The sodium-glucose cotransporter 2 inhibitor luseogliflozin can suppress muscle atrophy in Db/Db mice by suppressing the expression of foxo1

We investigated the effect of the sodium glucose cotransporter-2 inhibitor (SGLT-2i) luseogliflozin on skeletal muscle. Eight-week-old mice were fed a standard diet or the standard diet with added luseogliflozin for 8 weeks. The mice were divided into the following four genotype/dietary groups: Db/m mice without SGLT-2i, Db/m mice with SGLT-2i inhibitor, Db/Db without SGLT-2i, and Db/Db with SGLT-2i. Among the mice with and without SGLT-2i, the ratio of soleus and plantaris muscle to body weight in the Db/Db mice was significantly lower than that in the Db/m mice. The cross-sectional area of soleus muscle in the Db/Db mice without SGLT-2i was significantly higher than that in the Db/Db mice with SGLT-2i. The expression of foxo1 in soleus muscle of the Db/Db mice was significantly higher than that of the Db/m mice, and the foxo1 expression of the Db/Db mice with SGLT-2i was significantly lower than that of the mice without SGLT-2i. The fluorescence intensity of foxo1 in the Db/Db mice fed SGLT-2i was significantly lower than that in the Db/Db mice without SGLT-2i. The administration of luseogliflozin resulted in the suppression of both the increased foxo1 expression and the reduced muscle cross-sectional area in the soleus muscle of Db/Db mice.

Introduction T he numbers of individuals with type 2 diabetes are rapidly increasing worldwide. Complications of type 2 diabetes reduce a person's quality of life, and they add a heavy burden to the medical economy. (1) The prevention of the progression of diabetic complications is thus an important task. In recent years, muscle atrophy has been thought of as a complication of diabetes. (2) It has become clear that muscle atrophy, i.e., sarcopenia, and sarcopenic obesity are strongly associated with dietary pattern or metabolic disorder. (3,4) In fact, we demonstrated that muscle atrophy is present in diabetic patients. (5,6) Muscle atrophy is also a risk factor for both decreased daily life activity and mortality. (7,8) Several sodium glucose cotransporter-2 inhibitors (SGLT2i) have recently become available as anti-diabetic medications, and some of them have been reported to reduce the risk of incident cardiovascular disease. (9,10) The effects of SGLT2i on body composition have been described, (11,12) but the mechanisms underlying these effects on muscle have been unclear. We conducted the present study to investigate the effects of the SGLT2i luseogliflozin on muscle in Db/Db mice. We evaluated muscle atrophy using cross-sectional areas of muscle because this method has been often used as the best objective indicator of muscle atrophy. (13,14) We also evaluated the changes in gene expression in skeletal muscle following the administration of SGLT2i. The genes mstn, pgc1a, and foxo1 are related to muscle atrophy. (15)(16)(17) We focused on foxo1 in this study because the foxo1 expression of skeletal muscle in individuals with diabetes is accelerated, and this suppresses the glucose utilization and lipid synthesis in skeletal muscle. (18,19)

Materials and Methods
Animals and experimental design. All experimental procedures were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine. Six-week-old male non-diabetic heterozygous Db/m mice and 6-week-old male diabetic homozygous Db/Db mice were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). Starting when the mice were 8 weeks old, they were fed either a standard diet (SD; 344.9 kcal/ 100 g, fat kcal 4.6%; CLEA Japan, Tokyo, Japan) or the same standard diet with the SGLT2i luseogliflozin added (0.01% w/w in chow) for 8 weeks. We divided the mice into the following four groups: (1) Db/m without (w/o) SGLT2i, (2) Db/m with SGLT2i, (3) Db/Db w/o SGLT2i, and (4) Db/Db with SGLT2i. At 16 weeks old, after an overnight fast, all of the mice were killed by the administration of a combination anesthetic: 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol (Fig. 1A). (20) Glucose tolerance tests. Intraperitoneal glucose tolerance tests (iPGTTs) (2 g/kg) were performed in other 16-week-old mice that had been fasted for 5 h. Plasma glucose was measured from the tail vein using a glucometer (Gultest Neo Alpha; Sanwa Kagaku Kenkyusho, Nagoya, Japan).
Tissue collection and histological assessment of murine soleus and plantaris muscles. We used the soleus and plantaris muscles for the muscle samples. (21) The soleus muscle was either fixed with 10% buffered formaldehyde for the histological examination or immediately frozen in QIAzol Lysis reagent (Qiagen, Venlo, Netherlands) for mRNA extraction. We measured the weight and cross-sectional area of soleus and plantaris muscles of the four groups of mice described above. In this study, we used the anatomical cross-sectional area, which is the cross-sectional T area of a muscle perpendicular to its longitudinal axis of soleus muscle. (22) Soleus muscle sections were prepared and stained with hematoxylin and eosin or a monoclonal foxo1 (C29H4) antibody (Cell Signaling Technology, Beverly, MA) as a primary antibody, and a Texas-red-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA). Nuclei were stained with DAPI (Sigma-Aldrich, St. Louis, MO). Images were captured with a fluorescence microscope (BZ-X710, Keyence, Osaka, Japan), and the fluorescence intensity of the muscle tissue and the cell nuclei numbers were analyzed using Image J software. We measured the weights of the soleus and plantaris muscles and the cross-sectional areas of soleus muscle of the mice in the four groups described above. All images acquired using the BZ-X710 microscope and the cross-sectional areas of soleus muscle were measured using BZ-X analyzer software (Keyence).
Gene expression in soleus muscle. The soleus muscle of fasting mice were resected and immediately frozen using liquid nitrogen and homogenized in ice-cold QIAzol Lysis reagent, and total RNA was isolated as described in the manufacturer's instructions. We reverse-transcribed the total RNA (0.5 mg) by using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) for first-strand cDNA synthesis utilizing an oligonucleotide dT primer and random hexamer priming according to the manufacturer's recommendations. The reverse transcription (RT) reaction was performed for 120 min at 37°C, and the inactivation of RT was performed for 5 min at 85°C.
The mRNA expression levels of foxo1, myog, mstn, myod, pgc1a and ppara were quantified using a real-time reverse transcriptionpolymerase chain reaction (RT-PCR). The relative expression levels of each targeted gene was normalized to the gapdh threshold cycle (CT) values and quantified using the comparative threshold cycle 2 -DDC T method as described. (23) Signals from Db/m mice without SGLT2i feeding were assigned a relative value of 1.0. The RT-PCR was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems) according to the manufacturer's instructions. The following PCR conditions were used: 1 cycle for 2 min at 50°C and 20 s at 95°C, followed by 40 cycles for 1 s at 95°C, and s0 s at 60°C.
Statistical analysis. We analyzed the data using the JMP ver. 13.0 software (SAS, Cary, NC), and p values <0.05 were considered significant. Student's t test was used to compare the differences between pairs of groups.

Effect of SGLT 2i on body weight and glucose homeostasis
After the 8-week dietary treatment, the body weight and blood glucose in the two groups of Db/Db mice (those with and w/o the SGLT2i) were significantly higher than those of the two groups of Db/m mice. However, no significant reduction in body weight and no improvement in impaired glucose tolerance were observed following the administration of SGLT-2i (Fig. 1B-D).
Effect of SGLT 2i on skeletal muscle. In the mice treated with and without SGLT-2i, the weight of the soleus muscle of the Db/Db mice was significantly lower than that in the Db/m mice, whereas the weight of the plantaris muscle did not show a significant difference between the Db/Db and Db/m mice ( Fig. 2A and  B). Additionally, among the mice treated with and without SGLT-2i, the plantaris and soleus muscle to body weight ratio in the Db/Db mice was significantly lower than that in the Db/m mice ( Fig. 2C and D). The cross-sectional area of soleus muscle in the Db/Db mice without SGLT-2i was significantly less than that in the Db/Db mice with SGLT-2i (Fig. 3A-E).
SGLT 2i suppressed foxo1 expression in muscle. Our RT-PCR analyses revealed that the foxo1 expression in skeletal muscle of the Db/Db mice was significantly higher than that of the Db/m mice (Fig. 4A). However, the foxo1 expression in skeletal muscle of the Db/Db mice with SGLT-2i was significantly lower than that in the mice without SGLT-2i (Fig. 4A). The admin-   istration of SGLT-2i did not change the expressions of any other genes in the Db/m and Db/Db mice (Fig. 4B-F). In addition, the immunostaining of soleus muscle tissues demonstrated that the fluorescence intensity of foxo1 in the Db/Db w/o SGLT2i group was significantly higher than that of the Db/Db with SGLT2i group (Fig. 5A-E). Moreover, the number of cell nuclei per image in both the Db/m mice and the Db/Db mice treated with SGLT-2i were higher than those of the mice w/o SGLT-2i (Fig. 5F).

Discussion
Our findings demonstrated that the foxo1 expression in skeletal muscle of Db/Db mice is higher than that of Db/m mice and that an SGLT2i, luseogliflozin, suppressed this higher foxo1 expression in skeletal muscle of Db/Db mice. Increased foxo1 expression in skeletal muscle was reported to be associated with muscle atrophy. (24,25) Foxo1 could affect several metabolic pathways. Among them, proteolysis regulated by the ubiquitin-proteasome pathway, autophagy, and the repression of protein synthesis are dominant processes of muscle atrophy. (17,26) In addition, foxo1 has been thought to have a pivotal role in glycolysis in muscle. In fact, increased foxo1 expression resulted in the upregulation of pdk4 expression, which suppresses the glycolytic pathway. (18) Increased foxo1 expression represses the expression of srebp1c, which is mediated by nuclear receptors (such as liver X receptor and retinoid X receptor), and it upregulates the biosynthesis of fatty acid in skeletal muscle. (19) Therefore, increased foxo1 expression in skeletal muscle suppresses glucose utilization and lipid synthesis.
In the present study, the ratio of plantaris and soleus muscle to body weight in the Db/Db mice were significantly lower than that in the Db/m mice. Moreover, the cross-sectional area of soleus muscle in the Db/Db mice treated with SGLT2i was significantly higher than that of the mice w/o SGLT2i.
This study has some limitations. First, the sample size was small. Second, we did not investigate the biological mechanism of luseogliflozin in vitro. This issue is very important and should be addressed in future studies.

Conclusion
Taken together, our present findings suggest that increased foxo1 expression in skeletal muscle is associated with the muscle atrophy of Db/Db mice. This is the first study to demonstrate the increased expression of foxo1 in muscle tissue of Db/Db mice. The administration of luseogliflozin resulted in the suppression of both the increased foxo1 expression and the reduced muscle crosssectional area in the soleus muscle of Db/Db mice. Further studies investigating the association between the effect of an SGLT-2i on muscle and foxo1 in muscle are needed. grants from Sanofi, grants from Ono Pharmaceutical Co., and grants from Takeda Pharmaceutical Co., outside the submitted work. The sponsors were not involved in the study design; in the collection, analysis, interpretation of data; in the writing of this manuscript; or in the decision to submit the article for publication. The authors, their immediate families, and any research foundations with which they are affiliated have not received any financial payments or other benefits from any commercial entity related to the subject of this article. The authors declare that although they are affiliated with a department that is supported financially by pharmaceutical company, the authors received no current funding for this study and this does not alter their adherence to all the journal policies on sharing data and materials. The other authors have nothing to disclose.