Obesity is a risk factor for metabolic disorders, with its prevalence being increased in the world over the past several decades. Therapeutical interventions for obesity are thus urgently needed. In the present study, we investigated the effect of long-term treatment (0.51 and 5.1 g/kg/day, 5 days per week for a total of 40 doses) with an herbal formula MCC [which comprises the fruit of Momordica charantia (MC), the pericarpium of Citri reticulata and L-carnitine] in normal diet (ND) and high fat diet (HFD)-fed female ICR mice. Body weight change was monitored during the course of the experiment. Fat pad indices, plasma glucose and lipid contents, as well as metabolic enzyme activities and mitochondrial coupling efficiency in skeletal muscle were measured at 24 hours after the last dosing. Results showed that HFD increased the body weight, fat pad indices, plasma glucose and lipid contents as well as β-hydroxyacyl-Co A dehydrogenase (β-HAD) and carnitine palmitoyl CoA transferase (CPT) activities in skeletal muscle. However, the phosphofructokinase (PFK) activity was decreased in skeletal muscle. MCC treatment reduced the HFD-induced increases in body weight, fat pad indices and plasma lipid contents. MCC treatment only partially reversed the HFD-induced changes in β-HAD and CPT activities, but did not restore the HFD-induced decrease in PFK activity. MCC did not alter the plasma glucose level and mitochondrial coupling efficiency in skeletal muscle of ND and HFD-fed mice. Since MCC formula did not increase activities of energy metabolic enzymes or induce mitochondrial uncoupling, the weight loss effect of MCC is likely related to the reduction of intestinal lipid absorption in HFD-fed mice.
Obesity has become an overt worldwide epidemic. In 2005, 1.1 billion adults as well as 10% of children were regarded as overweight or obese over the world [
To mobilize the lipid reservoir in the body, skeletal muscle tissue plays a crucial role, wherein 90% of the energy expenditure of skeletal muscle is obtained from fatty acid oxidation, particularly in resting state [
In an attempt to develop an effective, palatable and safe intervention for obesity, herbal medicine, which has a long history of use in different cultures for weight reduction, may offer a promising prospect. Recently, a growing body of evidence has been accumulated showing the effectiveness of herbal medicine in controlling obesity. As such, the fruit of Momordica charantia (MC, also called bitter melon) was found to attenuate the high calorie diet-induced hypertrophy of adipose tissue in rodents [12,13], presumably through the down-regulation of lipogenic gene expression [
In the present study, we sought to evaluate the effect of MCC, an herbal formula comprising MC, CR and L-carnitine, on high fat diet-induced obesity in female ICR mice. The body weight, visceral (gonadal and mesenteric) and subcutaneous fat pads, plasma glucose level, plasma lipid contents [triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were measured. To examine the metabolic status of skeletal muscle, activities of myocellular phosphofructokinase (PFK), β-hydroxyacylCo A dehydrogenase (β-HAD), carnitine palmitoyl CoA transferase (CPT) and citrate synthase (CS) were also measured. To assess the mitochondrial functional status, state 3 and state 4 respiratory rates were measured using mitochondria isolated from skeletal muscle of mice. Coupling efficiency was estimated by computing the ratio of state 3 to state 4 mitochondrial respiratory rates.
PicoLabÒ Rodent diet 20 (normal diet) was purchased from LabDietÒ (City, State, USA). The “Original” High Fat Diet (Diet-induced Obesity Formula D12492, 60% energy from fat) was purchased from Research Diets, Inc. (New Brunswick, NJ, USA). TG and cholesterol assay kits were purchased from Wako Pure Chemical Industries, Ltd. (Okasa, Japan). HDL-C test kit was purchased from Wako Diagnostics (Richmond, VA, USA). Glucose assay reagent was obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.
MCC is mainly comprised of water extracts of MC (42%, w/w) and CR (14%) as well as L-carnitine (8%). The commercial preparation was manufactured and supplied by Infinitus (China) Company Ltd., Guangzhou, China.
Adult female ICR mice (8 - 10 weeks old, 20 - 25 g) were maintained under a 12-hour dark/light cycle at about 22˚C, and allowed food and water ad libitum in the Animal and Plant Care Facility at the Hong Kong University of Science and Technology (HKUST). All experimental protocols were approved by the University Committee on Research Practice at HKUST.
ICR mice were randomly assigned to 6 groups, with 10 - 15 mice in each: 1) Normal diet (ND, 13% energy from fat) control, 2) ND + low dose MCC (0.51 g/kg), 3) ND + high dose MCC (5.1 g/kg), 4) High fat diet (HFD, 60% energy from fat) control, 5) HFD + low dose MCC (0.51 g/kg) and 6) HFD + high dose MCC (5.1 g/kg). The low dose was estimated from the recommended oral dose for human usage after adjusting for the interspecies difference in drug distribution in the body [
Plasma samples were obtained by centrifuging whole blood samples at 1500 × g for 10 minutes at 4˚C. Plasma samples were then subjected to biochemical analysis.
Minced gastrocnemius muscle tissues were mixed with 10 mL collagenase solution [0.075% (w/v) in buffer], and the mixtures were incubated at 4˚C for 20 min. The digested tissue mixtures were centrifuged at 600 × g at 4˚C for 20 min. After removing the supernatant, the digested tissues were mixed with 20 mL of ice-cold homogenizing buffer (100 mM KCl, 50 mM MOPS, 10 mM EGTA, pH 7.2) and subjected to homogenization with a Teflon-glass homogenizer at 4,000 rpm for 25 - 30 complete strokes. Then the homogenates were centrifuged at 600 × g for 10 min at 4˚C. The resultant supernatant was nucleus-free fraction. For measurements of β-HAD, CS and CPT activities, nucleus-free fraction was diluted with 0.2% Triton X-100 (w/v in K2HPO4 buffer) [18,19].
Mitochondrial pellets were prepared from muscle homogenates by centrifugation at 9200 × g at 4˚C for 30 min. The mitochondrial pellets were then resuspended in a buffer containing 250 mM sucrose, 50 mM Tris, pH 7.5 [20,21].
Body weight of mice was measured once a week during the 8-week course of experiment. Gonadal, mesenteric and subcutaneous fat pads were weighed. The ratio of a particular fat pad weight to body weight was estimated and expressed as fat pad index [22,23].
Plasma glucose levels were measured using an assay kit basing on coupled hexokinase-catalyzed and glucose- 6-phosphate dehydrogenase-catalyzed reactions, with a resultant NAD reduction. Absorbance changes at 340 nm were monitored spectrophotometrically by Victor3 Multi- label Counter (Perkin Elmer, Turku, Finland).
Plasma levels of TG, HDL-C and TC levels were measured using assay kits. In brief, TG levels were measured spectrophotometrically by an enzymatic method using the quantitative production of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAOS, a blue pigment) via coupled reactions. HDL-C was measured spectrophotometrically by the method of phosphotungstate-magnesium salt precipitation. TC levels were measured by an enzymatic colorimetric method basing on the quantitative production of DAOS from coupled reactions. In the above assays, their end products were measured by monitoring the changes in absorbance at 600 nm. LDL-C level was estimated by Friedewald’s formula: LDL = TC - (HDL-C + TG/5) [
PFK activity was measured with a reaction cocktail containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 5 mM (NH4)2SO4, 1 mM fructose-6-phosphate, 1 mM ATP, 0.5 mM NADH, 2 mU/mL aldolase, 2 mU/mL triose phosphate isomerase, 2 mU/mL a-glycerophosphate dehydrogenase and 50 mg/mL of protein (muscle nucleusfree fraction) in a final volume of 200 mL. The reaction was initiated by the addition of nucleus-free fraction. NADH oxidation was measured by monitoring the changes in absorbance at 340 nm [
b-HAD activity was measured in 100 mM potassium phosphate buffer with 2 mM EDTA, pH 7.3. The reaction was started by the addition of 100 mM acetoacetyl-CoA and 100 mM β-NADH, and absorbance changes at 340 nm were monitored for 2 min at 30˚C [
A reaction cocktail 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 dithionitrobenezene (DTNB). The reaction was initiated by the addition of oxaloacetate (final concentration: 0.5 mM). Absorbance at 412 nm was recorded every 30 s for 3 min at 30˚C. The CS activity is also considered as an indirect measure of mitochondrial content in muscle tissue [26,27].
To measure the CPT activity, the reaction was initiated by adding L-carnitine (final concentration 6 mM) to the reaction mixture (16 mM Tris, 2.5 mM EDTA, 2 mM DTNB and 50 mM palmitoyl-CoA, pH 8.0). The absorbance at 412 nm was monitored for 180 s at 30˚C. The molar extinction coefficient of 13,600 (mol/L)−1cm−1 for 5’-thio-2-nitrobenzoate (end-product) 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 CoASH per min [14,28-30].
Mitochondrial respiratory was measured polarographically by a Clark-type oxygen electrode (Hansatech Instruments Ltd., Norfolk, UK) at 30˚C. Mitochondrial fraction (~0.5 mg protein/mL) was incubated in a buffer containing 30 mM KCl, 6 mM MgCl2, 75 mM sucrose, 1 mM EDTA, 20 mM KH2PO4 and 0.1% (w/v) fatty acidfree BSA, pH 7.0. Substrate solution containing 10 mM glutamate and 2.5 mM malate was added, and after a stable state 2 respiration had been established, state 3 respiration (coupling) was initiated by the addition of ADP (final concentration 0.6 mM). When all of the added ADP was used up for ATP generation, oligomycin (ATP synthase inhibitor) was added to induce the state 4 respiration (uncoupling). The respiratory control ratio (RCR) was estimated by calculating the ratio of state 3 to state 4 respirations [21,31,32].
Data were analyzed by one-way Analysis of Variance (ANOVA). Post-hoc multiple comparisons were performed using Least Significant Difference (LSD). P values < 0.05 were regarded as statistically significant.
The body weight of ND-fed mice was increased by 9% during the course of 8-week experiment. MCC treatment at the high dose (i.e., 5.1 g/kg/day) slightly but not significantly suppressed the body weight increase in ND-fed mice. HFD accelerated the body weight increase during the course of experiment, with the extent of stimulation being 219%, when compared with that of the ND-fed mice (
The slight reduction in body weight increase afforded by MCC treatment (5.1 g/kg/day) in ND-fed mice was associated with the suppression of total fat pad index, with the extent of suppression being 66% (
Treatment with MCC did not alter the plasma glucose level and lipid contents in ND-fed mice, except a reduction of plasma TG (25%) at the dose of 5.1 g/kg/day. HFD feeding increased plasma glucose (30%), TG (41%), HDL-C (58%) and LDL-C (124%) levels as well as plasma LDL/HDL ratio (54%) (
To evaluate the effect of MCC on the energy metabo-
Lism status of skeletal muscle, activities of PFK (the rate-limiting enzyme of glycolysis), b-HAD as well as CPT (two essential enzymes of fatty acid b-oxidation) and CS (an indirect measure of mitochondrial content in muscle tissue) were measured. Treatment with MCC (0.51 and 5.1 g/kg/day) suppressed the activities of bHAD (13% and 15%, respectively) and CPT (22% and 14%), but did not change the activities of PFK and CS in normal-diet fed mice. HFD feeding caused a decrease in PFK activity (19%) but increases in β-HAD (14.6%) and CPT (27.5%) activities. Treatment with MCC did not change PFK and CS activities in HFD-fed mice. While MCC treatment reversed the HFD-induced increases in activity of CPT (by 44% and 110%) in a dose-dependent manner, MCC treatment (5.1 g/kg/day) suppressed the activity of β-HAD (96%) in HFD-fed mice (
Coupling efficiency was indirectly assessed by estimating the ratio of state 3 to state 4 mitochondrial respiratory rates. HFD feeding did not affect coupling efficiency (data not shown). MCC treatment did not produce any detectable changes in the coupling efficiency in both ND and HFD-fed mice (data not shown).
In the present study, the HFD-induced body weight increase was associated with increases in subcutaneous and visceral fat pad indices, indicative of adipocyte hypertrophy in HFD-fed mice. In addition, HFD feeding increased plasma glucose and lipid contents, as well as decreased PFK activity in skeletal muscle of mice. In contrast, β-HAD and CPT activities were increased in skeletal muscle of HFD mice. As an excessive accumulation of visceral adipose tissue, but not subcutaneous abdominal fat, has been found to be associated with metabolic abnormalities in overweight/obese patients [
of intensive research. It has been shown that visceral adipose tissue, which secretes adipokines, adiponectins and cytokines, can lead to insulin resistance and early stages of inflammation, thrombosis and hypertension [
The herbal formula MCC was found to suppress the body weight increase and decrease fat pad indices in HFD-induced obese mice. Given the predominant role of skeletal muscle tissue in mobilizing the lipid reservoir in the body [
Overweight/obesity is found to be associated with metabolic syndrome (reviewed in [