Effect of annatto-extracted tocotrienols and green tea polyphenols on glucose homeostasis and skeletal muscle metabolism in obese male mice

Effect of annatto-extracted tocotrienols and green tea polyphenols on glucose homeostasis and skeletal muscle metabolism in obese male mice

Available online at www.sciencedirect.com ScienceDirect Journal of Nutritional Biochemistry 67 (2019) 36 – 43 Effect of annatto-extracted tocotrieno...

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Available online at www.sciencedirect.com

ScienceDirect Journal of Nutritional Biochemistry 67 (2019) 36 – 43

Effect of annatto-extracted tocotrienols and green tea polyphenols on glucose homeostasis and skeletal muscle metabolism in obese male mice☆ Eunhee Chung a , Salvatore N. Campise a , Hayli E. Joiner b, Michael D. Tomison c , Gurvinder Kaur d, e, Jannette M. Dufour f, g, Lillian Cole f , Latha Ramalingam e, g, h , Naima Moustaid-Moussa e, g, h , Chwan-Li Shen c, e, g,⁎ a

Department of Kinesiology, Health, and Nutrition, University of Texas at San Antonio, San Antonio, TX b Department of Kinesiology and Sport Management, Texas Tech University, Lubbock, TX c Department of Pathology, Texas Tech University Health Sciences Center, Lubbock, TX d Department of Medical Education, Texas Tech University Health Sciences Center, Lubbock, TX e Center of Excellence for Integrative Health, Texas Tech University Health Sciences Center, Lubbock, TX f Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX g Obesity Research Cluster, Texas Tech University, Lubbock, TX h Department of Nutritional Sciences, Texas Tech University, Lubbock, TX

Received 10 November 2018; received in revised form 18 January 2019; accepted 29 January 2019

Abstract Skeletal muscle is the major site for glucose uptake and thus plays an important role in initiating insulin resistance in type 2 diabetes mellitus. This study evaluated the effects of tocotrienols (TT) and green tea polyphenols (GTP) individually or in combination on glucose homeostasis and skeletal muscle metabolism in obese mice with insulin resistance and elevation of blood glucose. Forty-eight male mice were fed a high-fat diet and assigned to 4 groups in a 2 (no TT vs. 400 mg TT/kg diet) × 2 (no GTP vs. 0.5% vol/wt GTP in water) for 14 weeks. Both GTP and TT improved area under curve of insulin intolerance; while GTP increased serum insulin levels in obese mice, probably due to the addition of sweetener in drinking water. An interaction (TT×GTP) was observed in glucose tolerance test, total pancreas insulin concentration, and citrate synthase activity of soleus in mice. Neither TT nor GTP affected insulin and glucagon protein expression in pancreas based on immunohistochemistry. Both TT and GTP individually increased soleus muscle weight of mice; while only GTP increased gastrocnemius muscle weight of mice. The TT+GTP group had the greatest gastrocnemius muscle cross sectional area than other groups. GTP, not TT, induced cytochrome c oxidase activity and reduced thiobarbituric acid reactive substances levels in soleus muscle. Our results suggest that TT and GTP, individually or synergistically have the potential to improve skeletal muscle metabolism in obese mice by improving glucose homeostasis, reducing lipid peroxidation, and increasing rate limiting enzymes of oxidative phosphorylation. © 2019 Published by Elsevier Inc. Keywords: Dietary supplement; Aging; Sarcopenia; Animals; Diabetes; Obesity

1. Introduction Obesity induces ectopic lipid accumulation and desensitizes insulin signaling in skeletal muscle and adipose tissue, thus resulting in systemic insulin resistance and type 2 diabetes mellitus (T2DM) [1]. T2DM is characterized by peripheral insulin resistance with a variable degree of hyperglycemia (high blood sugar) and hyperinsulinemia (high insulin) [2], as well as reduced cellular response to insulin in skeletal muscle [3]. Patients with T2DM exhibit skeletal muscle ☆

dysfunction [4], including a greater decline of muscle mass [4–6] and strength [4,7]. The exact mechanisms underlying T2DM are still unclear; however, there is growing evidence to suggest that excess generation of reactive oxygen species (ROS), largely due to long-term hyperglycemia, results in increased oxidative stress [8,9] and an enhanced proinflammatory state [10]. Such excessive oxidative stress can impair insulin signaling and decrease glucose transport in skeletal muscle [11], and damage pancreatic beta cells [11]. Furthermore, the accumulation of toxic ROS targets mitochondria, the major site for

Conflicts of interest: None. ⁎ Corresponding author at: 1A092, 3601 4th street, Department of Pathology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430-8115, USA. Tel.: +1 806 743 2815; fax: +1 806 743 2117. E-mail addresses: [email protected] (E. Chung), [email protected] (S.N. Campise), [email protected] (H.E. Joiner), [email protected] (M.D. Tomison), [email protected] (G. Kaur), [email protected] (J.M. Dufour), [email protected] (L. Cole), [email protected] (L. Ramalingam), [email protected] (N. Moustaid-Moussa), [email protected] (C.-L. Shen). https://doi.org/10.1016/j.jnutbio.2019.01.021 0955-2863/© 2019 Published by Elsevier Inc.

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energy production, which could accelerate skeletal muscle dysfunction [3]. Yuzefovych et al. demonstrated that high-fat diet (HFD)induced mitochondrial DNA damage correlates with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver of C57BL/6J male mice [12]. In recent years, the use of dietary supplements, bioactive components, nutraceuticals, and botanicals (collectively called functional foods) have become an alternative approach to prevent and alleviate the complications of hyperglycemia in T2DM patients, to mitigate inflammation, and to maintain an oxidant-antioxidant balance. A review by Babu et al. proposed the potential benefits of flavonoids (bioactive polyphenolic compounds) in T2DM by (i) enhancing insulin secretion, reducing apoptosis, and promoting proliferation of pancreatic β-cells; (ii) decreasing hyperglycemia through regulation of glucose metabolism in hepatocytes; (iii) reducing insulin resistance, inflammation, and oxidative stress in muscle and fat; and (iv) increasing glucose uptake in skeletal muscle and white adipose tissue [13]. Among these alternatives, bioactive compounds, such as tocotrienols (TT) and green tea polyphenols (GTP) with strong anti-oxidant properties are good candidates for alleviating T2DM-related complications, such as hyperglycemia, hyperinsulinemia, and skeletal muscle disorders. Tocotrienols (TT), a subfamily of vitamin E with alpha (α), beta (β), delta (δ), and gamma (γ) isomers, is categorized as generally recognized as safe, and it is poised as a potential anti-diabetic agent. Siddiqui et al. reported that palm oil TT-rich fraction supplementation significantly improved glycemic status, serum lipid profiles, and renal function in streptozotocin (STZ)-treated type 1 DM rat [14]. Budin's study also showed the beneficial effects of palm oil TT-rich fraction supplementation on blood glucose control, oxidative stress and vascular wall in STZ-treated rats [15]. Lee and Lim recently demonstrated palm oil TT-rich fraction supplementation reduced hyperglycemia-induced skeletal muscle damage through regulating insulin signaling and suppressing oxidative stress in STZ-treated mice [16]. On the other hand, we recently reported annatto-extracted TT (consisting of 90% δ-TT and 10% γ-TT, free of tocopherol) improved glucose homeostasis via suppressing mRNA expression of proinflammation cytokines in obese mice with hyperglycemia and insulin resistance [17,18]. However, the beneficial effects of annattoextracted TT on skeletal muscle properties in high-fat diet induced obese mice with insulin resistance and elevation of blood glucose have not been previously evaluated. Besides TT, green tea has been proposed to have anti-diabetic properties [19]. In cellular studies, Li et al. reported that epigallocatechin 3-gallate (EGCG, the most abundant polyphenol in green tea) facilitates insulin secretion of isolated rat islets by inhibiting glutamate dehydrogenase, an enzyme in sensing mitochondrial energy supply and regulating insulin secretion [20]. Pournourmohammadi et al. demonstrated EGCG activates AMP-activated protein kinase through the inhibition of glutamate dehydrogenase in primary myotubes and pancreatic β-cells [21]. In animals, Chen et al. first reported green tea and EGCG improve glucose tolerance in HFD-fed rats [22]. Sae-tan et al. showed EGCG increased expression of genes related to fat oxidation in the skeletal muscle in HFD-fed mice [23]. Sundaram et al. reported green tea extract improves muscle and hepatic glycogen content in STZ and HFD-induced diabetic rats [24]. These studies suggest that regular consumption of green tea polyphenols (GTP, an extract of green tea, free of caffeine) maybe beneficial for the improvement of skeletal muscle properties in obese mice with hyperglycemic and insulin resistance. However, no study has evaluated such the potential benefit of GTP on skeletal muscle function in obese mice with hyperglycemic and insulin resistance. Long-term high-fat feeding (HFD) to C57BL/6J male mice has been widely used including by our team, as a model to study hyperglycemia and insulin resistance in obese mice [17,18,25]. In the present study,

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we investigated the potential benefit of two dietary bioactive components, annatto-extracted TT and GTP individually and in combination on skeletal muscle properties. We hypothesized that these protective effects are mediated in part by increased muscle cross-sectional area and mitochondrial enzyme activities in dietinduced hyperinsulinemic and glucose intolerant obese mice. Such changes in skeletal muscle may be mediated through improving glucose homeostasis and suppressing oxidative stress. Findings from these studies will advance the understanding of these bioactive compounds and their effects on skeletal muscle biology in humans with hyperglycemia and insulin resistance. 2. Materials and methods 2.1. Animals and treatments Forty-eight male 5-week-old C57BL/6J mice were purchased from Jackson Laboratory, Bar Harbor, ME, USA. After animal arrived at facility, they were fed chow diet and distilled water ad libitum for 5 days. Mice were housed (3 mice per cage) and maintained at a controlled temperature of 21±2 °C with a 12 h light–dark cycle. Each treatment group had 4 cages of mice. All animals were observed daily for clinical signs of disease. Body weight, food intake, and water consumption were recorded weekly. All conditions and handling of the animals were approved by the Texas Tech University Health Sciences Center Institutional Animal Care and Use Committee. All experiments were performed in accordance with the relevant guidelines and regulations. After 5-day acclimatization, mice were weighed, randomly stratified by weight and assigned to 4 groups (n=12/group) into a 2 (no TT vs 400 mg TT/kg diet) × 2 (no GTP vs 0.5% (wt/vol) GTP in drinking water) factorial design (namely, the control group, the TT group, the GTP group, the TT+GTP group) for 14 weeks. Throughout the study, all animals were fed with a HFD consisting of 20%, 22% and 58% of energy from carbohydrates, protein, and fat. Animals had free access to water and food during the 14-week study period. Annatto-extracted TT (gift from American River Nutrition, Inc., Hadley, MA) was extracted from 70% pure annatto oil, containing 90% δ -TT and 10% γ -TT. TT was premixed with tocopherol-stripped soybean oil (Dytes, Bethlehem, PA, USA) before adding to the high-fat diet. Distilled water mixed with GTP was prepared fresh daily and the amount of water consumed was recorded for each mice. GTP was purchased from the same source as that used in our previous studies (Shili Natural Product Company, Guangxi, China), with a purity higher than 98.5%. Every 1000 mg of GTP contained 464 mg of (−)epigallocatechin gallate, 112 mg of (−)epicatechin gallate, 100 mg of (−)epicatechin (EC), 78 mg of (−)epigallocatechin, 96 mg of (−) gallocatechin gallate and 44 mg of catechin according to the high performance liquid chromatography-electrochemical detection (HPLC-ECD) and high performance liquid chromatography-ultraviolet detection (HPLC-UV) analyses [26]. To avoid the bitter taste of GTP, we have added sweeter at 0.1% (weight/volume) into drinking water for the GTP and TT+GTP groups. Our previous animal studies showed the osteoprotective impact of δ-TT at 400 mg/kg diet [18] and GTP at 0.5% (wt/vol) in drinking water [26]. Our T400 mg/kg diet in mice corresponds to approximately 160 mg daily in humans for at 70-kg body weight. Our GTP at 0.5% vol/wt dosage in mice corresponds to 4–6 cups per day of tea equivalent in human consumption. 2.2. In vivo glucose and insulin tolerance tests After 12 weeks of treatment, mice (n=5–7 per treatment group) were fasted for 4 h and intraperitoneal glucose tolerance tests (GTT) were performed by intraperitoneal injection of 2 mg/g body weight of glucose. Blood glucose levels were measured 0, 15, 30, 60, and 120 mins following glucose injection. Additionally, after 12 weeks of treatment, intraperitoneal insulin tolerance tests (ITT) were performed on mice (n= 5–8 per treatment group) that were fasted for 4 h prior to intraperitoneal injection of 1 U/kg body weight insulin (Humulin, Abbott, Chicago, IL, USA). Blood glucose levels were analyzed 0, 15, 30, 60, and 120 mins following insulin injection. Total area under the curve (AUC) for both GTT and ITT were calculated by the trapezoidal method. For both GTT and ITT, blood was collected from the tail vein and measured using a glucometer (One touch ultra mini, Life Scan, Wayne, PA). 2.3. Sample collection At the end of the experiment, animals were fasted for 4-h and blood was collected from the abdominal aorta from mice anesthetized with isoflurane. Soleus and gastrocnemius muscles were harvested, cleaned of fat tissue, weighed, and stored at −80 °C for later analyses. Blood samples were centrifuged at 1500 × g for 20 min and serum samples were kept at−80 °C until analyzed. 2.4. Insulin measurement Pancreases (n=8 per treatment group) were collected at the end of the study and cellular insulin content was determined by acetic acid extraction followed by mouse

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2.5. Analysis of pancreatic tissue

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After 14 weeks of treatment, pancreases (n=4 per treatment group) were collected for histological assessment. Tissue was fixed in Z-fix, embedded in paraffin and tissue sections were immunostained as described previously (Dufour 2005). Primary antibodies were guinea pig anti-insulin (diluted 1:1000; Dako Agilent Pathology Solutions, Santa Clara, CA, USA) and mouse anti-glucagon (diluted 1:5000; Sigma). Appropriate biotinylated secondary antibodies and avidin–biotin–enzyme complexes were purchased from Vector Laboratories (Burlingame, CA, USA). Diaminobenzidine as the chromogen was purchased from BioGenex (Fermont, CA, USA). Tissue sections were counterstained with hematoxylin.

Body weight (g)

insulin ELISA (EMD Millipore, Billerica, MA, USA). Serum insulin (n=7 per treatment group) was also quantified using the same mouse insulin ELISA kit.

Control TT GTP TT+GTP

a a b b

30

25 At 14 weeks: TT: P=.622 GTP: P=.010 (a,b) TTxGTP: P=.517

20

2.6. Skeletal muscle cross-sectional area Gastrocnemius muscles (n=3 per treatment group) were fixed with 10% phosphate-buffered formalin for 24 h at room temperature and then placed into 70% ethanol at 4 °C until processed. Gastrocnemius muscles were embedded in paraffin, sectioned, and stained with hematoxylin–eosin to visualize tissue architecture and measure cross-sectional area (CSA). Tissue images were obtained at x20 using the cellSens Standard imaging software (Olympus, Tokyo, Japan). Fiber CSA was measured by tracing the outer perimeter of the cell with image J (NIH) over 1, 500 fibers per group (500 fibers/animals).

15 0

2

4

6

8

10

12

14

Weeks Fig. 1. Effect of annatto-extracted tocotrienols and green tea polyphenols on body weight. Different letters (a and b for GTP effect) are significantly different by two-way ANOVA and Fisher's LSD test (Pb.05). Values are mean (n=12/group) with the standard error of mean (S.E.M.) represented by vertical bars.

2.7. Biochemical assays in muscle The production of hydrogen peroxide (H2O2) was measured using an Amplex red H2O2 assay kit (A22188: Invitrogen) according to the manufacturer's instruction. Cytochrome c oxidase (COX) activity was determined by a colorimetric assay kit (CYTOCOX1; Sigma-Aldrich) and citrate synthase (CS) activity was determined by Citrate Synthase Assay Kit (CS0720; Sigma-Aldrich) according to the manufacturer's instruction. Malondialdehyde (MDA) levels were measured by Thiobarbituric Acid Reactive Substances (TBARS) assay according to manufacturer's instruction (Cayman Chemical) to evaluate lipid peroxidation. 2.8. Statistical analysis Results are presented as mean ± standard error of the mean (S.E.M.). All the other data were tested by two-way analysis of variance (ANOVA) followed by Fisher's Least Significant Difference (LSD) post hoc test with SigmaStat software, version 12.5 (Systat Software, San Jose, CA, USA). A significance level of Pb.05 applies to all statistical tests.

3. Results 3.1. Body weight, food intake, and water consumption Fig. 1 presents the mean body weight of different treatment groups. At baseline, there was no significant difference in body weight among all groups. Throughout the study period, body weight significantly increased in all animals regardless of treatment. Neither TT nor GTP supplementation affected body weight until 14 weeks. At the end of the 14-week study, only GTP supplementation (P=.010) significantly reduced body weight and no interaction between TT and GTP was observed (PN.05). Throughout the study, the average food intake by weight and water consumption were similar among all groups (both PN.05) (data not shown).

shown by a significant decrease in fasting blood glucose levels (Fig. 2A). Intriguingly, a post hoc Fisher's LSD analysis revealed that (i) in the absence of TT, GTP supplementation decreased blood sugar at 60 and 120 min after GTT test; and (ii) in the presence of TT, supplementation of GTP in drinking water did not further lower blood glucose. Similarly, in the absence of GTP, TT supplementation decreased blood sugar at both 60 and 120 min. However, in the presence of GTP, supplementation of TT into the diet did not further suppress blood sugar. The GTT AUC data followed the same trend showing that there was significant interaction (TTxGTP) in improved AUC (P=.035) (Fig. 2B). In terms of ITT results, 60 min after insulin administration, GTP supplementation decreased blood glucose levels in obese mice (P= .043, Fig. 2C). Similarly, GTP supplementation (P=.020) significantly decreased blood sugar 120 min after insulin injection, while TT (P= .059) effected a near-significance downward trend. For the ITT, no significant interaction between TT and GTP on blood glucose levels was observed (Fig. 2C). According to ITT AUC data, both TT (P=.038) and GTP (P=.025) significantly lowered ITT AUC (Fig. 2D). Fig. 2E shows GTP supplementation significantly increased serum insulin concentration in obese mice (Pb.001) and no interaction between TT and GTP was observed. Regarding total pancreatic insulin levels, there was a significant TT and GTP interaction (P=.015), resulting in the control group = the TT+GTP group N the GTP group = the TT group. Analysis of the pancreases for beta (insulin, Fig. 3A-D) and alpha (glucagon, Fig. 3E-H) cells via immunohistochemistry revealed that the pancreases from all of the animals, regardless of treatment, contained healthy glucagon producing alpha cells and insulin producing beta cells.

3.2. Glucose homeostasis and morphological analysis of the pancreas The effects of TT and GTP supplementation on glucose homeostasis were assessed by GTT (Fig. 2A and B) and ITT (Fig. 2C and D). Additionally, pancreatic β-cell function was assessed by measuring serum insulin levels (Fig. 2E) and pancreatic insulin levels (Fig. 2F). Prior to injection there was no significant difference in fasting blood glucose levels between groups. Fasting blood glucose levels were 178.8±6.4 mg/dL, 164.9±4.5 mg/dL, 164.6±7.0 mg/dL, and 162.4± 4.6 mg/dL for the HFD, HFD+TT, HFD+GTP, and HFD+TT+GTP group, respectively. For GTT, at both 60 and 120 min after glucose administration, there was a significant interaction between TT and GTP on improved glucose tolerance of HFD-treated mice (Pb.05), as

3.3. Muscle mass and cross-sectional area Fig. 4A illustrates both TT and GTP increased soleus muscle weight normalized by body weight (Sol/BW) after 14 weeks of feeding period. However, we did not find any interaction in Sol/BW between TT and GTP. Fig. 4B shows GTP, not TT, increased gastrocnemius muscle normalized body weight (Gas/BW). Next, based on the stained fiber CSA of gastrocnemius muscle (Fig. 5A–D), there was a significant difference in fiber CSA between TT and GTP (Pb.001, Fig. 5E) resulting in the order of the TT+GTP group N the GTP group ≥ the TT group ≥ the control group.

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B

A 600

Control TT GTP TT+GTP

60000

A

500 B B B

400 300

A B B B

200

Control TT GTP TT+GTP

A B

GTT AUC (mg/dL)

Blood glucose (mg/dL)

700

B

B

40000

20000

Effect: TT: P=.007 GTP: P=.043 TT x GTP: P=.035(A,B)

0

100 0

15

30

60

Treatment

120

Minutes

At 60 min TT:P=.070 GTP:P=.050 TTxGTP:P=.020(A,B)

At 120 min TT:P=.006 GTP:P<.001 TTxGTP:P=.020(A,B)

D 180

Control TT GTP TT+GTP

160 140 120 100

a

80

a a b b

60

a b

10000

ITT AUC (mg/dL)

C Blood glucose (mg/dL)

39

Control TT GTP TT+GTP

ax bx

ay by

8000 6000 4000 2000

b

Effect: TT: P=.038 GTP: P=.025 TT x GTP: P=.696

0

40 0

15

30

60

Treatment

120

Minutes At 60 min TT: P=.846 GTP: P=.043(a,b) TTxGTP: P=.497

At 120 min TT: P=.059 GTP: P=.020(a,b) TTxGTP: P=.491

F 40

Control TT GTP TT+GTP

a

a

30

20

b 10

b

Effect: TT: P=.915 GTP: P<.001(a,b) TT x GTP:P=.771

Pancreas insulin (ug/ml)

Serum insulin concentration (ng/ml)

E

400

Treatment

A A B

300

B

200

100

0

0

Control TT GTP TT+GTP

Effect: TT: P=.317 GTP: P=.660 TTxGTP: P=.015(A,B)

Treatment

Fig. 2. Effect of annatto-extracted tocotrienols and green tea polyphenols on blood glucose during GTT (2A), GTT AUC (2B), blood glucose during ITT (2C), ITT AUC (2D), serum insulin concentration (2E), and pancreas insulin concentration (2F). Different letters (x and y for TT effect; a and b for GTP effect; A and B for interaction effect) are significantly different by twoway ANOVA and Fisher's LSD test (Pb.05). Values are mean (n=6/group) with the standard error of mean (S.E.M.) represented by vertical bars.

3.4. Mitochondrial enzyme activity Citrate synthase (CS), an enzyme of the Kreb cycle, and cytochrome c oxidase (COX), an enzyme of the complex IV of electron transport

system were measured to estimate mitochondrial content of soleus muscle. Table 1 shows there was a significant interaction between TT and GTP supplementation in CS of soleus muscle (Pb.001) and the TT group had the highest citrate synthase activity among all treatment

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3.5. Oxidative stress As shown in Table 1, there was a significant interaction in soleus H2O2 levels between TT and GTP supplementation (Pb.001) and the TT +GTP group had the same H2O2 level as that of the control group. In terms of soleus TBARS (as measured by MDA, a marker of lipid peroxidation and an indirect index of oxidative stress), the GTPsupplemented group (the GTP and the TT+GTP groups) had a significant lower levels than those in the non-GTP supplemented groups (the control group and the TT group) (P=.022, Table 1). There was a tendency of interaction in soleus TBARS concentrations between TT and GTP supplementation (P=.060, Table 1). 4. Discussion In this study, we showed a beneficial effect of two dietary bioactive components, TT and GTP, on skeletal muscle properties of dietinduced obese male mice. To our knowledge, this is the first study demonstrating that the mice receiving TT plus GTP supplementation (the TT+GTP group) for 14 weeks had a synergistic effect with the higher value for fiber CSA of gastrocnemius muscles, when compared to those receiving TT (the TT group) or GTP (the GTP group) alone. In addition to assessing fiber CSA, the Sol/BW were also measured showing both TT and GTP treatments independently increased skeletal muscle weight normalized by body weight. The findings that TT increased skeletal muscle weight (Sol/BW) of obese mice corroborated with Lee's recent work using TT-rich fraction (consisting of 23.4% α-TT, 37.4% γ-TT, 21.8% α-tocopherol, and 1.0% γ-tocopherol) supplementation in diabetic mice [16]. Lee et al. reported TT-rich fraction supplementation mitigated muscle atrophy, plasma insulin concentration, and homeostatic model assessment estimated insulin resistance (HOMA-IR) in HFD-fed and STZ-injected diabetic mice via up-

A 0.4

Soleus muscle weight (mg/g BW)

groups. According to the post hoc LSD analysis, (i) in the presence of TT, GTP supplementation decreased CS activity, while in the absence of TT, GTP supplementation had no impact on it; (ii) in the presence of GTP, TT supplementation did not affect CS activity, while in the absence of GTP, TT supplementation increased CS activity. Table 1 illustrates GTP supplementation in the drinking water significantly increase the COX activity of soleus in the HFD-fed mice (both GTP group and TT+GTP group). There was no interaction between TT and GTP supplementation in soleus COX activity.

Control TT GTP TT+GTP

0.3

yb

xb

ya xa

0.2

0.1

Effect: TT: P=.039 (x,y) GTP: P=.019 (a,b) TT x GTP: P=.640

0.0 Treatment

B Gastronemius muscle weight (mg/g BW)

40

4

Control TT GTP TT+GTP

a b

a

b

3

2

1

Effect: TT: P=.911 GTP: P=.001 (a,b) TT x GTP: P=.830

0 Treatment Fig. 4. Effect of annatto-extracted tocotrienols and green tea polyphenols on soleus (4A) and gastrocnemius (4B) muscle weight normalized by body weight. Different letters (x and y for TT effect; a and b for GTP effect) are significantly different by two-way ANOVA and Fisher's LSD test (Pb.05). Values are mean (n=12/group) with the standard error of mean (S.E.M.) represented by vertical bars.

regulating IRS-1 and Akt levels accompanied by increased translation of GLUT4 [16]. Khor's study further demonstrated TT-rich fraction supplementation promotes myogenic differentiation in prevention of

Fig. 3. Effect of annatto-extracted tocotrienols and green tea polyphenols on immunohistochemical analysis of pancreatic tissue. Pancreatic tissue sections collected from mice fed HFD (3A and 3E), HFD supplemented with TT (3B and 3F, HFD supplemented with GTP (3C and 3G), and HFD supplemented with TT and GTP (3D and 3F) were immunostained for insulin (3A-3D) or glucagon (3E-3H) and counterstained with hematoxylin.

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A:Control

B:TT

C:GTP

41

D:TT+GTP

E: Statistical analysis 3000

Cell area (um2)

2500

Control TT GTP TT+GTP

A C

BC

B

2000

1500

1000 Effect: 500 TT: P<.001 GTP: P<.001 TT x GTP: P=<.001 (ABC) 0 Treatment Fig. 5. Effect of annatto-extracted tocotrienols and green tea polyphenols on gastrocnemius muscle cross sectional area. Representative images of HFD (5A), HFD+TT (5B), HFD+GTP (5C), and HFD+TT+GTP (5D) of gastrocnemius muscle sections stained with hematoxylin and eosin. The scale bar=100 μm. Over 1500 fibers per group (5E) were counted and analyzed by two-way ANOVA and Fisher's LSD test. Different letters (A, B, and C for interaction effect) are significantly different (Pb.05).

muscle, but not that of the slow soleus muscle, in aged rats [30]. Rodriguez et al. demonstrated that green tea extract protects against endoplasmic reticulum stress, oxidative stress, and protein degradation induced by a HFD in skeletal muscle [31]. Onishi et al. showed green tea extracts mitigated HFD- and aging-induced muscle atrophy, which is correlated with insulin resistance [32]. Taken together, these findings suggest that green tea extract provides the its' protective effects on fast twitch fibers, such as plantaris muscle, which is largely affected by aging process, but not for slow soleus muscle that is well maintained throughout the aging process [33]. The ability of TT to improve glucose tolerance and insulin resistance in this study are consistent with previous works in the STZ-induced type 1 diabetic rats using TT-rich fraction [15,34], HFDinduced T2DM mice using annatto-extracted TT [18], HFD- and high

replicative senescence of primary human myoblasts, potentially resulting in mitigating age-related phenotypes of skeletal muscle [27]. The observations that GTP benefits skeletal muscle in this study are supported by several previous studies. For example, Kim et al. showed EGCG administration to mice significantly increased muscle fiber size for regeneration by the induction of myogenic markers, including myogenin and muscle creatine kinase [28]. Always et al. reported that relative to the animals treated with vehicle, green tea extract increased satellite cell proliferation and differentiation in plantaris and soleus muscle during recovery from hind limb suspension in old rats and such changes led to attenuation of aging-induced muscle fiber CSA loss in both plantaris and soleus muscle [29]. Moreover, the impact of EGCG on muscle recovery after disuse in aged rats was muscle specific because EGCG only improved the recovery of plantaris

Table 1 Effect of annatto-extracted tocotrienols (TT) and green tea polyphenols (GTP) on CS activity, COX activity, H2O2, and TBARS of soleus muscle Parameter

CS activity (μmol/mg protein) COX activity (μU/mg protein) H2O2 activity (nmol/mg protein) TBARS (MDA nM/mg protein)

no TT

TT

Two-way ANOVA P value

no GTP (control group)

GTP (GTP group)

no GTP (TT group)

GTP (TT+GTP group)

TT

GTP

TT × GTP

389.5B±8.8 14.0b±1.4 36.3A±3.2 344.0a±54.3

401.6B±12.4 22.7a±1.8 15.4B±3.5 111.2b±33.6

633.4A±14.3 15.4b±1.4 19.1B±2.9 166.7a±66.8

417.9B±12.4 21.1a±1.8 32.1A±4.1 139.7b±72.2

b0.001 0.945 0.942 0.159

b0.001 b0.001 0.270 0.022

b0.001 0.406 b0.001 0.060

Different letters (a and b for GTP effect; A and B for interaction effect) are significantly different by two-way ANOVA and Fisher's LSD test (Pb.05). Values are mean (n=6/group) with the standard error of mean (S.E.M.) represented by vertical bars. CS, citrate synthase; COX, cytochrome c oxidase; H2O2, hydrogen peroxidase; TBARS, thiobarbituric acid reactive substances; MDA, malondialdehyde.

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carbohydrate diet-induced T2DM rats using individual TT isomer [35], and STZ+HFD-induced T2DM using TRF [16]. On the other hand, the findings that GTP improved both glucose tolerance and insulin resistance, and increased serum insulin levels in the present study is in the agreement with others [36–38]. For example, Li et al. reported EGCG attenuates free fatty acids-induced peripheral insulin resistance in rats through AMPK and insulin signaling pathway [36]. Bao et al. demonstrated EGCG improves insulin signaling by decreasing toll-like receptor 4- mediated inflammatory response in adipose tissue of HFDfed rats [37]. Gan et al. reported EGCG dose-dependently improves insulin resistance in non-alcoholic fatty liver mice [38] through reducing body weight and enhancing the insulin clearance by hepatic insulin-degrading enzyme. However, a recent meta-analysis of limited clinical studies did not provide evidence supporting the benefits of green tea in reducing hemoglobin A1c, HOMA-IR, fasting insulin, or fasting glucose in people with pre-diabetes/T2DM [39,40]. In the present study, we noted only GTP, not TT, increase serum insulin levels which may be due to the addition of sweetener into GTPsupplemented water. We also found that there was an interaction between TT and GTP supplementation in improving glucose tolerance of GTT; however, such an interaction effect was not found in the glucose levels of ITT results. Typically GTT shows more changes than ITT in general. GTT tests the ability of the pancreas to quickly sense glucose and secrete insulin to lower glucose, while ITT tests more sensitivity to insulin (though not directly). TT or GTP supplementation alone decreased pancreatic insulin concentration compared to the HFD, while the combination of TT+GTP had no effect on pancreatic insulin content when compared to the HFD group. There is evidence that obesity is associated with reduced skeletal muscle oxidative capacity [41–44]. The reduced oxidative capacity has been postulated to provoke perturbations in insulin signaling, leading to the eventual development of insulin resistance and T2DM [42,45]. CS activity is a validated biomarker for mitochondrial density in skeletal muscle to treatments [46]. In this study, TT supplementation increased CS activity of soleus, although the additional GTP supplementation counteracted such favorable effect. The beneficial effects of TT on CS activity in obese mice in the present study could be explained by the fact that TT regulates peroxisome proliferator-activated receptors (PPAR) [47,48]. PPAR-α, PPAR-γ, and PPAR-δ are ligandregulated transcription factors that play essential roles in lipid metabolism. TT-rich fraction has been shown to increase in fatty acid oxidation due to enhancing PPAR activity through PRAR receptors and subsequent mitochondrial biogenesis [47,48]). In reporter-based assays, Fang et al. reported both α-TT and γ-TT activated PPAR-α, while δ-TT activated PPAR-α, PPAR-γ, and PPAR-δ [47]. Allen et al. also showed annatto-extracted TT (90% δ-TT+10% γ-TT) activated both PPAR-α and PPAR-δ in adipose tissue [17]. COX activity, on the other hand, was increased by GTP supplementation, but not by TT. COX activity is used to estimate the maximal respiratory capacity of mitochondria and has been shown to be linked to the pathology of T2DM [49]. Increased COX activity by GTP supplementation seen in our study is in agreement with the previous study in human cultured neurons and astrocytes treated with EGCG [50]. The different regulation of mitochondrial enzyme by TT and GTP could be related to the genetic origin of these enzymes. CS is encoded by nuclear genes, while COX is encoded by both nuclear and the mitochondrial genomes [51]. Thus, increased COX activity by GTP suggests that GTP may stimulate mitochondrial genome transcript, which has shown in mice liver mitochondria after fasting [52]. Similar to GTP treatment, a previous study demonstrated that fasting increased COX activity but not CS activity in mice liver mitochondria by upregulating a mitochondrial T3 receptor p43 expression [52]. Both TT and GTP has antioxidant properties. TT is a major antioxidant in the cell membrane which can protect against oxidative damage, in here by H2O2 production, against HFD. Similar trend has

been shown in GTP treatment. However, the combination of TT and GTP negate reduction of H2O2 production, unlike independent treatment (TT or GTP). This may be due to a potential pro-oxidant property of GTP when TT was present [53]. However, the marker of lipid peroxidation was blunted with the combination of TT and GTP although H2O2 production is similar to HFD group. We measured MDA as a marker of lipid peroxidation through TBARS reaction by spectrophotometer, which could be a potential limitation of this study [54]. European Food Safety Authority recommends to measure MDA by high-performance lipid chromatography in addition to measurements of F2α-isoprostanes and LDL oxidation to accurately measure lipid peroxidation [54]. In conclusion, TT and GTP increased muscle fiber CSA with a synergistic manner, which could be used for therapeutic way to prevent muscle atrophy which is commonly seen in patients with insulin resistance and elevation of blood glucose. This phenotypic improvement may be partially through attenuation of oxidative stress and increased levels of major rate-limiting mitochondrial enzymes, but with different mechanisms. Acknowledgement This study was supported by American River Nutrition, Inc., Hadley, MA, and in part by the Obesity Research Cluster. EC was supported by startup funds from the University of Texas at San Antonio. SNC was supported by the Office of Undergraduate Research Scholarship from the University of Texas at San Antonio and HEJ was supported by the Undergraduate Project fund from Texas Tech University Center for Active Learning and Undergraduate Engagement (CALUE). JMD and GK were supported in part by The Ted Nash Long Life Foundation and The Jasper L. and Jack Denton Wilson Foundation. We thank Kassandra Gonzalez, Antonio Bollinger, and Analiza Morales for the assistance with this study. Author contributions statement EC and CLS designed the research, performed experiment, analyzed, and prepared figures and tables. SNC, HEJ, MDT, GK, LC, and LR performed experiments; EC, GK, JMD, NMM, and CLS wrote the manuscript; all authors approved the final version of the manuscript. References

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