Differing Endoplasmic Reticulum Stress Response to Excess Lipogenesis versus Lipid Oversupply in Relation to Hepatic Steatosis and Insulin Resistance

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been implicated in hepatic steatosis and insulin resistance. The present study investigated their roles in the development of hepatic steatosis and insulin resistance during de novo lipogenesis (DNL) compared to extrahepatic lipid oversupply. Male C57BL/6J mice were fed either a high fructose (HFru) or high fat (HFat) diet to induce DNL or lipid oversupply in/to the liver. Both HFru and HFat feeding increased hepatic triglyceride within 3 days (by 3.5 and 2.4 fold) and the steatosis remained persistent from 1 week onwards (p<0.01 vs Con). Glucose intolerance (iAUC increased by ∼60%) and blunted insulin-stimulated hepatic Akt and GSK3β phosphorylation (∼40–60%) were found in both feeding conditions (p<0.01 vs Con, assessed after 1 week). No impairment of mitochondrial function was found (oxidation capacity, expression of PGC1α, CPT1, respiratory complexes, enzymatic activity of citrate synthase & β-HAD). As expected, DNL was increased (∼60%) in HFru-fed mice and decreased (32%) in HFat-fed mice (all p<0.05). Interestingly, associated with the upregulated lipogenic enzymes (ACC, FAS and SCD1), two (PERK/eIF2α and IRE1/XBP1) of three ER stress pathways were significantly activated in HFru-fed mice. However, no significant ER stress was observed in HFat-fed mice during the development of hepatic steatosis. Our findings indicate that HFru and HFat diets can result in hepatic steatosis and insulin resistance without obvious mitochondrial defects via different lipid metabolic pathways. The fact that ER stress is apparent only with HFru feeding suggests that ER stress is involved in DNL per se rather than resulting from hepatic steatosis or insulin resistance

Effects of HFru and HFat on ER stress markers in the liver.

<p>After one week of HFru or HFat feeding, animals were fasted for 5-7 hours before tissue collection and liver homogenates were immunoblotted for markers of ER stress. (<b>A</b>) ATF6/β-actin (n = 4/group), (<b>B</b>) p-PERK, (<b>C</b>) p-eIF2α/t-eIF2α, (<b>D</b>) p-IRE1/t-IRE1, and (<b>E</b>) the post-transcriptional splicing of XBP1 transcript. Each lane represents a single mouse. Densitometry data are mean ± SE of 6 mice per group. ** <i>p</i><0.01.</p

Effects of HFru and HFat feeding on glucose tolerance and hepatic insulin signal transduction.

<p>The experiments were performed after one week of high fructose (HFru, ▪), high fat (HFat, □) or chow (CH, •) feeding. (<b>A</b>) Glucose tolerance test (GTT) was performed with an injection of glucose (3 g/kg, <i>ip</i>) after 5–7 hours of fasting. Data are mean ± SE, 6–10 mice per group. iAUC, incremental area under the curve for blood glucose level. Insulin signal transduction was assessed by immunoblotting of phosphorylated-Akt (Ser473) (<b>B</b>) and -GSK3β (Ser219) (<b>C</b>) in the liver in response to a bolus of insulin stimulation (2 U/kg, <i>ip</i>). Each lane represents a single mouse. Densitometry data are mean ± SE of 6 mice per group. ** <i>p</i><0.01.</p

Effects of HFru and HFat feeding on oxidative stress.

<p>After one week of HFru or HFat feeding, the activity of NADPH oxidase (<b>A</b>) and intracellular GSH content (<b>B</b>) were quantified in liver homogenates, data are mean ± SE of 6 to 8 mice per group. *** <i>p</i><0.001.</p

Effects of HFru and HFat on JNK and IKK activation.

<p>After one week of HFru or HFat feeding, liver homogenates were immunoblotted for p-JNK/t-JNK (<b>A</b>), and p-IKKα/β/t-IKKα/β (<b>B</b>) and t-IKBα (<b>C</b>). Each lane represents a single mouse. Densitometry data are mean ± SE of 6 mice per group. ** <i>p</i><0.01 vs CH fed mice.</p

Effects on HFru and HFat feeding on weight gain, adiposity and liver triglyceride.

<p>Male C57BL/6J mice (13–16 weeks old) were fed a high-fructose (HFru) or a high-fat (HFat) diet <i>ad libitum</i> as compared to a standard laboratory chow diet (CH). The body weight was similar among the three groups at the start of the feeding intervention (23.6±0.3, 23.4±0.5 and 23.7±0.5 g). (<b>A</b>) Body weight gain over the baseline. (<b>B</b>) Weight of epididymal fat depots. (<b>C</b>) Liver triglyceride content. Liver samples were freeze-clamped for the measurement of hepatic triglyceride content after 5–7 hour of fasting. Data are mean ± SE of 8–13 mice per group. ** <i>p</i><0.01; † <i>p</i><0.05, †† <i>p</i><0.01 vs HFru.</p

Basal metabolic parameters of HFru and HFat-fed mice.

<p>ND, not determined. Data are means ± SE of 10–15 mice per group.</p><p>*<i>p</i><0.05,</p><p>**<i>p</i><0.01 vs CH-fed mice.</p

Effects of HFru and HFat feeding on mitochondrial oxidation, key protein expressions and enzymatic activities.

<p>After one week of HFru or HFat feeding, the rates of hepatic oxidation of palmitate (<b>A</b>) and glutamate (<b>B</b>) were measured in liver homogenates using [1-¹⁴C]-palmitate or [1-¹⁴C]-glutamate as substrates. Data are mean ± SE from 6 mice per group. (<b>C</b>) Levels of key proteins for mitochondrial oxidation, biogenesis and numbers were measured by immunoblots. Each lane represents a single mouse, 6 mice per group. The specific activity of citrate synthase (<b>D</b>) and β-HAD (<b>E</b>), data are mean ± SE from 6 to 8 mice per group. * <i>p</i><0.05 vs CH.</p