Tert-Butylhydroquinone alleviates insulin resistance and liver steatosis in diabetes.

OBJECTIVES: This work aimed to determine tert-Butylhydroquinone (TBHQ)'s effects on insulin resistance (IR) and liver steatosis in diabetic animals and to explore the underpinning mechanisms.
MATERIALS AND METHODS: Male ApoE-/-mice underwent streptozocin (STZ) administration while receiving a sucrose/fat-rich diet for type 2 diabetes mellitus (T2DM) establishment. This was followed by a 6-week TBHQ administration. Body weight, fasting (FBG) and postprandial (PBG) blood glucose amounts, and insulin concentrations were measured, and the oral glucose tolerance test (OGTT) was carried out. Hematoxylin and eosin (H and E) staining and immunoblot were carried out for assessing histology and protein amounts in the liver tissue samples. In addition, cultured HepG2 cells were administered HClO and insulin for IR induction, and immunoblot was carried out for protein evaluation. Finally, the cells were stained with the Hoechst dye for apoptosis evaluation.
RESULTS: The model animals showed T2DM signs, and TBHQ decreased FBG, ameliorated glucose tolerance and reduced liver steatosis in these animals. In addition, TBHQ markedly upregulated AMPKα2, GLUT4 and GSK3 β, as well as phosphorylated PI3K and AKT in the liver of mice with T2DM. In agreement, TBHQ decreased HClO-and insulin-related IR in cells and suppressed apoptosis through AMPKα2/PI3K/AKT signaling.
CONCLUSIONS: TBHQ alleviates IR and liver steatosis in a mouse model of T2DM likely through AMPKα2/PI3K/AKT signaling.

Introduction

Roughly 425 million diabetic individuals have been reported worldwide, estimating approximately 700 diabetics by 2045. These include >90% type 2 diabetes mellitus (T2DM) patients.[1] T2DM results from insulin resistance (IR) that features abnormalities in the liver, fat tissue, and skeletal muscles under insulin induction.[23] IR, as the major factor in T2DM, is targeted in T2DM therapy.[4]

Tert-Butylhydroquinone (TBHQ), the most common antioxidant obtained synthetically,[5] enhances insulin's effect in cell cultures.[67] In addition, TBHQ markedly increases phosphorylated protein kinase B (AKT) amounts, reflecting insulin sensitivity in the liver and nerve cells, although the underpinning mechanism remains unknown.[89]

AMP-activated protein kinase (AMPK) comprises catalytic (α with isoforms 1α and 2α) and two modulatory (β and γ) subunits. AMPKα2 (or PRKAA2) is mostly expressed in the liver. In recent years, it was shown TBHQ activates AMPKα2,[10] which represses glucose biosynthesis and regulates insulin sensitivity in the liver.[111213]

Therefore, we speculated TBHQ protects liver function in diabetes, partly by upregulating the AMPKα2 factor.

Materials and Methods

Materials

Tert-Butylhydroquinone (TBHQ), streptozocin (STZ), insulin and HClO were provided by (Sigma, St. Louis, MO, USA).

Fetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) were provided by Gibco BRL (Gibico, Grand Island, NY, USA). The bicinchoninic acid (BCA) protein assay, hematoxylin and eosin (HE) and Hoechst staining kits were provided by Beyotime (Beijing, China). Antibodies targeting p-AKT, AMPKα2, glycogen synthase kinase-3 β (GSK3 β), and glucose transport-4 (GLUT4) were provided by Abcam (Cambridge, MA, USA). Anti-phosphatidylinositol 3-kinase (PI3K) and anti-AKT antibodies were provided by Sangon (Shanghai, China). Antibodies directed against p-PI3K were provided by Cell Signaling Technology (Danvers, MA, USA). Antibodies targeting β-actin were provided by Beyotime (Beijing, China). PRKAA2 siRNA and riboFECT™ CP transfection kit were provided by Guangzhou RiboBio (Guangzhou, China).

Mouse experiments

Male ApoE-/-mice were provided by Model Animal Research Center GemPharmatech, Nanjing University. The animal protocols had approval from the Xinxiang Medical University Veterinary Medicine Animal Care and Use Committee. The animals were housed at 25°C under a 12 h/12 h light-dark cycle. Following 7 days of adaptive housing, mice weighing 24 ± 0.5 g underwent randomization into five groups of 10 animals each [Table 1]:

Table 1

The treatment of each mice group

Group	Treatment
Control +TBHQ	Animal feeding using rodent chow for 11 weeks Animal feeding using rodent chow for 11 weeks. In the final 6 weeks, they were intragastrically treated with 60 mg/kg TBHQ[1415]
T2DM	Animal feeding with a sucrose/fat-rich diet for 14 days, followed by a 10-h fasting and STZ (50 mg/kg, pH=4.5) injection[16] Next, animal feeding with the same diet was carried out for 14 days for diabetes establishment. Finally, diabetic animals continued with this diet for the final 6 weeks
+TBHQ +Rosiglitazone	Animals with experimental T2DM were intragastrically administered 60 mg/kg TBHQ for the final 6 weeks Animals with experimental T2DM were intragastrically administered 7 mg/kg rosiglitazone for the final 6 weeks

TBHQ=Tert-butylhydroquinone, T2DM=Type 2 diabetes mellitus, STZ=Streptozocin

The sucrose/fat-rich diet followed a previous report.[17] Diabetic mice underwent the administration of TBHQ or rosiglitazone after β-cell damage [Figure 1a].

Figure 1

Tert-butylhydroquinone alleviates body weight, fasting and postprandial blood glucose amounts as well as oral glucose tolerance test changes in a mouse model of diabetes. (a) Schematic representation of the mouse study. (b) Fasting blood glucose. (c) Postprandial blood glucose. (d) Body weights. (e) Oral glucose tolerance test. **P < 0.01 versus Control group; ##P < 0.01 versus type 2 diabetes mellitus group. Values are mean ± standard deviation (n = 10)

Biochemical assays

Body weights and blood glucose levels were recorded weekly. Anesthesia was carried out by the intraperitoneal administration of sodium pentobarbital (30 mg/kg). The animals were euthanized through cervical dislocation after the 11 weeks. After retrobulbar bleeding, blood specimens underwent centrifugation (4000 rpm/15 min) at 4°C for serum preparation. Serum fasting insulin amounts were obtained with an enzyme-linked immunosorbent assay kit (Beyotime, Beijing, China). Fresh liver tissue specimens were utilized for analyzing the amounts of targeted mRNAs. Liver tissue specimens underwent fixation with 4% formalin for hematoxylin and eosin (HE) staining.

Fasting blood glucose and postprandial blood glucose detection and oral glucose tolerance test

Fasting blood glucose (FBG) was assessed in blood specimens obtained from the tail vein of mice following a 12-h fasting. For postprandial blood glucose (PBG) assessment, blood was collected following a 2-h feeding. Blood glucose amounts were determined with Glutest Pro (Sanwa Chemical, Japan).

At the endpoint, the oral glucose tolerance test (OGTT) was carried out after oral loading of glucose (1.0 g/kg) following a 12-h fasting. All blood glucose measurements followed previous protocols.[1718]

Hematoxylin and eosin staining

Fresh liver specimens underwent fixation with 4% formalin at ambient overnight, followed by dehydration, paraffin embedding, and sectioning at 4 μm. H and E staining was then carried out for histological analysis.[19]

Immunoblot

Total protein isolated from the liver tissue specimens or cultured HepG2 cells was obtained by incubation with chilled RIPA buffer with 1% PMSF (Beyotime, Beijing, China) for 30–60 min. A BCA Protein Assay kit (Beyotime, Beijing, China) was utilized for protein quantitation. Then, 20–60 μg of total protein underwent separation by 10% SDS-PAGE, and electro-transfer onto polyvinylidene fluoride membranes (Billerica, MA, USA) was next carried out. After a 1-h blocking (5% skimmed milk at ambient), successive incubations with primary (4°C, overnight) and HRP-linked anti-mouse (or anti-rabbit) (ABclonal; 1:5000 at ambient for 1 h) antibodies were performed. An ECL kit (Billerica, MA, USA) was utilized for detection, and densitometric quantitation used Image J 1.43 (National Institutes of Health, USA). The specific operation steps were outlined previously.[2021] Anti-GLUT4, anti-GSK3 β, and anti-p-AKT antibodies (1:1000, Abcam (Cambridge, MA, USA)), as well as anti-p-PI3K (1: 1000, Cell Signaling Technology (Danvers, MA,USA)), anti-AKT (1:500, Sangon (Shanghai, China)), anti-PI3K (1:500, Sangon (Shanghai, China)), anti-AMPKα2 (1:1000, Abcam (Cambridge, MA, USA)), and and anti-β-actin (1:1000, Beyotime (Beijing, China)) antibodies were probed.

Cells and treatments

HepG2 cells underwent culture in high-glucose DMEM with 10% FBS in an incubator with 5% CO2 at 37°C. For establishing an IR model in HepG2 cells, the latter underwent exposure to HClO (200 mM, 40 min) and insulin (25 min). PRKAA2 siRNA (5′-GTTTAGATGTTGTTGGAAAdTdT-3′) and negative control siRNA (5′-GACUACUGGUCGUUGAACUdTdT-3′), provided by Ribobio (China), were transfected with the ribo FECT™CP transfection kit (RiboBio, Guangzhou, China).

First, hypoxia's effect on PASMCs and AMPKα2's role were determined. To this end, the cells were assigned to eight groups, i.e., the control, TBHQ, HClO, insulin, HClO + TBHQ, HClO + insulin, TBHQ + insulin and HClO + TBHQ + insulin groups. Accordingly, GLUT4, GSK3 β, p-AKT, p-PI3K, AKT, PI3K, and AMPKα2 amounts were assessed, as well as apoptosis.

Then, AMPKα2's role in diabetic liver steatosis was assessed as well as AMPKα2/PI3K/AKT signaling involvement. The cells were assigned to 12 groups, i.e., the Control, PRKAA2 siRNA, TBHQ, TBHQ + PRKAA2 siRNA, TBHQ + Insulin, TBHQ + PRKAA2 siRNA + insulin, HClO, PRKAA2 siRNA + HClO, HClO + insulin, PRKAA2 siRNA + HClO + insulin, HClO + TBHQ + insulin and PRKAA2 siRNA + HClO + TBHQ + insulin groups. Accordingly, GLUT4, GSK3 β, p-AKT, p-PI3K, AKT, PI3K, and AMPKα2 were quantitated.

Hoechst staining

Cell seeding (roughly 3000 cells per well) was carried out in 96-well plates in DMEM plus 10% FBS for a 24-h pre-HClO and insulin administrations, followed by TBHQ administration for 30 min. The Hoechst staining kit (Beyotime, Beijing, China) was utilized for apoptosis quantitation as directed by the supplier.

Quantitative reverse transcription polymerase chain reaction

Quantitative reverse transcription polymerase chain reaction (RT-PCR) utilized the SYBR Premix Ex Taq (TaKaRa, Tkyo, Tapan), as directed by the manufacturer. AMPKα2 mRNA amounts were determined on an ABI step one plus Real-Time PCR System Applied Biosystems(Foster, CA, USA). The 2−ΔΔCt method[13] was utilized for analyzing data normalized to GAPDH expression. The primers were: Human PRKAA2, sense 5′-GGAGAACATCAATTAACAGGCC-3′ and antisense 5′-CCAACAACATCTAAACTGCGAA-3′; human GAPDH, sense 5′-CAAATTCCATGGCACCGTCA-3′and antisense 5′-GGTCATGAGTCCTTCCACGA-3′.

Statistical analysis

SPSS (Version X; IBM, Armonk, NY, USA) was utilized for the data analysis. All quantitative data are mean ± standard deviation and were compared by the one-way analysis of variance with the Newman-Student-Keuls test. Two-sided P < 0.05 indicated statistical significance.

Results

Tert-butylhydroquinone markedly alleviates glucose level changes in the mouse model of diabetes

Treating the animals with a sucrose/fat-rich diet and STZ resulted in T2DM modeling. Precisely, treated animals had marked elevations of serum FBG and PBG amounts [Figure 1c and d], increased OGTT values [Figure 1e], and decreased body weights [Figure 1b]. Interestingly, the rosiglitazone and TBHQ groups showed starkly reduced FBG, PBG and OGTT levels, compared with the T2DM group, while body weights were overtly elevated. In addition, TBHQ had similar or better effects in comparison with rosiglitazone. Jointly, the above results suggested the repeated administration of TBHQ alleviates glucose metabolic abnormalities as well as body weight increase in T2DM mice.

Tert-butylhydroquinone efficiently alleviates liver steatosis in the mouse model of diabetes and increases HepG2 cell viability

H and E staining [Figure 2a] showed no abnormal architectural and morphological changes of the liver tissue in control mice. Liver cells had a cord-like arrangement, surrounding the central vein. Meanwhile, in the T2DM group, an important amount of necrotic hepatocytes were detected, as well as macrovesicular steatosis. Meanwhile, rosiglitazone and TBHQ, respectively, markedly reduced these effects. The above findings demonstrated repeated TBHQ administration alleviated diabetes-related pathological alterations in the mouse liver.

Figure 2

Tert-butylhydroquinone alleviates liver steatosis in the diabetes mouse model and improves HepG2 cell survival. (a) H and E staining of liver tissue samples (read and blue represent glycogen and nuclear signals; ×200). (b) Hoechst staining of HepG2 cells (×200). Arrow, an apoptotic cell

In addition, HClO and insulin promoted cell apoptosis, as reflected by the amounts of cells showing nuclear condensation or fragmentation [Figure 2b]. In the TBHQ group, there were markedly less apoptotic HepG2 cells. The TBHQ groups had differing extents of apoptosis (64.65%, 71.56%, 23.05% and 20.08% in the HClO, HClO + insulin, HClO + TBHQ + insulin and HClO + TBHQ groups, respectively). The above data suggested TBHQ increased HepG2 cell survival.

Tert-butylhydroquinone modulates PI3K/AKT signaling in the mouse model of diabetes and IR in HepG2 cells

The proteins related to glucose metabolism, including GLUT4, GSK3 β, p-PI3K, and p-AKT, are indexes of diabetes and IR. In this study, TBHQ treatment resulted in higher protein amounts of GLUT4 [Figure 3a and b] and GSK3 β [Figure 3c and d] in comparison with diabetic mice and insulin-resistant HepG2 cells. In addition, phosphorylated [Figure 3e and f] PI3K and [Figure 3g and h] AKT amounts were increased in the in vivo and in vitro model groups by rosiglitazone or TBHQ. PI3K phosphorylation induces AKT phosphorylation.[2223]. Jointly, these findings suggested TBHQ reduced glucose uptake and increased glycogen biosynthesis through PI3K/AKT signaling.

Figure 3

Amounts of glucose metabolism-related proteins in the mouse liver and HepG2 cells. (a and b) GLUT4 protein amounts. (c and d) GSK3β protein amounts. (e and f) Phosphorylated PI3K amounts. (g and h) Phosphorylated AKT amounts. *P < 0.05 versus Control group; $P < 0.05 versus type 2 diabetes mellitus group; &P < 0.05 versus HClO + insulin group. Data are mean ± standard deviation (n = 3)

Tert-butylhydroquinone activates AMPKα2 in the mouse model of diabetes and insulin-resistant HepG2 cells

As an important cell energy modulator, AMPKα2 considerably affects glucose use.[12] As shown in Figure 4a and b, both in vivo and in vitro models showed markedly reduced AMPKα2 amounts, and this effect was reversed by TBHQ.

Figure 4

Tert-butylhydroquinone regulates AMPKα2. (a) AMPKα2 amounts in the mouse liver. (b) AMPKα2 amounts in HepG2 cells. *P < 0.05 versus Control group; #P < 0.05 versus type 2 diabetes mellitus group; &P < 0.05 versus HClO + insulin group. Data are mean ± standard deviation (n = 3)

Tert-butylhydroquinone activates AMPKα2 to enhance insulin sensitivity in HepG2 cells administered HClO and insulin

To examine whether TBHQ improves glucose metabolism through AMPKα2, AMPKα2 silencing was carried out in HepG2 cells, followed by p-AKT, p-PI3K, GLUT4, and GSK3 β level measurements. The results showed that HepG2 cells administered HClO and insulin had markedly reduced amounts of the above proteins. Interestingly, TBHQ upregulated these factors, and AMPKα2 blockade remarkably decreased TBHQ-related p-AKT, p-PI3K, GLUT4, and GSK3 β upregulation [Figure 5a-d]. Jointly, the above findings suggested TBHQ upregulated GLUT4, GSK3 β, p-PI3K, and p-AKT in IR HepG2 cells through AMPKα2 activation.

Figure 5

Tert-butylhydroquinone alleviates insulin resistance in HepG2 cells through AMPKα2/PI3K/AKT signaling. GLUT4 (a), GSK3β (b), phosphorylated PI3K (c) and phosphorylated AKT (d) amounts in HepG2 cells are shown. *P < 0.05 versus Control group; &P < 0.05 versus HClO + insulin group; #P < 0.05 versus siPRKAA2 + Control group; $P < 0.05 versus siPRKAA2 + HClO + insulin group; ^P < 0.05 versus siPRKAA2 + HClO + Tert-butylhydroquinone + insulin group. Data are mean ± standard deviation (n = 3)

Discussion

IR may cause T2DM, with liver impairment under insulin pressure.[23] TBHQ, an important plant antioxidant, increases insulin's effect, thereby decreasing IR.[567] However, whether TBHQ alleviates IR to control liver steatosis in diabetes is unknown. The above findings firstly showed TBHQ improved liver steatosis in diabetes, via AMPKα2/PI3K/AKT signaling. These results indicated repeated applications of TBHQ overtly reduced blood glucose amounts, increased animal weight, alleviated liver steatosis and activated AMPKα2. In vitro, TBHQ enhanced survival in HepG2 cells with HClO/insulin-related with IR, also involving AMPKα2/PI3K/AKT signaling. Therefore, TBHQ might be considered a T2DM drug that induces AMPKα2/PI3K/AKT signaling.

TBHQ, reducing oxidative stress and inflammation,[242526] protects cultured pancreatic islet cells and enhances insulin sensitivity for T2DM alleviation. The liver, a major organ for detoxification, is frequently impaired by metabolic overload, high lipid production and various parameters. Additionally, metabolic abnormalities are found in the liver during the development of T2DM. TBHQ inhibits liver cell apoptosis, preventing liver damage.[102728] Based on the above, we hypothesized TBHQ alleviates liver damage in T2DM, and the above findings supported this notion [Figure 2].

AMPKα2 represents an important regulator of insulin sensitivity,[2930] and AMPK-dependent decrease of insulin release might be essential for glucose homeostasis maintenance by reducing insulin secretion in case of low glucose amounts.[313233] TBHQ activates AMPKα2 and autophagic pathways in liver cells, thereby reducing fat amounts.[13] In agreement, AMPKα2 was downregulated in both in vitro and in vivo models of diabetes, as demonstrated above, and this effect was reversed by TBHQ. It is known insulin markedly decreases blood glucose by elevating the amounts of phosphorylated AKT and PI3K,[222331] which are associated with GSK3 β and GLUT4.[3233] AMPKα2 activates AKT via phosphorylation of Ser473 and Thr308.[34] Therefore, TBHQ may alleviate IR and liver steatosis via AMPKα2/PI3K/AKT signaling, although this has not been previously demonstrated. Based on siRNA-based AMPKα2 knockdown in HepG2 cells, TBHQ enhanced phosphorylated AKT and PI3K amounts, markedly upregulating GLUT4 and GSK3 β in liver samples from animals with diabetes and HepG2 cells with IR. In AMPKα2-silenced cells, PI3K/AKT signaling was markedly altered. The above findings indicate TBHQ suppresses PI3K/AKT signaling directly by inducing AMPKα2 in insulin-resistant cells.

Rosiglitazone must be cautiously utilized in individuals with reduced cardiac function, serious cardiovascular diseases and hypertension,[35] and could induce cardiovascular complications during diabetes therapy. Therefore, highly efficient products for preventing diabetes are urgently needed. TBHQ represents a synthetic phenolic antioxidant approved for clinical application by both the Food and Agriculture Organization and the World Health Organization.[363738] More importantly, this study demonstrated that TBHQ improved IR and liver steatosis besides the antioxidant effect. Therefore, TBHQ may constitute an ideal antidiabetic agent.

It is well-known that T2DM damages the liver, skeletal muscle and adipose tissue. This work only examined TBHQ's effect on liver damage, not probing TBHQ's inhibitory impact on skeletal muscle and adipose tissue injury, which a major limitation. In an upcoming work, C2C12 and 3T3-L1 cells will be used for assessing TBHQ's effects on the above organs in the disease setting. Since various tissues show distinct sensitivities to TBHQ, many TBHQ concentrations will be tested to determine its optimal dose in diabetes. Only then could TBHQ become an antidiabetic agent in clinic.

Conclusions

Overall, TBHQ ameliorates T2DM by inducing AMPKα2, which controls IR through AKT/PI3K signaling [Figure 6]. Thus, TBHQ could represent a potent drug for T2DM, with AMPKα2 being an important therapeutic target.

Figure 6

Tert-butylhydroquinone ameliorates type 2 diabetes mellitus through AMPKα2/AKT/PI3K signaling. In diabetes, Tert-butylhydroquinone induces AMPKα2 activation, which enhances phosphatidylinositol 3-kinase (PIK) and AKT phosphorylation. Meanwhile, p-AKT interacts with GLUT4 and GSK3β in hepatic cells for type 2 diabetes mellitus alleviation

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Effects of PRKAA2 siRNA on the expression of AMPK2. (a) The efficiency of the three small interference RNAs (siRNAs); (b) The effect of the chosen si RNA on the expression of AMPK2. **P < 0.01 versus Control, ##P < 0.01 versus type 2 diabetes mellitus. Data are represented as mean ± standard deviation (n = 3)

Click here for additional data file.