Unravelling the Carbohydrate-Binding Preferences of the Carbohydrate-Binding Modules of AMP-Activated Protein Kinase.

The ß subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which exists as two isoforms (ß1 and ß2) in humans, has a carbohydrate-binding module (CBM) that interacts with glycogen. Although the ß1- and ß2-CBMs are structurally similar, with strictly conserved ligand-contact residues, they show different carbohydrate affinities. ß2-CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into ß2-CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why ß2-CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for ß2 and Thr for ß1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant ß2-Thr101Δ/Val134Thr differs from that of ß1-CBM, and mutant ß1-Thr101ins/Thr134Val differs from that of ß2-CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild-type ß-CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant ß1-CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of ß2-CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.

When phosphorylated at Thr148, the ß2-subunit of AMP-activated kinase does not associate with glycogen in skeletal muscle.

The 5'-AMP-activated protein kinase (AMPK), a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, is activated in skeletal muscle in response to exercise and utilization of stored energy. The diffusibility properties of α- and ß-AMPK were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus and oxidative soleus muscles from which the surface membranes were removed by mechanical dissection. After the muscle segments were washed for 1 and 10 min, ∼60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. After in vitro stimulation of the muscle, which resulted in an ∼80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, as determined by addition of a wash step containing amylase. Stimulation of extensor digitorum longus muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target acetyl carboxylase-CoA. This, however, had no effect on the proportion of total ß2-AMPK that was phosphorylated in whole muscle homogenates measured by immunoprecipitation. These findings suggest that, in rat skeletal muscle, ß2-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate ß2-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of ß2-AMPK in skeletal muscle.

The recruitment of AMP-activated protein kinase to glycogen is regulated by autophosphorylation.

The mammalian AMP-activated protein kinase (AMPK) is an obligatory αßγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the ß-subunit (AMPKß) 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 ß1-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 AMPKß 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.

SnRK1 from Arabidopsis thaliana is an atypical AMPK.

SNF1-related protein kinase 1 (SnRK1) is the plant orthologue of the evolutionarily-conserved SNF1/AMPK/SnRK1 protein kinase family that contributes to cellular energy homeostasis. Functional as heterotrimers, family members comprise a catalytic α subunit and non-catalytic ß and γ subunits; multiple isoforms of each subunit type exist, giving rise to various isoenzymes. The Arabidopsis thaliana genome contains homologues of each subunit type, and, in addition, two atypical subunits, ß(3) and ßγ, with unique domain architecture, that are found only amongst plants, suggesting atypical heterotrimers. The AtSnRK1 subunit structure was determined using recombinant protein expression and endogenous co-immunoprecipitation, and six unique isoenzyme combinations were identified. Each heterotrimeric isoenzyme comprises a catalytic α subunit together with the unique ßγ subunit and one of three non-catalytic ß subunits: ß(1), ß(2) or the plant-specific ß(3) isoform. Thus, the AtSnRK1 heterotrimers contain the atypical ßγ subunit rather than a conventional γ subunit. Mammalian AMPK heterotrimers are phosphorylated on the T-loop (pThr175/176) within both catalytic a subunits. However, AtSnRK1 is insensitive to AMP and ADP, and is resistant to T-loop dephosphorylation by protein phosphatases, a process that inactivates other SNF1/AMPK family members. In addition, we show that SnRK1 is inhibited by a heat-labile, >30 kDa, soluble proteinaceous factor that is present in the lysate of young rosette leaves. Finally, none of the three SnRK1 carbohydrate-binding modules, located in the ß(1), ß(2) and ßγ subunits, associate with various carbohydrates, including starch, the plant analogue of glycogen to which AMPK binds in vitro. These data clearly demonstrate that AtSnRK1 is an atypical member of the SNF1/AMPK/SnRK1 family.

Determinants of oligosaccharide specificity of the carbohydrate-binding modules of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an αßγ heterotrimer that is important in regulating energy metabolism in all eukaryotes. The ß-subunit exists in two isoforms (ß1 and ß2) and contains a carbohydrate-binding module (CBM) that interacts with glycogen. The two CBM isoforms (ß1- and ß2-CBM) are near identical in sequence and structure, yet show differences in carbohydrate-binding affinity. ß2-CBM binds linear carbohydrates with 4-fold greater affinity than ß1-CBM and binds single α1,6-branched carbohydrates up to 30-fold tighter. To understand these affinity differences, especially for branched carbohydrates, we determined the NMR solution structure of ß2-CBM in complex with the single α1,6-branched carbohydrate glucosyl-ß-cyclodextrin (gBCD) which supported the dynamic nature of the binding site, but resonance broadening prevented defining where the α1,6 branch bound. We therefore solved the X-ray crystal structures of ß1- and ß2-CBM, in complex with gBCD, to 1.7 and 2.0 Å (1 Å=0.1 nm) respectively. The additional threonine (Thr101) of ß2-CBM expands the size of the surrounding loop, creating a pocket that accommodates the α1,6 branch. Hydrogen bonds are formed between the α1,6 branch and the backbone of Trp99 and Lys102 side chain of ß2-CBM. In contrast, the α1,6 branch could not be observed in the ß1-CBM structure, suggesting that it does not form a specific interaction. The orientation of gBCD bound to ß1- and ß2-CBM is supported by thermodynamic and kinetic data obtained through isothermal titration calorimetry (ITC) and NMR. These results suggest that AMPK containing the muscle-specific ß2-isoform may have greater affinity for partially degraded glycogen.

Myocardial glycogen dynamics: new perspectives on disease mechanisms.

Cardiac glycogen regulation involves a complex interplay between multiple signalling pathways, allosteric activation of enzymes, and sequestration for autophagic degradation. Signalling pathways appear to converge on glycogen regulatory enzymes via insulin (glycogen synthase kinase 3ß, protein phosphatase 1, allosteric action of glucose-6-phosphate), ß-adrenergic (phosphorylase kinase protein phosphatase 1 inhibitor), and 5' adenosine monophosphate-activated protein kinase (allosteric action of glucose-6-phosphate, direct glycogen binding, insulin receptor). While cytosolic glycogen synthesis and breakdown are relatively well understood, recent findings relating to phagic glycogen degradation highlight a new area of investigation in the heart. It has been recently demonstrated that a specific glycophagy pathway is operational in the myocardium. Proteins involved in recruiting glycogen to the forming phagosome have been identified. Starch-binding domain-containing protein 1 is involved in binding glycogen and mediating membrane anchorage via interaction with a homologue of the phagosomal protein light-chain 3. Specifically, it has been shown that starch-binding domain-containing protein 1 and light-chain 3 have discrete phagosomal immunolocalization patterns in cardiomyocytes, indicating that autophagic trafficking of glycogen and protein cargo in cardiomyocytes can occur via distinct pathways. There is strong evidence from glycogen storage diseases that phagic/lysosomal glycogen breakdown is important for maintaining normal cardiac glycogen levels and does not simply constitute a redundant 'alternative' breakdown route for glycogen. Advancing understanding of glycogen handling in the heart is an important priority with relevance not only to genetic glycogen storage diseases but also to cardiac metabolic stress disorders such as diabetes and ischaemia.

Cardiomyocyte glycophagy is regulated by insulin and exposure to high extracellular glucose.

Disturbed systemic glycemic and insulinemic status elicits cardiomyocyte metabolic stress and altered glucose handling. In diabetes, pathological myocardial glycogen accumulation occurs. Recently, evidence of a specific myocardial autophagic degradation pathway for glycogen ("glycophagy") has been reported, differentiated from the more well-characterized protein "macrophagy" pathway. The goal of this study was to identify potential mechanisms involved in cardiac glycogen accumulation, glycophagy, and macrophagy regulation using cultured neonatal rat ventricular myocytes (NRVMs). In NRVMs, insulin-induced Akt phosphorylation was evident with 5 mM-glucose conditions (∼2.3-fold increased). Under high-glucose (30 mM) conditions, insulin-augmented phosphorylation was not observed. Accumulation of glycogen was observed in response to insulin only in high-glucose conditions (∼2-fold increase). Increased expression of the glycophagy marker starch-binding domain-containing protein-1 (STBD1, 25% increase) was observed under high-glucose and insulin conditions. Expression levels of the macrophagy markers p62 and light chain protein 3BII:I were not increased by insulin at either glucose level. Preliminary results from hearts of streptozotocin-treated diabetic rats are supportive of the findings obtained in NRVMs, suggesting diabetes induced elevated expression of STBD1 and of an additional glycophagy marker GABA(A) receptor-associated protein-like 1. Confocal microscopy demonstrated that light chain protein 3B and STBD1 immunomarkers were not colocalized in NRVMs. These findings provide the first evidence that cardiomyocyte glycophagy induction occurs under the influence of insulin and is responsive to extracellular high glucose. This study suggests that the regulation of glycogen content and glycophagy induction in the cardiomyocyte may be linked, and it is speculated that glycogen pathology in diabetic cardiomyopathy has glycophagic involvement.

Changes in glycogen structure over feeding cycle sheds new light on blood-glucose control.

Liver glycogen, a highly branched polymer of glucose, is important for maintaining blood-glucose homeostasis. It was recently shown that db/db mice, a model for Type 2 diabetes, are unable to form the large composite glycogen α particles present in normal, healthy mice. In this study, the structure of healthy mouse-liver glycogen over the diurnal cycle was characterized using size exclusion chromatography and transmission electron microscopy. Glycogen was found to be formed as smaller ß particles, and then only assembled into large α particles, with a broad size distribution, significantly after the time when glycogen content had reached a maximum. This pathway, missing in diabetic animals, is likely to give optimal blood-glucose control during the daily feeding cycle. Lack of this control may contribute to, or result from, diabetes. This discovery suggests novel approaches to diabetes management.

Single fiber analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle.

To understand how glycogen affects skeletal muscle physiology, we examined enzymes essential for muscle glycogen synthesis and degradation using single fibers from quiescent and stimulated rat skeletal muscle. Presenting a shift in paradigm, we show these proteins are differentially associated with glycogen granules. Protein diffusibility and/or abundance of glycogenin, glycogen branching enzyme (GBE), debranching enzyme (GDE), phosphorylase (GP), and synthase (GS) were examined in fibers isolated from rat fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscle. GDE and GP proteins were more abundant (~10- to 100-fold) in fibers from EDL compared with SOL muscle. GS and glycogenin proteins were similar between muscles while GBE had an approximately fourfold greater abundance in SOL muscle. Mechanically skinned fibers exposed to physiological buffer for 10 min showed ~70% total pools of GBE and GP were diffusible (nonbound), whereas GDE and GS were considerably less diffusible. Intense in vitro stimulation, sufficient to elicit a ~50% decrease in intracellular glycogen, increased diffusibility of GDE, GP, and GS (~15-60%) and decreased GBE diffusibility (~20%). Amylase treatment, which breaks α-1,4 linkages of glycogen, indicated differential diffusibilities and hence glycogen associations of GDE and GS. Membrane solubilization (1% Triton-X-100) allowed a small additional amount of GDE and GS to diffuse from fibers, suggesting the majority of nonglycogen-associated GDE/GS is associated with myofibrillar/contractile network of muscle rather than membranes. Given differences in enzymes required for glycogen metabolism, the current findings suggest glycogen particles have fiber-type-dependent structures. The greater catabolic potential of glycogen breakdown in fast-twitch fibers may account for different contraction induced rates of glycogen utilization.

Molecular insights into glycogen α-particle formation.

Glycogen, a hyperbranched complex glucose polymer, is an intracellular glucose store that provides energy for cellular functions, with liver glycogen involved in blood-glucose regulation. Liver glycogen comprises complex α particles made up of smaller ß particles. The recent discovery that these α particles are smaller and fewer in diabetic, compared with healthy, mice highlights the need to elucidate the nature of α-particle formation; this paper tests various possibilities for binding within α particles. Acid hydrolysis effects, examined using dynamic light scattering and size exclusion chromatography, showed that the binding is not simple α-(1→4) or α-(1→6) glycosidic linkages. There was no significant change in α particle size after the addition of various reagents, which disrupt disulfide, protein, and hydrogen bonds and hydrophobic interactions. The results are consistent with proteinaceous binding between ß particles in α particles, with the inability of protease to break apart particles being attributed to steric hindrance.

Increased d-lactic Acid intestinal bacteria in patients with chronic fatigue syndrome.

Patients with chronic fatigue syndrome (CFS) are affected by symptoms of cognitive dysfunction and neurological impairment, the cause of which has yet to be elucidated. However, these symptoms are strikingly similar to those of patients presented with D-lactic acidosis. A significant increase of Gram positive facultative anaerobic faecal microorganisms in 108 CFS patients as compared to 177 control subjects (p<0.01) is presented in this report. The viable count of D-lactic acid producing Enterococcus and Streptococcus spp. in the faecal samples from the CFS group (3.5 x 10(7) cfu/L and 9.8 x 10(7) cfu/L respectively) were significantly higher than those for the control group (5.0 x 10(6) cfu/L and 8.9 x 10(4) cfu/L respectively). Analysis of exometabolic profiles of Enterococcus faecalis and Streptococcus sanguinis, representatives of Enterococcus and Streptococcus spp. respectively, by NMR and HPLC showed that these organisms produced significantly more lactic acid (p<0.01) from (13)C-labeled glucose, than the Gram negative Escherichia coli. Further, both E. faecalis and S. sanguinis secrete more D-lactic acid than E. coli. This study suggests a probable link between intestinal colonization of Gram positive facultative anaerobic D-lactic acid bacteria and symptom expressions in a subgroup of patients with CFS. Given the fact that this might explain not only neurocognitive dysfunction in CFS patients but also mitochondrial dysfunction, these findings may have important clinical implications.

Mas Receptor Activation Slows Tumor Growth and Attenuates Muscle Wasting in Cancer.

Cancer cachexia is a multifactorial syndrome characterized by a progressive loss of skeletal muscle mass associated with significant functional impairment. Cachexia robs patients of their strength and capacity to perform daily tasks and live independently. Effective treatments are needed urgently. Here, we investigated the therapeutic potential of activating the "alternative" axis of the renin-angiotensin system, involving ACE2, angiotensin-(1-7), and the mitochondrial assembly receptor (MasR), for treating cancer cachexia. Plasmid overexpression of the MasR or pharmacologic angiotensin-(1-7)/MasR activation did not affect healthy muscle fiber size in vitro or in vivo but attenuated atrophy induced by coculture with cancer cells in vitro. In mice with cancer cachexia, the MasR agonist AVE 0991 slowed tumor development, reduced weight loss, improved locomotor activity, and attenuated muscle wasting, with the majority of these effects dependent on the orexigenic and not antitumor properties of AVE 0991. Proteomic profiling and IHC revealed that mechanisms underlying AVE 0991 effects on skeletal muscle involved miR-23a-regulated preservation of the fast, glycolytic fibers. MasR activation is a novel regulator of muscle phenotype, and AVE 0991 has orexigenic, anticachectic, and antitumorigenic effects, identifying it as a promising adjunct therapy for cancer and other serious muscle wasting conditions. SIGNIFICANCE: These findings demonstrate that MasR activation has multiple benefits of being orexigenic, anticachectic, and antitumorigenic, revealing it as a potential adjunct therapy for cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/4/706/F1.large.jpg.See related commentary by Rupert et al., p. 699.

Integral membrane proteins in the mitochondrial outer membrane of Saccharomyces cerevisiae.

Mitochondria evolved from a bacterial endosymbiont ancestor in which the integral outer membrane proteins would have been beta-barrel structured within the plane of the membrane. Initial proteomics on the outer membrane from yeast mitochondria suggest that while most of the protein components are integral in the membrane, most of these mitochondrial proteins behave as if they have alpha-helical transmembrane domains, rather than beta-barrels. These proteins are usually predicted to have a single alpha-helical transmembrane segment at either the N- or C-terminus, however, more complex topologies are also seen. We purified the novel outer membrane protein Om14 and show it is encoded in the gene YBR230c. Protein sequencing revealed an intron is spliced from the transcript, and both transcription from the YBR230c gene and steady-state level of the Om14 protein is dramatically less in cells grown on glucose than in cells grown on nonfermentable carbon sources. Hydropathy predictions together with data from limited protease digestion show three alpha-helical transmembrane segments in Om14. The alpha-helical outer membrane proteins provide functions derived after the endosymbiotic event, and require the translocase in the outer mitochondrial membrane complex for insertion into the outer membrane.

Molecular Insights into the Enigmatic Metabolic Regulator, SnRK1.

Sucrose non-fermenting-1 (SNF1)-related kinase 1 (SnRK1) lies at the heart of metabolic homeostasis in plants and is crucial for normal development and response to stress. Evolutionarily related to SNF1 in yeast and AMP-activated kinase (AMPK) in mammals, SnRK1 acts protectively to maintain homeostasis in the face of fluctuations in energy status. Despite a conserved function, the structure and regulation of the plant kinase differ considerably from its relatively well-understood opisthokont orthologues. In this review, we highlight the known plant-specific modes of regulation involving SnRK1 together with new insights based on a 3D molecular model of the kinase. We also summarise how these differences from other orthologues may be specific adaptations to plant metabolism, and offer insights into possible avenues of future inquiry into this enigmatic enzyme.

ß-subunit myristoylation functions as an energy sensor by modulating the dynamics of AMP-activated Protein Kinase.

The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, ß and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the ß-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg2+ results in a global stabilization of myristoylated, but not non-myristoylated AMPK, and most notably for peptides of the activation loop of the α-kinase domain, the autoinhibitory sequence (AIS) and the ßCBM. AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high.

Muscle-specific deletion of SOCS3 increases the early inflammatory response but does not affect regeneration after myotoxic injury.

BACKGROUND: Muscles of old animals are injured more easily and regenerate poorly, attributed in part to increased levels of circulating pro-inflammatory cytokines. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade is a key mediator of inflammatory cytokine action, and signaling via this pathway is increased in muscles with aging. As a negative regulator of JAK/STAT signaling, a key mediator of myogenic proliferation and differentiation, altered expression of suppressor of cytokine signaling (SOCS3) is likely to have important consequences for muscle regeneration. To model this scenario, we investigated the effect of SOCS3 deletion within mature muscle fibers on injury and repair. We tested the hypothesis that reduced SOCS3 function would alter the inflammatory response and impair muscle regeneration after myotoxic injury. METHODS: Mice with a specific deletion of SOCS3 within mature skeletal muscle fibers were used to assess the effect of SOCS3 deletion on muscle injury and repair. Twelve-week-old or 24-month-old SOCS3 muscle-specific knockout (SOCS3 MKO) mice and littermate controls were either left uninjured or injured with a single injection of notexin (10 µg/ml) into the right tibialis anterior (TA) muscle. At 1, 2, 3, 5, 7, or 14 days post-injury, the right TA muscle was excised and subjected to histological, western immunoblotting, and gene expression analyses. Force production and fatigue were assessed in uninjured muscles and at 7 days post-notexin injury. RESULTS: In uninjured muscles, SOCS3 deletion decreased force production during fatigue but had no effect on the gross or histological appearance of the TA muscles. After notexin injury, deletion of SOCS3 increased STAT3 phosphorylation at day 1 and increased the mRNA expression of the inflammatory cytokine TNF-α, and the inflammatory cell markers F4/80 and CD68 at day 2. Gene expression analysis of the regeneration markers Pax7, MyoD, and Myogenin indicated SOCS3 deletion had no effect on the progression of muscle repair after notexin injury. Inflammation and regeneration were also unchanged in the muscles of 24-month-old SOCS3 MKO mice compared with control. CONCLUSIONS: Loss of SOCS3 expression in mature muscle fibers increased the inflammatory response to myotoxic injury but did not impair muscle regeneration in either adult or old mice. Therefore, reduced SOCS3 expression in muscle fibers is unlikely to underlie impaired muscle regeneration. Further investigation into the role of SOCS3 in other cell types involved in muscle repair is warranted.

FT011, a Novel Cardiorenal Protective Drug, Reduces Inflammation, Gliosis and Vascular Injury in Rats with Diabetic Retinopathy.

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.

Regulation of Starch Stores by a Ca(2+)-Dependent Protein Kinase Is Essential for Viable Cyst Development in Toxoplasma gondii.

Transmissible stages of Toxoplasma gondii store energy in the form of the carbohydrate amylopectin. Here, we show that the Ca(2+)-dependent protein kinase CDPK2 is a critical regulator of amylopectin metabolism. Increased synthesis and loss of degradation of amylopectin in CDPK2 deficient parasites results in the hyperaccumulation of this sugar polymer. A carbohydrate-binding module 20 (CBM20) targets CDPK2 to amylopectin stores, while the EF-hands regulate CDPK2 kinase activity in response to Ca(2+) to modulate amylopectin levels. We identify enzymes involved in amylopectin turnover whose phosphorylation is dependent on CDPK2 activity. Strikingly, accumulation of massive amylopectin granules in CDPK2-deficient bradyzoite stages leads to gross morphological defects and complete ablation of cyst formation in a mouse model. Together these data show that Ca(2+) signaling regulates carbohydrate metabolism in Toxoplasma and that the post-translational control of this pathway is required for normal cyst development.

Dysfunctional muscle and liver glycogen metabolism in mdx dystrophic mice.

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.

Tranilast administration reduces fibrosis and improves fatigue resistance in muscles of mdx dystrophic mice.

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.