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. 2018 Nov;175(22):4218-4228.
doi: 10.1111/bph.14482. Epub 2018 Oct 11.

Nuciferine Ameliorates Hepatic Steatosis in High-Fat Diet/Streptozocin-Induced Diabetic Mice Through a PPARα/PPARγ coactivator-1α Pathway

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Nuciferine Ameliorates Hepatic Steatosis in High-Fat Diet/Streptozocin-Induced Diabetic Mice Through a PPARα/PPARγ coactivator-1α Pathway

Chao Zhang et al. Br J Pharmacol. .
Free PMC article

Abstract

Background and purpose: Nuciferine, an alkaloid found in Nelumbo nucifera leaves, alleviates dyslipidemia in vivo. However, whether it improves liver injury in diabetic conditions and the underlying mechanism is unclear. The present study aimed to investigate the effects of nuciferine on lipid and glucose metabolism in a murine model of Type 2 diabetes mellitus (T2DM) and to determine the underlying mechanisms of these effects.

Experimental approach: A murine model of T2DM was induced by high-fat diet (HFD) feeding combined with streptozocin (STZ) injections, and the diabetic mice were treated with nuciferine in their food. The underlying mechanism of the anti-steatotic effect of nuciferine was further explored in HepG2 hepatocytes cultured with palmitic acid. Major signalling profiles involved in fatty acid oxidation were then evaluated, using Western blot, RT-qPCR and si-RNA techniques, along with immunohistochemistry.

Key results: Nuciferine restored impaired glucose tolerance and insulin resistance in diabetic mice. Hepatic levels of total cholesterol, triglycerides and LDL were decreased, as were the number of lipid droplets, by nuciferine treatment. Furthermore, nuciferine up-regulated β-oxidation related genes in livers of diabetic mice. Luciferase reporter cell assay showed that nuciferine directly reversed palmitic acid-induced inhibition of PPARα transcriptional activity. Silencing PPARγ coactivator-1α (PGC1α) expression in HepG2 cells abolished the effects of nuciferine in accelerating β-oxidation.

Conclusions and implications: Nuciferine improved lipid profile and attenuated hepatic steatosis in HFD/STZ-induced diabetic mice by activating the PPARα/PGC1α pathway. Nuciferine may be a potentially important candidate in improving hepatic steatosis and the management of T2DM.

Figures

Figure 1
Figure 1
Nuciferine improves glucose metabolism and insulin sensitivity on diabetic mice. (A) Chemical structure of nuciferine; (B and C) Results of IPGTT. Mice were fasted overnight for 12 h with only water available. Glucose (2.5 g·kg−1) was injected i.p. Blood glucose was determined before the injection and after 15, 30, 60 and 120 min. (D and E) Results of IPITT. Fasting mice (4 h) were given insulin (0.8 U·kg−1) by i.p. injection. Blood glucose was determined before the injection of insulin and after 15, 30, 45 and 60 min. (F) HOMA‐IR data. HOMA‐IR was calculated as follows: Fast glucose × fasting plasma insulin level/22.5. Data shown are means ± SEM; n = 6 per group. *P < 0.05, significantly different from NC group; # P < 0.05, significantly different from DC group.
Figure 2
Figure 2
Nuciferine attenuated weight gain, liver injury and lipid accumulation on diabetic mice. (A) The body weight growth rates of the mice. The weight growth rate was calculated as follows: Wn/W0 × 100%. Wn is body weight in n weeks, W0 is the body weight in day 0. (B) Serum levels of ALT. (C) Serum levels of AST. (D) Liver cholesterol pattern analysis of the mice. (E) Liver triglyceride levels of the mice. (F) Oil Red O staining and quantification of liver sections. Scale bar, 50 μm. Data shown are means ± SEM; n = 10 per group. *P < 0.05, significantly different from NC group; # P < 0.05, significantly different from DC group.
Figure 3
Figure 3
Nuciferine regulated PPARα expression and transactivation on diabetic mice and palmitic acid‐treated HepG2 cells. (A) Western blot analysis and qualification of PPARα in liver tissues of the mice. (B) Hepatic mRNA levels (expressed as fold change) of PPARα and its target genes involved in fatty acid oxidation were quantified by qRT‐PCR. n = 10 per group. (C) Protein levels and (D) mRNA levels of PPARα in HepG2 cells. HepG2 cells were pretreated with or without nuciferine (Nuci; 10 μM) for 12 h, then incubated with palmitic acid (PA; 150 μM) for 24 h. (E) The effect of nuciferine on PPARα transactivation. HepG2 cells were transfected with PPRE‐luc and PPARα plasmids and then treated with palmitic acid, with or without nuciferine. (F) The down‐regulation of PPARα protein expression in HepG2 cells after siRNA‐PPARα transfection. (G) PPARα silencing significantly reduced gene expression of Acox1 and Fgf21. Data shown are means ± SEM; n = 3 per group. *P < 0.05, significantly different from NC group or control group; # P < 0.05, significantly different from DC group or palmitic acid treated group.
Figure 4
Figure 4
Nuciferine promoted PGC1α expression on diabetic mice and PA‐treated HepG2 cells. (A) Western blot analysis and qualification of PGC1α in liver tissues of the mice. (B) Hepatic mRNA levels of PGC1α in liver tissues of the mice. N = 10 per group. (C) Immunohistochemistry staining and quantification for PGC1α in liver tissues. Scale bar: 20 μm. The (D) protein levels and (E) mRNA levels of PGC1α in HepG2 cells. HepG2 cells were pretreated with or without nuciferine (Nuci; 10 μM) for 12 h, then incubated with palmitic acid (PA; 150 μM) for 24 h. Data shown are means ± SEM; n = 3 per group *P < 0.05, significantly different from NC group or control group; # P < 0.05, significantly different from DC group or PA treatment group.
Figure 5
Figure 5
Acute knockdown of PGC1α in HepG2 cells alters the effects of nuciferine on gene expression of enzymes of fatty acid oxidation. (A) The down‐regulation of PGC1α expression in HepG2 cells after siRNA transfection. (B–D) PGC1α silencing abolished the effect of nuciferine (Nuci) on palmitic acid (PA)‐induced gene expression of Acox1, Ehhadh and Fgf21 in HepG2 cells. *P < 0.05, significantly different from control group; # P < 0.05, significantly different from PA treated group. (E) The effect of si‐PGC1α on PPARα transactivation. HepG2 cells were transfected with PPRE‐luc and PPARα plasmids with or without si‐PGC1α for 6 h. The medium was changed to DMEM and incubated for 36 h. Data shown are means ± SEM; n = 3 per group *P < 0.05, significantly different from control group.
Figure 6
Figure 6
Diagram of the possible molecular mechanisms involved in the attenuation of hepatic dyslipidemia in HFD/STZ‐induced diabetic mice. Nuciferine increased the expression of PGC1α and PPARα and activated their downstream target genes related to fatty acid oxidation.

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