Neuronal nitric oxide synthase is phosphorylated in response to insulin stimulation in skeletal muscle

doi:10.1016/j.bbrc.2013.05.020

Biochemical and Biophysical Research Communications

Volume 435, Issue 3, 7 June 2013, Pages 501-505

https://doi.org/10.1016/j.bbrc.2013.05.020 Get rights and content

Highlights

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nNOS is phosphorylated in response to insulin in C2C12 myotubes.
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nNOS is phosphorylated in response to insulin in mouse skeletal muscle.
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NO production increases in myotubes with insulin stimulation.

Abstract

Type 2 Diabetes (T2DM) is the seventh leading cause of death in the United States, and is quickly becoming a global pandemic. T2DM results from reduced insulin sensitivity coupled with a relative failure of insulin secretion. Reduced insulin sensitivity has been associated with reduced nitric oxide synthase (NOS) activity and impaired glucose uptake in T2DM skeletal muscle. Upon insulin stimulation, NO synthesis increases in normal adult skeletal muscle, whereas no such increase is observed in T2DM adults. Endothelial NOS is activated by phosphorylation in the C-terminal tail in response to insulin. Neuronal NOS (nNOS), the primary NOS isoform in skeletal muscle, contains a homologous phosphorylation site, raising the possibility that nNOS, too, may undergo an activating phosphorylation event upon insulin treatment. Yet it remains unknown if or how nNOS is regulated by insulin in skeletal muscle. Data shown herein indicate that nNOS is phosphorylated in response to insulin in skeletal muscle and that this phosphorylation event occurs rapidly in C2C12 myotubes, resulting in increased NO production. In vivo phosphorylation of nNOS was also observed in response to insulin in mouse skeletal muscle. These results indicate, for the first time, that nNOS is phosphorylated in skeletal muscle in response to insulin and in association with increased NO production.

Introduction

Type 2 Diabetes mellitus (T2DM) is characterized by insulin resistance and impaired insulin-stimulated nitric oxide (NO) generation in both endothelium and skeletal muscle. NO is an important gaseous regulatory molecule, produced by isoforms of nitric oxide synthase (NOS). NO acts variably as a vasodilator [1], [2], neurotransmitter [3] or cytotoxic agent [4], depending upon its source and location of production. NOS enzymes synthesize NO from l-arginine and molecular oxygen [5], utilizing electrons donated by NADPH. The homeostatic regulation of NO synthesis is crucial in biological systems, as the generation of insufficient amounts of NO can result in deleterious outcomes, such as increased blood pressure due to uncontrolled vasoconstriction. In contrast, overproduction of NO can yield toxic sequelae, such as endotoxic shock.

Neuronal NOS (nNOS) and endothelial NOS (eNOS) knockout mice demonstrate insulin resistance, suggesting that NO produced by nNOS plays an important role in mediating insulin action [6], [7]. However, the direct effect of insulin on skeletal muscle nNOS is unknown. One prior study by Kashyap et al. [8], using euglycemic/hyperinsulinemic clamps, showed that NO production in muscle biopsies is significantly reduced in obese, T2DM human subjects compared to obese, non-diabetic controls. Moreover, in the insulin-resistant subjects, insulin treatment failed to evoke increases in NO in skeletal muscle, whereas insulin stimulated a robust increase in NO in the control subjects [8]. These results suggest that insulin action in skeletal muscle involves NO production, thus implicating mechanisms regulating NO bioavailability. These mechanisms must be elucidated to understand the pathogenesis of muscle insulin resistance, which is an important component of the pathology of T2DM.

Phosphorylation plays a critical role in the potentiation of NO generation by vascular eNOS, which is activated by insulin-dependent AKT-catalyzed phosphorylation at eNOS Ser1177 [9], [10], potentially explaining the vasodilatory effects of insulin. While the role of eNOS in insulin action and its inactivation as a component of insulin resistance in the vasculature has been well-documented [2], [11], [12], [13], the metabolic role of NOS in other tissues, such as skeletal muscle, remains elusive. The predominant NOS isoform in skeletal muscle is nNOS, several splice variants of which, i.e., nNOSμ, nNOSβ and nNOSα [14], [15], may be present. In this report, we do not distinguish among the nNOS variants, hereafter collectively referred to as skeletal muscle nNOS.

The rapid increase in NO production observed in the human insulin clamp studies suggests that post-translational modification of skeletal muscle nNOS occurs in direct response to insulin rather than as insulin-regulated transcription [8]. Since skeletal muscle nNOS contains a homologous residue to the AKT-phosphorylated serine in eNOS (Ser1177), it was possible that nNOS could be phosphorylated in response to insulin in skeletal muscle. There is evidence of nNOS phosphorylation in response to insulin in brain: prior immunoblot studies in rat hypothalamus demonstrated an increase in nNOS Ser1416 phosphorylation [16], the rat equivalent of Ser1412 in mouse nNOS. In vitro kinase assays in rat brain nucleus Tractus solitarii additionally showed nNOS is phosphorylated at this residue in an insulin-dependent manner [17].

To examine nNOS phosphorylation by insulin treatment, the murine muscle C2C12 cell line, which can be differentiated from myoblasts to myotubes, was used to probe putative Ser1412 phosphorylation on the C-terminal tail of skeletal muscle nNOS in response to stimulation by insulin. Our results show that insulin stimulation resulted in significantly increased phosphorylation of skeletal muscle nNOS in C2C12 myotubes, as well as a concomitant increase in NO levels. In vivo, insulin-stimulated nNOS phosphorylation was also observed in WT mouse C57BL/6J muscle tissue, further validating that insulin-mediated phosphorylation of nNOS is an important component of the insulin-signaling cascade.

Section snippets

Cell culture and treatment

Murine-derived muscle cell line C2C12 (Cell Biology Labs) myoblasts were grown to near 100% confluency in high glucose (25 mM) DMEM (Sigma–Aldrich) with 10% fetal bovine serum (Gibco), hereafter called complete media. Myoblasts were then switched to high glucose DMEM media, 2% horse serum (Gibco), hereafter called horse serum media. After four days in serum starvation, cells were completely differentiated to myotubes, and were treated with either vehicle (3 μM HCl) or 100 nM insulin (Sigma

nNOS is phosphorylated in response to insulin in C2C12 myotubes

To determine whether nNOS is phosphorylated in skeletal muscle in response to insulin, the murine-derived in vitro muscle cell line C2C12 was used as a model system. Control and insulin-treated C2C12 cell lysates were probed for nNOS phosphorylated on residue S1412 via immunoblot analysis. As shown in Fig. 1A, insulin treatment resulted in increased nNOS phosphorylation at Ser1412 in a time-dependent manner, with significant (P < 0.05) phosphorylation occurring at 15, 30 and 60 min post-insulin

Acknowledgments

We would like to thank Srikanth Reddy Polusani, PhD, for his aid in all fluorescence experiments, as well as Satya P. Panda, PhD, for useful discussion. Supported by NIH GM052419 to LJR and BSM. BSM is the Robert A. Welch Distinguished Chair in Chemistry (AQ0012).

References (27)

P.L. Huang
eNOS metabolic syndrome and cardiovascular disease
Trends Endocrinol. Metab.
(2009)
J.B. Hibbs et al.
Nitric oxide: a cytotoxic activated macrophage effector molecule
Biochem. Biophys. Res. Commun.
(1988)
F. Silvagno et al.
Neuronal nitric-oxide synthase-mu, an alternatively spliced isoform expressed in differentiated skeletal muscle
J. Biol. Chem.
(1996)
H.T. Chiang et al.
Neuronal nitric oxide synthase activation is involved in insulin-mediated cardiovascular effects in the nucleus tractus solitarii of rats
Neuroscience
(2009)
J. Zielonka et al.
Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses
J. Biol. Chem.
(2012)
T. de Castro Barbosa et al.
L-arginine enhances glucose and lipid metabolism in rat L6 myotubes via the NO/c-GMP pathway
Metabolism
(2013)
A.A. Quyyumi et al.
Contribution of nitric oxide to metabolic coronary vasodilation in the human heart
Circulation
(1995)
M.J. Rand et al.
Nitric oxide as a neurotransmitter in peripheral nerves: nature of transmitter and mechanism of transmission
Annu. Rev. Physiol.
(1995)
P.J. Andrew et al.
Enzymatic function of nitric oxide synthases
Cardiovasc. Res.
(1999)
P. Turini et al.
Insulin resistance in mice lacking neuronal nitric oxide synthase is related to an alpha-adrenergic mechanism
Swiss Med. Wkly.
(2007)

R.R. Shankar et al.

Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance

Diabetes

(2000)

S.R. Kashyap et al.

Insulin resistance is associated with impaired nitric oxide synthase activity in skeletal muscle of type 2 diabetic subjects

J. Clin. Endocrinol. Metab.

(2005)

B.J. Michell et al.

The akt kinase signals directly to endothelial nitric oxide synthase

Curr. Biol.

(1999)

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Differential superoxide production in phosphorylated neuronal nitric oxide synthase mu and alpha variants
2024, Journal of Inorganic Biochemistry
Neuronal nitric oxide synthase (nNOS) is regulated by phosphorylation in vivo, yet the underlying biochemical mechanisms remain unclear, primarily due to difficulty in obtaining milligram quantities of phosphorylated nNOS protein; detailed spectroscopic and rapid kinetics investigations require purified protein samples at a concentration in the range of hundreds microM. Moreover, the functional diversity of the nNOS isoform is linked to its splice variants. Also of note is that determination of protein phosphorylation stoichiometry remains as a challenge. To address these issues, this study first expanded a recent genetic code expansion approach to produce phosphorylated rat nNOSμ and nNOSα holoproteins through site-specific incorporation of phosphoserine (pSer) at residues 1446 and 1412, respectively; this site is at the C-terminal tail region, a NOS-unique regulatory element. A quantitative mass spectrometric approach was then developed in-house to analyze unphosphorylated peptides in phosphatase-treated and -untreated phospho-nNOS proteins. The observed pSer-incorporation efficiency consistently exceeded 80%, showing high pSer-incorporation efficiency. Notably, EPR spin trapping results demonstrate that under l-arginine-depleted conditions, pSer1412 nNOSα presented a significant reduction in superoxide generation, whereas pSer1446 nNOSμ exhibited the opposite effect, compared to their unphosphorylated counterparts. This suggests that phosphorylation at the C-terminal tail has a regulatory effect on nNOS uncoupling that may differ between variant forms. Furthermore, the methodologies for incorporating pSer into large, complex protein and quantifying the percentage of phosphorylation in recombinant purified protein should be applicable to other protein systems.
Dietary L-arginine supplementation influences the muscle fiber characteristics and meat quality of Mongolian sheep through the NO/AMPK/PGC-1α pathway
2023, Food Bioscience
L-arginine serves as a substrate for the production of nitric oxide (NO) in animals, and it can also impact muscle fiber characteristics and meat quality in these animals. The present study was designed to explore the effects of adding 1% L-arginine to a basal diet regimen on the muscle fiber characteristics and meat quality of Mongolian sheep. Dietary L-arginine supplementation reduced shear force in the longissimus thoracis (LT) and increased a* in biceps femoris (BF) muscles (p < 0.05). L-arginine supplementation also increased the proportion of type IIA muscle fiber in the LT (p < 0.05) and type I muscle fiber in the BF (p < 0.05) while reducing both the diameter and CSA of type IIB muscle fiber in both the LT and BF (CSA in LT, p < 0.01; all others, p < 0.05). L-arginine treatment was also associated with the upregulation of MyHC IIa (LT), MyHC I (BF), nNOS (LT & BF), AMPKα1 (BF), PGC-1α (LT & BF) (PGC-1α in BF, p < 0.01; all others, p < 0.05), together with an increase in nNOS content (LT, p < 0.01; BF, p < 0.05). Dietary L-arginine supplementation was associated with a significant increase in the post-slaughter tenderness of lamb meat, which is related to transitions in muscle fiber types. The gene expression and nNOS analysis results generated herein further indicate that this effect is mediated by the NO/AMPK/PGC-1α pathway. Further studies are thus warranted to provide further insight into the role that NO signaling plays in controlling the associations between L-arginine, muscle ﬁber characteristics, and meat quality.
Evaluating the role of nitric oxide in myogenesis in vitro
2022, Biochimie
Citation Excerpt :
Our findings are congruent with the literature in that the inhibition of endogenous NO generation by C2C12 myoblast delays their differentiation and fusion into myoblasts [14]. Only maturing fusion-competent myoblasts express nNOS, which is possibly the source of the elevated NO levels reported in differentiating C2C12 myoblasts [19,55,56]. nNOS activity and elevated NO and calcium levels are required within 1 day after inflicted damage in order to induce eventual hypertrophy [57]; our results show the relevance of such an early steep elevation of NO produced by myoblasts at the onset of differentiation with subsequently sustained levels to result in effective myotube formation.
Skeletal muscle injury activates satellite cells to proliferate as myoblasts and migrate, differentiate and fuse with existing fibres at the site of injury. Nitric oxide (NO), a free radical produced by NO synthase, is elevated and supports healing after in vivo injury. NOS-independent elevation of NO levels in vitro is possible via donors such as molsidomine (SIN-1). We hypothesized that alterations in NO levels may directly influence myogenic processes critical for skeletal muscle wound healing. This study aimed to clarify the role of NO in myoblast proliferation, migration and differentiation. Baseline NO levels were established in vitro, whereafter NO levels were manipulated during myogenesis using l-NAME (NOS inhibitor) or SIN-1. Baseline NO levels generated by myoblasts in proliferation media did not change 1 h after stimulation. Addition of a pro-proliferative dose of HGF slightly elevated NO levels 1 h post-stimulation, whereas cell numbers assessed 24 h later increased significantly; l-NAME reduced the HGF-driven increase in NO and proliferation, reducing wound closure over 16 h. In differentiation media, NO levels increased significantly within 24 h, returning to baseline over several days. Regular addition of l-NAME to differentiating cells significantly reduced NO levels and fusion. SIN-1 increased NO levels in a dose-dependent manner, reaching maximal levels 16 h post-treatment. SIN-1, added at 0, 2 and 4 days, significantly increased myofiber area (26 ± 1.8% vs 18.6 ± 3.4% in control at 5 day, p < 0.0001), without affecting proliferation or migration. In conclusion, this study demonstrates that, during skeletal muscle regeneration, increased NO specifically stimulates myoblast differentiation.
Role of the macula densa sodium glucose cotransporter type 1-neuronal nitric oxide synthase-tubuloglomerular feedback pathway in diabetic hyperfiltration
2022, Kidney International
An increase of glomerular filtration rate (GFR) is a common observation in early diabetes and is considered a key risk factor for subsequent kidney injury. However, the mechanisms underlying diabetic hyperfiltration have not been fully clarified. Here, we tested the hypothesis that macula densa neuronal nitric oxide synthase (NOS1) is upregulated via sodium glucose cotransporter type 1 (SGLT1) in diabetes, which then inhibits tubuloglomerular feedback (TGF) promoting glomerular hyperfiltration. Therefore, we examined changes in cortical NOS1 expression and phosphorylation, nitric oxide production in the macula densa, TGF response, and GFR during the early stage of insulin-deficient (Akita) diabetes in wild-type and macula densa-specific NOS1 knockout mice. A set of sophisticated techniques including microperfusion of juxtaglomerular apparatus in vitro, micropuncture of kidney tubules in vivo, and clearance kinetics of plasma fluorescent-sinistrin were employed. Complementary studies tested the role of SGLT1 in SGLT1 knockout mice and explored NOS1 expression and phosphorylation in kidney biopsies of cadaveric donors. Diabetic mice had upregulated macula densa NOS1, inhibited TGF and elevated GFR. Macula densa-selective NOS1 knockout attenuated the diabetes-induced TGF inhibition and GFR elevation. Additionally, deletion of SGLT1 prevented the upregulation of macula densa NOS1 and attenuated inhibition of TGF in diabetic mice. Furthermore, the expression and phosphorylation levels of NOS1 were increased in cadaveric kidneys of diabetics and positively correlated with blood glucose as well as estimated GFR in the donors. Thus, our findings demonstrate that the macula densa SGLT1-NOS1-TGF pathway plays a crucial role in the control of GFR in diabetes.
Effects of chronic ethanol consumption on the expression of GLT-1 and neuroplasticity-related proteins in the nucleus accumbens of alcohol-preferring rats
2020, Brain Research Bulletin
Citation Excerpt :
Therefore, present and previous findings suggest that increases in glutamate neurotransmission are associated with increased BDNF-Arc signaling activity in the AcbSh, which may modulate anxiety-like behavior induced by ethanol consumption and subsequent withdrawal (Pandey et al., 2008). In the present study, the activity of nNOS was increased in the AcbSh after 6 weeks of ethanol intake as a result of increases in the p-NOS/nNOS ratio, which suggests increases in NO production (Hinchee-Rodriguez et al., 2013; Kar et al., 2015). nNOS-derived NO is known to regulate synaptic plasticity by induction of protein involved in synaptic changes through cGMP, protein kinase G and ERK pathways (Gallo and Iadecola, 2011a).
Chronic ethanol exposure induces impairments in CNS excitatory and inhibitory activity. These impairments are associated with glutamatergic dysfunction, including altered neuroplasticity. This study examined the effects of 6-week ethanol (15% and 30% v/v) consumption, by male alcohol-preferring P rats, on protein expression associated with neuroplasticity and glutamate transporter-1 (GLT-1) function. The latter regulates intra- and extra-synaptic glutamate levels. We focused on the shell and core subregions of the nucleus accumbens (Acb); i.e., shell (AcbSh) and core (AcbCo), for these measures. Chronic ethanol exposure increased the expression of BDNF, Arc and phosphorylated (p)-post-synaptic density protein-95 (p-PSD-95) in the AcbSh of P rats. Moreover, the ratio of phospho-neuronal nitric oxide synthase (p-nNOS) to total nNOS was also increased in the AcbSh. These changes in BDNF, Arc and p-nNOS/nNOS ratio were not observed in the AcbCo. Furthermore, chronic ethanol consumption reduced GLT-1 expression in the AcbSh. Alternatively, treatment with ceftriaxone (CEF), a known GLT-1 upregulator, abolished the effect of chronic ethanol consumption on BDNF expression in the AcbSh. Overall, the present findings confirm that chronic ethanol consumption modulates activity-associated synaptic proteins, including BDNF, Arc and nNOS in a subregion-specific (i.e., in the AcbSh but not AcbCo) manner. Thus, alterations in mesocorticolimbic glutamatergic homeostasis and neuroplasticity are possible functional targets for the treatment of alcohol use disorders.
Effects of inosine monophosphate and exercise training on neuronal nitric oxide synthase in the mouse brain
2020, Neuroscience Letters
Citation Excerpt :
The phosphorylation of nNOS is an important mechanism for modulating nNOS function and NO production. Previously, nNOS was reported to be phosphorylated by the PI3K/Akt pathway in the brain and skeletal muscle [31,36]. Consistent with this finding, the pnNOS and pAkt levels in both the dHp and vHp were positively correlated in this study.
Recently, the purine nucleoside inosine has been demonstrated to have several neuroprotective effects. Similarly, exercise training has well-known beneficial effects on mental health and cognitive function. Neuronal nitric oxide synthase (nNOS) is a key neuronal messenger in several brain regions, and the downregulation of nNOS has been shown to improve brain function. However, whether inosine and exercise training have combined effects on nNOS pathway-related proteins in the brain remains unknown. We found, for the first time, that inosine monophosphate (IMP), which is a precursor of inosine, decreases nNOS levels in the ventral hippocampus (vHp) and the cerebellum (Ce), but not in the dorsal hippocampus (dHp). In the vHp, the phosphorylation of cAMP response element-binding protein (CREB) was also upregulated, which negatively correlated with nNOS protein levels. In the cerebral cortex (Cx), no significant activation of the nNOS pathway was observed. In the dHp, vHp, Cx, and Ce, no interactions between the effects of IMP and exercise on nNOS protein and CREB phosphorylation levels were observed. The phosphorylation of nNOS was regulated by the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Although IMP induced minor changes in Akt phosphorylation, nNOS phosphorylation was unchanged by either IMP or exercise. In conclusion, in the vHp, which is associated with emotional behavior, IMP decreased nNOS levels and activated CREB, suggesting that IMP can elicit anxiolytic effects.

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¹: Present address: Department of Genetics and The Paul F. Glenn Laboratories for the Biological Mechanisms of Aging, Harvard Medical School, Boston, MA, USA.

²: Present address: Integrity Bio, Inc., Camarillo, CA 93012, USA.

³: Present address: Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245–3207, USA.

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Published by Elsevier Inc.

Neuronal nitric oxide synthase is phosphorylated in response to insulin stimulation in skeletal muscle

Highlights

Abstract

Introduction

Section snippets

Cell culture and treatment

nNOS is phosphorylated in response to insulin in C2C12 myotubes

Acknowledgments

Trends Endocrinol. Metab.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Neuroscience

J. Biol. Chem.

Metabolism

Contribution of nitric oxide to metabolic coronary vasodilation in the human heart

Circulation

Nitric oxide as a neurotransmitter in peripheral nerves: nature of transmitter and mechanism of transmission

Annu. Rev. Physiol.

Enzymatic function of nitric oxide synthases

Cardiovasc. Res.

Insulin resistance in mice lacking neuronal nitric oxide synthase is related to an alpha-adrenergic mechanism