Crocin ameliorates hepatic steatosis through activation of AMPK signaling in db/db mice

All animal experiments were approved by the Institutional Animal Care and Use Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (IACUCUH-2018-01-116) and in accordance with ARRIVE and NIH guidelines for animal welfare. C57BL/KsJ-Lepdb (db/db) mice and their lean littermates (wild type) were obtained from Mode Animal Research Center of Nanjing University. Eight-week-old male mice were maintained at 22 ± 2 °C with 60 ± 5% relative humidity, and under a 12 h light/dark cycle with free access to water and regular chow diet. db/db mice were randomly divided into three groups (db/db, Crocin, and Crocin+LV-shAMPK, n = 10 in each group). Mice in Crocin+LV-shAMPK group were injected with LV-shAMPK via tail vein at the age of 8 weeks. The injection of LV-shAMPK was performed once every two weeks for four weeks. Mice in db/db and Crocin group were injected with LV-shNC. After that, mice in Crocin and Crocin+LV-shAMPK groups were daily orally administrated with 20 mg/kg crocin for 8 weeks. Mice in db/db group was given vehicle administration. Meanwhile, WT mice was treated with vehicle in an identical manner as the normal control. After the treatment, all mice were anesthetized using 2% isoflurane and sacrificed. Blood samples were collected and tissue samples were snap-frozen in liquid nitrogen or collected for paraffin embedding.

Histological examination was performed to evaluated liver injury. Paraffin-embedded liver sections (5 μm) were prepared and stained with H&E. Images were captured using a light microscope (Olympus, Japan). Quantitative analysis of hepatic steatosis was performed using image-pro software and the percentage of vacuolar hepatocytes per field of vision was calculated. The results were shown as folds versus control.

Before the OGTT and IPITT examinations, mice were fasted for 12 h and 6 h, respectively. Blood glucose level was measured at 0, 30, 60, 90, and 120 min after the administration of glucose and insulin. Blood glucose level was determined using tail vein blood via Accu-Chek (Roche, Basel, Switzerland). At the end of the experiment, mice were fasted for 12 h and blood samples were collected from the orbital venous plexus immediately before the sacrifice. Serum levels of alanine aminotransferase (ALT), alanine aminotransferase (AST), triglyceride (TG), non-esterified fatty acid (NEFA) and total cholesterol (TC), and liver TG were quantified using commercial kits (Nanjing Jiancheng, Nangjing, China). Serum insulin levels were determined using an enzyme-linked-immunosorbent assay (ELISA) kit (Invitrogen, Carlsbad, CA, USA).

Total RNA from liver tissues was isolated using TRIzol (Life Technologies, Carlsbad, CA, USA). The RNA samples were treated with DNase and reverse-transcribed into cDNA using Superscript II (Life Technologies, Foster City, CA). Real-time RT-PCR was carried out with SYBR Green PCR Master Mix (Takara, Tokyo, Japan) in an ABI StepOnePlus Real-time PCR System. The expression of mRNA for each target gene was normalized relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Liver tissues were lysed in RIPA Lysis Buffer (Beyotime, Jiangsu, China). After the determination of protein concentration using BCA Protein Assay Kit (Beyotime, Jiangsu, China), the samples were separated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted onto polyvinylidene fluoride membranes (PVDF, Millipore). Subsequently, membranes were incubated with primary antibodies (diluted 1:1000; Cell Signaling Technology, USA) overnight at 4 °C. After washing with TBST, membranes were incubated with horseradish-peroxidase (HRP)-conjugated secondary antibody (diluted 1:5000; Thermo Fisher Scientific, USA) for 1 h at room temperature. Finally, the blots were visualized using chemiluminescence (ECL) detection reagents (Thermo Fisher Scientific, USA). Data were analyzed by calculation of integrated optical density.

Results were expressed as the mean ± SD. The significance of differences among groups was assessed by one-way ANOVA analysis followed by Dunnett’s test. Statistical significance was defined as p < 0.05.

We firstly evaluated the effect of crocin on AMPK signaling in liver of db/db diabetic mice. As shown in Fig. 1a and b, AMPK phosphorylation was decreased in liver of db/db mice, compared with that in WT mice. Crocin markedly upregulated the phosphorylation of AMPK in liver of db/db mice (Fig. 1a and b). Crocin-induced enhancement of AMPK phosphorylation was significantly inhibited by LV-shAMPK (Fig. 1a and b). LV-shAMPK abolished the total protein expression of AMPK (Fig. 1a and b). Additionally, mTOR phosphorylation was increased in liver of db/db mice, compared with that in WT mice (Fig. 1a and c). Crocin resulted in a significant decrease of mTOR phosphorylation in liver of db/db mice, whreas this effect was suppressed by LV-shAMPK injection (Fig. 1a and c). These results demonstrated that Crocin upregulated AMPK signaling in liver of diabetic mice.

In Fig. 2a, we showed that the increased liver weight in db/db mice was reduced by the treatment of crocin. Crocin-induced effect on liver weight was reversed by the injection of LV-shAMPK (Fig. 2a). The serum levels of AST and ALT in db/db mice were significantly higher than that in WT mice. Treatment of crocin remarkably reduced the serum levels of AST and ALT in db/db mice, which effect was inhibited by downregulation of AMPK (Fig. 2b and c). TG content was measured to evaluate lipid accumulation in liver. As shown in Fig. 2d, the high level of TG in liver of db/db mice was significantly reduced by crocin, whereas this effect of crocin was inhibited by downregulation of AMPK (Fig. 2d). Morphological changes of liver were observed and the results showed that there were marked vacuolar degradation and disorganized hepatocytes, and inflammatory infiltration in liver of db/db mice (Fig. 2e). Crocin significantly attenuated the morphological changes of liver and hepatic steatosis in db/db mice, which effect was inhibited by LV-shAMPK (Fig. 2e and f). These results suggested that crocin ameliorated hepatic steatosis and injury through activation of AMPK signaling in db/db mice.

To further evaluate the mechanism underlying the protective effects of crocin against hepatic steatosis and injury under diabetic condition, we determined the expression of a series of factors involved in the regulation of lipogenesis and β-oxidation of fatty acids. As shown in Fig. 3a-e, the mRNA expression of lipogenesis-related regulators, including sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1), peroxisome proliferator-activated receptor γ (PPARγ), and diacylglycerol acyltransferase (DGAT) in liver of db/db mice, was significantly reduced by crocin. Crocin-induced decrease of mRNA expression of these factors was inhibited by LV-shAMPK (Fig. 3a-e). Moreover, the mRNA expression of β-oxidation of fatty acids-associated regulators, including PPARα, acyl-CoA oxidase 1 (Acox1), carnitine palmitoyltransferase 1 (Cpt1), and 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) in liver of db/db mice, was significantly increased by crocin (Fig. 3f-i). LV-shAMPK significantly inhibited crocin-induced upregulation of β-oxidation-related factors (Fig. 3f-i). In addition, crocin decreased SREBP-1 protein expression but increased PPARα protein expression in liver of db/db mice, which effect was inhibited by LV-shAMPK (Fig. 3j). These data demonstrated that crocin inhibited lipogenesis and promoted β-oxidation of fatty acids through activation of AMPK signaling in liver in db/db mice.

Finally, we evaluated the effect of crocin on general metabolism of glucose and lipid in db/db diabetic and obese mice. In Fig. 4a,b and c, we showed that crocin significantly decreased the high level of body weight, fasting blood glucose, and serum insulin, whereas LV-shAMPK notably suppressed these effects of crocin. We used OGTT and IPITT tests to evaluate the glucose tolerance and insulin sensitivity. As shown in Fig. 4d-g, in both OGTT and IPITT tests, crocin decreased the glucose level at different time points after the load of glucose and insulin and reduced the area under the curve (AUC). Crocin-induced attenuation of glucose metabolic disorder was inhibited by LV-shAMPK (Fig. 4d-g). Additionally, the levels of serum TG, TC and NEFA in db/db mice were significantly decreased by crocin, which effect was inhibited by LV-shAMPK. These results demonstrated that crocin attenuates glucose and lipid metabolic disorder through regulation of AMPK signaling in db/db mice.

None.

The data will be available on request.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology and in accordance with ARRIVE and NIH guidelines for animal welfare.

Not applicable.

The authors declare no conflict of interest.

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The data will be available on request.