Relationship between Urinary Liver-Type Fatty Acid-Binding Protein (L-FABP) and Sarcopenia in Spontaneously Diabetic Torii Fatty Rats

Background Type 2 diabetes (T2D) is a known risk factor for diabetic kidney disease (DKD) and sarcopenia in older patients. Because there may be an interaction between DKD and sarcopenia, the aim of the present study is to investigate the relationship between urinary levels of liver-type fatty acid-binding protein (L-FABP) and sarcopenia using a novel rat model of T2D. Methods Male spontaneously diabetic Torii (SDT) fatty rats (n = 5) at 16 weeks of age were used as an animal model of T2D. Age- and sex-matched Sprague-Dawley (SD) rats (n = 7) were used as controls. Urine samples were obtained from the rats, and muscle strength was evaluated with the use of the forelimb grip test at 16, 20, and 24 weeks of age. Serum, kidney, soleus, and extensor digitorum longus (EDL) muscle samples were collected at 24 weeks of age. Urinary L-FABP levels were measured using dedicated enzyme-linked immunosorbent assays. Results Increased urinary L-FABP levels, focal glomerular sclerosis, moderate interstitial inflammation and fibrosis, and accumulation of renal oxidative proteins were significantly observed in the SDT fatty rats, compared to the SD rats. Muscle weight, muscle strength, cross-sectional areas of both type I and type IIb muscle fibers, and increasing rate of muscle strength were significantly decreased in the SDT fatty rats compared to the SD rats at 24 weeks. Urinary L-FABP levels at 20 and 24 weeks were significantly negatively correlated with muscle strength. Urinary L-FABP levels at 16 weeks were significantly negatively correlated with the increasing rate of muscle strength. Conclusions Urinary L-FABP reflects the degree of muscle strength and weight, as well as cross-sectional areas of muscle fibers. Although further clinical study is needed, urinary L-FABP may be useful to monitor the progression of sarcopenia and DKD in T2D patients.


Introduction
Chronic kidney disease (CKD) is a common disease in aging societies worldwide. Many studies have focused on the involvement of CKD in the onset of sarcopenia which is characterized by decreased skeletal muscle mass and strength and has been the focus of many studies in order to sustain satisfactory quality of life and prevent fatal diseases in the super-aging populations of advanced countries [1][2][3][4][5].
Type 2 diabetes (T2D) with insulin resistance is a common risk factor for diabetic kidney disease (DKD) and sarcopenia [1,2]. Furthermore, there may be an association between the development of DKD and sarcopenia. Therefore, comprehensive medical management of T2D to prevent both DKD and sarcopenia is needed in clinical practice. However, at present, there are no universally accepted markers to monitor the degree of sarcopenia in DKD patients.
There is a possibility that exercise not only is indispensable for the prevention of sarcopenia but may also be useful to inhibit the progression of DKD. Although the effects of aerobic training on renal morphometric abnormalities in a Zucker fatty rat model of impaired glucose tolerance remains controversial [3], a recent study with the Zucker diabetic fatty rat reported that chronic exercise more certainly prevented interstitial profibrotic change rather than glomerular sclerosis via decreased oxidative stress and improved renal microcirculation by upregulation of endothelial nitric oxide synthase expression [4]. Another study demonstrated the renal antifibrosis effects of exercise by promoting skeletal muscle growth in a model of advanced renal interstitial fibrosis [5]. A recent study by our group revealed that tubulointerstitial damage due to inappropriate activation of the renin-angiotensin-aldosterone system was attenuated by voluntary running exercise [6]. Based on these results, we hypothesize that there may be some sort of relationship between tubulointerstitial damage and sarcopenia.
Urinary liver-type fatty acid-binding protein (L-FABP) accurately reflects the degree of tubulointerstitial damage [7,8] and has been accepted as a tubular marker by the Ministry of Health, Labour and Welfare in Japan [9]. Moreover, a study by our group found that urinary L-FABP levels were inversely associated with exercise capacity or physical activity and that aerobic exercise training decreased urinary L-FABP levels in healthy middle-aged and older adults [10,11]. Therefore, urinary L-FABP may be useful to monitor the progression of sarcopenia.
Among the limited models of both DKD and sarcopenia, the spontaneously diabetic Torii (SDT) fatty rat is a unique experimental novel model of T2D with kidney disease and sarcopenia [12][13][14] that mimics the pathophysiology of human T2D. Therefore, the aim of the present study was to elucidate the relationship between urinary L-FABP and sarcopenia using the SDT fatty rat.

Materials and Methods
2.1. Animals. All animal studies were conducted in strict accordance with the St. Marianna University School of Medicine Institutional Guide for Animal Experiments and the Guide for the Care and Use of Laboratory Animals. All surgery was performed under 3% isoflurane anesthesia, and all efforts were made to minimize suffering.
In this study, male SDT fatty (SDT.Cg-Lepr fa /JttJcl) rats, derived from a Sprague-Dawley (SD) colony, were used as a model of T2D. Five-week-old male SDT fatty rats (n = 5) and SD rats (n = 7) of the same age (controls) were purchased from CLEA Japan, Inc. (Tokyo, Japan) and housed under standard conditions with free access to laboratory chow (CRF-2, Charles River Laboratories Japan, Inc., Yokohama, Japan) and water. Body weight, chow consumption, and muscle strength were measured every 4 weeks from 16 to 24 weeks of age, which is equivalent to a late middle-aged human patient. Urine was collected from the rats using metabolic cages. Muscle strength was measured using the forelimb grip test with a grip strength meter (MK-380CM/FM; Muromachi Kikai, Co., Ltd., Tokyo, Japan). The average of three measurements of muscle strength per animal per time point was recorded for comparative analysis.
For the experiments, 16-, 20-, and 24-week-old rats were individually housed overnight in metabolic cages with free access to tap water. Urine was collected for the measurement of urinary markers. Additionally, serum samples, leg muscle tissues, and kidney tissues were collected from rats that were sacrificed at the age of 24 weeks. The extracted kidneys were weighted and cut into pieces for various analyses. The extracted muscles specimens were sectioned into the soleus muscle as a slow muscle and the extensor digitorum longus (EDL) muscle as a fast muscle. After weighing, each muscle was frozen individually in liquid nitrogen and stored at -80°C for further analyses.
2.2. Blood Pressure Measurement. Systolic blood pressure (SBP) was measured using a tail-cuff apparatus (Softron BP-98A; Softron, Co., Ltd., Tokyo, Japan) every 4 weeks from 16 to 24 weeks of age. The average of three measurements of SBP per animal per time point was recorded for analysis.
2.5. Renal Immunohistochemical Analysis. Target antigens in the preprocessed sections were stained using the indirect immunoperoxidase method, as described previously [7]. Briefly, formalin-fixed, paraffin-embedded tissue specimens were used to stain for myofibroblasts and tubular cells exhibiting epithelial-mesenchymal transition using a mouse monoclonal antibody specific for α-smooth muscle actin (α-SMA; dilution, 1 : 800; Sigma-Aldrich Corporation, St. Louis, MO, USA). The tissue specimens fixed in methyl Carnoy's solution were assessed immunohistochemically for macrophages using a mouse monoclonal antibody (ED-1) specific for CD68 (1 : 100; Abcam, Tokyo, Japan) and goat polyclonal antibodies specific for type I and III collagen (1 : 200; Southern Biotech, Birmingham, AL, USA). Labeled proteins were visualized using polymeric horseradish peroxidase-conjugated secondary antibodies (ImmPRESS™ Polymer Detection Kit; Vector Laboratories, Burlingame, CA, USA). Peroxidase activity was detected via the diaminobenzidine reaction (Liquid DAB+; DAKO Japan, Inc., Tokyo, Japan), and sections were counterstained with hematoxylin. For quantification, images from 10 nonoverlapping fields throughout the cortical and outer medullary regions were captured at 100x magnification. The extent of macrophage and myofibroblast infiltration of the cortical and outer medullary interstitia was automatically measured using an image analyzer (WinRoof version 6.4; Mitani Corporation, Tokyo Japan). Briefly, the areas positively stained for CD68 and α-SMA were independently measured and expressed as ratios relative to the areas of the entire cortical and outer medullary regions. The same method was used to measure the expression levels of type I and III collagen.
2.6. Immunohistochemical Analysis of Muscle Tissues. The frozen excised soleus and EDL muscle tissues were cut centrally along the width and embedded in Tissue-Tek® O.C.T.™ Compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan). Continuous frozen sections sliced to a thickness of 10 μm were prepared using a cryostat (HM 550; Thermo Fisher Scientific, Rockford, IL, USA). Double-color immunofluorescent staining was performed in order to evaluate the diameter of each type I and IIb fiber.
The dried sections were washed three times with phosphate-buffered saline (PBS) for 5 min each time and soaked in methanol solution containing 0.3% hydrogen peroxide for 20 min. After washing three times with PBS for 5 min again, the sections were incubated with the following primary antibodies at 4°C overnight: anti-type I myosin heavy chain (MyHC) isoform IgG2b (MyHCI For quantification, tissue images of the soleus and EDL muscles were obtained using ZEN 2 pro imaging software (Carl Zeiss Microscopy Co., Ltd., Tokyo, Japan). The crosssectional areas of the specimens were evaluated using 300 fibers in two visual fields at 100x magnification per animal using an image analyzer (WinRoof).
The ratios of phosphorylated to total proteins were quantitated by densitometry analysis of immunoblots using ImageJ software (https://imagej.nih.gov/ij/). The same membranes that were incubated with antibodies against FoxO1 and phospho-FoxO1A were also incubated with rabbit monoclonal anti-α-tubulin antibody (1 : 4000; Abcam). After treatment with a stripping buffer (Wako), FoxO1 protein expression was normalized to that of α-tubulin.

Evaluation of Oxidative Stress in Muscle and Kidney
Tissues. After determining protein concentrations, to evaluate oxidative stress in the muscle and kidney tissues, protein carbonyls, which are the most common products of protein oxidation, were quantified using an OxiSelect™ Protein Carbonyl ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA) with 100 μg of protein extracted from both tissue types.
2.9. Statistical Analysis. All values are expressed as the mean ± standard error of the mean (SEM). A probability (p) value of <0.05 was considered statistically significant. Following the Kruskal-Wallis test, differences among each SD and SDT rat were identified using the Steel-Dwass test. The Mann-Whitney U test was used for comparisons between two groups. Spearman's rank correlation coefficient was used to evaluate nonparametric data and assess correlations between two parameters. Changes in muscle strength of each rat throughout the experimental period were defined as the slope of the regression line of a line-graph plot of muscle strength against experimental duration (weeks). All statistical analyses were performed using JMP® software, version 13.0.0 (SAS Institute, Cary, NC, USA).

Changes in Body
Weight, Blood Glucose Level, SBP, and Food Intake. The body weight, blood glucose level, SBP, and food intake of all rats in both groups were measured every 4 weeks between the ages of 16 and 24 weeks. As shown in Table 1, while the mean body weight in the SD group was significantly greater at 24 weeks than 16 weeks, that of the SDT group increased, but not significantly. There were no significant differences in the body weights between the two groups throughout the experimental period. Blood glucose levels were significantly higher in the SDT group than the SD group throughout the experimental period. Although there was no change over time in the SD group, SBP levels in the SDT group moderately increased throughout the experimental period. Food intake increased significantly at 20 and 24 weeks compared to 16 weeks in the SD group. Additionally, food intake at 16 and 20 weeks was significantly greater in the SDT group than the SD group. Table 2, at 24 weeks, serum creatinine and urea nitrogen levels were significantly increased in the SDT group compared to the SD group, while there was no significant difference in serum cystatin C levels. Serum insulin levels were comparable between the two groups, while serum IGF-1 levels were significantly lower in the SDT group. Both serum total cholesterol and triglyceride levels were significantly greater in the SDT group than the SD group.

Comparison of Kidney Function and Serum Parameters at 24 Weeks of Age. As shown in
3.3. Changes in Urinary Glucose, L-FABP, and Albumin Levels. The urinary glucose scores of the SDT and SD groups at 16, 20, and 24 weeks remained unchanged at 4 and 0, respectively. In the SD group, urinary L-FABP (Figure 1(a)) and urinary albumin (Figure 1(b)) levels remained unchanged throughout the experimental period. Conversely, urinary L-FABP at 24 weeks (Figure 1(a)) and urinary albumin at both 20 and 24 weeks (Figure 1(b)) were significantly increased in the SDT group compared to 16 weeks. Also, urinary L-FABP (Figure 1(a)) and urinary albumin (Figure 1(b)) levels were significantly higher in the SDT group than the SD group throughout the experimental period.

Immunohistological Analysis of Kidney Tissues.
To evaluate the degree of renal interstitial inflammation, immunohistochemical analysis of the macrophage marker CD68 was conducted. Macrophages were detected in the interstitium of the SDT fatty rats (Figure 2(a)), and the degree of macrophage infiltration was significantly greater in the SDT group than the SD group (Figure 2(b)).
Additionally, immunohistochemical analyses of α-SMA and type I and III collagen were performed to evaluate the extent of tubulointerstitial fibrosis. Areas positive for α-SMA were observed in the tubules and interstitium of the SDT group (Figure 2(c)) to a significantly greater extent compared to the SD group (Figure 2(d)). Further analysis revealed that the expression levels of type I (Figures 2(e) and 2(f)) and III (Figures 2(g) and 2(h)) collagen were significantly greater in the SDT group than the SD group.
3.5. Evaluation of Glomerular Sclerosis. The mean glomerular sclerosis score was significantly greater for the PAS-stained kidney tissues of the SDT group than the SD group throughout the experimental period (Figures 2(i) and 2(j)).

Changes in Muscle Strength.
Muscle strength significantly increased in the SD and SDT groups at 24 weeks compared to 16 and 20 weeks but was significantly lower in the SDT group compared to the SD group at 20 and 24 weeks (Figure 3(a)). The rate of increased muscle strength was significantly lower in the SDT group than the SD group (Figure 3(b)).

Comparison of Muscle Weight and Immunohistological
Analysis of Muscle Tissues. The muscle weights of the soleus and EDL muscles were significantly lower in the SDT group than the SD group (Figures 3(c) and 3(d)). Since the soleus is mainly composed of slow-twitch muscle fibers [17], a cross-sectional area of type I muscle fibers was evaluated (Figure 3(e)). The EDL muscle has a high proportion of 4 Journal of Diabetes Research fast-twitch muscle fibers [17]; thus, a cross-sectional area of type IIb muscle fibers was evaluated (Figure 3(f)). Type I muscle fiber was stained green (Figure 3(e)) and type IIb muscle fiber was stained red (Figure 3(f)). The crosssectional areas of muscle fiber types I (Figure 3(g)) and IIb (Figure 3(h)) were significantly decreased in the SDT group compared to the SD group.

Evaluation of Muscle Protein Synthesis or Degradation.
Maintenance of muscle mass is regulated by the balance between protein synthesis and degradation. Hence, the activation of insulin/IGF-1-Akt and mTOR-S6K signaling as markers of protein synthesis and the activation of insulin/IGF-1-Akt-FoxO signaling, phosphorylation of AMPK, and expression of E3 ubiquitin ligases as markers of protein degradation were evaluated.
The phosphorylation levels of Akt (Figure 4(a)), mTOR (Figure 4(b)), and S6K (Figure 4(c)) were significantly lower, while that of AMPK ( Figure 5(a)) was significantly higher in the soleus muscle of the SDT group compared to the SD group. Regarding the EDL muscle, although the phosphorylation levels of both Akt (Figure 4(a)) and AMPK ( Figure 5(a)) were not significantly different between the SDT and SD groups, those of mTOR (Figure 4(b)) and S6K (Figure 4(c)) were significantly lower in the SDT group.
FoxO1 protein expression was significantly higher (Figure 5(b)), while that of phospho-FoxO1 was significantly lower, in both the soleus and EDL muscles of the SDT group compared to the SD group ( Figure 5(c)).
Regarding the protein expression profiles of E3 ubiquitin ligases (Figures 5(d) and 5(e)), Atrogin-1 ( Figure 5(e)) expression levels were significantly higher in the EDL muscle of the SDT group than the SD group, but not MuRF-1 ( Figure 5(d)). There was no significant change in Atrogin-1 and MuRF-1 expression levels in the soleus muscle between the SDT and SD groups (Figures 5(d) and 5(e)).

Correlation between Renal Morphological Change and
Muscle Strength and Weight. The degrees of macrophage infiltration and α-SMA expression at 24 weeks were inversely Data are presented as the mean (standard error). * p < 0:05 and * * p < 0:01 vs. the SD rats at the same week; † p < 0:05 and ‡ p < 0:01 vs. the same group at 16 weeks of age.  (Table 3). Type I and III collagen expressions were inversely correlated with the weights of the soleus and EDL muscles, but not strength (Table 3). Glomerular sclerosis score was inversely correlated with the strength and weight of EDL muscles, but not the weight of the soleus (Table 3).

Evaluation of Oxidative Stress in Kidney and Muscle
Tissues. While protein carbonyl levels in kidney tissues were significantly higher in the SDT group (Figure 10(a)), those in the soleus muscle, but not the EDL muscle, were significantly lower compared to those in the SD group (Figures 10(b) and 10(c)).

Discussion
The results of the present study revealed that urinary L-FABP secretion reflected decreases in muscle strength, muscle weight, and cross-sectional areas of muscle fibers, and the high levels of urinary L-FABP at 16 weeks were associated with a lower increasing rate of muscle strength in mature SDT fatty rats with hyperglycemia, hyperlipidemia, low levels of serum IGF-1, and kidney disease. The current study indicated that urinary L-FABP might be useful to monitor the progression of sarcopenia in addition to DKD in T2D patients.
There is growing evidence of a correlation between sarcopenia and kidney disease, including DKD [18]. The results of the present study indicated a correlation between urinary markers and sarcopenia in mature rats with T2D and DKD. Our previous studies found that lower physical activity or weaker muscle strength was related to increased urinary L-FABP levels, but not urinary albumin [10], and that intervention with aerobic exercise training facilitated a reduction in increased urinary L-FABP in healthy middle-aged and older adults [11]. Furthermore, decreased muscle strength was independently associated with increased intrarenal vascular resistance [19], suggesting that sarcopenia might lead to impairments to the intrarenal vasculature. Considering that increased urinary L-FABP is induced by decreased renal microvascular blood flow [20] and that renal hypoxia was observed in the SDT fatty rats with DKD (unpublished data), urinary L-FABP might be useful for the detection of abnormalities in renal hemodynamics aggravated by sarcopenia in addition to glomerular sclerosis leading to renal hypoxia derived from reduction of postglomerular blood flow. On the contrary, accumulation of uremic toxins, metabolic acidosis, and anemia due to deterioration of renal function is reported to lead to sarcopenia [18]. Although renal dysfunction was mild in the SDT fatty rats in the present study, muscle mass loss was reported to appear in the early phase of CKD [21]. Therefore, in the present study, DKD might have influenced the progression of sarcopenia.
The progression of sarcopenia in T2D patients has been associated with lower renal function and higher urinary albumin levels [22]. As a marker of the severity of kidney disease, urinary albumin may reflect the degree of muscle weakness in patients with kidney disease. In the present study, urinary albumin at 16 weeks was significantly related to continued muscle growth in all rats used in the study, although the significant correlation disappeared in only SDT fatty rats. Hence, further studies are needed to determine whether urinary albumin, like urinary L-FABP, is a risk factor for the onset of sarcopenia.
In the skeletal muscle, there are four major kinds of muscle fiber: type I, type IIa, type IIx, and type IIb. The predominance of one over the other is dependent on the contractile rate and metabolic characteristics. While type I † SDT fatty  Journal of Diabetes Research fibers, which are slow-twitch and oxidative fibers, are influenced by insulin resistance in T2D [23], type IIb fibers, which are fast-twitch and glycolytic fibers, are influenced by aging [24]. Although serum insulin levels were similar in the SDT and SD groups, blood glucose levels were significantly higher in the SDT group, suggesting the presence of insulin resistance. Regarding the influence of muscle fiber on kidney disease, CKD induced insulin resistance [25], leading to a decrease in the proportion of type I fibers, as previously shown in the gastrocnemius muscle of a CKD model [26,27]. In the present study, type I fibers in the soleus muscle and type IIb fibers in the EDL muscle were assessed sepa-rately, which revealed decreases in both muscle weight and cross-sectional areas of type I and IIb fibers in the present study. Based on these results, insulin resistance, aging, and DKD might be related to the progression of sarcopenia in SDT fatty rats. Insulin or the IGF-1 signaling pathway plays a critical role in the regulation of muscle mass by controlling the balance between protein synthesis and degradation in muscle [28]. As a critical downstream effector of the insulin/IGF-1 pathway, phosphorylation of Akt leads to activation of protein synthesis via phosphorylation of both mTOR and S6K or inhibition of protein degradation via FoxO1 7 Journal of Diabetes Research phosphorylation, which results in downregulated expression levels of the E3 ligases Atrogin-1 and MuRF-1 [28]. Furthermore, AMPK, which is known to be a cellular fuel sensor activated by ATP depletion, is related to suppression of protein synthesis via decreased phosphorylation of mTOR [29] and activation of the FoxO-dependent protein degradation pathway [30]. In the present study, phosphorylation of Akt decreased and that of AMPK increased in the soleus muscle, but not in the EDL muscle, in the SDT fatty rats. Phosphorylation of mTOR, S6K, and FoxO1 was reduced in both the soleus and EDL muscles. Although increased Atrogin-1 expression was observed in the EDL muscle, there was no significant difference in MuRF-1 expression in the soleus and EDL muscles between the SDT and SD groups. Regarding the molecular mechanism underlying the progression of sarcopenia in the SDT group, disruption to insulin/IGF-1-Akt signaling due to insulin resistance and low serum IGF-1 levels was considered to provoke abnormalities in muscle strength and mass in the SDT group ( Figure 11). Furthermore, while decreased muscle protein synthesis was considered to contribute to weakness of both the soleus and EDL muscles in the SDT group, the involvement of muscle protein degradation signaling in muscle depression in the SDT group may be low (Figure 11). Although the results of this study support those of a previous study [14], further studies are needed to investigate the association of FoxOassociated downstream pathways with muscle depression in SDT fatty rats.
Oxidative stress is reported to promote muscle atrophy and weakness [31][32][33] in addition to CKD. However, decreased oxidative stress, as evaluated by carbonylation of muscle proteins, was observed in the soleus muscle of SDT fatty rats, despite increased oxidative stress in the kidney tissues. Antioxidants are reported to diminish the positive effects of exercise training via decreased generation of reactive oxidative species by exercise [34]. Misu et al. reported that selenoprotein P in the liver, which has antioxidative activities, was increased in T2D patients and contributed to the elimination of the beneficial effects of exercise on muscle [35]. Although the mechanism underlying the decreased oxidative stress in the soleus muscle of SDT fatty rats remains unknown, a certain degree of oxidative stress may be needed to maintain muscle mass and force mainly by type I oxidative fibers. Oxidative stress is not a common aggravating factor related to the progression of sarcopenia and DKD.
The results of the present study may be limited by several factors. First, we found no independent association between DKD and muscle atrophy in the SDT fatty rats. This point ‡ SDT fatty   Journal of Diabetes Research needs to be clarified in further studies aimed to determine whether muscle atrophy is prevented along with treatment of DKD. Second, while muscle strength decreases along with age and develops to sarcopenia in humans, muscle strength slightly increased during the experimental period and retention of muscle growth was observed in the SDT fatty rats from 16 to 24 weeks of age. Therefore, sarcopenia in humans was not completely reproduced in the present study; thus, further clinical studies of T2D patients are needed in order to reveal the clinical usefulness of urinary L-FABP as a marker of the progression of sarcopenia. Third, we did not determine the most suitable marker for sarcopenia because sample size was limited in the basic research. Future clinical research is needed to reveal the point. Finally, we did not investigate changes in urinary L-FABP levels in response to interventions that ameliorate renal hypoxia. Finally, we did not investigate changes in urinary L-FABP levels in response to interventions that prevent muscle atrophy. Because we recently demonstrated that habitual exercise led to a reduction in urinary L-FABP levels in healthy middle-aged and older adults [11], urinary L-FABP may respond to the relief of secondary sarcopenia due to diabetes and kidney disease.

Conclusion
In conclusion, urinary L-FABP reflects the degrees of muscle strength, muscle weight, and cross-sectional areas of muscle fibers. Although the detailed mechanisms of the interaction between sarcopenia and DKD was not revealed in this study, urinary L-FABP may be useful to monitor the progression of sarcopenia in addition to DKD in T2D patients. Nonetheless, future clinical studies of T2D patients are needed.    Figure 11: Molecular mechanism underlying the progression of sarcopenia in type 2 diabetes. Disruption to insulin/IGF-1-Akt signaling due to insulin resistance and low serum IGF-1 levels was considered to reduce muscle protein synthesis and to provoke abnormalities in muscle strength and mass in the SDT fatty rats. Furthermore, phosphorylation of AMPK due to reduced cellular uptake of glucose led to suppression of muscle protein synthesis. On the other hand, the involvement of muscle protein degradation signaling in muscle depression in the SDT group may be low.