The hypermetabolic state of burns is associated with uncontrolled catabolism of proteins, fat and carbohydrates, and affects morbidity and mortality. The associated major metabolic anomaly is resistance to the effects of insulin, the pivotal anabolic hormone. Among the signaling cascades activated by insulin, the insulin receptor (IR), insulin receptor substrates (IRSs), phosphatidylinositol-3-phosphate kinase (PI 3-K) and Akt/PKB are central for energy metabolism and glucose homeostasis. Activated Akt/PKB in turn inhibits its downstream molecule, glucose synthase kinase-3 (GSK-3), resulting in increased protein and glycogen synthesis. Altered activation of all these signaling molecules occurs following burn injury, but the molecular mechanisms inducing these changes have not been elucidated. Many cytokines are expressed locally and systematically following burn injury, leading to increased expression of inducible nitric oxide (iNOS), and release of high levels of nitric oxide (NO). Based on compelling and convincing preliminary data, we hypothesize that iNOS, via release of NO with superoxide, plays an important role in insulin resistance of burn by altered signaling via IR, IRSs, PI 3-K, Akt/PKB and GSK-3. The following Specific Aims will test the above hypothesis in burn/sham-injured rodents in vivo, in cultured cells and in reconstituted in vitro systems:
Specific Aim 1 will test the hypothesis that iNOS is required for insulin resistance.
Specific Aim 2 will test the hypothesis that the exaggerated production of NO by iNOS decreases tyrosine kinase activity of IR and tyrosyl phosphorylation of IRSs. The molecular mechanism of inactivation of JR and IRSs (S-nitrosylation vs. nitration) will also be identified.
Specific Aim 3 will test the hypothesis that the exaggerated production of NO by iNOS alters the kinase activity of Akt/PKB, the further downstream molecule of PI 3-K, independent of IR and IRSs. The molecular mechanisms responsible for inactivation (post-translational modifications) of Ak/IPKB by NO will also be identified.
Specific Aim 4 will test the hypothesis that exaggerated production of NO by iNOS increases activity of GSK-3, due to effects related to both decreased AktJPKB activity and direct effects of NO on GSK-3. The direct role of NO on activation of GSK-3 (independent of AktIPKB) will be tested with NO donors and scavengers. The in vivo studies will include the use of burn and sham-injured rats, and iNOS knock out (-/-) and wild type (+/+) mice. Insulin mediated signaling changes, and the post-translational modifications in the signaling molecules enumerated above with and without specific iNOS inhibitor (1400W) will be evaluated. Functional changes, evaluated using 2-deoxyglucose uptake in muscle and adipocyte, will be correlated to signaling changes. Using adipocyte and myocyte cell lines and primary cultures from iNOS -/- and iNOS +/+ mice, the role of iNOS/NO will be evaluated with and without NO donors or scavengers. The role of NO will be confirmed in in vitro reconstitution system containing active signaling molecules. The post-translational modifications (nitration vs. S-nitrosylation) associated with 1NOS/NO will be studied by biochemical, spectrophotometric and immunoblot techniques. Several lines of evidence suggest that protein S-nitrosylationl-denitrosylation and tyrosine nitration/denitration may serve as regulatory components. The involvement of NO in insulin resistance will be assessed in the light of this new concept. The immediate short-term goals of these studies are, therefore, to characterize the molecular and biochemical mechanisms inducing insulin resistance, so that in the long-term, insulin resistance of burn injury in humans can be reversed. The studies together will thus provide significant insights into the pathogenesis of insulin resistance and provide information on novel therapeutic strategies to treat burn, and other stress or inflammation-induced insulin resistance.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Somers, Scott D
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Massachusetts General Hospital
United States
Zip Code
Lee, Sangseok; Yang, Hong-Seuk; Sasakawa, Tomoki et al. (2014) Immobilization with atrophy induces de novo expression of neuronal nicotinic ?7 acetylcholine receptors in muscle contributing to neurotransmission. Anesthesiology 120:76-85
Kaneki, Masao; Fukushima, Yuji; Shinozaki, Shohei et al. (2013) iNOS inhibitor, L-NIL, reverses burn-induced glycogen synthase kinase-3? activation in skeletal muscle of rats. Metabolism 62:341-6
Nagashima, Michio; Yasuhara, Shingo; Martyn, J A Jeevendra (2013) Train-of-four and tetanic fade are not always a prejunctional phenomenon as evaluated by toxins having highly specific pre- and postjunctional actions. Anesth Analg 116:994-1000
Zhu, Shimei; Nagashima, Michio; Khan, Mohammed A S et al. (2013) Lack of caspase-3 attenuates immobilization-induced muscle atrophy and loss of tension generation along with mitigation of apoptosis and inflammation. Muscle Nerve 47:711-21
Yasuda, Yoshikazu; Fukushima, Yuji; Kaneki, Masao et al. (2013) Anesthesia with propofol induces insulin resistance systemically in skeletal and cardiac muscles and liver of rats. Biochem Biophys Res Commun 431:81-5
Frick, Christiane G; Fink, Heidrun; Blobner, Manfred et al. (2012) A single injection of botulinum toxin decreases the margin of safety of neurotransmission at local and distant sites. Anesth Analg 114:102-9
Khan, Mohammed Abdul Sattar; Farkhondeh, Mina; Crombie, Jennifer et al. (2012) Lipopolysaccharide upregulates ?7 acetylcholine receptors: stimulation with GTS-21 mitigates growth arrest of macrophages and improves survival in burned mice. Shock 38:213-9
Sugita, Michiko; Sugita, Hiroki; Kim, Minhye et al. (2012) Inducible nitric oxide synthase deficiency ameliorates skeletal muscle insulin resistance but does not alter unexpected lower blood glucose levels after burn injury in C57BL/6 mice. Metabolism 61:127-36
Lee, Hyung-yul; Kaneki, Masao; Andreas, Jonathan et al. (2011) Novel mitochondria-targeted antioxidant peptide ameliorates burn-induced apoptosis and endoplasmic reticulum stress in the skeletal muscle of mice. Shock 36:580-5
Frick, Christiane G; Helming, Marc; Martyn, J A Jeevendra et al. (2010) Continuous administration of pyridostigmine improves immobilization-induced neuromuscular weakness. Crit Care Med 38:922-7

Showing the most recent 10 out of 45 publications