Publications by authors named "Darren Henstridge"

68 Publications

Fine-tuning the cardiac O-GlcNAcylation regulatory enzymes governs the functional and structural phenotype of the diabetic heart.

Cardiovasc Res 2021 Feb 8. Epub 2021 Feb 8.

School of Biosciences, Parkville, Victoria, Australia, 3010.

Aims: The glucose-driven enzymatic modification of myocardial proteins by the sugar moiety, β-N-acetylglucosamine (O-GlcNAc), is increased in pre-clinical models of diabetes, implicating protein O-GlcNAc modification in diabetes-induced heart failure. Our aim was to specifically examine cardiac manipulation of the two regulatory enzymes of this process on the cardiac phenotype, in the presence and absence of diabetes, utilising cardiac-targeted recombinant-adeno-associated viral-vector-6 (rAAV6)-mediated gene delivery.

Methods And Results: In human myocardium, total protein O-GlcNAc modification was elevated in diabetic relative to non-diabetic patients, and correlated with left ventricular (LV) dysfunction. The impact of rAAV6-delivered O-GlcNAc transferase (rAAV6-OGT, facilitating protein O-GlcNAcylation), O-GlcNAcase (rAAV6-OGA, facilitating de-O-GlcNAcylation) and empty vector (null) were determined in non-diabetic and diabetic mice. In non-diabetic mice, rAAV6-OGT was sufficient to impair LV diastolic function and induce maladaptive cardiac remodelling, including cardiac fibrosis and increased Myh-7 and Nppa pro-hypertrophic gene expression, recapitulating characteristics of diabetic cardiomyopathy. In contrast, rAAV6-OGA (but not rAAV6-OGT) rescued LV diastolic function and adverse cardiac remodelling in diabetic mice. Molecular insights implicated impaired cardiac PI3K(p110α)-Akt signalling as a potential contributing mechanism to the detrimental consequences of rAAV6-OGT in vivo. In contrast, rAAV6-OGA preserved PI3K(p110α)-Akt signalling in diabetic mouse myocardium in vivo and prevented high glucose-induced impairments in mitochondrial respiration in human cardiomyocytes in vitro.

Conclusion: Maladaptive protein O-GlcNAc modification is evident in human diabetic myocardium, and is a critical regulator of the diabetic heart phenotype. Selective targeting of cardiac protein O-GlcNAcylation to restore physiological O-GlcNAc balance may represent a novel therapeutic approach for diabetes-induced heart failure.

Translational Perspective: There remains a lack of effective clinical management of diabetes-induced cardiac dysfunction, even via conventional intensive glucose-lowering approaches. Here we reveal that the modification of myocardial proteins by O-GlcNAc, a glucose-driven process, is not only increased in human diabetic myocardium, but correlates with reduced cardiac function in affected patients. Moreover, manipulation of the two regulatory enzymes of this process exert opposing influences on the heart, whereby increasing O-GlcNAc transferase (OGT) is sufficient to replicate the cardiac phenotype of diabetes (in the absence of this disease), while increasing O-GlcNAc-ase (OGA) rescues diabetes-induced impairments in both cardiac dysfunction and remodelling. Cardiac O-GlcNAcylation thus represents a novel therapeutic target for diabetes-induced heart failure.
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http://dx.doi.org/10.1093/cvr/cvab043DOI Listing
February 2021

Deletion of Trim28 in committed adipocytes promotes obesity but preserves glucose tolerance.

Nat Commun 2021 01 4;12(1):74. Epub 2021 Jan 4.

Baker Heart & Diabetes Institute, Melbourne, VIC, Australia, 3004.

The effective storage of lipids in white adipose tissue (WAT) critically impacts whole body energy homeostasis. Many genes have been implicated in WAT lipid metabolism, including tripartite motif containing 28 (Trim28), a gene proposed to primarily influence adiposity via epigenetic mechanisms in embryonic development. However, in the current study we demonstrate that mice with deletion of Trim28 specifically in committed adipocytes, also develop obesity similar to global Trim28 deletion models, highlighting a post-developmental role for Trim28. These effects were exacerbated in female mice, contributing to the growing notion that Trim28 is a sex-specific regulator of obesity. Mechanistically, this phenotype involves alterations in lipolysis and triglyceride metabolism, explained in part by loss of Klf14 expression, a gene previously demonstrated to modulate adipocyte size and body composition in a sex-specific manner. Thus, these findings provide evidence that Trim28 is a bona fide, sex specific regulator of post-developmental adiposity and WAT function.
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http://dx.doi.org/10.1038/s41467-020-20434-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782476PMC
January 2021

Characterization of the circulating and tissue-specific alterations to the lipidome in response to moderate and major cold stress in mice.

Am J Physiol Regul Integr Comp Physiol 2021 02 11;320(2):R95-R104. Epub 2020 Nov 11.

Baker Heart and Diabetes Institute, Melbourne, Australia.

This study analyzed the effects of 24 h of cold stress (22°C or 5°C . mice maintained at 30 °C) on the plasma, brown adipose tissue (BAT), subcutaneous (SubQ) and epididymal (Epi) white adipose tissue (WAT), liver, and skeletal muscle lipidome of mice. Using mass spectrometry-lipidomics, 624 lipid species were detected, of which 239 were significantly altered in plasma, 134 in BAT, and 51 in the liver. In plasma, acylcarnitines and free fatty acids were markedly increased at 5°C. Plasma triacylglycerols (TGs) were reduced at 22°C and 5°C. We also identified ether lipids as a novel, cold-induced lipid class. In BAT, TGs were the principal lipid class affected by cold stress, being significantly reduced at both 22°C and 5°C. Interestingly, although BAT TG species were uniformly affected at 5°C, at 22°C we observed species-dependent effects, with TGs containing longer and more unsaturated fatty acids particularly sensitive to the effects of cold. In the liver, TGs were the most markedly affected lipid class, increasing in abundance at 5 °C. TGs containing longer and more unsaturated fatty acids accumulated to a greater degree. Our work demonstrates the following: ) acute exposure to moderate (22°C) cold stress alters the plasma and BAT lipidome; although this effect is markedly less pronounced than at 5°C. ) Cold stress at 5°C dramatically alters the plasma lipidome, with ether lipids identified as a novel lipid class altered by cold exposure. ) Cold-induced alterations in liver and BAT TG levels are not uniform, with changes being influenced by acyl chain composition.
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http://dx.doi.org/10.1152/ajpregu.00112.2020DOI Listing
February 2021

Fructose stimulated de novo lipogenesis is promoted by inflammation.

Nat Metab 2020 10 24;2(10):1034-1045. Epub 2020 Aug 24.

Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, School of Medicine, University of California San Diego, La Jolla, CA, USA.

Benign hepatosteatosis, affected by lipid uptake, de novo lipogenesis and fatty acid (FA) oxidation, progresses to non-alcoholic steatohepatitis (NASH) on stress and inflammation. A key macronutrient proposed to increase hepatosteatosis and NASH risk is fructose. Excessive intake of fructose causes intestinal-barrier deterioration and endotoxaemia. However, how fructose triggers these alterations and their roles in hepatosteatosis and NASH pathogenesis remain unknown. Here we show, using mice, that microbiota-derived Toll-like receptor (TLR) agonists promote hepatosteatosis without affecting fructose-1-phosphate (F1P) and cytosolic acetyl-CoA. Activation of mucosal-regenerative gp130 signalling, administration of the YAP-induced matricellular protein CCN1 or expression of the antimicrobial peptide Reg3b (beta) peptide counteract fructose-induced barrier deterioration, which depends on endoplasmic-reticulum stress and subsequent endotoxaemia. Endotoxin engages TLR4 to trigger TNF production by liver macrophages, thereby inducing lipogenic enzymes that convert F1P and acetyl-CoA to FA in both mouse and human hepatocytes.
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http://dx.doi.org/10.1038/s42255-020-0261-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018782PMC
October 2020

MCL-1 is essential for survival but dispensable for metabolic fitness of FOXP3 regulatory T cells.

Cell Death Differ 2020 Dec 1;27(12):3374-3385. Epub 2020 Jul 1.

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, Australia.

FOXP3 regulatory T (Treg) cells are essential for maintaining immunological tolerance. Given their importance in immune-related diseases, cancer and obesity, there is increasing interest in targeting the Treg cell compartment therapeutically. New pharmacological inhibitors that specifically target the prosurvival protein MCL-1 may provide this opportunity, as Treg cells are particularly reliant upon this protein. However, there are two distinct isoforms of MCL-1; one located at the outer mitochondrial membrane (OMM) that is required to antagonize apoptosis, and another at the inner mitochondrial membrane (IMM) that is reported to maintain IMM structure and metabolism via ATP production during oxidative phosphorylation. We set out to elucidate the relative importance of these distinct biological functions of MCL-1 in Treg cells to assess whether MCL-1 inhibition might impact upon the metabolism of cells able to resist apoptosis. Conditional deletion of Mcl1 in FOXP3 Treg cells resulted in a lethal multiorgan autoimmunity due to the depletion of the Treg cell compartment. This striking phenotype was completely rescued by concomitant deletion of the apoptotic effector proteins BAK and BAX, indicating that apoptosis plays a pivotal role in the homeostasis of Treg cells. Notably, MCL-1-deficient Treg cells rescued from apoptosis displayed normal metabolic capacity. Moreover, pharmacological inhibition of MCL-1 in Treg cells resistant to apoptosis did not perturb their metabolic function. We conclude that Treg cells require MCL-1 only to antagonize apoptosis and not for metabolism. Therefore, MCL-1 inhibition could be used to manipulate Treg cell survival for clinical benefit without affecting the metabolic fitness of cells resisting apoptosis.
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http://dx.doi.org/10.1038/s41418-020-0585-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853142PMC
December 2020

Fecal microbiota transplantation from high caloric-fed donors alters glucose metabolism in recipient mice, independently of adiposity or exercise status.

Am J Physiol Endocrinol Metab 2020 07 9;319(1):E203-E216. Epub 2020 Jun 9.

Baker Heart and Diabetes Institute, Melbourne, Australia.

Studies suggest the gut microbiota contributes to the development of obesity and metabolic syndrome. Exercise alters microbiota composition and diversity and is protective of these maladies. We tested whether the protective metabolic effects of exercise are mediated through fecal components through assessment of body composition and metabolism in recipients of fecal microbiota transplantation (FMT) from exercise-trained (ET) mice fed normal or high-energy diets. Donor C57BL/6J mice were fed a chow or high-fat, high-sucrose diet (HFHS) for 4 wk to induce obesity and glucose intolerance. Mice were divided into sedentary (Sed) or ET groups (6 wk treadmill-based ET) while maintaining their diets, resulting in four donor groups: chow sedentary (NC-Sed) or ET (NC-ET) and HFHS sedentary (HFHS-Sed) or ET (HFHS-ET). Chow-fed recipient mice were gavaged with feces from the respective donor groups weekly, creating four groups (NC-Sed-R, NC-ET-R, HFHS-Sed-R, HFHS-ET-R), and body composition and metabolism were assessed. The HFHS diet led to glucose intolerance and obesity in the donors, whereas exercise training (ET) restrained adiposity and improved glucose tolerance. No donor group FMT altered recipient body composition. Despite unaltered adiposity, glucose levels were disrupted when challenged in mice receiving feces from HFHS-fed donors, irrespective of donor-ET status, with a decrease in insulin-stimulated glucose clearance into white adipose tissue and large intestine and specific changes in the recipient's microbiota composition observed. FMT can transmit HFHS-induced disrupted glucose metabolism to recipient mice independently of any change in adiposity. However, the protective metabolic effect of ET on glucose metabolism is not mediated through fecal factors.
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http://dx.doi.org/10.1152/ajpendo.00037.2020DOI Listing
July 2020

Serine administration as a novel prophylactic approach to reduce the severity of acute pancreatitis during diabetes in mice.

Diabetologia 2020 09 8;63(9):1885-1899. Epub 2020 May 8.

Swiss Hepato-Pancreato-Biliary Center, Department of Visceral and Transplantation Surgery, University Hospital Zurich, Zurich, Switzerland.

Aims/hypothesis: Compared with the general population, individuals with diabetes have a higher risk of developing severe acute pancreatitis, a highly debilitating and potentially lethal inflammation of the exocrine pancreas. In this study, we investigated whether 1-deoxysphingolipids, atypical lipids that increase in the circulation following the development of diabetes, exacerbate the severity of pancreatitis in a diabetic setting.

Methods: We analysed whether administration of an L-serine-enriched diet to mouse models of diabetes, an established method for decreasing the synthesis of 1-deoxysphingolipids in vivo, reduced the severity of acute pancreatitis. Furthermore, we elucidated the molecular mechanisms underlying the lipotoxicity exerted by 1-deoxysphingolipids towards rodent pancreatic acinar cells in vitro.

Results: We demonstrated that L-serine supplementation reduced the damage of acinar tissue resulting from the induction of pancreatitis in diabetic mice (average histological damage score: 1.5 in L-serine-treated mice vs 2.7 in the control group). At the cellular level, we showed that L-serine decreased the production of reactive oxygen species, endoplasmic reticulum stress and cellular apoptosis in acinar tissue. Importantly, these parameters, together with DNA damage, were triggered in acinar cells upon treatment with 1-deoxysphingolipids in vitro, suggesting that these lipids are cytotoxic towards pancreatic acinar cells in a cell-autonomous manner. In search of the initiating events of the observed cytotoxicity, we discovered that 1-deoxysphingolipids induced early mitochondrial dysfunction in acinar cells, characterised by ultrastructural alterations, impaired oxygen consumption rate and reduced ATP synthesis.

Conclusions/interpretation: Our results suggest that 1-deoxysphingolipids directly damage the functionality of pancreatic acinar cells and highlight that an L-serine-enriched diet may be used as a promising prophylactic intervention to reduce the severity of pancreatitis in the context of diabetes.
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http://dx.doi.org/10.1007/s00125-020-05156-xDOI Listing
September 2020

Intravascular Follistatin gene delivery improves glycemic control in a mouse model of type 2 diabetes.

FASEB J 2020 04 5;34(4):5697-5714. Epub 2020 Mar 5.

Centre for Muscle Research, Department of Physiology, The University of Melbourne, Parkville, VIC, Australia.

Type 2 diabetes (T2D) manifests from inadequate glucose control due to insulin resistance, hypoinsulinemia, and deteriorating pancreatic β-cell function. The pro-inflammatory factor Activin has been implicated as a positive correlate of severity in T2D patients, and as a negative regulator of glucose uptake by skeletal muscle, and of pancreatic β-cell phenotype in mice. Accordingly, we sought to determine whether intervention with the Activin antagonist Follistatin can ameliorate the diabetic pathology. Here, we report that an intravenous Follistatin gene delivery intervention with tropism for striated muscle reduced the serum concentrations of Activin B and improved glycemic control in the db/db mouse model of T2D. Treatment reversed the hyperglycemic progression with a corresponding reduction in the percentage of glycated-hemoglobin to levels similar to lean, healthy mice. Follistatin gene delivery promoted insulinemia and abundance of insulin-positive pancreatic β-cells, even when treatment was administered to mice with advanced diabetes, supporting a mechanism for improved glycemic control associated with maintenance of functional β-cells. Our data demonstrate that single-dose intravascular Follistatin gene delivery can ameliorate the diabetic progression and improve prognostic markers of disease. These findings are consistent with other observations of Activin-mediated mechanisms exerting deleterious effects in models of obesity and diabetes, and suggest that interventions that attenuate Activin signaling could help further understanding of T2D and the development of novel T2D therapeutics.
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http://dx.doi.org/10.1096/fj.201802059RRRDOI Listing
April 2020

Sex-specific adipose tissue imprinting of regulatory T cells.

Nature 2020 03 26;579(7800):581-585. Epub 2020 Feb 26.

Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia.

Adipose tissue is an energy store and a dynamic endocrine organ. In particular, visceral adipose tissue (VAT) is critical for the regulation of systemic metabolism. Impaired VAT function-for example, in obesity-is associated with insulin resistance and type 2 diabetes. Regulatory T (T) cells that express the transcription factor FOXP3 are critical for limiting immune responses and suppressing tissue inflammation, including in the VAT. Here we uncover pronounced sexual dimorphism in T cells in the VAT. Male VAT was enriched for T cells compared with female VAT, and T cells from male VAT were markedly different from their female counterparts in phenotype, transcriptional landscape and chromatin accessibility. Heightened inflammation in the male VAT facilitated the recruitment of T cells via the CCL2-CCR2 axis. Androgen regulated the differentiation of a unique IL-33-producing stromal cell population specific to the male VAT, which paralleled the local expansion of T cells. Sex hormones also regulated VAT inflammation, which shaped the transcriptional landscape of VAT-resident T cells in a BLIMP1 transcription factor-dependent manner. Overall, we find that sex-specific differences in T cells from VAT are determined by the tissue niche in a sex-hormone-dependent manner to limit adipose tissue inflammation.
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http://dx.doi.org/10.1038/s41586-020-2040-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241647PMC
March 2020

Delineating a role for the mitochondrial permeability transition pore in diabetic kidney disease by targeting cyclophilin D.

Clin Sci (Lond) 2020 Jan;134(2):239-259

Department of Diabetes, Central Clinical School, Alfred Medical Research and Education Precinct, Monash University, Melbourne, Victoria, Australia.

Mitochondrial stress has been widely observed in diabetic kidney disease (DKD). Cyclophilin D (CypD) is a functional component of the mitochondrial permeability transition pore (mPTP) which allows the exchange of ions and solutes between the mitochondrial matrix to induce mitochondrial swelling and activation of cell death pathways. CypD has been successfully targeted in other disease contexts to improve mitochondrial function and reduced pathology. Two approaches were used to elucidate the role of CypD and the mPTP in DKD. Firstly, mice with a deletion of the gene encoding CypD (Ppif-/-) were rendered diabetic with streptozotocin (STZ) and followed for 24 weeks. Secondly, Alisporivir, a CypD inhibitor was administered to the db/db mouse model (5 mg/kg/day oral gavage for 16 weeks). Ppif-/- mice were not protected against diabetes-induced albuminuria and had greater glomerulosclerosis than their WT diabetic littermates. Renal hyperfiltration was lower in diabetic Ppif-/- as compared with WT mice. Similarly, Alisporivir did not improve renal function nor pathology in db/db mice as assessed by no change in albuminuria, KIM-1 excretion and glomerulosclerosis. Db/db mice exhibited changes in mitochondrial function, including elevated respiratory control ratio (RCR), reduced mitochondrial H2O2 generation and increased proximal tubular mitochondrial volume, but these were unaffected by Alisporivir treatment. Taken together, these studies indicate that CypD has a complex role in DKD and direct targeting of this component of the mPTP will likely not improve renal outcomes.
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http://dx.doi.org/10.1042/CS20190787DOI Listing
January 2020

Complement C5a Induces Renal Injury in Diabetic Kidney Disease by Disrupting Mitochondrial Metabolic Agility.

Diabetes 2020 01 17;69(1):83-98. Epub 2019 Oct 17.

Department of Diabetes, Central Clinical School, Alfred Medical Research and Education Precinct, Monash University, Melbourne, Victoria, Australia

The sequelae of diabetes include microvascular complications such as diabetic kidney disease (DKD), which involves glucose-mediated renal injury associated with a disruption in mitochondrial metabolic agility, inflammation, and fibrosis. We explored the role of the innate immune complement component C5a, a potent mediator of inflammation, in the pathogenesis of DKD in clinical and experimental diabetes. Marked systemic elevation in C5a activity was demonstrated in patients with diabetes; conventional renoprotective agents did not therapeutically target this elevation. C5a and its receptor (C5aR1) were upregulated early in the disease process and prior to manifest kidney injury in several diverse rodent models of diabetes. Genetic deletion of C5aR1 in mice conferred protection against diabetes-induced renal injury. Transcriptomic profiling of kidney revealed diabetes-induced downregulation of pathways involved in mitochondrial fatty acid metabolism. Interrogation of the lipidomics signature revealed abnormal cardiolipin remodeling in diabetic kidneys, a cardinal sign of disrupted mitochondrial architecture and bioenergetics. In vivo delivery of an orally active inhibitor of C5aR1 (PMX53) reversed the phenotypic changes and normalized the renal mitochondrial fatty acid profile, cardiolipin remodeling, and citric acid cycle intermediates. In vitro exposure of human renal proximal tubular epithelial cells to C5a led to altered mitochondrial respiratory function and reactive oxygen species generation. These experiments provide evidence for a pivotal role of the C5a/C5aR1 axis in propagating renal injury in the development of DKD by disrupting mitochondrial agility, thereby establishing a new immunometabolic signaling pathway in DKD.
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http://dx.doi.org/10.2337/db19-0043DOI Listing
January 2020

Glycolysis Is Required for LPS-Induced Activation and Adhesion of Human CD14CD16 Monocytes.

Front Immunol 2019 6;10:2054. Epub 2019 Sep 6.

Division of Immunometabolism, Baker Heart and Diabetes Institute, Melbourne, VIC, Australia.

Monocytes in humans consist of 3 subsets; CD14CD16 (classical), CD14CD16 (intermediate) and CD14CD16 (non-classical), which exhibit distinct and heterogeneous responses to activation. During acute inflammation CD14CD16 monocytes are significantly elevated and migrate to the sites of injury via the adhesion cascade. The field of immunometabolism has begun to elucidate the importance of the engagement of specific metabolic pathways in immune cell function. Yet, little is known about monocyte metabolism and the role of metabolism in mediating monocyte activation and adherence to vessels. Accordingly, we aimed to determine whether manipulating the metabolism of CD14CD16 monocytes alters their ability to become activated and adhere. We discovered that LPS stimulation increased the rate of glycolysis in human CD14CD16 monocytes. Inhibition of glycolysis with 2-deoxy-D-glucose blunted LPS-induced activation and adhesion of monocytes. Mechanistically, we found that increased glycolysis was regulated by mTOR-induced glucose transporter (GLUT)-1. Furthermore, enhanced glycolysis increased accumulation of reactive oxygen species (ROS) and activation of p38 MAPK, which lead to activation and adhesion of monocytes. These findings reveal that glycolytic metabolism is critical for the activation of CD14CD16 monocytes and contributes to our understanding of the interplay between metabolic substrate preference and immune cell function.
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http://dx.doi.org/10.3389/fimmu.2019.02054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742687PMC
October 2020

Treatment of type 2 diabetes with the designer cytokine IC7Fc.

Nature 2019 10 25;574(7776):63-68. Epub 2019 Sep 25.

The Garvan Institute of Medical Research, Sydney, New South Wales, Australia.

The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.
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http://dx.doi.org/10.1038/s41586-019-1601-9DOI Listing
October 2019

CORP: Practical tools for improving experimental design and reporting of laboratory studies of cardiovascular physiology and metabolism.

Am J Physiol Heart Circ Physiol 2019 09 26;317(3):H627-H639. Epub 2019 Jul 26.

Baker Heart and Diabetes Institute, Melbourne, Australia.

The exercise consisted of: ) a short survey to acquire baseline data on current practices regarding the conduct of animal studies, ) a series of presentations for promoting awareness and providing advice and practical tools for improving experimental design, and ) a follow-up survey 12 mo later to assess whether practices had changed. The surveys were compulsory for responsible investigators ( = 16; paired data presented). Other investigators named on animal ethics applications were encouraged to participate (2017, total of 36 investigators; 2018, 37 investigators). The major findings to come from the exercise included ) a willingness of investigators to make changes when provided with knowledge/tools and solutions that were relatively simple to implement (e.g., proportion of responsible investigators showing improved practices using a structured method for randomization was 0.44, 95% CI (0.19; 0.70), = 0.003, and deidentifying drugs/interventions was 0.40, 95% CI (0.12; 0.68), = 0.010); ) resistance to change if this involved more personnel and time (e.g., as required for allocation concealment); and ) evidence that changes to long-term practices ("habits") require time and follow-up. Improved practices could be verified based on changes in reporting within publications or documented evidence provided during laboratory visits. In summary, this exercise resulted in changed attitudes, practices, and reporting, but continued follow-up, monitoring, and incentives are required. Efforts to improve experimental rigor will reduce bias and will lead to findings with the greatest translational potential. The goal of this exercise was to encourage preclinical researchers to improve the quality of their cardiac and metabolic animal studies by ) increasing awareness of concerns, which can arise from suboptimal experimental designs; ) providing knowledge, tools, and templates to overcome bias; and ) conducting two short surveys over 12 mo to monitor change. Improved practices were identified for the uptake of structured methods for randomization, and de-identifying interventions/drugs.Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/experimental-design-survey-training-practical-tools/.
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http://dx.doi.org/10.1152/ajpheart.00327.2019DOI Listing
September 2019

The Zinc Transporter Zip7 Is Downregulated in Skeletal Muscle of Insulin-Resistant Cells and in Mice Fed a High-Fat Diet.

Cells 2019 07 1;8(7). Epub 2019 Jul 1.

College of Health and Medicine, School of Health Sciences, University of Tasmania, Hobart, Tasmania 7005, Australia.

Background: The zinc transporter Zip7 modulates zinc flux and controls cell signaling molecules associated with glucose metabolism in skeletal muscle. The present study evaluated the role of Zip7 in cell signaling pathways involved in insulin-resistant skeletal muscle and mice fed a high-fat diet.

Methods: Insulin-resistant skeletal muscle cells were prepared by treatment with an inhibitor of the insulin receptor, HNMPA-(AM)3 or palmitate, and Zip7 was analyzed along with pAkt, pTyrosine and Glut4. Similarly, mice fed normal chow (NC) or a high-fat diet (HFD) were also analyzed for protein expression of Glut4 and Zip7. An overexpression system for Zip7 was utilized to determine the action of this zinc transporter on several genes implicated in insulin signaling and glucose control.

Results: We identified that Zip7 is upregulated by glucose in normal skeletal muscle cells and downregulated in insulin-resistant skeletal muscle. We also observed (as expected) a decrease in pAkt and Glut4 in the insulin-resistant skeletal muscle cells. The overexpression of Zip7 in skeletal muscle cells led to the modulation of key genes involved in the insulin signaling axis and glucose metabolism including , , , , , , , and . In an mouse model, we identified a reduction in Glut4 and Zip7 in the skeletal muscle of mice fed a HFD compared to NC controls.

Conclusions: These data suggest that Zip7 plays a role in skeletal muscle insulin signaling and is downregulated in an insulin-resistant, and HFD state. Understanding the molecular mechanisms of Zip7 action will provide novel opportunities to target this transporter therapeutically for the treatment of insulin resistance and type 2 diabetes.
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http://dx.doi.org/10.3390/cells8070663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678147PMC
July 2019

Respiratory syncytial virus co-opts host mitochondrial function to favour infectious virus production.

Elife 2019 06 27;8. Epub 2019 Jun 27.

Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.

Although respiratory syncytial virus (RSV) is responsible for more human deaths each year than influenza, its pathogenic mechanisms are poorly understood. Here high-resolution quantitative imaging, bioenergetics measurements and mitochondrial membrane potential- and redox-sensitive dyes are used to define RSV's impact on host mitochondria for the first time, delineating RSV-induced microtubule/dynein-dependent mitochondrial perinuclear clustering, and translocation towards the microtubule-organizing centre. These changes are concomitant with impaired mitochondrial respiration, loss of mitochondrial membrane potential and increased production of mitochondrial reactive oxygen species (ROS). Strikingly, agents that target microtubule integrity the dynein motor protein, or inhibit mitochondrial ROS production strongly suppresses RSV virus production, including in a mouse model with concomitantly reduced virus-induced lung inflammation. The results establish RSV's unique ability to co-opt host cell mitochondria to facilitate viral infection, revealing the RSV-mitochondrial interface for the first time as a viable target for therapeutic intervention.
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http://dx.doi.org/10.7554/eLife.42448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598784PMC
June 2019

Metabolic control and sex: A focus on inflammatory-linked mediators.

Br J Pharmacol 2019 11 21;176(21):4193-4207. Epub 2019 Apr 21.

Division of Diabetes & Metabolism, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.

Men and women have many differing biological and physiological characteristics. Thus, it is no surprise that the control of metabolic processes and the mechanisms underlying metabolic-related diseases have sex-specific components. There is a clear metabolic sexual dimorphism in that up until midlife, men have a far greater likelihood of acquiring cardio-metabolic disease than women. Following menopause, however, this difference is reduced, suggestive of a protective role of the female sex hormones. Inflammatory processes have been implicated in the pathogenesis of cardio-metabolic disease with human studies correlating metabolic disease acquisition or risk with levels of various inflammatory markers. Rodent studies employing genetic modifications or novel pharmacological approaches have provided mechanistic insight into the role of these inflammatory mediators. Sex differences impact inflammatory processes and the subsequent biological response. As a consequence, this may affect how inflammation alters metabolic processes between the sexes. Recently, some of our work in the field of inflammatory genes and metabolic control identified a sexual dimorphism in a preclinical model and caused us to question the frequency and scale of such findings in the literature. This review concentrates on inflammatory-related signalling in relation to obesity, insulin resistance, and type 2 diabetes and highlights the differences observed between males and females. Differences in the activation and signalling of various inflammatory genes and proteins present another reason why studying both male and female patients or animals is important in the context of understanding and finding therapeutics for metabolic-related disease. LINKED ARTICLES: This article is part of a themed section on The Importance of Sex Differences in Pharmacology Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.21/issuetoc.
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http://dx.doi.org/10.1111/bph.14642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877797PMC
November 2019

The E3 ligase MARCH5 is a PPARγ target gene that regulates mitochondria and metabolism in adipocytes.

Am J Physiol Endocrinol Metab 2019 02 4;316(2):E293-E304. Epub 2018 Dec 4.

Baker Heart and Diabetes Institute , Melbourne, Victoria , Australia.

Mitochondrial dynamics refers to the constant remodeling of mitochondrial populations by multiple cellular pathways that help maintain mitochondrial health and function. Disruptions in mitochondrial dynamics often lead to mitochondrial dysfunction, which is frequently associated with disease in rodents and humans. Consistent with this, obesity is associated with reduced mitochondrial function in white adipose tissue, partly via alterations in mitochondrial dynamics. Several proteins, including the E3 ubiquitin ligase membrane-associated RING-CH-type finger 5 (MARCH5), are known to regulate mitochondrial dynamics; however, the role of these proteins in adipocytes has been poorly studied. Here, we show that MARCH5 is regulated by peroxisome proliferator-activated receptor-γ (PPARγ) during adipogenesis and is correlated with fat mass across a panel of genetically diverse mouse strains, in ob/ob mice, and in humans. Furthermore, manipulation of MARCH5 expression in vitro and in vivo alters mitochondrial function, affects cellular metabolism, and leads to differential regulation of several metabolic genes. Thus our data demonstrate an association between mitochondrial dynamics and metabolism that defines MARCH5 as a critical link between these interconnected pathways.
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http://dx.doi.org/10.1152/ajpendo.00394.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397360PMC
February 2019

Protein Kinase C Epsilon Deletion in Adipose Tissue, but Not in Liver, Improves Glucose Tolerance.

Cell Metab 2019 01 11;29(1):183-191.e7. Epub 2018 Oct 11.

Diabetes and Metabolism Division, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia; St Vincent's Clinical School, University of New South Wales, Sydney, NSW 2010, Australia. Electronic address:

Protein kinase C epsilon (PKCɛ) activation in the liver is proposed to inhibit insulin action through phosphorylation of the insulin receptor. Here, however, we demonstrated that global, but not liver-specific, deletion of PKCɛ in mice protected against diet-induced glucose intolerance and insulin resistance. Furthermore, PKCɛ-dependent alterations in insulin receptor phosphorylation were not detected. Adipose-tissue-specific knockout mice did exhibit improved glucose tolerance, but phosphoproteomics revealed no PKCɛ-dependent effect on the activation of insulin signaling pathways. Altered phosphorylation of adipocyte proteins associated with cell junctions and endosomes was associated with changes in hepatic expression of several genes linked to glucose homeostasis and lipid metabolism. The primary effect of PKCɛ on glucose homeostasis is, therefore, not exerted directly in the liver as currently posited, and PKCɛ activation in this tissue should be interpreted with caution. However, PKCɛ activity in adipose tissue modulates glucose tolerance and is involved in crosstalk with the liver.
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http://dx.doi.org/10.1016/j.cmet.2018.09.013DOI Listing
January 2019

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice.

J Vis Exp 2018 05 24(135). Epub 2018 May 24.

Baker Heart and Diabetes Institute;

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.
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http://dx.doi.org/10.3791/57280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101377PMC
May 2018

The effect of oxygen in Sirt3-mediated myocardial protection: a proof-of-concept study in cultured cardiomyoblasts.

J Thromb Thrombolysis 2018 Jul;46(1):102-112

Department of Cardiology, University Heart Center Zurich, University Hospital Zurich, Ramistr. 100, 8098, Zurich, Switzerland.

Sirtuin 3 is a nicotinamide adenine dinucleotide dependent mitochondrial deacetylase that governs mitochondrial metabolism and oxidative defense. The demise in myocardial function following myocardial ischemia has been associated with mitochondrial dysfunction. Sirt3 maintains myocardial contractile function and protects from cardiac hypertrophy. The role of Sirt3 in ischemia is controversial. Our objective was to understand, under what circumstances Sirt3 is protective in different facets of ischemia, using an in vitro proof-of-concept approach based on simulated ischemia in cultured cardiomyoblasts. Cultured H9c2 cardiomyoblasts were subjected to hypoxia and/or serum deprivation, the combination of which we refer to as simulated ischemia. Apoptosis, as assessed by Annexin V staining in life-cell imaging and propidium-iodide inclusion in flow cytometry, was enhanced following simulated ischemia. Interestingly, serum deprivation was a stronger trigger of apoptosis than hypoxia. Knockdown of Sirt3 further increased apoptosis upon serum deprivation, whereas no such effect occurred upon additional hypoxia. Similarly, only upon serum deprivation but not upon simulated ischemia, silencing of Sirt3 led to a deterioration of mitochondrial function in extracellular flux analysis. In the absence of oxygen these Sirt3-dependent effects were abolished. These data indicate, that Sirt3-mediated myocardial protection is oxygen-dependent. Thus, mitochondrial respiration takes center-stage in Sirt3-dependent prevention of stress-induced myocardial damage.
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http://dx.doi.org/10.1007/s11239-018-1677-3DOI Listing
July 2018

APP deficiency results in resistance to obesity but impairs glucose tolerance upon high fat feeding.

J Endocrinol 2018 06 19;237(3):311-322. Epub 2018 Apr 19.

Metabolic Research UnitSchool of Medicine and Centre for Molecular and Medical Research, Deakin University, Geelong, Victoria, Australia

The amyloid precursor protein (APP) generates a number of peptides when processed through different cleavage mechanisms, including the amyloid beta peptide that is implicated in the development of Alzheimer's disease. It is well established that APP via its cleaved peptides regulates aspects of neuronal metabolism. Emerging evidence suggests that amyloidogenic processing of APP can lead to altered systemic metabolism, similar to that observed in metabolic disease states. In the present study, we investigated the effect of APP deficiency on obesity-induced alterations in systemic metabolism. Compared with WT littermates, APP-deficient mice were resistant to diet-induced obesity, which was linked to higher energy expenditure and lipid oxidation throughout the dark phase and was associated with increased spontaneous physical activity. Consistent with this lean phenotype, APP-deficient mice fed a high-fat diet (HFD) had normal insulin tolerance. However, despite normal insulin action, these mice were glucose intolerant, similar to WT mice fed a HFD. This was associated with reduced plasma insulin in the early phase of the glucose tolerance test. Analysis of the pancreas showed that APP was required to maintain normal islet and β-cell mass under high fat feeding conditions. These studies show that, in addition to regulating aspects of neuronal metabolism, APP is an important regulator of whole body energy expenditure and glucose homeostasis under high fat feeding conditions.
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http://dx.doi.org/10.1530/JOE-18-0051DOI Listing
June 2018

Skeletal muscle-specific overexpression of heat shock protein 72 improves skeletal muscle insulin-stimulated glucose uptake but does not alter whole body metabolism.

Diabetes Obes Metab 2018 08 3;20(8):1928-1936. Epub 2018 May 3.

Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia.

Aims: The induction of heat shock protein 72 (Hsp72) via heating, genetic manipulation or pharmacological activation is metabolically protective in the setting of obesity-induced insulin resistance across mammalian species. In this study, we set out to determine whether the overexpression of Hsp72, specifically in skeletal muscle, can protect against high-fat diet (HFD)-induced obesity and insulin resistance.

Materials And Methods: An Adeno-Associated Viral vector (AAV), designed to overexpress Hsp72 in skeletal muscle only, was used to study the effects of increasing Hsp72 levels on various metabolic parameters. Two studies were conducted, the first with direct intramuscular (IM) injection of the AAV:Hsp72 into the tibialis anterior hind-limb muscle and the second with a systemic injection to enable body-wide skeletal muscle transduction.

Results: IM injection of the AAV:Hsp72 significantly improved skeletal muscle insulin-stimulated glucose clearance in treated hind-limb muscles, as compared with untreated muscles of the contralateral leg when mice were fed an HFD. Despite this finding, systemic administration of AAV:Hsp72 did not improve body composition parameters such as body weight, fat mass or percentage body fat, nor did it lead to an improvement in fasting glucose levels or glucose tolerance. Furthermore, no differences were observed for other metabolic parameters such as whole-body oxygen consumption, energy expenditure or physical activity levels.

Conclusions: At the levels of Hsp72 over-expression reported herein, skeletal muscle-specific Hsp72 overexpression via IM injection has the capacity to increase insulin-stimulated glucose clearance in this muscle. However, upon systemic injection, which results in lower muscle Hsp72 overexpression, no beneficial effects on whole-body metabolism are observed.
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http://dx.doi.org/10.1111/dom.13319DOI Listing
August 2018

Transcription Factor IRF4 Promotes CD8 T Cell Exhaustion and Limits the Development of Memory-like T Cells during Chronic Infection.

Immunity 2017 12 12;47(6):1129-1141.e5. Epub 2017 Dec 12.

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia; Department of Microbiology and Immunology, The University of Melbourne, Parkville, VIC 3010, Australia; The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000, Australia. Electronic address:

During chronic stimulation, CD8 T cells acquire an exhausted phenotype characterized by expression of inhibitory receptors, down-modulation of effector function, and metabolic impairments. T cell exhaustion protects from excessive immunopathology but limits clearance of virus-infected or tumor cells. We transcriptionally profiled antigen-specific T cells from mice infected with lymphocytic choriomeningitis virus strains that cause acute or chronic disease. T cell exhaustion during chronic infection was driven by high amounts of T cell receptor (TCR)-induced transcription factors IRF4, BATF, and NFATc1. These regulators promoted expression of inhibitory receptors, including PD-1, and mediated impaired cellular metabolism. Furthermore, they repressed the expression of TCF1, a transcription factor required for memory T cell differentiation. Reducing IRF4 expression restored the functional and metabolic properties of antigen-specific T cells and promoted memory-like T cell development. These findings indicate that IRF4 functions as a central node in a TCR-responsive transcriptional circuit that establishes and sustains T cell exhaustion during chronic infection.
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http://dx.doi.org/10.1016/j.immuni.2017.11.021DOI Listing
December 2017

Distinct lipidomic profiles in models of physiological and pathological cardiac remodeling, and potential therapeutic strategies.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 Mar 6;1863(3):219-234. Epub 2017 Dec 6.

Baker Heart and Diabetes Institute, Melbourne, VIC 3004, Australia; Department of Medicine, Monash University, Clayton, VIC 3800, Australia; Department of Physiology, Monash University, Clayton, VIC 3800, Australia. Electronic address:

Cardiac myocyte membranes contain lipids which remodel dramatically in response to heart growth and remodeling. Lipid species have both structural and functional roles. Physiological and pathological cardiac remodeling have very distinct phenotypes, and the identification of molecular differences represent avenues for therapeutic interventions. Whether the abundance of specific lipid classes is different in physiological and pathological models was largely unknown. The aim of this study was to determine whether distinct lipids are regulated in settings of physiological and pathological remodeling, and if so, whether modulation of differentially regulated lipids could modulate heart size and function. Lipidomic profiling was performed on cardiac-specific transgenic mice with 1) physiological cardiac hypertrophy due to increased Insulin-like Growth Factor 1 (IGF1) receptor or Phosphoinositide 3-Kinase (PI3K) signaling, 2) small hearts due to depressed PI3K signaling (dnPI3K), and 3) failing hearts due to dilated cardiomyopathy (DCM). In hearts of dnPI3K and DCM mice, several phospholipids (plasmalogens) were decreased and sphingolipids increased compared to mice with physiological hypertrophy. To assess whether restoration of plasmalogens could restore heart size or cardiac function, dnPI3K and DCM mice were administered batyl alcohol (BA; precursor to plasmalogen biosynthesis) in the diet for 16weeks. BA supplementation increased a major plasmalogen species (p18:0) in the heart but had no effect on heart size or function. This may be due to the concurrent reduction in other plasmalogen species (p16:0 and p18:1) with BA. Here we show that lipid species are differentially regulated in settings of physiological and pathological remodeling. Restoration of lipid species in the failing heart warrants further examination.
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http://dx.doi.org/10.1016/j.bbalip.2017.12.003DOI Listing
March 2018

High-density lipoprotein delivered after myocardial infarction increases cardiac glucose uptake and function in mice.

Sci Transl Med 2017 Oct;9(411)

Baker Heart and Diabetes Institute, Melbourne, Australia.

Protecting the heart after an acute coronary syndrome is a key therapeutic goal to support cardiac recovery and prevent progression to heart failure. A potential strategy is to target cardiac glucose metabolism at the early stages after ischemia when glycolysis is critical for myocyte survival. Building on our discovery that high-density lipoprotein (HDL) modulates skeletal muscle glucose metabolism, we now demonstrate that a single dose of reconstituted HDL (rHDL) delivered after myocardial ischemia increases cardiac glucose uptake, reduces infarct size, and improves cardiac remodeling in association with enhanced functional recovery in mice. These findings applied equally to metabolically normal and insulin-resistant mice. We further establish direct effects of HDL on cardiomyocyte glucose uptake, glycolysis, and glucose oxidation via the Akt signaling pathway within 15 min of reperfusion. These data support the use of infusible HDL preparations for management of acute coronary syndromes in the setting of primary percutaneous interventions.
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http://dx.doi.org/10.1126/scitranslmed.aam6084DOI Listing
October 2017

Metabolically active CD4+ T cells expressing Glut1 and OX40 preferentially harbor HIV during in vitro infection.

FEBS Lett 2017 10 11;591(20):3319-3332. Epub 2017 Oct 11.

Centre for Biomedical Research, Burnet Institute, Melbourne, Australia.

High glucose transporter 1 (Glut1) surface expression is associated with increased glycolytic activity in activated CD4+ T cells. Phosphatidylinositide 3-kinases (PI3K) activation measured by p-Akt and OX40 is elevated in CD4+Glut1+ T cells from HIV+ subjects. TCR engagement of CD4+Glut1+ T cells from HIV+ subjects demonstrates hyperresponsive PI3K-mammalian target of rapamycin signaling. High basal Glut1 and OX40 on CD4+ T cells from combination antiretroviral therapy (cART)-treated HIV+ patients represent a sufficiently metabolically active state permissive for HIV infection in vitro without external stimuli. The majority of CD4+OX40+ T cells express Glut1, thus OX40 rather than Glut1 itself may facilitate HIV infection. Furthermore, infection of CD4+ T cells is limited by p110γ PI3K inhibition. Modulating glucose metabolism may limit cellular activation and prevent residual HIV replication in 'virologically suppressed' cART-treated HIV+ persons.
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http://dx.doi.org/10.1002/1873-3468.12843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658250PMC
October 2017

Over-expressing the soluble gp130-Fc does not ameliorate methionine and choline deficient diet-induced non alcoholic steatohepatitis in mice.

PLoS One 2017 20;12(6):e0179099. Epub 2017 Jun 20.

Cellular and Molecular Metabolism Laboratory, Baker Heart & Diabetes Institute, Melbourne, Australia.

Non-alcoholic steatohepatitis (NASH) is a liver disease with the potential to lead to cirrhosis and hepatocellular carcinoma. Interleukin-6 (IL-6) has been implicated in the pathogenesis of NASH, with the so-called IL-6 'trans-signaling' cascade being responsible for the pro-inflammatory actions of this cytokine. We aimed to block IL-6 'trans-signaling', using a transgenic mouse that overexpresses human soluble glycoprotein130 (sgp130Fc Tg mice) fed a commonly used dietary model of inducing NASH (methionine and choline deficient-diet; MCD diet) and hypothesized that markers of NASH would be ameliorated in such mice. Sgp130Fc Tg and littermate control mice were fed a MCD or control diet for 4 weeks. The MCD diet induced many hallmarks of NASH including hepatomegaly, steatosis, and liver inflammation. However, in contrast with other mouse models and, indeed, human NASH, the MCD diet model did not increase the mRNA or protein expression of IL-6. Not surprisingly, therefore, markers of MCD diet-induced NASH were unaffected by sgp130Fc transgenic expression. While the MCD diet model induces many pathophysiological markers of NASH, it does not induce increased IL-6 expression in the liver, a key hallmark of human NASH. We, therefore, caution the use of the MCD diet as a viable mouse model of NASH.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179099PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478123PMC
September 2017