Genetic deletion of Rheb1 in the brain reduces food intake and causes hypoglycemia with altered peripheral metabolism

Abstract

Excessive food/energy intake is linked to obesity and metabolic disorders, such as diabetes. The hypothalamus in the brain plays a critical role in the control of food intake and peripheral metabolism. The signaling pathways in hypothalamic neurons that regulate food intake and peripheral metabolism need to be better understood for developing pharmacological interventions to manage eating behavior and obesity. Mammalian target of rapamycin (mTOR), a serine/threonine kinase, is a master regulator of cellular metabolism in different cell types. Pharmacological manipulations of mTOR complex 1 (mTORC1) activity in hypothalamic neurons alter food intake and body weight. Our previous study identified Rheb1 (Ras homolog enriched in brain 1) as an essential activator of mTORC1 activity in the brain. Here we examine whether central Rheb1 regulates food intake and peripheral metabolism through mTORC1 signaling. We find that genetic deletion of Rheb1 in the brain causes a reduction in mTORC1 activity and impairs normal food intake. As a result, Rheb1 knockout mice exhibit hypoglycemia and increased lipid mobilization in adipose tissue and ketogenesis in the liver. Our work highlights the importance of central Rheb1 signaling in euglycemia and energy homeostasis in animals.

Figure 1.

Genetic deletion of Rheb1 in the brain disrupts food intake. (A) Western blotting showing that Rheb1 and pS6 (Ser240/244) protein levels are reduced in Rheb1 ko hypothalamus; (B) Histograms showing the drastic reduction of food intake in Rheb1 ko mice compared to controls (N = 4). Results are averages of four independent animals. Data represent mean ± SEM. *** p < 0.001.

Figure 2.

Genetic deletion of Rheb1 in the brain leads to hypoglycemia. (A) Biochemical assays showing the robust reduction of blood glucose level in Rheb1 ko mice compared controls (N = 4). Results are averages of four independent animals. Data represent mean ± SEM. ** p < 0.01; (B) Real-time PCR assays showing the increasing mRNA levels of G6pc and Pck1 in livers of Rheb1 ko mice (N = 3). Results are averages of three independent animals. Data represent mean ± SEM. * p < 0.05, *** p < 0.001.

Figure 3.

Genetic deletion of Rheb1 in the brain induces ketogenesis in the liver. (A) Biochemical assays showing increased blood ketone levels in 4-week-old Rheb1 ko mice (N = 3). Results are averages of three independent animals. Data represent mean ± SEM. * p < 0.05; (B) Real-time PCR results showing the increasing levels of ketogenesis genes Hmgcs2 and Hmgcl in livers of Rheb1 ko mice (N = 3). Results are averages of three independent animals. Data represent mean ± SEM. * p < 0.05; (C,D) Western blots and histograms showing the increasing of HMGCS2 protein level in livers of Rheb1 ko mice (N = 3). Results are averages of three independent animals. Data represent mean ± SEM. ** p < 0.01, *** p < 0.001; (E,F) Western blots and histograms showing the increasing of ATGL protein level in adipose of Rheb1 ko mice (N = 3). Results are averages of three independent animals. Data represent mean ± SEM. * p < 0.05.

Figure 4.

Genetic deletion of Rheb1 in the brain increases liver lipid droplets and beta oxidation. (A) Representative oil red O staining images showing increased number of lipid droplets in livers of Rheb1 ko mice. Bar 25 μm; (B) Real-time PCR assays showing that hepatic lipogenesis genes Srebp1c, Acl and Fasn are not increased in livers of Rheb1 ko mice (N = 3). Results are averages of three independent animals. Data represent mean ± SEM. * p < 0.05; (C) Real-time PCR assays showing the increasing of mRNA levels of beta-oxidation genes, Atgl, Pparα, Cpt1-a, Mlycd and Mcad in livers of Rheb1 ko mice (N = 3). Results are averages of three independent animals. Data represent mean ± SEM. * p < 0.05, ** p < 0.01; (D,E) Western blots and histograms showing the increasing of protein levels of ATGL, PPARα and CPT1-A in livers of Rheb1 ko mice (N = 3). Results are averages of three independent animals. Data represent mean ± SEM. * p < 0.05, ** p < 0.01.