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Acute and neurotoxic regimens of METH are known to increase reactive oxygen species (ROS), affect redox homeostasis, and lead to cellular damage in dopamine neurons. However, functional changes induced by long-term METH self-administration on mitochondrial respiratory metabolism and redox homeostasis are less known. To fill this gap in our knowledge, we implanted adult mice with a jugular catheter and trained them to nose poke for METH infusions in operant chambers. After completing several weeks of METH self-administration exposure, we collected samples of the ventral striatum (vSTR) and the ventral midbrain (vMB), containing the nucleus accumbens (NAc) and the ventral tegmental area (VTA), respectively. We used HPLC to determine the levels of the ROS scavenger glutathione in its reduced (GSH) and oxidized (GSSG) forms. Then, we used high-resolution respirometry to determine the oxygen consumption rate (OCR) of mitochondrial complexes under several substrates and inhibitors. Finally, we used in vivo single-unit extracellular recordings to assess changes in dopamine neuron firing activity in the VTA. METH self-administration produces a progressive decrease of the GSH pool in vST, which correlates with METH lifetime intake. We observed increased mitochondrial respiration across the two mesolimbic regions, but only vMB OCR correlates with METH lifetime intake. We recorded an increased number of spontaneously active dopamine neurons with decreased firing rate and burst activity in the VTA. METH lifetime intake inversely correlates with firing rate, the percentage of spikes in a burst, and directly correlates with the number of neurons per track. We conclude that METH self-administration progressively decreased the antioxidant pool in sites of higher dopamine release and produced an increased 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 in response to the prolonged effects of METH on dopamine release and circuitry feedback.

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11357-020-00189-x) contains supplementary material, which is available to authorized users.

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11357-020-00172-6) contains supplementary material, which is available to authorized users.

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.

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.

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, β-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.

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., Graphical abstract fx1, Highlights • Oxidative stress is related to SERCA dysfunction and muscle defect in Sod1-/- mice. • CDN1163 restores SERCA and prevents oxidative stress and muscle defect in Sod1-/- mice. • CDN1163 offers a promising therapy for muscle defect related to oxidative stress.

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.

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.

Summary Sarcopenia, the loss of skeletal muscle mass and function during aging, is a major contributor to disability and frailty in the elderly. Previous studies found a protective effect of reduced histone deacetylase activity in models of neurogenic muscle atrophy. Because loss of muscle mass during aging is associated with loss of motor neuron innervation, we investigated the potential for the histone deacetylase (HDAC) inhibitor butyrate to modulate age‐related muscle loss. Consistent with previous studies, we found significant loss of hindlimb muscle mass in 26‐month‐old C57Bl/6 female mice fed a control diet. Butyrate treatment starting at 16 months of age wholly or partially protected against muscle atrophy in hindlimb muscles. Butyrate increased muscle fiber cross‐sectional area and prevented intramuscular fat accumulation in the old mice. In addition to the protective effect on muscle mass, butyrate reduced fat mass and improved glucose metabolism in 26‐month‐old mice as determined by a glucose tolerance test. Furthermore, butyrate increased markers of mitochondrial biogenesis in skeletal muscle and whole‐body oxygen consumption without affecting activity. The increase in mass in butyrate‐treated mice was not due to reduced ubiquitin‐mediated proteasomal degradation. However, butyrate reduced markers of oxidative stress and apoptosis and altered antioxidant enzyme activity. Our data is the first to show a beneficial effect of butyrate on muscle mass during aging and suggests HDACs contribute to age‐related muscle atrophy and may be effective targets for intervention in sarcopenia and age‐related metabolic disease.

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 3β (GSK3β) in renal cells and in the kidneys of diabetic mice. We tested whether activation of GSK3β 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 GSK3β and inhibiting Akt, mTORC1, and events in mRNA translation regulated by mTORC1 and ERK. NONOate, an NO donor, also activated GSK3β, indicating that NO may mediate SNP stimulation of GSK3β. 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 GSK3β by activation of Src, Pyk2, Akt, and ERK; GSK3β inhibition activated mTORC1 and downstream events in mRNA translation in the kidney cortex. These reactions were abrogated by SNP. We conclude that activation of GSK3β by SNP ameliorates kidney injury induced by diabetes.

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 β-oxidation and increased adipogenesis markers, reduced insulin receptor beta (IR-β 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.

We explored molecular events associated with aging-induced matrix changes in the kidney. C57BL6 mice were studied in youth, middle age, and old age. Albuminuria and serum cystatin C level (an index of glomerular filtration) increased with aging. Renal hypertrophy was evident in middle-aged and old mice and was associated with glomerulomegaly and increase in mesangial fraction occupied by extracellular matrix. Content of collagen types I and III and fibronectin was increased with aging; increment in their mRNA varied with the phase of aging. The content of ZEB1 and ZEB2, collagen type I transcription inhibitors, and their binding to the collagen type Iα2 promoter by ChIP assay also showed age-phase-specific changes. Lack of increase in mRNA and data from polysome assay suggested decreased degradation as a potential mechanism for kidney collagen type I accumulation in the middle-aged mice. These changes occurred with increment in TGFβ mRNA and protein and activation of its SMAD3 pathway; SMAD3 binding to the collagen type Iα2 promoter was also increased. TGFβ-regulated microRNAs (miRs) exhibited selective regulation. The renal cortical content of miR-21 and miR-200c, but not miR-192, miR-200a, or miR-200b, was increased with aging. Increased miR-21 and miR-200c contents were associated with reduced expression of their targets, Sprouty-1 and ZEB2, respectively. These data show that aging is associated with complex molecular events in the kidney that are already evident in the middle age and progress to old age. Age-phase-specific regulation of matrix protein synthesis occurs and involves matrix protein-specific transcriptional and post-transcriptional mechanisms.

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.

We tested a pharmacological intervention to modulate sarcopenia, i.e, activation of the sarcoendoplasmic reticulum (SR) calcium ATPase (SERCA), that returns Ca2+ back into the SR following muscle contraction. We asked whether CDN1163, an allosteric activator of SERCA, could modulate muscle mass and function in a mouse model of sarcopenia (Sod1-/- mice). Two month old Sod1-/- mice were treated for 7 weeks with CDN1163 (50 mg/kg, i.p., 3x/week). After treatment, gastrocnemius mass of CDN1163-treated Sod1-/- mice was 23% greater than untreated Sod1-/- mice and the 22% reduction in specific force in untreated Sod1-/- versus wild type mice was reversed. CDN1163 also prevented the increase in muscle mitochondrial ROS generation in mitochondria from Sod1-/-mice and reduced muscle oxidative damage measured as F2-isoprostanes by 50% compared to untreated Sod1-/- mice. These findings suggest that pharmacological stabilization of SERCA with CDN1163 may be a powerful tool to counter age- and oxidative stress-associated muscle impairment.

Background Reactive oxygen species (ROS) are known to impair contractile function and contribute to muscle atrophy, and one of the primary sources of ROS is mitochondria. The goal of this study is to test whether muscle specific overexpression of mitochondrial hydrogen peroxide scavenger, peroxiredoxin3 (Prdx3), can prevent loss of muscle mass and function associated with sarcopenia. Methods We overexpressed prdx3 by flanking a prdx3 transgene with a stop codon that can be released by skeletal muscle specific expression of cre recombinase driven by the human skeletal actin (HSA) promotor. Muscle specific cre expression allows the transcription of the prdx3 transgene and leads to increased prdx3 expression in skeletal muscle. The prdx3 muscle specific transgenic mice were crossed with an established mouse model of accelerated sarcopenia (Sod1KO) to generate Sod1KO/mPrd3Tg mice. Results In agreement with our previous results, the basal rate of ROS generation was significantly elevated in isolated mitochondria from Sod1KO mice but normalized by prdx3 overexpression in the Sod1KO/mPrd3Tg mice. The reduction in oxygen consumption rate from complex II present in mitochondria from Sod1KO muscle was also restored by prdx3 overexpression. To determine the effects of mitochondrial ROS on skeletal muscle weakness, we tested in vitro contractile properties of extensor digitorum longus (EDL). Maximum isometric specific force was decreased by ~20% in Sod1KO mice, but the reduction in force was rescued inSod1KO/mPrd3Tg mice. Importantly, the loss in gastrocnemius mass that occurs in the Sdo1KO mice was fully restored by prdx3 overexpression. Conclusion Our data demonstrate, for the first time, a direct role of skeletal muscle-specific mitochondrial H2O2 in promoting contractile abnormalities and atrophy of skeletal muscle. This study implicates mitochondrial H2O2 generation as a novel therapeutic target to ameliorate skeletal muscle pathologies.

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-α and also displayed increased expression of vascular endothelial growth factor; on the other hand, rapamycin inhibited RT-Tg expression of both hypoxia-inducible factor-α 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.

Ambient protein levels are affected by both synthesis and degradation. Synthesis of a protein is regulated by transcription and messenger RNA (mRNA) translation. Translation has emerged as an important site of regulation of protein expression during development and disease. It is under the control of distinct factors that regulate initiation, elongation and termination phases. Regulation of translation occurs via signaling reactions, guanosine diphosphate–guanosine triphosphate binding and by participation of non-coding RNA species such as microRNA. Recent work has revealed an important role for translation in hypertrophy, matrix protein synthesis, elaboration of growth factors in in vivo and in vitro models of diabetic nephropathy. Studies of translation dysregulation in diabetic nephropathy have enabled identification of novel therapeutic targets. Translation of mRNA is a fertile field for exploration in investigation of kidney disease.

High glucose and high insulin, pathogenic factors in type 2 diabetes, induce rapid synthesis of the matrix protein laminin-β1 in renal proximal tubular epithelial cells by stimulation of initiation phase of mRNA translation. We investigated if elongation phase of translation also contributes to high glucose and high insulin induction of laminin-β1 synthesis in proximal tubular epithelial cells. High glucose or high insulin rapidly increased activating Thr56 dephosphorylation of eEF2 and inactivating Ser366 phosphorylation of eEF2 kinase, events that facilitate elongation. Studies with inhibitors showed that PI3 kinase-Akt-mTOR-p70S6 kinase pathway controlled changes in phosphorylation of eEF2 and eEF2 kinase induced by high glucose or high insulin. Renal cortical homogenates from db/db mice in early stage of type 2 diabetes showed decrease in eEF2 phosphorylation and increment in eEF2 kinase phosphorylation in association with renal hypertrophy and glomerular and tubular increase in laminin-β1 content. Rapamycin, an inhibitor of mTOR, abolished diabetes-induced changes in phosphorylation of eEF2, eEF2 kinase, and p70S6 kinase and ameliorated renal hypertrophy and laminin-β1 protein content, without affecting hyperglycemia. These data show that mTOR is an attractive target for amelioration of diabetes-induced renal injury.

Ambient protein levels are under coordinated control of transcription, mRNA translation, and degradation. Whereas transcription and degradation mechanisms have been studied in depth in renal science, the role of mRNA translation, the process by which peptide synthesis occurs according to the genetic code that is present in the mRNA, has not received much attention. mRNA translation occurs in three phases: Initiation, elongation, and termination. Each phase is controlled by unique eukaryotic factors. In the initiation phase, mRNA and ribosomal subunits are brought together. During the elongation phase, amino acids are added to the nascent peptide chain in accordance with codon sequences in the mRNA. During the termination phase, the fully synthesized peptide is released from the ribosome for posttranslational processing. Signaling pathways figure prominently in regulation of mRNA translation, particularly the phosphatidylinositol 3 kinase-Akt-mammalian target of rapamycin pathway, the AMP-activated protein kinase-tuberous sclerosis complex protein 1/tuberous sclerosis complex protein 2-Rheb pathway, and the extracellular signal-regulated kinase 1/2 type mitogen-activated protein kinase signaling pathway; there is significant cross-talk among these pathways. Regulation by mRNA translation is suggested when changes in mRNA and protein levels do not correlate and in the setting of rapid protein synthesis. Ongoing work suggests an important role for mRNA translation in compensatory renal growth, hypertrophy and extracellular matrix synthesis in diabetic nephropathy, growth factor synthesis by kidney cells, and glomerulonephritis. Considering that mRNA translation plays an important role in cell growth, development, malignancy, apoptosis, and response to stress, its study should provide novel insights in renal physiology and pathology.

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 β. 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.

The study was undertaken to explore the effect of rapamycin, an anti-inflammatory agent, on the metabolic profile of type 2 diabetic mice. Seven-month-old diabetic db/db mice and their lean littermate non-diabetic controls (db/m) were randomized to receive control chow or chow mixed with rapamycin (2.24 mg/kg/day) (each group n=20, males and females) for 4 months and sacrificed. Serum samples were analyzed for the measurement of glucose, creatinine, blood urea nitrogen (BUN), alkaline phosphatase (ALP), alanine aminotransferase (ALT), total cholesterol, total triglyceride, and total protein, using the automated dry chemistry analysis. Rapamycin elevated serum glucose in female diabetic mice. Serum creatinine tended to be higher in diabetic mice but was not affected by rapamycin; there was no difference in BUN levels among the groups. Serum ALP was elevated in diabetic mice and rapamycin lowered it only in female diabetic mice; serum ALT levels were increased in female diabetic mice, unaffected by rapamycin. Serum total protein was elevated in diabetic mice of both genders but was not affected by rapamycin. Diabetic mice from both genders had elevated serum cholesterol and triglycerides; rapamycin did not affect serum cholesterol but decreased serum total triglycerides in male diabetic mice. We conclude that rapamycin elicits complex metabolic responses in aging diabetic mice, worsening hyperglycemia in females but improving ALP in female diabetic and total triglycerides in male diabetic mice, respectively. The metabolic effects of rapamycin should be considered while performing studies with rapamycin in mice. Keywords: alkaline phosphatase; alanine aminotransferase; cholesterol; triglycerides (Published: 23 April 2012) Citation: Pathobiology of Aging & Age-related Diseases 2012, 2 : 15896 - http://dx.doi.org/10.3402/pba.v2i0.15896

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 β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-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.

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 γ1and fibronectin, and rDNA transcription in glomerular epithelial cells (GECs) compared with 5 mM glucose. High glucose induced Ser388phosphorylation 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 γ1synthesis, 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.

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.

High glucose-induced protein synthesis in the glomerular epithelial cell (GEC) is partly dependent on reduction in phosphorylation of AMP-activated protein kinase (AMPK). We evaluated the effect of resveratrol, a phytophenol known to stimulate AMPK, on protein synthesis. Resveratrol completely inhibited high glucose stimulation of protein synthesis and synthesis of fibronectin, an important matrix protein, at 3 days. Resveratrol dose-dependently increased AMPK phosphorylation and abolished high glucose-induced reduction in its phosphorylation. We examined the effect of resveratrol on critical steps in mRNA translation, a critical event in protein synthesis. Resveratrol inhibited high glucose-induced changes in association of eIF4E with eIF4G, phosphorylation of eIF4E, eEF2, eEF2 kinase and, p70S6 kinase, indicating that it affects important events in both initiation and elongation phases of mRNA translation. Upstream regulators of AMPK in high glucose-treated GEC were explored. High glucose augmented acetylation of LKB1, the upstream kinase for AMPK, and inhibited its activity. Resveratrol prevented acetylation of LKB1 and restored its activity in high glucose-treated cells; this action did not appear to depend on SIRT1, a class III histone deacetylase. Our data show that resveratrol ameliorates protein synthesis by regulating the LKB1-AMPK axis.

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.

High glucose (30 mM) and high insulin (1 nM), pathogenic factors of type 2 diabetes, increased mRNA expression and synthesis of lamininbeta1 and fibronectin after 24 h of incubation in kidney proximal tubular epithelial (MCT) cells. We tested the hypothesis that inactivation of glycogen synthase kinase 3beta (GSK3beta) by high glucose and high insulin induces increase in synthesis of laminin beta1 via activation of eIF2Bepsilon. Both high glucose and high insulin induced Ser-9 phosphorylation and inactivation of GSK3beta at 2 h that lasted for up to 48 h. This was associated with dephosphorylation of eIF2Bepsilon and eEF2, and increase in phosphorylation of 4E-BP1 and eIF4E. Expression of the kinase-dead mutant of GSK3beta or constitutively active kinase led to increased and diminished laminin beta1 synthesis, respectively. Incubation with selective kinase inhibitors showed that high glucose- and high insulin-induced laminin beta1 synthesis and phosphorylation of GSK3beta were dependent on PI 3-kinase, Erk, and mTOR. High glucose and high insulin augmented activation of Akt, Erk, and p70S6 kinase. Dominant negative Akt, but not dominant negative p70S6 kinase, inhibited GSK3beta phosphorylation induced by high glucose and high insulin, suggesting Akt but not p70S6 kinase was upstream of GSK3beta. Status of GSK3beta was examined in vivo in renal cortex of db/db mice with type 2 diabetes at 2 weeks and 2 months of diabetes. Diabetic mice showed increased phosphorylation of renal cortical GSK3beta and decreased phosphorylation of eIF2Bepsilon, which correlated with renal hypertrophy at 2 weeks, and increased laminin beta1 and fibronectin protein content at 2 months. GSK3beta and eIF2Bepsilon play a role in augmented protein synthesis associated with high glucose- and high insulin-stimulated hypertrophy and matrix accumulation in renal disease in type 2 diabetes.

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.

We tested the hypothesis that AMP-activated protein kinase (AMPK), an energy sensor, regulates diabetes-induced renal hypertrophy. In kidney glomerular epithelial cells, high glucose (30 mM), but not equimolar mannitol, stimulated de novo protein synthesis and induced hypertrophy in association with increased phosphorylation of eukaryotic initiation factor 4E binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2, regulatory events in mRNA translation. These high-glucose-induced changes in protein synthesis were phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin (mTOR) dependent and transforming growth factor-β independent. High glucose reduced AMPK α-subunit theronine (Thr) 172 phosphorylation, which required Akt activation. Changes in AMP and ATP content could not fully account for high-glucose-induced reductions in AMPK phosphorylation. Metformin and 5-aminoimidazole-4-carboxamide-1β-riboside (AICAR) increased AMPK phosphorylation, inhibited high-glucose stimulation of protein synthesis, and prevented high-glucose-induced changes in phosphorylation of 4E binding protein 1 and eukaryotic elongation factor 2. Expression of kinase-inactive AMPK further increased high-glucose-induced protein synthesis. Renal hypertrophy in rats with Type 1 diabetes was associated with reduction in AMPK phosphorylation and increased mTOR activity. In diabetic rats, metformin and AICAR increased renal AMPK phosphorylation, reversed mTOR activation, and inhibited renal hypertrophy, without affecting hyperglycemia. AMPK is a newly identified regulator of renal hypertrophy in diabetes.

Kidney injury is one of the most serious complications of diabetes. Current therapies only slow down but do not stop its progression to end stage kidney failure; thus, there is a need for novel approaches. Accumulation of extracellular matrix proteins, e.g., laminin, fibronectin, contributes to the loss of functioning parenchyma and progressive loss of kidney function in diabetes. We have recently reported that H2S inhibits high glucose-induced increase in synthesis of proteins including matrix proteins (H.J. Lee et al., J. Biol. Chem. 287 (2012) 4451). We wish to identify H2S generating agents that can be used in humans to treat diabetic kidney disease. We hypothesized that tadalafil, a H2S donor and phosphodiesterase 5 (PDE5) inhibitor, inhibits high glucose stimulation of synthesis of proteins including ECM proteins by generating H2S in mouse kidney podocytes. High glucose (30 mM), but not equimolar mannitol, significantly promoted global protein synthesis, cellular hypertrophy and expression of laminin-γ1 and fibronectin. These events were inhibited by 10 μM tadalafil. Because mRNA translation is rate-limiting for protein synthesis, we examined tadalafil effect on events in translation. Tadalafil abrogated high glucose-induced mTORC1 activation as indicated by phosphorylation of p70S6 kinase and 4E-BP1; both these events are important for the initiation phase of translation. Tadalafil reversed high glucose-induced changes in the phosphorylation of eEF2 and eEF2 kinase, which regulate the elongation phase of translation. Upstream regulators of mTORC1 were investigated. Tadalafil stimulated activating phosphorylation of AMPK in a dose dependent manner, peaking at 10 μM. Pre-incubation with Compound C, an AMPK inhibitor, completely abolished tadalafil effect on high glucose stimulation of events in mRNA translation as well as global protein and laminin synthesis. Tadalafil-induced AMPK phosphorylation was abolished by dl -propargylglycine (PAG), an inhibitor of cystathionine γ lyase (CSE), a H2S generating enzyme. Tadalafil rapidly increased CSE protein expression in 1 h which was inhibited by cycloheximide but not actinomycin-D, suggesting regulation at the level of mRNA translation. In an enzymatic assay, PAG abrogated tadalafil stimulation of generation of H2S. PAG completely abolished tadalafil effect on high glucose stimulation of events in mRNA translation as well as global protein and laminin synthesis. Tadalafil is a PDE5 inhibitor that increases cGMP content. ODQ, an inhibitor of guanylyl cyclase that generates cGMP, abolished tadalafil induction of AMPK phosphorylation. ODQ abrogated tadalafil inhibition of high glucose-induced increase in laminin expression. Renal cortical content of cystathionine β synthase (CBS) and CSE, H2S generating enzymes, was significantly reduced in the kidney in mice with type 1 or type 2 diabetes. Our data show that tadalafil stimulates H2S-AMPK axis to inhibit mTORC1 and mRNA translation leading to amelioration of high glucose-induced global and matrix protein increment in renal podocytes. It is likely that tadalafil stimulation of H2S is cGMP-dependent in the podocyte. Future work is needed to define whether tadalafil can ameliorate kidney injury in animal models of diabetes.