Small heterodimer partner deletion prevents hepatic steatosis and when combined with farnesoid X receptor loss protects against type 2 diabetes in mice

Abstract

Nuclear receptors farnesoid X receptor (FXR) and small heterodimer partner (SHP) are important regulators of bile acid, lipid, and glucose homeostasis. Here, we show that global Fxr ^-/- Shp^-/- double knockout (DKO) mice are refractory to weight gain, glucose intolerance, and hepatic steatosis when challenged with high-fat diet. DKO mice display an inherently increased capacity to burn fat and suppress de novo hepatic lipid synthesis. Moreover, DKO mice were also very active and that correlated well with the observed increase in phosphoenolpyruvate carboxykinase expression, type IA fibers, and mitochondrial function in skeletal muscle. Mechanistically, we demonstrate that liver-specific Shp deletion protects against fatty liver development by suppressing expression of peroxisome proliferator-activated receptor gamma 2 and lipid-droplet protein fat-specific protein 27 beta.

Conclusion: These data suggest that Fxr and Shp inactivation may be beneficial to combat diet-induced obesity and uncover that hepatic SHP is necessary to promote fatty liver disease. (Hepatology 2017;66:1854-1865).

Figure 1. Loss of Fxr and Shp in mice results in better glucose handling

(a) DKO mice exhibit lower body weight on normal chow diet. (n= 6–7 mice per group). (b) Gross pictures of WT and DKO mice on normal chow diet. (c) DKO mice have increased lean mass and decreased fat mass compared to WT, as measured by DEXA scan. (n=6 mice per group). (d) Difference in food intake between DKO and WT mice. (n=6 mice per group). (e–f) DKO mice are more glucose tolerant and insulin sensitive as determined by glucose and insulin tolerance tests. (n>9 per group). Student t-test was used to compare two groups. Mean± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to WT mice.

Figure 2. DKO mice exhibit resistance to diet-induced obesity

(a) DKO mice gain less weight compared to WT mice and gross mice pictures depict this reduced weight gain in DKO mice after 8 weeks HFD. (b) DKO mice accumulate less fat mass than the WT mice when challenged with HFD. (c) Basal serum glucose levels are decreased in DKO (n=16) mice compared to WT (n=12) despite similar food intake (d) between DKO and WT mice (n=6/group). DKO mice show robust glucose tolerance (e) and insulin sensitivity (f) when maintained on HFD (n>12/group). Student’s t-test was used to compare two groups. Mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to WT mice.

Figure 3. Fxr and Shp deficiency increases overall energy expenditure

(a) Oxygen consumption (VO₂) and (b) carbon dioxide production (CO₂) at rest. (c) Lower respiratory exchange ratio (RER) in DKO on normal chow suggests preference for fat whereas WT mice exhibit preference for glucose as their fuel source. On HFD, WT mice shift to fat metabolism and DKO further increase their fat utilization. Mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 compared to WT mice.

Figure 4. DKO mice have increased physical activity and fatty acid oxidation. (a–c)

There is increased physical activity in DKO mice on normal chow and high fat diet compared to WT. Increased mitochondrial complexes of the electron transport in DKO mice on normal chow indicating increased supply of energy compared to WT. (d) Gene expression as determined by RT-qPCR shows increase in genes involved in fatty acid oxidation and protection against insulin resistance. (n=4–6 mice per group) Mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 compared to WT mice.

Figure 5. DKO liver display decreased glucose and fatty acid metabolism and increased oxidative metabolism

Expression of genes involved in (a) glycolytic metabolism, (b) oxidative metabolism and (c) fatty acid metabolism (n= 6 mice per group). (d) H&E staining of liver tissues shows hepatic steatosis is less severe in DKO mice compared to WT animals fed HFD. (e) Oil red O staining confirms the reduction in hepatic steatosis in DKO compared to WT. Mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to WT mice.

Figure 6. Loss of Shp in the liver protects against HFD-induced hepatic steatosis

(a) L-ShpKO and f/f Shp mice on HFD exhibit similar weight gain. Liver to body weight ratio did not change but a modest decrease in white adipose tissue to body weight ratio was observed in L-ShpKO compared to f/f Shp control. (b) Despite HFD challenge, improved lipid metabolic profile was seen in L-ShpKO mice compared to f/f Shp control. (c) L-ShpKO mice exhibit improved glucose tolerance but did not show enhanced insulin sensitivity on HFD. (d) The neutral lipid staining revealed a striking reduction in hepatic fat in the L-ShpKO mice when compared to f/f Shp mice. (e) Hepatic gene expression showed reduction in lipogenesis and lipid droplet stability when Shp is deleted (n= 3–5 mice per group) Mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 compared to f/f Shp mice. (f) Schematic describing the underlying mechanism(s) that protect DKO and L-ShpKO mice from high-fat diet induced steatosis.