Integrative effects of dystrophin loss on metabolic function of the mdx mouse.

Duchenne muscular dystrophy (DMD) is a disease marked by the development of skeletal muscle weakness and wasting. DMD results from mutations in the gene for the cytoskeletal protein dystrophin. The loss of dystrophin expression is not limited to muscle weakness but has multiple systemic consequences. Managing the nutritional requirements is an important aspect of the clinical care of DMD patients and is complicated by the poor understanding of the role of dystrophin, and dystrophic processes, in regulating metabolism. Here, we show that mdx mice, a genetic model of DMD, have significantly reduced fat mass relative to wild type C57BL/10. The alteration in body composition is independent of the presence of skeletal muscle disease, as it is still present in mice with transgenic expression of a fully-functional dystrophin in skeletal muscle. Furthermore, mdx mice do not increase their fat mass or body weight when housed under thermoneutral conditions, in marked contrast to C57BL/10 mice. We also demonstrated that mdx mice have significantly reduced fat metabolism and altered glucose uptake. These significant metabolic changes in dystrophic mice implicate dystrophin as an important regulator of metabolism. Understanding the metabolic functions of dystrophin is important for managing the nutritional needs of DMD patients.

Hypoxia-induced cardiac injury in dystrophic mice.

Duchenne muscular dystrophy (DMD) is a disease of progressive destruction of striated muscle, resulting in muscle weakness with progressive respiratory and cardiac failure. Respiratory and cardiac disease are the leading causes of death in DMD patients. Previous studies have suggested an important link between cardiac dysfunction and hypoxia in the dystrophic heart; these studies aim to understand the mechanism underlying this connection. Here we demonstrate that anesthetized dystrophic mice display significant mortality following acute exposure to hypoxia. This increased mortality is associated with a significant metabolic acidosis, despite having significantly higher levels of arterial Po2 Chronic hypoxia does not result in mortality, but rather is characterized by marked cardiac fibrosis. Studies in isolated hearts reveal that the contractile function of dystrophic hearts is highly susceptible to short bouts of ischemia, but these hearts tolerate prolonged acidosis better than wild-type hearts, indicating an increased sensitivity of the dystrophic heart to hypoxia. Dystrophic hearts display decreased cardiac efficiency and oxygen extraction. Isolated dystrophic cardiomyocytes and hearts have normal levels of FCCP-induced oxygen consumption, and mitochondrial morphology and content are normal in the dystrophic heart. These studies demonstrate reductions in cardiac efficiency and oxygen extraction of the dystrophic heart. The underlying cause of this reduced oxygen extraction is not clear; however, the current studies suggest that large disruptions of mitochondrial respiratory function or coronary flow regulation are not responsible. This finding is significant, as hypoxia is a common and largely preventable component of DMD that may contribute to the progression of the cardiac disease in DMD patients.

Rational steering of insulin binding specificity by intra-chain chemical crosslinking.

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone's B-chain C-terminus for its IR-B specificity.

Dystrobrevin increases dystrophin's binding to the dystrophin-glycoprotein complex and provides protection during cardiac stress.

Duchenne muscular dystrophy is a fatal progressive disease of both cardiac and skeletal muscle resulting from the mutations in the DMD gene and loss of the protein dystrophin. Alpha-dystrobrevin (α-DB) tightly associates with dystrophin but the significance of this interaction within cardiac myocytes is poorly understood. In the current study, the functional role of α-DB in cardiomyocytes and its implications for dystrophin function are examined. Cardiac stress testing demonstrated significant heart disease in α-DB null (adbn(-/-)) mice, which displayed mortality and lesion sizes that were equivalent to those seen in dystrophin-deficient mdx mice. Despite normal expression and subcellular localization of dystrophin in the adbn(-/-) heart, there is a significant decrease in the strength of dystrophin's interaction with the membrane-bound dystrophin-associated glycoprotein complex (DGC). A similar weakening of the dystrophin-membrane interface was observed in mice lacking the sarcoglycan complex. Cardiomyocytes from adbn(-/-) mice were smaller and responded less to adrenergic receptor induced hypertrophy. The basal decrease in size could not be attributed to aberrant Akt activation. In addition, the organization of the microtubule network was significantly altered in adbn(-/-) cardiac myocytes, while the total expression of tubulin was unchanged in adbn(-/-) hearts. These studies demonstrate that α-DB is a multifunctional protein that increases dystrophin's binding to the dystrophin-glycoprotein complex, and is critical for the full functionality of dystrophin.

Concurrent effects of lexical status and letter-rotation during early stage visual word recognition: evidence from ERPs.

Recent studies report that the occipito-temporal N170 component of the ERP is enhanced by letter strings, relative to non-linguistic strings of similar visual complexity, with a left-lateralized distribution. This finding is consistent with underlying mechanisms that serve visual word recognition. Conclusions about the level of analysis reflected within the N170 effects, and therefore the timecourse of word recognition, have been mixed. Here, we investigated the timing and nature of brain responses to putatively low- and high-level processing difficulty. Low-level processing difficulty was modulated by manipulating letter-rotation parametrically at 0°, 22.5°, 45°, 67.5°, and 90°. Higher-level processing difficulty was modulated by manipulating lexical status (words vs. word-like pseudowords). Increasing letter-rotation enhanced the N170 led to monotonic increases in P1 and N170 amplitude up to 67.5° but then decreased amplitude at 90°. Pseudowords enhanced the N170 over left occipital-temporal sites, relative to words. These combined findings are compatible with a cascaded, interactive architecture in which lower-level analysis (e.g., word-form feature extraction) leads higher-level analysis (e.g., lexical access) in time, but that by approximately 170 ms, the brain's response to a visual word includes parallel, interactive processing at both low-level feature extraction and higher-order lexical access levels of analysis.

Inhibition of betaine-homocysteine S-methyltransferase in rats causes hyperhomocysteinemia and reduces liver cystathionine ß-synthase activity and methylation capacity.

Methylation of homocysteine (Hcy) by betaine-Hcy S-methyltransferase (BHMT) produces methionine, which is required for S-adenosylmethionine (SAM) synthesis. We have recently shown that short-term dietary intake of S-(Δ-carboxybutyl)-dl-Hcy (D,L-CBHcy), a potent and specific inhibitor of BHMT, significantly decreases liver BHMT activity and SAM concentrations but does not have an adverse affect on liver histopathology, plasma markers of liver damage, or DNA methylation in rats. The present study was designed to investigate the hypothesis that BHMT is required to maintain normal liver and plasma amino acid and glutathione profiles, and liver SAM and lipid accumulation. Rats were fed an adequate (4.5 g/kg methionine and 3.7 g/kg cystine), cysteine-devoid (4.5 g/kg methionine and 0 g/kg cystine), or methionine-deficient (1.5 g/kg methionine and 3.7 g/kg cystine) diet either with or without L-CBHcy for 3 or 14 days. All rats fed L-CBHcy had increased total plasma Hcy (2- to 5-fold) and reduced liver BHMT activity (>90%) and SAM concentrations (>40%). S-(Δ-carboxybutyl)-l-Hcy treatment slightly reduced liver glutathione levels in rats fed the adequate or cysteine-devoid diet for 14 days. Rats fed the methionine-deficient diet with L-CBHcy developed fatty liver. Liver cystathionine ß-synthase activity was reduced in all L-CBHcy-treated animals, and the effect was exacerbated as time on the L-CBHcy diet increased. Our data indicate that BHMT activity is required to maintain adequate levels of liver SAM and low levels of total plasma Hcy and might be critical for liver glutathione and triglyceride homeostasis under some dietary conditions.

Involvement of CTP:phosphocholine cytidylyltransferase-ß2 in axonal phosphatidylcholine synthesis and branching of neurons.

In the brain, phosphatidylcholine (PC) is synthesized by the CDP-choline pathway in which the rate-limiting step is catalyzed by two isoforms of CTP:phosphocholine cytidylyltransferase (CT): CTα and CTß2. In mice, CTß2 mRNA is more highly expressed in the brain than in other tissues, and several observations suggest that CTß2 plays an important role in the nervous system. We, therefore, investigated the importance of CTß2 for PC synthesis as well as for axon formation, growth and branching of primary sympathetic neurons. We show that in cultured primary neurons nerve growth factor increases the amount of CTß2, but not CTα, mRNA and protein. The brains of mice lacking CTß2 had normal PC content despite having 35% lower CT activity than wild-type brains. CTß2 mRNA and protein are abundant in distal axons of mouse sympathetic neurons whereas CTα mRNA and protein were not detected. Moreover, CTß2 deficiency in distal axons reduced the incorporation of [(3)H]choline into PC by 95% whereas PC synthesis in cell bodies/proximal axons was unaltered. These data suggest that CTß2 is the major CT isoform involved in PC synthesis in axons. Axons of CTß2-deficient sympathetic neurons contained 32% fewer branch points than did wild-type neurons although the number of axons/neuron and the rate of axon extension were the same as in wild-type neurons. We conclude that in distal axons of primary sympathetic neurons CTß2 is a major contributor to PC synthesis and promotes axon branching, whereas CTα appears to be the major CT isoform involved in PC synthesis in cell bodies/proximal axons.

Validated sandwich ELISA for the quantification of von Willebrand factor in rabbit plasma.

von Willebrand Factor (vWF) is a multimeric plasma protein important for platelet plug formation. As part of its haemostatic role, it is released from endothelial cells during vascular stress or injury and is considered an excellent biomarker of endothelial function. Currently, there are no validated kits available to measure vWF in rabbits. We developed a sensitive and reproducible sandwich enzyme-linked immunosorbent assay (ELISA) for detection of vWF in rabbit plasma using commercially available antibodies and reagents. Purified human vWF was used as a calibrator standard with a dynamic range of 1.56-100 ng/mL. The Minimum Required Dilution for rabbit plasma was 1:100. When plasma was spiked with 3.76 or 10 ng/mL vWF, recovery was 108 ± 2% and 93 ± 2%, respectively. Intra- and inter-assay precision for 8 rabbit plasma samples were 3% and 4%, respectively. The Minimum Detectable Concentration was 254 pg/mL for purified human vWF and 1:10,700 dilution of cholesterol-fed rabbit plasma, and the Reliable Detection Limits were 457 pg/mL and 1:5940. Three freeze-thaw cycles significantly decreased vWF concentrations for purified human vWF and 2 of 3 plasma samples assayed. This ELISA provides sensitive and reproducible measurements of rabbit plasma vWF, which is an important biomarker for cardiovascular research.

Dietary intake of S-(alpha-carboxybutyl)-DL-homocysteine induces hyperhomocysteinemia in rats.

Betaine homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to homocysteine (Hcy), forming dimethylglycine and methionine. We previously showed that inhibiting BHMT in mice by intraperitoneal injection of S-(alpha-carboxybutyl)-DL-homocysteine (CBHcy) results in hyperhomocysteinemia. In the present study, CBHcy was fed to rats to determine whether it could be absorbed and cause hyperhomocysteinemia as observed in the intraperitoneal administration of the compound in mice. We hypothesized that dietary administered CBHcy will be absorbed and will result in the inhibition of BHMT and cause hyperhomocysteinemia. Rats were meal-fed every 8 hours an L-amino acid-defined diet either containing or devoid of CBHcy (5 mg per meal) for 3 days. The treatment decreased liver BHMT activity by 90% and had no effect on methionine synthase, methylenetetrahydrofolate reductase, phosphatidylethanolamine N-methyltransferase, and CTP:phosphocholine cytidylyltransferase activities. In contrast, cystathionine beta-synthase activity and immunodetectable protein decreased (56% and 26%, respectively) and glycine N-methyltransferase activity increased (52%) in CBHcy-treated rats. Liver S-adenosylmethionine levels decreased by 25% in CBHcy-treated rats, and S-adenosylhomocysteine levels did not change. Furthermore, plasma choline decreased (22%) and plasma betaine increased (15-fold) in CBHcy-treated rats. The treatment had no effect on global DNA and CpG island methylation, liver histology, and plasma markers of liver damage. We conclude that CBHcy-mediated BHMT inhibition causes an elevation in total plasma Hcy that is not normalized by the folate-dependent conversion of Hcy to methionine. Furthermore, metabolic changes caused by BHMT inhibition affect cystathionine beta-synthase and glycine N-methyltransferase activities, which further deteriorate plasma Hcy levels.

Inhibition of betaine-homocysteine S-methyltransferase causes hyperhomocysteinemia in mice.

Inhibitors and methyl donor substrates for betaine-homocysteine S-methyltransferase (BHMT) were used to study the role of this enzyme in the regulation of plasma total homocysteine (tHcy). Mice were administered an i.p. injection of S-(delta-carboxybutyl)-dl-homocysteine (CBHcy; 1 mg), a specific and potent inhibitor of BHMT, and tHcy and hepatic BHMT protein and activity levels were monitored over a 24-h period. Compared with saline-injected control mice, at 2 h postinjection, the CBHcy-treated mice had 87% lower BHMT activity and a 2.7-fold increase (11.1 vs. 3.0 micromol/L) in tHcy, effects that lasted nearly 8 h but returned to normal by 24 h. The level of BHMT protein remained constant over the 24-h period. After 6 CBHcy (1 mg) injections (one every 12 h), the mice had 7-fold higher tHcy, a 65% reduction in the liver S-adenosylmethionine:S-adenosylhomocysteine ratio, and a marked upregulation of BHMT protein expression. At 2 h after injection of the sulfoxide derivative of CBHcy (10 mg) into mice, there was a modest reduction in BHMT activity and a 90% increase in tHcy. When given an injection of Met (3 mg) or Met plus CBHcy (1 mg), post-Met load tHcy levels were 2.2-fold higher (128 vs. 40 micromol/L) at 2 h postinjection in the mice given CBHcy. Like betaine, dimethylsulfoniopropionate was an effective tHcy-lowering agent when given with a Met load. These studies are the first to show that transient inhibition of BHMT in vivo causes transient hyperhomocysteinemia, and that dimethylsulfoniopropionate can reduce a post-Met load rise in tHcy.

A mammalian ortholog of Saccharomyces cerevisiae Vac14 that associates with and up-regulates PIKfyve phosphoinositide 5-kinase activity.

Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P(2), produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P(2) production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P(2) conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P(2) synthesis and endomembrane homeostasis in mammalian cells.

Role for a novel signaling intermediate, phosphatidylinositol 5-phosphate, in insulin-regulated F-actin stress fiber breakdown and GLUT4 translocation.

The cellular functions and regulation of phosphatidylinositol (PtdIns) 5-phosphate (5-P), the newest addition to the family of phosphoinositides (PIs), are still elusive. Here we have examined a plausible role of PtdIns 5-P as a signaling intermediate in acute insulin action. A wortmannin-insensitive transient increase of PtdIns 5-P mass levels that peaked at 10 min, and declined 20-30 min after insulin stimulation, was observed in both Chinese hamster ovary (CHO)-T cells stably expressing the insulin receptor and 3T3-L1 adipocytes. Similarly to insulin, found to induce a rapid disassembly of Texas-Red phalloidin-labeled actin stress fibers in CHO-T cells, microinjected PtdIns 5-P, but not other PIs, decreased the number and length of F-actin stress fibers in this cell type to a magnitude seen in response to insulin. Likewise, increases of PtdIns 5-P by ectopic expression of the PtdIns 5-P-producing enzyme PIKfyve yielded a similar effect. As with insulin, the PtdIns 5-P-induced loss of actin stress fibers was independent of PI 3-kinase activation. Furthermore, sequestration of functional PtdIns 5-P, either by ectopic expression of 3xPHD domains that bind selectively PtdIns 5-P or by microinjecting the GST-3xPHD fusion peptide, abrogated insulin-induced F-actin stress fiber disassembly in CHO-T cells. In 3T3-L1 adipocytes, microinjected PtdIns 5-P, but not other PIs, partially mimicked insulin's effect of translocating enhanced green fluorescent protein-GLUT4 to the cell surface. Conversely, insulin-induced myc-GLUT4 vesicle dynamics was arrested in the presence of coexpressed enhanced green fluorescent protein-3xPHD. Involvement of PIKfyve membrane recruitment, but not activation, and/or a decrease in PtdIns 4,5-bisphosphate levels are likely to be among the mechanisms underlying the insulin-induced PtdIns 5-P increase. Together, these results identify PtdIns 5-P as a novel key intermediate for insulin signaling in F-actin remodeling and GLUT4 translocation.