-specific imprinted expression, Nesp is expressed only from the maternal allele, and Gnasxl is expressed only from the paternal allele. The mechanisms controlling these expression patterns are not known. To identify potential imprinting regulatory regions, particularly for the reciprocally expressed Nesp and Gnasxl promoters, we examined epigenetic properties of the locus in gametes, embryonic stem cells, and fetal and adult tissues. The Nesp and Gnasxl promoter regions are contained in extensive CpG islands with methylation of the paternal allele at Nesp and the maternal allele at Gnasxl. Parental allele-specific DNase I-hypersensitive sites were found at these regions, which correlate with hypomethylation rather than actual expression status. A germ line methylation mark was identified covering the promoters for Gnasxl and the antisense transcript Nespas. Prominent DNase I-hypersensitive sites present on paternal alleles in embryonic stem cells are contained within this mark. This is the second gametic mark identified at Gnas and suggests that the Nesp and Gnasxl promoters are under separate control from the Gnas promoter. We propose models to account for the regulation of imprinting at the locus.
Project description:Pseudohypoparathyroidism type I (PHP-I) is divided into PHP-Ia with Albright hereditary osteodystrophy and PHP-Ib, which usually shows no Albright hereditary osteodystrophy features. Although PHP-Ia and PHP-Ib are typically caused by genetic defects involving ? subunit of the stimulatory G protein (Gs?)-coding GNAS exons and methylation defects of the GNAS differentially methylated regions (DMRs) on the maternal allele, respectively, detailed phenotypic characteristics still remains to be examined.To clarify phenotypic characteristics according to underlying (epi)genetic causes.We performed (epi)genotype-phenotype analysis in 69 Japanese patients with PHP-I; that is, 28 patients with genetic defects involving Gs?-coding GNAS exons (group 1) consisting of 12 patients with missense variants (subgroup A) and 16 patients with null variants (subgroup B), as well as 41 patients with methylation defects (group 2) consisting of 21 patients with broad methylation defects of the GNAS-DMRs (subgroup C) and 20 patients with an isolated A/B-DMR methylation defect accompanied by the common STX16 microdeletion (subgroup D).Although (epi)genotype-phenotype findings were grossly similar to those reported previously, several important findings were identified, including younger age at hypocalcemic symptoms and higher frequencies of hyperphosphatemia in subgroup C than in subgroup D, development of brachydactyly in four patients of subgroup C, predominant manifestation of subcutaneous ossification in subgroup B, higher frequency of thyrotropin resistance in group 1 than in group 2, and relatively low thyrotropin values in four patients with low T4 values and relatively low luteinizing hormone/follicle-stimulating hormone values in five adult females with ovarian dysfunction.The results imply the presence of clinical findings characteristic of each underlying cause and provide useful information on the imprinting status of Gs?.