Optimized Effects of Fisetin and Hydroxychloroquine on ER Stress and Autophagy in Nonalcoholic Fatty Pancreas Disease in Mice

Abstract

Background: Fat accumulation in the pancreas, known as nonalcoholic fatty pancreatic disease (NAFPD), is associated with obesity and may lead to prediabetes and Type 2 diabetes. Reducing endoplasmic reticulum stress and enhancing autophagy could offer therapeutic benefits. This study examines the effects of fisetin (FSN) and hydroxychloroquine (HCQ) on NAFPD. Method: Forty-eight Male C57BL/6 J mice were assigned to a standard chow diet (SCD) or a high-fat diet (HFD) for 16 weeks. The HFD group was divided into five subgroups; each group contains eight mice: HFD, HFD + V (vehicle), HFD + FSN, HFD + HCQ, and HFD + FSN + HCQ. FSN was given daily at 80 mg/kg, and HCQ was injected IP at 50 mg/kg twice weekly for more 8 weeks. Insulin resistance was assessed through OGTT and HOMA-IR. Histological analysis of pancreatic tissue was conducted, and the protein and mRNA levels of molecules associated with ER stress and autophagy were assessed using PCR and immunoblotting techniques. Result: FSN and HCQ significantly reduced weight gain, pancreatic adipocyte accumulation, and insulin resistance caused by HFD in obese mice, with the combination of the two compounds producing even more pronounced effects. Additionally, the HFD increased the expression of UPR markers ATF4 and CHOP, a response that was further intensified by HCQ. In contrast, FSN attenuated the UPR by regulating GRP78 levels. Furthermore, the HFD resulted in a significant decrease in the LC3II/LC3I ratio and an accumulation of p62 protein due to reduced p-AMPK levels. Following treatment with FSN, these alterations were reversed, leading to decreased mTOR expression and increased levels of autophagy markers such as ATG5 and Beclin1. Conclusion: Our study reveals that FSN and HCQ effectively combat HFD-induced NAFPD, improving insulin sensitivity and addressing pancreatic fat deposition linked to metabolic syndrome. While HCQ may cause endoplasmic reticulum stress, FSN offers protective effects, supporting their combined use for better treatment outcomes.

Keywords: ER stress; autophagy; fatty pancreas; fisetin; hydroxychloroquine; metabolic syndrome.

Figure 1

Improvement of HFD-induced obesity and NAFPD in mice by fisetin and hydroxychloroquine. (a) Schematic diagram of the experimental procedure. (b) Change of body weight during a 24-week study period. (c) The pancreas weight (milligrams). (d) The representative histological images of H&E-stained sections (100× magnification in up and 400× magnification in down). A 24-week period of HFD resulted in interlobular adipocyte infiltration (which showed by green arrows), interstitial edema (enlarged interlobular space area) (which showed by blue arrows), as well as inflammation indicated by the presence of inflammatory cells (which showed by red arrows) in pancreatic tissue of HFD and HFD + V mice compared to SCD mice. However, HFD + FSN and HFD + HCQ groups showed a moderate improvement, and HFD + FSN + HCQ mice exhibited a noticeable change near the SCD histology (n = 7). Values are presented as means ± SEM. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. NAFPD, nonalcoholic fatty pancreatic disease; HFD, high-fat diet; V, vehicle; SCD, standard chow diet; FSN, fisetin; HCQ, hydroxychloroquine; H&E, hematoxylin and eosin.

Figure 2

Restoration of glucose homeostasis and insulin resistance by fisetin and hydroxychloroquine in HFD mice. (a) OGTT, (b) AUC of blood glucose level during OGTT, Serum (c) insulin and (d) glucose in mice after 10 h of fasting, (e) HOMA-IR (n = 5). Values are presented as means ± SEM. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. HFD, high-fat diet; V, vehicle; SCD, standard chow diet; FSN, fisetin; HCQ, hydroxychloroquine; OGTT, oral glucose tolerance test; AUC, area under the curve; HOMA-IR, homeostasis model assessment of insulin resistance.

Figure 3

Reinforcement of HFD-induced ER stress by hydroxychloroquine and inhibition by fisetin in mouse pancreas. The mRNA level of (a) ATF4, (b) CHOP, and (c) GRP78 genes was measured by real-time PCR (n = 5). Values are presented as means ± SEM. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. HFD, high-fat diet; V, vehicle; SCD, standard chow diet; FSN, fisetin; HCQ, hydroxychloroquine; ER, endoplasmic reticulum; ATF4, activating transcription Factor 4; CHOP, C/EBP homologous protein; GRP78, glucose-regulated Protein 78.

Figure 4

Restoration of HFD-reduced autophagy in the pancreas of mice by fisetin. The results from the western blot of proteins involved in autophagy: (a) LC3II/LC3I, (b) p62, and (c) AMPK phosphorylation in mice (n = 3). (d) The expression level of p62 gene, (e) p62 protein/mRNA ratio. The expression levels of (f) Beclin1, (g) ATG5, and (h) mTOR (n = 5). Data are presented as mean ± SEM. Values are significantly different between groups as determined using one-way ANOVA. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. HFD, high-fat diet; V, vehicle; SCD, standard chow diet; FSN, fisetin; HCQ, hydroxychloroquine; LC3, microtubule-associated Protein 1A/1B light chain 3; p62, Sequestosome 1; AMPK, AMP-activated protein kinase; Beclin1, Beclin 1; ATG5, autophagy-related Protein 5; mTOR, mechanistic target of rapamycin.