Type 2 diabetes-related sarcopenia: role of nitric oxide

Abstract

Sarcopenia, characterized by progressive and generalized loss of skeletal muscle (SkM) mass, strength, and physical performance, is a prevalent complication in type 2 diabetes (T2D). Nitric oxide (NO), a multifunctional gasotransmitter involved in whole-body glucose and insulin homeostasis, plays key roles in normal SkM physiology and function. Here, we highlight the role of NO in SkM mass maintenance and its potential contribution to the development of T2D-related sarcopenia. Physiologic NO level, primarily produced by sarcolemmal neuronal nitric oxide synthase (nNOSμ isoform), is involved in protein synthesis in muscle fibers and maintenance of SkM mass. The observed effect of nNOSμ on SkM mass is muscle-type specific and sex-dependent. Impaired NO homeostasis [due to a diminished nNOSμ-NO availability and excessive NO production through inducible NOS (iNOS) in response to atrophic stimuli, e.g., inflammatory cytokines] in SkM occurred during the development and progression of T2D, may cause sarcopenia. Theoretically, restoration of NO through nNOS overexpression, supplying NOS substrates (e.g., L-arginine and L-citrulline), phosphodiesterase (PDE) inhibition, and supplementation with NO donors (e.g., inorganic nitrate) may be potential therapeutic approaches to preserve SkM mass and prevents sarcopenia in T2D.

Introduction

Sarcopenia, from the Greek sarx (flesh) and penia (loss), was first introduced by Irwin Rosenberg in 1989 to describe age-related muscle mass loss, but it now encompasses progressive, generalized loss of skeletal muscle (SkM) mass, strength, and physical function [1]. Primarily associated with ageing (primary sarcopenia), it can also develop alongside chronic diseases, malnutrition, cachexia, or inactivity, known as secondary sarcopenia [2, 3]. Sarcopenia negatively impacts quality of life, mobility, and independence while imposing significant healthcare costs [2]. In healthy adults, SkM mass comprises approximately 38.4% of body weight in men and 30.6% in women [4], corresponding to an SkM mass index [SMMI, muscle mass (kg)/height (m)²] of 8.98–8.31 and 6.80–6.78 kg/m², respectively [5]. Sarcopenia is diagnosed based on SMMI thresholds of 5.86–7.40 kg/m² in men and 4.42–5.67 kg/m² in women [6]. The mechanisms underlying sarcopenia involve a reduction in muscle fiber number and size, decreased cross-sectional area (CSA), impaired innervation [7, 8], and changes in SkM protein turnover (i.e., an imbalanced rate of protein synthesis and degradation) [9].

Secondary sarcopenia is a prevalent complication in type 2 diabetes (T2D), affecting 16–30% of patients [10,11,12]. Compared to individuals with normoglycemia, patients with T2D have a 1.5 to 3.0-fold greater risk of sarcopenia [10,11,12]. Older adults with T2D experienced ~ 1.5-fold greater annual muscle mass loss and a 33% greater annual decline in muscle strength than aged-matched non-T2D subjects [13].

Nitric oxide (NO), a multifunctional gasotransmitter involved in whole-body glucose and insulin homeostasis [14,15,16], plays critical roles in normal SkM physiology and function. Impaired NO homeostasis in SkM, which occurred during the development and progression of T2D [17,18,19,20], may cause SkM atrophy and developing sarcopenia. Here, we highlight the role of NO in SkM mass maintenance and its potential contribution to the development of T2D-related sarcopenia. We will discuss how boosting the NO system in SkM may emerge as a promising therapeutic approach targeting sarcopenia in patients with T2D.

Type 2 diabetes-related sarcopenia

Sarcopenia is increasingly viewed as both a cause and consequence of T2D, with common genetic signatures and shared pathophysiological pathways between the two conditions [21]. However, T2D-related sarcopenia is distinct from primary sarcopenia and disuse muscle atrophy (in case of etiology, age characteristics, pathological changes, and underlying mechanisms) [22]. For instance, unlike primary sarcopenia, T2D-related sarcopenia may occur not only in the elderly but also in younger individuals [22]. While primary sarcopenia is predominantly characterized by atrophy of type II fibers [including fast-oxidative glycolytic (FOG) and fast-glycolytic (FG)] [23, 24], T2D-related sarcopenia primarily affects type I or slow-oxidative (SO) fibers [25]. SO fibers have higher expression of glucose transporter 4 (GLUT4) [26] and are more insulin-sensitive and more insulin-responsive compared with type II fibers [27, 28]. A reduced proportion of SO fibers, along with the downregulation of GLUT4 in SO fibers [29], has been suggested as a primary mechanism of T2D-related sarcopenia. Furthermore, insulin resistance (IR), a hallmark of T2D, significantly impacts SkM mass by reducing protein synthesis and increasing protein degradation [30]. Impaired insulin signaling in SkM disrupts the insulin-driven anabolic signaling pathway [i.e., insulin receptor substrate (IRS)-phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB/Akt)-mammalian target of rapamycin (mTOR)], which is responsible for SkM protein synthesis and maintaining muscle mass [30, 31]. Furthermore, IR activates the ubiquitin–proteasome system (UPS) and autophagy-lysosome system (ALS), promoting protein degradation [32]. Beyond IR, adiposity, hyperglycemia, inflammation, oxidative stress, and advanced glycation end products (AGEs) are other critical mediators for impaired SkM function and mass loss, leading to sarcopenia in T2D [33]. On the other hand, SkM loss in sarcopenia contributes to developing IR and T2D through the decreased production of myokines that play a crucial role in glucose homeostasis [34].

Nitric oxide system in skeletal muscle

Two main sources of NO exist in SkM cells, including NO synthase (NOS) isoforms synthesizing NO from L-arginine (L-Arg) and the nitrate (NO₃)-nitrite (NO₂)-NO pathway [35]. The L-Arg-NOS pathway corresponds to about two-thirds of NO production; the remaining is derived from the NO₃-NO₂-NO pathway. This estimation is supported by data from different SkM indicating ~ 57–77% reduced NO release [36,37,38,39] and a 78% decreased NO₃ content of muscle [40] following NOS inhibition. At low oxygen (O₂) tension and slightly acidic conditions (pH ~ 5.5–6.5), the NO₃-NO₂-NO plays a NOS-independent essential role in maintaining NO bioavailability in SkM [41].

L-arginine-NOS pathway

All three NOS isoforms, including neuronal NOS (nNOS), endothelial NO (eNOS), and inducible NOS (iNOS), are found in SkM (Table 1) [42]. Based on tissue mass, SkM is the major source of nNOS in mammalians [43]. Among five known splice variants of nNOS (nNOSα, nNOSβ, nNOSγ, nNOSμ, and nNOS-2), two transcripts, including nNOSµ and nNOSβ are translated into structural and functionally distinct protein isoforms in fully differentiated SkM cells [44]. nNOSµ is the major source of NO in SkM [45]. In humans, 80% of nNOSµ is localized in sarcolemma (called sarcolemmal nNOSµ) [46], which binds to the scaffold protein α-syntrophin, a component of the dystrophin-associated glycoprotein complex (DGC) [45]. Loss of dystrophin prevents expression of nNOSμ [~ 100% in humans and 80% in mice with Duchenne muscular dystrophy (DMD)] and its sarcolemmal scaffolding, resulting in inhibited NO signaling in SkM [46]. nNOSµ displays various functions, including matching blood supply to the O₂ and metabolic demands of active muscle, glucose homeostasis regulation, and muscle mass control [47, 48]. A fraction of cytoplasmic nNOSμ has been suggested to be associated with the ryanodine receptor 1 Ca²⁺ release channel (RyR1) at the sarcoplasmic reticulum (SR) [44]. The function of cytoplasmic nNOSµ remains to be elucidated [49]. A distinct PDZ (an amino-terminal scaffolding domain)-lacking nNOS isoform, nNOSβ localizing at the Golgi complex in close to soluble guanylate cyclase (sGC) and protein kinase G (PKG), is a critical regulator of the structural and functional integrity of SkM [50]. nNOSβ is essential to maintain contractile force generation during and after exercise [50]. A nNOS isoform (nNOSα) located inside mitochondria has been detected in SkM and is called mitochondrial NOS (mtNOS) [51]. This isoform of nNOS controls the activity of the electron transport chain and inhibits mitochondrial respiration by reversible inhibition of cytochrome c oxidase [45]. mtNOS interacts with the peroxisome proliferator activated receptor-γ co-activator 1α (PGC-1α) and contributes to mitochondrial biogenesis and SkM metabolism [45]. mtNOS is the downstream target of the insulin/Akt pathway, which increases glucose uptake by SkM [52].

Table 1 Characteristics of skeletal muscle nitric oxide synthase (NOS) isoforms [37, 42, 44, 50, 54, 64, 65]

eNOS is highly expressed in the endothelium of vessels in SkM and is modified by fatty acylation (myristoylation and palmitoylation) to interact with caveolin-1 and localize in cell membrane caveolae [42, 53]. eNOS is also distributed in the sarcoplasm of all fibers in close vicinity of succinate dehydrogenase (complex II, a key component of both the mitochondrial respiratory chain and the tricarboxylic acid cycle) [37, 54]. In rodents, eNOS expression in FG-rich fiber muscles is lower than that of SO-rich fiber muscles [55]. eNOS appears to have a key role in SkM energy metabolism [56], glucose and insulin homeostasis [57], and keeping muscle mass [56]. eNOS^−/− soleus muscle in mice showed ~ 30% lower mass, lower number of mitochondria, and a decrease in creatine kinase, citrate synthase, adenylate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and pyruvate kinase (PK) activity [56], while gastrocnemius muscle remained unaffected by lacking eNOS [56]. Moreover, eNOS^−/− plantaris muscle in mice showed a decreased CSA in type I but not type II fibers [58].

In contrast to cNOSs, iNOS is hardly detectable in normal human SkM and is expressed in response to inflammatory cytokines and oxidative stress [59]. iNOS is co-localized with the sarcolemmal caveola membrane protein, caveolin-3 [60].

No compensatory relationship exists between NOS isoforms for NO production in SkM; lacking nNOS did not increase mRNA and protein expression of eNOS or iNOS [61,62,63].

The nitrate-nitrite-nitric oxide pathway

SkM is taught to be a significant NO₃ reservoir that essentially maintains whole-body NO homeostasis [41, 66]. The NO₃ content of SkM is provided endogenously through oxidation of nNOS-NO by oxymyoglobin (as the primary source) and exogenously by active NO₃ uptake from the circulation via sialin transporter and chloride voltage-gated channel 1 (CLC1) [35, 40, 67]. Upon rapid uptake by myoblasts and myotubes, a portion of the NO₃ is converted to NO₂ (dose-dependently) through the enzymatic activity of NO₃ reductases within myotubes, with maximum NO₂ concentrations achieved after 40 min following NO₃ availability [35]. Intracellular NO₃ reduction to NO₂ is primarily catalyzed by xanthine oxidoreductase (XOR), a mammalian NO₃ reductase [35]. The XOR seems to be localized in vascular tissue surrounding muscle fibers and possibly within the sub-sarcolemma of SkM [41]. Additional molybdenum-containing enzymes, i.e., mitochondrial amidoxime-reducing components (MARC-1 and MARC-2) and aldehyde oxidase (AO), are expressed within cellular environments and may potentially catalyze the reduction of both NO₃ and NO₂ to NO [35]. Although the liver is much more potent in NO₃ reductase activity than SkM, probably due to the much higher XOR expression levels, SkM significantly contribute to systemic NO₂ and NO formation because of its large total mass and high NO₃ content [68]. XOR-NO generation capacity differ in type I and II fibers, because of their different O₂ tension (type II fibers are more hypoxic than type I) [69] and possibly different expression levels of XOR. The muscles predominantly comprised of type IIb-highly glycolytic fibers appear to be the main target of and take more advantage of NO₃ supplementation [69, 70].

The cross-talk between NO₃-NO₂-NO and NOS pathways in controlling NO homeostasis in SkM has remained to be elucidated. When the NOS system is compromised, the NO₃-NO₂-NO pathway acts as a compensatory pathway for NO production in the SkM [61]. In the nNOS-knockout (KO) mice, NO₃-NO₂ reductive pathway-associated genes (i.e., sialin, CLC1, and XOR) were significantly upregulated, resulting in NO₃ accumulation in the SkM approximately twofold higher than the wild mice, following a high-NO₃ diet [61]. Negative feedback between the NO₃-NO₂-NO and NOS pathways has also been suggested [66], an idea supported by evidence indicating that nNOS expression slightly decreased (0.7-fold) in response to high-NO₃ loading and then elevated by 2.4-fold at day 21 after NO₃ cessation, where NO₃ content of SkM reached below its basal values [71]. In contrast, some evidence indicated that the NOS system did not compensate for a diminished NO₃-NO₂ reductive pathway [61]; mRNA and protein expression of NOS isoforms remained unaffected upon a low NO₃ diet [61].

Mechanisms of NO action in the skeletal muscle

NO primarily acts through its specific receptor, sGC [72, 73]. The activated catalytic domain of sGC converts guanosine triphosphate (GTP) to 3',5'-cyclic guanosine monophosphate (cGMP); cGMP mediates physiological actions of NO by activating the downstream elements of the signaling pathway, including cGMP-dependent protein kinases G [72, 73]. NO also exerts its physiological or pathological actions via post-translational modifications of proteins (e.g., nitration [74, 75] and S-nitrosylation [76]). Due to high concentrations of NO scavengers, including myoglobin [77] and glutathione [78] in the SkM which potentially minimize NO diffusion and long-lasting effects [79], cGMP‐dependent and independent signaling effects of NO appear to be in the close vicinity of target proteins.

Change of skeletal muscle nitric oxide system in type 2 diabetes

As shown in Table 2, SkM NOSs’ expression and activity significantly change in prediabetes and T2D [19, 80,81,82,83]. The nNOS expression was significantly reduced in subjects with T2D [19]. Insulin-stimulated NOS activity in SkM is impaired in subjects with T2D [80]. Impaired insulin signaling and dysregulated network of protein phosphorylation in SkM [84] and decreased adiponectin levels [80] in T2D are potential mechanisms underlying downregulated nNOS. A significant loss of the α-syntrophin and syntrophin-nNOS complex (~ 50%) in muscle fibers in T2D [85] might, at least in part, be responsible for the decreased nNOS-NO, leading to SkM atrophy in T2D.

Table 2 Changes of NOSs’ expression/activity and NO metabolites in SkM in human subjects with prediabetes and T2D^*

Human studies reported an elevated iNOS expression in the SkM of patients with T2D [19, 82]. In T2D patients with good glycemic control (glycosylated hemoglobin = 6.6%), no significant difference between iNOS immunoreactivity was detected in the vastus lateralis muscle compared to controls [81]. Upregulated iNOS in T2D was associated with elevated circulating levels of interferon-γ (IFNγ), lipopolysaccharide-binding protein (LBP), thiobarbituric acid-reactive substances (TBARS), and tumor necrosis factor-α (TNF-α), as well as decreased glutathione levels (GSH) [18, 19].

NO metabolites (NOx) and nitrotyrosine concentrations were also reported to be significantly higher in SkM of subjects with T2D [19, 82]. Variation in NOx concentrations in SkM of subjects with T2D was explained by variation (99%) in CD163 (i.e., macrophage membrane protein) and TNF-α, an observation indicating a role for macrophages in activation of iNOS-NO production [82]. Furthermore, nitrotyrosine levels in SkM were highly correlated with SkM iNOS and CD163 proteins, as well as glycosylated hemoglobin [82]. An immune-inflammatory reaction is suggested to be responsible for iNOS upregulation in the SkM in T2D [82]. Activated T cells produce IFN-γ, which in turn upregulates macrophage interleukin (IL)-1β, IL-6, and TNF-α; iNOS expression in SkM requires both IL-1β and IFN-γ to induce nuclear factor kappa B (NFκB) activation, which is essential for iNOS transcription [86]. Cytokines-induced iNOS causes NO overproduction, which in turn reacts with superoxide to produce peroxynitrite (ONOO⁻) and, consequently, nitrotyrosine generation in SkM [82].

Regulation of skeletal muscle mass: Role of nitric oxide

NOS- KO [48, 56, 58] and -overexpressed (OE) models [87] provide interesting insights into the physiologic relevance of NO action in SkM mass regulation. Table 3 summarizes evidence of NOS-KO mice models in relation to SkM atrophy (i.e., indicated as decreased muscle mass and fiber CSA). The nNOS(μ)-KO is the most studied model, and soleus, tibialis anterior (TA) and extensor digitorum longus (EDL) are frequently studied muscles. The observed effect of nNOS(μ) on SkM mass is muscle-type specific and sex-dependent. Lack of nNOS(μ) resulted in decreased soleus mass [in male [48, 88, 89] but not in female mice [48]], TA mass [in male [48, 89, 90] but not female mice [48]], and EDL muscle mass in male mice [88,89,90,91,92]. Overexpression of nNOS resulted in increased soleus and quadriceps mass (22 and 17%, respectively) in older (96 weeks old) but not younger (48 weeks old) female mice [87]. The soleus fibers of nNOS-OE female mice also displayed an increased CSA [87].

Table 3 Changes of SkM mass and CSA in NOSs-knockout mice models

The effect of eNOS-NO on the SkM mass has remained inconclusive. eNOS deficiency (partially or entirely) decreased total SkM mass [57]. One study indicates that eNOS-mediated muscle mass regulation may be fiber-type specific since lacking eNOS affects SO-rich fiber muscle more than FG-rich fiber muscle mass (28% vs. 11% in soleus compared to plantaris) [56]. Furthermore, EDL muscle mass was not affected by eNOS deficiency [88, 92]. Table 3 indicates that stimulated iNOS activity contributes to muscle atrophy. Lack of iNOS prevents the lipopolysaccharide (LPS)‐induced wasting of TA and quadriceps muscles but not the soleus and gastrocnemius muscles and preserves normal CSA in TA [93]. Inactivity-induced atrophy is also prevented in the quadriceps and gastrocnemius muscles of iNOS-KO mice [94].

Taken together, evidence obtained from genetically modified NOS models indicates that: 1) nNOS(μ) is the predominant NOS isoform responsible for preserving muscle mass; 2) the role of nNOS(μ)-NO on muscle mass appears to be fiber-type and sex-specific, and age-dependent; the SO-rich fiber muscle, male sex, and older-aged rodents appear to be more sensitive to diminished nNOS(μ)-NO; 3) iNOS mediates muscle atrophy in response to atrophic stimuli.

Potential mechanisms underlying NO contribution to muscle mass regulation

SkM mass regulation is governed by a balance between anabolic and catabolic pathways, with key signaling molecules summarized in Fig. 1. The mammalian target of rapamycin (mTOR) pathway, particularly mTOR complexes 1 and 2 (mTORC1 and mTORC2), plays a central role in promoting muscle hypertrophy [96,97,98]. mTORC1, activated by signals such as insulin-like growth factor 1 (IGF-1), insulin, and amino acids, drives protein synthesis by phosphorylating ribosomal S6 kinase (S6K1) and inhibiting the translational repressor 4E-binding protein 1 (4EBP1), thereby enhancing ribosome biogenesis [96,97,98]. Mechanical overload, independent of PI3K/Akt signaling, activates mTORC1 through phospholipase D (PLD) and phosphatidic acid (PA), facilitating protein synthesis. Additionally, mechanosensors like Ca²⁺-permeable ion channels stimulate hypertrophy by triggering the Ca²⁺-calmodulin (CaM)/CaM-dependent protein kinase (CaMK)/c-Jun N-terminal kinase (JNK) axis, which further activates S6K1 and inhibits SMAD2/3 (mothers against decapentaplegic homolog2/3), a suppressor of mTORC1 [99,100,101]. The pathway also suppresses protein degradation, aiding muscle maintenance [102, 103].

Fig. 1

Main anabolic and catabolic pathways coordinating protein balance and skeletal muscle (SkM) mass regulation and the role of nitric oxide (NO). Signaling pathways are initiated through various hormones (e.g., insulin, IGF-I, EP, NEP), nutrients (amino acids), energy state and physical activity (e.g., AMP-to-ATP ratio, AMPK), environmental stress (e.g., cytokines, LPS). Key signaling molecules that integrate these stimuli into protein synthesis and degradation are PI3K/Akt, mTORC1, GSK-3β and FoxO. α-Syn, α-syntrophin; AAs, amino acids; ActIIβ, activin type IIβ; AMP, adenosine monophosphate; AMPK; AMP-activated protein kinase; ATP, adenosine triphosphate; β₂-AR, β₂- adrenergic receptor; CaM, calmodulin; CaMK, CaM-dependent protein kinase; cGMP, cyclic guanosine monophosphate; CLC1, chloride voltage-gated channel 1; DGC, dystrophin-associated glycoprotein complex; EP, epinephrine; 4EBP1, 4E-binding protein 1; eNOS, endothelial nitric oxide synthase; eIF3f, eukaryotic translation initiation factor 3 subunit F; FoxO; forkhead box O; GTP, guanosine triphosphate; iNOS; inducible nitric oxide synthase; IGF-1, insulin-like growth factor 1; GSK-3β, glycogen synthase kinase-3β; IKKβ, inhibitory kappa B kinase β; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharides; MAFbx, muscle atrophy F-box; mTOR, mammalian target of rapamycin; MOV, mechanical overload; MuRF1, muscle ring finger 1; MA ion channels, mechanically-activated ion channels; NEP, norepinephrine; NFAT, nuclear factor of activated T-cells; NR, nitrate reductase; NFκB, nuclear factor kappa B; nNOS, neuronal nitric oxide synthase; NO₃, nitrate; ONOO⁻, peroxynitrite; PI3K, phosphoinositide 3-kinase; PKG, protein kinase G; PLD, phospholipase D; sGC, soluble guanylate cyclase; RNS, reactive nitrogen species; ROS, reactive oxygen species; RyR1; ryanodine receptor 1 Ca²⁺ release channel; SIRT1, silent mating type information regulation 2 homolog 1; SR, sarcoplasmic reticulum; SMAD, small mothers against decapentaplegic-related protein; TNF-α, tumor necrosis factor-α; TNFR, tumor necrosis factor-α receptor; TRPV1, transient receptor potential cation channel, subfamily V, member 1; TSC2, tuberous sclerosis complex 2; XOR, xanthine oxidoreductase

Catabolic processes that lead to SkM atrophy are primarily regulated by the transcription factor FoxO, which is linked to proteolytic systems such as the ubiquitin–proteasome system (UPS) and the autophagy-lysosome system (ALS) [104]. Upon activation by catabolic stimuli like myostatin, TNF-α, and elevated adenosine monophosphate (AMP)-to-adenosine triphosphate (ATP) ratios, forkhead box O (FoxO) induces the expression of atrophy markers such as muscle atrophy F-box (MAFbx) and muscle ring finger 1 (MuRF1), shifting the SkM protein balance toward degradation [105, 106]. Myostatin, for example, inhibits Akt-mTORC1 signaling, while TNF-α induces nuclear factor κB (NF-κB), which promotes MuRF1 expression (107, 108). Energy stress, sensed by AMP-activated protein kinase (AMPK), also activates FoxO and reduces mTORC1 activity, thereby contributing to muscle atrophy [109,110,111]. Together, these pathways highlight the complex regulation of SkM mass, where anabolic and catabolic signals converge to finely tune protein synthesis and degradation.

The potential mechanisms underlying NO-mediated effects on the maintenance of SkM mass are illustrated in Fig. 1. Sarcolemmal nNOSμ prevents sarcopenia by enhancing protein synthesis and inhibiting protein catabolism. Sarcolemmal nNOSμ acts as a paracrine signal, supplying blood flow [112, 113] and maintaining appropriate O₂ and nutrients (e.g., amino acids) delivery to the active SkM, which can facilitate protein synthesis and myofibril development. Loss of contraction-induced sarcolemmal nNOSμ activity causes functional ischemia [114]. nNOSμ-derived NO can also trigger SkM hypertrophy signaling via Ca²⁺-mediated mTORC1 activation [115]. In response to MOV, NO reacts with superoxide to generate ONOO⁻, which in turn activates TRPV1 (transient receptor potential cation channel, subfamily V, member 1), leading to Ca²⁺ release from the sarcoplasmic reticulum (SR), elevated intracellular Ca²⁺ [Ca²⁺]_i concentration and mTORC1 activation [115]. This NO action is independent of the sGC-cGMP pathway [115]. Other sources of elevated [Ca²⁺]_i concentration may also trigger SkM hypertrophy via Ca²⁺-mediated mTORC1 activation. In SkM, RyR1 is S-nitrosylated (preferentially at the low O₂ tension of ~ 10 mm Hg in active muscles) at a single cysteine residue (Cys³⁶³⁵) by nanomolar NO concentrations and that modification enhances its activity and increased [Ca²⁺]_i [76, 116]. This NO action is Ca²⁺/CaM dependent [76, 116], and sarcoplasmic nNOSμ is the candidate for the NO-mediated S-nitrosylation of RyR1 [44, 117]. RyR1-Cys³⁶³⁶ mutation results in diminished myofiber diameter and muscle mass, a finding that indicates the role of NO-mediated S-nitrosylation of RyR1 on SkM mass [117]. NO (probably nNOSμ-derived) can also act as a negative regulator of glycogen synthase kinase-3β (GSK-3β) by its phosphorylation via sGC-cGMP-PKG pathway [72]. Active GSK-3β suppresses several anabolic signaling pathways involving translational efficiency and translational capacity, and inhibitory phosphorylation of GSK-3β promotes protein synthesis [105].

nNOSμ protects against protein degradation by inhibiting pathways that promote muscle atrophy, including FoxO3. nNOSμ inhibits FoxO3 through its phosphorylation by Akt [89]. nNOSμ-KO muscles contain significantly lower levels of phosphorylated FoxO3 [89]. Inhibition of the nNOSμ-NO-cGMP-PKG pathway induces the transcriptional activity of FoxO3 and upregulates UPS [89].

On the other hand, nNOSμ acts as an inactivity sensor in SkM and initiates a number of atrophic pathways [118]. In response to long-term inactivity, delocalization (dissociation from sarcolemmal DGC complex and accumulation in the sarcoplasm) and downregulation of nNOS protein occurs, triggering FoxO-dependent muscle atrophy [119, 120]. Delocalization of nNOSμ is suggested to lead first to the loss of NO physiologic effects, mostly exerted via sGC-cGMP signaling, and second to overproduction of NO, which subsequently leads to nitration and hyper-S-nitrosylation of several proteins such as RyR1 [119]. Upregulation of two atrophy-related proteins (i.e., MuRF-1 and MAFbx) is a downstream event of nNOSμ dislocation in inactivity-induced muscle atrophy [119]. FoxO3 protein content and its phosphorylated level (p-FoxO3) remained unchanged in nNOS-KO mice, making them resistant to inactivity-induced muscle atrophy [119]. Two proposed mechanisms may explain the role of delocalized nNOSμ-NO signal in the activation of FoxO3-induced atrophy. First, supra-physiologic NO concentrations, probably S-nitrosylate inhibitor of kappa B kinase β (IKKβ) and Akt, preventing FoxO3 phosphorylation and degradation and accelerating its translocation (from the cytoplasm to the nucleus) followed by the enhancement of UPS [119]. Second, delocalized nNOSμ-NO might decrease the nuclear export of FoxO3, facilitating its accumulation in myonuclei and protecting it from phosphorylation by Akt [119]. Furthermore, accumulated NO in sarcoplasm is suggested to cause hyper-S-nitrosylation of RyR1, followed by Ca²⁺ leakage from the SR (probably due to displacement of calstabin, i.e., a subunit of RyR1 stabilizing the closed state of the channel) [121,122,123]. Such Ca²⁺ sparks may trigger mitochondrial ROS generation [124] and activate proteases such as calpain [125], altogether with reactive nitrogen species (RNS) overproduction (due to excessive free NO in the sarcoplasm) [91] induce UPS. On the other hand, excessive amounts of NO can react with superoxide anions to form ONOO⁻ which in turn results in nitration of tyrosine residues, forming 3-nitrotyrosine proteins, and induction of proteasome-mediated degradation of the modified proteins (e.g., HCM) [74]. Increased 3-nitrotyrosine levels in SkM are associated with an activated UPS and muscle wasting [126]. Furthermore, ONOO⁻ activates NFκB and stimulates ubiquitination (i.e., an enzymatic post-translational modification) of muscle proteins, leading to their subsequent degradation [75].

Involvement of eNOS-NO in protein synthesis-degradation in SkM is less documented. eNOS-NO may increase muscle mass via sGC-cGMP-PI3K-Akt pathway, which in turn results in inhibitory phosphorylation of GSK-3β and nuclear translocation of the nuclear factor of activated T-cells (NFAT)-c1 transcription factor [58]. eNOS-NO can be activated through silent mating type information regulation 2 homolog 1 (SIRT1) in response to MOV, and that produced NO mediates a signaling cascade via sGC-cGMP pathway to induce follistatin (FST) expression, an antagonist of myostatin [127], a negative regulator of SkM mass.

The mechanisms by which iNOS-NO contributes to muscle atrophy are still poorly understood; iNOS, i.e., highly expressed in atrophic muscle, is a downstream effector of the NF-kB pathway [128]. On the other hand, LPS-induced iNOS impairs cellular energy production (due to decreased glycolysis and disrupting the entry of pyruvate and fatty acids into the TCA cycle), thereby increasing AMP/ATP levels and AMPK activation, events that were not observed in iNOS-KO mice [93].

In contrast, some evidence indicates that an elevated iNOS expression [in response to L-citrulline (L-Cit) and S-nitroso-glutathione (GSNO)] in myotubes and SkM fibers protects muscle cells against atrophic stimuli through the preservation of protein synthesis and inhibition of protein degradation pathways [129, 130]. GSNO (i.e., a NO donor) and L-Cit (a precursor of NO) inhibit inflammatory (LPS)- and oxidative stress-(H₂O₂)-induced muscle atrophy by upregulating iNOS (but not eNOS or nNOS) in SkM [129, 130]. The protective effect of iNOS, i.e., evident in oxidative but not in glycolytic fibers, is also attributed to the induction of antioxidant genes, including superoxide dismutase (SOD) and catalase, and reduces the expression MAFbx [129]. Previously observed resistance of oxidative myofibers to atrophic muscle stimuli, including LPS and TNF-α [131], has been attributed to iNOS-NO signaling [129]. The discrepancy in the role of iNOS-NO in muscle atrophy can be at least in part due to divergent atrophy-induced stimuli and NO precursor (L-Cit vs. L-Arg) used in different studies.

Of note, vascular NO resistance and endothelial dysfunction commonly observed in T2D [132, 133] may impair nutrient and O₂ delivery to SkM, exacerbating muscle wasting, and hindering muscle growth and leading to sarcopenia. The interplay between endothelial dysfunction and sarcopenia has been well-documented in some studies. Endothelial function (i.e., measured by reactive hyperemia index) was significantly lower in T2D patients with sarcopenia [134]. A lower vascular reactivity index (VRI, 0.83 vs. 1.08) was also observed in patients with chronic kidney disease who had sarcopenia [135]. Endothelial dysfunction (i.e., defined as low VRI) was associated with a higher probability of having both sarcopenia and impaired SkM function [135, 136]. Furthermore, vascular endothelial dysfunction has been identified as an early predictor of physical frailty and sarcopenia [137]. This evidence collectively highlights that endothelial dysfunction, by compromising vascular function and impairing nutrient delivery, may play a role in the pathogenesis of sarcopenia in T2D.

Potential NO-based therapeutic approaches for T2D-related sarcopenia

Since NO plays an important role in maintenance of SkM mass, restoring its bioavailability through various approaches, including nNOS overexpression, supplying NOS substrates (e.g., L-Arg and L-Cit administration), phosphodiesterase (PDE) inhibition, and supplementation with NO donors (e.g., inorganic NO₃) represent promising therapeutic approaches to preserve SkM mass and prevents sarcopenia in T2D, a state of impaired NO homeostasis in SkM. No direct evidence is available to support such theoretical approaches in T2D. However, the observed effect of NO restoration on various states of muscle atrophy (e.g., DMD, age-related and disuse-mediated muscle atrophy) may provide some insights into the development of mechanism-based target-directed therapeutics in T2D-related sarcopenia because of partially shared pathophysiology, including the downregulation/mislocalization of nNOSμ and consequent abrogation of NO signaling in SkM [85]. Furthermore, the existence of compensatory mechanisms in T2D (e.g., upregulated sialin expression in soleus muscle of male rats with T2D [138]) that provide sustain NO bioavailability from exogenous NO₃ suggests a potential entry site to preserve SkM mass in the state of the impaired nNOSμ-NO signaling in T2D.

Restoration of nNOSμ protein has been suggested as a therapeutic candidate for boosting NO availability and reversing muscle atrophy in DMD [139]. Re-localization of nNOSμ to the sarcolemma (e.g., using transgenic expression of full-length dystrophin or somatic gene transfer of full-length dystrophin) appears to be essential for restoration of nNOSμ-NO signaling [140], since nNOS overexpression per se did not prevent muscle atrophy in dko mice (i.e., dystrophin/utrophin double-KO model) [141].

Upregulation of L-Arg transporter expression and function in DMD suggests a compensatory mechanism and a potential entry site to preserve SkM mass in the state of the impaired nNOSμ-NO signaling [142]. L-Arg supplementation (oral dose of 2.5 g, 3 times/day) combined with metformin (250 mg, twice daily, as pharmacological AMPK activator and indirect nNOS inducer in SkM [143]) for 16 weeks in human subjects with DMD increased muscle mass (1.8%) in quadriceps and all thigh muscles (4.9 and 6.2%, respectively) [144]. This therapeutic approach significantly increased nitrotyrosine and cGMP concentrations in SkM biopsies of patients, indicating an increased NO formation in DMD muscles [144]. Administration of L-Arg-butyrate (dose of 250 mg/kg/day for 6 months) in mdx mice (an animal model of DMD, i.e., dystrophin-deficient) significantly increased soleus mass but not gastrocnemius or EDL mass [145]. Furthermore, L-Arg consistently resulted in improvement of myopathological hallmarks (indicated by decreased fatty and fibrotic tissue, collagen deposition, inflammatory cell infiltration, and necrosis) and SkM function (indicated by increased grip strength, contractile function, capacity to exercise) in treated mdx mice [145,146,147,148]. L-Arg did not provide protection from myotube wasting; however, L-Arg deficiency limits NO production and the rate of protein synthesis [130].

L-Cit supplementation (3 g/d for 6 weeks) in older adults showed potential benefits for improving walking speed and mobility, though it had no significant effect on strength, endurance, or markers of SkM damage [149]. L-Cit protects myotubes from various atrophic stimuli (e.g., growth factor deprivation, inflammation, and oxidative stress), and that effect is dependent on iNOS expression and activity, and is mediated through preserving the phosphorylation status of mTORC1 and 4EBP1, mTORC1 activation, and rate of protein synthesis [130].

While PDE inhibition, amplifying NO-dependent cGMP signal, may theoretically mitigate dystrophic phenotype of SkM, treatment with sildenafil and tadalafil failed to show efficacy in human clinical trials in subjects with DMD [150].

Chronic (6-week) administration of isosorbide dinitrate (ISDN) combined with exercise preserved age-related decreased mass of quadriceps muscle (but not TA and gastrocnemius muscles) in 20-old mice, while its administration per se did not affect muscle mass [151]. Fiber diameter in the quadriceps also increased in mice treated with ISDN + excercise, and fiber distribution was shifted to larger fibers in that group [151]. The observed hypertrophy in the ISDN-treated group was accompanied by modifications in the DGC, i.e., increased ratio and localization of NOS-1 and β-dystroglycan protein [151]. Oral administration of NO donors, guaifenesin dinitrate and ISDN in mdx mice for 18 days increased quadriceps but not triceps surae muscle mass [152]. A 6-month treatment of mdx mice with MMPN (a NO donor, i.e., nitrate ester of 2-methyl-2-n-propyl-1,3-propanediol) significantly increased fiber size and CSA in TA muscle and improved muscle function [153]. Evidence of NO₃ tolerance in SkM in response to nitroglycerin [154] may limit the implication of organic NO-releasing compounds for long-term treatment. However, cyclooxygenase (COX)-inhibiting NO donors (CINODs) cover this limitation because they release NO at low concentrations for prolonged periods [155]. A 7-month treatment of mdx mice with Naproxcinod (i.e., a compound belonging to CINODs) significantly reduced inflammatory infiltrates and fibrotic area in TA and diaphragm muscles and improved SkM function [156]. NO donation may preserve muscle atrophy by inhibiting calpain‐mediated SkM proteolysis via S‐nitrosylation of the cysteine residue of calpain [125].

While inorganic NO₃ supplementation increased SkM mass in normal conditions [157], its effects in dystrophic states remain inconclusive. Administration of inorganic NO₃ (1 mM sodium nitrate, NaNO₃) did not preserve SkM mass (soleus, gastrocnemius, plantaris) or myofibrillar protein synthesis rates and could not prevent disuse-mediated muscle atrophy (i.e., mainly attributed to delocalization of nNOSμ) [158]. Furthermore, compensating lack of muscle nNOS-NO signaling in mdx mice using 8-week supplementation with NaNO₃ (85 mg/L in drinking water) had no effect on muscle mass (EDL, TA, gastrocnemius, and soleus) and displayed detrimental effects on SkM (i.e., indicated as induction of mitochondrial uncoupling, and increased muscle ONOO⁻) [159]. Pseudo-hypertrophy (i.e., a histopathological hallmark of dystrophin-deficient muscle) and damaged area in muscle (indicated by increased areas of inflammatory cell/nuclei infiltration and nitrotyrosine content by 27-fold), were exacerbated by NO₃ supplementation [159]. Such evidence may imply that the lack of nNOSμ and its translocational capacity to deliver NO to specific sarcoplasmic sites cannot be compensated by systemic inorganic NO₃ supplementation.

Taken together, the NO₃-NO₂-NO pathway does not appear to be a viable therapeutic approach for muscle atrophy due to its detrimental effects on dystrophin-deficient muscle [159]. NO₃ supplementation in dystrophin-deficient muscle is suggested to require a concomitant upregulation of sarcolemmal nNOS protein expression/activity to obtain the same benefits it does in healthy muscle [150]. This idea may be logically true in T2D atrophic muscle, where α-dystrophin and nNOSμ are lost by approximately half. Given that the beneficial effect of L-Arg on SkM mass and function can be augmented in the presence of active sarcolemmal nNOSμ [144], a combination of L-Arg with a nNOS inducer like metformin (i.e., a biguanide used as first-line treatment of T2D) may be more effective. Notably, elevated synthesis of asymmetric dimethylarginine (ADMA, an endogenous inhibitor of NO synthesis via downregulation and uncoupling of NOS) in T2D [160, 161] may minimize L-Arg utilization for NO restoration in subjects with T2D. Further investigations are required to fully elucidate these knowledge gaps. Targeting NO restoration in SkM through L-Arg-metformin and CINODs appears to be a logical future direction in T2D-related sarcopenia.

In summary, while NO-based interventions, such as L-Arg supplementation and using NO donors, show theoretical promise for preventing SkM atrophy, the current evidence remains inconclusive. Despite the role of NO in muscle maintenance, especially in models of muscle atrophy like DMD and age-related sarcopenia, there is no clear, direct evidence supporting the efficacy of these approaches in humans, particularly in the context of conditions like T2D, where NO signaling is often impaired. Preclinical studies have shown some benefits of L-Arg and NO donors in improving muscle function and mass in animal models, but these effects are limited and inconsistent across different models. Additionally, the failure of certain NO-based treatments, such as inorganic NO₃ supplementation, to preserve muscle mass in dystrophic models further complicates the potential therapeutic application. Therefore, while NO restoration remains a promising area of research, its application as a standalone treatment for muscle atrophy, particularly in T2D, requires further investigation and refinement to better understand its potential and limitations.

Conclusion

NO is essential for maintaining SkM mass since it is tightly connected to various relevant pathways, including PI3K/Akt, mTORC1, GSK-3β and FoxO. This signaling molecule integrates various hypertrophic/atrophic stimuli (e.g., hormones, energy state, mechanical stimuli, and environmental stress) into protein synthesis and degradation pathways. Impaired NO signaling in T2D, mainly due to nNOSμ downregulation/mislocalization and iNOS induction leading to decreased physiologic NO availability and pathologic NO/RNS generation, activates catabolic signaling pathways and induces muscle atrophy via UPS and autophagy. Further research investigating the interplay between NO and T2D-related atrophic pathways in SkM may yield a more comprehensive understanding of the complex regulatory networks in T2D-related sarcopenia. When considering therapeutic NO-based approaches for T2D-related sarcopenia, it is essential to note that NO restoration, whether beneficial or harmful, may significantly be affected by its mode of administration (L-Arg, L-Cit, inorganic NO₃, and NO donors), concentration and distribution (being in close vicinity to its target molecules). Notably, the effect of NO restoration on SkM mass preservation depends on fiber type, sex, and age. Advances in this field depend on further understanding of the addressed knowledge gaps.