Cold-induced expression of a truncated adenylyl cyclase 3 acts as rheostat to brown fat function.

Promoting brown adipose tissue (BAT) activity innovatively targets obesity and metabolic disease. While thermogenic activation of BAT is well understood, the rheostatic regulation of BAT to avoid excessive energy dissipation remains ill-defined. Here, we demonstrate that adenylyl cyclase 3 (AC3) is key for BAT function. We identified a cold-inducible promoter that generates a 5' truncated AC3 mRNA isoform (Adcy3-at), whose expression is driven by a cold-induced, truncated isoform of PPARGC1A (PPARGC1A-AT). Male mice lacking Adcy3-at display increased energy expenditure and are resistant to obesity and ensuing metabolic imbalances. Mouse and human AC3-AT are retained in the endoplasmic reticulum, unable to translocate to the plasma membrane and lack enzymatic activity. AC3-AT interacts with AC3 and sequesters it in the endoplasmic reticulum, reducing the pool of adenylyl cyclases available for G-protein-mediated cAMP synthesis. Thus, AC3-AT acts as a cold-induced rheostat in BAT, limiting adverse consequences of cAMP activity during chronic BAT activation.

Gsα-dependent signaling is required for postnatal establishment of a functional ß-cell mass.

OBJECTIVE: Early postnatal life is a critical period for the establishment of the functional ß-cell mass that will sustain whole-body glucose homeostasis during the lifetime. ß cells are formed from progenitors during embryonic development but undergo significant expansion in quantity and attain functional maturity after birth. The signals and pathways involved in these processes are not fully elucidated. Cyclic adenosine monophosphate (cAMP) is an intracellular signaling molecule that is known to regulate insulin secretion, gene expression, proliferation, and survival of adult ß cells. The heterotrimeric G protein Gs stimulates the cAMP-dependent pathway by activating adenylyl cyclase. In this study, we sought to explore the role of Gs-dependent signaling in postnatal ß-cell development. METHODS: To study Gs-dependent signaling, we generated conditional knockout mice in which the α subunit of the Gs protein (Gsα) was ablated from ß-cells using the Cre deleter line Ins1Cre. Mice were characterized in terms of glucose homeostasis, including in vivo glucose tolerance, glucose-induced insulin secretion, and insulin sensitivity. ß-cell mass was studied using histomorphometric analysis and optical projection tomography. ß-cell proliferation was studied by ki67 and phospho-histone H3 immunostatining, and apoptosis was assessed by TUNEL assay. Gene expression was determined in isolated islets and sorted ß cells by qPCR. Intracellular cAMP was studied in isolated islets using HTRF-based technology. The activation status of the cAMP and insulin-signaling pathways was determined by immunoblot analysis of the relevant components of these pathways in isolated islets. In vitro proliferation of dissociated islet cells was assessed by BrdU incorporation. RESULTS: Elimination of Gsα in ß cells led to reduced ß-cell mass, deficient insulin secretion, and severe glucose intolerance. These defects were evident by weaning and were associated with decreased proliferation and inadequate expression of key ß-cell identity and maturation genes in postnatal ß-cells. Additionally, loss of Gsα caused a broad multilevel disruption of the insulin transduction pathway that resulted in the specific abrogation of the islet proliferative response to insulin. CONCLUSION: We conclude that Gsα is required for ß-cell growth and maturation in the early postnatal stage and propose that this is partly mediated via its crosstalk with insulin signaling. Our findings disclose a tight connection between these two pathways in postnatal ß cells, which may have implications for using cAMP-raising agents to promote ß-cell regeneration and maturation in diabetes.

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism.

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease.

LincRNA H19 protects from dietary obesity by constraining expression of monoallelic genes in brown fat.

Increasing brown adipose tissue (BAT) thermogenesis in mice and humans improves metabolic health and understanding BAT function is of interest for novel approaches to counteract obesity. The role of long noncoding RNAs (lncRNAs) in these processes remains elusive. We observed maternally expressed, imprinted lncRNA H19 increased upon cold-activation and decreased in obesity in BAT. Inverse correlations of H19 with BMI were also observed in humans. H19 overexpression promoted, while silencing of H19 impaired adipogenesis, oxidative metabolism and mitochondrial respiration in brown but not white adipocytes. In vivo, H19 overexpression protected against DIO, improved insulin sensitivity and mitochondrial biogenesis, whereas fat H19 loss sensitized towards HFD weight gains. Strikingly, paternally expressed genes (PEG) were largely absent from BAT and we demonstrated that H19 recruits PEG-inactivating H19-MBD1 complexes and acts as BAT-selective PEG gatekeeper. This has implications for our understanding how monoallelic gene expression affects metabolism in rodents and, potentially, humans.

Effects of sex steroids on the pattern of methylation and expression of the promoter region of estrogen and androgen receptors in people with gender dysphoria under cross-sex hormone treatment.

Cross-sex hormone therapy (CHT) is critical for phenotypical and physiological transition in adults with gender dysphoria (GD). However, the impact of the CHT onto the molecular level/epigenetic regulation has not been comprehensively addressed. We postulate that CHT in GD could drive changes at the androgen receptor (AR), estrogen receptor alpha (ESR1) and estrogen receptor beta (ESR2), affecting their DNA methylation pattern and mRNA expression that may influence in the phenotypical changes associated to CHT. We carried out a prospective observational study on individuals with a diagnosis of GD. 18 subjects (no previous CHT): 12 female to male (FtoM) and 6 male to female (MtoF). An Epityper Mass array TM method was used to study the DNA methylation and Real-time PCR quantitative reverse transcription PCR (qRT-PCR) was used to quantify the gene expression. The analysis of AR, ESR1 and ESR2 receptor was performed at baseline, 6 and 12 months after CHT. No differences in DNA methylation of ESR were found in MtoF, while DNA methylation was increased in FtoM at 6 and 12 months of CHT. The AR showed a significant increase of methylation in MtoF group after 12 months of estrogenic treatment. Regarding the expression analysis, AR expression was significantly decreased in FtoM upon CHT treatment. AR, ESR1 and ESR2 methylation were correlated with anthropometric, metabolic and hormonal parameters in FtoM and MtoF. Our results support that CHT is associated to epigenetic changes that might affect the response to treatment with sex steroids.

Dicer1-miR-328-Bace1 signalling controls brown adipose tissue differentiation and function.

Activation of brown adipose tissue (BAT) controls energy homeostasis in rodents and humans and has emerged as an innovative strategy for the treatment of obesity and type 2 diabetes mellitus. Here we show that ageing- and obesity-associated dysfunction of brown fat coincides with global microRNA downregulation due to reduced expression of the microRNA-processing node Dicer1. Consequently, heterozygosity of Dicer1 in BAT aggravated diet-induced-obesity (DIO)-evoked deterioration of glucose metabolism. Analyses of differential microRNA expression during preadipocyte commitment and mouse models of progeria, longevity and DIO identified miR-328 as a regulator of BAT differentiation. Reducing miR-328 blocked preadipocyte commitment, whereas miR-328 overexpression instigated BAT differentiation and impaired muscle progenitor commitment-partly through silencing of the ß-secretase Bace1. Loss of Bace1 enhanced brown preadipocyte specification in vitro and was overexpressed in BAT of obese and progeroid mice. In vivo Bace1 inhibition delayed DIO-induced weight gain and improved glucose tolerance and insulin sensitivity. These experiments reveal Dicer1-miR-328-Bace1 signalling as a determinant of BAT function, and highlight the potential of Bace1 inhibition as a therapeutic approach to improve not only neurodegenerative diseases but also ageing- and obesity-associated impairments of BAT function.

New splice site acceptor mutation in AIRE gene in autoimmune polyendocrine syndrome type 1.

Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison's disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the AIRE gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases.

Role of the single nucleotide polymorphism rs7903146 of TCF7L2 in inducing nonsense-mediated decay.

BACKGROUND: The single nucleotide polymorphism (SNP) rs7903146 (C/T), located in intron 4 of the transcription factor 7-like 2 gene (TCF7L2), has been associated with an increased risk of developing Type 2 Diabetes, although the molecular mechanism remain elusive. The TCF7L2 gene is alternatively spliced but an association between genotype and splice variants has not been shown convincingly. We hypothesized that a yet unknown extra exon, containing either the C or T genotype of the SNP rs7903146, could introduce a premature stop codon and consequently result in nonsense-mediated decay (NMD). FINDINGS: Running the sequences C and T of the SNP region in different servers we found that the two alleles could display differential recognition by splicing factors. The C variant showed the possible inclusion of an unknown exon. This unknown exon contained a stop codon and thus could induce NMD. We then determined that the splicing pattern in isolated mouse islets and MIN6 cells was similar to that in human pancreatic islets. Therefore, we used MIN6 cells to study the splicing of human intron 4: two mini-genes of intron 4 containing either the C/C genotype or the T/T genotype were transfected into MIN6 cells. Our constructs were spliced normally, excluding intron 4, but we did not observe the presence of an extra exon with either construct. CONCLUSIONS: We found that an extra exon could theoretically exist, although we were not able to capture it in our model. A better model is needed to determine whether a theoretical extra exon can induce NMD.