Inhibition of mTOR improves malnutrition induced hepatic metabolic dysfunction

Abstract

Severe malnutrition accounts for half-a-million deaths annually in children under the age of five. Despite improved WHO guidelines, inpatient mortality remains high and is associated with metabolic dysfunction. Previous studies suggest a correlation between hepatic metabolic dysfunction and impaired autophagy. We aimed to determine the role of mTORC1 inhibition in a murine model of malnutrition-induced hepatic dysfunction. Wild type weanling C57/B6 mice were fed a 18 or 1% protein diet for two weeks. A third low-protein group received daily rapamycin injections, an mTORC1 inhibitor. Hepatic metabolic function was assessed by histology, immunofluorescence, gene expression, metabolomics and protein levels. Low protein-fed mice manifested characteristics of severe malnutrition, including weight loss, hypoalbuminemia, hypoglycemia, hepatic steatosis and cholestasis. Low protein-fed mice had fewer mitochondria and showed signs of impaired mitochondrial function. Rapamycin prevented hepatic steatosis, restored ATP levels and fasted plasma glucose levels compared to untreated mice. This correlated with increased content of LC3-II, and decreased content mitochondrial damage marker, PINK1. We demonstrate that hepatic steatosis and disturbed mitochondrial function in a murine model of severe malnutrition can be partially prevented through inhibition of mTORC1. These findings suggest that stimulation of autophagy could be a novel approach to improve metabolic function in severely malnourished children.

Figure 1

Low protein fed mice have altered morphometrics and liver function markers, with only fasting glucose levels improving by rapamycin. (a) Weight curve over 14 days, (b) Cumulative weigh change from weaning to sacrifice, %. (c) Photo of mice, from bottom 18% diet, 1% diet, 1% diet + Rapa. (d) Length at sacrifice, cm. (e) Liver weight (g). (f) Liver weight/body weight ratio. (g) ALT, U/L. (h) Plasma albumin, g/dl. (i) 15 h fasted glucose, mg/dl. (j) Total bile acids, uM. n = 6. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Figure 2

Mice on a low protein diet develop hepatic steatosis, which is partially mitigated by rapamycin. (a) BODIPY staining, scale bar 49um. (b) Area fraction of lipid droplets in percent. (c) Average lipid count. (d) Average lipid droplet size, nm. (e) Triglyceride quantification, mg/g, n = 6. (f) Oil Red O staining, scale bar 100 um. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Figure 3

Low protein fed mice illustrate changed mitochondrial morphology and markers of function which were mitigated by rapamycin. (a) TEM magnification × 4000, scale bar 2um, red square encircles a mitochondria with an inclusion body, # indicate autophagosomes. (b) HSP60 IF staining in red, scale bar 10um. (c) mitochondrial DNA. (d) TOM20 quantification of western blot relative to 18% diet. (e) Westernblots. (f) Relative expression Tfam. (g) Relative expression NRF1. (h) Complex I quantification of western blot relative to 18% diet. (i) Hepatic ATP levels, nmol/g wet weight. (j) PINK1 quantification of western blot relative to 18% diet. The westerns were cut prior to incubation with the primary antibodies, the full size blots can be seen in Supplementary Fig. S7. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Figure 4

Rapamycin treatment significantly increased autophagy markers. (a). Western blots LC3, p62, GAPDH. Western blot quantification of and shown as a relative expression to 18% control diet (b) total LC3-II/GAPDH. (c) LC3-II to I ratio. (d) p62/GAPDH. (e) Western blots S6K, GAPDH, phos-S6K, beta-actin. Western blot quantification of and shown as a relative expression to 18% control diet (f) S6K/GAPDH. (g) phos-S6K. (h) phos-S6K/S6K ratio. (i) Western blots ULK1, GAPDH, phos-ULK1 (j) ULK1/GAPDH. (k) phos-ULK1/GAPDH. (l) phos-ULK1:ULK ratio. The westerns were cut prior to incubation with the primary antibodies, the full size blots can be seen in Supplementary Fig. S9. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Figure 5

A low protein diet impairs the TCA cycle, which is in part prevented by rapamycin treatment. (a) Left panel, score plot of PLS-DA showing group separation based on all measured metabolites (groups coded as per legend); Right panel, correlation plot presenting the relationships between the top distinctive metabolites (VIP > 1); the metabolites that significantly contribute to component-1 (i.e., control vs. low protein fed) are indicated with black lines, those in grey show metabolites that only contribute to component-2 (i.e., difference between rapamycin treated vs. untreated low protein fed animals); while metabolites that contribute to both component-1 and -2 are indicated by orange lines. (b) Bar plots presenting mean and SEM of selected top metabolites that most discriminate between groups, nmol/g. n = 5. (c) Schematic overview of the changes in metabolites seen in the low protein fed-mice, red arrow indicating the increase or decrease in metabolites compared to control mice, and blue arrow compared to rapamycin. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.