Juzentaihoto Suppresses Muscle Atrophy in KKAy Mice

Abstract

In obese patients with type 2 diabetes, reduced insulin sensitivity, increased production of inflammatory cytokines, and increased oxidative stress were observed, which lead to decreased protein synthesis and increased proteolysis in the skeletal muscles. Juzentaihoto (JTT) is herbal medicine and we have previously reported that the administration of JTT hot water extract alleviates skeletal muscle atrophy in a mouse model with streptozotocin-induced type 1 diabetes. In this study, we evaluated the inhibitory effects of JTT on muscle atrophy in a mouse model with obesity and type 2 diabetes. JTT was administered to KKAy mice with type 2 diabetic obesity and its effects on the skeletal muscles were evaluated. After JTT administration in KKAy mice, the wet weight and muscle fibre cross-sectional area of gastrocnemius increased and the time duration of exercise in the rotarod test improved. In addition, the serum levels of tumour necrosis factor-α and interleukin-6 decreased, adiponectin levels increased, and homeostasis model assessment for insulin resistance improved. Furthermore, JTT administration decreased the mRNA levels of ubiquitin ligase (atrogin-1, muscle RING-finger protein-1), increased the mRNA levels of Sirtuin1 in gastrocnemius. Our results suggest that JTT improves insulin resistance, suppresses inflammation, and reduces oxidative stress in KKAy mice, thereby suppressing skeletal muscle atrophy. JTT administration in clinical practice is expected to improve muscle atrophy in patients with obesity and type 2 diabetes.

INTRODUCTION

Skeletal muscle atrophy progresses in obese patients with type 2 diabetes because of increased insulin resistance, increased inflammation, and oxidative stress.^1–4) Muscle atrophy reduces motor function and significantly impairs the QOL.^5–9)

In obesity and type 2 diabetes, increased insulin resistance leads to a decrease in protein synthesis in skeletal muscles.^1,2) Inflammatory cytokines such as tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) are produced in adipose tissues^10,11) and can cause skeletal muscle atrophy via the increased level of ubiquitin ligase.^12,13) Oxidative stress is enhanced by fat cell hypertrophy and hyperglycaemia that are associated with obesity. Oxidative stress causes myoblast apoptosis and myotube atrophy that leads to skeletal muscle atrophy.^14,15)

KKAy mice are used as an obesity and type 2 diabetes model in which early chronic obesity and hyperglycaemia are produced by introducing an obesity gene. In KKAy mice, increased insulin resistance, overproduction of inflammatory cytokines, and increased oxidative stress cause symptoms similar to those observed in patients with obesity and type 2 diabetes.^16,17)

Juzentaihoto (JTT) is an herbal medicine that is used to improve the physical strength of individuals suffering from weakness and fatigue. JTT exhibits anti-inflammatory, anti-oxidative, anti-cancer and immunostimulatory properties.^18–21) In our previous study, we reported that the administration of JTT hot water extract alleviates skeletal muscle atrophy in the mouse model with streptozotocin-induced type 1 diabetes.^22,23) However, no studies have been performed to determine the effects of JTT on skeletal muscle atrophy in mouse models with obesity and type 2 diabetes. In the present study, we evaluated the inhibitory effect of JTT on skeletal muscle atrophy in the KKAy mouse model with type 2 diabetic obesity.

MATERIALS AND METHODS

Preparation of Chow Containing JTT Extracts

The JTT hot water extracts used as the spray-dried product by Tsumura (Tokyo, Japan). Around 5.0 g of the extract was obtained from 28.5 g of crude drug mixture, as shown in Table 1. The JTT extract was mixed with normal feed (CLEA, Shizuoka, Japan) at a concentration of 4% (w/w).^22,23) The three dimensional (3D)-HPLC chart of JTT hot water extract is shown in the Supplementary Fig. 1.

Table 1. Parts of Plants Containing Juzentaihoto (JTT)^a)

Name of the crude drug	Japanese name	Dosage (g/d)
Astragali Radix (root of Astragalus mongholicus)	Ogi	3.0
Atractylodis Lanceae Rhizoma (rhizome of Atractylodes lancea)	Sojutsu	3.0
Cinnamomi Cortex (bark of Cinnamomum cassia)	Keihi	3.0
Angelica Radix (root of Angelica acutiloba)	Toki	3.0
Rehmanniae Radix (root of Rehmannia glutinosa)	Jio	3.0
Ginseng Radix (root of Panax ginseng)	Ninjin	3.0
Paeoniae Radix (root of Paeonia lactiflora)	Shakuyaku	3.0
Poria (sclerotium of Poria cocos)	Bukuryo	3.0
Cnidii Rhizoma (rhizome of Cnidium officinale)	Senkyu	3.0
Glycyrrhizae Radix (root of Glycyrrhiza uralensis)	Kanzo	1.5

a) Source: The Japanese Pharmacopoeia, 17th Edition (JP17).³⁹⁾

Animals

All animal experiments were conducted in accordance with the NIH guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Kochi University (Approval No. L-00090, Approval Date: 29 June, 2017). The KKAy mice were purchased as a type 2 diabetic obesity model and the C57BL/6 mice were purchased as a control mouse from CLEA Japan (Tokyo, Japan).

Blood glucose and body weight were measured at the beginning of JTT administration (day 0). KKAy mice (8 weeks old, male) and C57BL/6 mice (8 weeks old, male) were randomly divided into two groups (KKAy: n = 7 and C57BL/6: n = 6), to ensure that the average blood glucose levels and body weight were the same, and each group received normal feed (CE-2) or 4% JTT-mixed feed. Each mouse was maintained for 56 d, and blood glucose level and body weight were measured every 7 d. At the time of blood glucose measurement, the mice fasted for 6 h before blood collection, and the blood was collected from the tail vein and tested using Glu-TEST-STRIPS (Nova Biomedical, Tokyo, Japan).

Rotarod Performance Test

In the rotarod test, the speed of the rotor was increased at a rate of 0.3 rotations/s until it was maintained at 80 rotations/min. The time until the mouse was felled from the rota was measured. The mice were tested three times at 10-min intervals, and the mean of the three tests was calculated. The rotarod test was performed every 7 d during the 56-d experimental period.

Measurement of Skeletal Muscle Weight

The mice underwent laparotomy under anaesthesia 56 d after the beginning of JTT administration, and the whole blood was collected from the descending vena cava. The gastrocnemius and soleus muscles were then extracted from the lower limbs, and the wet weight of each muscle tissue was measured.

Measurement of Myocyte Cross-Sectional Area

The excised gastrocnemius muscles were fixed with 10% formalin, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin. A cross-sectional area of the gastrocnemius muscles was analysed using the computer analysis software WinROOF (Mitani Corporation, Ohtsu, Japan), and the cross-sectional area of each fibre was measured. The cross-sectional areas of 100 muscle fibres from each mouse were randomly measured, and the average value was used in further analyses.

Quantitative RT-PCR of mRNA in Gastrocnemius Muscles

The excised gastrocnemius muscles were cut in 0.1-cm sized pieces and homogenised with POLYTRON RT-MR 3100 (Central Science Trade, Tokyo, Japan). The homogenate was centrifuged (8000 × g, 15 min, 4 °C) and the supernatant was collected. Total mRNA present in the supernatant was extracted using RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed using Prime Script RT reagent Kit (TaKaRa Bio, Otsu, Japan) to obtain the corresponding cDNA. Quantitative RT-PCR was performed using Step One Plus Real-Time PCR System (Applied Biosystems, CA, U.S.A.). The mRNA levels of sirt1 and ubiquitin ligases (murf1 and atrogin1) were corrected using the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. GAPDH (Mm99999915_g1), sirt1 (Mm01168521_m1), muscle RING-finger protein-1 (murf1) (Mm01185221_m1) and atrogin1 (Mm00499523_m1) were used as TaqMan probes. TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, U.S.A.) was used for PCR.

Enzyme-Linked Immunosorbent Assay (ELISA)

The serum level of TNF-α was evaluated using a TNF-α ELISA kit (R&D Systems, Minneapolis, MN, U.S.A.); IL-6 level was evaluated using an IL-6 ELISA kit (R&D Systems); adiponectin level was evaluated using a mouse/rat adiponectin ELISA kit (Otsuka, Tokushima, Japan), and insulin level was evaluated using a mouse insulin kit (Morinaga, Yokohama, Japan). Homeostasis model assessment for insulin resistance (HOMA-R) was calculated from the fasting blood glucose level and serum insulin level using the following formula:

  

Statistical Analysis

The results are expressed as the mean ± standard deviation. Two-way ANOVA was performed to analyse the blood glucose levels, body weight, and the rotarod test results. When the F value was statistically significant (p < 0.05), the data were analysed by Bonferroni’s test for each measurement day. For the other results, one-way ANOVA was performed. Statistical analyses were performed using the Stat Flex programme (View Flex, Tokyo, Japan). The statistical significance was set at p < 0.05.

RESULTS

Effect of JTT on Body Weight and Food Intake

To determine the effect of JTT, body weight was measured after every 7 d from the beginning of JTT administration. The body weight of C57BL/6 mice was not affected from the beginning (day 0) to the end (day 56) of JTT administration (Fig. 1). In KKAy mice, the body weight was significantly decreased by JTT administration on days 42–56 (p < 0.05) (Fig. 1). Two-way repeated ANOVA results indicated that these effects were significant with respect to time (F8,199  =  365, p < 0.01), group (F3,199  =  834, p < 0.01), and the interaction of time and the group (F24,199  =  14.4, p < 0.01). There was no significant difference by JTT administration in dietary intake in C57BL/6 and KKAy mice for all time periods (Supplementary Table 1).

Fig. 1. Juzentaihoto (JTT) Suppressed Weight Gain

Body weight was measured from 0–56 d after the beginning of JTT administration. The mice were fasted for 12 h before the measurement. Data are expressed as means ± standard deviation (S.D.) (C57/BL6 mice, n = 6; KKAy mice, n = 7) and analysed by two-way repeated ANOVA followed by Bonferroni test. * p < 0.05, vs. JTT no treatment group in the same strain of mice.

Effect of JTT on Rotarod Test Performance

The rotarod test was performed after every 7 d from the beginning of JTT administration to determine the effect of JTT on motor function. In C57BL/6 mice, the endurance time was not changed from the beginning (day 0) to the end (day 56) of JTT administration in the rotarod test period (Fig. 2). In KKAy mice, the endurance time increased significantly by JTT administration on day 56 (p < 0.05) (Fig. 2). Two-way ANOVA results indicated that these effects were significant with respect to time (F8,199  =  5.8, p < 0.01), group (F3,199  =  4.7, p < 0.01), and the interaction of time and the group (F24,199  =  3.9, p < 0.05).

Fig. 2. JTT Improved Motor Function

The rotarod tolerance test was performed from 0–56 d after the beginning of JTT administration. Data are expressed as means ± S.D. (C57/BL6 mice, n = 6; KKAy mice, n = 7) and analysed by two-way repeated ANOVA followed by Bonferroni test. * p < 0.05, vs. JTT no treatment group in the same strain of mice.

Effect of JTT on Blood Glucose Levels

The blood glucose levels were evaluated after every 7 d from the beginning of JTT administration. In C57BL/6 mice, no change in the blood glucose levels was observed from the beginning (day 0) to the end (day 56) of JTT administration (Fig. 3). In KKAy mice, blood glucose levels decreased significantly after JTT administration on days 49 and 56 (p < 0.05) (Fig. 3). Two-way ANOVA results indicated that these effects were significant with respect to time (F8,199  =  3.1, p < 0.05), group (F3,199  =  173, p < 0.01), and association between time and the group (F24,199  =  3.8, p < 0.05).

Fig. 3. JTT Improved Hyperglycaemic Condition

Blood glucose levels were evaluated from 0–56 d after the beginning of JTT administration. The mice were fasted for 12 h before the measurement. Data are expressed as means ± S.D. (C57/BL6 mice, n = 6; KKAy mice, n = 7) and analysed by two-way repeated ANOVA followed by Bonferroni test. * p < 0.05, vs. JTT no treatment group in the same strain of mice.

Effect of JTT on Skeletal Muscle Weight

The weights of gastrocnemius and soleus muscles extracted 56 d after JTT administration (day 56) were measured to determine the effect of JTT on skeletal muscle weight. In C57BL/6 mice, JTT administration did not affect the weights of gastrocnemius and soleus muscles (Table 2). In KKAy mice, JTT administration did not affect soleus muscle weight but gastrocnemius weight was significantly increased (p < 0.05) (Table 2). One-way ANOVA results indicated that these effects were significant (gastrocnemius: F3,22  =  3.6, p < 0.05, soleus: F3,22  =  17.0, p < 0.01).

Table 2. Effect of JTT on the Skeletal Muscle Weight^a)

Muscle	C57BL/6	C57BL/6+ JTT	KKAy	KKAy + JTT
Gastrocnemius	136 ± 19	139 ± 8	129 ± 12	145 ± 8*
Soleus	8.2 ± 0.8	7.9 ± 0.4	6.0 ± 0.8	5.7 ± 0.7

a) Gastrocnemius and soleus muscles were removed from mice at 56 d after the beginning of JTT administration and wet weights were measured. Data are expressed as means ± standard deviation (S.D.) (C57/BL6 mice, n = 6; KKAy mice, n = 7) and analysed by one-way ANOVA followed by Bonferroni test. * p < 0.05, vs. JTT no treatment group in the same strain of mice.

Effect of JTT on Insulin Resistance

Insulin levels in the serum collected 56 d after the beginning of JTT administration (day 56) were evaluated to determine the effect of JTT on insulin level and insulin resistance. In C57BL/6 mice, the insulin levels in serum and HOMA-R were unchanged after JTT administration (Table 3). In KKAy mice, HOMA-R was significantly decreased after JTT administration (p < 0.05). The serum insulin levels also decreased after JTT administration (p = 0.056) (Table 3). One-way ANOVA results indicated that these effects were significant (insulin: F3,22  =  9.7, p < 0.01, HOMA-R: F3,22  =  15.0, p < 0.01).

Table 3. Effect of JTT on Serum Insulin Resistance^a)

Measurement	C57BL/6	C57BL/6+ JTT	KKAy	KKAy + JTT
Insulin	0.39 ± 0.22	0.49 ± 0.21	2.73 ± 0.76	2.08 ± 0.26
HOMA-R	0.17 ± 0.12	0.21 ± 0.09	2.28 ± 0.87	1.33 ± 0.25*

a) Blood was collected from mice 56 d after the beginning of JTT administration. Serum insulin levels were measured, and the homeostasis model assessment ratio (HOMA-R) was calculated from serum insulin and glucose levels. Data are expressed as means ± S.D. (C57/BL6 mice, n = 6; KKAy mice, n = 7) and analysed by one-way ANOVA followed by Bonferroni test. * p < 0.05, vs. JTT no treatment group in the same strain of mice.

Effect of JTT on Inflammatory Cytokines and Adiponectin in Serum

The levels of TNF-α, IL-6 and adiponectin in the serum collected 56 d after the beginning of JTT administration were evaluated to determine the effect of JTT on inflammatory cytokines and adiponectin. In C57BL/6 mice, JTT administration did not affect the serum levels of TNF-α, IL-6 and adiponectin (Fig. 4). In KKAy mice, the serum levels of TNF-α and IL-6 were decreased (p < 0.05) (Fig. 4) and those of adiponectin increased after JTT administration (p = 0.051) (Fig. 4). One-way ANOVA results were significant (TNF-α: F3,22  =  41.9, p < 0.01, IL-6: F3,22  =  11.2, p < 0.01, adiponectin: F3,22  =  39.9, p < 0.01).

Fig. 4. JTT Decreased Inflammatory Cytokines and Increased Adiponectin in the Serum

The blood was collected from mice after 56 d from the beginning of JTT administration, and (A) TNF-α, (B) IL-6, and (C) adiponectin levels in serum were measured. Data are expressed as means ± S.D. (C57/BL6 mice, n = 6; KKAy mice, n = 7) and analysed by one-way ANOVA followed by Bonferroni test. * p < 0.05, vs. JTT no treatment group in the same strain of mice.

Effect of JTT on Myocyte Cross-Sectional Area

The cross-sectional areas of muscle fibres in gastrocnemius extracted 56 d after the beginning of JTT administration were measured to determine the effect of JTT on inhibiting the progression of skeletal muscle atrophy. In C57BL/6 mice, JTT administration did not affect the cross-sectional areas of muscle fibres in gastrocnemius (Fig. 5). In KKAy mice, the cross-sectional areas of muscle fibres in gastrocnemius were significantly increased after JTT administration (p < 0.05) (Fig. 5). One-way ANOVA results indicated that these effects were significant (F3,22  =  8.2, p < 0.01).

Fig. 5. Skeletal Muscle Atrophy in Mice

Gastrocnemius muscles were removed from the mice 56 d after the beginning of juzentaihoto (JTT) administration and stained with hematoxylin and eosin. (A) Myocyte cross-sections of gastrocnemius (B) Summary data of myocyte cross-sectional areas. Data are expressed as means ± S.D. (C57/BL6 mice, n = 6; KKAy mice, n = 7) and analysed by one-way ANOVA followed by Bonferroni test. * p < 0.05, vs. JTT no treatment group in the same strain of mice.

Effect of JTT on the mRNA Levels of Sirtuin1 (sirt1) and Ubiquitin Ligase in Gastrocnemius

The mRNA levels of sirt1 and ubiquitin ligase in gastrocnemius extracted 56 d after the beginning of JTT administration were measured. In C57BL/6 mice, JTT administration did not affect sirt1 and ubiquitin ligase mRNA levels (Fig. 6). In KKAy mice, JTT administration increased the sirt1 mRNA level (p < 0.05) and decreased the mRNA levels of murf1 in gastrocnemius (p < 0.05) (Fig. 6). The mRNA levels of atrogin1 in gastrocnemius decreased after JTT administration (p = 0.060) (Fig. 6). One-way ANOVA results indicated that these effects were significant (sirt1: F3,22  =  6.8, p < 0.01, murf1: F3,22  =  9.9, p < 0.01, atrogin1: F3,22  =  12.6, p < 0.01).

Fig. 6. JTT Increased the mRNA Level of sirt1 and Decreased the mRNA Level of Ubiquitin Ligase

Gastrocnemius muscles were removed from mice 56 d after the beginning of JTT administration, total mRNA in gastrocnemius was isolated, and the mRNA levels of (A) sirt1, (B) murf1, and (C) atrogin1 were evaluated. Data are expressed as means ± S.D. (C57/BL6 mice, n = 6; KKAy mice, n = 7) and analysed by one-way ANOVA followed by Bonferroni test. * p < 0.05, vs. JTT no treatment group in the same strain of mice.

DISCUSSION

Our study showed that JTT administration in KKAy mice with obesity and type 2 diabetes improved skeletal muscle atrophy and motor function depression (Table 2, Figs. 2, 5). JTT administration increased sirt1 mRNA levels in KKAy mice. In addition, insulin resistance was decreased, adiponectin level was increased, inflammatory cytokine level was decreased (Table 3, Figs. 4, 6), which may lead to the increase in muscle mass.

Hyperglycaemia leads to increased inflammatory cytokine production, oxidative stress, and insulin resistance, which causes skeletal muscle atrophy, predominantly in fast-twitch muscles.²⁴⁾ In the present study, the administration of JTT in KKAy mice resulted in a significant increase in the wet weight and muscle fibre cross-sectional area of gastrocnemius, which is composed of fast and slow-twitch muscles. However, the wet weight of soleus muscle, which is mainly composed of slow-twitch muscles, was not changed after JTT administration. The results of the present study suggest that JTT administration suppresses the atrophy of fast-twitch skeletal muscles but does not affect the atrophy of slow-twitch skeletal muscles.

Overproduction of inflammatory cytokines increases the production of ubiquitin ligases (murf1 and atrogin1) and enhances the activity of the ubiquitin–proteasome system. The increased activity of the ubiquitin–proteasome system leads to skeletal muscle atrophy.^12,13,25) In this study, JTT administration in KKAy mice resulted in the decreased serum levels of TNF-α and IL-6 and decreased mRNA levels of murf1 and atrogin1 in gastrocnemius (Figs. 4, 6). The results suggest that JTT administration suppresses skeletal muscle atrophy by decreasing the production of inflammatory cytokines and suppressing the increased activity of the ubiquitin–proteasome system.

We evaluated fat mass in KKAy mice treated with JTT in the past study. JTT administration reduced fat mass, inhibited adipocyte hypertrophy.²⁶⁾ Adiposity is related to insulin resistance and chronic inflammation of the living body. Inflammatory cytokines from adipocyte affects insulin resistance and muscle atrophy.^10,11) Conversely, adiponectin is an adipokine also produced by adipocyte that improves insulin resistance.^27,28) In this study, the administration of JTT decreased the production of TNF-α and IL-6 and increased the production of adiponectin in KKAy mice (Fig. 4). JTT administration may improve insulin resistance and muscle atrophy by reduction fat mass and regulating the level of adipokines. Previous studies have shown that some active ingredients contained in JTT such as ginsenosides exhibit anti-obesity effects by increase energy expenditure by stimulating the adenosine monophosphate-activated kinase pathway,^29,30) and cinnamaldehyde exhibit anti-obesity effects by uncoupling protein 1 (UCP-1).³¹⁾ The anti-obesity effect of JTT may be due to these components. However, the mechanism and active components on obesity inhibition by JTT administration is not clearly in this study and needs further investigation.

Insulin resistance decreases protein synthesis in skeletal muscles and leads to skeletal muscle atrophy.^1,2,17) In the present study, insulin resistance was improved after JTT administration in KKAy mice, which suggests that JTT suppresses skeletal muscle atrophy by improving insulin resistance. Sirt1 exhibits anti-inflammatory effects and improve insulin resistance,^32,33) thus suppressing muscle atrophy.^34,35) In the present study, sirt1 mRNA expression was increased by JTT administration in KKAy mice, and inflammation and insulin sensitivity may have been improved via sirt1 after JTT administration. Improvement of insulin resistance by sirt1 is known to involve activation of insulin signal via Akt/ forkhead box protein O1.³⁴⁾ In previous studies, panaxatriol increased insulin receptor expression and improved insulin sensitivity via phosphoinositide 3-kinase (PI3K)/Akt signal pathway,³⁶⁾ and cinnamon extract activates AMP activated protein kinase (AMPK) in C2C12 cells; derived from mouse skeletal muscle,³⁷⁾ and insulin signal pathway may be enhanced by these compounds contained in JTT. Sirt1 is also known to activate and upregulate the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α); involved in mitochondrial synthesis, angiogenesis, and changes in muscle fiber composition.³⁸⁾ In the present study, JTT administration may be an effect related to PGC-1α via Sirt1. However, this study did not evaluate insulin and PGC-1α signal, and it is unclear whether JTT has act in these factors. Further studies are needed to clarify the detailed mechanisms.

JTT is composed of 10 crude herbal medicines.³⁹⁾ In our previous study, we observed that the hot water extracts of Cinnamomi Cortex, Astragali Radix, and Glycyrrhizae Radix increase the transcriptional activity of Sirt1 in C2C12 cells.²²⁾ These findings suggest that the ameliorating effect of JTT on muscle atrophy is attributable to its constituent herbs. However, to what extent do these components contribute to the activity of JTT remains elusive and warrants further studies.

This study has some limitations. 1) JTT dose was also determined by referring to past pharmacokinetic studies of kampo medicine in experimental animals.^22,23) The daily clinical dosage for human as a base powder is 5.0 g, and the dosage of 4% (w/w) was high by the dose considered by conversion by the body surface area of human to mice. In the future, it needs to be verified whether the same results can be obtained with normal doses of the drug in clinical practice. 2) Some studies reported that used C57BL/6 as a control for KKAy mice to evaluate anti muscle atrophy effect on obese type 2 diabetic.^40,41) The present study was conducted based on these reports. However, a model on muscle atrophy in obese type 2 diabetic has been reported that C57BL/6 mice fed high-fat diet or normal diet was used,⁴²⁾ and this study design may be clearly examined the effects of drugs on muscle atrophy affected by obesity and insulin sensitivity. Future studies using such a model may be needed. 3) Recent studies have shown that hyperglycaemia directly stimulates protein catabolism in skeletal muscle without altering circulating insulin levels.^43,44) In addition, suppression of fat mass reduces the expression of inflammation-related cytokines and prevents muscle atrophy.^45,46) In this study, JTT inhibits hyperglycaemia, suppresses body weight, and decreases the expression of cytokines associated with inflammation. It remains unknown whether JTT directly improved skeletal muscle atrophy or indirectly prevented by these factors. In the future, the directly effects of JTT on muscle cells should be examined to resolve these questions. 4) Myostatin is one of the factors that regulate skeletal muscle growth, and its expression is upregulated in diabetes and has been reported to be associated with progression of obesity, diabetes and muscle atrophy.⁴⁷⁾ Although not evaluated in this study.

Our results suggest that JTT improves insulin sensitivity and suppresses inflammation in KKAy mice, thereby suppressing skeletal muscle atrophy. However, our study is non-clinical. Further studies on human subjects are required in the future. The administration of JTT in clinical practice is expected to improve muscle atrophy in obese patients with type 2 diabetes.

The treatment for skeletal muscle atrophy in obese patients with type 2 diabetes has not been established in clinical settings. Therefore, the use of natural products such as JTT can greatly contribute to the development of skeletal muscle atrophy treatment.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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