**5. ECM and the metabolic function of skeletal muscle**

days of training. MMP-2 and 9 proteins were both present in the ECM, around myofibers and

Mechanical loading induces the secretion of TGF-beta, PDGF, and bFGF in tendon fibroblasts; moreover, it increases the expression of collagen and other ECM components, such as proteo‐ glycans. TGF-beta stimulates collagen formation and reduces its degradation, also via activation of the TIMPs, together with a suppression of MMPs, leading to the ECM accumu‐ lation. TGF-beta is known to function as a modulator of ECM proteins and to induce both collagen gene activation and protein formation. In a human model of microdialysis of the Achilles tendon, both local and systemic levels of TGF-beta increase in response to 1 h of running, proving a release of this cytokine from tissues that are mechanically activated during exercise and suggesting a role in the response to mechanical loading in vivo [1]. Mechanical loading induces FGF release from skeletal muscle cells in vitro. Several isoforms of FGFs exist; of these, basic FGF (or FGF2) and, to a lesser extent, the acidic FGF (FGF1) stimulate fibroblast proliferation and collagen synthesis. Interleukin-6 (IL-6) is considered as a physical activityassociated myokine released from working muscles [45]. It can stimulate fibroblasts to increase the synthesis of collagens, glycosaminoglycans, hyaluronic acid, and chondroitin sulfates. Increased expression of IL-6 is necessary for the regulation of ECM remodeling during the hypertrophic response of skeletal muscle to overload [46]. Mechanical activity increases expression of IL-1beta in human and rabbit tendon cells, leading to increased MMPs activity, diminished collagen synthesis, and initiating tissue degradation and remodeling in response to loading. IGF-I is directly involved in skeletal muscle ECM synthesis after mechanical loading. This growth factor increases the expression of types I and III collagen in intramuscular fibroblasts. Bioavailability of IGF-I is controlled by IGF-binding proteins, and increased proteolysis of IGFBPs occurs in response to prolonged training in humans. Interestingly, MMPs can degrade IGFBPs, which provides a possible mechanism of regulation of the free

The blood flow in skeletal muscle is tightly coupled with the metabolic demands of contracting myofibers. During exercise, local mechanisms cause rapid dilation of muscle arterioles to increase the flow of blood to the working muscle. It appears that fibronectin fibrils in the extracellular matrix transduce signals from actively shortening skeletal muscle fibers to local blood vessels to increase blood flow. Skeletal muscle contraction alters the conformation of ECM fibronectin, which results in transient exposure of specific matricryptic sites. These sequences are not exposed in the soluble form of ECM molecules, but may be expressed due to structural or conformational changes, providing "a reserve" of signaling sites activated during ECM remodeling. Matricryptic fibronectin sites (FNIII-1) interact with FNIII-1H receptors on smooth muscle cells and/or skeletal muscle fibers. This activates the neuronal nitrogen oxide (NO) synthase to release NO, which leads to smooth muscle relaxation, vasodilation, and increased blood flow. Thus, FNIII-1 sites in ECM fibronectin serve as important mechanical coupling between skeletal muscle contraction and arteriolar dilation [47]. **Figure 3** summarizes the cellular mechanisms activated during exercise leading to skeletal

capillaries, but MMP-2 was also visible within the skeletal muscle fibers [44].

12 Composition and Function of the Extracellular Matrix in the Human Body

IGF-I in skeletal muscle tissue and circulation.

muscle ECM remodeling.

Skeletal muscle is a key insulin-sensitive tissue, important in maintaining homeostasis, due to its relatively large mass and energy needs [48,49]. Postprandial, insulin-stimulated glucose disposal in skeletal muscle results from the activation of a complex signaling network with multiple alternative and complementary pathways. Insulin binding to the insulin receptor causes tyrosine autophosphorylation of the receptor beta-subunit, activation of its intrinsic tyrosine kinase, and subsequent phosphorylation of several intracellular proteins, including insulin receptor substrate (IRS) proteins [50]. This leads to the recruitment of further signaling components such as phosphatidylinositol-3 kinase (PI-3 kinase), the tyrosine phosphatase SHPTP2, the growth factor receptor-binding protein-2 (GRB-2), as well as protein serine/ threonine kinases: phosphoinositide-dependent protein kinase (PDK1), protein kinase B (PKB), atypical isoforms of protein kinase C (PKC) lambda and zeta, mitogen-activated protein kinase (MAPK), and others, which support the signal divergency and function as messengers for various biological effects of insulin. Regarding postprandial glucose uptake in skeletal muscle, the activation of insulin signaling leads to the translocation of the insulin-responsive glucose transporter, Glut4, from intracellular storage sites to cell surface membrane, which is a critical step in cellular glucose utilization. Dysregulation of any step of this process in skeletal muscle results in insulin resistance, predisposing for diabetes.

There is an important cross-talk between extracellular matrix and insulin signaling in skeletal muscle. Integrin engagement stimulates both IRS-1-associated PI-3 kinase activity and PKB/Akt pathway. Integrin receptor beta1 subunit increases insulin-stimulated IRS phos‐ phorylation, IRS-associated PI-3 kinase, and activation of PKB (summarized in [51]). Regula‐ tion of focal adhesion kinase (FAK) by integrin receptors modulates insulin-dependent cytoskeleton organization, glucose transport, and glycogen synthesis in myoblasts [4]. FAK can interact with IRS-1, PI-3 kinase, PKC, and glycogen synthase kinase-3beta, leading to translocation of Glut4. A decrease in tyrosine phosphorylation and activation of FAK was reported in skeletal muscle of insulin-resistant Spraque-Dowley rats fed with a high-fat diet, as well as in insulin-resistant C2C12 myoblasts [52]. The expression of IRS-1 mRNA is abolished in FAK knockout mouse fibroblasts. Apart from the regulation of skeletal muscle insulin signaling and action by FAK, the reciprocal interaction is documented. It appears that FAK tyrosine phosphorylation, essential for skeletal muscle differentiation, is modulated by insulin. Insulin causes an increase in FAK phosphorylation in proliferating myoblasts, while in differentiating cells, there is an inhibition of FAK phosphorylation [53]. Under insulin resistance, the phosphatase PTEN and SHIP2, usually recognized as negative regulators of insulin signaling, are up-regulated, and they impair insulin action through FAK dephosphor‐ ylation [54]. The integrin-linked kinase (ILK) can phosphorylate and activate PKB, and function as its potential upstream regulator. Integrin beta1 knockout mice manifest an impairment of insulin-stimulated skeletal muscle glucose uptake and glycogen synthesis in skeletal muscle, resulting from marked reduction in ILK expression and concomitant decrease in PKB phosphorylation.

Insulin resistance is tightly associated with the ECM remodeling in muscle, and the ECM defects predisposing to diabetes-related symptoms are known. The deposition of collagens, the most abundant structural ECM components, is increased in insulin-resistant muscles, both in humans and rodent experimental models [55]. Synthesis of fibronectin, laminin, and collagen IV is up-regulated by high glucose and diabetes [56], which may lead to basement membrane thickening and the development of diabetes-associated microangiopathy. Similarly, a high-fat diet causes an increase in collagen IV in skeletal muscle [57]. As MMPs are responsible for the degradation of all components of the ECM, their dysregulation is also implicated in the pathology of diabetes and obesity. MMP-9 activity in skeletal muscle is decreased in high fat-fed mice, and it is related inversely to muscle collagen deposition and directly to muscle insulin resistance [58]. The genetic deletion of MMP-9 worsens diet-induced muscle insulin resistance, indicating that this metalloproteinase is necessary to protect against more serious metabolic disturbances associated with high fat feeding. Collagen V, widely expressed and a less abundant fibrillar protein, which regulates collagen fibril geometry and strength, is important for skeletal muscle glucose homeostasis. Mutant mice lacking *col5a3* gene manifest hyperglycemia, glucose intolerance, and insulin resistance [59]. Skeletal muscle of these mutants is defective in glucose uptake and mobilization of intracellular Glut4 glucose transporter to the plasma membrane in response to insulin.

components such as phosphatidylinositol-3 kinase (PI-3 kinase), the tyrosine phosphatase SHPTP2, the growth factor receptor-binding protein-2 (GRB-2), as well as protein serine/ threonine kinases: phosphoinositide-dependent protein kinase (PDK1), protein kinase B (PKB), atypical isoforms of protein kinase C (PKC) lambda and zeta, mitogen-activated protein kinase (MAPK), and others, which support the signal divergency and function as messengers for various biological effects of insulin. Regarding postprandial glucose uptake in skeletal muscle, the activation of insulin signaling leads to the translocation of the insulin-responsive glucose transporter, Glut4, from intracellular storage sites to cell surface membrane, which is a critical step in cellular glucose utilization. Dysregulation of any step of this process in skeletal

There is an important cross-talk between extracellular matrix and insulin signaling in skeletal muscle. Integrin engagement stimulates both IRS-1-associated PI-3 kinase activity and PKB/Akt pathway. Integrin receptor beta1 subunit increases insulin-stimulated IRS phos‐ phorylation, IRS-associated PI-3 kinase, and activation of PKB (summarized in [51]). Regula‐ tion of focal adhesion kinase (FAK) by integrin receptors modulates insulin-dependent cytoskeleton organization, glucose transport, and glycogen synthesis in myoblasts [4]. FAK can interact with IRS-1, PI-3 kinase, PKC, and glycogen synthase kinase-3beta, leading to translocation of Glut4. A decrease in tyrosine phosphorylation and activation of FAK was reported in skeletal muscle of insulin-resistant Spraque-Dowley rats fed with a high-fat diet, as well as in insulin-resistant C2C12 myoblasts [52]. The expression of IRS-1 mRNA is abolished in FAK knockout mouse fibroblasts. Apart from the regulation of skeletal muscle insulin signaling and action by FAK, the reciprocal interaction is documented. It appears that FAK tyrosine phosphorylation, essential for skeletal muscle differentiation, is modulated by insulin. Insulin causes an increase in FAK phosphorylation in proliferating myoblasts, while in differentiating cells, there is an inhibition of FAK phosphorylation [53]. Under insulin resistance, the phosphatase PTEN and SHIP2, usually recognized as negative regulators of insulin signaling, are up-regulated, and they impair insulin action through FAK dephosphor‐ ylation [54]. The integrin-linked kinase (ILK) can phosphorylate and activate PKB, and function as its potential upstream regulator. Integrin beta1 knockout mice manifest an impairment of insulin-stimulated skeletal muscle glucose uptake and glycogen synthesis in skeletal muscle, resulting from marked reduction in ILK expression and concomitant decrease

Insulin resistance is tightly associated with the ECM remodeling in muscle, and the ECM defects predisposing to diabetes-related symptoms are known. The deposition of collagens, the most abundant structural ECM components, is increased in insulin-resistant muscles, both in humans and rodent experimental models [55]. Synthesis of fibronectin, laminin, and collagen IV is up-regulated by high glucose and diabetes [56], which may lead to basement membrane thickening and the development of diabetes-associated microangiopathy. Similarly, a high-fat diet causes an increase in collagen IV in skeletal muscle [57]. As MMPs are responsible for the degradation of all components of the ECM, their dysregulation is also implicated in the pathology of diabetes and obesity. MMP-9 activity in skeletal muscle is decreased in high fat-fed mice, and it is related inversely to muscle collagen deposition and

muscle results in insulin resistance, predisposing for diabetes.

14 Composition and Function of the Extracellular Matrix in the Human Body

in PKB phosphorylation.

High-ambient glucose markedly elevates the level of fibronectin in myogenic cells in vitro and causes a decrease in cellular content of the full length 100 kDa form of ADAM12, without affecting integrin alpha5 and integrin beta1 subunit expressions [60]. Such alterations could result in the disturbances in ECM remodeling and accumulation, which in turn contribute to the impairment of the myogenic differentiation, manifested by decrease in MyoD, myogenin,

**Figure 4.** Proposed schema illustrating the cross-talk between insulin signaling and ECM signaling in skeletal muscle. To clarify the picture, both the insulin signaling pathway and ECM signaling are markedly simplified, as they present only the most important linkings and biological effects. Solid lines mean direct connections, dashed lines mean indirect effects. → means activation/stimulation, ┤means inhibition. Green lines indicate interactions between insulin- and in‐ tegrin-activated pathways (direct or indirect) reported in skeletal muscle. Blue lines indicate interactions described in other cell types [61], and only potentially functioning in skeletal muscle tissue.

myosin heavy chain levels, and fusion index. In view of an important cross-talk between ECM and insulin signaling [4,51], the high-glucose-induced alterations in ECM can, at least partly, contribute to the attenuated insulin and growth factors' action in skeletal muscle under hyperglycemia and diabetes. The ECM turnover also plays a role in the metabolic regulation of skeletal muscle in the pathology of diet-induced insulin resistance. **Figure 4** illustrates the most important points of the cross-talk between insulin signaling and the ECM-related signaling cascades in skeletal muscle.
