Effects of constitutive activation of the PI3K-mTORC2-Akt-mTORC1 pathway on the morphology and glucose and lipid metabolism of white and brown adipocytes: studying the individual contributions of mTORC1 and mTORC2.

Abstract

The PI3K-mTORC2-Akt-mTORC1 pathway regulates lipid and glucose metabolism in white and brown adipocytes through unknown mechanisms that involves the transcriptional factors SREBP1 e PPARgama, among others. One of the main issues to study these mechanisms is related to the intricate crosstalk between mTORC2-Akt and mTORC1 (mTORC2-Akt activates mTORC1, while mTORC1 inhibits mTORC2-Akt), impairing the evaluation of each individual complex biological effects. To advance our understanding of the individual role of mTORC2-Akt and mTORC1 in the regulation of lipid and glucose metabolism in white and brown adipocytes, we will use in this study the following genetically modified mice bearing: 1- constitutive activation of the PI3KmTORC2-Akt-mTORC1 pathway due to Pten deletion in adipocytes; 2- constitutive activation of mTORC2-Akt and mTORC1 deficiency due to double deletion of Pten and Raptor in adipocytes; 3- constitutive activation of mTORC1 and mTORC2-Akt deficiency due to double deletion of Rictor and Tsc1 in adipocytes. These mice will be fed either a chow or a high fat diet (HFD, 60% calories from lipids), supplemented or not with the PPARgamma agonist pioglitazone (Pio, 30 mg/kg/day) during 8 weeks or exposed to thermoneutrality (30°C) or cold (10°C) during 2 weeks and evaluated for energy balance, thermogenic capacity, glucose homeostasis, adiposity, adipocyte number and diameter, glucose and lipid metabolism, adipokine secretion, autophagy, inflammation, insulin signaling, PPARgamma and SREBP1 content and transcriptional activity and gene expression profile of their target genes. Cultured white and brown adipocytes from these mice will be evaluated for glucose and lipid metabolism, mitochondrial respiration, autophagy, insulin signaling, PPARgamma and SREBP1 content and transcriptional activity, and gene expression profile of their target genes. Data will be analyzed using GraphPad Prism version 9 for normality with the Shapiro-Wilk test, for homogeneity of variances using the Bartlett test followed by ANOVA test or Kruskal-Wallis. A significance level of P < 0.05 will be used.Keywords: mTORC2, mTORC1, adipose tissue, lipid metabolism