Taxifolin and Sorghum Ethanol Extract Protect against Hepatic Insulin Resistance via the miR-195/IRS1/PI3K/AKT and AMPK Signalling Pathways

This study aimed to evaluate the effects of taxifolin and sorghum ethanol extract on free fatty acid (FFA)-induced hepatic insulin resistance. FFA treatment decreased glucose uptake by 16.2% compared with that in the control, whereas taxifolin and sorghum ethanol extract increased the glucose uptake. Additionally, taxifolin and sorghum ethanol extract increased the expression of p-PI3K, p-IRS1, p-AKT, p-AMPK, and p-ACC in FFA-induced hepatocytes. Furthermore, FFA treatment increased the expression of miR-195. However, compared with the FFA treatment, treatment with taxifolin and sorghum ethanol extract decreased miR-195 expression in a dose-dependent manner. Taxifolin and sorghum ethanol extract enhanced p-IRS1, p-PI3K, p-AMPK, p-AKT, and p-ACC expression by suppressing miR-195 levels in miR-195 mimic- or inhibitor-transfected cells. These results indicate that taxifolin and sorghum ethanol extract attenuate insulin resistance by regulating miR-195 expression, which suggests that taxifolin and sorghum ethanol extract may be useful antidiabetic agents.

The liver is an important organ for glucose production and utilization as well as metabolic homeostasis. Insulin resistance leads to hyperglycaemia and abnormalities in the hepatic glucose output [11]. Free fatty acids (FFAs) have been shown to directly impair insulin signaling in hepatocytes [12]. In general, two main signaling pathways are involved in the regulation of glucose metabolism: the insulin receptor substrate-1 (IRS1)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and AMP-activated protein kinase (AMPK) pathways [13]. IRS1 has been linked to the treatment of hepatic insulin resistance [14]. Phosphorylation of IRS1 activates PI3K, regulates the downstream factor AKT, and promotes glucose uptake [15]. Activation of AMPK coordinates glucose

Cell Culture and Cytotoxicity
Human-derived hepatic cell lines (HepG2; HB-8065, ATCC, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium with 10% heat-inactivated foetal bovine serum and 1% penicillin-streptomycin at 37 • C in 5% CO 2 humidified air. Cytotoxicity was evaluated by the MTT assay. HepG2 cells were seeded onto plates at a density of 1 × 10 5 cells/mL and treated with an FFA mixture (500 µM) for 18 h. After FFA exposure, the hepatocytes were immediately treated with taxifolin or sorghum ethanol extract in RPMI1640 containing 0.2% BSA for an additional 24 h. After culture, MTT reagent (1 mg/mL) was added to each well and incubated for 2 h. The medium was removed, and the blue crystalized formazans were dissolved in DMSO. Absorbance at 550 nm was measured using a microplate reader (BioTek, Inc., Winooski, VT, USA).

Glucose Uptake
Glucose uptake assay was performed as stated before with slight modifications [28]. HepG2 cells were cultured in a serum-free medium containing an FFA mixture with taxifolin or sorghum ethanol extract for 18 h and then treated with taxifolin or sorghum ethanol extract in RPMI 1640 containing 0.2% BSA and insulin (100 nM) for 15 min. The culture medium was harvested and glucose colorimetric assay kit II (BioVision, Inc., San Francisco, CA, USA) was used. Absorbance at 450 nm was measured (BioTek, Inc., Winooski, VT, USA).

Western Blotting
Protein expression levels were confirmed as previously described [29]. Equal amounts of proteins were separated on a 10% sodium dodecyl sulphate polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK). The membranes were blocked with Tris-buffered saline/Tween 20 (TBST) containing 5% skim milk and incubated for 12 h with primary antibodies (1:1000). After washing with TBST, horseradish peroxidase-labelled secondary antibodies (1:2000) were added, and the blots were incubated for 2 h. Protein bands were activated by chemiluminescence and visualized on an X-ray film.

Statistical Analysis
Data are presented as mean ± standard error from at least three independent experiments. Student's t-test using SAS (version 8.1; SAS Institute, Cary, NC, USA) and Tukey's post-hoc test using GraphPad Prism software 5.0 (GraphPad Software Inc., La Jolla, CA, USA) were used.

Identification of Taxifolin in Sorghum Ethanol Extract
The antidiabetic activity of plant materials correlates with the content of bioactive compounds such as flavonoids and polyphenols [31]. UPLC-ESI-Q-TOF/MS was used to determine the composition of sorghum ethanol extract according to its retention time, standard, and pseudo-molecular ion formation ([M − H] − ). According to the UPLC and PDA chromatograms (at λ = 280 nm) of the sorghum ethanol extract shown in Figure 1, the peak was detected at a retention time of 2.78 min with formula of C 15 H 12 O 7 , neutral mass (Da) of 304.0583, observed ion (m/z) of 303.0546, and adduct of −H. Based on the MS/MS spectra and comparisons of the proposed fragment pattern in previous studies [32,33], the peak was identified as taxifolin. Taxifolin content (195 mg/100 g sorghum) was calculated by plotting a calibration curve using a standard. Taxifolin is a unique dihydroflavonol, which is the main flavonoid detected in sorghum. A recent study reported that the taxifolin content in sorghum grains ranges from 1.37 to 44.62 mg/100 g dry weight [1]. In recent years, because of its medicinal value, taxifolin has been widely used in the treatment of atherosclerosis, dyslipidemia, cardiovascular diseases, and other chronic diseases. Taxifolin treatment did not induce any apparent systemic abnormalities in TgSwDI mice [34]. A previous study showed that taxifolin at 25 mg/kg (equivalent to 240 mg/60kg human) can improve homeostasis of glucose in male spontaneously hypertensive (SHR) rats [11]. The absolute bioavailability of taxifolin was reported as 0.17% in rats. The bioavailability of taxifolin was 36% in rabbits upon detection of total conjugated and free taxifolin in plasma following enzymatic hydrolysis [35]. In the metabolite study, 191 metabolites from taxifolin were identified and 17 metabolites among them had similar bioactivities to taxifolin. This indicated that the effective form of taxifolin is the metabolites arising from it in vivo as well as the parent form. Moreover, taxifolin and its metabolites including quercetin, isorhamnetin disulfate, eriodictyol, luteolin-7-O-glucuronide, 3/4-hydroxyphenylpropionic acid, dihydroxyphenylacetic acid, and dihydrocaffeic acid have been reported to exert similar anti-diabetic activity [35]. Therefore, taxifolin and sorghum ethanol extract could be promising candidates for the treatment or prevention of diabetes.

Effects of Taxifolin and Sorghum Ethanol Extract on Cell Viability and Glucose Uptake
Inducing glucose absorption in the tissues and simultaneously supplying antioxidants and α-glucosidase inhibitors through nutrients could be a feasible and potential strategy to manage T2D [36]. Several studies showed that cereal grains and their phytochemicals possess antidiabetic activity. Polyphenols improve glucose consumption and upregulate insulin-dependent signals in many cell types [31]. Chung et al. (2011) reported that sorghum extract decreases serum fasting glucose and cholesterol levels [37]. Treatment with taxifolin (25 and 50 μM) or sorghum ethanol extract (5 and 10 μg/mL) along with FFA (500 μM) did not result in significant changes in cell viability ( Figure 2A). As shown in Figure 2B, FFA treatment significantly reduced glucose uptake by 16.2% compared with that in the control. Exposure to taxifolin (50 μM) and sorghum ethanol extract (10 μg/mL) enhanced glucose uptake by 24.1% and 27.6%, respectively, compared with that with the FFA treatment. Our results indicated that taxifolin and sorghum ethanol extract exerts an important role in the consumption of glucose in insulin resistance conditions.

Effects of Taxifolin and Sorghum Ethanol Extract on Cell Viability and Glucose Uptake
Inducing glucose absorption in the tissues and simultaneously supplying antioxidants and α-glucosidase inhibitors through nutrients could be a feasible and potential strategy to manage T2D [36]. Several studies showed that cereal grains and their phytochemicals possess antidiabetic activity. Polyphenols improve glucose consumption and upregulate insulin-dependent signals in many cell types [31]. Chung et al. (2011) reported that sorghum extract decreases serum fasting glucose and cholesterol levels [37]. Treatment with taxifolin (25 and 50 µM) or sorghum ethanol extract (5 and 10 µg/mL) along with FFA (500 µM) did not result in significant changes in cell viability ( Figure 2A). As shown in Figure 2B, FFA treatment significantly reduced glucose uptake by 16.2% compared with that in the control. Exposure to taxifolin (50 µM) and sorghum ethanol extract (10 µg/mL) enhanced glucose uptake by 24.1% and 27.6%, respectively, compared with that with the FFA treatment. Our results indicated that taxifolin and sorghum ethanol extract exerts an important role in the consumption of glucose in insulin resistance conditions. Antioxidants 2021, 10, x FOR PEER REVIEW 6 of 13

Effects of Taxifolin and Sorghum Ethanol Extract on the IRS1/PI3K/AKT and AMPK Signalling Pathways
The PI3K/AKT signaling pathway is a classic insulin signaling pathway that can modulate glucose transport by activating insulin [38]. AMPK is an evolutionarily conserved serine/threonine kinase whose activation induces insulin-sensitizing effects, making it an ideal therapeutic target for T2D [39]. Administration of sorghum extract significantly decreased the expression of p-p38 and PEPCK while increasing p-AMPK expression [40]. Gao et al. (2020) showed that taxifolin modulates insulin signaling in the kidneys via the PI3K/AKT pathway [11]. To investigate the effects of taxifolin and sorghum ethanol extract on the insulin signaling pathway, we confirmed IRS1, PI3K, AKT, AMPK, and ACC phosphorylation levels by western blotting. Compared with the control, FFA treatment in hepatocytes reduced the phosphorylation of IRS1 tyr ( Figure 3A). However, sorghum ethanol extract significantly increased IRS1 tyr phosphorylation at concentration of 10 μg/mL. Similarly, PI3K and AKT phosphorylation levels were decreased following FFA treatment in hepatocytes, whereas treatment with taxifolin and sorghum ethanol extract rescued the reduced phosphorylation in a dose-dependent manner ( Figure 3B,C). As shown in Figure 3D,E, the phosphorylation of AMPK was significantly enhanced by sorghum ethanol extract (10 μg/mL) administration and the phosphorylation of ACC was increased by taxifolin (50 μM) and sorghum ethanol extract (10 μg/mL). Consistent with the findings of previous reports, regulation of the IRS1/PI3K/AKT and AMPK pathways is a possible mechanism by which sorghum ethanol extract alleviates insulin resistance.

Effects of Taxifolin and Sorghum Ethanol Extract on the IRS1/PI3K/AKT and AMPK Signalling Pathways
The PI3K/AKT signaling pathway is a classic insulin signaling pathway that can modulate glucose transport by activating insulin [38]. AMPK is an evolutionarily conserved serine/threonine kinase whose activation induces insulin-sensitizing effects, making it an ideal therapeutic target for T2D [39]. Administration of sorghum extract significantly decreased the expression of p-p38 and PEPCK while increasing p-AMPK expression [40]. Gao et al. (2020) showed that taxifolin modulates insulin signaling in the kidneys via the PI3K/AKT pathway [11]. To investigate the effects of taxifolin and sorghum ethanol extract on the insulin signaling pathway, we confirmed IRS1, PI3K, AKT, AMPK, and ACC phosphorylation levels by western blotting. Compared with the control, FFA treatment in hepatocytes reduced the phosphorylation of IRS1 tyr ( Figure 3A). However, sorghum ethanol extract significantly increased IRS1 tyr phosphorylation at concentration of 10 µg/mL. Similarly, PI3K and AKT phosphorylation levels were decreased following FFA treatment in hepatocytes, whereas treatment with taxifolin and sorghum ethanol extract rescued the reduced phosphorylation in a dose-dependent manner ( Figure 3B,C). As shown in Figure 3D,E, the phosphorylation of AMPK was significantly enhanced by sorghum ethanol extract (10 µg/mL) administration and the phosphorylation of ACC was increased by taxifolin (50 µM) and sorghum ethanol extract (10 µg/mL). Consistent with the findings of previous reports, regulation of the IRS1/PI3K/AKT and AMPK pathways is a possible mechanism by which sorghum ethanol extract alleviates insulin resistance.

Effects of Taxifolin and Sorghum Ethanol Extract on the IRS1/PI3K/AKT and AMPK Signalling Pathways
The PI3K/AKT signaling pathway is a classic insulin signaling pathway that can modulate glucose transport by activating insulin [38]. AMPK is an evolutionarily conserved serine/threonine kinase whose activation induces insulin-sensitizing effects, making it an ideal therapeutic target for T2D [39]. Administration of sorghum extract significantly decreased the expression of p-p38 and PEPCK while increasing p-AMPK expression [40]. Gao et al. (2020) showed that taxifolin modulates insulin signaling in the kidneys via the PI3K/AKT pathway [11]. To investigate the effects of taxifolin and sorghum ethanol extract on the insulin signaling pathway, we confirmed IRS1, PI3K, AKT, AMPK, and ACC phosphorylation levels by western blotting. Compared with the control, FFA treatment in hepatocytes reduced the phosphorylation of IRS1 tyr ( Figure 3A). However, sorghum ethanol extract significantly increased IRS1 tyr phosphorylation at concentration of 10 μg/mL. Similarly, PI3K and AKT phosphorylation levels were decreased following FFA treatment in hepatocytes, whereas treatment with taxifolin and sorghum ethanol extract rescued the reduced phosphorylation in a dose-dependent manner ( Figure 3B,C). As shown in Figure 3D,E, the phosphorylation of AMPK was significantly enhanced by sorghum ethanol extract (10 μg/mL) administration and the phosphorylation of ACC was increased by taxifolin (50 μM) and sorghum ethanol extract (10 μg/mL). Consistent with the findings of previous reports, regulation of the IRS1/PI3K/AKT and AMPK pathways is a possible mechanism by which sorghum ethanol extract alleviates insulin resistance.

Effects of Taxifolin and Sorghum Ethanol Extract on IRS1, PI3K, AKT, AMPK, and ACC Phosphorylation in miR-195 Mimic-and Inhibitor-Transfected Cells
miR-195 is known to directly target the insulin receptor, which is IRS1 upstream [22]. Searching the TargetScan (http://www.targetscan.org accessed on 1 September 2020) database reveals putative miR-195-binding sites on the 3′-UTR of IRS1, PI3K, AMPK, and ACC mRNA. To evaluate the involvement of miR-195 in the effects of taxifolin and sorghum ethanol extract on the modulation of the IRS1/PI3K/AKT signaling pathway in the FFA-treated HepG2 cells, we confirmed the changes in miR-195 expression levels and investigated p-IRS1, p-PI3K, p-AKT, p-AMPK, and p-ACC expression levels. As shown in Figure 4, FFA treatment increased the miR-195 level. However, treatment with taxifolin or sorghum ethanol extract decreased miR-195 levels in a dose-dependent manner. We overexpressed or inhibited miR-195 using a mimic (50 nM) or an inhibitor (50 nM), respectively. The miR-195 level was significantly enhanced in overexpressed cells and decreased in inhibited cells compared with that in negative control-treated cells ( Figure S1).
Overexpression or inhibition of miR-195 affected the phosphorylation of IRS1, PI3K, AKT, AMPK, and ACC. Transfection with the miR-195 mimic partly aggravated the FFAinduced downregulation of p-IRS1 ( Figure 5A). However, taxifolin and sorghum ethanol extract enhanced the phosphorylation of IRS1. Similarly, PI3K, AKT, AMPK, and ACC phosphorylation levels were inhibited following FFA with miR-195 mimic treatment,

Effects of Taxifolin and Sorghum Ethanol Extract on IRS1, PI3K, AKT, AMPK, and ACC Phosphorylation in miR-195 Mimic-and Inhibitor-Transfected Cells
miR-195 is known to directly target the insulin receptor, which is IRS1 upstream [22]. Searching the TargetScan (http://www.targetscan.org accessed on 1 September 2020) database reveals putative miR-195-binding sites on the 3 -UTR of IRS1, PI3K, AMPK, and ACC mRNA. To evaluate the involvement of miR-195 in the effects of taxifolin and sorghum ethanol extract on the modulation of the IRS1/PI3K/AKT signaling pathway in the FFA-treated HepG2 cells, we confirmed the changes in miR-195 expression levels and investigated p-IRS1, p-PI3K, p-AKT, p-AMPK, and p-ACC expression levels. As shown in Figure 4, FFA treatment increased the miR-195 level. However, treatment with taxifolin or sorghum ethanol extract decreased miR-195 levels in a dose-dependent manner. We overexpressed or inhibited miR-195 using a mimic (50 nM) or an inhibitor (50 nM), respectively. The miR-195 level was significantly enhanced in overexpressed cells and decreased in inhibited cells compared with that in negative control-treated cells ( Figure S1).

Conclusions
The present study showed that taxifolin and sorghum ethanol extract attenuated FFA-induced hepatic insulin resistance by suppressing miR-195 expression. Sorghum is a rich source of phytochemicals such as phenolic acids and flavonoids, which may contribute to its antidiabetic activity. Moreover, taxifolin is a bioactive flavonoid of dietary origin that performs several functions. The antidiabetic activity of taxifolin and sorghum ethanol extract was demonstrated through the regulation of miR-195 expression and the IRS1/PI3K/AKT, and AMPK signaling pathways. Hence, our results suggest that taxifolin and sorghum ethanol extract may be useful alternatives to miR-based therapeutics.