Targeting IRE1α and PERK in the endoplasmic reticulum stress pathway attenuates fatty acid-induced insulin resistance in bovine hepatocytes

Endoplasmic reticulum (ER) stress can be induced by various stimuli and triggers the unfolded protein re-sponse to activate intracellular signaling pathways that are mediated by 3 ER-resident sensors: inositol requir-ing protein-1 alpha (IRE1 alpha), PKR-like ER kinase (PERK), and activating transcription factor-6 (ATF6). In nonru-minants, ER stress plays a critical role in hepatic insulin resistance. However, whether ER stress plays a role in nonesterified fatty acid (NEFA)-induced hepatic insulin resistance in dairy cows is still unknown. Experiments were conducted using primary bovine hepatocytes iso-lated from 5 healthy calves (body weight: 30-40 kg; 1 d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h. Treatment with NEFA elevated abundance of phosphorylated IRE1 alpha and PERK, and cleavage of ATF6, along with the ER stress-associated genes XBP1, ATF4, and DNAJC3, resulting in both linear and quadratic effects. Further-more, ER Tracker red staining and transmission electron microscopy results indicated that ER was dilated and degranulated in response to NEFA treatment, suggest-ing that ER stress was induced by NEFA treatment in bovine hepatocytes. Second, to assess the effect of ER stress on NEFA-induced insulin resistance, hepatocytes were treated with different concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 5 h with or without taurourso-deoxycholic acid (TUDCA, a canonical inhibitor of ER stress). Here, NEFA induced insulin resistance by in-creasing the abundance of insulin receptor substrate-1 (IRS1) phosphorylation at the inhibitory residue Ser 307 (S307) and decreasing the abundance of phosphor-ylated protein kinase B (AKT) and glycogen synthase kinase-30 (GSK30) in a dose-dependent manner. This was accompanied by upregulation of an abundance of gluconeogenic genes [phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6-Pase)]. These detrimental effects of NEFA on insulin signaling could be reversed with TUDCA treatment, indicating a mechanistic link between ER stress and NEFA-induced insulin resistance. In a third experiment, pGPU6/GFP/ Neo vectors containing short hairpin RNA targeting IRE1 alpha were used to silence IRE1 alpha transcription, and GSK2656157 (PERK phosphorylation inhibitor) and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF; an inhibitor of ATF6) were used to block PERK and ATF6 branches, respectively. Notably, the silencing of the IRE1 alpha branch improved NEFA-induced insulin re-sistance by decreasing phosphorylation of IRS1 (S307) and increasing phosphorylation of AKT and GSK30, and reducing PEPCK and G6-Pase mRNA abundance, which was likely dependent on IRE1 alpha kinase activ-ity. Similarly, blockage of the PERK branch increased phosphorylation of AKT and GSK30, and reduced PEPCK and G6-Pase mRNA abundance, but had no effect on phosphorylation of IRS1 (S307). However, re-sults showed that inhibition of the ATF6 branch had no effects on phosphorylation of IRS1, AKT, and GSK30, and instead found increasing PEPCK and G6-Pase mRNA abundance. Taken together, data in the present study found that impeding IRE1 alpha and PERK signaling might aid in relieving hepatic insulin resistance. How-ever, the more detailed mechanisms of how IRE1 alpha and PERK signaling contribute to hepatic insulin resistance in dairy cows remain to be determined.