Tadalafil Integrates Nitric Oxide-Hydrogen Sulfide Signaling to Inhibit High Glucose-induced Matrix Protein Synthesis in Podocytes.

Diabetes-induced kidney cell injury involves an increase in matrix protein expression that is only partly alleviated by current treatment, prompting a search for new modalities. We have previously shown that hydrogen sulfide (H2S) inhibits high glucose-induced protein synthesis in kidney podocytes. We tested whether tadalafil, a phosphodiesterase 5 inhibitor used to treat erectile dysfunction, ameliorates high glucose stimulation of matrix proteins by generating H2S in podocytes. Tadalafil abrogated high glucose stimulation of global protein synthesis and matrix protein laminin γ1. Tadalafil inhibited high glucose-induced activation of mechanistic target of rapamycin complex 1 and laminin γ1 accumulation in an AMP-activated protein kinase (AMPK)-dependent manner. Tadalafil increased AMPK phosphorylation by stimulating calcium-calmodulin kinase kinase ß. Tadalafil rapidly increased the expression and activity of the H2S-generating enzyme cystathionine γ-lyase (CSE) by promoting its translation. dl-Propargylglycine, a CSE inhibitor, and siRNA against CSE inhibited tadalafil-induced AMPK phosphorylation and abrogated the tadalafil effect on high glucose stimulation of laminin γ1. In tadalafil-treated podocytes, we examined the interaction between H2S and nitric oxide (NO). N(ω)-Nitro-L-arginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, inhibitors of NO synthase (NOS) and soluble guanylyl cyclase, respectively, abolished tadalafil induction of H2S and AMPK phosphorylation. Tadalafil rapidly augmented inducible NOS (iNOS) expression by increasing its mRNA, and siRNA for iNOS and 1400W, an iNOS blocker, inhibited tadalafil stimulation of CSE expression and AMPK phosphorylation. We conclude that tadalafil amelioration of high glucose stimulation of synthesis of proteins including matrix proteins in podocytes requires integration of the NO-H2S-AMPK axis leading to the inhibition of high glucose-induced mechanistic target of rapamycin complex 1 activity and mRNA translation.

Activation of glycogen synthase kinase 3ß ameliorates diabetes-induced kidney injury.

Increase in protein synthesis contributes to kidney hypertrophy and matrix protein accumulation in diabetes. We have previously shown that high glucose-induced matrix protein synthesis is associated with inactivation of glycogen synthase kinase 3ß (GSK3ß) in renal cells and in the kidneys of diabetic mice. We tested whether activation of GSK3ß by sodium nitroprusside (SNP) mitigates kidney injury in diabetes. Studies in kidney-proximal tubular epithelial cells showed that SNP abrogated high glucose-induced laminin increment by stimulating GSK3ß and inhibiting Akt, mTORC1, and events in mRNA translation regulated by mTORC1 and ERK. NONOate, an NO donor, also activated GSK3ß, indicating that NO may mediate SNP stimulation of GSK3ß. SNP administered for 3 weeks to mice with streptozotocin-induced type 1 diabetes ameliorated kidney hypertrophy, accumulation of matrix proteins, and albuminuria without changing blood glucose levels. Signaling studies showed that diabetes caused inactivation of GSK3ß by activation of Src, Pyk2, Akt, and ERK; GSK3ß inhibition activated mTORC1 and downstream events in mRNA translation in the kidney cortex. These reactions were abrogated by SNP. We conclude that activation of GSK3ß by SNP ameliorates kidney injury induced by diabetes.

Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells.

Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase ß, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase ß inhibitor, had the same effect. Renal cortical content of cystathionine ß-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

An evolutionarily conserved TNF-alpha-responsive enhancer in the far upstream region of human CCL2 locus influences its gene expression.

Comparative cross-species genomic analysis has served as a powerful tool to discover novel noncoding regulatory regions that influence gene expression in several cytokine loci. In this study, we have identified several evolutionarily conserved regions (ECRs) that are shared between human, rhesus monkey, dog, and horse and that are upstream of the promoter regions that have been previously shown to play a role in regulating CCL2 gene expression. Of these, an ECR that was ~16.5 kb (-16.5 ECR) upstream of its coding sequence contained a highly conserved NF-κB site. The region encompassing the -16.5 ECR conferred TNF-α responsiveness to homologous and heterologous promoters. In vivo footprinting demonstrated that specific nucleotide residues in the -16.5 ECR were protected or became hypersensitive after TNF-α treatment. The footprinted regions were found to bind NF-κB subunits in vitro and in vivo. Mutation/deletion of the conserved NF-κB binding site in the -16.5 ECR led to loss of TNF-α responsiveness. After TNF-α stimulation, the -16.5 ECR showed increased sensitivity to nuclease digestion and loss of histone signatures that are characteristic of a repressive chromatin. Chromosome conformation capture assays indicated that -16.5 ECR physically interacts with the CCL2 proximal promoter after TNF-α stimulation. Taken together, these results suggest that the -16.5 ECR may play a critical role in the regulation of CCL2.

Long-term methamphetamine self-administration increases mesolimbic mitochondrial oxygen consumption and decreases striatal glutathione.

Neurotoxic regimens of methamphetamine (METH) are known to increase reactive oxygen species (ROS), affect redox homeostasis, and lead to damage in dopamine neurons. Functional changes induced by long-term METH self-administration on mitochondrial respiratory metabolism and redox homeostasis are less known. To fill this gap, we implanted a jugular catheter into adult male mice and trained them to nose poke for METH infusions. After several weeks of METH exposure, we collected samples of the ventral striatum (vST) and the ventral midbrain (vMB). We used HPLC to determine the levels of the ROS scavenger glutathione in its reduced (GSH) and oxidized forms. Then, we used high-resolution respirometry to determine the oxygen consumption rate (OCR) of mitochondrial complexes. Finally, using in vivo electrophysiology, we assessed changes in dopamine neuron firing activity in the VTA. METH self-administration produced a decrease of the GSH pool in vST, correlating with lifetime METH intake. We observed increased mitochondrial respiration across the two mesolimbic regions. METH self-administration decreases firing rate and burst activity but increases the number of spontaneously active dopamine neurons per track. We conclude that METH self-administration progressively decreased the antioxidant pool in sites of higher dopamine release and produced an increase in mitochondrial metabolism in the mesolimbic areas, probably derived from the increased number of dopamine neurons actively firing. However, dopamine neuron firing activity is decreased by METH self-administration, reflecting a new basal level of dopamine neurotransmission.

Crossroads between Autoimmunity and COVID-19 in Lung Transplant Recipients.

The presence of a certain group of auto-antibodies (AAbs) is known to correlate with the severity of COVID-19. It is, however, unknown if such AAbs are prevalent and impact COVID-19-related outcomes in lung transplant recipients (LTRs) who are immunosuppressed. We performed a retrospective study of LTRs with COVID-19 and analyzed samples before and after COVID-19 for IgG AAbs. AAbs analysis was carried out using autoimmune and coronavirus microarray and the resulting cross-sectional differences in Ab-scores and clinical variables were analyzed using Fischer's Exact test for categorical variables and a paired t-test for continuous variables. Linear regression was used to analyze the differences in Ab-scores and COVID-19 severity. LTRs with non-severe [NS gp (n = 10)], and severe [S gp (n = 8)] COVID-19 disease were included. Ferritin and acute respiratory failure were higher in the S group (p = 0.03; p < 0.0001). Among the AAbs analyzed, interferon-related AAbs (IFN-alpha2, IFN-beta, IFN lamba, IFN-epsilon), eight interleukin-related AAbs, and several tissue-related AAbs were also found to be changed significantly from pre- to post-COVID-19 (p < 0.05). IFN-lambda (p = 0.03) and IL-22 (p = 0.002) were significantly associated with COVID-19 severity and remained significant in linear regression analysis while controlling for other variables. AAbs are common in LTRs, and certain groups of antibodies are particularly enhanced in LTRs with severe COVID-19. Preliminary observations of this study need to be confirmed by a larger sample size.

Ribosomal biogenesis induction by high glucose requires activation of upstream binding factor in kidney glomerular epithelial cells.

Diabetes promotes protein synthesis to induce kidney hypertrophy and increase renal matrix proteins. Increased capacity for mRNA translation by way of ribosomal biogenesis facilitates sustained stimulation of protein synthesis. We tested the hypothesis that high glucose induces ribosomal biogenesis as indicated by an increase in rRNA synthesis in the setting of augmented protein synthesis. High glucose (30 mM) increased global protein synthesis, expression of matrix proteins, laminin γ1 and fibronectin, and rDNA transcription in glomerular epithelial cells (GECs) compared with 5 mM glucose. High glucose induced Ser388 phosphorylation of upstream binding factor (UBF), an rDNA transcription factor, along with increased phosphorylation of Erk and p70S6 kinase. Inactivation of Erk and p70S6 kinase either by their respective chemical inhibitors or by expression of their inactive mutant constructs blocked high-glucose-induced UBF phosphorylation. High glucose reduced nuclear content of p19ARF and promoted dissolution of inactive UBF-p19ARF complex. High glucose also promoted association of UBF with RPA194, a subunit of RNA polymerase I. Inhibition of Erk, p70S6 kinase, and UBF1 by transfecting GECs with their respective inactive mutants abolished laminin γ1 synthesis, protein synthesis, and rDNA transcription. Renal cortex from type 1 diabetic rats and type 2 diabetic db/db mice showed increased phosphorylation of UBF, Erk, and p70S6 kinase coinciding with renal hypertrophy and onset of matrix accumulation. Our data suggest that augmented ribosome biogenesis occurs in an UBF-dependent manner during increased protein synthesis induced by high glucose in the GECs that correlates with UBF activation and renal hypertrophy in rodents with type 1 and type 2 diabetes.

HIV-associated nephropathy: role of mammalian target of rapamycin pathway.

Both glomerular and tubular lesions are characterized by a proliferative phenotype in HIV-associated nephropathy. We hypothesized that mammalian target of rapamycin (mTOR) contributes to the development of the HIVAN phenotype. Both glomerular and tubular epithelial cells showed enhanced expression of phospho (p)-mTOR in HIV-1 transgenic mice (Tgs). In addition, renal tissues of transgenic mice (RT-Tg) showed enhanced phosphorylation of p70S6 kinase and an associated diminished phosphorylation of eEF2. Moreover, RT-Tgs showed enhanced phosphorylation of 4EBP1 and eIF4B; these findings indicated activation of the mTOR pathway in RT-Tgs. To test our hypothesis, age- and sex-matched control mice and Tgs were administered either saline or rapamycin (an inhibitor of the mTOR pathway) for 4 weeks. Tgs receiving rapamycin not only showed inhibition of the mTOR-associated downstream signaling but also displayed attenuated renal lesions. RT-Tgs showed enhanced expression of hypoxia-inducible factor-alpha and also displayed increased expression of vascular endothelial growth factor; on the other hand, rapamycin inhibited RT-Tg expression of both hypoxia-inducible factor-alpha and vascular endothelial growth factor. We conclude that the mTOR pathway contributes to the HIVAN phenotype and that inhibition of the mTOR pathway can be used as a therapeutic strategy to alter the course of HIVAN.

Targeting cPLA2 derived lipid hydroperoxides as a potential intervention for sarcopenia.

Defects in neuromuscular innervation contribute significantly to the age-related decline in muscle mass and function (sarcopenia). Our previous studies demonstrated that denervation induces muscle mitochondrial hydroperoxide production (H2O2 and lipid hydroperoxides (LOOHs)). Here we define the relative contribution of mitochondrial electron transport chain (ETC) derived H2O2 versus cytosolic phospholipase A2 (cPLA2) derived LOOHs in neurogenic muscle atrophy. We show that denervation increases muscle cPLA2 protein content, activity, and metabolites downstream of cPLA2 including LOOHs. Increased scavenging of mitochondrial H2O2 does not protect against denervation atrophy, suggesting ETC generated H2O2 is not a critical player. In contrast, inhibition of cPLA2 in vivo mitigates LOOH production and muscle atrophy and maintains individual muscle fiber size while decreasing oxidative damage. Overall, we show that loss of innervation in several muscle atrophy models including aging induces generation of LOOHs produced by arachidonic acid metabolism in the cPLA2 pathway contributing to loss of muscle mass.

Molecular changes associated with spinal cord aging.

Age-related muscle weakness and loss of muscle mass (sarcopenia) is a universal problem in the elderly. Our previous studies indicate that alpha motor neurons (α-MNs) play a critical role in this process. The goal of the current study is to uncover changes in the aging spinal cord that contribute to loss of innervation and the downstream degenerative processes that occur in skeletal muscle. The number of α-MNs is decreased in the spinal cord of wildtype mice during aging, beginning in middle age and reaching a 41% loss by 27 months of age. There is evidence for age-related loss of myelin and mild inflammation, including astrocyte and microglia activation and an increase in levels of sICAM-1. We identified changes in metabolites consistent with compromised neuronal viability, such as reduced levels of N-acetyl-aspartate. Cleaved caspase-3 is more abundant in spinal cord from old mice, suggesting that apoptosis contributes to neuronal loss. RNA-seq analysis revealed changes in the expression of a number of genes in spinal cord from old mice, in particular genes encoding extracellular matrix components (ECM) and a 172-fold increase in MMP-12 expression. Furthermore, blood-spinal cord barrier (BSCB) permeability is increased in old mice, which may contribute to alterations in spinal cord homeostasis and exacerbate neuronal distress. Together, these data show for the first time that the spinal cord undergoes significant changes during aging, including progressive α-MNs loss that is associated with low-grade inflammation, apoptosis, changes in ECM, myelination, and vascular permeability.

Molecular changes in transcription and metabolic pathways underlying muscle atrophy in the CuZnSOD null mouse model of sarcopenia.

Mice lacking the superoxide anion scavenger CuZn superoxide dismutase (Sod1-/- mice) develop a number of age-related phenotypes, including an early progression of muscle atrophy and weakness (sarcopenia) associated with loss of innervation. The purpose of this study was to delineate the early development of sarcopenia in the Sod1-/- mice and to measure changes in the muscle transcriptome, proteome, and eicosanoid profile at the stage when sarcopenia is markedly induced in this model (7-9 months of age). We found a strong correlation between muscle atrophy and mitochondrial state 1 hydroperoxide production, which was 40% higher in isolated mitochondria from Sod1-/- mouse gastrocnemius muscle by 2 months of age. The primary pathways showing altered gene expression in Sod1-/- mice identified by RNA-seq transcriptomic analysis are protein ubiquitination, synaptic long-term potentiation, calcium signaling, phospholipase C signaling, AMPK, and TWEAK signaling. Targeted proteomics shows elevated expression of mitochondrial proteins, fatty acid metabolism enzymes, tricarboxylic acid (TCA) cycle enzymes, and antioxidants, while enzymes involved in carbohydrate metabolism are downregulated in Sod1-/- mice. LC-MS analysis of lipids in gastrocnemius muscle detected 78 eicosanoids, of which 31 are significantly elevated in muscle from Sod1-/- mice. These data suggest that mitochondrial hydroperoxide generation is elevated prior to muscle atrophy and may be a potential driving factor of changes in the transcriptome, proteome, and eicosanoid profile of the Sod1-/- mice. Together, these analyses revealed important molecular events that occur during muscle atrophy, which will pave the way for future studies using new approaches to treat sarcopenia.

Regulation of mRNA translation in renal physiology and disease.

Translation, a process of generating a peptide from the codons present in messenger RNA, can be a site of independent regulation of protein synthesis; it has not been well studied in the kidney. Translation occurs in three stages (initiation, elongation, and termination), each with its own set of regulatory factors. Mechanisms controlling translation include small inhibitory RNAs such as microRNAs, binding proteins, and signaling reactions. Role of translation in renal injury in diabetes, endoplasmic reticulum stress, acute kidney injury, and, in physiological adaptation to loss of nephrons is reviewed here. Contribution of mRNA translation to physiology and disease is not well understood. Because it is involved in such diverse areas as development and cancer, it should prove a fertile field for investigation in renal science.

PKCdelta regulates the stimulation of vascular endothelial factor mRNA translation by angiotensin II through hnRNP K.

Angiotensin II (Ang II)-induced renal injury is partly mediated by growth factors such as VEGF. We have previously shown that Ang II rapidly increases VEGF protein synthesis in proximal tubular epithelial (MCT) cells by augmenting mRNA translation, which is partly dependent on activation and binding of hnRNP K to 3' untranslated region (UTR) of VEGF mRNA. Regulation of hnRNP K activation by PKCdelta was studied in MCT cells. Transfection with a PKCdelta siRNA inhibited hnRNP K Ser302 phosphorylation and activation, and reduced Ang II stimulation of VEGF synthesis. Inhibition of PKCdelta with röttlerin also prevented binding of hnRNP K to VEGF mRNA and reduced the efficiency of VEGF mRNA translation. In db/db mice at 2 weeks of type 2 diabetes, VEGF expression was increased, which was due not to increase in transcription but to augmented translation of VEGF mRNA. Augmented VEGF expression was associated with increased binding of hnRNP K to VEGF mRNA. c-src and PKCdelta activities and hnRNP K phosphorylation on Ser302 in renal cortex of db/db mice were increased compared to control mice. We conclude: Ang II-induced VEGF mRNA translation is associated with activation of hnRNP K in MCT cells. In the signaling pathway leading to hnRNP K activation induced by Ang II, PKCdelta is downstream of c-src. PKCdelta-mediated phosphorylation of hnRNP K is required for Ang II stimulation of VEGF mRNA translation. In mice with type 2 diabetes, src and PKCdelta activation and hnRNP K phosphorylation correlate with increased VEGF mRNA translation and kidney hypertrophy. 3' UTR events are important in regulation of VEGF expression in models of renal injury.

Restoration of SERCA ATPase prevents oxidative stress-related muscle atrophy and weakness.

Molecular targets to reduce muscle weakness and atrophy due to oxidative stress have been elusive. Here we show that activation of Sarcoplasmic Reticulum (SR) Ca2+ ATPase (SERCA) with CDN1163, a novel small molecule allosteric SERCA activator, ameliorates the muscle impairment in the CuZnSOD deficient (Sod1-/-) mouse model of oxidative stress. Sod1-/- mice are characterized by reduced SERCA activity, muscle weakness and atrophy, increased oxidative stress and mitochondrial dysfunction. Seven weeks of CDN1163 treatment completely restored SERCA activity and reversed the 23% reduction in gastrocnemius mass and 22% reduction in specific force in untreated Sod1-/- versus wild type mice. These changes were accompanied by restoration of autophagy protein markers to the levels found in wild-type mice. CDN1163 also reversed the increase in mitochondrial ROS generation and oxidative damage in muscle tissue from Sod1-/- mice. Taken together our findings suggest that the pharmacological restoration of SERCA is a promising therapeutic approach to counter oxidative stress-associated muscle impairment.

Metabolic and Stress Response Changes Precede Disease Onset in the Spinal Cord of Mutant SOD1 ALS Mice.

Many Amyotrophic Lateral Sclerosis (ALS) patients experience hypermetabolism, or an increase in measured vs. calculated metabolic rate. The cause of hypermetabolism and the effects on neuronal metabolism in ALS are currently unknown, but the efficacy of dietary interventions shows promise for metabolism as an ALS therapeutic target. The goal of this study is to measure changes in metabolic pathways as a function of disease progression in spinal cords of the SOD1G93A mouse model of ALS. We conducted a comprehensive assessment of protein expression for metabolic pathways, antioxidants, chaperones, and proteases in lumbar spinal cord from male SOD1G93A mice at pre-onset, onset, and end-stages of the disease using targeted proteomic analysis. These results reveal that protein content of metabolic proteins including proteins involved in glycolysis, ß-oxidation, and mitochondrial metabolism is altered in SOD1G93A mouse spinal cord well before disease onset. The changes in mitochondrial metabolism proteins are associated with decreased maximal respiration and glycolytic flux in SOD1G93A dermal fibroblasts and increased hydrogen peroxide and lipid hydroperoxide production in mitochondria from sciatic nerve and gastrocnemius muscle fibers at end stage of disease. Consistent with redox dysregulation, expression of the glutathione antioxidant system is decreased, and peroxiredoxins and catalase expression are increased. In addition, stress response proteases and chaperones, including those involved in the mitochondrial unfolded protein response (UPRmt), are induced before disease onset. In summary, we report that metabolic and stress response changes occur in SOD1G93A lumbar spinal cord before motor symptom onset, and are primarily caused by SOD1G93A expression and do not vary greatly as a function of disease course.

Role of Signaling Molecules in Mitochondrial Stress Response.

Mitochondria are established essential regulators of cellular function and metabolism. Mitochondria regulate redox homeostasis, maintain energy (ATP) production through oxidative phosphorylation, buffer calcium levels, and control cell death through apoptosis. In addition to these critical cell functions, recent evidence supports a signaling role for mitochondria. For example, studies over the past few years have established that peptides released from the mitochondria mediate stress responses such as the mitochondrial unfolded protein response (UPRMT) through signaling to the nucleus. Mitochondrial damage or danger associated molecular patterns (DAMPs) provide a link between mitochondria, inflammation and inflammatory disease processes. Additionally, a new class of peptides generated by the mitochondria affords protection against age-related diseases in mammals. In this short review, we highlight the role of mitochondrial signaling and regulation of cellular activities through the mitochondrial UPRMT that signals to the nucleus to affect homeostatic responses, DAMPs, and mitochondrial derived peptides.

Protein imbalance in the development of skeletal muscle wasting in tumour-bearing mice.

BACKGROUND: Cancer cachexia occurs in approximately 80% of cancer patients and is a key contributor to cancer-related death. The mechanisms controlling development of tumour-induced muscle wasting are not fully elucidated. Specifically, the progression and development of cancer cachexia are underexplored. Therefore, we examined skeletal muscle protein turnover throughout the development of cancer cachexia in tumour-bearing mice. METHODS: Lewis lung carcinoma (LLC) was injected into the hind flank of C57BL6/J mice at 8 weeks age with tumour allowed to develop for 1, 2, 3, or 4 weeks and compared with PBS injected control. Muscle size was measured by cross-sectional area analysis of haematoxylin and eosin stained tibialis anterior muscle. 2 H2 O was used to assess protein synthesis throughout the development of cancer cachexia. Immunoblot and RT-qPCR were used to measure regulators of protein turnover. TUNEL staining was utilized to measure apoptotic nuclei. LLC conditioned media (LCM) treatment of C2C12 myotubes was used to analyse cancer cachexia in vitro. RESULTS: Muscle cross-sectional area decreased ~40% 4 weeks following tumour implantation. Myogenic signalling was suppressed in tumour-bearing mice as soon as 1 week following tumour implantation, including lower mRNA contents of Pax7, MyoD, CyclinD1, and Myogenin, when compared with control animals. AchRδ and AchRε mRNA contents were down-regulated by ~50% 3 weeks following tumour implantation. Mixed fractional synthesis rate protein synthesis was ~40% lower in 4 week tumour-bearing mice when compared with PBS controls. Protein ubiquitination was elevated by ~50% 4 weeks after tumour implantation. Moreover, there was an increase in autophagy machinery after 4 weeks of tumour growth. Finally, ERK and p38 MAPK phosphorylations were fourfold and threefold greater than control muscle 4 weeks following tumour implantation, respectively. Inhibition of p38 MAPK, but not ERK MAPK, in vitro partially rescued LCM-induced loss of myotube diameter. CONCLUSIONS: Our findings work towards understanding the pathophysiological signalling in skeletal muscle in the initial development of cancer cachexia. Shortly following the onset of the tumour-bearing state alterations in myogenic regulatory factors are apparent, suggesting early onset alterations in the capacity for myogenic induction. Cancer cachexia presents with a combination of a loss of protein synthesis and increased markers of protein breakdown, specifically in the ubiquitin-proteasome system. Also, p38 MAPK may be a potential therapeutic target to combat cancer cachexia via a p38-FOX01-atrogene-ubiquitin-proteasome mechanism.

Sco2 deficient mice develop increased adiposity and insulin resistance.

Cytochrome c oxidase (COX) is an essential transmembrane protein complex (Complex IV) in the mitochondrial respiratory electron chain. Mutations in genes responsible for the assembly of COX are associated with Leigh syndrome, cardiomyopathy, spinal muscular atrophy and other fatal metabolic disorders in humans. Previous studies have shown that mice lacking the COX assembly protein Surf1 (Surf1-/- mice) paradoxically show a number of beneficial metabolic phenotypes including increased insulin sensitivity, upregulation of mitochondrial biogenesis, induction of stress response pathways and increased lifespan. To determine whether these effects are specific to the Surf1 mutation or a more general effect of reduced COX activity, we asked whether a different mutation causing reduced COX activity would have similar molecular and physiologic changes. Sco2 knock-in/knock-out (KI/KO) mice in which one allele of the Sco2 gene that encodes a copper chaperone required for COX activity is deleted and the second allele is mutated, have previously been shown to be viable despite a 30-60% reduction in COX activity. In contrast to the Surf1-/- mice, we show that Sco2 KI/KO mice have increased fat mass, associated with reduced ß-oxidation and increased adipogenesis markers, reduced insulin receptor beta (IR-ß levels in adipose tissue, reduced muscle glucose transporter 4 (Glut4) levels and a impaired response to the insulin tolerance test consistent with insulin resistance. COX activity and protein are reduced approximately 50% in adipose tissue from the Sco2 KI/KO mice. Consistent with the increase in adipose tissue mass, the Sco2 KI/KO mice also show increased hepatosteatosis, elevated serum and liver triglyceride and increased serum cholesterol levels compared to wild-type controls. In contrast to the Surf1-/- mice, which show increased mitochondrial number, upregulation of the mitochondrial unfolded protein response (UPRMT) pathway and no significant change in mitochondrial respiration in several tissues, Sco2 KI/KO mice do not upregulate the UPRMT, and tissue oxygen consumption and levels of several proteins involved in mitochondrial function are reduced in adipose tissue compared to wild type mice. Thus, the metabolic effects of the Sco2 and Surf1-/- mutations are opposite, despite comparable changes in COX activity, illuminating the complex impact of mitochondrial dysfunction on physiology and pointing to an important role for complex IV in regulating metabolism.

Ablation of the mitochondrial complex IV assembly protein Surf1 leads to increased expression of the UPR(MT) and increased resistance to oxidative stress in primary cultures of fibroblasts.

Mice deficient in the electron transport chain (ETC) complex IV assembly protein SURF1 have reduced assembly and activity of cytochrome c oxidase that is associated with an upregulation of components of the mitochondrial unfolded protein response (UPR(MT)) and increased mitochondrial number. We hypothesized that the upregulation of proteins associated with the UPR(MT) in response to reduced cytochrome c oxidase activity in Surf1(-/-) mice might contribute to increased stress resistance. To test this hypothesis we asked whether primary cultures of fibroblasts from Surf1(-/-) mice exhibit enhanced resistance to stressors compared to wild-type fibroblasts. Here we show that primary dermal fibroblasts isolated from Surf1(-/-) mice have increased expression of UPR(MT) components ClpP and Hsp60, and increased expression of Lon protease. Fibroblasts from Surf1(-/-) mice are significantly more resistant to cell death caused by oxidative stress induced by paraquat or tert-Butyl hydroperoxide compared to cells from wild-type mice. In contrast, Surf1(-/-) fibroblasts show no difference in sensitivity to hydrogen peroxide stress. The enhanced cell survival in response to paraquat or tert-Butyl hydroperoxide in Surf1(-/-) fibroblasts compared to wild-type fibroblasts is associated with induced expression of Lon, ClpP, and Hsp60, increased maximal respiration, and increased reserve capacity as measured using the Seahorse Extracellular Flux Analyzer. Overall these data support a protective role for the activation of the UPR(MT) in cell survival.

Rapamycin Increases Mortality in db/db Mice, a Mouse Model of Type 2 Diabetes.

We examined the effect of rapamycin on the life span of a mouse model of type 2 diabetes, db/db mice. At 4 months of age, male and female C57BLKSJ-lepr (db/db) mice (db/db) were placed on either a control diet, lacking rapamycin or a diet containing rapamycin and maintained on these diets over their life span. Rapamycin was found to reduce the life span of the db/db mice. The median survival of male db/db mice fed the control and rapamycin diets was 349 and 302 days, respectively, and the median survival of female db/db mice fed the control and rapamycin diets was 487 and 411 days, respectively. Adjusting for gender differences, rapamycin increased the mortality risk 1.7-fold in both male and female db/db mice. End-of-life pathological data showed that suppurative inflammation was the main cause of death in the db/db mice, which is enhanced slightly by rapamycin treatment.