Background: SGLT2 (sodium/glucose cotransporter 2) inhibitors exert robust cardioprotective effec... more Background: SGLT2 (sodium/glucose cotransporter 2) inhibitors exert robust cardioprotective effects against heart failure in patients with diabetes, and there is intense interest to identify the underlying molecular mechanisms that afford this protection. Because the induction of the late component of the cardiac sodium channel current (late- I Na ) is involved in the etiology of heart failure, we investigated whether these drugs inhibit late- I Na . Methods: Electrophysiological, in silico molecular docking, molecular, calcium imaging, and whole heart perfusion techniques were used to address this question. Results: The SGLT2 inhibitor empagliflozin reduced late- I Na in cardiomyocytes from mice with heart failure and in cardiac Nav1.5 sodium channels containing the long QT syndrome 3 mutations R1623Q or ΔKPQ. Empagliflozin, dapagliflozin, and canagliflozin are all potent and selective inhibitors of H 2 O 2 -induced late- I Na (half maximal inhibitory concentration = 0.79, 0.58, an...
ABSTRACTPediatric obstructive sleep apnea (OSA), a relatively common sleep-related breathing diso... more ABSTRACTPediatric obstructive sleep apnea (OSA), a relatively common sleep-related breathing disorder affecting ∼1-5% of children, is often caused by anatomical obstruction and/or collapse of the nasal and/or pharyngeal airways. The resulting sleep disruption and intermittent hypoxia lead to various systemic morbidities. Predicting the development of OSA from craniofacial features alone is currently not possible, and controversy remains as to whether upper-airway obstruction facilitates reduced midfacial growth or vice versa. Currently, there is no rodent model that recapitulates both the development of craniofacial abnormalities and upper-airway obstruction to address these questions. Here, we describe that mice with a neural crest-specific deletion of Bmp7 (Bmp7ncko) present with a shorter, more acute-angled cranial base, midfacial hypoplasia, nasal septum deviation, turbinate swelling and branching defects, and nasal airway obstruction. Interestingly, several of these craniofacia...
The Na+/H+ exchanger is a pH-regulatory protein that extrudes one H+ ion in exchange for one Na+ ... more The Na+/H+ exchanger is a pH-regulatory protein that extrudes one H+ ion in exchange for one Na+ ion when intracellular pH declines. A number of studies have shown phorbol ester stimulation of activity in intact cells, leading to the idea that the exchanger is regulated by protein kinase C-mediated phosphorylation in vivo. cDNA encoding the protein has been cloned, and a recent model suggests a large internal cytoplasmic C-terminal domain that may be a site of regulation of the exchanger [Sardet, Franchi & Pouyssegur (1989) Cell 56, 271-280]. We examined this region of the protein using a rabbit cardiac Na+/H+ exchanger cDNA clone. cDNA of the Na+/H+ exchanger, coding for the C-terminal 178 amino acid residues, was cloned into the expression vector pEX-1 and expressed as a fusion protein with beta-galactosidase. The fusion protein reacted with an antibody produced against a synthetic peptide of the C-terminal 13 amino acid residues of the Na+/H+ exchanger, confirming the identity of...
Although multiple regions of the cerebral cortex have been implicated in swallowing, the function... more Although multiple regions of the cerebral cortex have been implicated in swallowing, the functional contributions of each brain area remain unclear. The present study sought to clarify the roles of these cortical foci in swallowing by comparing brain activation associated with voluntary saliva swallowing and voluntary tongue elevation. Fourteen healthy right-handed subjects were examined with single-event–related functional magnetic resonance imaging (fMRI) while laryngeal movements associated with swallowing and tongue movement were simultaneously recorded. Both swallowing and tongue elevation activated 1) the left lateral pericentral and anterior parietal cortex, and 2) the anterior cingulate cortex (ACC) and adjacent supplementary motor area (SMA), suggesting that these brain regions mediate processes shared by swallowing and tongue movement. Tongue elevation activated a larger total volume of cortex than swallowing, with significantly greater activation within the ACC, SMA, righ...
We examined the myocardial form of the Na+ /H+ exchanger. A partial length cDNA clone was isolate... more We examined the myocardial form of the Na+ /H+ exchanger. A partial length cDNA clone was isolated from a rabbit cardiac library and it encoded for a Na+ /H+ exchange protein. In comparison with the human Na+ /H+ exchanger, the sequence of the 5′ end of the cDNA was highly conserved, much more than the 3/t' region, while the deduced amino acid sequence was also highly conserved. To further characterize the myocardial Na+ /H+ exchange protein, we examined Western blots of isolated sarcolemma with antibody produced against a fusion protein of the Na+ /H+ exchanger. The antibodies reacted with a sarcolemma protein of 50 kDa and with a protein of 70 kDa. The results show that the rabbit myocardium does possess a Na+ /H+ exchanger protein homologous to the known human Na+ /H+ exchanger.
Transgenic mice with selective induction of calreticulin transgene expression in cardiomyocytes (... more Transgenic mice with selective induction of calreticulin transgene expression in cardiomyocytes (Cardiac CRT+) were analyzed. Cardiac CRT+ cardiomyocytes showed increased contractility and Ca 2+ transients. Yet, in vivo assessment of cardiac performance, and ischemic tolerance of Cardiac CRT+ mice demonstrated right ventricle dilation and reduced cardiac output, increased QT interval and decreased P amplitude. Paradoxically, ex vivo working hearts from Cardiac CRT+ mice showed enhanced ischemic cardio-protection and cardiac efficiency. Under aerobic conditions, Cardiac CRT+ hearts showed less efficient cardiac function than sham control hearts due to an increased ATP production from glycolysis relative to glucose oxidation. During reperfusion, this inefficiency was reversed, with Cardiac CRT+ hearts exhibiting better functional recovery and increased cardiac efficiency compared to sham control hearts. On the other hand, mechanical stretching of isolated cardiac fibroblasts activated the IRE1α branch of the unfolded protein response pathway as well as induction of Col1A2 and TGFβ gene expression ex vivo, which were all suppressed by tauroursodeoxycholic acid.
American Journal of Physiology-Heart and Circulatory Physiology, 1998
Malonyl-CoA is a potent inhibitor of fatty acid uptake into the mitochondria. Although the synthe... more Malonyl-CoA is a potent inhibitor of fatty acid uptake into the mitochondria. Although the synthesis of malonyl-CoA in the heart by acetyl-CoA carboxylase (ACC) has been well characterized, no information is available as to how malonyl-CoA is degraded. We demonstrate that malonyl-CoA decarboxylase (MCD) activity is present in the heart. Partial purification revealed a protein of ∼50 kDa. The role of MCD in regulating fatty acid oxidation was also studied using isolated, perfused hearts from newborn rabbits and adult rats. Fatty acid oxidation in rabbit hearts increased dramatically between 1 day and 7 days after birth, which was accompanied by a decrease in both ACC activity and malonyl-CoA levels and a parallel increase in MCD activity. When adult rat hearts were aerobically reperfused after a 30-min period of no-flow ischemia, levels of malonyl-CoA decreased dramatically, which was accompanied by a decrease in ACC activity, a maintained MCD activity, and an increase in fatty acid ...
Subcutaneous white adipose tissue (scWAT) is the major fat depot in humans and is a central playe... more Subcutaneous white adipose tissue (scWAT) is the major fat depot in humans and is a central player in regulating whole body metabolism. Skin exposure to UV wavelengths from sunlight is required for Vitamin D synthesis and pigmentation, although it is plausible that longer visible wavelengths that penetrate the skin may regulate scWAT function. In this regard, we discovered a novel blue light-sensitive current in human scWAT that is mediated by melanopsin coupled to transient receptor potential canonical cation channels. This pathway is activated at physiological intensities of light that penetrate the skin on a sunny day. Daily exposure of differentiated adipocytes to blue light resulted in decreased lipid droplet size, increased basal lipolytic rate and alterations in adiponectin and leptin secretion. Our results suggest that scWAT function may be directly under the influence of ambient sunlight exposure and may have important implications for our current understanding of adipocyte...
American journal of physiology. Heart and circulatory physiology, Jan 30, 2017
Cardiac ATP-sensitive potassium (KATP) channels couple changes in cellular metabolism to membrane... more Cardiac ATP-sensitive potassium (KATP) channels couple changes in cellular metabolism to membrane excitability and are activated during metabolic stress. Although under basal aerobic conditions, KATP channels are thought to be predominately closed. Despite intense research into the roles of KATP channels during metabolic stress, their contribution to aerobic basal cardiac metabolism has not been investigated previously. Hearts from Kir6.2+/+ and Kir6.2 -/- mice were perfused in working mode and rates of glycolysis, fatty acid oxidation and glucose oxidation were measured. Changes in activation/expression of proteins regulating metabolism were probed by Western blot analysis. Despite cardiac mechanical function and metabolic efficiency being similar in both groups, hearts from Kir6.2-/- mice displayed a ~2-fold increase in fatty acid oxidation, and a 0.45-fold reduction in glycolytic rates but similar glucose oxidation rates compared with Kir6.2+/+ hearts. Kir6.2-/- hearts also posse...
BACKGROUND AND DESIGN: Adiponectin is an adipokine secreted primarily from adipose tissue that ca... more BACKGROUND AND DESIGN: Adiponectin is an adipokine secreted primarily from adipose tissue that can influence circulating plasma glucose and lipid levels through multiple mechanisms involving a variety of organs. In humans, reduced plasma adiponectin levels induced by obesity are associated with insulin resistance and type 2 diabetes, suggesting that low adiponectin levels may contribute the pathogenesis of obesity-related insulin resistance. METHODS AND RESULTS: The objective of the present study was to investigate whether gene therapy designed to elevate circulating adiponectin levels is a viable strategy for ameliorating insulin resistance in mice fed a high-fat, high-sucrose (HFHS) diet. Electroporation-mediated gene transfer of mouse adiponectin plasmid DNA into gastrocnemius muscle resulted in elevated serum levels of globular and high-molecular weight adiponectin compared with control mice treated with empty plasmid. In comparison to HFHS-fed mice receiving empty plasmid, mice receiving adiponectin gene therapy displayed significantly decreased weight gain following 13 weeks of HFHS diet associated with reduced fat accumulation, and exhibited increased oxygen consumption and locomotor activity as measured by indirect calorimetry, suggesting increased energy expenditure in these mice. Consistent with improved whole-body metabolism, mice receiving adiponectin gene therapy also had lower blood glucose and insulin levels, improved glucose tolerance and reduced hepatic gluconeogenesis compared with control mice. Furthermore, immunoblot analysis of livers from mice receiving adiponectin gene therapy showed an increase in insulin-stimulated phosphorylation of insulin signaling proteins. CONCLUSION: Based on these data, we conclude that adiponectin gene therapy ameliorates the metabolic abnormalities caused by feeding mice a HFHS diet and may be a potential therapeutic strategy to improve obesity-mediated impairments in insulin sensitivity.
Background During and following myocardial ischemia, glucose oxidation rates are low and fatty ac... more Background During and following myocardial ischemia, glucose oxidation rates are low and fatty acids dominate as a source of oxidative metabolism. This metabolic phenotype is associated with contractile dysfunction during reperfusion. To determine the mechanism of this reliance on fatty acid oxidation as a source of ATP generation, a functional proteomics approach was utilized. Results 2-D gel electrophoresis of mitochondria from working rat hearts subjected to 25 minutes of global no flow ischemia followed by 40 minutes of aerobic reperfusion identified 32 changes in protein abundance compared to aerobic controls. Of the five proteins with the greatest change in abundance, two were increased (long chain acyl-coenzyme A dehydrogenase (48 ± 1 versus 39 ± 3 arbitrary units, n = 3, P < 0.05) and α subunit of ATP synthase (189 ± 15 versus 113 ± 23 arbitrary units, n = 3, P < 0.05)), while two were decreased (24 kDa subunit of NADH-ubiquinone oxidoreductase (94 ± 7 versus 127 ± 9 a...
In the heart, insulin stimulates a variety of kinase cascades and controls glucose utilization. B... more In the heart, insulin stimulates a variety of kinase cascades and controls glucose utilization. Because insulin is able to activate Akt and inactivate AMP-activated protein kinase (AMPK) in the heart, we hypothesized that Akt can regulate the activity of AMPK. To address the potential existence of this novel signaling pathway, we used a number of experimental protocols to activate Akt in cardiac myocytes and monitored the activation status of AMPK. Mouse hearts perfused in the presence of insulin demonstrated accelerated glycolysis and glucose oxidation rates as compared with non-insulin-perfused hearts. In addition, insulin caused an increase in Akt phosphorylation and a decrease in AMPK phosphorylation at its major regulatory site (threonine 172 of the ␣ catalytic subunit). Transgenic mice overexpressing a constitutively active mutant form of Akt1 displayed decreased phosphorylation of cardiac ␣-AMPK. Isolated neonatal cardiac myocytes infected with an adenovirus expressing constitutively active mutant forms of either Akt1 or Akt2 also suppressed AMPK phosphorylation. However, Akt-dependent depression of ␣-AMPK phosphorylation could be overcome in the presence of the AMPK activator, metformin, suggesting that an override mechanism exists that can restore AMPK activity. Taken together, this study suggests that there is crosstalk between the AMPK and Akt pathways and that Akt activation can lead to decreased AMPK activity. In addition, our data suggest that the ability of insulin to inhibit AMPK may be controlled via an Akt-mediated mechanism.
Functional closure of the human ductus arteriosus (DA) is initiated within minutes of birth by O ... more Functional closure of the human ductus arteriosus (DA) is initiated within minutes of birth by O 2 constriction. It occurs by an incompletely understood mechanism that is intrinsic to the DA smooth muscle cell (DASMC). We hypothesized that O 2 alters the function of an O 2 sensor (the mitochondrial electron transport chain, ETC) thereby increasing production of a diffusible redox-mediator (H 2 O 2 ), thus triggering an effector mechanism (inhibition of DASMC voltage-gated K + channels, Kv). O 2 constriction was evaluated in 26 human DAs (12 female, aged 9±2 days) studied in their normal hypoxic state or after normoxic tissue culture. In fresh, hypoxic DAs, 4-aminopyridine (4-AP), a Kv inhibitor, and O 2 cause similar constriction and K + current inhibition ( I K ). Tissue culture for 72 hours, particularly in normoxia, causes ionic remodeling, characterized by decreased O 2 and 4-AP constriction in DA rings and reduced O 2 - and 4-AP–sensitive I K in DASMCs. Remodeled DAMSCs are dep...
Objetive: Malonyl CoA is an important regulator of fatty acid oxidation in the heart secondary to... more Objetive: Malonyl CoA is an important regulator of fatty acid oxidation in the heart secondary to its ability to inhibit camitine palmitoyltransferase 1 (CFT 1). Malonyl CoA is produced from acetyl CoA in a reaction catalyzed by acetyl CoA carboxylase (ACC). In this study we determined if alterations in malonyl CoA regulation of fatty acid metabolism are involved in the increase in energy transduction seen following an increase in cardiac work. Methods: Anesthetized, (open-chest, domestic swine were subjected to a 30 min control period followed by a 30 min treatment period with either dobutamine (15 pg. kg-'. min-' i.v.> (n-6) or saline (n = 6). Results: Heart rate, left ventricular peak dp/dt, and MVO,, were significantly increased in the dobutamine group compared to the saline group during the treatment period. Free fatty acid and glucose uptake were increased 210 and 248%, respectively, in the dobutamine group during the treatment period. Malonyl CoA content was decreased by 55 % (from 0.40 f 0.05 to 0.18 St 0.12 nmol/g wet wt; P < 0.05) with dobutamine treatment, but was not affected by saline treatment. ACC activity was not significantly different between groups (0.3 1 * 0.02 vs. 0.30 + 0.04 nmol. min-'. mg protein-' , respectively). The activity of AMP-dependent protein kinase (AMPK), which phosphorylates and inactivates ACC, was also not significantly different in the dobutamine hearts compared to the saline hearts (322 f 26 vs. 338 f 39 pmol. min-'. mg protein-', respectively). Conclusion: The increased cardiac work following dobutamine infusion is accompanied by a decrease in malonyl CoA levels and an increase in fatty acid uptake. However, the decrease in malonyl CoA cannot be explained by a decrease in ACC activity.
In the liver, malonyl-CoA is central to many cellular processes, including both fatty acid biosyn... more In the liver, malonyl-CoA is central to many cellular processes, including both fatty acid biosynthesis and oxidation. Malonyl-CoA decarboxylase (MCD) is involved in the control of cellular malonyl-CoA levels, and functions to decarboxylate malonyl-CoA to acetyl-CoA. MCD may play an essential role in regulating energy utilization in the liver by regulating malonyl-CoA levels in response to various nutritional or pathological states. The purpose of the present study was to investigate the role of liver MCD in the regulation of fatty acid oxidation in situations where lipid metabolism is altered. A single MCD enzyme of molecular mass 50.7 kDa was purified from rat liver using a sequential column chromatography procedure and the cDNA was subsequently cloned and sequenced. The liver MCD cDNA was identical to rat pancreatic β-cell MCD cDNA, and contained two potential translational start sites, producing proteins of 50.7 kDa and 54.7 kDa. Western blot analysis using polyclonal antibodies generated against rat liver MCD showed that the 50.7 kDa isoform of MCD is most abundant in heart and liver, and of relatively low abundance in skeletal muscle (despite elevated MCD transcript levels in skeletal muscle). Tissue distribution experiments demonstrated that the pancreas is the only rat tissue so far identified that contains both the 50.7 kDa and 54.7 kDa isoforms of MCD. In addition, transfection of the full-length rat liver MCD cDNA into COS cells produced two isoforms of MCD. This indicated either that both initiating methionines are functionally active, generating two proteins, or that the 54.7 kDa isoform is the only MCD protein translated and removal of the putative mitochondrial targeting pre-sequence generates a protein of approx. 50.
American Journal of Respiratory Cell and Molecular Biology, 2011
Bronchopulmonary dysplasia (BPD) is the main complication of extreme prematurity, resulting in pa... more Bronchopulmonary dysplasia (BPD) is the main complication of extreme prematurity, resulting in part from mechanical ventilation and oxygen therapy. Currently, no specific treatment exists for BPD. BPD is characterized by an arrest in alveolar development and increased apoptosis of alveolar epithelial cells (AECs). Type 2 AECs are putative distal lung progenitor cells, capable of regenerating alveolar homeostasis after injury. We hypothesized that the protection of AEC2 death via the activation of the prosurvival Akt pathway prevents arrested alveolar development in experimental BPD. We show that the pharmacologic inhibition of the prosurvival factor Akt pathway with wortmannin during the critical period of alveolar development impairs alveolar development in newborn rats, resulting in larger and fewer alveoli, reminiscent of BPD. Conversely, in an experimental model of BPD induced by oxygen exposure of newborn rats, alveolar simplification is associated with a decreased activation of lung Akt. In vitro studies with rat lung epithelial (RLE) cells cultured in hyperoxia (95% O 2) showed decreased apoptosis and improved cell survival after the forced expression of active Akt by adenovirus-mediated gene transfer. In vivo, adenovirus-mediated Akt gene transfer preserves alveolar architecture in the newborn rat model of hyperoxia-induced BPD. We conclude that inhibition of the prosurvival factor Akt disrupts normal lung development, whereas the expression of active Akt in experimental BPD preserves alveolar development. We speculate that the modulation of apoptosis may have therapeutic potential in lung diseases characterized by alveolar damage.
AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte. Recent... more AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte. Recently, LKB1 was identified as a kinase that regulates AMPK. Using immunoblot analysis, we confirmed high expression of LKB1 in isolated rat cardiac myocytes but show that, under basal conditions, LKB1 is primarily localized to the nucleus, where it is inactive. We examined the role of LKB1 in cardiac myocytes, using adenoviruses that express LKB1, and its binding partners Ste20-related adaptor protein (STRADα) and MO25α. Infection of neonatal rat cardiac myocytes with all three adenoviruses substantially increased LKB1/STRADα/MO25α expression, LKB1 activity, and AMPKα phosphorylation at its activating phosphorylation site (threonine-172). Since activation of AMPK can inhibit hypertrophic growth and since LKB1 is upstream of AMPK, we hypothesized that expression of an active LKB1 complex would also inhibit protein synthesis associated with hypertrophic growth. Expression of the LKB1/STRADα/M...
Light PE, Human islets contain a subpopulation of glucagon-like peptide-1 secreting α cells that ... more Light PE, Human islets contain a subpopulation of glucagon-like peptide-1 secreting α cells that is increased in type 2 diabetes, Molecular Metabolism,
Objetive: Malonyl CoA is an important regulator of fatty acid oxidation in the heart secondary to... more Objetive: Malonyl CoA is an important regulator of fatty acid oxidation in the heart secondary to its ability to inhibit camitine palmitoyltransferase 1 (CFT 1). Malonyl CoA is produced from acetyl CoA in a reaction catalyzed by acetyl CoA carboxylase (ACC). In this study we determined if alterations in malonyl CoA regulation of fatty acid metabolism are involved in the increase in energy transduction seen following an increase in cardiac work. Methods: Anesthetized, (open-chest, domestic swine were subjected to a 30 min control period followed by a 30 min treatment period with either dobutamine (15 pg. kg-' . min-' i.v.> (n -6) or saline (n = 6).
Background: SGLT2 (sodium/glucose cotransporter 2) inhibitors exert robust cardioprotective effec... more Background: SGLT2 (sodium/glucose cotransporter 2) inhibitors exert robust cardioprotective effects against heart failure in patients with diabetes, and there is intense interest to identify the underlying molecular mechanisms that afford this protection. Because the induction of the late component of the cardiac sodium channel current (late- I Na ) is involved in the etiology of heart failure, we investigated whether these drugs inhibit late- I Na . Methods: Electrophysiological, in silico molecular docking, molecular, calcium imaging, and whole heart perfusion techniques were used to address this question. Results: The SGLT2 inhibitor empagliflozin reduced late- I Na in cardiomyocytes from mice with heart failure and in cardiac Nav1.5 sodium channels containing the long QT syndrome 3 mutations R1623Q or ΔKPQ. Empagliflozin, dapagliflozin, and canagliflozin are all potent and selective inhibitors of H 2 O 2 -induced late- I Na (half maximal inhibitory concentration = 0.79, 0.58, an...
ABSTRACTPediatric obstructive sleep apnea (OSA), a relatively common sleep-related breathing diso... more ABSTRACTPediatric obstructive sleep apnea (OSA), a relatively common sleep-related breathing disorder affecting ∼1-5% of children, is often caused by anatomical obstruction and/or collapse of the nasal and/or pharyngeal airways. The resulting sleep disruption and intermittent hypoxia lead to various systemic morbidities. Predicting the development of OSA from craniofacial features alone is currently not possible, and controversy remains as to whether upper-airway obstruction facilitates reduced midfacial growth or vice versa. Currently, there is no rodent model that recapitulates both the development of craniofacial abnormalities and upper-airway obstruction to address these questions. Here, we describe that mice with a neural crest-specific deletion of Bmp7 (Bmp7ncko) present with a shorter, more acute-angled cranial base, midfacial hypoplasia, nasal septum deviation, turbinate swelling and branching defects, and nasal airway obstruction. Interestingly, several of these craniofacia...
The Na+/H+ exchanger is a pH-regulatory protein that extrudes one H+ ion in exchange for one Na+ ... more The Na+/H+ exchanger is a pH-regulatory protein that extrudes one H+ ion in exchange for one Na+ ion when intracellular pH declines. A number of studies have shown phorbol ester stimulation of activity in intact cells, leading to the idea that the exchanger is regulated by protein kinase C-mediated phosphorylation in vivo. cDNA encoding the protein has been cloned, and a recent model suggests a large internal cytoplasmic C-terminal domain that may be a site of regulation of the exchanger [Sardet, Franchi & Pouyssegur (1989) Cell 56, 271-280]. We examined this region of the protein using a rabbit cardiac Na+/H+ exchanger cDNA clone. cDNA of the Na+/H+ exchanger, coding for the C-terminal 178 amino acid residues, was cloned into the expression vector pEX-1 and expressed as a fusion protein with beta-galactosidase. The fusion protein reacted with an antibody produced against a synthetic peptide of the C-terminal 13 amino acid residues of the Na+/H+ exchanger, confirming the identity of...
Although multiple regions of the cerebral cortex have been implicated in swallowing, the function... more Although multiple regions of the cerebral cortex have been implicated in swallowing, the functional contributions of each brain area remain unclear. The present study sought to clarify the roles of these cortical foci in swallowing by comparing brain activation associated with voluntary saliva swallowing and voluntary tongue elevation. Fourteen healthy right-handed subjects were examined with single-event–related functional magnetic resonance imaging (fMRI) while laryngeal movements associated with swallowing and tongue movement were simultaneously recorded. Both swallowing and tongue elevation activated 1) the left lateral pericentral and anterior parietal cortex, and 2) the anterior cingulate cortex (ACC) and adjacent supplementary motor area (SMA), suggesting that these brain regions mediate processes shared by swallowing and tongue movement. Tongue elevation activated a larger total volume of cortex than swallowing, with significantly greater activation within the ACC, SMA, righ...
We examined the myocardial form of the Na+ /H+ exchanger. A partial length cDNA clone was isolate... more We examined the myocardial form of the Na+ /H+ exchanger. A partial length cDNA clone was isolated from a rabbit cardiac library and it encoded for a Na+ /H+ exchange protein. In comparison with the human Na+ /H+ exchanger, the sequence of the 5′ end of the cDNA was highly conserved, much more than the 3/t' region, while the deduced amino acid sequence was also highly conserved. To further characterize the myocardial Na+ /H+ exchange protein, we examined Western blots of isolated sarcolemma with antibody produced against a fusion protein of the Na+ /H+ exchanger. The antibodies reacted with a sarcolemma protein of 50 kDa and with a protein of 70 kDa. The results show that the rabbit myocardium does possess a Na+ /H+ exchanger protein homologous to the known human Na+ /H+ exchanger.
Transgenic mice with selective induction of calreticulin transgene expression in cardiomyocytes (... more Transgenic mice with selective induction of calreticulin transgene expression in cardiomyocytes (Cardiac CRT+) were analyzed. Cardiac CRT+ cardiomyocytes showed increased contractility and Ca 2+ transients. Yet, in vivo assessment of cardiac performance, and ischemic tolerance of Cardiac CRT+ mice demonstrated right ventricle dilation and reduced cardiac output, increased QT interval and decreased P amplitude. Paradoxically, ex vivo working hearts from Cardiac CRT+ mice showed enhanced ischemic cardio-protection and cardiac efficiency. Under aerobic conditions, Cardiac CRT+ hearts showed less efficient cardiac function than sham control hearts due to an increased ATP production from glycolysis relative to glucose oxidation. During reperfusion, this inefficiency was reversed, with Cardiac CRT+ hearts exhibiting better functional recovery and increased cardiac efficiency compared to sham control hearts. On the other hand, mechanical stretching of isolated cardiac fibroblasts activated the IRE1α branch of the unfolded protein response pathway as well as induction of Col1A2 and TGFβ gene expression ex vivo, which were all suppressed by tauroursodeoxycholic acid.
American Journal of Physiology-Heart and Circulatory Physiology, 1998
Malonyl-CoA is a potent inhibitor of fatty acid uptake into the mitochondria. Although the synthe... more Malonyl-CoA is a potent inhibitor of fatty acid uptake into the mitochondria. Although the synthesis of malonyl-CoA in the heart by acetyl-CoA carboxylase (ACC) has been well characterized, no information is available as to how malonyl-CoA is degraded. We demonstrate that malonyl-CoA decarboxylase (MCD) activity is present in the heart. Partial purification revealed a protein of ∼50 kDa. The role of MCD in regulating fatty acid oxidation was also studied using isolated, perfused hearts from newborn rabbits and adult rats. Fatty acid oxidation in rabbit hearts increased dramatically between 1 day and 7 days after birth, which was accompanied by a decrease in both ACC activity and malonyl-CoA levels and a parallel increase in MCD activity. When adult rat hearts were aerobically reperfused after a 30-min period of no-flow ischemia, levels of malonyl-CoA decreased dramatically, which was accompanied by a decrease in ACC activity, a maintained MCD activity, and an increase in fatty acid ...
Subcutaneous white adipose tissue (scWAT) is the major fat depot in humans and is a central playe... more Subcutaneous white adipose tissue (scWAT) is the major fat depot in humans and is a central player in regulating whole body metabolism. Skin exposure to UV wavelengths from sunlight is required for Vitamin D synthesis and pigmentation, although it is plausible that longer visible wavelengths that penetrate the skin may regulate scWAT function. In this regard, we discovered a novel blue light-sensitive current in human scWAT that is mediated by melanopsin coupled to transient receptor potential canonical cation channels. This pathway is activated at physiological intensities of light that penetrate the skin on a sunny day. Daily exposure of differentiated adipocytes to blue light resulted in decreased lipid droplet size, increased basal lipolytic rate and alterations in adiponectin and leptin secretion. Our results suggest that scWAT function may be directly under the influence of ambient sunlight exposure and may have important implications for our current understanding of adipocyte...
American journal of physiology. Heart and circulatory physiology, Jan 30, 2017
Cardiac ATP-sensitive potassium (KATP) channels couple changes in cellular metabolism to membrane... more Cardiac ATP-sensitive potassium (KATP) channels couple changes in cellular metabolism to membrane excitability and are activated during metabolic stress. Although under basal aerobic conditions, KATP channels are thought to be predominately closed. Despite intense research into the roles of KATP channels during metabolic stress, their contribution to aerobic basal cardiac metabolism has not been investigated previously. Hearts from Kir6.2+/+ and Kir6.2 -/- mice were perfused in working mode and rates of glycolysis, fatty acid oxidation and glucose oxidation were measured. Changes in activation/expression of proteins regulating metabolism were probed by Western blot analysis. Despite cardiac mechanical function and metabolic efficiency being similar in both groups, hearts from Kir6.2-/- mice displayed a ~2-fold increase in fatty acid oxidation, and a 0.45-fold reduction in glycolytic rates but similar glucose oxidation rates compared with Kir6.2+/+ hearts. Kir6.2-/- hearts also posse...
BACKGROUND AND DESIGN: Adiponectin is an adipokine secreted primarily from adipose tissue that ca... more BACKGROUND AND DESIGN: Adiponectin is an adipokine secreted primarily from adipose tissue that can influence circulating plasma glucose and lipid levels through multiple mechanisms involving a variety of organs. In humans, reduced plasma adiponectin levels induced by obesity are associated with insulin resistance and type 2 diabetes, suggesting that low adiponectin levels may contribute the pathogenesis of obesity-related insulin resistance. METHODS AND RESULTS: The objective of the present study was to investigate whether gene therapy designed to elevate circulating adiponectin levels is a viable strategy for ameliorating insulin resistance in mice fed a high-fat, high-sucrose (HFHS) diet. Electroporation-mediated gene transfer of mouse adiponectin plasmid DNA into gastrocnemius muscle resulted in elevated serum levels of globular and high-molecular weight adiponectin compared with control mice treated with empty plasmid. In comparison to HFHS-fed mice receiving empty plasmid, mice receiving adiponectin gene therapy displayed significantly decreased weight gain following 13 weeks of HFHS diet associated with reduced fat accumulation, and exhibited increased oxygen consumption and locomotor activity as measured by indirect calorimetry, suggesting increased energy expenditure in these mice. Consistent with improved whole-body metabolism, mice receiving adiponectin gene therapy also had lower blood glucose and insulin levels, improved glucose tolerance and reduced hepatic gluconeogenesis compared with control mice. Furthermore, immunoblot analysis of livers from mice receiving adiponectin gene therapy showed an increase in insulin-stimulated phosphorylation of insulin signaling proteins. CONCLUSION: Based on these data, we conclude that adiponectin gene therapy ameliorates the metabolic abnormalities caused by feeding mice a HFHS diet and may be a potential therapeutic strategy to improve obesity-mediated impairments in insulin sensitivity.
Background During and following myocardial ischemia, glucose oxidation rates are low and fatty ac... more Background During and following myocardial ischemia, glucose oxidation rates are low and fatty acids dominate as a source of oxidative metabolism. This metabolic phenotype is associated with contractile dysfunction during reperfusion. To determine the mechanism of this reliance on fatty acid oxidation as a source of ATP generation, a functional proteomics approach was utilized. Results 2-D gel electrophoresis of mitochondria from working rat hearts subjected to 25 minutes of global no flow ischemia followed by 40 minutes of aerobic reperfusion identified 32 changes in protein abundance compared to aerobic controls. Of the five proteins with the greatest change in abundance, two were increased (long chain acyl-coenzyme A dehydrogenase (48 ± 1 versus 39 ± 3 arbitrary units, n = 3, P < 0.05) and α subunit of ATP synthase (189 ± 15 versus 113 ± 23 arbitrary units, n = 3, P < 0.05)), while two were decreased (24 kDa subunit of NADH-ubiquinone oxidoreductase (94 ± 7 versus 127 ± 9 a...
In the heart, insulin stimulates a variety of kinase cascades and controls glucose utilization. B... more In the heart, insulin stimulates a variety of kinase cascades and controls glucose utilization. Because insulin is able to activate Akt and inactivate AMP-activated protein kinase (AMPK) in the heart, we hypothesized that Akt can regulate the activity of AMPK. To address the potential existence of this novel signaling pathway, we used a number of experimental protocols to activate Akt in cardiac myocytes and monitored the activation status of AMPK. Mouse hearts perfused in the presence of insulin demonstrated accelerated glycolysis and glucose oxidation rates as compared with non-insulin-perfused hearts. In addition, insulin caused an increase in Akt phosphorylation and a decrease in AMPK phosphorylation at its major regulatory site (threonine 172 of the ␣ catalytic subunit). Transgenic mice overexpressing a constitutively active mutant form of Akt1 displayed decreased phosphorylation of cardiac ␣-AMPK. Isolated neonatal cardiac myocytes infected with an adenovirus expressing constitutively active mutant forms of either Akt1 or Akt2 also suppressed AMPK phosphorylation. However, Akt-dependent depression of ␣-AMPK phosphorylation could be overcome in the presence of the AMPK activator, metformin, suggesting that an override mechanism exists that can restore AMPK activity. Taken together, this study suggests that there is crosstalk between the AMPK and Akt pathways and that Akt activation can lead to decreased AMPK activity. In addition, our data suggest that the ability of insulin to inhibit AMPK may be controlled via an Akt-mediated mechanism.
Functional closure of the human ductus arteriosus (DA) is initiated within minutes of birth by O ... more Functional closure of the human ductus arteriosus (DA) is initiated within minutes of birth by O 2 constriction. It occurs by an incompletely understood mechanism that is intrinsic to the DA smooth muscle cell (DASMC). We hypothesized that O 2 alters the function of an O 2 sensor (the mitochondrial electron transport chain, ETC) thereby increasing production of a diffusible redox-mediator (H 2 O 2 ), thus triggering an effector mechanism (inhibition of DASMC voltage-gated K + channels, Kv). O 2 constriction was evaluated in 26 human DAs (12 female, aged 9±2 days) studied in their normal hypoxic state or after normoxic tissue culture. In fresh, hypoxic DAs, 4-aminopyridine (4-AP), a Kv inhibitor, and O 2 cause similar constriction and K + current inhibition ( I K ). Tissue culture for 72 hours, particularly in normoxia, causes ionic remodeling, characterized by decreased O 2 and 4-AP constriction in DA rings and reduced O 2 - and 4-AP–sensitive I K in DASMCs. Remodeled DAMSCs are dep...
Objetive: Malonyl CoA is an important regulator of fatty acid oxidation in the heart secondary to... more Objetive: Malonyl CoA is an important regulator of fatty acid oxidation in the heart secondary to its ability to inhibit camitine palmitoyltransferase 1 (CFT 1). Malonyl CoA is produced from acetyl CoA in a reaction catalyzed by acetyl CoA carboxylase (ACC). In this study we determined if alterations in malonyl CoA regulation of fatty acid metabolism are involved in the increase in energy transduction seen following an increase in cardiac work. Methods: Anesthetized, (open-chest, domestic swine were subjected to a 30 min control period followed by a 30 min treatment period with either dobutamine (15 pg. kg-'. min-' i.v.> (n-6) or saline (n = 6). Results: Heart rate, left ventricular peak dp/dt, and MVO,, were significantly increased in the dobutamine group compared to the saline group during the treatment period. Free fatty acid and glucose uptake were increased 210 and 248%, respectively, in the dobutamine group during the treatment period. Malonyl CoA content was decreased by 55 % (from 0.40 f 0.05 to 0.18 St 0.12 nmol/g wet wt; P < 0.05) with dobutamine treatment, but was not affected by saline treatment. ACC activity was not significantly different between groups (0.3 1 * 0.02 vs. 0.30 + 0.04 nmol. min-'. mg protein-' , respectively). The activity of AMP-dependent protein kinase (AMPK), which phosphorylates and inactivates ACC, was also not significantly different in the dobutamine hearts compared to the saline hearts (322 f 26 vs. 338 f 39 pmol. min-'. mg protein-', respectively). Conclusion: The increased cardiac work following dobutamine infusion is accompanied by a decrease in malonyl CoA levels and an increase in fatty acid uptake. However, the decrease in malonyl CoA cannot be explained by a decrease in ACC activity.
In the liver, malonyl-CoA is central to many cellular processes, including both fatty acid biosyn... more In the liver, malonyl-CoA is central to many cellular processes, including both fatty acid biosynthesis and oxidation. Malonyl-CoA decarboxylase (MCD) is involved in the control of cellular malonyl-CoA levels, and functions to decarboxylate malonyl-CoA to acetyl-CoA. MCD may play an essential role in regulating energy utilization in the liver by regulating malonyl-CoA levels in response to various nutritional or pathological states. The purpose of the present study was to investigate the role of liver MCD in the regulation of fatty acid oxidation in situations where lipid metabolism is altered. A single MCD enzyme of molecular mass 50.7 kDa was purified from rat liver using a sequential column chromatography procedure and the cDNA was subsequently cloned and sequenced. The liver MCD cDNA was identical to rat pancreatic β-cell MCD cDNA, and contained two potential translational start sites, producing proteins of 50.7 kDa and 54.7 kDa. Western blot analysis using polyclonal antibodies generated against rat liver MCD showed that the 50.7 kDa isoform of MCD is most abundant in heart and liver, and of relatively low abundance in skeletal muscle (despite elevated MCD transcript levels in skeletal muscle). Tissue distribution experiments demonstrated that the pancreas is the only rat tissue so far identified that contains both the 50.7 kDa and 54.7 kDa isoforms of MCD. In addition, transfection of the full-length rat liver MCD cDNA into COS cells produced two isoforms of MCD. This indicated either that both initiating methionines are functionally active, generating two proteins, or that the 54.7 kDa isoform is the only MCD protein translated and removal of the putative mitochondrial targeting pre-sequence generates a protein of approx. 50.
American Journal of Respiratory Cell and Molecular Biology, 2011
Bronchopulmonary dysplasia (BPD) is the main complication of extreme prematurity, resulting in pa... more Bronchopulmonary dysplasia (BPD) is the main complication of extreme prematurity, resulting in part from mechanical ventilation and oxygen therapy. Currently, no specific treatment exists for BPD. BPD is characterized by an arrest in alveolar development and increased apoptosis of alveolar epithelial cells (AECs). Type 2 AECs are putative distal lung progenitor cells, capable of regenerating alveolar homeostasis after injury. We hypothesized that the protection of AEC2 death via the activation of the prosurvival Akt pathway prevents arrested alveolar development in experimental BPD. We show that the pharmacologic inhibition of the prosurvival factor Akt pathway with wortmannin during the critical period of alveolar development impairs alveolar development in newborn rats, resulting in larger and fewer alveoli, reminiscent of BPD. Conversely, in an experimental model of BPD induced by oxygen exposure of newborn rats, alveolar simplification is associated with a decreased activation of lung Akt. In vitro studies with rat lung epithelial (RLE) cells cultured in hyperoxia (95% O 2) showed decreased apoptosis and improved cell survival after the forced expression of active Akt by adenovirus-mediated gene transfer. In vivo, adenovirus-mediated Akt gene transfer preserves alveolar architecture in the newborn rat model of hyperoxia-induced BPD. We conclude that inhibition of the prosurvival factor Akt disrupts normal lung development, whereas the expression of active Akt in experimental BPD preserves alveolar development. We speculate that the modulation of apoptosis may have therapeutic potential in lung diseases characterized by alveolar damage.
AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte. Recent... more AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte. Recently, LKB1 was identified as a kinase that regulates AMPK. Using immunoblot analysis, we confirmed high expression of LKB1 in isolated rat cardiac myocytes but show that, under basal conditions, LKB1 is primarily localized to the nucleus, where it is inactive. We examined the role of LKB1 in cardiac myocytes, using adenoviruses that express LKB1, and its binding partners Ste20-related adaptor protein (STRADα) and MO25α. Infection of neonatal rat cardiac myocytes with all three adenoviruses substantially increased LKB1/STRADα/MO25α expression, LKB1 activity, and AMPKα phosphorylation at its activating phosphorylation site (threonine-172). Since activation of AMPK can inhibit hypertrophic growth and since LKB1 is upstream of AMPK, we hypothesized that expression of an active LKB1 complex would also inhibit protein synthesis associated with hypertrophic growth. Expression of the LKB1/STRADα/M...
Light PE, Human islets contain a subpopulation of glucagon-like peptide-1 secreting α cells that ... more Light PE, Human islets contain a subpopulation of glucagon-like peptide-1 secreting α cells that is increased in type 2 diabetes, Molecular Metabolism,
Objetive: Malonyl CoA is an important regulator of fatty acid oxidation in the heart secondary to... more Objetive: Malonyl CoA is an important regulator of fatty acid oxidation in the heart secondary to its ability to inhibit camitine palmitoyltransferase 1 (CFT 1). Malonyl CoA is produced from acetyl CoA in a reaction catalyzed by acetyl CoA carboxylase (ACC). In this study we determined if alterations in malonyl CoA regulation of fatty acid metabolism are involved in the increase in energy transduction seen following an increase in cardiac work. Methods: Anesthetized, (open-chest, domestic swine were subjected to a 30 min control period followed by a 30 min treatment period with either dobutamine (15 pg. kg-' . min-' i.v.> (n -6) or saline (n = 6).
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Papers by Amy Barr