Role of Protein Farnesylation in Burn-Induced Metabolic Derangements and Insulin Resistance in Mouse Skeletal Muscle

Objective: Metabolic derangements, including insulin resistance and hyperlactatemia, are a major complication of major trauma (e.g., burn injury) and affect the prognosis of burn patients. Protein farnesylation, a posttranslational lipid modification of cysteine residues, has been emerging as a potential component of inflammatory response in sepsis. However, farnesylation has not yet been studied in major trauma. To study a role of farnesylation in burn-induced metabolic aberration, we examined the effects of farnesyltransferase (FTase) inhibitor, FTI-277, on burn-induced insulin resistance and metabolic alterations in mouse skeletal muscle. Methods: A full thickness burn (30% total body surface area) was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP) or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated. Results: Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt and GSK-3β was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling), PTEN (a negative regulator of Akt-mediated signaling), protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277. Conclusions: Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a pivotal role in burn-induced metabolic dysfunction and inflammatory response. Our study identifies FTase as a novel potential molecular target to reverse or ameliorate metabolic derangements in burn patients

Battery of behavioral tests in mice to study postoperative delirium

Postoperative delirium is associated with increased morbidity, mortality and cost. However, its neuropathogenesis remains largely unknown, partially owing to lack of animal model(s). We therefore set out to employ a battery of behavior tests, including natural and learned behavior, in mice to determine the effects of laparotomy under isoflurane anesthesia (Anesthesia/Surgery) on these behaviors. The mice were tested at 24 hours before and at 6, 9 and 24 hours after the Anesthesia/Surgery. Composite Z scores were calculated. Cyclosporine A, an inhibitor of mitochondria permeability transient pore, was used to determine potential mitochondria-associated mechanisms of these behavioral changes. Anesthesia/Surgery selectively impaired behaviors, including latency to eat food in buried food test, freezing time and time spent in the center in open field test, and entries and duration in the novel arm of Y maze test, with acute onset and various timecourse. The composite Z scores quantitatively demonstrated the Anesthesia/Surgery-induced behavior impairment in mice. Cyclosporine A selectively ameliorated the Anesthesia/Surgery-induced reduction in ATP levels, the increases in latency to eat food, and the decreases in entries in the novel arm. These findings suggest that we could use a battery of behavior tests to establish a mouse model to study postoperative delirium

Farnesyltransferase inhibitors prevent HIV protease inhibitor (lopinavir/ritonavir)-induced lipodystrophy and metabolic syndrome in mice

Highly active antiretroviral therapy (HAART) has successfully reduced the mortality rate of patients with human immune deficiency virus (HIV) and HIV protease inhibitors (HIV PIs) are key components of HAART. Complications of HAART, particularly those associated with HIV PIs including lipodystrophy and metabolic disturbance, have emerged as an important public health issue. No specific treatment is available to prevent and/or treat HIV PI-associated lipodystrophy and metabolic syndrome. The present study demonstrated that a relatively low-dose of farnesyltransferase inhibitor (FTI), tipifarnib (3 mg/kg/day, subcutaneous injection) and lonafarnib (5 mg/kg/day, subcutaneous injection), prevented the onset of lipodystrophy and metabolic syndrome induced by the combination of two HIV PIs, lopinavir (50 mg/kg/day, intraperitoneal injection) and ritonavir (12.5 mg/kg/day, intraperitoneal injection), in mice. Consistent with previous studies, treatment with lopinavir/ritonavir for 2 weeks decreased body weight, adipose tissue mass, levels of plasma adiponectin and leptin, and increased plasma levels of triglycerides, total cholesterol and insulin. Tipifarnib and lonafarnb prevented or ameliorated all of these alterations in the HIV PI-treated mice. These data identify FTIs as a novel potential strategy to prevent or treat HIV PI-associated lipodystrophy and metabolic syndrome in HIV-infected patients on HAART

Farnesysltransferase Inhibitor Prevents Burn Injury-Induced Metabolome Changes in Muscle

Burn injury remains a significant public health issue worldwide. Metabolic derangements are a major complication of burn injury and negatively affect the clinical outcomes of severely burned patients. These metabolic aberrations include muscle wasting, hypermetabolism, hyperglycemia, hyperlactatemia, insulin resistance, and mitochondrial dysfunction. However, little is known about the impact of burn injury on the metabolome profile in skeletal muscle. We have previously shown that farnesyltransferase inhibitor (FTI) reverses burn injury-induced insulin resistance, mitochondrial dysfunction, and the Warburg effect in mouse skeletal muscle. To evaluate metabolome composition, targeted quantitative analysis was performed using capillary electrophoresis mass spectrometry in mouse skeletal muscle. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and hierarchical cluster analysis demonstrated that burn injury induced a global change in metabolome composition. FTI treatment almost completely prevented burn injury-induced alterations in metabolite levels. Pathway analysis revealed that the pathways most affected by burn injury were purine, glutathione, β-alanine, glycine, serine, and threonine metabolism. Burn injury induced a suppressed oxidized to reduced nicotinamide adenine dinucleotide (NAD+/NADH) ratio as well as oxidative stress and adenosine triphosphate (ATP) depletion, all of which were reversed by FTI. Moreover, our data raise the possibility that burn injury may lead to increased glutaminolysis and reductive carboxylation in mouse skeletal muscle

Hereditary Neuropathy with Liability to Pressure Palsy and Vocal Cord Paralysis After Pulmonary Lobectomy: A Case Report.

Hereditary neuropathy with liability to pressure palsy (HNPP) is a rare peripheral neurological disorder that manifests with increased sensitivity to pressure. In people with this disorder, the peripheral nerves are unusually sensitive to pressure. Minor trauma or compression causing paralysis in the extremities is a hallmark of this disorder. Ensuring there is no pressure on the extremities is recommended as a preventive measure. We describe for the first time, postoperative vocal cord paralysis in a patient with HNPP due to left recurrent laryngeal nerve palsy. Anesthesiologists and surgeons should be aware of this possible complication in patients with HNPP

iNOS as a Driver of Inflammation and Apoptosis in Mouse Skeletal Muscle after Burn Injury: Possible Involvement of Sirt1 S-Nitrosylation-Mediated Acetylation of p65 NF-κB and p53

Inflammation and apoptosis develop in skeletal muscle after major trauma, including burn injury, and play a pivotal role in insulin resistance and muscle wasting. We and others have shown that inducible nitric oxide synthase (iNOS), a major mediator of inflammation, plays an important role in stress (e.g., burn)-induced insulin resistance. However, it remains to be determined how iNOS induces insulin resistance. Moreover, the interrelation between inflammatory response and apoptosis is poorly understood, although they often develop simultaneously. Nuclear factor (NF)-κB and p53 are key regulators of inflammation and apoptosis, respectively. Sirt1 inhibits p65 NF-κB and p53 by deacetylating these transcription factors. Recently, we have shown that iNOS induces S-nitrosylation of Sirt1, which inactivates Sirt1 and thereby increases acetylation and activity of p65 NF-κB and p53 in various cell types, including skeletal muscle cells. Here, we show that iNOS enhances burn-induced inflammatory response and apoptotic change in mouse skeletal muscle along with S-nitrosylation of Sirt1. Burn injury induced robust expression of iNOS in skeletal muscle and gene disruption of iNOS significantly inhibited burn-induced increases in inflammatory gene expression and apoptotic change. In parallel, burn increased Sirt1 S-nitrosylation and acetylation and DNA-binding capacity of p65 NF-κB and p53, all of which were reversed or ameliorated by iNOS deficiency. These results indicate that iNOS functions not only as a downstream effector but also as an upstream enhancer of burn-induced inflammatory response, at least in part, by Sirt1 S-nitrosylation-dependent activation (acetylation) of p65 NF-κB. Our data suggest that Sirt1 S-nitrosylation may play a role in iNOS-mediated enhanced inflammatory response and apoptotic change, which, in turn, contribute to muscle wasting and supposedly to insulin resistance after burn injury

FTI-277 treatment prevented burn-induced increased protein farnesylation and FTase expression in skeletal muscle.

<p>(A) The amount of farnesylated proteins was increased in skeletal muscle at 3 days after burn compared with sham-burn. FTI-277 treatment prevented burn-induced increase in farnesylated proteins. n = 10 mice per group. (B) Burn increased farnesyltransferase (FTase) protein expression compared with sham-burn. FTI-277 treatment significantly inhibited burn-induced increased FTase expression. n = 6 mice per group. **p<0.01, ***p<0.001.</p

Increases in farnesyltransferase (FTase), PTP-1B and PTEN expression were associated with hyperlactatemia following burn injury in mice.

<p>Muscle and blood samples were collected from naïve mice (Control), and at 6 h, 1 (D1), 3 (D3) and 7 days (D7) after burn injury. Immunoblot analysis revealed that FTase protein expression was significantly increased at 3 days after burn compared with naïve mice (B). PTP-1B and PTEN protein expression were significantly increased at 3 days after burn compared with naïve mice (C, D). FTase, PTP-1B and PTEN expression were normalized to that of GAPDH. Plasma lactate concentration was significantly increased at 3 days after burn compared with naïve mice (E). *p<0.05, **p<0.01, ***p<0.001 vs. Control. n = 4 mice at each time point.</p