STING, Tmem173, is involved in liver injury caused by both infectious and sterile inflammatory models. Its role in toxic liver injury and non-alcoholic fatty liver disease (NAFLD), however, is less clear. While a few groups have investigated its role in NAFLD pathogenesis, results have been conflicting. The objective of this study was to clarify the exact role of STING in toxic liver injury and NAFLD models. Goldenticket mice (Tmem173^gt), which lack STING protein, were subjected to either a toxic liver injury with tunicamycin (TM) or one of two dietary models of non-alcoholic fatty liver disease: high fructose feeding or Fructose-Palmitate-Cholesterol (FPC) feeding. Three days after TM injection, Tmem173^gt mice demonstrated less liver injury (average ALT of 54 ± 5 IU/L) than control mice (average ALT 108 ± 24 IU/L). In contrast, no significant differences in liver injury were seen between WT and Tmem173^gt mice fed either high fructose or FPC. Tmem173^gt mice only distinguished themselves from WT mice in their increased insulin resistance. In conclusion, while STING appears to play a role in toxic liver injury mediated by TM, it plays little to no role in two dietary models of NAFLD. The exact role of STING appears to be stimulus-dependent.

Neutrophils and macrophages are important constituents of the hepatic inflammatory infiltrate in non-alcoholic steatohepatitis. These innate immune cells express CD18, an adhesion molecule that facilitates leukocyte activation. In the context of fatty liver, activation of infiltrated leukocytes is believed to enhance hepatocellular injury. The objective of this study was to determine the degree to which activated innate immune cells promote steatohepatitis by comparing hepatic outcomes in wild-type and CD18-mutant mice fed a methionine-choline-deficient (MCD) diet. After 3 weeks of MCD feeding, hepatocyte injury, based on serum ALT elevation, was 40% lower in CD18-mutant than wild-type mice. Leukocyte infiltration into the liver was not impaired in CD18-mutant mice, but leukocyte activation was markedly reduced, as shown by the lack of evidence of oxidant production. Despite having reduced hepatocellular injury, CD18-mutant mice developed significantly more hepatic steatosis than wild-type mice after MCD feeding. This coincided with greater hepatic induction of pro-inflammatory and lipogenic genes as well as a modest reduction in hepatic expression of adipose triglyceride lipase. Overall, the data indicate that CD18 deficiency curbs MCD-mediated liver injury by limiting the activation of innate immune cells in the liver without compromising intrahepatic cytokine activation. Reduced liver injury occurs at the expense of increased hepatic steatosis, which suggests that in addition to damaging hepatocytes, infiltrating leukocytes may influence lipid homeostasis in the liver.

X-box binding protein-1 (XBP1) is a transcription factor that plays a central role in controlling cellular responses to endoplasmic reticulum (ER) stress. Under stress conditions, the transcriptionally active form of XBP1 is generated via splicing of Xbp1 mRNA by the ER-resident protein inositol-requiring enzyme-1 (IRE1α). Genetic deletion of XBP1 has multiple consequences: some resulting from the loss of the transcription factor per se, and others related to compensatory activation of IRE1α. The objective of the current study was to investigate the effects of XBP1 deletion in adult mouse liver and determine to what extent they are direct or indirect. XBP1 was deleted from hepatocytes in adult Xbp1^fl/fl mice using AAV8-Transthyretin-Cre (Xbp1^Δhep). Xbp1^Δhep mice exhibited no liver disease at baseline, but developed acute biochemical and histologic liver injury in response to a dietary challenge with fructose for 4 weeks. Fructose-mediated liver injury in Xbp1^Δhep mice coincided with heightened IRE1α activity, as demonstrated by Xbp1 mRNA splicing, JNK activation, and regulated IRE1α-dependent RNA decay (RIDD). Activation of eIF2α was also evident, with associated up-regulation of the pro-apoptotic molecules CHOP, BIM, and PUMA. To determine whether the adverse consequences of liver-specific XBP1 deletion were due to XBP1 loss or heightened IRE1α activity, we repeated a fructose challenge in mice with liver-specific deletion of both XBP1 and IRE1α (Xbp1^Δhep;Ire1a^Δhep). Xbp1^Δhep;Ire1a^Δhep mice were protected from fructose-mediated liver injury and failed to exhibit any of the signs of ER stress seen in mice lacking XBP1 alone. The protective effect of IRE1α deletion persisted even with long-term exposure to fructose. Xbp1^Δhep mice developed liver fibrosis at 16 weeks, but Xbp1^Δhep;Ire1a^Δhep mice did not. Overall, the results indicate that the deleterious effects of hepatocyte-specific XBP1 deletion are due primarily to hyperactivation of IRE1α. They support further exploration of IRE1α as a contributor to acute and chronic liver diseases.

Diets containing excess carbohydrate and fat promote hepatic steatosis and steatohepatitis in mice. Little is known, however, about the impact of specific carbohydrate/fat combinations on liver outcome. This study was designed to determine whether high-energy diets with identical caloric density but different carbohydrate and fat composition have unique effects on the liver. Four experimental diets were formulated with 60%kcal carbohydrate and 20%kcal fat, each in nearly pure form from a single source: starch-oleate, starch-palmitate, sucrose-oleate and sucrose-palmitate. The diets were fed to mice for 3 or 12 weeks for analysis of lipid metabolism and liver injury. All mice developed hepatic steatosis over 12 weeks, but mice fed the sucrose-palmitate diet accumulated more hepatic lipid than those in the other three experimental groups. The exaggerated lipid accumulation in sucrose-palmitate-fed mice was attributable to a disproportionate rise in hepatic de novo lipogenesis. These mice accrued more hepatic palmitate and exhibited more evidence of liver injury than any of the other experimental groups. Interestingly, lipogenic gene expression in mice fed the custom diets did not correlate with actual de novo lipogenesis. In addition, de novo lipogenesis rose in all mice between 3 and 12 weeks, without feedback inhibition from hepatic steatosis. The pairing of simple sugar (sucrose) and saturated fat (palmitate) in a high-carbohydrate/moderate-fat diet induces more de novo lipogenesis and liver injury than other carbohydrate/fat combinations. Diet-induced liver injury correlates positively with hepatic de novo lipogenesis and is not predictable by isolated analysis of lipogenic gene expression.

Background & aims

The factors that distinguish metabolically healthy obesity from metabolically unhealthy obesity are not well understood. Diet has been implicated as a determinant of the unhealthy obesity phenotype, but which aspects of the diet induce dysmetabolism are unknown. The goal of this study was to investigate whether specific macronutrients or macronutrient combinations provoke dysmetabolism in the context of isocaloric, high-energy diets.

Methods

Mice were fed 4 high-energy diets identical in calorie and nutrient content but different in nutrient composition for 3 weeks to 6 months. The test diets contained 42% carbohydrate (sucrose or starch) and 42% fat (oleate or palmitate). Weight and glucose tolerance were monitored; blood and tissues were collected for histology, gene expression, and immunophenotyping.

Results

Mice gained weight on all 4 test diets but differed significantly in other metabolic outcomes. Animals fed the starch-oleate diet developed more severe hepatic steatosis than those on other formulas. Stable isotope incorporation showed that the excess hepatic steatosis in starch-oleate-fed mice derived from exaggerated adipose tissue lipolysis. In these mice, adipose tissue lipolysis coincided with adipocyte necrosis and inflammation. Notably, the liver and adipose tissue abnormalities provoked by starch-oleate feeding were reproduced when mice were fed a mixed-nutrient Western diet with 42% carbohydrate and 42% fat.

Conclusions

The macronutrient composition of the diet exerts a significant influence on metabolic outcome, independent of calories and nutrient proportions. Starch-oleate appears to cause hepatic steatosis by inducing progressive adipose tissue injury. Starch-oleate phenocopies the effect of a Western diet; consequently, it may provide clues to the mechanism whereby specific nutrients cause metabolically unhealthy obesity.