Asprosin contributes to nonalcoholic fatty liver disease through regulating lipid accumulation and inflammatory response via AMPK signaling

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) is a primary contributor to liver-related morbidity and mortality. Asprosin has been reported to be implicated in NAFLD.

Aims: This work is to illuminate the effects of Asprosin on NAFLD and the possible downstream mechanism.

Materials & methods: The weight of NAFLD mice induced by a high-fat diet was detected. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) examined serum Asprosin expression. RT-qPCR and western blot analysis examined Asprosin expression in mice liver tissues. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were implemented. Biochemical kits tested liver enzyme levels in mice serum and liver tissues. Hematoxylin and eosin staining evaluated liver histology. Liver weight was also tested and oil red O staining estimated lipid accumulation. RT-qPCR and western blot analysis analyzed the expression of gluconeogenesis-, fatty acid biosynthesis-, fatty acid oxidation-, and inflammation-associated factors. Besides, western blot analysis examined the expression of AMP-activated protein kinase (AMPK)/p38 signaling-associated factors. In palmitic acid (PA)-treated mice hepatocytes, RT-qPCR and western blot analysis examined Asprosin expression. Lipid accumulation, gluconeogenesis, fatty acid biosynthesis, fatty acid oxidation, and inflammation were appraised again.

Results: Asprosin was overexpressed in the serum and liver tissues of NAFLD mice and PA-treated mice hepatocytes. Asprosin interference reduced mice body and liver weight, improved glucose tolerance and diminished liver injury in vivo. Asprosin knockdown alleviated lipid accumulation and inflammatory infiltration both in vitro and in vivo. Additionally, Asprosin absence activated AMPK/p38 signaling and AMPK inhibitor Compound C reversed the impacts of Asprosin on lipid accumulation and inflammatory response.

Conclusion: Collectively, Asprosin inhibition suppressed lipid accumulation and inflammation to obstruct NAFLD through AMPK/p38 signaling.

Keywords: AMPK signaling; Asprosin; inflammatory response; lipid accumulation; nonalcoholic fatty liver disease.

Figure 1

Asprosin expression is increased in the serum and liver tissues of NAFLD mice (n = 5/group). (A) Examination of mice body weight. (B) RT‐qPCR tested Asprosin expression in the mice serum. (C) RT‐qPCR and western blot tested Asprosin expression in mice liver tissues. Data are presented as mean ± SD. ***p < .001 versus Control. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HFD, high‐fat diet; NAFLD, nonalcoholic fatty liver disease; RT‐qPCR, quantitative reverse‐transcription polymerase chain reaction; SD, standard deviation.

Figure 2

Asprosin interference reduces mice body weight, improves glucose tolerance, and insulin sensitivity in HFD‐induced NAFLD mice model (n = 5/group). (A) RT‐qPCR tested Asprosin expression in the mice serum following transduction of Lenti‐Asprosin. (B) RT‐qPCR and western blot tested Asprosin expression in mice liver tissues following transduction of Lenti‐Asprosin. (C) Examination of mice body weight. (D) IPGTT and (E) IPITT tested glucose tolerance and insulin tolerance. Data are presented as mean ± SD. *p < .05, ***p < .001 versus Control. ^## p < .01, ^### p < .001 versus HFD+Lenti‐vector. AUC, area under curve; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HFD, high‐fat diet; IPGTT, intraperitoneal glucose tolerance test; ITT; insulin tolerance test; NAFLD, nonalcoholic fatty liver disease; RT‐qPCR, quantitative reverse‐transcription polymerase chain reaction; SD, standard deviation.

Figure 3

Asprosin absence alleviates hepatic function injury and pathological damage of liver tissues of HFD‐induced NAFLD mice (n = 5/group). Related kits tested (A) ALT, AST, (B) TG, and TC levels in the mice serum. (C) H&E staining evaluated pathological alternations of mice liver tissues (×400). Data are presented as mean ± SD. ***p < .001 versus Control. ^### p < .001 versus HFD+Lenti‐vector. ALT, alanine aminotransferase; AST, aspartate aminotransferase; H&E, hematoxylin and eosin; HFD, high‐fat diet; NAFLD, nonalcoholic fatty liver disease; SD, standard deviation; TC, total cholesterol; TG, triglyceride.

Figure 4

Asprosin absence mitigates lipid accumulation in HFD‐stimulated NAFLD mice (n = 5/group). (A) Examination of mice liver weight. (B) Oil red O staining estimated lipid deposition (×400). (C) Related kits tested TG and TC levels in mice liver tissues. (D) RT‐qPCR and (E) western blot tested the expression of gluconeogenesis‐, fatty acid biosynthesis‐ and fatty acid oxidation‐associated factors. Data are presented as mean ± SD. ***p < .001 versus Control. ^## p < .01, ^### p < .001 versus HFD+Lenti‐vector. CPT1A, carnitine palmitoyltransferase 1A; FABP1, fatty acid binding protein‐1; FAS, fatty acid synthase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HFD, high‐fat diet; HMGCR, 3‐hydroxy‐3‐methylglutaryl‐coA reductase; NAFLD, nonalcoholic fatty liver disease; RT‐qPCR, quantitative reverse‐transcription polymerase chain reaction; SD, standard deviation; TC, total cholesterol; TG, triglyceride.

Figure 5

Asprosin insufficiency ameliorates inflammatory infiltration in HFD‐stimulated NAFLD mice (n = 5/group). (A) RT‐qPCR and (B) western blot tested the expression of inflammatory enzymes. (C) Western blot tested the expression of p‐p65 and p65. Data are presented as mean ± SD. ***p < .001 versus Control. ^### p < .001 versus HFD+Lenti‐vector. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HFD, high‐fat diet; IL‐1β, interleukin‐1beta. IL‐6, interleukin‐6; NAFLD, nonalcoholic fatty liver disease; p‐p65, phosphorylated‐p65; RT‐qPCR, quantitative reverse‐transcription polymerase chain reaction; SD, standard deviation; TNF‐α, Tumor necrosis factor‐alpha.

Figure 6

Asprosin insufficiency activates AMPK‐p38 signaling in HFD‐stimulated NAFLD mice (n = 5/group). Western blot analyzed the expression of AMPK‐p38 signaling‐associated proteins. Data are presented as mean ± SD. ***p < .001 versus Control. ^### p < .001 versus HFD+Lenti‐vector. AMPK, AMP‐activated protein kinase; HFD, high‐fat diet; NAFLD, nonalcoholic fatty liver disease; SD, standard deviation.

Figure 7

Asprosin interference suppresses lipid deposition in PA‐exposed AML‐12 cells. (A) RT‐qPCR and western blot tested Asprosin expression in AML‐12 cells challenged with PA. (B) RT‐qPCR and western blot tested Asprosin expression in PA‐exposed AML‐12 cells following transduction of shRNA‐Asprosin. (C) Related kits tested TG level. (D) Oil red O staining estimated lipid deposition (×400). (E) RT‐qPCR and (F) western blot tested the expression of gluconeogenesis‐, fatty acid biosynthesis‐, and fatty acid oxidation‐associated factors. Data are presented as mean ± SD. **p < .01, ***p < .001 versus Control. ^## p < .01, ^### p < .001 versus PA+shRNA‐NC. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; CPT1A, carnitine palmitoyltransferase 1A; FABP1, fatty acid binding protein‐1; FAS, fatty acid synthase; HMGCR, 3‐hydroxy‐3‐methylglutaryl‐coA reductase; PA, palmitic acid; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction; SD, standard deviation; shRNA, short hairpin RNA; TG, triglyceride.

Figure 8

Asprosin interference eases inflammatory response in PA‐challenged AML‐12 cells. (A) RT‐qPCR tested the expression of inflammatory enzymes. (B) Western blot tested the expression of p‐p65 and p65. Data are presented as mean ± SD. ***p < .001 versus Control. ^## p < .01, ^### p < .001 versus PA+shRNA‐NC. PA, palmitic acid; p‐p65, phosphorylated‐p65; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction; SD, standard deviation.

Figure 9

AMPK inhibitor reverses the impacts of Asprosin inhibition on AMPK‐p38 signaling in AML‐12 cells exposed to PA. (A–C) Western blot analyzed the expression of AMPK‐p38 signaling‐associated proteins. Data are presented as mean ± SD. ***p < .001 versus Control. ^### p < .001 versus PA+shRNA‐NC. ⁺ p < .05 versus PA+shRNA‐Asprosin. AMPK, AMP‐activated protein kinase; PA, palmitic acid; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction; SD, standard deviation; shRNA, short hairpin RNA.

Figure 10

Asprosin absence activates AMPK‐p38 signaling to diminish PA‐evoked lipid aggregation in AML‐12 cells. (A) Related kits tested TG level. (B) Oil red O staining estimated lipid deposition (×400). (C) RT‐qPCR and (D) western blot tested the expression of gluconeogenesis‐, fatty acid biosynthesis‐, and fatty acid oxidation‐associated factors. Data are presented as mean ± SD. ***p < .001 versus Control. ^### p < .001 versus PA+shRNA‐NC. ⁺ p < .05, ⁺⁺ p < .01, ⁺⁺⁺ p < .001 versus PA+shRNA‐Asprosin. AMPK, AMP‐activated protein kinase; CPT1A, carnitine palmitoyltransferase 1A; FABP1, fatty acid binding protein‐1; FAS, fatty acid synthase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HMGCR, 3‐hydroxy‐3‐methylglutaryl‐coA reductase; PA, palmitic acid; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction; SD, standard deviation; shRNA, short hairpin RNA; TG, triglyceride.

Figure 11

Asprosin absence activates AMPK‐p38 signaling to relieve PA‐elicited inflammatory response in AML‐12 cells. (A) RT‐qPCR tested the expression of inflammatory enzymes. (B) Western blot tested the expression of p‐p65 and p65. Data are presented as mean ± SD. ***p < .001 versus Control. ^### p < .001 versus PA+shRNA‐NC. ⁺⁺ p < .01, ⁺⁺⁺ p < .001 versus PA+shRNA‐Asprosin. AMPK, AMP‐activated protein kinase; IL‐1β, interleukin‐1beta; IL‐6, interleukin‐6; PA, palmitic acid; p‐p65, phosphorylated‐p65; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction; SD, standard deviation; shRNA, short hairpin RNA; TNF‐α, tumor necrosis factor‐alpha.