Animal diets: PicoLab Rodent Diet 20 model 5053 was used for normal diet (ND) (24% kcal from protein, 13.2% kcal from fat, and 62% kcal from carbohydrate; kcal per gram of diet was 3.02), and Research Diets Formula D12492 was used for high-fat diet (HFD) (20% kcal from protein, 60% kcal from fat, and 20% kcal from carbohydrate; kcal per gram of diet was 5.24) in the animal model experiments. ND was purchased from LabDiet (St. Louis, MO, USA), and HFD was purchased from Research Diets, Inc. (New Brunswick, NJ, USA).

Cell culture reagents: Gibco Dulbecco’s modified eagle medium (DMEM, high glucose), fetal bovine serum (FBS), heat-inactivated hours serum (HIHS) were purchased from Life Technologies Limited (Carlsbad, CA, USA). Sodium pyruvate (P) and sodium palmitate (SP) in were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and TCI (Shanghai) Development Co., Ltd. (Shanghai, China). Free fatty acid bovine serum albumin (FFABSA) was bought from Gold Biotechnology®, Inc. (St. Louis, MO, USA). Sigma-Aldrich-brand (Burlington, MA, USA) insulin was used.

Kits: Glucose Liqui-UV kit (Hexokinase method), Glucose Liquicolor® kit, Glycohemoglobin kit, Triglyceride Liquicolor®kit and Cholesterol Liquicolor® kit were purchased from Stanbio Laboratory (Boerne, TX, USA). LifeScan OneTouch Ultra blood glucose monitoring system was obtained from Johnson & Johnson company (Milpitas, CA, USA).

Herbal formulation: SSEM consisted of four component herbs, namely Cynomorii Herba, Schisandrae Fructus, Eucommia Ulmoides, and Momordica Fructus. Standardized herbal extracts, namely, Cynomorium Herba extract (4:1; water: ethanol, v/v), Momordica Fructus extract (4:1; water: ethanol, v/v), Schisandra extract (4:1; water: ethanol, v/v), and Eucommia Floret extract (4:1; water: ethanol, v/v), were purchased from Shaanxi Jiahe Phytochem Co., Ltd. (Xi’an, China). They were mixed in a specific ratio and formed the SSEM.

Male ICR mice (8 weeks old) were obtained from the Laboratory Animal Facility at the Hong Kong University of Science and Technology (HKUST). Animals were maintained under a 12-hour dark/light cycle at approximately 22 ̊C and allowed food and water ad libitum. The experimental protocol was approved by the Animal Ethics Committee at HKUST (2018-016).

Eight-week-old male ICR mice were randomly divided into two control groups and two co-treatment groups, with 6 - 13 mice per group. A previous study found that having five animals in each experimental group was adequate for detecting differences in endpoint measurements compared to the control group [13]. Each group was fed either normal diet (ND) control (n = 6), HFD control (n = 11), HFD + 0.21 g/kg SSEM (n = 13), or HFD + 0.63 g/kg (n = 12) SSEM formulation. The control groups were fed ND or HFD ad libitum, while SSEM formulation in the co-treatment groups was delivered via intragastric administration simultaneously with HFD feeding for 8 weeks. Control mice received vehicle only. The low dose of SSEM (0.21 g/kg) was the human equivalent dose. Body weight was measured once a week during the 8 weeks. At the end of the experiment, the mice were fasted overnight and sacrificed by cervical dislocation. Blood samples were then drawn using syringes containing 0.5% heparin in saline as an anticoagulant by cardiac puncture. Various fat pads, including posterior subcutaneous, epididymal, and mesenteric fat, were dissected and weighed. Samples of livers were excised and then subjected to further biochemical analysis. Biochemical analyses were done in a non-biased manner with established protocols. A blinding approach was not adopted in dosing, tissue processing, and endpoint readouts [13].

Food consumption per cage was measured weekly during the whole period of the experiment. Briefly, each cage was provided with 200 g of ND or HFD according to the assigned grouping. The weight of diet per cage consumed was calculated by (200-gram of diet left in the cage after a week). The food consumption (g of diet/mouse/day) = gram of diet per cage consumed/no. of mice in the cage/7. The endpoint food consumption of each group at week 8 (g of diet/mouse/day) was ND control = 5.48 ± 0.53; HDF control = 3.42 ± 0.19*; HFD + 0.21g/kg SSEM = 3.66 ± 0.79; HFD + 0.63 g/kg SSEM = 2.81 ± 0.17. If in terms of kcal of diet/mouse/day: ND control = 16.5 ± 1.60; HDF control = 17.9 ± 0.98; HFD + 0.21 g/kg SSEM = 19.2 ± 4.13; HFD + 0.63 g/kg SSEM = 14.7 ± 0.90.

Plasma samples were obtained by centrifuging the collected whole blood samples at 2000 × g for 10 min at 4˚C and then stored at 4˚C before use for biochemical analysis. Plasma glucose, cholesterol and triglyceride levels were determined using corresponding assay kits mentioned in Chemicals and Reagents section [13][14].

After 8 weeks of feeding, oral glucose tolerance test (OGTT) was performed. Briefly, mice were fasted for 6 hours and intragastrically administered glucose (2 g/kg). Blood samples were then collected at 15, 30, 60, and 120-minute intervals from the tip of the tail vein and used to measure blood glucose using blood glucose monitoring system. The extent of glucose tolerance impairment was estimated by computing the area under the curve (AUC) of the graph plotting glucose concentrations against time [13][14].

Whole blood samples from the mice were hemolyzed, and HbA1c was determined using an assay kit (Stanbio Laboratory Glycohemoglobin) [13][14].

Liver homogenates were prepared with phosphate buffered saline (without calcium and magnesium, PBS-A) in 10% (w/v). The lipid content was extracted by methanol/chloroform method. The extracted content was dissolved in isopropanol for lipid measurements with Stanbio Laboratory kits mentioned in Chemicals and Reagents section [13][14].

2.8.1. Reagents Preparation

SP was dissolved in 0.1M NaOH at 75 mM, then heated to 70˚C to dissolve. The intermediate stock of SP was then prepared with DMEM+ 1 mM P + 10% FFBSA in 5 mM. The working SP medium (0.75 mM) was prepared by dilution of intermediate stock with DMEM (+P) to induce insulin resistance in differentiated cells. Insulin was dissolved in 0.1 M HCl to make 100 μM stock solution. SSEM formulation was dissolved in DMSO to a stock concentration of 150 mg/mL.

2.8.2. C2C12 Myotubes

C2C12 mouse immortalized myoblast cell line was maintained in DMEM + 1 mM P + 10% HIFBS. Cell differentiation was initiated by differentiation medium containing DMEM + 1 mM P + 2% HI Heat-inactivated Horse Serum (HG2%HIHS). Then the working concentration of SSEM was prepared and diluted with medium (finally each group containing 0.2% DMSO (v/v)). The non-toxic concentration for SSEM was determined by crystal violet toxicity test with 24 h incubation, and the assay result indicated that 300 μg/mL of SSEM was non-toxic.

2.8.3. Glucose Uptake Assay

The C2C12 myotubes (passage 14 - 24 were used in this study) were seeded and grown in 6-well tissue culture plates for 3 days before being treated with HG2%HIHS over 4 days to differentiate the myoblasts into myotubules. The cells were dispensed into different treatment groups, namely, SSEM (0, 3, 10, 300 μg/mL), SSEM (0, 3, 10, 300 μg/mL) + SP (0.75 mM), SSEM (0, 3, 10, 300 μg/mL) in the presence of insulin, and SSEM (0, 3, 10, 300 μg/mL) + SP (0.75 mM) in the presence of insulin. All groups were first incubated with either SSEM only or SSEM + SP for 18 hours. After 18 hours, cells were washed with PBS-A before changing the glucose uptake assay medium. Insulin and non-insulin groups were then incubated with 100 nM of insulin (in DMEM without glucose, P and phenol red, all no DMEM) or all no DMEM, respectively for 10 minutes. An aliquot (4.8 μL) of 2-NBDG (stock: 25mM in DMSO, then 60 μM working) was then added to the wells of all samples before incubating for another 50 min at 37˚C in the dark. Samples were washed thrice with PBS-A, and trypsinized (with 400 μL 0.25 mM Trypsin with 0.53 mM Na2EDTA in PBS-A) at 37˚C for 10 minutes. The reaction was stopped with 300 μL PBS-A, and samples were collected into centrifuge tubes. Aliquots (200 μL) of the samples were put into wells of a black microplate, and then glucose uptake was analyzed with the Flex Station 3 plate reader (read three times, excitation at 468 nm, emission at 540 nm).

Protein content was measured to standardize the glucose uptake readings. Centrifuged the leftover of collected samples in 20,000 × g for 5 min, then removed the supernatant. The cell pellet was lysed with 200 μL 0.1% (v/v) TritonX in PBS-A and then with 10 min sonication. The cell debris was precipitated by centrifugation at 20,000 × g for 5 min. The supernatant was used for protein measurement by Braford assay.

The effect of SSEM on glucose uptake in myotubules was then estimated. One-way ANOVA with LSD test was performed to detect significant differences in glucose uptake in different experimental groups (0 μL SSEM, 0 μL SSEM in the presence of insulin, 0 μL SSEM + SP in the presence of insulin), as compared with the respective control group.

Data were expressed as the mean ± standard deviation (SD). Unless otherwise specified, data from different groups at the same time point were analyzed by one-way analysis of variance (one-way ANOVA), and intergroup differences were detected by post hoc LSD tests, with a value of P < 0.05.

Weekly body weight measurements were taken over the 8-week experimental period. As presented in Figure 1, HFD-fed mice demonstrated a substantial (31.7%) and statistically significant increase in body weight compared to the incremental gain (8.33%) in ND-fed mice. Crucially, SSEM co-treatment, administered at either 0.21 g/kg or 0.63 g/kg, significantly mitigated the body weight escalation in HFD-fed mice throughout the 8-week study.

Figure 1.Effect of SSEM on body weight in HFD-fed mice. Data were expressed as a percentage of initial body weight. Each data point represents mean ± standard deviation (SD), with n = 6 - 13 for each group. *Significantly different from the ND control; # Significantly different from the HFD control.

SSEM co-treatment (0.21 or 0.63 g/kg) completely prevented HFD-induced weight gain during the whole course of the experiment, with no significant difference when compared with that of ND-fed mice. There was no dose-dependent effect of SSEM on the body weight in HFD-treated mice, with significant suppression of HFD-induced weight gain by 76.9 and 80.5% at doses of 0.21 and 0.63 g/kg, respectively, by the end of the 8^th week.

The HFD-induced body weight gain was independent of food consumption volume, given that HFD mice consumed less food than their ND counterparts. Additionally, SSEM administration did not alter feeding behavior in HFD mice (data not shown), indicating its efficacy in mitigating weight gain is driven by metabolic modulation rather than appetite suppression.

HFD feeding markedly increased the posterior subcutaneous, epididymal, and mesenteric fat indices by 290, 282, and 365%, respectively. In HFD-fed mice, SSEM treatment significantly reduced posterior subcutaneous fat and visceral fat (epididymal plus mesenteric) by 67.1 and 66.7 at 0.21 g/kg, and by 58.7 and 47.8% at 0.63 g/kg, respectively, resulting in overall reductions in total fat mass of 66.9 and 51.1% (Figure 2).

Figure 2.Effect of SSEM on adiposity in HFD-fed mice. Posterior subcutaneous fat, epididymal fat, and mesenteric fat were weighed. Values given are means ± SD, with n = 6 - 13. *Significantly different from the ND control; #Significantly different from the HFD control.

The effect of SSEM co-treatment on glucose tolerance in HFD-fed mice was assessed via an OGTT performed at the 8-week mark of the experiment.Figure 3 illustrates that HFD feeding led to substantial glucose intolerance compared to ND-fed controls. Crucially, SSEM co-treatment achieved a complete reversal of this diet-induced glucose intolerance. The AUC values in HFD-fed mice receiving SSEM were reduced to 70.8 and 72.7% of the untreated HFD group at SSEM doses of 0.21 and 0.63 g/kg, respectively, with the values not being different from that of the ND group.

Figure 3. Effects of SSEM on HFD-induced glucose intolerance in mice. Glucose tolerance (estimated by AUC) was expressed as a percentage of ND-fed mice as a control. Each data point represents mean ± standard deviation (SD), with n = 6 - 13. The value for glucose tolerance (AUC) in the ND control was 1364 ± 217. *Significantly different from the ND control; #Significantly different from the HFD control.

We investigated the long-term effect of SSEM on blood glucose control in high-fat diet (HFD)-fed mice by measuring HbA1c levels after an 8-week experimental period. While HFD feeding induced a substantial 35.8% increase in HbA1c compared to normal diet (ND) controls, co-administration of SSEM at daily doses of 0.21 or 0.63 g/kg failed to ameliorate this hyperglycemia, as HbA1c levels remained unchanged in SSEM-treated HFD mice (Table 1).

Table 1.Effects of SSEM on fasting blood/plasma glucose and lipid contents in HFD-fed mice. Data were expressed as a percentage of ND-fed mice. Each data point represents mean ± SD, with n = 6 - 13. The ND control value for HbA1c (%) was 3.10 ± 0.31; plasma glucose (mg/dL) was 76.3 ± 5.19; plasma TG (mg/dL) was 34.7 ± 2.86; plasma TC (mg/dL) was 86.2 ± 5.69; plasma HDL-C (mg/dL) was 57.5 ± 2.92; plasma LDL-C (mg/dL) was 38.8 ± 34.2 and TC/HDL-C ratio was 1.50 ± 0.10. *Significantly different from the ND control; ^#Significantly different from the HFD control.

Fasting plasma glucose levels were also measured at the end of the 8-week experimental period. Table 1 reveals that HFD feeding significantly elevated fasting plasma glucose by 42% in mice compared to the ND group. Notably, SSEM co-treatment (0.21 or 0.63 g/kg) exacerbated this effect, leading to further increases of 64% and 63%, respectively, above the levels observed in ND-fed mice.

HFD feeding significantly elevated plasma lipid profiles, increasing TG, TC, HDL-C, and LDL-C levels by 47% - 124%. Treatment with a high dose of SSEM (0.63 g/kg) effectively attenuated the HFD-induced elevation in TG while further increasing HDL-C levels. Consequently, SSEM administration improved the TC/HDL-C ratio, mitigating the elevated ratio observed in the HFD-control group (Table 1).

HFD feeding significantly increased hepatic TC by approximately 195% compared to the ND control. SSEM treatments 0.21 g/kg and 0.63 g/kg effectively counteracted this HFD-induced TC elevation, reducing it by 44.2 and 36.5%, respectively (Figure 4(a)), to a similar extent when compared to untreated HFD mice. HFD also markedly raised hepatic TG levels by 175%. In HFD-fed mice, 0.21 and 0.63 g/kg SSEM treatments significantly reduced these elevated hepatic TG levels by 25.3 and 25.9%, respectively (Figure 4(b)).

(a) (b)

Figure4. Effects of SSEM on hepatic lipid contents in ND-fed and HFD-fed mice. Hepatic TG and TC level were measured. Data were expressed in percent control with respect to the ND control [control hepatic TC level (μg/mg protein): 0.68 ± 0.03; control hepatic TG level (μg/mg protein): 2.81 ± 0.24]. Values given are means ± SD, with n = 6 - 13. *Significantly different from the ND control; #Significantly different from the HFD control.

To investigate the cellular mechanism by which SSEM ameliorates HFD-induced glucose intolerance, we examined glucose uptake in differentiated C2C12 myotubules. Insulin resistance was experimentally induced using SP, a free fatty acid, to replicate HFD conditions within skeletal muscle cells. Figure 5 reveals that SSEM treatment alone had no significant impact on glucose uptake in control myotubules. In contrast, co-incubation with SP and SSEM at concentrations of 100 and 300 μg/mL significantly enhanced glucose uptake by 18.6 and 26.7%, respectively, relative to SP-treated cells. When insulin was added, overall glucose uptake increased by 73.3% compared to the non-insulin control. Importantly, SSEM co-incubation in the presence of insulin consistently boosted glucose uptake by 12% - 14% at all tested concentrations, relative to the insulin control. The incubation of SP alone, however, significantly attenuated insulin-stimulated glucose uptake by 32.3%. Notably, SSEM demonstrated a dose-dependent reversal of SP-induced suppression. At 30 and 100 μg/mL SSEM, a partial reversal of SP’s inhibitory effect was observed, while at 300 μg/mL SSEM, the effect was fully reversed.

Figure 5.Effect of SSEM on glucose uptake in SP-induced insulin resistance in myotubules. Data were expressed as percentages of initial control values (0 μg/mL SSEM). Each bar represents mean ± standard error (SEM), with n = 6. The value of glucose uptake in the initial control was 150 ± 4.70 signal/mg protein. *Significantly different from the initial control; #Significantly different from the SP control. $Significantly different from insulin control. ^Significantly different from the SP insulin control.