Recent Advances in Pre-Clinical Development of Adiponectin Receptor Agonist Therapies for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by genetic mutations in the cytoskeletal-sarcolemmal anchor protein dystrophin. Repeated cycles of sarcolemmal tearing and repair lead to a variety of secondary cellular and physiological stressors that are thought to contribute to weakness, atrophy, and fibrosis. Collectively, these stressors can contribute to a pro-inflammatory milieu in locomotor, cardiac, and respiratory muscles. Given the many unwanted side effects that accompany current anti-inflammatory steroid-based approaches for treating DMD (e.g., glucocorticoids), there is a need to develop new therapies that address inflammation and other cellular dysfunctions. Adiponectin receptor (AdipoR) agonists, which stimulate AdipoR1 and R2 isoforms on various cell types, have emerged as therapeutic candidates for DMD due to their anti-inflammatory, anti-fibrotic, and pro-myogenic properties in pre-clinical human and rodent DMD models. Although these molecules represent a new direction for therapeutic intervention, the mechanisms through which they elicit their beneficial effects are not yet fully understood, and DMD-specific data is limited. The overarching goal of this review is to investigate how adiponectin signaling may ameliorate pathology associated with dystrophin deficiency through inflammatory-dependent and -independent mechanisms and to determine if current data supports their future progression to clinical trials.

The adiponectin-derived peptide ALY688 protects against the development of metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is the severe form of non-alcoholic fatty liver disease which has a high potential to progress to cirrhosis and hepatocellular carcinoma, yet adequate effective therapies are lacking. Hypoadiponectinemia is causally involved in the pathogenesis of MASH. This study investigated the pharmacological effects of adiponectin replacement therapy with the adiponectin-derived peptide ALY688 (ALY688-SR) in a mouse model of MASH. Human induced pluripotent stem (iPS) cell-derived hepatocytes were used to test cytotoxicity and signaling of unmodified ALY688 in vitro. High-fat diet with low methionine and no added choline (CDAHF) was used to induce MASH and test the effects of ALY688-SR in vivo. Histological MASH activity score (NAS) and fibrosis score were determined to assess the effect of ALY688-SR. Transcriptional characterization of mice through RNA sequencing was performed to indicate potential molecular mechanisms involved. In cultured hepatocytes, ALY688 efficiently induced adiponectin-like signaling, including the AMP-activated protein kinase and p38 mitogen-activated protein kinase pathways, and did not elicit cytotoxicity. Administration of ALY688-SR in mice did not influence body weight but significantly ameliorated CDAHF-induced hepatic steatosis, inflammation, and fibrosis, therefore effectively preventing the development and progression of MASH. Mechanistically, ALY688-SR treatment markedly induced hepatic expression of genes involved in fatty acid oxidation, whereas it significantly suppressed the expression of pro-inflammatory and pro-fibrotic genes as demonstrated by transcriptomic analysis. ALY688-SR may represent an effective approach in MASH treatment. Its mode of action involves inhibition of hepatic steatosis, inflammation, and fibrosis, possibly via canonical adiponectin-mediated signaling.

MitoNEET preserves muscle insulin sensitivity during iron overload by regulating mitochondrial iron, reactive oxygen species and fission.

Iron overload (IO) is known to contribute to metabolic dysfunctions such as type 2 diabetes and insulin resistance. Using L6 skeletal muscle cells overexpressing the CDGSH iron-sulfur domain-containing protein 1 (CISD1, also known as mitoNEET) (mitoN) protein, we examined the potential role of MitoN in preventing IO-induced insulin resistance. In L6 control cells, IO resulted in insulin resistance which could be prevented by MitoN as demonstrated by western blot of p-Akt and Akt biosensor cells. Mechanistically, IO increased; mitochondrial iron accumulation, mitochondrial reactive oxygen species (ROS), Fis1-dependent mitochondrial fission, mitophagy, FUN14 domain-containing protein 1 (FUNDC1) expression, and decreased Parkin. MitoN overexpression was able to reduce increases in mitochondrial iron accumulation, mitochondrial ROS, mitochondrial fission, mitophagy and FUNDC1 upregulation due to IO. MitoN did not have any effect on the IO-induced downregulation of Parkin. MitoN alone also upregulated peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) protein levels, a master regulator of mitochondrial biogenesis. The use of mitochondrial antioxidant, Skq1, or fission inhibitor, Mdivi-1, prevented IO-induced insulin resistance implying both mitochondrial ROS and fission play a causal role in the development of insulin resistance. Taken together, MitoN is able to confer protection against IO-induced insulin resistance in L6 skeletal muscle cells through regulation of mitochondrial iron content, mitochondrial ROS, and mitochondrial fission.

Salubrinal promotes phospho-eIF2α-dependent activation of UPR leading to autophagy-mediated attenuation of iron-induced insulin resistance.

Identification of new mechanisms mediating insulin sensitivity is important to allow validation of corresponding therapeutic targets. In this study, we first used a cellular model of skeletal muscle cell iron overload and found that endoplasmic reticulum (ER) stress and insulin resistance occurred after iron treatment. Insulin sensitivity was assessed using cells engineered to express an Akt biosensor, based on nuclear FoxO localization, as well as western blotting for insulin signaling proteins. Use of salubrinal to elevate eIF2α phosphorylation and promote the unfolded protein response (UPR) attenuated iron-induced insulin resistance. Salubrinal induced autophagy flux and its beneficial effects on insulin sensitivity were not observed in autophagy-deficient cells generated by overexpressing a dominant-negative ATG5 mutant or via knockout of ATG7. This indicated the beneficial effect of salubrinal-induced UPR activation was autophagy-dependent. We translated these observations to an animal model of systemic iron overload-induced skeletal muscle insulin resistance where administration of salubrinal as pretreatment promoted eIF2α phosphorylation, enhanced autophagic flux in skeletal muscle and improved insulin responsiveness. Together, our results show that salubrinal elicited an eIF2α-autophagy axis leading to improved skeletal muscle insulin sensitivity both in vitro and in mice.

A novel blood-based bioassay to monitor adiponectin signaling.

The diverse beneficial effects of adiponectin-receptor signaling, including its impact on the regulation of inflammatory processes in vivo, have resulted in development of adiponectin receptor agonists as a treatment for metabolic disorders. However, there are no established non-invasive bioassays for detection of adiponectin target engagement in humans or animal models. Here, we designed an assay using small amounts of blood to assess adiponectin action. Specifically, we tested effects of the small 10-amino acid peptide adiponectin receptor agonist, ALY688, in a sublethal LPS endotoxemia model in mice. LPS-induced pro-inflammatory cytokine levels in serum were significantly reduced in mice treated with ALY688, assessed via multiplex ELISA in flow cytometry. Furthermore, ALY688 alone significantly induced TGF-ß release in serum 1 h after treatment and was elevated for up to 24 h. Additionally, using a flow-cytometry panel for detection of changes in circulating immune cell phenotypes, we observed a significant increase in absolute T cell counts in mice after ALY688 treatment. To assess changes in intracellular signaling effectors downstream of adiponectin, phospho-flow cytometry was conducted. There was a significant increase in phosphorylation of AMPK and p38-MAPK in mice after ALY688 treatment. We then used human donor immune cells (PBMCs) treated with ALY688 ex vivo and observed elevation of AMPK and p38-MAPK phosphorylation from baseline in response to ALY688. Together, these results indicate we can detect adiponectin action on immune cells in vivo by assessing adiponectin signaling pathway for AMPK and p38-MAPK, as well as pro-inflammatory cytokine levels. This new approach provides a blood-based bioassay for screening adiponectin action.

Ischemia-induced cardiac dysfunction is exacerbated in adiponectin-knockout mice due to impaired autophagy flux.

Strategies to enhance autophagy flux have been suggested to improve outcomes in cardiac ischemic models. We explored the role of adiponectin in mediating cardiac autophagy under ischemic conditions induced by permanent coronary artery ligation. We studied the molecular mechanisms underlying adiponectin's cardio-protective effects in adiponectin knockout (Ad-KO) compared with wild-type (WT) mice subjected to ischemia by coronary artery ligation and H9c2 cardiomyocyte cell line exposed to hypoxia. Systemic infusion of a cathepsin-B activatable near-infrared probe as a biomarker for autophagy and detection via noninvasive three-dimensional fluorescence molecular tomography combined with computerized tomography to quantitate temporal changes, indicated increased activity in the myocardium of WT mice after myocardial infarction which was attenuated in Ad-KO. Seven days of ischemia increased myocardial adiponectin accumulation and elevated ULK1/AMPK phosphorylation and autophagy assessed by Western blotting for LC3 and p62, an outcome not observed in Ad-KO mice. Cell death, assessed by TUNEL analysis and the ratio of Bcl-2:Bax, plus cardiac dysfunction, measured using echocardiography with strain analysis, were exacerbated in Ad-KO mice. Using cellular models, we observed that adiponectin stimulated autophagy flux in isolated primary adult cardiomyocytes and increased basal and hypoxia-induced autophagy in H9c2 cells. Real-time temporal analysis of caspase-3/7 activation and caspase-3 Western blot indicated that adiponectin suppressed activation by hypoxia. Hypoxia-induced mitochondrial reactive oxygen species production and cell death were also attenuated by adiponectin. Importantly, the ability of adiponectin to reduce caspase-3/7 activation and cell death was not observed in autophagy-deficient cells generated by CRISPR-mediated deletion of Atg7. Collectively, our data indicate that adiponectin acts in an autophagy-dependent manner to attenuate cardiomyocyte caspase-3/7 activation and cell death in response to hypoxia in vitro and ischemia in mice.

Cardioprotection by the adiponectin receptor agonist ALY688 in a preclinical mouse model of heart failure with reduced ejection fraction (HFrEF).

AIMS: Adiponectin has been shown to mediate cardioprotective effects and levels are typically reduced in patients with cardiometabolic disease. Hence, there has been intense interest in developing adiponectin-based therapeutics. The aim of this translational research study was to examine the functional significance of targeting adiponectin signaling with the adiponectin receptor agonist ALY688 in a mouse model of heart failure with reduced ejection fraction (HFrEF), and the mechanisms of cardiac remodeling leading to cardioprotection. METHODS AND RESULTS: Wild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricular pressure overload (PO), or sham surgery, with or without daily subcutaneous ALY688-SR administration. Temporal analysis of cardiac function was conducted via weekly echocardiography for 5 weeks and we observed that ALY688 attenuated the PO-induced dysfunction. ALY688 also reduced cardiac hypertrophic remodeling, assessed via LV mass, heart weight to body weight ratio, cardiomyocyte cross sectional area, ANP and BNP levels. ALY688 also attenuated PO-induced changes in myosin light and heavy chain expression. Collagen content and myofibroblast profile indicated that fibrosis was attenuated by ALY688 with TIMP1 and scleraxis/periostin identified as potential mechanistic contributors. ALY688 reduced PO-induced elevation in circulating cytokines including IL-5, IL-13 and IL-17, and the chemoattractants MCP-1, MIP-1ß, MIP-1alpha and MIP-3α. Assessment of myocardial transcript levels indicated that ALY688 suppressed PO-induced elevations in IL-6, TLR-4 and IL-1ß, collectively indicating anti-inflammatory effects. Targeted metabolomic profiling indicated that ALY688 increased fatty acid mobilization and oxidation, increased betaine and putrescine plus decreased sphingomyelin and lysophospholipids, a profile indicative of improved insulin sensitivity. CONCLUSION: These results indicate that the adiponectin mimetic peptide ALY688 reduced PO-induced fibrosis, hypertrophy, inflammation and metabolic dysfunction and represents a promising therapeutic approach for treating HFrEF in a clinical setting.

The slow-release adiponectin analog ALY688-SR modifies early-stage disease development in the D2.mdx mouse model of Duchenne muscular dystrophy.

Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-ß1 (TGF-ß1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.

Perturbations in lipid metabolism and gut microbiota composition precede cardiac dysfunction in a mouse model of thalassemia.

Cardiomyopathy is a major complication of thalassemia, yet the precise underlying molecular mechanisms remain unclear. We examined whether altered lipid metabolism is an early driving factor in the development of cardiomyopathy using the Th3/+ mouse model of thalassemia. At age 20 weeks, male and female Th3/+ mice manifested anemia and iron overload; however, only males displayed metabolic defects and altered cardiac function. Untargeted lipidomics indicated that the circulating levels of 35 lipid species were significantly altered in Th3/+ mice compared to wild-type controls: triglycerides (TGs) with saturated fatty acids (FAs; TG42:0 and TG44:0) were elevated, while TGs with unsaturated FAs (TG(18:2_20:5_18:2 and TG54:8)) were reduced. Similarly, phosphatidylcholines (PCs) with long chain FAs (palmitic (16:0) or oleic (18:1)) were increased, while PCs with polyunsaturated FAs decreased. Circulating PC(16:0_14:0), GlcCer(d18:1/24:0) correlated significantly with iron overload and cardiac hypertrophy. 16S rRNA gene profiling revealed alterations in the intestinal microbiota of Th3/+ mice. Differentially abundant bacterial genera correlated with PC(39:6), PC(18:1_22:6), GlcCer(d18:1/24:1) and CE(14:0). These results provide new knowledge on perturbations in lipid metabolism and the gut microbiota of Th3/+ mice and identify specific factors which may represent early biomarkers or therapeutic targets to prevent development of cardiomyopathy in ß-thalassemia.

Copper infused fabric attenuates inflammation in macrophages.

While inflammation is an important immune response for protection from infections, excessive or prolonged inflammation can lead to a variety of debilitating diseases including skin disease, diabetes, heart disease, stroke, autoimmune diseases and cancer. Inflammation is a graded response that is typically initiated when resident macrophages sense the presence of pathogens or damage in the tissue and produce inflammatory cytokines and chemokines to kill the pathogen, clear debris and dead tissue, and initiate tissue repair. Here we show that copper-infused fabrics can prevent inflammation by blocking the production of inflammatory cytokines from macrophages after being exposed to LPS, a component of bacterial cell wall. Mechanistically, we show that copper-infused fabrics can significantly reduce the NF-κB and IRF3 activation in LPS-stimulated macrophages. Given the importance of excessive inflammation in diabetes, we show that copper can reduce insulin resistance mediated by inflammatory cytokines in muscle cells. Our data show that copper infused fabrics may be useful to reduce excessive inflammation in macrophages and improve insulin sensitivity in skeletal muscles.

Memory impairment in the D2.mdx mouse model of Duchenne muscular dystrophy is prevented by the adiponectin receptor agonist ALY688.

NEW FINDINGS: What is the central question of this study? Can adiponectin receptor agonism improve recognition memory in a mouse model of Duchenne muscular dystrophy? What is the main finding and its importance? Short-term treatment with the new adiponectin receptor agonist ALY688 improves recognition memory in D2.mdx mice. This finding suggests that further investigation into adiponectin receptor agonism is warranted, given that there remains an unmet need for clinical approaches to treat this cognitive dysfunction in people with Duchenne muscular dystrophy. ABSTRACT: Memory impairments have been well documented in people with Duchenne muscular dystrophy (DMD). However, the underlying mechanisms are poorly understood, and there is an unmet need to develop new therapies to treat this condition. Using a novel object recognition test, we show that recognition memory impairments in D2.mdx mice are completely prevented by daily treatment with the new adiponectin receptor agonist ALY688 from day 7 to 28 of age. In comparison to age-matched wild-type mice, untreated D2.mdx mice demonstrated lower hippocampal mitochondrial respiration (carbohydrate substrate), greater serum interleukin-6 cytokine content and greater hippocampal total tau and Raptor protein contents. Each of these measures was partly or fully preserved after treatment with ALY688. Collectively, these results indicate that adiponectin receptor agonism improves recognition memory in young D2.mdx mice.

Sterile inflammation and the NLRP3 inflammasome in cardiometabolic disease.

Inflammation plays an important role in the pathophysiology of cardiometabolic diseases. Sterile inflammation, a non-infectious and damage-associated molecular pattern (DAMP)-induced innate response, is now well-established to be closely associated with development and progression of cardiometabolic diseases. The NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome is well-established as a major player in sterile inflammatory responses. It is a multimeric cytosolic protein complex which regulates the activation of caspase-1 and subsequently promotes cleavage and release of interleukin (IL)-1 family cytokines, which have a deleterious impact on the development of cardiometabolic diseases. Therefore, targeting NLRP3 itself or the downstream consequences of NLRP3 activation represent excellent potential therapeutic targets in inflammatory cardiometabolic diseases. Here, we review our current understanding of the role which NLRP3 inflammasome regulation plays in cardiometabolic diseases such as obesity, diabetes, non-alcoholic steatohepatitis (NASH), atherosclerosis, ischemic heart disease and cardiomyopathy. Finally, we highlight the potential of targeting NLPR3 or related signaling molecules as a therapeutic approach.

MitoNEET prevents iron overload-induced insulin resistance in H9c2 cells through regulation of mitochondrial iron.

Iron overload (IO) induces insulin resistance in H9c2 cardiomyoblast cells. Here, we used H9c2 cells overexpressing MitoNEET to examine the potential for protection against iron accumulation in the mitochondria and subsequent insulin resistance. In control H9c2 cells, IO was observed to increase mitochondrial iron content, reactive oxygen species (ROS) production, mitochondrial fission, and reduced insulin-stimulated Akt and ERK1/2 phosphorylation. IO did not significantly affect mitophagy, or mitochondrial content, however, an increase in peroxisome-proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) protein expression, a key regulator of mitochondrial biogenesis, was observed. MitoNEET overexpression was able to attenuate the effects of IO on mitochondrial iron content, reactive oxygen species, mitochondrial fission, and insulin signaling. MitoNEET overexpression also upregulated levels of PGC1α protein. The mitochondria-targeted antioxidant, Skq1, prevented IO-induced ROS production and insulin resistance in control cells, indicating mitochondrial ROS plays a causal role in the onset of insulin resistance. The selective mitochondrial fission inhibitor, Mdivi-1, prevented IO-induced mitochondrial fission, however, it did not alleviate IO-induced insulin resistance. Collectively, IO causes insulin resistance in H9c2 cardiomyoblasts and this can be averted by reduction of mitochondrial iron accumulation and ROS production by overexpression of the MitoNEET protein.

Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells.

Iron overload is associated with various pathological changes which contribute to metabolic syndrome, many of which have been proposed to occur via damaging tissue through an excessive amount of reactive oxygen species (ROS) production. In this study, we established a model of iron overload in L6 skeletal muscle cells and observed that iron enhanced cytochrome c release from depolarized mitochondria, assayed by immunofluorescent colocalization of cytochrome c with Tom20 and the use of JC-1, respectively. This subsequently elevated apoptosis, determined via use of a caspase-3/7 activatable fluorescent probe and western blotting for cleaved caspase-3. Using CellROX deep red and mBBr, we observed that iron increased generation of reactive oxygen species (ROS), and that pretreatment with the superoxide dismutase mimetic MnTBAP reduced ROS production and attenuated iron-induced intrinsic apoptosis and cell death. Furthermore, using MitoSox Red we observed that iron enhanced mROS and the mitochondria-targeted anti-oxidant SKQ1 reduced iron-induced ROS generation and cell death. Western blotting for LC3-II and P62 levels as well as immunofluorescent detection of autophagy flux with LC3B and P62 co-localization indicated that iron acutely (2-8 h) activated and later (12-24 h) attenuated autophagic flux. We used autophagy-deficient cell models generated by overexpressing a dominant-negative Atg5 mutant or CRISPR-mediated ATG7 knock out to test the functional significance of autophagy and observed that autophagy-deficiency exacerbated iron-induced ROS production and apoptosis. In conclusion, our study showed that high iron levels promoted ROS production, blunted the self-protective autophagy response and led to cell death in L6 skeletal muscle cells.

Discovery-Based Proteomics Identify Skeletal Muscle Mitochondrial Alterations as an Early Metabolic Defect in a Mouse Model of ß-Thalassemia.

Although metabolic complications are common in thalassemia patients, there is still an unmet need to better understand underlying mechanisms. We used unbiased global proteomics to reveal molecular differences between the th3/+ mouse model of thalassemia and wild-type control animals focusing on skeletal muscles at 8 weeks of age. Our data point toward a significantly impaired mitochondrial oxidative phosphorylation. Furthermore, we observed a shift from oxidative fibre types toward more glycolytic fibre types in these animals, which was further supported by larger fibre-type cross-sectional areas in the more oxidative type fibres (type I/type IIa/type IIax hybrid). We also observed an increase in capillary density in th3/+ mice, indicative of a compensatory response. Western blotting for mitochondrial oxidative phosphorylation complex proteins and PCR analysis of mitochondrial genes indicated reduced mitochondrial content in the skeletal muscle but not the hearts of th3/+ mice. The phenotypic manifestation of these alterations was a small but significant reduction in glucose handling capacity. Overall, this study identified many important alterations in the proteome of th3/+ mice, amongst which mitochondrial defects leading to skeletal muscle remodelling and metabolic dysfunction were paramount.

MitoNEET Provides Cardioprotection via Reducing Oxidative Damage and Conserving Mitochondrial Function.

Cardiometabolic diseases exert a significant health impact, leading to a considerable economic burden globally. The metabolic syndrome, characterized by a well-defined cluster of clinical parameters, is closely linked to an elevated risk of cardiovascular disease. Current treatment strategies often focus on addressing individual aspects of metabolic syndrome. We propose that exploring novel therapeutic approaches that simultaneously target multiple facets may prove more effective in alleviating the burden of cardiometabolic disease. There is a growing body of evidence suggesting that mitochondria can serve as a pivotal target for the development of therapeutics aimed at resolving both metabolic and vascular dysfunction. MitoNEET was identified as a binding target for the thiazolidinedione (TZD) class of antidiabetic drugs and is now recognized for its role in regulating various crucial cellular processes. Indeed, mitoNEET has demonstrated promising potential as a therapeutic target in various chronic diseases, encompassing cardiovascular and metabolic diseases. In this review, we present a thorough overview of the molecular mechanisms of mitoNEET, with an emphasis on their implications for cardiometabolic diseases in more recent years. Furthermore, we explore the potential impact of these findings on the development of novel therapeutic strategies and discuss potential directions for future research.

FGF21-Sirtuin 3 Axis Confers the Protective Effects of Exercise Against Diabetic Cardiomyopathy by Governing Mitochondrial Integrity.

BACKGROUND: Exercise is an effective nonpharmacological strategy to alleviate diabetic cardiomyopathy (DCM) through poorly defined mechanisms. FGF21 (fibroblast growth factor 21), a peptide hormone with pleiotropic benefits on cardiometabolic homeostasis, has been identified as an exercise responsive factor. This study aims to investigate whether FGF21 signaling mediates the benefits of exercise on DCM, and if so, to elucidate the underlying mechanisms. METHODS: The global or hepatocyte-specific FGF21 knockout mice, cardiomyocyte-selective ß-klotho (the obligatory co-receptor for FGF21) knockout mice, and their wild-type littermates were subjected to high-fat diet feeding and injection of streptozotocin to induce DCM, followed by a 6-week exercise intervention and assessment of cardiac functions. Cardiac mitochondrial structure and function were assessed by electron microscopy, enzymatic assays, and measurements of fatty acid oxidation and ATP production. Human induced pluripotent stem cell-derived cardiomyocytes were used to investigate the receptor and postreceptor signaling pathways conferring the protective effects of FGF21 against toxic lipids-induced mitochondrial dysfunction. RESULTS: Treadmill exercise markedly induced cardiac expression of ß-klotho and significantly attenuated diabetes-induced cardiac dysfunction in wild-type mice, accompanied by reduced mitochondrial damage and increased activities of mitochondrial enzymes in hearts. However, such cardioprotective benefits of exercise were largely abrogated in mice with global or hepatocyte-selective ablation of FGF21, or cardiomyocyte-specific deletion of ß-klotho. Mechanistically, exercise enhanced the cardiac actions of FGF21 to induce the expression of the mitochondrial deacetylase SIRT3 by AMPK-evoked phosphorylation of FOXO3, thereby reversing diabetes-induced hyperacetylation and functional impairments of a cluster of mitochondrial enzymes. FGF21 prevented toxic lipids-induced mitochondrial dysfunction and oxidative stress by induction of the AMPK/FOXO3/SIRT3 signaling axis in human induced pluripotent stem cell-derived cardiomyocytes. Adeno-associated virus-mediated restoration of cardiac SIRT3 expression was sufficient to restore the responsiveness of diabetic FGF21 knockout mice to exercise in amelioration of mitochondrial dysfunction and DCM. CONCLUSIONS: The FGF21-SIRT3 axis mediates the protective effects of exercise against DCM by preserving mitochondrial integrity and represents a potential therapeutic target for DCM. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03240978.

Copper fabric improves the metabolic profile of obese mice: Potential role of the gut microbiota.

Copper and copper alloys have antimicrobial activity through the rapid contact killing of viruses, bacteria and yeasts on their surface. Dysregulation of host microbiota can contribute to the pathogenesis of inflammatory diseases such as obesity, diabetes and cardiovascular disease. Anecdotal evidence noted improved overall well-being in individuals sleeping on copper-containing fabric bedding. We hypothesized that the beneficial effect of copper-infused fabric bedding on cardiometabolic health is linked to changes in gut microbiota composition. This study utilized a mouse model of diet-induced obesity to assess the beneficial effects of copper-infused fabric bedding on metabolic health. Body composition, inflammatory markers, metabolic and cardiovascular status and changes in the faecal microbiota composition were evaluated for up to 2 months in mice fed with a normal chow diet or high fat high cholesterol diet in the presence of bedding made with and without copper-infused fabric. Results showed that mice subjected to diet-induced obesity and housed in cages with copper-infused fabric liner displayed less body weight gain than mice in cages with control fabric. Mice housed with copper-infused fabric also displayed improved glucose tolerance and reduced inflammation biomarker lipocalin-2. We also observed a beneficial shift in gut bacterial composition of obese mice housed with copper fabric bedding. Taken in conjunction, our study provides direct animal-based evidence supporting the beneficial effects of copper fabric on metabolic health.

ALY688 elicits adiponectin-mimetic signaling and improves insulin action in skeletal muscle cells.

Adiponectin is well established to mediate many beneficial metabolic effects, and this has stimulated great interest in development and validation of adiponectin receptor agonists as pharmaceutical tools. This study investigated the effects of ALY688, a peptide-based adiponectin receptor agonist, in rat L6 skeletal muscle cells. ALY688 significantly increased phosphorylation of several adiponectin downstream effectors, including AMPK, ACC, and p38MAPK, assessed by immunoblotting and immunofluorescence microscopy. Temporal analysis using cells expressing an Akt biosensor demonstrated that ALY688 enhanced insulin sensitivity. This effect was associated with increased insulin-stimulated Akt and IRS-1 phosphorylation. The functional metabolic significance of these signaling effects was examined by measuring glucose uptake in myoblasts stably overexpressing the glucose transporter GLUT4. ALY688 treatment increased basal glucose uptake and enhanced insulin-stimulated glucose uptake. In the model of high-glucose/high-insulin (HGHI)-induced insulin-resistant cells, both temporal studies using the Akt biosensor as well as immunoblotting to assess Akt and IRS-1 phosphorylation indicated that ALY688 significantly reduced insulin resistance. Importantly, we observed that ALY688 administration to high-fat high-sucrose-fed mice also improves glucose handling, validating its efficacy in vivo. In summary, these data indicate that ALY688 activates adiponectin signaling pathways in skeletal muscle, leading to improved insulin sensitivity and beneficial metabolic effects.

Role of Mitochondrial Iron Overload in Mediating Cell Death in H9c2 Cells.

Iron overload (IO) is associated with cardiovascular diseases, including heart failure. Our study's aim was to examine the mechanism by which IO triggers cell death in H9c2 cells. IO caused accumulation of intracellular and mitochondrial iron as shown by the use of iron-binding fluorescent reporters, FerroOrange and MitoFerroFluor. Expression of cytosolic and mitochondrial isoforms of Ferritin was also induced by IO. IO-induced iron accumulation and cellular ROS was rapid and temporally linked. ROS accumulation was detected in the cytosol and mitochondrial compartments with CellROX, DCF-DA and MitoSOX fluorescent dyes and partly reversed by the general antioxidant N-acetyl cysteine or the mitochondrial antioxidant SkQ1. Antioxidants also reduced the downstream activation of apoptosis and lytic cell death quantified by Caspase 3 cleavage/activation, mitochondrial Cytochrome c release, Annexin V/Propidium iodide staining and LDH release of IO-treated cells. Finally, overexpression of MitoNEET, an outer mitochondrial membrane protein involved in the transfer of Fe-S clusters between mitochondrial and cytosol, was observed to lower iron and ROS accumulation in the mitochondria. These alterations were correlated with reduced IO-induced cell death by apoptosis in MitoNEET-overexpressing cells. In conclusion, IO mediates H9c2 cell death by causing mitochondrial iron accumulation and subsequent general and mitochondrial ROS upregulation.