Roles of ADP-Ribosylation during Infection Establishment by Trypanosomatidae Parasites.

ADP-ribosylation is a reversible post-translational protein modification, which is evolutionarily conserved in prokaryotic and eukaryotic organisms. It governs critical cellular functions, including, but not limited to cellular proliferation, differentiation, RNA translation, and genomic repair. The addition of one or multiple ADP-ribose moieties can be catalysed by poly(ADP-ribose) polymerase (PARP) enzymes, while in eukaryotic organisms, ADP-ribosylation can be reversed through the action of specific enzymes capable of ADP-ribose signalling regulation. In several lower eukaryotic organisms, including Trypanosomatidae parasites, ADP-ribosylation is thought to be important for infection establishment. Trypanosomatidae encompasses several human disease-causing pathogens, including Trypanosoma cruzi, T. brucei, and the Leishmania genus. These parasites are the etiological agents of Chagas disease, African trypanosomiasis (sleeping sickness), and leishmaniasis, respectively. Currently, licenced medications for these infections are outdated and often result in harmful side effects, and can be inaccessible to those carrying infections, due to them being classified as neglected tropical diseases (NTDs), meaning that many infected individuals will belong to already marginalised communities in countries already facing socioeconomic challenges. Consequently, funding to develop novel therapeutics for these infections is overlooked. As such, understanding the molecular mechanisms of infection, and how ADP-ribosylation facilitates infection establishment by these organisms may allow the identification of potential molecular interventions that would disrupt infection. In contrast to the complex ADP-ribosylation pathways in eukaryotes, the process of Trypanosomatidae is more linear, with the parasites only expressing one PARP enzyme, compared to the, at least, 17 genes that encode human PARP enzymes. If this simplified pathway can be understood and exploited, it may reveal new avenues for combatting Trypanosomatidae infection. This review will focus on the current state of knowledge on the importance of ADP-ribosylation in Trypanosomatidae during infection establishment in human hosts, and the potential therapeutic options that disrupting ADP-ribosylation may offer to combat Trypanosomatidae.

PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response.

The ADP-ribosyltransferase, PARP1 enzymatically generates and applies the post-translational modification, ADP-Ribose (ADPR). PARP1 roles in genome maintenance are well described, but recent work highlights roles in many fundamental processes including cellular identity and energy homeostasis. Herein, we show in both mouse and human skeletal muscle cells that PARP1-mediated PARylation is a regulator of the myogenic program and the muscle transcriptional response to steroid hormones. Chemical PARP1 modulation impacts the expression of major myocellular proteins, including troponins, key in dictating muscle contractile force. Whilst PARP1 in absence of DNA damage is often assumed to be basally inactive, we show PARylation to be acutely sensitive to extracellular glucose concentrations and the steroid hormone class, glucocorticoids which exert considerable authority over muscle tissue mass. Specifically, we find during myogenesis, a transient and significant rise in PAR. This early-stage differentiation event, if blocked with PARP1 inhibition, reduced the abundance of important muscle proteins in the fully differentiated myotubes. This suggests that PAR targets during early-stage differentiation are central to the proper development of the muscle contractile unit. We also show that reduced PARP1 in myoblasts impacts a variety of metabolic pathways in line with the recorded actions of glucocorticoids. Currently, as both regulators of myogenesis and muscle mass loss, glucocorticoids represent a clinical conundrum. Our work goes on to identify that PARP1 influences transcriptional activation by glucocorticoids of a subset of genes critical to human skeletal muscle pathology. These genes may therefore signify a regulatory battery of targets through which selective glucocorticoid modulation could be achieved. Collectively, our data provide clear links between PARP1-mediated PARylation and skeletal muscle homeostatic mechanisms crucial to tissue mass maintenance and endocrine response.

Rapid isolation of respiring skeletal muscle mitochondria using nitrogen cavitation.

Methods of isolating mitochondria commonly utilise mechanical force and shear stress to homogenize tissue followed by purification by multiple rounds of ultracentrifugation. Existing protocols can be time-consuming with some physically impairing integrity of the sensitive mitochondrial double membrane. Here, we describe a method for the recovery of intact, respiring mitochondria from murine skeletal muscle tissue and cell lines using nitrogen cavitation. This protocol results in high-yield, pure and respiring mitochondria without the need for purification gradients or ultracentrifugation. The protocol takes under an hour and requires limited specialised equipment. Our methodology is successful in extracting mitochondria of both cell extracts and skeletal muscle tissue. This represents an improved yield in comparison to many of the existing methods. Western blotting and electron microscopy demonstrate the enrichment of mitochondria with their ultrastructure well-preserved and an absence of contamination from cytoplasmic or nuclear fractions. Using respirometry analysis we show that mitochondria extracted from murine skeletal muscle cell lines (C2C12) and tibialis anterior tissue have an appropriate respiratory control ratio. These measures are indicative of healthy coupled mitochondria. Our method successfully demonstrates the rapid isolation of functional mitochondria and will benefit researchers studying mitochondrial bioenergetics as well as providing greater throughput and application for time-sensitive assays.

Physiological Roles of Carnosine in Myocardial Function and Health.

Carnosine is a pleiotropic histidine-containing dipeptide synthesized from ß-alanine and l-histidine, with the intact dipeptide and constituent amino acids being available from the diet. The therapeutic application of carnosine in myocardial tissue is promising, with carnosine playing a potentially beneficial role in both healthy and diseased myocardial models. This narrative review discusses the role of carnosine in myocardial function and health, including an overview of the metabolic pathway of carnosine in the myocardial tissue, the roles carnosine may play in the myocardium, and a critical analysis of the literature, focusing on the effect of exogenous carnosine and its precursors on myocardial function. By so doing, we aim to identify current gaps in the literature, thereby identifying considerations for future research.

NAD+ Degrading Enzymes, Evidence for Roles During Infection.

Declines in cellular nicotinamide adenine dinucleotide (NAD) contribute to metabolic dysfunction, increase susceptibility to disease, and occur as a result of pathogenic infection. The enzymatic cleavage of NAD+ transfers ADP-ribose (ADPr) to substrate proteins generating mono-ADP-ribose (MAR), poly-ADP-ribose (PAR) or O-acetyl-ADP-ribose (OAADPr). These important post-translational modifications have roles in both immune response activation and the advancement of infection. In particular, emergent data show viral infection stimulates activation of poly (ADP-ribose) polymerase (PARP) mediated NAD+ depletion and stimulates hydrolysis of existing ADP-ribosylation modifications. These studies are important for us to better understand the value of NAD+ maintenance upon the biology of infection. This review focuses specifically upon the NAD+ utilising enzymes, discusses existing knowledge surrounding their roles in infection, their NAD+ depletion capability and their influence within pathogenic infection.

Nicotinamide riboside has minimal impact on energy metabolism in mouse models of mild obesity.

Supplementation with precursors of NAD has been shown to prevent and reverse insulin resistance, mitochondrial dysfunction, and liver damage in mouse models of diet-induced obesity. We asked whether the beneficial effects of supplementation with the NAD precursor nicotinamide riboside (NR) are dependent on mouse strain. We compared the effects of NR supplementation on whole-body energy metabolism and mitochondrial function in mildly obese C57BL/6N and C57BL/6J mice, two commonly used strains to investigate metabolism. Male C57BL/6N and C57BL/6J mice were fed a high-fat diet (HFD) or standard chow with or without NR supplementation for 8 weeks. Body and organ weights, glucose tolerance, and metabolic parameters as well as mitochondrial O2 flux in liver and muscle fibers were assessed. We found that NR supplementation had no influence on body or organ weight, glucose metabolism or hepatic lipid accumulation, energy expenditure, or metabolic flexibility but increased mitochondrial respiration in soleus muscle in both mouse strains. Strain-dependent differences were detected for body and fat depot weight, fasting blood glucose, hepatic lipid accumulation, and energy expenditure. We conclude that, in mild obesity, NR supplementation does not alter metabolic phenotype in two commonly used laboratory mouse strains.

Carnosine protects stimulus-secretion coupling through prevention of protein carbonyl adduction events in cells under metabolic stress.

Type 2 diabetes is characterised by failure to control glucose homeostasis, with numerous diabetic complications attributable to the resulting exposure of cells and tissues to chronic elevated concentrations of glucose and fatty acids. This, in part, results from formation of advanced glycation and advanced lipidation end-products that are able to modify protein, lipid, or DNA structure, and disrupt normal cellular function. Herein we used mass spectrometry to identify proteins modified by two such adduction events in serum of individuals with obesity, type 2 diabetes, and gestational diabetes, along with similar analyses of human and mouse skeletal muscle cells and mouse pancreatic islets exposed to glucolipotoxic stress. We also report that carnosine, a histidine containing dipeptide, prevented 65-90% of 4-hydroxynonenal and 3-nitrotyrosine adduction events, and that this in turn preserved mitochondrial function and protected stimulus-secretion coupling in cells exposed to metabolic stress. Carnosine therefore offers significant therapeutic potential against metabolic diseases.

Induction of the nicotinamide riboside kinase NAD+ salvage pathway in a model of sarcoplasmic reticulum dysfunction.

BACKGROUND: Hexose-6-Phosphate Dehydrogenase (H6PD) is a generator of NADPH in the Endoplasmic/Sarcoplasmic Reticulum (ER/SR). Interaction of H6PD with 11ß-hydroxysteroid dehydrogenase type 1 provides NADPH to support oxo-reduction of inactive to active glucocorticoids, but the wider understanding of H6PD in ER/SR NAD(P)(H) homeostasis is incomplete. Lack of H6PD results in a deteriorating skeletal myopathy, altered glucose homeostasis, ER stress and activation of the unfolded protein response. Here we further assess muscle responses to H6PD deficiency to delineate pathways that may underpin myopathy and link SR redox status to muscle wide metabolic adaptation. METHODS: We analysed skeletal muscle from H6PD knockout (H6PDKO), H6PD and NRK2 double knockout (DKO) and wild-type (WT) mice. H6PDKO mice were supplemented with the NAD+ precursor nicotinamide riboside. Skeletal muscle samples were subjected to biochemical analysis including NAD(H) measurement, LC-MS based metabolomics, Western blotting, and high resolution mitochondrial respirometry. Genetic and supplement models were assessed for degree of myopathy compared to H6PDKO. RESULTS: H6PDKO skeletal muscle showed adaptations in the routes regulating nicotinamide and NAD+ biosynthesis, with significant activation of the Nicotinamide Riboside Kinase 2 (NRK2) pathway. Associated with changes in NAD+ biosynthesis, H6PDKO muscle had impaired mitochondrial respiratory capacity with altered mitochondrial acylcarnitine and acetyl-CoA metabolism. Boosting NAD+ levels through the NRK2 pathway using the precursor nicotinamide riboside elevated NAD+/NADH but had no effect to mitigate ER stress and dysfunctional mitochondrial respiratory capacity or acetyl-CoA metabolism. Similarly, H6PDKO/NRK2 double KO mice did not display an exaggerated timing or severity of myopathy or overt change in mitochondrial metabolism despite depression of NAD+ availability. CONCLUSIONS: These findings suggest a complex metabolic response to changes in muscle SR NADP(H) redox status that result in impaired mitochondrial energy metabolism and activation of cellular NAD+ salvage pathways. It is possible that SR can sense and signal perturbation in NAD(P)(H) that cannot be rectified in the absence of H6PD. Whether NRK2 pathway activation is a direct response to changes in SR NAD(P)(H) availability or adaptation to deficits in metabolic energy availability remains to be resolved.

Nicotinamide Riboside Augments the Aged Human Skeletal Muscle NAD+ Metabolome and Induces Transcriptomic and Anti-inflammatory Signatures.

Nicotinamide adenine dinucleotide (NAD+) is modulated by conditions of metabolic stress and has been reported to decline with aging in preclinical models, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD+ metabolome and if it can alter muscle mitochondrial bioenergetics. We supplemented 12 aged men with 1 g NR per day for 21 days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD+ metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways, without altering mitochondrial bioenergetics. NR also depressed levels of circulating inflammatory cytokines. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR.

Nicotinamide Nucleotide Transhydrogenase as a Novel Treatment Target in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is an aggressive malignancy with poor response to chemotherapy. In this study, we evaluated a potential new treatment target for ACC, focusing on the mitochondrial reduced form of NAD phosphate (NADPH) generator nicotinamide nucleotide transhydrogenase (NNT). NNT has a central role within mitochondrial antioxidant pathways, protecting cells from oxidative stress. Inactivating human NNT mutations result in congenital adrenal insufficiency. We hypothesized that NNT silencing in ACC cells will induce toxic levels of oxidative stress. To explore this, we transiently knocked down NNT in NCI-H295R ACC cells. As predicted, this manipulation increased intracellular levels of oxidative stress; this resulted in a pronounced suppression of cell proliferation and higher apoptotic rates, as well as sensitization of cells to chemically induced oxidative stress. Steroidogenesis was paradoxically stimulated by NNT loss, as demonstrated by mass spectrometry-based steroid profiling. Next, we generated a stable NNT knockdown model in the same cell line to investigate the longer lasting effects of NNT silencing. After long-term culture, cells adapted metabolically to chronic NNT knockdown, restoring their redox balance and resilience to oxidative stress, although their proliferation remained suppressed. This was associated with higher rates of oxygen consumption. The molecular pathways underpinning these responses were explored in detail by RNA sequencing and nontargeted metabolome analysis, revealing major alterations in nucleotide synthesis, protein folding, and polyamine metabolism. This study provides preclinical evidence of the therapeutic merit of antioxidant targeting in ACC as well as illuminating the long-term adaptive response of cells to oxidative stress.

Metabolic tracing reveals novel adaptations to skeletal muscle cell energy production pathways in response to NAD + depletion.

Background: Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD + availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD + as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD + homeostasis on these pathways is unknown. Methods: We utilised skeletal muscle myotube models to induce NAD + depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD + metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U- 13C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. Results: NAD + excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells resulted in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD + depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity was increased in response to low NAD +, which was rapidly reversed with repletion of the NAD + pool using NR. NAD + depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reversed these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD + depletion remains unclear, and requires further investigation. Conclusions: These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD + precursor usage in models of skeletal muscle physiology.

Glucocorticoids Reprogram ß-Cell Signaling to Preserve Insulin Secretion.

Excessive glucocorticoid exposure has been shown to be deleterious for pancreatic ß-cell function and insulin release. However, glucocorticoids at physiological levels are essential for many homeostatic processes, including glycemic control. We show that corticosterone and cortisol and their less active precursors 11-dehydrocorticosterone (11-DHC) and cortisone suppress voltage-dependent Ca2+ channel function and Ca2+ fluxes in rodent as well as in human ß-cells. However, insulin secretion, maximal ATP/ADP responses to glucose, and ß-cell identity were all unaffected. Further examination revealed the upregulation of parallel amplifying cAMP signals and an increase in the number of membrane-docked insulin secretory granules. Effects of 11-DHC could be prevented by lipotoxicity and were associated with paracrine regulation of glucocorticoid activity because global deletion of 11ß-hydroxysteroid dehydrogenase type 1 normalized Ca2+ and cAMP responses. Thus, we have identified an enzymatically amplified feedback loop whereby glucocorticoids boost cAMP to maintain insulin secretion in the face of perturbed ionic signals. Failure of this protective mechanism may contribute to diabetes in states of glucocorticoid excess, such as Cushing syndrome, which are associated with frank dyslipidemia.

Cellular and genetic models of H6PDH and 11ß-HSD1 function in skeletal muscle.

Glucocorticoids are important for skeletal muscle energy metabolism, regulating glucose utilization, insulin sensitivity, and muscle mass. Nicotinamide adenine dinucleotide phosphate-dependent 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1)-mediated glucocorticoid activation in the sarcoplasmic reticulum (SR) is integral to mediating the detrimental effects of glucocorticoid excess in muscle. 11ß-Hydroxysteroid dehydrogenase type 1 activity requires glucose-6-phosphate transporter (G6PT)-mediated G6P transport into the SR for its metabolism by hexose-6-phosphate dehydrogenase (H6PDH) for NADPH generation. Here, we examine the G6PT/H6PDH/11ß-HSD1 triad in differentiating myotubes and explore the consequences of muscle-specific knockout of 11ß-HSD1 and H6PDH. 11ß-Hydroxysteroid dehydrogenase type 1 expression and activity increase with myotube differentiation and in response to glucocorticoids. Hexose-6-phosphate dehydrogenase shows some elevation in expression with differentiation and in response to glucocorticoid, while G6PT appears largely unresponsive to these particular conditions. When examining 11ß-HSD1 muscle-knockout mice, we were unable to detect significant decrements in activity, despite using a well-validated muscle-specific Cre transgene and confirming high-level recombination of the floxed HSD11B1 allele. We propose that the level of recombination at the HSD11B1 locus may be insufficient to negate basal 11ß-HSD1 activity for a protein with a long half-life. Hexose-6-phosphate dehydrogenase was undetectable in H6PDH muscle-knockout mice, which display the myopathic phenotype seen in global KO mice, validating the importance of SR NADPH generation. We envisage these data and models finding utility when investigating the muscle-specific functions of the 11ß-HSD1/G6PT/H6PDH triad.

Nicotinamide riboside kinases display redundancy in mediating nicotinamide mononucleotide and nicotinamide riboside metabolism in skeletal muscle cells.

OBJECTIVE: Augmenting nicotinamide adenine dinucleotide (NAD+) availability may protect skeletal muscle from age-related metabolic decline. Dietary supplementation of NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) appear efficacious in elevating muscle NAD+. Here we sought to identify the pathways skeletal muscle cells utilize to synthesize NAD+ from NMN and NR and provide insight into mechanisms of muscle metabolic homeostasis. METHODS: We exploited expression profiling of muscle NAD+ biosynthetic pathways, single and double nicotinamide riboside kinase 1/2 (NRK1/2) loss-of-function mice, and pharmacological inhibition of muscle NAD+ recycling to evaluate NMN and NR utilization. RESULTS: Skeletal muscle cells primarily rely on nicotinamide phosphoribosyltransferase (NAMPT), NRK1, and NRK2 for salvage biosynthesis of NAD+. NAMPT inhibition depletes muscle NAD+ availability and can be rescued by NR and NMN as the preferred precursors for elevating muscle cell NAD+ in a pathway that depends on NRK1 and NRK2. Nrk2 knockout mice develop normally and show subtle alterations to their NAD+ metabolome and expression of related genes. NRK1, NRK2, and double KO myotubes revealed redundancy in the NRK dependent metabolism of NR to NAD+. Significantly, these models revealed that NMN supplementation is also dependent upon NRK activity to enhance NAD+ availability. CONCLUSIONS: These results identify skeletal muscle cells as requiring NAMPT to maintain NAD+ availability and reveal that NRK1 and 2 display overlapping function in salvage of exogenous NR and NMN to augment intracellular NAD+ availability.

11ß-HSD1 Modulates the Set Point of Brown Adipose Tissue Response to Glucocorticoids in Male Mice.

Glucocorticoids (GCs) are potent regulators of energy metabolism. Chronic GC exposure suppresses brown adipose tissue (BAT) thermogenic capacity in mice, with evidence for a similar effect in humans. Intracellular GC levels are regulated by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activity, which can amplify circulating GC concentrations. Therefore, 11ß-HSD1 could modulate the impact of GCs on BAT function. This study investigated how 11ß-HSD1 regulates the molecular architecture of BAT in the context of GC excess and aging. Circulating GC excess was induced in 11ß-HSD1 knockout (KO) and wild-type mice by supplementing drinking water with 100 µg/mL corticosterone, and the effects on molecular markers of BAT function and mitochondrial activity were assessed. Brown adipocyte primary cultures were used to examine cell autonomous consequences of 11ß-HSD1 deficiency. Molecular markers of BAT function were also examined in aged 11ß-HSD1 KO mice to model lifetime GC exposure. BAT 11ß-HSD1 expression and activity were elevated in response to GC excess and with aging. 11ß-HSD1 KO BAT resisted the suppression of uncoupling protein 1 (UCP1) and mitochondrial respiratory chain subunit proteins normally imposed by GC excess. Furthermore, brown adipocytes from 11ß-HSD1 KO mice had elevated basal mitochondrial function and were able to resist GC-mediated repression of activity. BAT from aged 11ß-HSD1 KO mice showed elevated UCP1 protein and mitochondrial content, and a favorable profile of BAT function. These data reveal a novel mechanism in which increased 11ß-HSD1 expression, in the context of GC excess and aging, impairs the molecular and metabolic function of BAT.

PARP Inhibitors: Staying on Target?

Successful phase III trials with poly-ADP-ribose (PARP) inhibitors will have implications for stratified cancer therapy. In this issue of Cell Chemical Biology, Knezevic et al. (2016) demonstrate that the existing collection of PARP inhibitors each display distinctive protein interaction profiles, reaching beyond their intended therapeutic target, with implications for metabolic and other disease.

11ß-Hydroxysteroid dehydrogenase type 1 within muscle protects against the adverse effects of local inflammation.

Muscle wasting is a common feature of inflammatory myopathies. Glucocorticoids (GCs), although effective at suppressing inflammation and inflammatory muscle loss, also cause myopathy with prolonged administration. 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is a bidirectional GC-activating enzyme that is potently upregulated by inflammation within mesenchymal-derived tissues. We assessed the regulation of this enzyme with inflammation in muscle, and examined its functional impact on muscle. The expression of 11ß-HSD1 in response to proinflammatory stimuli was determined in a transgenic murine model of chronic inflammation (TNF-Tg) driven by overexpression of tumour necrosis factor (TNF)-α within tissues, including muscle. The inflammatory regulation and functional consequences of 11ß-HSD1 expression were examined in primary cultures of human and murine myotubes and human and murine muscle biopsies ex vivo. The contributions of 11ß-HSD1 to muscle inflammation and wasting were assessed in vivo with the TNF-Tg mouse on an 11ß-HSD1 null background. 11ß-HSD1 was significantly upregulated within the tibialis anterior and quadriceps muscles from TNF-Tg mice. In human and murine primary myotubes, 11ß-HSD1 expression and activity were significantly increased in response to the proinflammatory cytokine TNF-α (mRNA, 7.6-fold, p < 0.005; activity, 4.1-fold, p < 0.005). Physiologically relevant levels of endogenous GCs activated by 11ß-HSD1 suppressed proinflammatory cytokine output (interkeukin-6, TNF-α, and interferon-γ), but had little impact on markers of muscle wasting in human myotube cultures. TNF-Tg mice on an 11ß-11ß-HSD1 knockout background developed greater muscle wasting than their TNF-Tg counterparts (27.4% less; p < 0.005), with smaller compacted muscle fibres and increased proinflammatory gene expression relative to TNF-Tg mice with normal 11ß-HSD1 activity. This study demonstrates that inflammatory stimuli upregulate 11ß-HSD1 expression and GC activation within muscle. Although concerns have been raised that excess levels of GCs may be detrimental to muscle, in this inflammatory TNF-α-driven model, local endogenous GC activation appears to be an important anti-inflammatory response that protects against inflammatory muscle wasting in vivo. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Male 11ß-HSD1 Knockout Mice Fed Trans-Fats and Fructose Are Not Protected From Metabolic Syndrome or Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) defines a spectrum of conditions from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis and is regarded as the hepatic manifestation of the metabolic syndrome. Glucocorticoids can promote steatosis by stimulating lipolysis within adipose tissue, free fatty acid delivery to liver and hepatic de novo lipogenesis. Glucocorticoids can be reactivated in liver through 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) enzyme activity. Inhibition of 11ß-HSD1 has been suggested as a potential treatment for NAFLD. To test this, male mice with global (11ß-HSD1 knockout [KO]) and liver-specific (LKO) 11ß-HSD1 loss of function were fed the American Lifestyle Induced Obesity Syndrome (ALIOS) diet, known to recapitulate the spectrum of NAFLD, and metabolic and liver phenotypes assessed. Body weight, muscle and adipose tissue masses, and parameters of glucose homeostasis showed that 11ß-HSD1KO and LKO mice were not protected from systemic metabolic disease. Evaluation of hepatic histology, triglyceride content, and blinded NAFLD activity score assessment indicated that levels of steatosis were similar between 11ß-HSD1KO, LKO, and control mice. Unexpectedly, histological analysis revealed significantly increased levels of immune foci present in livers of 11ß-HSD1KO but not LKO or control mice, suggestive of a transition to NASH. This was endorsed by elevated hepatic expression of key immune cell and inflammatory markers. These data indicate that 11ß-HSD1-deficient mice are not protected from metabolic disease or hepatosteatosis in the face of a NAFLD-inducing diet. However, global deficiency of 11ß-HSD1 did increase markers of hepatic inflammation and suggests a critical role for 11ß-HSD1 in restraining the transition to NASH.

Glucocorticoids and 11ß-HSD1 are major regulators of intramyocellular protein metabolism.

The adverse metabolic effects of prescribed and endogenous glucocorticoid excess, 'Cushing's syndrome', create a significant health burden. While skeletal muscle atrophy and resultant myopathy is a clinical feature, the molecular mechanisms underpinning these changes are not fully defined. We have characterized the impact of glucocorticoids upon key metabolic pathways and processes regulating muscle size and mass including: protein synthesis, protein degradation, and myoblast proliferation in both murine C2C12 and human primary myotube cultures. Furthermore, we have investigated the role of pre-receptor modulation of glucocorticoid availability by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in these processes. Corticosterone (CORT) decreased myotube area, decreased protein synthesis, and increased protein degradation in murine myotubes. This was supported by decreased mRNA expression of insulin-like growth factor (IGF1), decreased activating phosphorylation of mammalian target of rapamycin (mTOR), decreased phosphorylation of 4E binding protein 1 (4E-BP1), and increased mRNA expression of key atrophy markers including: atrogin-1, forkhead box O3a (FOXO3a), myostatin (MSTN), and muscle-ring finger protein-1 (MuRF1). These findings were endorsed in human primary myotubes, where cortisol also decreased protein synthesis and increased protein degradation. The effects of 11-dehydrocorticosterone (11DHC) (in murine myotubes) and cortisone (in human myotubes) on protein metabolism were indistinguishable from that of CORT/cortisol treatments. Selective 11ß-HSD1 inhibition blocked the decrease in protein synthesis, increase in protein degradation, and reduction in myotube area induced by 11DHC/cortisone. Furthermore, CORT/cortisol, but not 11DHC/cortisone, decreased murine and human myoblast proliferative capacity. Glucocorticoids are potent regulators of skeletal muscle protein homeostasis and myoblast proliferation. Our data underscores the potential use of selective 11ß-HSD1 inhibitors to ameliorate muscle-wasting effects associated with glucocorticoid excess.

Knockdown of AKR1C3 exposes a potential epigenetic susceptibility in prostate cancer cells.

BACKGROUND: The aldo-keto reductase 1C3 (AKR1C3) has been heavily implicated in the propagation of prostate malignancy. AKR1C3 protein is elevated within prostate cancer tissue, it contributes to the formation of androgens and downstream stimulation of the androgen receptor (AR). Elevated expression of AKR1C3 is also reported in acute myeloid leukemia but the target nuclear receptors have been identified as members of the peroxisome-proliferator activated receptor (PPARs) subfamily. Thus, AKR1C3 cancer biology is likely to be tissue dependent and hormonally linked to the availability of ligands for both the steroidogenic and non-steroidogenic nuclear receptors. METHODS: In the current study we investigated the potential for AKR1C3 to regulate the availability of prostaglandin-derived ligands for PPARg mainly, prostaglandin J2 (PGJ2). Using prostate cancer cell lines with stably reduced AKR1C3 levels we examined the impact of AKR1C3 upon proliferation mediated by PPAR ligands. RESULTS: These studies revealed knockdown of AKR1C3 had no effect upon the sensitivity of androgen receptor independent prostate cancer cells towards PPAR ligands. However, the reduction of levels of AKR1C3 was accompanied by a significantly reduced mRNA expression of a range of HDACs, transcriptional co-regulators, and increased sensitivity towards SAHA, a clinically approved histone deacetylase inhibitor. CONCLUSIONS: These results suggest a hitherto unidentified link between AKR1C3 levels and the epigenetic status in prostate cancer cells. This raises an interesting possibility of a novel rational to target AKR1C3, the utilization of AKRIC3 selective inhibitors in combination with HDAC inhibition as part of novel epigenetic therapies in androgen deprivation therapy recurrent prostate cancer.