Cistanches Herba Reduces The Weight Gain in High Fat Diet-induced Obese Mice Possibly Through Mitochondrial Uncoupling

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Hoi Shan Wong, Jihang Chen, Pou Kuan Leong, Hoi Yan Leung, Wing Man Chan, Kam Ming Ko

* Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China.

Abstract:

A semi-purified fraction isolated from Cistanches Herba (HCF1), was previously found to induce mitochondrial uncoupling in H9c2 cells and in rat hearts. We, therefore, hypothesized that HCF1 would produce a weight loss effect against a high-fat diet (HFD)-induced obesity. To test this hypothesis, a mouse model of HFD-induced obesity was established and the effects of HCF1 on a normal diet (ND)-fed and HFD-fed mice were examined. In the study, long-term HCF1 treatment produced a weight reduction effect against HFD-induced obesity in male and female mice. The HCF1-induced weight loss was associated with improved insulin sensitivity in HFD-fed animals. To understand the action mechanism underlying the weight reduction effect afforded by HCF1, its effects on mitochondrial uncoupling were examined. A comparative study with cholestyramine (CT), a bile acid sequestrant, was also conducted. The findings demonstrated that HCF1-induced weight loss was likely mediated by the increase in energy consumption, probably via the induction of mitochondrial uncoupling in mouse skeletal muscle. Thus, our findings suggest the potential use of HCF1 to prevent obesity and the associated health consequences such as diabetes, cardiovascular diseases, and metabolic syndrome.

Keywords: Cistanches Herba, Weight control, Mitochondrial uncoupling, Mitochondrial uncoupling protein 3

cistanche tubolosa health benefits

Chengdu Wecistanche mainly produces Cistanche Products for a healthy life.

Introduction

Obesity is characterized by the abnormal accumulation of fat in adipose tissue and other organs that produces adverse effects on health. Current findings indicated that the sedentary lifestyle and the changes in dietary composition in affluent societies caused an obesity epidemic, with the concomitant increase in the incidences of various non-communicable obesity-related chronic diseases such as metabolic syndrome (Shi et al., 2012). National Health and Nutrition Examination Survey (NHANES) conducted in 2007 showed that the global obesity prevalence was over 30%, and it was expected to increase by a further 33% over the next two decades (Finkelstein et al., 2012). The onset of obesity will also shift to the young populations, as reflected by the trends in overweight and obesity in children and adolescents (McTigue, Garrett, & Popkin, 2002). These findings forecast the potential burden on the medical care for obesity and the associated health consequences, implicating an immediate need to control the size of the obese population.

The management of obesity necessitates a long-term intervention and a multi-disciplinary approach. Given the obesity epidemic, the search for feasible non-therapeutic and/or therapeutic interventions has been an area of intensive research. The strategy is mainly focused on the establishment of energy balance by increasing energy expenditure or reducing energy intake. Among various approaches which include a dietary replacement, exercise, inhibition of intestinal absorption and activation of the sympathetic nervous system, the induction of mitochondrial uncoupling, either by the use of chemical uncoupler or by the induction of mitochondrial uncoupling proteins (Cerqueira, Laurindo, & Kowaltowski, 2011; Clapham et al., 2000), is proven to be an effective means for weight loss (Clapham et al., 2000; Diehl & Hoek, 1999; Harper, Dickinson, & Brand, 2001; Li et al., 2000). Mitochondrial uncoupling refers to a process causing the uncoupling between the energy-yielding electron transport processes and the phosphorylation of ADP in the mitochondrion. By offering an alternative proton conductance pathway, mitochondrial uncoupling causes the dissipation of the proton gradient and a faster rate of oxygen consumption (Brand et al., 2005). Under the uncoupling condition, the decrease in ATP synthesis is associated with a reduction in mitochondrial membrane potential, with the potential energy stored in the proton gradient being dissipated as heat. This futile cycle of proton transport consumes a high proportion of metabolic energy in various tissues, especially in skeletal muscle which consumes around 20% of total energy generated in metabolism (Chan, Wei, Chigurupati, & Tu, 2010; Korshunov, Skulachev, & Starkoc, 1997). The modulation of mitochondrial proton leakage increases the basal metabolic rate, which contributes to a significant portion of the daily energy expenditure, with subsequent elevation in the use of fuel molecules (such as fatty acids) and therefore causes weight loss (Ricquier & Bouillaud, 2000).

Cistanches Herba, a dried whole plant of Cistanche deserticola YC Ma, is a ‘Yang-invigorating’ tonic herb in traditional Chinese medicine. The herb has also been used as a healthy food for the treatment of kidney defificiency for hundreds of years in China. Our recent findings showed that a semipurified fraction of Cistanches Herba (HCF1) induced mitochondrial uncoupling in H9c2 cells and in rat hearts (Wong & Ko, 2013). It was also demonstrated that long term HCF1 treatment (at daily doses of 1.14 and 11.4 mg/kg; 14 consecutive days) produced an uncoupling effect on mitochondria isolated from kidney and liver in rats, as indirectly evidenced by the reduction in ATP levels in these tissues (unpublished data). Given that the induction of mitochondrial uncoupling is an effective means for weight loss (Clapham et al., 2000; Diehl & Hoek, 1999; Harper et al., 2001; Li et al., 2000), we hypothesized that HCF1 would also produce mitochondrial uncoupling in skeletal muscle, with resultant weight reduction. To test the hypothesis, a mouse model of high-fat diet (HFD)-induced obesity was established and the effects of HCF1 on a normal diet (ND)-fed and HFD-fed mice were investigated. In addition, a comparative study between the effects of HCF1 and a bile acid sequestrant cholestyramine (CT) (Chen et al., 2010; Yamato et al., 2012) was also conducted to characterize the weight reduction effect afforded by HCF1.

cistanche stem

2. Materials and methods

2.1. Herbal extraction

Cistanches Herba, the dried whole plant of Cistanches deserticola YC Ma (Orobanchaceae), was purchased from a local herbal dealer (Lee Hoong Kee). The herb was authenticated by the supplier and a voucher specimen (HKUST00301) was deposited in the Division of Life Science, the Hong Kong University of Science and Technology (HKUST). Cistanches Herba ethanol extract was obtained by ethanol extraction of ground herbal material by heating under reflux at 78 °C for 2 h, as previously described (Leung & Ko, 2008), with the yield being 14% (w/w). The extract was further fractionated using silica gel chromatography (Wong & Ko, 2013). HCF1, with the extraction yield being 1.14%, was obtained. The extract was dried by evaporating the solvent under reduced pressure at 50 °C, and the dried extract was stored at −20 °C until use.

2.2. Chemicals

Bio-Rad assay reagent was purchased from Bio-Rad Laboratories (Richmond, CA, USA). LabAssay™ Triglyceride (290-63701), LabAssay™ Cholesterol (294-65801), and HDL-Cholesterol E test kit (431-52501) were purchased from Wako (Osaka, Japan). AntiUCP3 (E-18) antibody (catalog # sc-31387) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA).

2.3. Animal care

ICR mice (8 weeks; 30–35 g for males and 25–30 g for females) were maintained under a 12-h dark/light cycle in an air/ humidity-controlled room at about 22 °C and allowed food and water ad libitum in the Animal and Plant Care Facilities (APCF) at HKUST. All experimental procedures were approved by the Research Practice Committee (HKUST) (Animal protocol approval no. 2013049; approved date: 25 September 2013; experiment duration: 3 years).

cistanche life extension

2.4. Treatment protocol

To examine the effect of HCF1 on HFD-induced obesity, male or female ICR mice were randomly assigned to eight groups, with 10–15 mice in each: (1) Normal diet (ND, 13% energy derived from fat) control; (2) ND + low dose of HCF1 (HCF1 L) (at a daily dose of 1.5 mg/kg); (3) ND + medium dose of HCF1 (HCF1 M) (at a daily dose of 15 mg/kg); (4) ND + high dose of HCF1 (HCF1 H) (at a daily dose of 45 mg/kg); (5) HFD (60% energy derived from fat; purchased from Research Diet Inc., product no. D12492) control; (6) HFD + HCF1 L; (7) HFD + HCF1 M; and (8) HFD + HCF1 H. As gender differences in the responsiveness towards dietinduced body weight changes and weight reduction interventions were reported in rats and humans (Rodriguez & Palou, 2004; Rodriguez, Quevedo-Coli, Roca, & Palou, 2001), we were interested in investigating the effects of HCF1 on both male and female mice. The doses of HCF1 were derived from its effective doses to induce mitochondrial uncoupling rat hearts (Wong & Ko, 2013). In HCF1 co-treatment groups, mice were intragastrically administered with HCF1, 5 days per week for 8 weeks (i.e., 40 doses). Control mice received vehicle (olive oil) only. Body weights and food consumption of the mice were monitored weekly during the course of the experiment. The changes in body weight were quantified by calculating the area under the curve (AUC) of the graph plotting percent initial body weight against time (week 1–8) and expressed in arbitrary units. Blood samples were drawn 24 h after the last dosing with HCF1 from overnight fasted, phenobarbital-anesthetized mice by cardiac puncture. The mice were then sacrificed by cervical dislocation. Samples of gastrocnemius muscle were excised for further biochemical analysis. Fat pads (gonadal, retroperitoneal, and mesenteric fat) were dissected and weighed. The ratio of a particular fat pad weight to body weight was estimated and expressed as fat pad index. To examine the effect of CT on HFD-fed obese male mice, male mice were intragastrically administered with CT (suspended in water) at a daily dose of 300 mg/kg.

2.5. Sample preparations

Plasma samples, skeletal muscle homogenates (nucleus-free fraction), and skeletal muscle mitochondria were obtained as described previously (Leong et al., 2013).

2.6. Biochemical analysis

2.6.1. Plasma glucose and lipid contents

Plasma glucose levels were measured by a glucose (hexokinase) assay kit (Sigma, St. Louis, MO, USA). The amount of glucose was assessed by the addition of a reaction mixture containing 1.5 mM NAD, 1.0 mM ATP, 1.0 unit/mL hexokinase, and 1.0 unit/mL glucose-6-phosphate dehydrogenase. Absorbance changes at 340 nm were monitored spectrophotometrically by Victor V3 Multi-Label Counter (Perkin Elmer, Santa Clara, CA, USA).

Plasma triglyceride (TG), total cholesterol (TC), and high-density lipoprotein-cholesterol (HDL) levels were measured using assay kits: TG: LabAssayTM Triglyceride, TC: LabAssayTM Cholesterol; HDL-C: HDL-Cholesterol E test kit. The TG, TC, and HDL contents were assessed by adding the chromogen reagents provided by the corresponding assay kits. Absorbance changes at 600 nm were monitored (Siddiqua, Hamid, Ar-Rashid, Akther, & Choudhuri, 2010). Low-density lipoprotein-cholesterol (LDL) level was estimated by Friedewald’s formula:

LDL= TC-( HDL + TG/5 )

2.6.2. Phosphofructokinase (PFK) activity in skeletal muscle

The PFK activity was assessed by mixing a reaction mixture containing 1 mM fructose-6-phosphate, 1 mM ATP, 0.5 mM NADH, 2 mU/mL aldolase, 2 mU/mL triosephosphate isomerase, 2 mU/mL glycerophosphate dehydrogenase in the assay buffer (50 mM Tris-HCl, 5 mM MgCl2, 5 mM (NH4)2SO4, pH 7.4) with the nucleus-free fraction (50 μg protein/mL) of skeletal muscle in a fifinal volume of 200 μL. NADH oxidation was then measured by monitoring the absorbance changes at 340 nm (Coelho, Costa, & Sola-Penna, 2007).

2.6.3. Citrate synthase (CS) activity

A reaction mixture for measuring CS activity was prepared by mixing 0.1 M Tris buffer (pH 8.0), 0.058 mM acetyl-CoA, and 0.1 mM 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB). The reaction was initiated by the addition of oxaloacetate (fifinal concentration: 0.5 mM). Absorbance at 412 nm was recorded every 30 s at 30 °C for 3 min (Carter, Rennie, Hamilton, & Tarnopolsky, 2001; Holloway, Bonen, & Spriet, 2009).

cistanche tubolosa health benefits

2.6.4. β-hydroxy acyl-coenzyme A dehydrogenase (β-HAD) activity

β-HAD activity was measured by mixing the reaction mixture containing 100 μM acetoacetyl-CoA and 100 μM β-NADH in assay buffer (100 mM potassium phosphate buffer with 2 mM ethylenediaminetetraacetic acid (EDTA), pH 7.3). The absorbance changes at 340 nm were monitored at 30 °C for 2 min (Holloway et al., 2006).

2.6.5. Carnitine palmitoyltransferase (CPT) activity

The assessment of CPT activity was based on CPT-catalyzed production of CoA-SH from palmityl-CoA. The reaction was initiated by adding L-carnitine (fifinal concentration 6 μM) to the reaction mixture (16 mM Tris, 2.5 mM EDTA, 2 mM DTNB and 50 μM palmitoyl-CoA, pH 8.0). The absorbance at 412 nm was monitored at 30 °C for 180 s. The millimolar extinction coefficient of 13.6 mM−1 cm−1 for 5’-thio-2-nitrobenzoate (endproduct) was used for the estimation of enzyme activity. One unit of CPT activity is defined as the amount of enzyme catalyzing the release of 1 nmol CoA-SH in 1 min (Karlic, Lohninger, Koeck, & Lohninger, 2002).

2.6.6. Mitochondrial respiration

The respiration rates in mitochondria isolated from mouse skeletal muscle were determined as described by Wong and Ko (2013). In brief, mitochondrial respiration was measured polarographically by a Clark-type electrode (Hansatech Instruments, Norfolk) at 30 °C. Mitochondrial fraction (1 mg protein/ mL) was incubated in the respiration buffer (30 mM KCl, 6 mM MgCl2, 75 mM sucrose, 1 mM EDTA, 20 mM KH2PO4, pH 7.0). Substrate solution containing 15 mM sodium pyruvate and 5 mM sodium malate was added. After equilibration, state 3 respiration was initiated by the addition of ADP (fifinal concentration 0.6 mM). When all of the added ADP was used up for ATP generation, oligomycin was added to induce the state 4 respiration. The respiratory control ratio (RCR) was estimated by calculating the ratio of state 3 to state 4 respiration rates (Jiang et al., 2009).

2.6.7. UCP3 expression

The UCP3 level was estimated by Western blot analysis using anti-UCP3 (E-18) antibody following SDS-PAGE analysis of the mitochondrial fraction, using a separating gel of 10% acrylamide. Mitochondrial fractions isolated from mouse skeletal muscle (100  g) were loaded and cytochrome c oxidase (COX) was used as a marker for reference. The immune-stained protein bands were analyzed by densitometry (Quantiscan, Biosoft, Cambridge, GB, UK), and the amounts (arbitrary units) of UCP3 were normalized with reference to COX level (arbitrary units) in the sample.

2.7. Protein assay

Protein concentration was determined using a Bio-Rad protein assay kit. Protein concentration was determined from a calibration curve using bovine serum albumin (BSA) as standard.

2.8. Statistical analysis

All data were expressed as mean ± standard error of the mean (SEM) unless otherwise specified. Data were analyzed by one-way analysis of variance (one-way ANOVA) and the inter-group difference was detected by Tukey range test when p < 0.05.

Cistanche herba

3. Results

3.1. Effects of HCF1 on body weight in ND-fed and HFD-fed mice

ND diet feeding caused a slight increase in body weight (by 13% and 10%, respectively) in male and female mice during the course of the 8-week experiment (Fig. 1a). HFD hastened the increase in body weight, with the extent of stimulation being 269% and 320%, respectively, in male and female mice, when compared with the respective ND animals (Fig. 1a). The changes in body weight during the course of the 8-week experiment were quantified and expressed in arbitrary units. HCF1 treatment completely suppressed the body weight gain in ND-fed male mice (Fig. 1b). HCF1treatment also dose-dependently suppressed the body weight gain in HFD-fed mice, with the degree of inhibition being 100% and 61%, respectively, at 45 mg/kg in both male and female mice (Fig. 1b). No significant change in the food consumption was observed upon HFD feeding and/ or HCF1 co-treatments when compared with the untreated NDfed mice (data not shown).

Fig. 1 – Effects of HCF1 on body weight changes in ND-fed and HFD-fed mice. The body weight was monitored weekly during the course of the 8-week experiment. (a) The time course of body weight changes was analyzed by a mixed-design ANOVA and the intergroup difference was detected by the Tukey range test. Data were expressed in percent control with respect to the respective initial body weight. (b) The body weight changes were quantified as described in Materials and Methods. Data were expressed in percent control with respect to the ND control [control AUC value (arbitrary unit): male = 777.6 ± 10.4; female = 742.0 ± 8.0]. Values given are means ± SEM, with n = 15. * Significantly different from the ND control; # Signifificantly different from the HFD control (p < 0.05).

3.2. Effects of HCF1 on fat pad indices in ND-fed and HFD-fed mice

The effects of HCF1 on fat accumulation were also examined. HFD induced significant increases in subcutaneous and visceral fat indices in male (by 346% and 325%, respectively) and female (by 248% and 257%, respectively) mice (Fig. 2). While HCF1 treatment did not produce any effect on subcutaneous and visceral fat in ND-fed mice, the ability of HCF1 to inhibit the HFD-induced gain in body weight was associated with the decrease in the body fat mass, as indicated by reductions in both subcutaneous and visceral fat in HFD-fed male (by 42% and 49%, respectively, at 45 mg/kg) and in visceral fat in HFDfed female mice (by 53%, at 45 mg/kg) (Fig. 2).

Fig. 2 – Effects of HCF1 on fat pad indices in ND-fed and HFD-fed mice. The mass of subcutaneous fat and visceral fat were measured as described in Materials and Methods. Data were expressed in percent control with respect to the ND control. Values given are means ± SEM, with n = 15. * Significantly different from the ND control; # Signifificantly different from the HFD control (p < 0.05).

3.3. Effects of HCF1 on plasma glucose and lipid contents in ND-fed and HFD-fed mice

HCF1 treatment did not alter plasma glucose and lipid contents in ND-fed male and female mice (Fig. 3). HFD feeding caused a significant elevation in plasma glucose level (by 39% and 29%, respectively) in male and female mice when compared with the respective ND control (Fig. 3a). HCF1 H reversed the HFD-induced elevation in plasma glucose level (by 41 and 64%, respectively) in male and female mice (Fig. 3a). HFD also significantly elevated plasma TG levels (by 38 and 53%, respectively) in male and female mice, when compared with the respective ND control (Fig. 3b). HCF1 L and HCF1 M significantly inhibited the HFD-induced elevation in plasma TG levels in HFD-fed male (by 135 and 192, respectively) and female (by 103 and 146%, respectively) mice (Fig. 3b). HCF1 H caused a significant elevation in plasma TG level (by 21%) in HFD-fed female mice when compared with the untreated HFD control (Fig. 3b). In addition to plasma TG, significant increases in plasma TC levels were also observed after HFD feeding, with the extent of increases being 73% and 100%, respectively, in male and female mice (Fig. 3c). The increases in plasma TC level were associated with a significant decline in plasma HDL/LDL ratio in both HFD-fed male (by 29%) and female (by 49%) mice (Fig. 3d). HCF1 M and HCF1 H reduced the HFD-induced increase in plasma TC level in HFD-fed male mice, with a concomitant elevation in the plasma HDL/LDL ratio (by 255% and 212%, respectively), when compared with the untreated HFD control (Fig. 3c and 3d). HCF1 L and HCF1 M suppressed the HFD-induced elevation in plasma TC level (by 22% and 32%, respectively) in female mice, with no change in the plasma HDL/ LDL ratio being observed (Fig. 3c and 3d).

Fig. 3 – Effects of HCF1 on plasma glucose and lipid contents in ND-fed and HFD-fed mice. Plasma glucose, TG and TC level were measured as described in Materials and Methods. Data were expressed in percent control with respect to the ND control [control plasma glucose level (mg/dL): male = 91.1 ± 3.3, female = 86.0 ± 2.3; control plasma TG level (mg/dL): male = 52.0 ± 1.7, female = 95.8 ± 4.1; control plasma TC level (mg/dL): male = 137.8 ± 4.3, female = 118.6 ± 5.4]. Values given are means ± SEM, with n = 15. * Signifificantly different from the ND control; # Signifificantly different from the HFD control (p < 0.05).

3.4. Effects of HCF1 on hepatic lipid contents in ND-fed and HFD-fed mice

HCF1 treatment did not change the hepatic TG and TC level in ND-fed mice (Fig. 4). The consumption of HFD significantly elevated hepatic TG levels (by 108% and 135%, respectively) in male and female mice, when compared with the respective ND control (Fig. 4a). HCF1 H, on one hand, suppressed the HFDinduced elevation in hepatic TG level (by 54%) in male mice, when compared with the untreated HFD control (Fig. 4a). On the other hand, HCF1 H further increased hepatic TG level (by 14%) in HFD-fed female mice, when compared with the respective untreated HFD control (Fig. 4a). HFD also caused significant increases in hepatic TC levels (by 48% and 26%, respectively) in male and female mice (Fig. 4b). While HCF1 L and HCF1 H significantly suppressed the HFD-induced elevation (by 42 and 44%, respectively) in hepatic TC levels in HFD-fed male mice, only HCF1 H could produce a similar effect on HFD-fed female mice, with the degree of inhibition being 62% (Fig. 4b).

Fig. 4 – Effects of HCF1 on hepatic lipid contents in ND-fed and HFD-fed mice. Hepatic TG and TC levels were measured as described in Materials and Methods. Data were expressed in percent control with respect to the ND control [control hepatic TG level ( g/mg protein): male = 31.7 ± 1.6, female = 38.5 ± 1.7; control hepatic TC level ( g/mg protein): male = 12.8 ± 0.4, female = 17.9 ± 1.2]. Values given are means ± SEM, with n = 15. * Significantly different from the ND control; # Signifificantly different from the HFD control (p < 0.05).

3.5. Effects of HCF1 on metabolic enzyme activities in skeletal muscle isolated from ND-fed and HFD-fed mice

HCF1 treatment stimulated the PFK activity in skeletal muscle of either ND-fed male or female mice, with the effect on male mice being more prominent (55% vs 20% increase at 45 mg/ kg) (Fig. 5a). The consumption of HFD caused significant suppressions in PFK activity (by 16% and 17%, respectively) in male and female mice (Fig. 5a). HCF1 H reversed the HFD-induced suppressions of PFK activity (by 153% and 100%, respectively) in HFD-fed male and female mice (Fig. 5a). Both HFD and HCF1 treatment produced no detectable effects on the CS activity in skeletal muscle of male or female mice when compared with the respective untreated ND control (Fig. 5b). HCF1 did not produce any effect on the β-HAD activity of skeletal muscle of ND-fed male or female mice (Fig. 5c). HFD feeding stimulated the β-HAD activity (by 47 and 21%, respectively) in male and female mice when compared with the untreated ND control (Fig. 5c). HCF1, at all tested doses, significantly inhibited the HFD-induced stimulation in β-HAD activity in male mice, with the degree of inhibition being 62%, when compared with the untreated HFD control (Fig. 5c). HCF1 treatment produced no significant alterations in β-HAD activity in ND-fed and HFD-fed female mice (Fig. 5c). While HCF1 H caused a significant decrease in the CPT activity (by 16%) of skeletal muscle in ND-fed male mice (Fig. 5d), HCF1 L and HCF1 M significantly increased the CPT activity by 24% and 22% in NDfed female mice when compared with the ND control (Fig. 5d). HFD feeding also increased the CPT activity (by 13% and 18%, respectively) in male and female mice. HCF1 reversed the HFDinduced increase in CPT activity, with the degree of suppression being 72% in male mice (at all tested doses) and 100% in female mice (HCF1 L) (Fig. 5d).

Fig. 5 – Effects of HCF1 on metabolic enzyme activities in skeletal muscle isolated from ND-fed and HFD-fed mice. Skeletal muscle PFK, CS, β-HAD and CPT activities were measured as described in Materials and Methods. Data were expressed in percent control with respect to the ND control [control PFK activity (mU/mg protein): male = 19.5 ± 1.1, female = 14.1 ± 0.7; control CS activity (mU/mg protein): male = 30.6 ± 1.3, female = 36.8 ± 3.6; control β-HAD activity (mU/mg protein): male = 13.5 ± 0.6, female = 16.8 ± 0.6; control CPT activity (mU/mg protein): male = 5.0 ± 0.3, female = 3.0 ± 0.1]. Values given are means ± SEM, with n = 15. * Signifificantly different from the ND control; # Signifificantly different from the HFD control (p < 0.05).

3.6. Effects of HCF1 on mitochondrial RCR in mouse skeletal muscle

HFD produced no detectable effect on mitochondrial RCR in mouse skeletal muscle (Fig. 6). HCF1 treatment-induced mitochondrial uncoupling in mouse skeletal muscle, as evidenced by significant reductions in mitochondrial RCR in both ND-fed (61–69% in male; 66% in female) and HFD-fed (73–78% in male; 67–70% in female) mice, when compared with the respective untreated animals (Fig. 6).

Fig. 6 – Effects of HCF1 on mitochondrial RCR in skeletal muscle of ND-fed and HFD-fed male and female mice. The mitochondrial RCR was measured as described in Materials and Methods. Data were expressed in percent control with respect to the ND control. Values given are means ± SEM, with n = 15. * Significantly different from the ND control; # Signifificantly different from the HFD control (p < 0.05).

3.7. Effects of HCF1 on UCP3 expression in mitochondria isolated from mouse skeletal muscle

The effects of HCF1 on UCP3 expression were also examined to explore the possible involvement of mitochondrial uncoupling in HCF1-induced weight loss. A 2-week treatment of HCF1 H significantly elevated UCP3 level in mitochondria isolated from mouse skeletal muscle in male mice, with the extent of stimulation being 67% when compared with the untreated control (Fig. 7).

Fig. 7 – Effects of HCF1 on UCP3 expression in mitochondria isolated from mouse skeletal muscle. Male mice were intragastrically administered with HCF1 H for 14 consecutive days. Mitochondria were isolated as described in Materials and Methods, and the UCP3 expression was measured by Western blot analysis. Data were quantified and expressed in percent control with respect to the untreated control. Values given are means ± SEM, with n = 4. * Significantly different from the untreated control.

3.8. Comparisons between HCF1 and CT on their effects on ND-fed and HFD-fed mice

To better understand the mechanism underlying the weight reduction effect afforded by HCF1, the effects of CT, a bile acid sequestrant, on ND-fed and HFD-fed male mice were also studied. Both CT and HCF1 completely inhibited the ND-induced weight gain during the 8-week course of the experiment. Both treatments also produced suppressive effects on the HFD-induced increase in body weight, visceral fat index, plasma glucose/TG/TC as well as hepatic TG/TC level, with the effect of CT being more prominent (Table 1). Unlike HCF1, which increased the plasma HDL/LDL ratio only in HFD-fed mice, CT significantly elevated plasma HDL/LDL ratio in both ND-fed and HFD-fed mice (by 58% and 17%, respectively), when compared with the respective untreated ND control (Table 1). In addition, while HCF1 increased the PFK activity in skeletal muscle of both ND-fed (54%) and HFD-fed (14%) male mice when compared with the untreated ND control, CT produced no detectable effect in ND-fed mice and could only restore the HFD-induced decrease in the PFK activity to the control value of ND-fed mice (Table 1). Both CT and HCF1 treatments reversed the HFD-induced increases in β-HAD (by 61% and 60%, respectively) and CPT (by 122% and 72%, respectively) activities in male mice (Table 1). While HCF1 induced mitochondrial uncoupling, as evidenced by significant decreases in mitochondrial RCR value in both ND- and HFD-fed male mice, CT produced no detectable effect on mitochondrial uncoupling (Table 1).

For more information, click here to get Part II

From: 'Cistanches Herba reduces the weight gain in high fat diet-induced obese mice possibly through mitochondrial uncoupling' by Hoi Shan Wong et al.

---journal of functional foods 10 (2014) 292–304