Flaxseed Oil Alleviates Chronic HFD-Induced Insulin Resistance

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Flaxseed Oil Alleviates Chronic HFD-Induced Insulin Resistance through Remodeling Lipid Homeostasis in Obese Adipose Tissue Xiao Yu, Yuhan Tang, Peiyi Liu, Lin Xiao, Liegang Liu, Rui-Ling Shen, Qianchun Deng, and Ping Yao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03325 • Publication Date (Web): 07 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017

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Journal of Agricultural and Food Chemistry

Schematic diagram of the potential mechanisms that underlie the protective effect of ALA-rich flaxseed oil against HFD-induced inflammation and insulin resistance in mice 254x190mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Flaxseed Oil Alleviates Chronic HFD-Induced Insulin Resistance through

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Remodeling Lipid Homeostasis in Obese Adipose Tissue

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Xiao Yu†,‡ , Yuhan Tang§, Peiyi Liu§, Lin Xiao§, Liegang Liu§,, Ruiling Shen†,‡, Qianchun

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Deng*,&,#,ǁ, Ping Yao*,§

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†

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Zhengzhou 450002, China

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‡

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China

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§

College of Food and Biological Engineering, Zhengzhou University of Light Industry,

Henan Collaborative Innovation Center for Food Production and Safety, Zhengzhou 450002,

Department of Nutrition and Food Hygiene and MOE Key Laboratory of Environment and

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Health, School of Public Health, Tongji Medical College, Huazhong University of Science and

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Technology, Wuhan, 430030, China

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&

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#

Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China;

Hubei Key Laboratory of Lipid Chemistry and Nutrition, Wuhan 430062, China

ǁ

Key Laboratory of Oilseeds processing, Ministry of Agriculture, Wuhan 430062, China


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ABSTRCT:

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Emerging evidence suggested that higher circulating long-chain n-3 polyunsaturated

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fatty acids (n-3PUFA) levels were intimately associated with lower prevalence of

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obesity and insulin resistance. However, the understanding of bioactivity and potential

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mechanism of α-linolenic acid-rich flaxseed oil (ALA-FO) against insulin resistance

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was still limited. This study evaluated the effect of FO on high-fat diets

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(HFD)-induced insulin resistance in C57BL/6J mice focused on adipose tissue

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lipolysis. Mice after HFD feeding for 16 weeks (60% fat-derived calories) exhibited

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systemic insulin resistance in mice, which was greatly attenuated by medium dose of

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FO (M-FO), paralleling with differential accumulation of ALA and its n-3 derivatives

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across serum lipid fractions. Moreover, M-FO was sufficient to effectively block the

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metabolic activation of adipose tissue macrophages (ATM), thereby improving

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adipose tissue insulin signaling. Importantly, suppression of hypoxia-inducible factors

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HIF-1α and HIF-2α were involved in FO-mediated modulation of adipose tissue

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lipolysis, accompanied by specific reconstitution of n-3PUFA within adipose tissue

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lipid fractions.

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KEYWORDS: flaxseed oil, insulin resistance, adipose tissue lipolysis, ATM

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metabolic activation, hypoxia

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INTRODCTION

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Obesity-induced insulin resistance is the dominant factor underlying the rising

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tide of Type 2 diabetes mellitus (T2DM), now affecting 387 million people worldwide

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estimated by the International Diabetes Federation (IDF). The “Western-style diet”,

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characterized by high-calorie, high-saturated-fat diet and micronutrients deficiency,

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contributes to the growing prevalence of obesity and insulin resistance. Now it has

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become more common particularly in developing and newly developed countries due

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to the global diet and life-style acculturation.1,2 In China, the overall prevalence of

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T2DM in 2010 is estimated to be 11.6% in adult, accounting for 25% of T2DM cases

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worldwide.3 More importantly, the prevalence of T2DM is still growing rapidly

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accompanied by the explosive increase in obesity and insulin resistance.4 Therefore, it

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is imperative to seek an effective dietary intervention strategy and further explore its

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potential mechanism against the progression of obesity-induced insulin resistance.

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Multiple mechanisms, including chronic low-grade inflammation, activation of

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unfolded protein response and dysregulation of autophagy, are implicated in the

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pathogenesis of obesity-induced insulin resistance.5 However, these pathways are

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closely linked to the abnormal lipid metabolism as manifested by specific lipid

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signature in circulation and local tissues during the development of obesity-induced

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insulin resistance.6,7 The white adipose tissue, as an important endocrine organ, plays

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a critical role in maintaining whole-body lipid homeostasis by releasing lipid

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mediators, inflammatory cytokines and hormones to exert diverse local and systemic

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effects.8 The over-flowed lipids due to disordered fat storage and mobilization are

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buffered by adipose tissue macrophages (ATMs) in an anti-inflammatory

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lysosomal-dependent lipid metabolism pathawy.9 Moreover, the free fatty acids

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released from dramatically enlarged adipocyte activate a pro-inflammatory Toll-like

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receptor 4 (TLR4)-dependent pathway in ATMs, leading to adipose tissue insulin

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resistance.10 Although available evidence has substantiated that excessive adipocyte

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lipolysis drives the metabolic activation of ATMs, the paradoxical role of ATMs in a

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lipid-rich microenvironment indicates that the potential mechanism underlying ATM

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metabolic activation might be specific during different progression stages of


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obesity-induced insulin resistance, which needs to be explored.9

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The favorable effects of n-3 polyunsaturated fatty acids (n-3PUFAs), especially

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marine-derived eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on

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obesity-related metabolic diseases have been widely recognized from animal to

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epidemiologic studies.11,12 Alpha-linolenic acid (ALA), as an essential n-3PUFA, is

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commonly available in vegetable oils, especially flaxseed oil.13 Although the

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metabolic benefits defined by animal and human studies, the role of ALA-rich

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flaxseed oil against obesity-induced insulin resistance and risk of T2DM remains

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inconsistent.14-18 The ALA intake and n-6/n-3 fatty acid ratio in dietary background

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and the disease progression may contribute to these discrepant antagonistic effects. If

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so, the magnitude of flaxseed oil for exerting its effective bioactivities against the

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progression of obesity-induced insulin resistance remains unknown. Moreover,

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enough data have been accumulated to explore the potentially independent

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mechanism of long chain n-3PUFA against obesity-related metabolic diseases,

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involving the remodeling of membrane phospholipids, activation of n-3PUFA

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receptor/sensor-mediated anti-inflammatory signaling and production of biologically

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active resolvins and protectins.19 Nevertheless, the mechanism responsible for the

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potential bioactivities of ALA is not as extensive as EPA and DHA.20 Importantly,

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ALA can be converted sequentially into EPA, docosapentaenoic acid (DPA) and DHA

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in mammals in a tissue-selective manner. However, the conversion efficiency of ALA,

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particularly into DPA and DHA, is largely affected by the background diet and the

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pathophysiological status in mammals.21 Thus, it is still elusive whether the

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synergistic effects of ALA and its n-3 series of derivatives are involved in improving

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adipose tissue lipid homeostasis during the progression of obesity-induced insulin

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resistance. In current study, the circulating lipid profiles were analyzed to explore the

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specific lipid signature coupled with different degrees of systemic insulin resistance

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mediated by lard-rich HFD without or with flaxseed oil replacement. Moreover, the

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remodeling of lipid homeostasis and potential mechanism for flaxseed oil against

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obesity-induced adipose tissue insulin resistance were further explored.

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MATERIAL AND METHODS



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Experimental Material and Chemicals

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Seventy-nine flaxseed cultivars were collected from the major planting areas in

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China. The crude flaxseed oil was obtained by cold pressing and partial refining and

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further assessed based on the contents of ALA and micronutrients. Flaxseed oil with

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the highest ALA but lowest micronutrients was selected as the experimental materials.

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The main fatty acid composition of selected flaxseed oil was 6.08%, 4.36%, 19.97%,

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9.90% and 59.69% for C16:0, C18:0, C18:1n-9, C18:2n-6 and C18:3n-3, respectively.

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Fatty acid methyl ester blends (GLC-463), cholesteryl ester (CE), triglyceride (TG),

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diglyceride (DG), free fatty acid (FFA) and phospholipid (PL) standards and

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acetylchloride were purchased from NU-CHEK-PREP, Avanti polar lipids and

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Sigma-Aldrich respectively. 50% Glucose injection was obtained from CR

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Double-Crane (Beijing, China). Recombinant human insulin (100 UI/mL) was

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provided by Novo Nordisk (Copenhagen, Denmark). Aminopropyl SPE column was

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provided by Sepax Technologies, Inc. (Suzhou, China). Chloroform, methanol and

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n-hexane (HPLC grade) were provided by Merck (Darmstadt, Germany). Other

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solvents were purchased from local reagent retailer. Anti-hormone-sensitive lipase

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(HSL), anti-phospho-hormone-sensitive lipase (p-HSL), anti-adipose triglyceride

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lipase (ATGL), anti-protein kinase Akt, anti-phospho-Akt (p-Akt), anti-p62/SQSTM1,

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anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-rabbit IgG and

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anti-mouse IgG were obtained from Cell Signaling Technology. Anti-peroxisome

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proliferator-activated receptor γ (PPARγ), anti-hypoxia-inducible factors HIF1α and

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HIF2α were purchased from Abcam. Western blotting detecting reagents (ECL) and

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reblot buffer were provided by Chemicon (Temecula, CA, USA).

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Experimental Animals and Treatments

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Male C57BL/6 mice (18–20 g) were obtained from Sino-British Sippr/BK

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(Shanghai, China) and randomly divided into 5 groups of 16 animals in each diet. The

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mice were fed either low fat diet (LFD, 10% fat-derived calories) or isocaloric

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high-fat diets (HFD, 60% fat-derived calories) containing 35.3% fat (wt/wt; Corn oil:

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3.2%; Lard: 32.1%) without or with 10%, 20% and 30% (wt/wt) flaxseed oil

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replacement for 16 weeks. The detailed fatty acid contents in the experimental diets


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were shown in Table S1. The animals were taken care of according to the Guiding

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Principles in the Care and Use of Laboratory Animals published by the US National

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Institutes of Health. The animal experiments were approved by the Tongji Medical

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College Council on Animal Care Committee. The animals had free access to food and

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water, and were housed in a temperature/humidity-regulated room with a 12-h

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light/dark cycle. Body weight and average food intake were recorded twice per week.

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Calorie intake was calculated by food intake and calorie contents in each diet. After

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16 weeks of feeding, the animals were anesthetized by an intraperitoneal injection of

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sodium pentobarbital (50 mg/kg) and sacrificed by cervical dislocation following an

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overnight fasting. Serum was prepared from blood by centrifuge at 3500 g for 10 min

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at 4 °C. Epididymal adipose tissue was fixed for histopathology examination and

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quickly frozen by liquid nitrogen, respectively. All samples were stored at –80 °C for

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further analysis.

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Intraperitoneal Glucose and Insulin Tolerance Tests (IPGTT and IPITT)

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IPGTT and IPITT were performed one week before the end of experiment as

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previously described.22 Briefly, mice were fasted overnight and intraperitoneally

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injected with glucose (1g/kg b.w.), or fasted for 6 hours and intraperitoneally injected

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with insulin (0.75 U/kg b.w.) for IPGTT and IPITT, respectively. The blood glucose

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levels were measured from tail veins by using Accu-Chek glucometer (Roche

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Diagnostics, Germany) at 0, 15, 30, 60, and 120 min after injection, respectively.

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Serum Biochemical Analysis

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Fasting serum glucose concentrations were determined by enzymatic colorimetric

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methods using commercial kits (Biosino Bio-Technology, Beijing, China). Fasting

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serum insulin levels were analyzed with commercialized radioimmunoassay kits

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(Chinese Academy of Atomic Energy, Beijing, China). The homeostasis model

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assessment of insulin resistance (HOMA-IR) was calculated as [glucose (mmol/L) ×

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insulin (mU/L)]/22.5.

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Histopathological Examination

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For light microscopy, fresh epididymal adipose tissue was cut into 3 mm-thick

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slices, fixed in 4% paraformaldehyde and embedded in paraffin wax. Tissue sections



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(5 µm thick) obtained from the paraffin blocks were stained with haematoxylin and

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eosin (H&E) and then observed with a BX50 Olympus light microscope. For

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transmission electron microscopy, fresh epididymal adipose tissue fragments were

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carefully collected, rapidly fixed in 2.5% glutaraldehydein phosphate buffer (pH 7.4),

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fixed in 1% osmium tetraoxide, dehydrated by gradually increasing concentrations of

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ethanol and embedded with Epon 812 resin. Ultrathin sections were stained with

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uranyl acetate and lead citrate and then visualized with a FEI Tecnai G2 electron

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microscope.

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Lipid Extraction, Separation and Fatty Acid Composition Analysis

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Total lipids from serum and epididymal adipose tissue were extracted into

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chloroform–methanol (2:1, v/v) according to Folch et al.23 The different lipid fractions

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(CE/TG/DG/FFA/PL) were separated using aminopropyl SPE column as described by

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Agren et al.24 Individual lipid fraction was transmethylated based on the method of

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Bicalho et al.25 Fatty acid methyl esters were separated using Agilent 6890 GC with a

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HP-INNOWAX fused silica capillary column (60 m×0.25 mm i.d.×0.25 µm) and a

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flame ionization detector. The oven temperature started at 175 °C and held for 10 min

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and then increased to 250 °C at 1 °C /min. Both the injector and detector temperatures

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were set at 250 °C. Helium was used as carrier gas (1.5 mL/min). The injection

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volume was 1µL in a splitless mode. The fatty acid methyl esters were identified by

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comparing with authentic standards (GL-463). Known concentrations of internal

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standards were used to monitor the accuracy of lipid extraction, methylation and

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quantification.

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Western Blot Analysis

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Epididymal adipose tissue was lysed in RIPA lysis buffer (1% Triton X-100, 1%

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deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with protease and

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phosphatases inhibitors. Protein quantification was performed using a BCA Protein

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Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein extracts were

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subjected to heat denaturation in loading buffer (3:1, v/v), separated by SDS-PAGE,

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electroblotted onto polyvinylidene fluoride membrane and blotted with specific

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primary antibodies overnight at 4 °C. After washing, membrane was incubated with


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species-specific second antibody. The chemiluminescence intensity of membrane was

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detected using ECL Western Blotting Detection System (Amersham Biosciences,

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Little Chalford, UK). The optical densities of bands were quantified by Gel Pro 3.0

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software (Biometra, Goettingen, Germany). All data were standardized to GAPDH as

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optical density (OD/mm2).

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Real-Time Quantitative Reverse Transcriptase PCR (qRT-PCR) Analysis

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Total RNA was extracted from epididymal adipose tissue using the TRIzol

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reagent (Ambion®, Austin, TX, USA). The mRNA expressions of target genes were

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quantified by SYBR green-based qRT-PCR assay with specific primers using a

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RT-PCR machine (Applied Biosystems, Forster, CA, USA). GAPDH was used as an

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endogenous control. All results were calculated by a comparative 2–∆∆Ct method.

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Statistical Analysis

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Data were presented as mean ± SD and subjected to one-way ANOVA followed

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by Tukey's test using SPSS12.0 statistical software. Statistical differences between

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groups were considered significant at p

Flaxseed Oil Alleviates Chronic HFD-Induced Insulin Resistance

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