Insulin Protects Hepatic Lipotoxicity by Regulating ER Stress through the PI3K/Akt/p53 Involved Pathway Independently of Autophagy Inhibition

Abstract

The detrimental role of hepatic lipotoxicity has been well-implicated in the pathogenesis of NAFLD. Previously, we reported that inhibiting autophagy aggravated saturated fatty acid (SFA)-induced hepatotoxicity. Insulin, a physiological inhibitor of autophagy, is commonly increased within NAFLD mainly caused by insulin resistance. We therefore hypothesized that insulin augments the sensitivity of hepatocyte to SFA-induced lipotoxicity. The present study was conducted via employing human and mouse hepatocytes, which were exposed to SFAs, insulin, or their combination. Unexpectedly, our results indicated that insulin protected hepatocytes against SFA-induced lipotoxicity, based on the LDH, MTT, and nuclear morphological measurements, and the detection from cleaved-Parp-1 and -caspase-3 expressions. We subsequently clarified that insulin led to a rapid and short-period inhibition of autophagy, which was gradually recovered after 1 h incubation in hepatocytes, and such extent of inhibition was insufficient to aggravate SFA-induced lipotoxicity. The mechanistic study revealed that insulin-induced alleviation of ER stress contributed to its hepatoprotective role. Pre-treating hepatocytes with insulin significantly stimulated phosphorylated-Akt and reversed SFA-induced up-regulation of p53. Chemical inhibition of p53 by pifithrin-α robustly prevented palmitate-induced cell death. The PI3K/Akt pathway blockade by its special antagonist abolished the protective role of insulin against SFA-induced lipotoxicity and p53 up-regulation. Furthermore, we observed that insulin promoted intracellular TG deposits in hepatocytes in the present of palmitate. However, blocking TG accumulation via genetically silencing DGAT-2 did not prevent insulin-protected lipotoxicity. Our study demonstrated that insulin strongly protected against SFA-induced lipotoxicity in hepatocytes mechanistically through alleviating ER stress via a PI3K/Akt/p53 involved pathway but independently from autophagy.

Matrine attenuates endoplasmic reticulum stress and mitochondrion dysfunction in nonalcoholic fatty liver disease by regulating SERCA pathway

“…Palmitate exhibited significant lipotoxicity in a dose-dependent manner in human hepatocytes [ 24 ]. Using oil red O staining, profound lipid droplets were found in PA-induced L02 cells.…”

Is p53 Involved in Tissue‐Specific Insulin Resistance Formation?

“…p53 level was upregulated by the treatment of hepatocytes with saturated fatty acids (SFA) along with its inhibition resulting in the prevention of SFA-induced cell death [ 197 ]. Once again, the antagonism between p53 and insulin activities was strongly highlighted, as insulin treatment antagonized the deleterious effects of p53.…”

Tumor necrosis factor-inducible gene 6 protein ameliorates chronic liver damage by promoting autophagy formation in mice

“…In addition, we assessed whether TSG-6 protected hepatocytes from lipotoxicity-induced ER stress. To induce fatty acid toxicity in hepatocytes, AML12 cells were treated with PA, which has been reported to induce lipotoxicity, 26 , 27 , 28 and TSG-6 or vehicle CON was added to these cells at 3 h post PA treatment. TSG-6 improved the number of viable cells in PA-treated cells, whereas 3-MA suppressed this TSG-6-mediated effect on cell viability ( Supplementary Figure 2a ).…”

“… 26 , 27 To perform biochemical analyses to assess growth and gene expression, AML12 cells at 70–80% confluence were serum-starved in medium containing no fetal bovine serum overnight. After starvation, cells were cultured with tunicamycin (TM) (5 μg ml −1 ) (ME-654380l; EMD Millipore, Billerica, MA, USA) or palmitic acid (400 μ M ) (P0500; Sigma-Aldrich) 28 in order to induce cell damage for 24 h, and the cell medium was then changed after washing with phosphate-buffered saline (PBS) to withdraw TM. These cells were treated with 3-MA (10 n M ) (M9281; Sigma-Aldrich) or PBS for 3 h, and they were then stimulated with TSG-6 (10 ng ml −1 ) for 24 and 48 h. These experiments were repeated at least three times.…”