Canagliflozin Attenuates Hepatic Steatosis and Atherosclerosis Progression in Western Diet-Fed ApoE-Knockout Mice

Abstract

Purpose To investigate the effect of canagliflozin (20 mg/kg) on hepatic steatosis and atherosclerosis, and further to explore its possible mechanism. Methods Blood glucose, blood lipid, oxidative stress response and inflammatory cytokines were examined by intraperitoneal glucose tolerance test and ELISA assay. HE and Oil Red O staining were used to estimate the extent of hepatic steatosis and atherosclerosis. RNA-seq and qRT-PCR were used to further investigate the potential mechanism. The effects of canagliflozin on autophagy were detected using transmission electron microscopy and Western blotting. The endothelial function-related markers were determined by qRT-PCR. Results Canagliflozin notably alleviated the elevation in blood glucose and insulin resistance in western diet-fed ApoE-/- mice. In ApoE-/-+Cana group, ApoE-/- mice had lower levels of TG, TC, LDL-C, TNF-α, IL-6, IL-1β, and MCP-1. HE and Oil Red O staining presented that canagliflozin restrained the atherosclerotic plaque development and lipid accumulation. RNA-seq showed that 87 DEGs were relevant to improvement of hepatic steatosis and atherosclerosis by canagliflozin. Among them, CPS1, ASS1, ASL, ARG1, MATLA, GLS2, GOT1, SREBP1, Plin5, Retreg1, and C/EBPβ were verified. KEGG enrichment analysis indicated that DEGs were mainly involved in amino acid metabolism. Besides, we observed that canagliflozin reduced the contents of aspartic acid and citrulline in liver. Western blotting showed that ASS1 and p-AMPK/AMPK was remarkably elevated after administration of canagliflozin. Correspondingly, canagliflozin down-regulated SREBP1, FAS, ACC1, HMGCR, p-mTOR/m-TOR, p-ULK1/ULK1 and p62, but up-regulated CPT1, Beclin 1 and LC3 II/LC3I. TEM showed that canagliflozin reduced the number of lipid droplets and increased the autophagosomes. Moreover, we found that canagliflozin elevated the aortic endothelial function-associated markers including ASS1, ASL and eNOS. Conclusion Canagliflozin may attenuate hepatic steatosis by improving lipid metabolism, enhancing autophagy, and reducing inflammatory response through ASS1/AMPK pathway. Besides, canagliflozin further effectively improves the aortic endothelial function, thereby suppressing atherosclerosis development.

Canagliflozin Alleviates Atherosclerosis Progression through Inflammation, Oxidative Stress, and Autophagy in Western Diet-fed ApoE−/− Mice

“…The methods and steps were the same as described in our previous study [21]. The slides were incubated first with anti-F4/80 (GB113373, 1:800, Affinity, Wuhan, China) overnight, then with antirabbit IgG-HRP (GB23303, 1:200, Servicebio, Wuhan, China), after being washed three times with PBS [21]. Next, the sections were stained with a 3,3′-diaminobenzidine kit (ZLI-9017, Zhongshan, Beijing, China) [21].…”

“…The slides were incubated first with anti-F4/80 (GB113373, 1:800, Affinity, Wuhan, China) overnight, then with antirabbit IgG-HRP (GB23303, 1:200, Servicebio, Wuhan, China), after being washed three times with PBS [21]. Next, the sections were stained with a 3,3′-diaminobenzidine kit (ZLI-9017, Zhongshan, Beijing, China) [21]. The relative F4/80 level was quantified in Image-pro plus 6.0 software (Media Cybermetrics, Rockville, MD, USA).…”

“…The PCR assay was as described in our previous article [21]. Total RNA was extracted with TRIzol Universal Reagent (Tiangen, Beijing, China), and RNase-free DNase I (Tiangen, Beijing, China) was used to remove DNA [21].…”

“…The PCR assay was as described in our previous article [21]. Total RNA was extracted with TRIzol Universal Reagent (Tiangen, Beijing, China), and RNase-free DNase I (Tiangen, Beijing, China) was used to remove DNA [21]. After quality assessment, cDNA was synthesized with ReverTra Ace reverse transcriptase (Tiangen, Beijing, China), and real-time PCR was conducted with SuperReal PreMix Plus (Tiangen, Beijing, China) [21].…”

Advances in the Utilization of Zebrafish for Assessing and Understanding the Mechanisms of Nano-/Microparticles Toxicity in Water

“…Therefore, glucose and pyruvate production were insufficient, resulting in glucose metabolism disorders [94][95][96]. At the same time, alterations in several genes that regulate hepatic lipogenesis, for example, decreased expression of peroxisome proliferator-activated receptor (PPAR) family genes, acetyl-coa carboxylase 1 (ACC1), and fatty acid synthase (FAS) genes, have been associated with downregulation of triglyceride (TG) [97]. In addition, the reduction of α-ketoglutarate and isocitrate dehydrogenase (ICD), which are involved in tricarboxylic acid cycle energy metabolism, implies that MP exposure reduced energy metabolism in zebrafish.…”