Alexander, C. M. | Search bioRxiv

bioRxiv2022

Regulation of metabolic health by dietary histidine in mice

Low protein (LP) diets are associated with a decreased risk of diabetes in humans, and a low protein diet promotes leanness and glycemic control in both rodents and humans. While the effects of a LP diet on glycemic control are mediated by reduced dietary levels of the branched- chain amino acids (BCAAs), we have observed that reducing dietary levels of the other six essential amino acids leads to changes in body composition. Here, we find that dietary histidine plays a key role in the response to a LP diet in male C57BL/6J mice. Specifically reducing dietary levels of histidine by 67% reduces weight gain of young, lean male mice, reducing both adipose and lean mass gain, without altering glucose metabolism. Specifically reducing dietary histidine rapidly reverses diet-induced obesity and hepatic steatosis in diet-induced obese male mice, increasing insulin sensitivity; this normalization of metabolic health was associated not with caloric restriction or increased activity, but with increased energy expenditure. We find that the effects of histidine restriction surprisingly does not require the energy balance hormone Fgf21. Histidine restriction started in mid-life promoted leanness and glucose tolerance in aged males but not females, but did not affect frailty or lifespan in either sex. Finally, we demonstrate that variation in dietary histidine levels helps to explain body mass index differences in humans. Overall, our findings demonstrate that dietary histidine is a key regulator of weight and body composition in male mice and in humans, and suggest that reducing dietary levels of histidine may be a highly translatable option for the treatment of obesity. Key PointsO_LIProtein restriction (PR) promotes metabolic health in rodents and humans and extends rodent lifespan. C_LIO_LIRestriction of specific individual essential amino acids can recapitulate the benefits of PR. C_LIO_LIReduced histidine promotes leanness and increased energy expenditure in mice. C_LIO_LIReduced histidine does not extend the lifespan of mice when begun in mid-life. C_LIO_LIDietary levels of histidine are positively associated with BMI in humans. C_LI

physiology↗

bioRxiv2021

Enhanced Immunoprecipitation Techniques for the Identification of RNA Binding Protein Partners: CRD-BP interactions in mammary epithelial cells

RNA binding proteins (RBPs) regulate expression of large cohorts of RNA species to affect programmatic changes in cellular phenotypes. In order to describe the function of RBPs within a cell, it is key to identify their mRNA binding partners. This is often done by cross-linking nucleic acids to RBPs, followed by chemical release of the nucleic acid fragments for analysis. However, this methodology is lengthy, involves complex processing leading to extraordinary losses, requires large amounts of starting materials, and is prone to artifacts due to the labile nature of mRNA. To evaluate potential alternative technologies, we tested "exclusion-based" purification of immunoprecipitates (oil-based IFAST or air-based SLIDE), and report here that these methods can efficiently, rapidly and specifically isolate RBP-RNA complexes with minimal handling. The analysis starts with >100x less material than for techniques that include cross-linking. Depending on the specific antibody used, 50-100% of starting protein is retrieved, allowing the assay of endogenous levels of RBP instead of tagged and over-expressed ectopic proteins. Isolated protein and nucleic acid components are purified and analyzed using standard techniques to provide a comprehensive portrait of RBP complexes. Using exclusion-based techniques, we show that the mRNA binding partners for CRD-BP/IMP1/IGF2BP1/ZBP1 in cultured mammary epithelial cells are enriched in mRNAs important for de-toxifying superoxides (glutathione metabolic enzymes) and other mRNAs encoding mitochondrial proteins.

molecular biology↗