Nrg4 promotes fuel oxidation and a healthy adipokine profile to ameliorate diet-induced metabolic disorders.

OBJECTIVE: Brown and white adipose tissue exerts pleiotropic effects on systemic energy metabolism in part by releasing endocrine factors. Neuregulin 4 (Nrg4) was recently identified as a brown fat-enriched secreted factor that ameliorates diet-induced metabolic disorders, including insulin resistance and hepatic steatosis. However, the physiological mechanisms through which Nrg4 regulates energy balance and glucose and lipid metabolism remain incompletely understood. The aims of the current study were: i) to investigate the regulation of adipose Nrg4 expression during obesity and the physiological signals involved, ii) to elucidate the mechanisms underlying Nrg4 regulation of energy balance and glucose and lipid metabolism, and iii) to explore whether Nrg4 regulates adipose tissue secretome gene expression and adipokine secretion. METHODS: We examined the correlation of adipose Nrg4 expression with obesity in a cohort of diet-induced obese mice and investigated the upstream signals that regulate Nrg4 expression. We performed metabolic cage and hyperinsulinemic-euglycemic clamp studies in Nrg4 transgenic mice to dissect the metabolic pathways regulated by Nrg4. We investigated how Nrg4 regulates hepatic lipid metabolism in the fasting state and explored the effects of Nrg4 on adipose tissue gene expression, particularly those encoding secreted factors. RESULTS: Adipose Nrg4 expression is inversely correlated with adiposity and regulated by pro-inflammatory and anti-inflammatory signaling. Transgenic expression of Nrg4 increases energy expenditure and augments whole body glucose metabolism. Nrg4 protects mice from diet-induced hepatic steatosis in part through activation of hepatic fatty acid oxidation and ketogenesis. Finally, Nrg4 promotes a healthy adipokine profile during obesity. CONCLUSIONS: Nrg4 exerts pleiotropic beneficial effects on energy balance and glucose and lipid metabolism to ameliorate obesity-associated metabolic disorders. Biologic therapeutics based on Nrg4 may improve both type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) in patients.

Whole Exome Sequencing Reveals Novel PHEX Splice Site Mutations in Patients with Hypophosphatemic Rickets.

OBJECTIVE: Hypophosphatemic rickets (HR) is a heterogeneous genetic phosphate wasting disorder. The disease is most commonly caused by mutations in the PHEX gene located on the X-chromosome or by mutations in CLCN5, DMP1, ENPP1, FGF23, and SLC34A3. The aims of this study were to perform molecular diagnostics for four patients with HR of Indian origin (two independent families) and to describe their clinical features. METHODS: We performed whole exome sequencing (WES) for the affected mother of two boys who also displayed the typical features of HR, including bone malformations and phosphate wasting. B-lymphoblast cell lines were established by EBV transformation and subsequent RT-PCR to investigate an uncommon splice site variant found by WES. An in silico analysis was done to obtain accurate nucleotide frequency occurrences of consensus splice positions other than the canonical sites of all human exons. Additionally, we applied direct Sanger sequencing for all exons and exon/intron boundaries of the PHEX gene for an affected girl from an independent second Indian family. RESULTS: WES revealed a novel PHEX splice acceptor mutation in intron 9 (c.1080-3C>A) in a family with 3 affected individuals with HR. The effect on splicing of this mutation was further investigated by RT-PCR using RNA obtained from a patient's EBV-transformed lymphoblast cell line. RT-PCR revealed an aberrant splice transcript skipping exons 10-14 which was not observed in control samples, confirming the diagnosis of X-linked dominant hypophosphatemia (XLH). The in silico analysis of all human splice sites adjacent to all 327,293 exons across 81,814 transcripts among 20,345 human genes revealed that cytosine is, with 64.3%, the most frequent nucleobase at the minus 3 splice acceptor position, followed by thymidine with 28.7%, adenine with 6.3%, and guanine with 0.8%. We generated frequency tables and pictograms for the extended donor and acceptor splice consensus regions by analyzing all human exons. Direct Sanger sequencing of all PHEX exons in a sporadic case with HR from the Indian subcontinent revealed an additional novel PHEX mutation (c.1211_1215delACAAAinsTTTACAT, p.Asp404Valfs*5, de novo) located in exon 11. CONCLUSIONS: Mutation analyses revealed two novel mutations and helped to confirm the clinical diagnoses of XLH in two families from India. WES helped to analyze all genes implicated in the underlying disease complex. Mutations at splice positions other than the canonical key sites need further functional investigation to support the assertion of pathogenicity.