Physiology - Research Publications
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ItemNo Preview AvailableMolecular Structural Differences between Type-2-Diabetic and Healthy Glycogen(AMER CHEMICAL SOC, 2011-06)Glycogen is a highly branched glucose polymer functioning as a glucose buffer in animals. Multiple-detector size exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis were used to examine the structure of undegraded native liver glycogen (both whole and enzymatically debranched) as a function of molecular size, isolated from the livers of healthy and db/db mice (the latter a type 2 diabetic model). Both the fully branched and debranched levels of glycogen structure showed fundamental differences between glycogen from healthy and db/db mice. Healthy glycogen had a greater population of large particles, with more α particles (tightly linked assemblages of smaller β particles) than glycogen from db/db mice. These structural differences suggest a new understanding of type 2 diabetes.
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ItemA Purpose-Synthesised Anti-Fibrotic Agent Attenuates Experimental Kidney Diseases in the Rat(PUBLIC LIBRARY SCIENCE, 2012-10-10)BACKGROUND AND PURPOSE: Locally-active growth factors have been implicated in the pathogenesis of many diseases in which organ fibrosis is a characteristic feature. In the setting of chronic kidney disease (CKD), two such pro-fibrotic factors, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) have emerged as lead potential targets for intervention. Given the incomplete organ protection afforded by blocking the actions of TGF-β or PDGF individually, we sought to determine whether an agent that inhibited the actions of both may have broader effects in ameliorating the key structural and functional abnormalities of CKD. EXPERIMENTAL APPROACH: Accordingly, we studied the effects of a recently described, small molecule anti-fibrotic drug, 3-methoxy-4-propargyloxycinnamoyl anthranilate (FT011, Fibrotech Therapeutics, Australia), which should have these effects. KEY RESULTS: In the in vitro setting, FT011 inhibited both TGF-β1 and PDGF-BB induced collagen production as well as PDGF-BB-mediated mesangial proliferation. Consistent with these in vitro actions, when studied in a robust model of non-diabetic kidney disease, the 5/6 nephrectomised rat, FT011 attenuated the decline in GFR, proteinuria and glomerulosclerosis (p<0.05 for all). Similarly, in the streptozotocin-diabetic Ren-2 rat, a model of advanced diabetic nephropathy, FT011 reduced albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. CONCLUSIONS AND IMPLICATIONS: Together these studies suggest that broadly antagonising growth factor actions, including those of TGF-β1 and PDGF-BB, has the potential to protect the kidney from progressive injury in both the diabetic and non-diabetic settings.
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ItemThe Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2015-05-01)The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.
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ItemMolecular Basis of Impaired Glycogen Metabolism during Ischemic Stroke and Hypoxia(PUBLIC LIBRARY SCIENCE, 2014-05-23)BACKGROUND: Ischemic stroke is the combinatorial effect of many pathological processes including the loss of energy supplies, excessive intracellular calcium accumulation, oxidative stress, and inflammatory responses. The brain's ability to maintain energy demand through this process involves metabolism of glycogen, which is critical for release of stored glucose. However, regulation of glycogen metabolism in ischemic stroke remains unknown. In the present study, we investigate the role and regulation of glycogen metabolizing enzymes and their effects on the fate of glycogen during ischemic stroke. RESULTS: Ischemic stroke was induced in rats by peri-vascular application of the vasoconstrictor endothelin-1 and forebrains were collected at 1, 3, 6 and 24 hours post-stroke. Glycogen levels and the expression and activity of enzymes involved in glycogen metabolism were analyzed. We found elevated glycogen levels in the ipsilateral hemispheres compared with contralateral hemispheres at 6 and 24 hours (25% and 39% increase respectively; P<0.05). Glycogen synthase activity and glycogen branching enzyme expression were found to be similar between the ipsilateral, contralateral, and sham control hemispheres. In contrast, the rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 58% lower activity (P<0.01) in the ipsilateral hemisphere (24 hours post-stroke), which corresponded with a 48% reduction in cAMP-dependent protein kinase A (PKA) activity (P<0.01). In addition, glycogen debranching enzyme expression 24 hours post-stroke was 77% (P<0.01) and 72% lower (P<0.01) at the protein and mRNA level, respectively. In cultured rat primary cerebellar astrocytes, hypoxia and inhibition of PKA activity significantly reduced glycogen phosphorylase activity and increased glycogen accumulation but did not alter glycogen synthase activity. Furthermore, elevated glycogen levels provided metabolic support to astrocytes during hypoxia. CONCLUSION: Our study has identified that glycogen breakdown is impaired during ischemic stroke, the molecular basis of which includes reduced glycogen debranching enzyme expression level together with reduced glycogen phosphorylase and PKA activity.
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ItemFT011, a Novel Cardiorenal Protective Drug, Reduces Inflammation, Gliosis and Vascular Injury in Rats with Diabetic Retinopathy(PUBLIC LIBRARY SCIENCE, 2015-07-29)Diabetic retinopathy features inflammation as well as injury to glial cells and the microvasculature, which are influenced by hypertension and overactivity of the renin-angiotensin system. FT011 is an anti-inflammatory and anti-fibrotic agent that has been reported to attenuate organ damage in diabetic rats with cardiomyopathy and nephropathy. However, the potential therapeutic utility of FT011 for diabetic retinopathy has not been evaluated. We hypothesized that FT011 would attenuate retinopathy in diabetic Ren-2 rats, which exhibit hypertension due to an overactive extra-renal renin-angiotensin system. Diabetic rats were studied for 8 and 32 weeks and received intravitreal injections of FT011 (50 μM) or vehicle (0.9% NaCl). Comparisons were to age-matched controls. In the 8-week study, retinal inflammation was examined by quantitating vascular leukocyte adherence, microglial/macrophage density and the expression of inflammatory mediators. Macroglial Müller cells, which exhibit a pro-inflammatory and pro-angiogenic phenotype in diabetes, were evaluated in the 8-week study as well as in culture following exposure to hyperglycaemia and FT011 (10, 30, 100 μM) for 72 hours. In the 32-week study, severe retinal vasculopathy was examined by quantitating acellular capillaries and extracellular matrix proteins. In diabetic rats, FT011 reduced retinal leukostasis, microglial density and mRNA levels of intercellular adhesion molecule-1 (ICAM-1). In Müller cells, FT011 reduced diabetes-induced gliosis and vascular endothelial growth factor (VEGF) immunolabeling and the hyperglycaemic-induced increase in ICAM-1, monocyte chemoattractant protein-1, CCL20, cytokine-induced neutrophil chemoattractant-1, VEGF and IL-6. Late intervention with FT011 reduced acellular capillaries and the elevated mRNA levels of collagen IV and fibronectin in diabetic rats. In conclusion, the protective effects of FT011 in cardiorenal disease extend to key elements of diabetic retinopathy and highlight its potential as a treatment approach.
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ItemTranilast administration reduces fibrosis and improves fatigue resistance in muscles of mdx dystrophic mice(BMC, 2014)BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe and progressive muscle-wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilising protein dystrophin. Dystrophic muscle fibres are susceptible to injury and degeneration, and impaired muscle regeneration is associated with fibrotic deposition that limits the efficacy of potential pharmacological, cell- and gene-based therapies. Novel treatments that can prevent or attenuate fibrosis have important clinical merit for DMD and related neuromuscular diseases. We investigated the therapeutic potential for tranilast, an orally bioavailable anti-allergic agent, to prevent fibrosis in skeletal muscles of mdx dystrophic mice. RESULTS: Three-week-old C57Bl/10 and mdx mice received tranilast (~300 mg/kg) in their food for 9 weeks, after which fibrosis was assessed through histological analyses, and functional properties of tibialis anterior muscles were assessed in situ and diaphragm muscle strips in vitro. Tranilast administration did not significantly alter the mass of any muscles in control or mdx mice, but it decreased fibrosis in the severely affected diaphragm muscle by 31% compared with untreated mdx mice (P < 0.05). A similar trend of decreased fibrosis was observed in the tibialis anterior muscles of mdx mice (P = 0.10). These reductions in fibrotic deposition were not associated with improvements in maximum force-producing capacity, but we did observe small but significant improvements in the resistance to fatigue in both the diaphragm and TA muscles of mdx mice treated with tranilast. CONCLUSION: Together these findings demonstrate that administration of potent antifibrotic compounds such as tranilast could help preserve skeletal muscle structure, which could ultimately increase the efficacy of pharmacological, cell and gene replacement/correction therapies for muscular dystrophy and related disorders.
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ItemDysfunctional Muscle and Liver Glycogen Metabolism in mdx Dystrophic Mice(PUBLIC LIBRARY SCIENCE, 2014-03-13)BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe, genetic muscle wasting disorder characterised by progressive muscle weakness. DMD is caused by mutations in the dystrophin (dmd) gene resulting in very low levels or a complete absence of the dystrophin protein, a key structural element of muscle fibres which is responsible for the proper transmission of force. In the absence of dystrophin, muscle fibres become damaged easily during contraction resulting in their degeneration. DMD patients and mdx mice (an animal model of DMD) exhibit altered metabolic disturbances that cannot be attributed to the loss of dystrophin directly. We tested the hypothesis that glycogen metabolism is defective in mdx dystrophic mice. RESULTS: Dystrophic mdx mice had increased skeletal muscle glycogen (79%, (P<0.01)). Skeletal muscle glycogen synthesis is initiated by glycogenin, the expression of which was increased by 50% in mdx mice (P<0.0001). Glycogen synthase activity was 12% higher (P<0.05) but glycogen branching enzyme activity was 70% lower (P<0.01) in mdx compared with wild-type mice. The rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 62% lower activity (P<0.01) in mdx mice resulting from a 24% reduction in PKA activity (P<0.01). In mdx mice glycogen debranching enzyme expression was 50% higher (P<0.001) together with starch-binding domain protein 1 (219% higher; P<0.01). In addition, mdx mice were glucose intolerant (P<0.01) and had 30% less liver glycogen (P<0.05) compared with control mice. Subsequent analysis of the enzymes dysregulated in skeletal muscle glycogen metabolism in mdx mice identified reduced glycogenin protein expression (46% less; P<0.05) as a possible cause of this phenotype. CONCLUSION: We identified that mdx mice were glucose intolerant, and had increased skeletal muscle glycogen but reduced amounts of liver glycogen.
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ItemAttenuation of Armanni-Ebstein lesions in a rat model of diabetes by a new anti-fibrotic, anti-inflammatory agent, FT011(SPRINGER, 2013-03)AIMS/HYPOTHESIS: A key morphological feature of diabetic nephropathy is the accumulation and deposition of glycogen in renal tubular cells, known as Armanni-Ebstein lesions. While this observation has been consistently reported for many years, the molecular basis of these lesions remains unclear. METHODS: Using biochemical and histochemical methods, we measured glycogen concentration, glycogen synthase and glycogen phosphorylase enzyme activities, and mRNA expression and protein levels of glycogenin in kidney lysates from control and transgenic (mRen-2)27 rat models of diabetes that had been treated with and without a new anti-fibrotic agent, FT011. RESULTS: Diabetic nephropathy was associated with increased glycogen content, increased glycogen synthase activity and decreased glycogen phosphorylase activity. Glycogenin, the key protein responsible for initiating the synthesis of each glycogen particle, had very high levels in the diabetic kidney together with increased mRNA expression compared with control kidneys. Treatment with FT011 did not change glycogen synthase or glycogen phosphorylase enzyme activities but prevented both glycogenin mRNA synthesis and accumulation of Armanni-Ebstein lesions in the diabetic kidney. CONCLUSIONS/INTERPRETATION: Armanni-Ebstein lesions found in diabetic nephropathy are due to aberrant glycogenin protein levels and mRNA expression, providing an explanation for the increased glycogen concentration found within the diabetic kidney. FT011 treatment in diabetic rats reduced glycogenin levels and, subsequently, renal glycogen concentration.
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ItemNo Preview AvailableAMP-Activated Protein Kinase β-Subunit Requires Internal Motion for Optimal Carbohydrate Binding(CELL PRESS, 2012-01-18)AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β(1) and β(2)). Muscle-specific β(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β(2)-CBM is concurrent with an increase in flexibility in β(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to β(1)-CBM, unbound β(2)-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β(2)-CBM mutant. Insertion of a threonine into the background of β(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.
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ItemMyocardial glycophagy - A specific glycogen handling response to metabolic stress is accentuated in the female heart(ELSEVIER SCI LTD, 2013-12)Cardiac metabolic stress is a hallmark of many cardiac pathologies, including diabetes. Cardiac glycogen mis-handling is a frequent manifestation of various cardiopathologies. Diabetic females have a higher risk of heart disease than males, yet sex disparities in cardiac metabolic stress settings are not well understood. Oestrogen acts on key glycogen regulatory proteins. The goal of this study was to evaluate sex-specific metabolic stress-triggered cardiac glycogen handling responses. Male and female adult C57Bl/6J mice were fasted for 48h. Cardiac glycogen content, particle size, regulatory enzymes, signalling intermediates and autophagic processes were evaluated. Female hearts exhibited 51% lower basal glycogen content than males associated with lower AMP-activated-kinase (AMPK) activity (35% decrease in pAMPK:AMPK). With fasting, glycogen accumulated in female hearts linked with decreased particle size and upregulation of Akt and AMPK signalling, activation of glycogen synthase and inactivation of glycogen phosphorylase. Fasting did not alter glycogen content or regulatory proteins in male hearts. Expression of glycogen autophagy marker, starch-binding-protein-domain-1 (STBD1), was 63% lower in female hearts than males and increased by 69% with fasting in females only. Macro-autophagy markers, p62 and LC3BII:I ratio, increased with fasting in male and female hearts. This study identifies glycogen autophagy ('glycophagy') as a potentially important component of the response to cardiac metabolic stress. Glycogen autophagy occurs in association with a marked and selective accumulation of glycogen in the female myocardium. Our findings suggest that sex-specific differences in glycogen handling may have cardiopathologic consequences in various settings, including diabetic cardiomyopathy.