Endoplasmic reticulum (ER) stress can be induced by various stimuli and triggers the unfolded protein response to activate intracellular signaling pathways that are mediated by 3 ER-resident sensors:
inositol requiring protein-1α (IRE1α), PKR-like ER
kinase (PERK), and activating transcription factor-6 (ATF6). In nonruminants, 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 isolated 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 IRE1α and PERK, and cleavage of ATF6, along with the ER stress-associated genes XBP1, ATF4, and DNAJC3, resulting in both linear and quadratic effects. Furthermore, ER Tracker red staining and transmission electron microscopy results indicated that ER was dilated and degranulated in response to
NEFA treatment, suggesting 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
tauroursodeoxycholic acid (
TUDCA, a canonical inhibitor of ER stress). Here,
NEFA induced
insulin resistance by increasing the abundance of
insulin receptor substrate-1 (IRS1) phosphorylation at the inhibitory residue Ser 307 (S307) and decreasing the abundance of phosphorylated
protein kinase B (AKT) and
glycogen synthase kinase-3β (GSK3β) 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 IRE1α were used to silence IRE1α 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 IRE1α branch improved
NEFA-induced
insulin resistance by decreasing phosphorylation of IRS1 (S307) and increasing phosphorylation of AKT and GSK3β, and reducing PEPCK and G6-Pase
mRNA abundance, which was likely dependent on IRE1α
kinase activity. Similarly, blockage of the PERK branch increased phosphorylation of AKT and GSK3β, and reduced PEPCK and G6-Pase
mRNA abundance, but had no effect on phosphorylation of IRS1 (S307). However, results showed that inhibition of the ATF6 branch had no effects on phosphorylation of IRS1, AKT, and GSK3β, and instead found increasing PEPCK and G6-Pase
mRNA abundance. Taken together, data in the present study found that impeding IRE1α and PERK signaling might aid in relieving hepatic
insulin resistance. However, the more detailed mechanisms of how IRE1α and PERK signaling contribute to hepatic
insulin resistance in dairy cows remain to be determined.