D-Chiro-inositol Ameliorates High Fat Diet-Induced Hepatic Steatosis

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Bioactive Constituents, Metabolites, and Functions

D-Chiro-inositol Ameliorates High Fat Diet-Induced Hepatic Steatosis and Insulin Resistance via PKC#-PI3K/AKT Pathway feier cheng, Lin Han, Yao Xiao, Chuanying Pan, Yunlong LI, xinhui Ge, Yao Zhang, Shaoqing Yan, and Min Wang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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Journal of Agricultural and Food Chemistry

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D-Chiro-inositol Ameliorates High Fat Diet-Induced Hepatic Steatosis and Insulin

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Resistance via PKCε-PI3K/AKT Pathway

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Feier Cheng @, Lin Han @, Yao Xiao @, Chuanying Pan $, Yunlong Li &, Xinhui Ge @, Yao Zhang @,

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@

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College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China

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College of Food Science and Engineering, Northwest A&F University, Yangling, 712100, P. R. China

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Shaoqing Yan @, Min Wang @*

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Institute of Agricultural Products Processing, Shanxi Academy of Agriculture Sciences,

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Taiyuan, China

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* Corresponding Author

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College of Food Science and Engineering, Northwest A&F University, Yangling, 712100, P. R.

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China

ACS Paragon Plus Environment


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ABSTRACT: D-chiro-inositol (DCI) is a biologically active component found in tartary buckwheat,

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which can reduce hyperglycemia and ameliorate insulin resistance. However, the mechanism

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underlying the anti-diabetic effects of DCI remains largely unclear. This study investigated the

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effects and underlying molecular mechanisms of DCI on hepatic gluconeogenesis in mice fed a

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high-fat diet and saturated palmitic acid-treated hepatocytes. DCI attenuated free fatty acid uptake

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by the liver via lipid trafficking inhibition, reduced diacylglycerol deposition and hepatic PKCε

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translocation. Thus, DCI could improve insulin sensitivity by suppressing hepatic gluconeogenesis.

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Subsequent analyses revealed that DCI decreased hepatic glucose output and the expression levels

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of PEPCK and G6Pase in insulin resistant mice through PKCε-IRS/PI3K/AKT signaling pathway.

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Likewise, such effects of DCI were confirmed in HepG2 cells with palmitate-induced insulin

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resistance. These findings indicate a novel pathway by which DCI prevents hepatic gluconeogenesis,

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reduces lipid deposition and ameliorates insulin resistance via regulation of PKCε-PI3K/AKT axis.

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KEYWORDS: D-Chiro-inositol; liver; insulin resistance; gluconeogenesis


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INTRODUCTION

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Data from the International Diabetes Federation Atlas (2015) indicate that at least 415 million

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individuals have been diagnosed with diabetes, a figure projected to rise to more than 642 million

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by 2040. Of them, type 2 diabetes mellitus (T2DM) accounts for approximately 90%. Clinically, it

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is difficult to determine the onset of diabetes.1 Insulin resistance is responsible for the reduction in

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glucose disposal, and thus leading to hyperglycaemia in diabetes.2 The liver plays an essential role

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in the maintenance of normal glucose levels by controlling glucose production (gluconeogenesis)

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and glycogen breakdown (glycogenolysis). Hepatic gluconeogenesis is the main source of

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endogenous glucose production to supply energy to all cells in the body, especially during fasting

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state. However, excessive production of glucose in the liver can result in hyperglycemia. Insulin

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resistance has been defined as the impaired insulin-dependent regulation of glucose and lipid

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metabolism in the targeted tissues, such as adipocytes, skeletal muscle and liver, even though the

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circulating levels of insulin are normal. In response to the aberrant regulation of insulin signaling,

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hepatic insulin resistance can enhance lipid accumulation and increase glucose production, resulting

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in dyslipidemia and hyperglycemia. Therefore, drug targeting hepatic insulin resistance may be an

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attractive therapeutic approach to treat T2DM and hepatic steatosis.

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Blunted responses of adipocytes may result in decreased uptake of free fatty acids (FFAs) and

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impaired glucose utilization, thus leading to ectopic fat deposition in insulin-target tissues.3 The

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ectopic accumulation of fat in the liver is closely related to insulin resistance.4 Moreover, excess

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FFAs elevate hepatic acetyl-CoA content and in turn activate pyruvate carboxylase, which is

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necessary to initiate gluconeogenesis5. In addition, FFAs increase diacylglycerol (DAG) formation,

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and thus activating protein kinase C (PKC).6 PKC is the most important intracellular target of DAG,



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which contributes to insulin resistance by inhibiting the insulin-induced phosphorylation level of

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insulin receptor substrate (IRS).7 Binding of IRS1/2 to the insulin receptor (InsR) is crucial for the

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regulation of insulin signaling and energy homeostasis.8 In insulin-sensitive tissues, the activation

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of InsR recruits IRS by tyrosine phosphorylation, and in turn activates downstream effectors.9 It is

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well established that tyrosine-phosphorylated IRS recruits phosphoinositide-3-kinase (PI3K) and

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activate protein B (AKT) to trigger insulin action.9-14 High concentrations of FFAs can diminish the

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effects of insulin receptor signaling by suppressing the insulin-stimulated phosphorylation level of

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AKT. Forkhead box protein O1 (FOXO1) is a particularly well-characterized AKT target in

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hepatocytes.15-19 FOXO1 is a transcription factor that modulates the expression levels of genes

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encoding glucose-6-phosphatase (G6Pase) and cytosolic phosphoenolpyruvate carboxykinase

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(PEPCK), and thus facilitating gluconeogenesis.

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inactivate FOXO1 through phosphorylation, resulting in the elimination of FOXO1 from the nucleus.

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D-chiro-inositol (DCI) is primarily absorbed from the diet, and has been commonly found in

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leguminous plants, including buckwheat, carob pod, mung bean, soybean, soy whey and tartary

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buckwheat.22-27 Among them, tartary buckwheat (Fagopyrum tataricum (L.) Gaench) seeds are a

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major source of DCI. Additionally, mung bean seeds may contain higher amounts of DCI compared

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to buckwheat28. Besides, the majority of DCIs occur in the form of its galactosides, namely

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fagopyritols. Notably, 5 different types of fagopyritols have been found in buckwheat, which is

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higher than other plant materials, including chickpea, lupine, lentil and soybean.29 As high as 2.2

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mg/g of DCI is found in the embryo tissues of buckwheat seeds, whereas lower concentration of

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DCI (1.05 mg/g) is detected in the mature seeds. In addition, a total level of 192 mg/100 g of DCI

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In fact, AKT mediates insulin action to


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has been observed in tartary buckwheat.30 As a dual-use food and medicinal plant, tartary buckwheat

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has drawn more and more attention, due to its therapeutic effects on metabolic disorders31 such as

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diabetes32 and hypertension.33 DCI has been implicated to ameliorate endothelial dysfunction34 and

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enhance insulin sensitization.35,36 Our previous work demonstrates that DCI regulates energy

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metabolism and improves endothelial dysfunction.37 DCI is shown to induce pyruvate

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dehydrogenase activity by inactivating pyruvate dehydrogenase kinase (PDK),38 and this regulation

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raises the possibility that DCI shifts mitochondrial pyruvate towards oxidation in hepatocytes, and

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thus limiting pyruvate carboxylation for gluconeogenesis.39 Given the critical role of enhanced

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gluconeogenesis in diabetes,40 the present study aimed to investigate the effects of DCI on glucose

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and lipid metabolism, with the focus on hepatic gluconeogenesis.

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MATERIALS AND METHODS

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Animals. Six-week-old male C57BL/6 mice were supplied by the Xi’an Jiaotong University Health

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Science Center. The mice were housed in groups of three in a cage under a 12 h light/dark cycles at

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22 ± 2oC. They were given ad libitum access to water and food, and were randomly categorized into

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four groups (n=9, in each group). Normal control-diet (NCD; TP 23302, 10% Fat), high fat-diet

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(HFD; TP 23300, 60% HFD for diet-induced obesity, only fat has different calories) were obtained

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from TROPHIC Animal Feed High-Tech Co., Ltd. (Nanjing, China). The mice in NCD and HFD

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group were intragastrically administered with carboxymethylcellulose sodium (CMC-Na; 0.3%),

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tartary buckwheat DCI extract (purity>95%, NewGenco Biotech Co., Ltd., Shanghai, China) and

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metformin (MET; Beyotime Institute of Biotechnology, Shanghai, China) suspended in 0.3% CMC-

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Na. The HFD-fed mice in MET and DCI groups were intragastrically administered with 50 mg/kg

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bw/day of DCI 41,42 and 200 mg/kg bw/day of MET, respectively, for 8 weeks.



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The study protocol was approved by the Faculty Animal Policy and Welfare Committee of

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Northwest A&F University, China, and the animal experiments were conducted in accordance with

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the laws, regulations, guidelines, and standards on animal care. The mice were anesthetized, and

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every effort was made to minimize their suffering. Following cardiac perfusion with phosphate

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buffer solution (PBS), the liver was immediately resected, washed again in PBS, fixed in 4%

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paraformaldehyde at 4°C and stored at -80°C until further analyses. After centrifugation, serum was

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separated from the whole blood collected by retro-orbital bleeding. These samples were determined

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immediately or otherwise stored at -80°C for plasma content analysis.

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Insulin resistance index. For the purposes of insulin tolerance test (ITT) and oral glucose tolerance

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test (OGTT), all animals starved for 6 h up to overnight. For pyruvate tolerance test (PTT), all

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animals were starved for 16 h up to overnight. After starvation, the mice were intraperitoneally

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injected with insulin (0.75 units/kg bw), pyruvic acid sodium (2.0 g/kg bw; Sigma-Aldrich, USA)

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or oral glucose (2.0 g/kg bw; Aladdin, Shanghai, China).43 The glucose levels in tail-vein blood

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samples were determined using a blood glucometer (OneTouch) at 15, 30, 60 and 120 min after the

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injection.

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Estimation of serum FFAs. The levels of FFAs were quantificationally measured using

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JYM0246Mo FFA ELISA Kit (Wuhan Genemei Biotechnology Co., Ltd., Wuhan, China) according

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to the manufacturer’s protocol.

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Extraction and analysis of liver lipids. Hepatic lipids were extracted from the liver samples using

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the chloroform-methanol (Chron chemicals, Chengdu, China) admixture (2:1, v/v) method as

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described previously44. The hepatic content of DAG in lipid extracts was determined by a specific

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ELISA kit (SBJ-M0093, SenBeiJia Biological Co., Ltd., Nanjing, China).


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Histopathology. The liver samples were subjected to 4% paraformaldehyde fixing, gradient ethanol

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dehydration, paraffin embedding, and 5-μm sectioning with a microtome (Leica RM2235,

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Germany). Subsequently, the tissue sections were dried at 37°C overnight. For histopathology and

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muscle glycogen staining, the sections were heated at 60°C for 1 h, deparaffinized and rehydrated,

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followed by hematoxylin and eosin (H&E) staining (Solarbio, Beijing, China). Thereafter, the

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stained sections were dehydrated in gradient ethanol and xylene, sealed with neutral balsam, and

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air-dried at room temperature. After mounting, the sections were examined using an optical

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microscope (Olympus, Tokyo, Japan) at a magnification of 400×.

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Oil Red O Staining. To determined the lipid content, the liver sections were prepared for Oil Red

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O staining. Briefly, the tissue samples were rinsed with PBS and fixed in 10% buffered formalin,

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followed by Oil Red O (0.5 g in 100 ml isopropanol) staining for 60 min. After discarding the

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staining solution, isopropanol was added in the samples to elute the retaining dyes. Finally, the

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optical density was determined at 520 nm, and the images were acquired using an EVOS microscope

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(Thermo Fisher Scientific, Waltham, MA, USA).

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HepG2 cell culture and insulin resistance model. Human hepatoma HepG2 cell line was

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purchased from Shanghai Institute of Cell Biology (Shanghai, China). The cells were cultured in

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RPMI medium (Hyclone, Logan, Utah, USA) supplemented with 10% fetal bovine serum (Gibco,

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Grand Island, USA), 100 units/mL penicillin and 100 μg/mL streptomycin, and maintained in a

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humidified atmosphere containing 5% CO2 at 37oC. To construct a cell model of insulin resistance,

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the HepG2 cells were incubated with serum-free 1640 medium containing high free fatty acid (100

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μM palmitate acid [PA]) for 24 h. DCI (purity>98%, Yuanye Bio-Technology Co., Shanghai, China)

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and MET were suspended in PBS before cell treatment. All cell experiments were performed



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between passages 3 and 6 only.

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Glucose production. The medium of HepG2 cells in six-well plates was substituted with 2 ml of

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glucose production buffer containing glucose-free DMEM (without phenol red) with 2 mM sodium

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pyruvate and 20 mM sodium lactate. After incubating for 3 h, 1 mL of glucose production buffer

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was collected, and the glucose content was analyzed using a glucose oxidase–peroxidase assay kit

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(Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The obtained values were

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normalized to total protein level determined by the BCA Protein Assay Kit (Beyotime

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Biotechnology, Shanghai, China)45.

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RNA extraction and quantitative RT-PCR. TRIzol (Trizol, Takata, Japan) was used to isolate

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total RNA from the cell samples. After cDNA synthesis, qRT-PCR reactions were carried out with

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the following primers: G6Pase F: 5’-CTGTTTGGACAACGCCCGTAT-3’; G6Pase R:

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AGGTGACAGGGAACTGCTTTA-3’; PEPCK F: 5’-TGACAGACTCGCCCTATGTG-3’; and

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PEPCK R: 5’-CCCAGTTGTTGACCAAAGGC-3’.

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Western blot analysis. Anti-PI3K, anti-PKCε (Abcam, Cambridge, MA, USA), anti-AKT, anti-

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phospho-AKT, anti-FOXO1, anti-phospho-FOXO1 (Cell Signaling Technology, Inc., Beverly, MA.,

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USA), and other antibodies (Bioworld Technology, Co, Ltd, Nanjing, China) as well as a Molecular

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Imager ChemiDOC XRS System (Bio-Rad, Shanghai, China) were used for Western blot analysis.

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Briefly, the cell and liver samples were rinsed with PBS and lysed in RIPA buffer (Beyotime

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Institute of Biotechnology, Shanghai, China) containing 1 mmol/Lof PMSF (Beyotime Institute of

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Biotechnology, Shanghai, China). Subsequently, the cell and tissue lysates were separated with 10%

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SDS-PAGE, and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After

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primary and secondary antibody incubation, the membranes were stained with the


5’-

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chemiluminescent reagents (Western Bright ECL Kit). Finally, the Bio-Rad Chemidoc (Bio-Rad,

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Shanghai, China) was used to visualize the protein bands.

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Statistical analysis. All experiments were performed at least in triplicate. Data are presented as the

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mean ± SEM of at least 3 independent experiments. The differences in the measurement results

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between control and treatment groups were compared with one-way ANalysis Of VAriance

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(ANOVA), followed by Tukey's test (Graphpad Prism 6). P values of less than 0.05 were regarded

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as statistically significant.

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RESULTS

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DCI enhances glucose tolerance in HFD-fed mice. The effects of intragastric administration of

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DCI on insulin sensitivity and glucose metabolism were examined in HFD-fed mice. DCI (50 mg/kg

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• d-1) and MET remarkably decreased body weight gain in HFD-fed mice, probably due to the

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reduction in food intake (Fig. 1A and B). The results of OGTT and ITT showed that HFD feeding

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induced glucose intolerance, but such alternation was reversed by DCI treatment (Fig. 1C-F). These

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findings suggest that DCI can improve insulin sensitivity. Fasting level of blood glucose is mainly

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controlled by endogenous glucose production. Considering that pyruvate is a major substrate for

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improving hepatic glucose production via gluconeogenesis, PTT can be used as a determinant of

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endogenous glucose production. Notably, DCI reduced fasting blood glucose level and attenuated

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the hyperglycemic response after pyruvate load, suggesting its role in limiting endogenous glucose

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production (Fig. 1G-I).

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DCI reduces lipid accumulation in the liver. In H&E stained sections (Fig. 2A and B), vesicular

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steatosis was observed in the cytoplasm of hepatocytes in HFD group, and such vacuolization was

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attenuated by DCI intervention. Consistently, the results of Oil Red O staining showed that HFD



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caused an excessive accumulation of fat in the liver, and DCI administration markedly reduced the

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abnormal hepatic lipid accumulation (Fig. 2C and D).

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DCI inhibits PKCε activation in the liver. During the hepatic uptake of FFAs, a series of

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esterification processes could result in an increase in intracellular diglyceride content. DCI

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decreased the values of liver index and reduced hepatic FFAs (Fig. 3A and B). DAG is an

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intermediate of the transesterification reaction, and PKCε is a target protein of DAG. Abnormal

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DAG/PKCε signaling pathway can cause lipid metabolism disorder and insulin resistance. DIC

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reduced hepatic DAG generation, and prevented ectopic PKCε translocation to the membrane,

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indicating its role in the suppression of PKCε activation (Fig. 3C and D).

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DCI activates PI3K/AKT signaling pathway in insulin resistant mice. Insulin signaling is

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activated by binding to its cell surface receptors via intramolecular trans phosphorylation. Our

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results showed that the expression levels of insulin receptor (IRβ) and PI3Kp85 were downregulated

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in HFD-fed mice, and DCI increased the low levels of IRβ and PI3Kp85. In addition, the

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phosphorylation levels of IRS-2 and AKT were reduced in HFD-fed mice, and such downregulation

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could be reversed by DCI treatment (Fig. 4). These in vivo results provide evidence that DCI

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mediates the activation of PI3K/AKT signaling.

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DCI improves glucose metabolism in hepatocytes. Palmitic acid (PA) was used to trigger insulin

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resistance in HepG2 cells. PEPCK and G6Pase are two important rate-limiting enzymes that

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regulate hepatic gluconeogenesis.46 PA exposure increased the mRNA expression levels of PEPCK

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and G6Pase in HepG2 cells (Fig. 5A and B). On the contrary, co-incubation with DCI significantly

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decreased the mRNA expression levels of PEPCK and G6Pase in HepG2 cells (P

D-Chiro-inositol Ameliorates High Fat Diet-Induced Hepatic Steatosis

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