Hypoglycemic And Hypolipidemic Effects Of Blueberry Anthocyanins By AMPK Activation: In Vitro And in Vivo Studies Part 1

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Abstract: Blueberries are rich in bioactive anthocyanins, with a high level of malvidin, which is associated with antioxidant benefits that contribute to reducing the risk of diabetes. The objective of this study was to investigate the hypoglycemic and hypolipidemic effects of blueberry anthocyanin extract (BAE), malvidin (Mv), malvidin-3-glucoside (Mv-3-GLC), and malvidin-3-galactoside (Mv-3-gal) in both human hepatocarcinoma cell line HepG2 and in a high-fat diet combining streptozotocin-induced diabetic mice. High glucose treatment significantly increased hepatic oxidative stress up to 6-fold and decreased HepG2 cell viability. Pretreatment with BAE, Mv, Mv-3-glc, and Mlv-3-gal significantly mitigated these damages by lowering the reactive oxygen species (ROS) by 87,80,76, and 91%, and increasing cell viability by 88,79,73, and 98%, respectively. These pretreatments also effectively inhibited hyperglycemia and hyperlipidemia, respectively by reducing the expression levels of enzymes participating in gluconeogenesis and lipogenesis and enhancing those involved in glycogenolysis and lipolysis, via the adenosine monophosphate-activated protein kinase(AMPK)signaling pathway in HepG2 cells. To determine, the role of AMPK in BAE-induced reaction of glucose and lipid metabolism in vivo, doses of 100 mg/kg (blueberry anthocyanin extracts-low concentration, BAE-L)and 400 mg/kg (blueberry anthocyanin extracts -high concentration, BAE-H) were administrated per day to diabetic mice for 5 weeks. BAE treatments had a significant (P<0.05) effect on body weight and increased the AMPK activity, achieving the decrease of blood and urine-glucose, as well as triglyceride and total cholesterol. This research suggested that anthocyanins contributed to the blueberry extract-induced hypoglycemia and hypolipidemic effects in diabetes and BAE could be a promising functional food or medicine for diabetes treatment.

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1. Introduction

Diabetes mellitus (DM) is a well-known disease typically characterized by an inability to maintain normal blood glucose levels. This chronic metabolic disorder causes serious harm to human health and imposes a heavy financial burden on worldwide health care systems [1]. This disease is classified into two main types based on the cause of blood glucose dysregulation. Type 1 DM is caused by autoimmune destruction of beta-cells leading to an inability to produce insulin whereas type 2 diabetes is characterized by insulin resistance insulin receptors, having reduced function, do not allow cells to respond adequately to rising blood glucose.

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In addition to hyperglycemia, diabetics tend to have hyperlipidemia both conditions of which can result in damage to organs and tissues via increased glycoxidative stress [2]. Insulin production and insulin receptor damage are among the factors that cause hyperglycemia in diabetics. In the liver, over-expression of gluconeogenesis and glycogenolysis re-leases glucose into the bloodstream [3] whereas, in the small intestine, over-expression of glucose transporter 2(GLUT2) causes an increase in glucose transport [4]. Hyperlipidemia caused by the over-expression of lipogenesis in adipose tissue [5] may also cause impaired insulin-stimulated glucose uptake and glycogen synthesis leading to insulin resistance and thus contributing to the progression of diabetes 6]. Long-term complications related to diabetes affect various organs and include hypertension, atherosclerosis, retinopathy, nephropathy, foot ulcers, and peripheral neuropathy [7].

There is currently no cure for diabetes, however, its effects can be alleviated [8]. For people diagnosed with type 1 diabetes, a combination of diet regulation, and appropriate intake of insulin according to carbohydrates consumed is the common treatment. Current challenges for type 1 diabetics include inability or difficulties in estimating total carbohydrates and appropriate insulin dose; however, near-ideal glycemic levels are possible given technological progress including insulin pumps [9]. For patients suffering from type 2 diabetes, treatment may be more complex including combinations of diet regulation, exercise, insulin, and other drugs with a variety of mechanisms of action that work to lower blood glucose[10].The common dietary recommendation is to follow a diet of low carbohydrates [11,12].

Blueberry is one of the fruits that diabetic patients can consume and could diminish the risk of type 2 diabetes mellitus (T2DM)[13,14]. Blueberries are rich in a wide variety of compounds beneficial to human health including minerals, fiber organic acids, phenolic acids, and flavonoids including flavonols and anthocyanins [15,16]. However, typical anthocyanin profiles include the galactosides, glucosides, and arabinoside of delphinidin, malvidin, cyanidin, petunidin, and peonidin [17]with malvidin and delphinidins usually being the major contributor to total anthocyanin content [18,19]. In our previous study [20], malvidin was the most abundant anthocyanidin found in rabbiteye blueberry fruits extract along with delphinidin, cyanidin, petunidin, and peonidin which is in agreement with other authors who also found malvidin to be an important contributor to total anthocyanin content in different types of blueberries [17,21].

Blueberry anthocyanins may improve insulin sensitivity through antioxidant capacity and through other regulatory interactions as their effects cannot be explained entirely by the former [13,22]. Studies have also found the benefits of blueberry anthocyanins for other diabetes-related concerns. In an in vitro study, Huang et al. [23] found that blueberry anthocyanins had anti-inflammatory and antioxidant effects on human retinal cells in high glucose conditions. Cells exposed to high glucose had 64% viability whereas when exposed to blueberry anthocyanin extract, malvidin, malvidin-glucoside, or malvidin-galactoside, their viability was7-9%,83%,91-%, or 86%, respectively. On the other hand, Song et al. [24] found that blueberry anthocyanins reduced oxidative stress and inflammation in diabetic rat retina. Diabetic rats were given 20, 40, and 80 mg/kg of blueberry anthocyanins orally for 12 weeks. Rats with high doses of blueberry anthocyanins had lower blood glucose, higher antioxidant capacity and lower inflammation in the retina, and lower reactive oxygen species compared to those without blueberry anthocyanins.

Although blueberry anthocyanins deliver health benefits to the liver and other organs for diabetics, hypoglycemic and hypolipidemic actions of blueberry anthocyanin extract (BAE)in human hepatic cells are unclear. In the present study, it was speculated that BAE, as well as Mv, Mv-3-glc, and Mv-3-gal, have hypoglycemic and hypolipidemic activities in human hepatocarcinoma cells (HepG2)and mice and that they could maintain glucolipid homeostasis thus alleviating the development of diabetes.

2. Materials and methods

2.1. Chemical and reagents

Brightwell rabbiteye blueberries(Vaccinium ashei were harvested from Fujiabian Orchard Picking (Nanjing, China) and their anthocyanin extracts were then obtained and stored in the dark at-18°C. The re-agent 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl-2H-tetrazolium bromide (MTT) and the standards malvidin (Mv), malvidin-3-glucose (Mv-3-glc), and malvidin-3-galactose(Mv-3-gal)were procured from Sigma Aldrich (Shanghai, China). HepG2 primary cells, bicinchoninic acid (BCA)protein assay kit, and enhanced chemiluminescence (ECL)western blotting detection reagents were acquired from CW Biotechnology (Beijing, China). Fetal bovine serum (FBS), Dulbecco's modified Eagle medium (DMEM), and penicillin-streptomycin were procured from Gibco (Auckland, New Zealand). BioFroxx streptozotocin (STZ) was purchased from Saiguo Biotech (Guangzhou, China). Dichloro-dihydro-fluorescein diacetate (DCFH-DA) detection kit was bought from Beyo-time Institute of Technology(Shanghai, China). Bovine serum albumin (BSA) was acquired from Shyuanye (Shanghai, China). The high-fat and high-sugar feed (fat 35.5%,protein 20%, carbohydrate 36.4%,0.1%cellulose) and the normal feed (fat 4.5%,protein 23%, carbohydrate 31.9%,3.7% fructose,and 5.3% cellulose)for mice were obtained from Xietong Bioengineering Co., Ltd (Nanjing, China). The insulin, triglyceride (TG), total cholesterol (TCHD, glutathione peroxidase(GSH-Px), and superoxide dismutase (SOD) assay kits were bought from Jiancheng Bioengineering Research Institute (Nanjing, China). AndyGene human Forkhead Box O1(FOXO1), glucose-6-phosphatase(G6Pase), glycogen synthase-phosphorylation (p-GS), glycogen synthase kinase-3 beta-phosphorylation (p-GSK3β), glucose transporter 2(GLUT2), acetyl coenzyme A carboxylase (ACC), hormone-sensitive triglyceride lipase (HSL),3-hydroxy-3-methylglutaryl-coenzyme reductase (HMGCR) and mouse fasting insulin enzyme-linked immunosorbent assay(ELISA)kits were all purchased from Bluegene Biotech(Shanghai, China). The chemicals and reagents used in this study were all of the pure analytical grades.

2.2. Antibodies

Primary antibodies against GS, p-GS (Ser641), sterol regulatory element-binding protein-1c(SREBP-1c), and phosphoenolpyruvate carboxykinase (PEPCK) were acquired from Abcam (Cambridge, United Kingdom). Primary antibodies against adenosine monophosphate-activated protein kinase alpha (AMPKa), p-AMPKαa (Thr172), peroxisome-proliferator-activated receptor-y-coactivator-1α (PGC-1α), ACC, p-ACC (Ser79), and horseradish peroxidase(HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against glyceraldehyde 3-phosphate dehydrogenase(GAPDH) was purchased from Nanjing Beidi Biomed Technology (Nanjing, China)while antibody against β-actin was bought from Sigma Aldrich(St. Louis, MO, USA). Primary antibodies were used at 1:1000 dilutions whereas 1:4000 dilutions were used for secondary antibodies.

2.3. Preparation of blueberry anthocyanin extract (BAE)

The extraction of blueberry anthocyanins was performed using our previous method of Huang et al. [25]. The frozen rabbiteye blueberries were held at room temperature until defrosted. Then, they were beaten at 10 000 rpm for 30 s using a T18 basic ULTRA-TURRAX homogenizer (IKA Works Guangzhou, China). An amount of 250 g of blueberries was then soaked in a solution of 1000 mL of methanol containing 1% HCl for 24 h. The extract was then collected following centrifugation at 5000×g for 15 min. After evaporation of the solvent at 40°C in a vacuum, the residue was extracted with 1:1 (v/v)ethyl acetate three times. The water phase containing anthocyanins was collected and concentrated using vacuo evaporation to obtain the crude anthocyanin extract. The extract was further purified with AB-8 macroporous resin (Sigma Aldrich, Shanghai, China). To remove fructose and protein, the extract was subjected to column chromatography on 1000 g AB-8 macroporous resin for 24 h absorption and then eluted with double distilled water. The anthocyanin fraction was eluted with 80% ethanol containing 1% HCl solution, concentrated in vacuo, and then dried using an Eyela FDU-1200 freeze dryer (Tokyo Rikakikai, Japan) in order to get a blueberry anthocyanin extract (BAE) powder.

2.4. Cell culture and treatments

HepG2 cell lines, derived from a specific-distinguished human hepatocellular carcinoma, have a high proportion of liver-specific proteins. For this reason, they are commonly used as laboratory models for human liver cell studies [26]. HepG2 cells were grown in DMEM containing normal D-glucose(5.5 mM) supplemented with 10% FBS and 1%penicillin-streptomycin and kept at 37℃C and 5% CO2 atmosphere incubator (Thermo Scientific, Waltham, MA, USA). After reaching 70-80% confluence, the HepG2 cells were subcultured. Four hours before the experiment, HepG2 cells were quiesced in a reduced serum medium. After pretreatment with 5 ug/mLof Mv, Mv-3-glc, Mv-3-gal, or BAE for 24 h, the cells were exposed to normal (5.5 mM) or high (30 mM)glucose concentrations to mimic normal and diabetic conditions. The cells were prepared for Western Blot analysis and the supernatants were collected for ELISA analysis.

2.5. Animals and experimental design

C57BL/6J healthy male mice (20 ± 2 g) from GemPharmatech (Nanjing, Jiangsu, China) were kept in standard laboratory conditions, including a constant temperature of 25°C and a 12-h day, and night alternation. The Jiangsu Academy of Agricultural Sciences Subcommittee on Research Animal Care and Use Committee had previously approved all animal experimental procedures. A high-fat diet and streptozotocin (STZ) were used to induce T2DM in the animal models according to the method used by Islam and Loots [27]. All mice were fed with a high-fat and high-sugar diet except for the control group which consumed a normal diet. After 4 weeks, the mice were fasted for 12 h and injected intraperitoneally with 100 mg/kg STZ dissolved in sodium citrate buffer solution (pH 4.2-4.5). One week after STZ induction, blood samples were taken from the tail vein of the mice who had fasted overnight. Mice that presented diabetic symptoms including polyuria, polydipsia, and hyperglycemia (fasting blood glucose level > 11.1 mmol/L)were recognized as T2DM and used for future study. To check the model's T2DM stability, all mice were fed with a normal diet for one week. The normal mice were used as the control group. The diabetic mice were then randomly divided into three groups: model, low-dose BAE(100 mg/kg),and high-dose BAE (400 mg/kg). For 6 consecutive days, intragastric administration of either the same volume of solvent （100 μL），100 mg/kg or 400 mg/kg of BAE were given to the control group, the model group, the low dose BAE and high-dose BAE respectively. On the seventh day, body weight and fasting blood glucose were measured. The same intragastric administration continued for 5 weeks. Mice were then fasted overnight, and blood samples were collected for serum preparation from the inferior vena cava and stored at-20°C. After blood sampling, all the mice were anesthetized and sacrificed. The mice liver, spleen, kidney, and thymus tissues were removed, weighed, and stored at-80 °C for further experiments.

2.6. Cell viability detection

The cell viability was determined by the MTT method. Five μg/mL of Mv， Mv-3-glc, Mv-3-gal or BAE were used for pretreatment of cells for 24 h. The cells were then treated with 5 mMor 30 mM glucose for 24 h and 10 μL 0.5%（5 mg/mL）of MTT was added to the cells which were then cultured again. After 4 h， the MTT solution was removed， and 100 μL dimethyl sulfoxide(DMSO)was added before the mixture was shaken slowly for 10 min to dissolve the cell crystal. The absorbance at 490 nm was measured on a StatFax-2100 microplate reader(Awareness Tech-nology Inc., Plam, FL, USA) to obtain the optical density (OD) values. Cells cultured with normalglucose levels (5.5 mmol/L)were used as the control group, whereas wells without cells were used as the blank. The following formula was used to determine cell viability: Cell viability(%)=(sample group OD value-blank group OD value)/(control group OD value-blank group OD value)x 100%.

2.7. ROS fluorescence visualization

The reactive oxygen species (ROS) in HepG2 cells were assessed using the DCFH-DA detection kit. After an initial treatment with the 5 μg/mL of Mv， Mv-3-glc， Mv-3-gal， or BAE for 24 h which was later followed by a 5 mM or 30 mMglucose treatment for 24 h, the cells were washed with PBS， and then 10 μM DCFH-DA was added to each well and allowed to react for 20 min at 37°C. The cells were again washed thoroughly with PBS and then a group of these cells was immediately observed under an IX53 inverted fluorescent microscope(Olympus, Tokyo, Japan) at 530 nm emission and 485 nm excitation filters. The images are presented under 200× magnification. After dissociation, another group of cells was collected and their fluorescence was recorded by a Synergy H4 multi-mode microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The total fluorescence intensity of cells in each well was noted, and ROS generation was measured as a fold of the Control.

2.8. ELISA

ELISA kits were used to quantify proteins involved in gluconeogenesis (FOXO1, G6Pase, p-GS), glycogenolysis (p-GSK3β), glucose transporter(GLUT2), lipogenesis (ACC and HMGCR), and lipolysis (HSL) in the supernatants of HepG2 cells. The quantity of GLUT2 in mice livers was also determined. The BCA protein assay kit was used to quantify the total cell protein of the supernatant. The absorbance at 450 nm was measured at 37°C on a StatFax-2100 microplate reader (Awareness Technology Inc., Plam, FL, USA) to determine protein levels.

2.9. Western blotting analysis

Western blotting was performed on HepG2 lysates in order to measure the protein level of AMPK, p-AMPK, gluconeogenesis (PGCl, PEPCK, glycogenolysis(GS, p-GS), and lipolysis (ACC, p-ACC, SREBP-1c)in the cells. Western blotting was also performed to determine the quantities of AMPK,p-AMPK on the livers of diabetes-induced mice. Either GAPDH or β-Actin was used as a loading control. LAS-3000 imaging system (Fuji, Tokyo, Japan) was used to observe the protein bands, and their density was quantified using Bio Profile 1D++(Vilbert Lour-mat, Marne La Vallee, France) software. All data were expressed as a fold change to the control.

2.10. Fasting blood glucose and glucose tolerance assay

Fasting blood glucose was measured once a week for a period of 5 weeks after a 12-h fasting period. For glucose tolerance assay, mice were fasted for 16 h and fed with 2 g/kg of 20% glucose （200 μL）intra-gastrically(i.g.)to determine the blood glucose at 0,0.5,1,1.5, and 2h. The blood was collected from the tail vein and the glucose levels were measured using a glucometer (Sinocare, Changsha, China). The glucose level in the urine of mice who received i.g. was also measured for a period of five weeks.

2.11.Estimation of serum biochemical indexes and enzyme activity in mice liver

The quantity of insulin, triglyceride and total cholesterol in the serum was measured, while SOD and GSH-PX enzyme activity was estimated using the commercial kits according to the manufacturer's protocol. Absorbance at 500 nm for triglycerides (TG) and total cholesterol (TCH), and 450 nm for the others (insulin, SOD, and GSH-PX)was measured at 37 °C on a StatFax-2100 microplate reader (Awareness Technology InC., Plam, FL, USA).

2.12. Statistical analysis

All data are presented as the mean value±standard deviation(SD) of at least three independent experiments. The figures were obtained using GraphPad Prism Version 8(GraphPad Software, Inc., CA, USA). One-way analysis of variance(ANOVA), t-tests, or Sidak's multiple comparisons test were performed to determine statistical differences among different groups. Differences were considered significant at P<0.05.

This article is extracted from https://doi.org/10.1016/j.redox.2021.102100 Received 21 July 2021; Received in revised form 9 August 2021; Accepted 11 August 2021