Introduction
Type 2 diabetes mellitus (T2DM) is one of the most common metabolic diseases worldwide [
1,
2]. The pathophysiology of T2DM includes disorders of carbohydrate, lipid, and protein metabolism resulting from defects in insulin secretion and action [
2,
3]. Insulin resistance leads to the disruption of glucose homeostasis, which increased glucose production in the liver and reduced glucose uptake in skeletal muscle and adipose tissue [
2,
4]. Fasting and postprandial hyperglycemia cause secondary complications in various organs, such as the heart, brain, kidneys, and eyes [
3‐
5]. Target drugs for diabetes have been developed, but their side effects and disadvantages are still controversial [
6]. Therefore, it is essential to research using natural functional materials with low side effects for diabetes.
Skeletal muscle, a significant determinant of resting metabolic rate, accounts for 40%–50% of the total body weight [
7] and consumes approximately 80% glucose stimulated by insulin [
8‐
10]. An essential function of the muscle is to increase glucose transport activity via the insulin signaling cascade [
11]. The binding of insulin to the insulin receptor induces insulin receptor substrate (IRS)-1 tyrosine phosphorylation. This results in the activation of Akt phosphorylation, which facilitates the translocation of glucose transporter type 4 (GLUT4) and subsequent glucose uptake into muscle cells [
12,
13].
Insulin resistance decreases protein synthesis and increases protein degradation, leading to muscle atrophy and sarcopenia [
14,
15]. In skeletal muscle, impaired mitochondrial function causes lipid oxidation, resulting in insulin resistance [
16]. AMP-activated protein kinase-α (AMPKα), a central regulator of mitochondrial biogenesis, controls intracellular energy balance and is an essential factor in skeletal muscle atrophy [
17,
18]. Deacetylation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) plays an essential role in mitochondrial biogenesis, which is regulated by sirtuin-1 (SIRT1) [
19]. In addition, hyperglycemia-induced oxidative stress contributes to the reduction in insulin action [
20]. The abnormal cell signaling resulting from a decreased ability to eliminate oxidative stress can lead to insulin resistance [
21]. Furthermore, a persistent state of oxidative stress induces apoptosis [
22], which in itself can also cause muscle atrophy [
23,
24].
Stevia (
Stevia rebaudiana) is a natural sweetener used as a substitute for sucrose worldwide [
25]. Stevia leaves have been used as tea and medicines in many parts of the world for centuries [
25‐
28]. Several steviol glycosides, which are four-ring diterpenes, such as stevioside (SV), rebaudioside A, D, C, D, and E, dulcoside A and B, have been identified from stevia leaves [
29]. This non-caloric sweetener passes through the body without being metabolized, which is why it is favored bydiet-conscious consumers and can be effectively used to treat diabetes, obesity, and cardiovascular disease [
25]. SV, an abundant glycoside found in stevia leaves, is 300 times sweeter than sugar [
25,
30]. Several studies have reported the therapeutic benefits of stevia and SV, lowering blood sugar, cholesterol, and triglyceride levels and exhibiting [
31‐
34], antioxidant, anti-inflammatory, and anti-cancerous properties [
35‐
37]. In addition, in vivo studies have investigated the antidiabetic effect of SV, suggesting alleviation of hyperglycemia [
33,
38]. However, few studies have focused on whether stevia and SV improve insulin resistance in skeletal muscle, essential for insulin-stimulated glucose absorption. Therefore, in this study, we investigated the effect of stevia extract (SE) and its glycoside, SV, on insulin resistance and improvement mechanism in skeletal muscle in diabetic
db/db mice.
In this study, we used
db/db mice with severe obesity caused by leptin receptor defects to model T2DM [
39]. Metformin, a T2DM medication, was used as a positive control (PC) verifying a significant reduction in blood glucose in mice [
40]. This study shows that the effect of SE and SV administration on insulin resistance in skeletal muscle of diabetic mice will occur through the regulation of mitochondrial function and oxidative stress, and suggests that SE and SV administration may improve diabetic muscles compared to metformin administration.
Materials and methods
Materials
Stevia rebaudiana was registered in plant variety protection right as grant No. 6485 by Korea Seed & Variety Service in 2017, according to Plant Variety Protection Act (application No. 2015–257). Experimental research on plants was performed following the relevant guidelines and legislation [
41]. Lyophilized powder of stevia leaves was obtained from Pharminogen Co. (Yongin, Kyunggi-do, Korea). Briefly, stevia leaves were dried and extracted with water at 100 °C for 3 h using a heating mantle. Next, the extract was filtered through 500 mesh filter and concentrated using vacuum evaporation. The concentrate was lyophilized to remove moisture entirely and stored at -20 °C in a powder. Stevioside (C
38H
60O
18) was purchased from ChemFaces Biochemical Co. Ltd. (Wuhan, China).
Animal studies
Eight-week-old BKS.Cg-Dock7
m + / + Leprdb/J (Heterozygous for Dock7
m/ Heterozygous for
Leprdb) (
db/m + , negative controls) and
db/db mice (BKS.Cg-Dock7
m + / +
Leprdb/J, homozygote) were obtained from Jackson Laboratories (Sacramento, CA, USA). Mice were allowed to adjust to a controlled environment with a 12 h light/dark cycle at 20–25 °C for two weeks. Following the acclimatization period, six non-diabetic mice (
db/m +) were assigned to the control group, and 30 diabetic mice (
db/db) were randomly divided into six groups as follows: (1) Negative control (N +) group, non-diabetic mice + saline; (2) Normal control (NC) group, diabetic mice + saline; (3) PC group, diabetic mice + metformin (200 mg/kg BW, p.o.); (4) SE200 group, diabetic mice + SE (SE, 200 mg/kg BW, p.o.); (5) SE500 group, diabetic mice + SE (500 mg/kg BW, p.o.); (6) SV group, diabetic mice + stevioside (SV, 40 mg/kg BW, p.o.). Saline, metformin, SE, and SV were administered by oral gavage daily for 35 days (Figure S
1). Mice were weighed twice a week. All animal experiments were performed following the Guide for the Care and Use of Laboratory Animals and were approved by Gachon University (GI-ACUC-R2020012).
Fasting blood glucose levels (FBGLs), oral glucose tolerance test (OGTT), and insulin tolerance test (ITT)
Mice fasted for 3 h and the FBGLs were measured using Accu-Chek Performa (Roche Diagnostics Korea Co., Ltd., Seoul, Korea). Blood was collected from the tail vein of each mouse. On day 21 of administration, overnight-fasted mice were orally administered glucose (Dai Han Pharm. Co. Ltd., Seoul, Korea) at a concentration of 2 g/kg BW. Blood glucose levels were measured at 0, 15, 30, 60, and 120 min after oral glucose gavage. In addition, ITT was performed on day 35 in mice following fasting for 3 h. Mice were administered an intraperitoneal injection of Humulin (1 U/kg BW; Eli Lilly, Indianapolis, IN, USA) and the blood glucose level was checked at 0, 15, 30, and 60 min. Blood glucose and insulin tolerance levels were determined using the area under the curve (AUC).
Biochemical analysis
Serum triglyceride (TG) and total cholesterol (TC) levels were determined using commercial kits (Asan Pharm, Seoul, Korea), and serum insulin level was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Thermo Fisher, Waltham, MA, USA). The homeostatic model assessment for insulin resistance (HOMA-IR) was calculated using the following formula:
\(\mathrm{HOMA}-\mathrm{IR}=\{\mathrm{fasting insulin}\left[\frac{\mathrm{U}}{\mathrm{mL}}\right]\times \mathrm{fasting glucose}\left[\frac{\mathrm{mg}}{\mathrm{dL}}\right]\}/405\) [
42]. The concentration of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in mouse serum was measured using Bluegene ELISA kits (Shanghai Bluegene Biotech Co. Ltd., Shanghai, China). The biochemical experiments were conducted according to the manufacturer’s recommendations.
To determine malondialdehyde (MDA) levels, liver homogenates dissolved in 1.15% KCl (Sigma-Aldrich, St Louis, MO) were obtained from one portion of 0.05 g liver of each mouse. Then, the samples were blended with a mixed solution containing 8.1% sodium dodecyl sulfate (SDS) (iNtRON Biotechnology, Seongnam, Korea) 200 μL, 20% acetic acid (Daejung Chemical, Suwon, Korea), 1.5 mL 0.8% thiobarbituric acid (TBA) (Sigma-Aldrich, St Louis, MO) 1.5 mL, distilled water 700 μL. A 5 ml volume of n-butanol (SAMCHUN, Gyeonggi-do, Korea) and 1 ml volume of distilled water were added to mixtures, followed by incubating in a 95 °C water bath for 30 min and ice for 10 min. After centrifuging at 4,000 rpm for 10 min, MDA levels of each supernatant were measured at 532 nm.
Hematoxylin and eosin (H&E) staining and analysis of the cross-sectional area of skeletal muscle fibers
Skeletal muscle tissue samples of 3 mice in each group were fixed in 10% formalin (Sigma-Aldrich, St. Louis, MO, USA). The samples were embedded in paraffin and stained with H&E. Stained sections were visualized using an Olympus Provis AX70 microscope (Olympus, Tokyo, Japan). To analyze muscle fiber size, the cross-sectional area of the skeletal muscle in mice was calculated using ImageJ software (rsb.info.nih.gov/ij).
Western blot analysis
Gastrocnemius tissue (20 mg) was homogenized in PRO-PREP™ solution (iNtRON Biotechnology, Seongnam, Korea) containing a phosphatase inhibitor (Thermo Fisher) and incubated for 30 min on ice. Following lysis, the tissue suspension was centrifuged at 13,000 × g for 5 min at 4 °C to obtain the supernatant. Nuclear and cytoplasmic fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher) according to the manufacturer’s instructions. Protein concentrations were measured at 595 nm using the PRO-MEASURE™ solution (iNtRON Biotechnology). Concentrations were calculated according to the manufacturer’s instructions.
Equal amounts of total protein (30 μg) were electrophoresed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% skim milk for 1 h and incubated with the following primary antibodies: p-Akt, Akt, p-IRS, IRS, p-AMPKα, AMPKα, citrate synthase (CS) (Cell Signaling Technology, Beverly, MA, USA, 1:1000), SIRT1, 4-hydroxynonenal (4-HNE), Bax, Bcl-2 (Abcam, Cambridge, MA, USA, 1:1000), Lamin B1 (Abcam, 1:10,000), α-tubulin (Abcam, 1:5000), heme oxygenase 1 (HO-1), SOD, GPx (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:200), PGC-1α (Bioss Antibodies, Woburn, MA, USA, 1:1000), and GLUT4 (Thermo Fisher, 1:1000). After incubation with secondary antibodies for 1 h, specific bands were visualized using an enhanced chemiluminescence method (iNtRON Biotechnology) and a Quant LAS 500 system (GE Healthcare Bio-Sciences AB, Björkgatan, Uppsala, Sweden).
Statistical analysis
GraphPad Prism 5.03 (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analysis using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. Data were presented as the mean ± standard deviation (SD). Statistical significance was set at p < 0.05.
Discussion
In the current study, we investigated the effects of SE and SV administration on insulin resistance and its underlying mechanism in the skeletal muscle of diabetic mice. First, treatment of SE and SV significantly reduced hyperglycemia. Additionally, the treatment raised the levels of antioxidant enzymes while decreasing serum TG, TC, and insulin levels. The administration of metformin showed the same results. Interestingly, only mice given SE500 and SV experienced an increase in muscle fiber size. In the skeletal muscle of diabetic mice, PC, SE500, and SV administration triggered insulin signaling. In particular, SE500 administration had a notable impact on elevated mitochondrial activity. Furthermore, the study demonstrated that PC and SV treatments were associated with lowering oxidative stress. Overall, this study found that supplying SE and SV to db/db mice’s skeletal muscles ameliorated their insulin resistance and was associated with regulating mitochondrial function and oxidative stress.
The incidence of T2DM, a severe metabolic disorder, is increasing worldwide. T2DM, characterized by high blood glucose and insulin dysfunction, can damage the cardiovascular system, spleen, retina, and skeletal muscles [
2,
5,
45]. Given the side effects of anti-diabetic drugs, managing diabetes with herbal medicines based on plants and natural phytochemicals such as alkaloids, polysaccharides, and flavonoids is gaining more attention [
46]. In the present study, we investigated the effect of stevia, a natural sweetener, on diabetes in
db/db mice.
Stevia has been used as a sweetener for decades and has been reported to have various health benefits. Some studies have investigated the health risks of stevia, including the allergenic potential of purified stevia in rats [
47] and its effect in reducing fertility in female rats [
48]. Nevertheless, stevia and SV which have non-caloric properties are consistently proposed as a sugar substitute in obesity and diabetes [
30,
31,
33,
49,
50]. In the present study, PC, SE, and SV administration significantly suppressed the increased blood glucose, serum TG, and serum TC levels in
db/db mice. In addition, we performed an OGTT to monitor peripheral disposal after oral glucose loading and insulin secretion over time [
51]. The AUC of the OGTT in the treated mice was significantly lower than that in the NC group, indicating greater glucose intolerance. The results of ITT, reflecting whole-body insulin action, showed a decrease in treated mice, similar to the results of the OGTT. NC mice show hyperinsulinemia, one of the symptoms of diabetes. SE administration has been reported to reduce serum insulin levels and HOMA-IR, an indicator of hepatic insulin action [
52].
Our results manifested that SE500 and SV administration increased muscle fiber size while ameliorating muscle damage. It is unclear why PC mice treated with metformin did not exhibit a substantial increase; nonetheless, a recent study found that metformin causes induces atrophy by regulating myostatin [
53]. To determine whether the effects of SE and SV on muscle are related to insulin action, we investigated insulin signaling in the skeletal muscle of
db/db mice. Insulin binds to IR, resulting in glucose uptake and tyrosine kinase activation, leading to IRS phosphorylation. Subsequent activation of the PI3K/Akt pathway results in the translocation of GLUT4 to the membrane, thereby mediating glucose transport [
54]. One study substantiated that SV activated IR/IRS-1/Akt/GLUT 4 signaling, promoting glucose uptake in the gastrocnemius muscle of diabetic rats [
55]. In this study, consistent with the metformin group, SE and SV administration was found to upregulate the expression of proteins related to insulin activity. Insulin resistance reduces muscle quality and strength, resulting in muscle atrophy [
56]. It is necessary to confirm the significant overlap in molecular pathways in which sarcopenia and muscle IR are abnormally regulated [
57]. Previous studies have reported that treatment with rosiglitazone and anti-myostatin antibodies increased insulin sensitivity and suppressed muscle protein degradation, and increased skeletal muscle mass and strength in mice [
58,
59]. Likewise, our data showed that SE and SV administration improved insulin resistance, increasing muscle fiber size and alleviating muscle damage. Meanwhile, insulin resistance is closely associated with mitochondrial dysfunction [
60]. One study reported that the enzymatic activity of mitochondrial complexes involved in oxidative phosphorylation was reduced by approximately 40% in human skeletal muscle in T2DM [
61]. In mitochondrial biogenesis, PGC-1α is essential in increasing cellular ATP [
62], and SIRT1-mediated PGC-1α functions to promote metabolic adaptations in tissue [
63]. In the current study, PC, SE, and SV administration upregulated the protein expression of p-AMPKα/AMPKα ratio and SIRT1 and consequently activated the PGC-1α in the skeletal muscle of
db/db mice. In particular, a high dose of SE effectively activated mitochondrial function. To further support the effect on mitochondria, we measured the protein expression levels of CS. PC, SE, and SV administration increased CS levels, which increased mitochondrial function and content [
64]. Consequently, the administration improved insulin resistance by activating the AMPK/SIRT1/PGC-1α pathway in the skeletal muscle of
db/db mice.
The activity of antioxidant proteins increases under high oxidative stress [
65], such as T2DM, Alzheimer’s disease, and cardiovascular disease. SOD and GPx are key cellular antioxidant enzymes that scavenge toxic free radicals and neutralize ROS [
44]. Therefore, we assessed the expression of proteins related to oxidative stress, which can affect impaired insulin action [
20]. NC mice exhibited increased HO-1, SOD, and GPx protein levels, indicating that severe diabetic conditions induce oxidative stress. In contrast, PC, SE500, and SV administration suppressed the expression of these proteins. In particular, SV administration showed a strong antioxidant effect, similar to the results of other studies [
20,
66,
67]. Furthermore, we investigated aberrant apoptosis, which may be caused by excessive free radicals [
68]. The Bax/Bcl
-2 ratio, a key role in the apoptotic pathway, was increased in diabetic mice and decreased in PC, SE500, and SV mice. These findings indicated that SE500 and SV administration decreased oxidative stress and apoptosis, which enhanced insulin sensitivity.
Several studies have been reported [
69‐
71] on the antihyperglycemic effects of stevia, or SV, in general adults. We used the skeletal muscle of diabetic mice to examine their antihyperglycemic mechanisms. According to a recent study, SV attenuated insulin resistance in rats fed a high-fat diet by activating the insulin signaling pathway in their muscles [
55]. We did, however, confirm the effects of SE and SV together and found that they activated insulin signaling while also controlling mitochondrial function and oxidative stress. However, given that this study only involved a small number of animals, translating its findings into clinical practice is challenging. Future research on deeper mechanisms, including inflammation and fatty acid-induced insulin resistance, using sufficiently large numbers of animals is needed in order to completely comprehend the effects of SE and SV in diabetic muscle. Furthermore, future research may also need to be tailored for the muscles of patients with type 2 diabetes.
In this study, SE and SV administration improves insulin resistance by activating mitochondrial function through the activation AMPK/SIRT1/PGC-1α pathway and reducing oxidative stress, which increases muscle fiber size and alleviates degenerated fibers in db/db mice. Therefore, our data suggest that SE and SV may be potential nutraceuticals for the management of diabetic muscle.
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