**4.3 AGEs and insulin resistance in skeletal muscle**

In skeletal muscle, the initial insulin signaling events include insulin binding to the extracellular α-subunit of the insulin receptor, rapid phosphorylation of the receptor (auto-phosphorylation) and insulin receptor substrate (IRS)-1 on tyrosine residues, and recruitment and activation of class IA phosphatidylinositol 3-kinase. These lead to the generation of the critical second messenger PI-3,4,5-triphosphate, which in turn triggers the activation of Akt [51]. TBC1 domain (TBC1D) family member 1 and TBC1D4 act as downstream mediators of Akt. TBC1D1 and TBC1D4 contain a Rab-GTPase-activating protein domain that prevents glucose transporter 4 (GLUT4) translocation by inactivating Rab proteins. TBC1D1 and TBC1D4 dissociate from GLUT4 vesicles in the phosphorylated state, and thereby facilitating GLUT4 translocation and glucose transport [52, 53].

Many evidence have shown that AGEs impair insulin signaling transduction and induce insulin resistance in skeletal muscle. Miele et al. [54] showed that exposure of glycated albumin (0.1–0.2 mg/ml) to skeletal muscle cells for 24 hours impaired insulin-stimulated 2-deoxyglucose uptake, accompanied by reduced IRS-1 tyrosine phosphorylation, Akt activity, but not insulin receptor kinase activity, suggesting that AGEs affect factors downstream from insulin receptor. AGEsinduced inhibition of glucose transport was supported by the work of Wu et al. [55] that exposure of glyoxal-derived AGEs (0.1 mg/ml) to skeletal muscle cells for 8–48 hours completely abolished 2-deoxyglucose uptake. Their subsequent research found that AGEs-induced impairment of insulin action might be mediated by the formation of multimolecular complex among RAGE/IRS-1/src and protein kinase C [56]. Animal study by Rai et al. [57] demonstrated that fructose intake (20% in drinking water) for 16 weeks decreased insulin-stimulated Akt phosphorylation accompanied by elevated serum and muscle AGEs level and RAGE mRNA level in rat skeletal muscle. However, these changes were suppressed by co-ingestion of AGEs inhibitor aminoguanidine (100 mg/kg). Pinto-Junior et al. [58] showed that injection of glycolaldehyde-derived AGEs (20 mg/kg/day) to rat for 12 weeks led to whole-body insulin resistance and decreased GLUT4 mRNA and protein levels in skeletal muscle. Furthermore, they demonstrated that exposure of glycolaldehydederived AGEs (1.0 mg/ml) to skeletal muscle cells for 2.5 hours increased nuclear factor (NF)-κB expression and nuclear protein binding activity into a GLUT4 gene promoter NF-κB binding site, suggesting that AGEs reduce GLUT4 transcription through NF-κB signaling. These AGEs-induced aggravating effect on insulin signaling may induce skeletal muscle insulin resistance, and thereby contributing to impairment of whole-body glucose homeostasis with aging or diabetes.

#### **4.4 The effect of AGEs on myogenesis, development, atrophy of skeletal muscle**

As described above, AGEs are a crucial factor affecting skeletal muscle functions, especially skeletal muscle mass. Considering the formation process of AGEs derived from sugar, it has been initially investigated the effects of AGEs on diabetic muscle atrophy. Snow et al. [59] observed distribution patterns of AGEs in skeletal muscle of diabetic rats and found the presence of CML intracellularly and at sites along the muscle fiber periphery. However, there was no difference in muscle fiber cross-sectional area between AGEs-positive and -negative fibers in both control and diabetic rats, indicating the indirect effect of intracellular AGEs on muscle size. Alternatively, a more detailed study by Chiu et al. [60] demonstrated that decreased muscle mass and fiber cross-sectional area in diabetic rats was attenuated by the 4-week treatment with AGEs inhibitor, alagebrium chloride, accompanied by decreased AGEs and RAGE expressions. They also investigated the direct effect of AGEs on muscle atrophy and found that exposure of glucose-derived AGEs (0.025– 0.2 mg/ml) to human primary skeletal muscle cells for 48 hours induced myotube atrophy via RAGE, 5'AMP-activated protein kinase, and Akt signaling-mediated upregulation of ubiquitin-proteasome system.

Our recent study supports the involvement of RAGE in skeletal muscle atrophy. In addition to diabetes, muscle disuse due to injury, casting, and bedrest is a potent inducer of muscle mass loss [61]. However, there was no evidence that glycation stress was involved in disuse-induced skeletal muscle atrophy. Therefore, we investigated the contribution of RAGE to disuse-induced skeletal muscle atrophy [62]. Our study showed that 1-week hindlimb suspension procedure to mice led to muscle atrophy accompanied by intracellular MG-H1 and CML accumulations. However, treatment with RAGE antagonist during the suspension attenuated the atrophic response (**Figure 3**), and muscle mass inversely correlated with the accumulation of MG-H1 and CML in skeletal muscle (**Figure 4**). RAGE inhibition also suppressed the atrophy-associated expression of proinflammatory cytokines and activation of ubiquitin-proteasome system. These findings suggest the contribution of RAGE to

#### **Figure 3.**

*Soleus weight normalized to body weight and muscle fiber cross sectional area (CSA) after hindlimb suspension and/or receptor for AGEs (RAGE) antagonist treatment. Mice in the HS group were subjected to continuous hindlimb suspension for 1 week. Age-matched mice that did not undergo hindlimb suspension were used as controls (CON). Mice in the HS + FPS-ZM1 group were injected daily intraperitoneally with 1 mg/kg FPS-ZM1, a RAGE antagonist, during hindlimb suspension. Data are expressed as means ± SE; n = 7–9 per group. Individual data points are indicated on the bar graph. Representative images of immunofluorescence are shown. Scale bars, 50* μ*m. The value of effect size is listed in parentheses. Statistical significance was analyzed using Tukey–Kramer multiple comparison tests. This figure was adapted from Egawa et al. [62] with permission from the publisher.*

*The Effect of Glycation Stress on Skeletal Muscle DOI: http://dx.doi.org/10.5772/intechopen.97769*

**Figure 4.**

*The correlation between methylglyoxal (MG)- or N*ε*-(carboxymethyl) lysine (CML)-modified protein level and muscle weight. For the quantification of MG-modified and CML-modified proteins, the signal intensity of full-molecular-weight was quantified after western blotting. n = 8 per group. Statistical significance was assessed using Pearson's correlation. This figure was adapted from Egawa et al. [62] with permission from the publisher.*

disuse-induced skeletal muscle atrophy. Furthermore, in this study, RAGE expression was increased in response to suspension, and this was limited to atrophied soleus and plantaris muscles but not unatrophied extensor digitorum longus muscle. Therefore, muscle disuse itself but not systemic mediators may regulate RAGE expression.

The effect of glycation stress on muscle growth was first reported in our research [63]. We evaluated the differences in muscle mass, contractile properties and molecular responses between mice that received a diet containing high-AGEs and low-AGEs for 16 weeks [63]. As a result, exposure to a high-AGEs promoted CML accumulation in skeletal muscle, suppressed muscle growth, and induced skeletal muscle dysfunctions including suppression of muscle strength, fatigue resistance, and force production (**Figure 5**). In addition, the expression of myogenic factors and phosphorylation of p70 s6 kinase, an enzyme playing a key role in the regulation of protein synthesis, were decreased in the high-AGEs treated group. These results suggest that exposure to AGEs impairs postnatal growth and muscle development.

To clarify the underlying mechanism of AGEs-induced inhibition of muscle growth, we next carried out the comprehensive analysis of protein phosphorylation status by using the reverse phase protein array method [64]. In the study, the average level of phosphorylation of skeletal muscle cells exposed to various kinds of AGEs (glyoxylic-, pyruvate, glycolaldehyde, and glucose-derived AGEs, 0.1 mg/ml) was increased at eight phosphorylation sites and decreased at 64. The most upregulated phosphorylation sites were signal transducer and activator of transcription 3 (STAT3) Tyr705. The most downregulated phosphorylation sites were extracellular signal-regulated kinase (ERK) Thr202/Tyr204. Almost all of the phosphorylation sites related to insulin/insulin-like growth factor 1 (IGF-1) signaling were also downregulated by AGEs (**Figure 6**). Increased STAT3 Tyr705 phosphorylation and decreased ERK Thr202/Tyr204 phosphorylation were also confirmed in the skeletal muscles of mice treated with a diet high in AGEs for 16 weeks. These results suggest that systemic AGEs modulate cellular signaling transduction pathways, such as STAT3 and insulin/IGF-1 signaling, and thereby contribute to the impairment of skeletal muscle growth and development. Accordingly, Adachi et al. demonstrated that IGF-1 treatment protected AGEs-induced deterioration of myogenic differentiation in skeletal muscle cells [65].

#### **Figure 5.**

*The grip strength test, wire-hanging test, and in vitro force production of plantaris muscles in mice fed a diet low in AGEs (L-AGEs) or a diet high in AGEs (H-AGEs). The grip strength test and wire-hanging test was conducted 5 and 4 days before the end of the 16-week study, respectively. For measuring in vitro forth production, isolated plantaris muscle was allowed to rest for 30 min and the muscle was tetanically contracted at frequencies of 0, 5, 10, 25, 50, 75, and 100 Hz with a 2 min rest between contractions. Data are expressed as mean ± SE, n = 10 per group. \*P < 0.05 vs. L-AGEs mice. Statistical significance was analyzed using Student's t test or Tukey–Kramer multiple comparisons tests. This figure was adapted from Egawa et al. [63] with permission from the publisher.*
