This study investigates the mechanism of hepatic insulin resistance in non-alcoholic fatty liver disease (NAFLD) using a rat model of short-term high-fat feeding. Key findings include: 1. **Hepatic Fat Accumulation**: Short-term high-fat feeding in rats led to a significant increase in liver triglyceride and total fatty acyl-CoA content, without significant changes in visceral or skeletal muscle fat content. 2. **Insulin Resistance**: The ability of insulin to suppress endogenous glucose production (EGP) was diminished in the high-fat feeding (FF) group, despite normal basal EGP and insulin-stimulated peripheral glucose disposal. 3. **Insulin Signaling**: Hepatic insulin resistance was attributed to impaired insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2, which was associated with activation of PKC-ε and JNK1. 4. **Glycogen Synthase and Gluconeogenesis**: Hepatic fat accumulation decreased insulin activation of glycogen synthase and increased gluconeogenesis, leading to impaired glucose storage and increased glucose production. 5. **Treatment with 2,4-Dinitrophenol**: Treatment with low doses of 2,4-dinitrophenol, a mitochondrial uncoupler, prevented the development of fatty liver, hepatic insulin resistance, activation of PKC-ε and JNK1, and defects in insulin signaling. 6. **Conclusion**: The data support the hypothesis that hepatic steatosis leads to hepatic insulin resistance by stimulating gluconeogenesis and activating PKC-ε and JNK1, which interfere with IRS-1 and IRS-2 tyrosine phosphorylation and impair insulin activation of glycogen synthase.This study investigates the mechanism of hepatic insulin resistance in non-alcoholic fatty liver disease (NAFLD) using a rat model of short-term high-fat feeding. Key findings include: 1. **Hepatic Fat Accumulation**: Short-term high-fat feeding in rats led to a significant increase in liver triglyceride and total fatty acyl-CoA content, without significant changes in visceral or skeletal muscle fat content. 2. **Insulin Resistance**: The ability of insulin to suppress endogenous glucose production (EGP) was diminished in the high-fat feeding (FF) group, despite normal basal EGP and insulin-stimulated peripheral glucose disposal. 3. **Insulin Signaling**: Hepatic insulin resistance was attributed to impaired insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2, which was associated with activation of PKC-ε and JNK1. 4. **Glycogen Synthase and Gluconeogenesis**: Hepatic fat accumulation decreased insulin activation of glycogen synthase and increased gluconeogenesis, leading to impaired glucose storage and increased glucose production. 5. **Treatment with 2,4-Dinitrophenol**: Treatment with low doses of 2,4-dinitrophenol, a mitochondrial uncoupler, prevented the development of fatty liver, hepatic insulin resistance, activation of PKC-ε and JNK1, and defects in insulin signaling. 6. **Conclusion**: The data support the hypothesis that hepatic steatosis leads to hepatic insulin resistance by stimulating gluconeogenesis and activating PKC-ε and JNK1, which interfere with IRS-1 and IRS-2 tyrosine phosphorylation and impair insulin activation of glycogen synthase.