Dietary Protein and Amino Acid Deficiency Inhibit Pancreatic Digestive Enzyme mRNA Translation by Multiple Mechanisms.

BACKGROUND & AIMS: Chronic amino acid (AA) deficiency, as in kwashiorkor, reduces the size of the pancreas through an effect on mammalian target of rapamycin complex 1 (mTORC1). Because of the physiological importance of AAs and their role as a substrate, a stimulant of mTORC1, and protein synthesis, we studied the effect of acute protein and AA deficiency on the response to feeding. METHODS: ICR/CD-1 mice were fasted overnight and refed for 2 hours with 4 different isocaloric diets: control (20% Prot); Protein-free (0% Prot); control (AA-based diet), and a leucine-free (No Leu). Protein synthesis, polysomal profiling, and the activation of several protein translation factors were analyzed in pancreas samples. RESULTS: All diets stimulated the Protein Kinase-B (Akt)/mTORC1 pathway, increasing the phosphorylation of the kinase Akt, the ribosomal protein S6 (S6) and the formation of the eukaryotic initiation factor 4F (eIF4F) complex. Total protein synthesis and polysome formation were inhibited in the 0% Prot and No Leu groups to a similar extent, compared with the 20% Prot group. The 0% Prot diet partially reduced the Akt/mTORC1 pathway and the activity of the guanine nucleotide exchange factor eIF2B, without affecting eIF2α phosphorylation. The No Leu diet increased the phosphorylation of eIF2α and general control nonderepressible 2, and also inhibited eIF2B activity, without affecting mTORC1. Essential and nonessential AA levels in plasma and pancreas indicated a complex regulation of their cellular transport mechanisms and their specific effect on the synthesis of digestive enzymes. CONCLUSIONS: These studies show that dietary AAs are important regulators of postprandial digestive enzyme synthesis, and their deficiency could induce pancreatic insufficiency and malnutrition.

Enhanced dimethylarginine degradation improves coronary flow reserve and exercise tolerance in Duchenne muscular dystrophy carrier mice.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates postexercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac ß-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in patients with DMD.NEW & NOTEWORTHY Duchenne muscular dystrophy (DMD) carriers are at risk for cardiomyopathy. The nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is released from damaged muscle in DMD and impairs exercise performance. Transgenic expression of dimethylarginine dimethylaminohydrolase to degrade ADMA prevents cardiac hypertrophy, improves cardiac function, and improves exercise tolerance in DMD carrier mice. These findings highlight the relevance of ADMA to muscular dystrophy and have important implications for therapies targeting nitric oxide in patients with DMD and DMD carriers.

Constitutive release of CPS1 in bile and its role as a protective cytokine during acute liver injury.

Carbamoyl phosphate synthetase-1 (CPS1) is the major mitochondrial urea cycle enzyme in hepatocytes. It is released into mouse and human blood during acute liver injury, where is has a short half-life. The function of CPS1 in blood and the reason for its short half-life in serum are unknown. We show that CPS1 is released normally into mouse and human bile, and pathologically into blood during acute liver injury. Other cytoplasmic and mitochondrial urea cycle enzymes are also found in normal mouse bile. Serum, bile, and purified CPS1 manifest sedimentation properties that overlap with extracellular vesicles, due to the propensity of CPS1 to aggregate despite being released primarily as a soluble protein. During liver injury, CPS1 in blood is rapidly sequestered by monocytes, leading to monocyte M2-polarization and homing to the liver independent of its enzyme activity. Recombinant CPS1 (rCPS1), but not control r-transferrin, increases hepatic macrophage numbers and phagocytic activity. Notably, rCPS1 does not activate hepatic macrophages directly; rather, it activates bone marrow and circulating monocytes that then home to the liver. rCPS1 administration prevents mouse liver damage induced by Fas ligand or acetaminophen, but this protection is absent in macrophage-deficient mice. Moreover, rCPS1 protects from acetaminophen-induced liver injury even when given therapeutically after injury induction. In summary, CPS1 is normally found in bile but is released by hepatocytes into blood upon liver damage. We demonstrate a nonenzymatic function of CPS1 as an antiinflammatory protective cytokine during acute liver injury.

Differential impact of type-1 and type-2 diabetes on control of heart rate in mice.

AIMS: Cardiac autonomic dysfunction is a serious complication of diabetes. One consequence is disruption of the normal beat-to-beat regulation of heart rate (HR), i.e. HR variability (HRV). However, our understanding of the disease process has been limited by inconsistent HR/HRV data from previous animal studies. We hypothesized that differences in the method of measurement, time of day, and level of stress account for the differing results across studies. Thus, our aim was to systematically assess HR and HRV in two common diabetic mouse models. METHODS: ECG radiotelemetry devices were implanted into db/db (type-2 diabetic), STZ-treated db/+ (type-1 diabetic), and control db/+ mice (n=4 per group). HR and HRV were analyzed over 24 h and during treadmill testing. RESULTS: 24 h analysis revealed that db/db mice had an altered pattern of circadian HR changes, and STZ-treated mice had reduced HR throughout. HRV measures linked to sympathetic control were reduced in db/db mice in the early morning and early afternoon, and partially reduced in STZ-treated mice. HR response to treadmill testing was blunted in both models. CONCLUSIONS: It is important to consider both time of day and level of stress when assessing HR and HRV in diabetic mice. db/db mice may have altered circadian rhythm of sympathetic control of HR, whereas STZ-treated mice have a relative reduction. This study provides baseline data and a framework for HR analysis that may guide future investigations.

Ambient Light Promotes Selective Subcellular Proteotoxicity after Endogenous and Exogenous Porphyrinogenic Stress.

Hepatic accumulation of protoporphyrin-IX (PP-IX) in erythropoietic protoporphyria (EPP) or X-linked-dominant protoporphyria (XLP) cause liver damage. Hepatocyte nuclear lamin aggregation is a sensitive marker for PP-IX-mediated liver injury. We tested the hypothesis that extracellular or intracellular protoporphyria cause damage to different subcellular compartments, in a light-triggered manner. Three hepatoma cell lines (HepG2, Hepa-1, and Huh-7) were treated with exogenous PP-IX (mimicking XLP extrahepatic protoporphyria) or with the iron chelator deferoxamine and the porphyrin precursor 5-aminolevulinic acid (ALA) (mimicking intracellular protoporphyrin accumulation in EPP). Exogenous PP-IX accumulated predominantly in the nuclear fraction and caused nuclear shape deformation and cytoplasmic vacuoles containing electron-dense particles, whereas ALA+deferoxamine treatment resulted in higher PP-IX in the cytoplasmic fraction. Protein aggregation in the nuclear and cytoplasmic fractions paralleled PP-IX levels and, in cell culture, the effects were exclusively ambient light-mediated. PP-IX and ALA caused proteasomal inhibition, whereas endoplasmic reticulum protein aggregation was more prominent in ALA-treated cells. The enhanced ALA-related toxicity is likely due to generation of additional porphyrin intermediates including uroporphyrin and coproporphyrin, based on HPLC analysis of cell lysates and the culture medium, as well as cell-free experiments with uroporphyrin/coproporphyrin. Mouse livers from drug-induced porphyria phenocopied the in vitro findings, and mass spectrometry of liver proteins isolated in light/dark conditions showed diminished (as compared with light-harvested) but detectable aggregation under dark-harvested conditions. Therefore, PP-IX leads to endoplasmic reticulum stress and proteasome inhibition in a manner that depends on the source of porphyrin buildup and light exposure. Porphyrin-mediated selective protein aggregation provides a potential mechanism for porphyria-associated tissue injury.

Packed red blood cells are an abundant and proximate potential source of nitric oxide synthase inhibition.

OBJECTIVE: We determined, for packed red blood cells (PRBC) and fresh frozen plasma, the maximum content, and ability to release the endogenous nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA) and monomethylarginine (LNMMA). BACKGROUND: ADMA and LNMMA are near equipotent NOS inhibitors forming blood's total NOS inhibitory content. The balance between removal from, and addition to plasma determines their free concentrations. Removal from plasma is by well-characterized specific hydrolases while formation is restricted to posttranslational protein methylation. When released into plasma they can readily enter endothelial cells and inhibit NOS. Fresh rat and human whole blood contain substantial protein incorporated ADMA however; the maximum content of ADMA and LNMMA in PRBC and fresh frozen plasma has not been determined. METHODS: We measured total (free and protein incorporated) ADMA and LNMMA content in PRBCs and fresh frozen plasma, as well as their incubation induced release, using HPLC with fluorescence detection. We tested the hypothesis that PRBC and fresh frozen plasma contain substantial inhibitory methylarginines that can be released chemically by complete in vitro acid hydrolysis or physiologically at 37°C by enzymatic blood proteolysis. RESULTS: In vitro strong-acid-hydrolysis revealed a large PRBC reservoir of ADMA (54.5 ± 9.7 µM) and LNMMA (58.9 ± 28.9 µM) that persisted over 42-d at 6° or -80°C. In vitro 5h incubation at 37°C nearly doubled free ADMA and LNMMNA concentration from PRBCs while no change was detected in fresh frozen plasma. CONCLUSION: The compelling physiological ramifications are that regardless of storage age, 1) PRBCs can rapidly release pathologically relevant quantities of ADMA and LNMMA when incubated and 2) PRBCs have a protein-incorporated inhibitory methylarginines reservoir 100 times that of normal free inhibitory methylarginines in blood and thus could represent a clinically relevant and proximate risk for iatrogenic NOS inhibition upon transfusion.

Nitric oxide synthase, ADMA, SDMA, and nitric oxide activity in the paraventricular nucleus throughout the etiology of renal wrap hypertension.

Within the paraventricular nucleus (PVN), there is a balance between the excitatory and inhibitory neurotransmitters that regulate blood pressure; in hypertension, the balance shifts to enhanced excitation. Nitric oxide (NO) is an atypical neurotransmitter that elicits inhibitory effects on cardiovascular function. We hypothesized that reduced PVN NO led to elevations in blood pressure during both the onset and sustained phases of hypertension due to decreased NO synthase (NOS) and increased asymmetrical dimethylarginine (ADMA; an endogenous NOS inhibitor) and symmetric dimethylarginine (SDMA). Elevated blood pressure, in response to PVN bilateral microinjections of a NO inhibitor, nitro-L-arginine methyl ester, was blunted in renal wrapped rats during the onset of hypertension (day 7) and sustained renal wrap hypertension (day 28) compared with sham-operated rats. Adenoviruses (Ad) encoding endothelial NOS (eNOS) or LacZ microinjected into the PVN [1 × 10(9) plaque-forming units, bilateral (200 nl/site)] reduced mean arterial pressure compared with control (Day 7, Ad LacZ wrap: 144 ± 7 mmHg and Ad eNOS wrap: 117 ± 5 mmHg, P ≤ 0.05) throughout the study (Day 28, Ad LacZ wrap: 123 ± 1 mmHg and Ad eNOS wrap: 108 ± 4 mmHg, P ≤ 0.05). Western blot analyses of PVN NOS revealed significantly lower PVN neuronal NOS during the onset of hypertension but not in sustained hypertension. Reduced SDMA was found in the PVN during the onset of hypertension; however, no change in ADMA was observed. In conclusion, functional indexes of NO activity indicated an overall downregulation of NO in renal wrap hypertension, but the mechanism by which this occurs likely differs throughout the development of hypertension.

Pathogenic cycle between the endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine and the leukocyte-derived hemoprotein myeloperoxidase.

BACKGROUND: The nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) and the leukocyte-derived hemoprotein myeloperoxidase (MPO) are associated with cardiovascular diseases. Activation of monocytes and polymorphonuclear neutrophils (PMNs) with concomitant release of MPO is regulated in a nitric oxide-dependent fashion. The aim of the study was to investigate a potential 2-way interaction between ADMA and MPO. METHODS AND RESULTS: Ex vivo, ADMA uptake by isolated human PMNs, the principal source of MPO in humans, significantly impaired nitric oxide synthase activity determined by gas chromatography-mass spectrometry. In humans, short-term ADMA infusion (0.0125 mg · kg(-1) · min(-1)) significantly increased MPO plasma concentrations. Functionally, PMN exposure to ADMA enhanced leukocyte adhesion to endothelial cells, augmented NADPH oxidase activity, and stimulated PMN degranulation, resulting in release of MPO. In vivo, a 28-day ADMA infusion (250 µmol · kg(-1) · d(-1)) in C57Bl/6 mice significantly increased plasma MPO concentrations, whereas this ADMA effect on MPO was attenuated by human dimethylarginine dimethylaminohydrolase1 (hDDAH1) overexpression. Moreover, the MPO-derived reactive molecule hypochlorous acid impaired recombinant hDDAH1 activity in vitro. In MPO(-/-) mice, the lipopolysaccharide-induced increase in systemic ADMA concentrations was abrogated. CONCLUSIONS: ADMA profoundly impairs nitric oxide synthesis of PMNs, resulting in increased PMN adhesion to endothelial cells, superoxide generation, and release of MPO. In addition, MPO impairs DDAH1 activity. Our data reveal an ADMA-induced cycle of PMN activation, enhanced MPO release, and subsequent impairment of DDAH1 activity. These findings not only highlight so far unrecognized cytokine-like properties of ADMA but also identify MPO as a regulatory switch for ADMA bioavailability under inflammatory conditions.

Secretin is not necessary for exocrine pancreatic development and growth in mice.

Adaptive exocrine pancreatic growth is mediated primarily by dietary protein and the gastrointestinal hormone cholecystokinin (CCK). Feeding trypsin inhibitors such as camostat (FOY-305) is known to induce CCK release and stimulate pancreatic growth. However, camostat has also been reported to stimulate secretin release and, because secretin often potentiates the action of CCK, it could participate in the growth response. Our aim was to test the role of secretin in pancreatic development and adaptive growth through the use of C57BL/6 mice with genetic deletion of secretin or secretin receptor. The lack of secretin in the intestine or the secretin receptor in the pancreas was confirmed by RT-PCR. Other related components, such as vasoactive intestinal polypeptide (VIP) receptors (VPAC(1) and VPAC(2)), were not affected. Secretin increased cAMP levels in acini from wild-type (WT) mice but had no effect on acini from secretin receptor-deleted mice, whereas VIP and forskolin still induced a normal response. Secretin in vivo failed to induce fluid secretion in receptor-deficient mice. The pancreas of secretin or secretin receptor-deficient mice was of normal size and histology, indicating that secretin is not necessary for normal pancreatic differentiation or maintenance. When WT mice were fed 0.1% camostat in powdered chow, the pancreas doubled in size in 1 wk, accompanied by parallel increases in protein and DNA. Camostat-fed littermate secretin and secretin receptor-deficient mice had similar pancreatic mass to WT mice. These results indicate that secretin is not required for normal pancreatic development or adaptive growth mediated by CCK.

Murine prolylcarboxypeptidase depletion induces vascular dysfunction with hypertension and faster arterial thrombosis.

Prolylcarboxypeptidase (PRCP) activates prekallikrein to plasma kallikrein, leading to bradykinin liberation, and degrades angiotensin II. We now identify PRCP as a regulator of blood vessel homeostasis. ß-Galactosidase staining in PRCP(gt/gt) mice reveals expression in kidney and vasculature. Invasive telemetric monitorings show that PRCP(gt/gt) mice have significantly elevated blood pressure. PRCP(gt/gt) mice demonstrate shorter carotid artery occlusion times in 2 models, and their plasmas have increased thrombin generation times. Pharmacologic inhibition of PRCP with Z-Pro-Prolinal or plasma kallikrein with soybean trypsin inhibitor, Pro-Phe-Arg-chloromethylketone or PKSI 527 also shortens carotid artery occlusion times. Aortic and renal tissues have uncoupled eNOS and increased reactive oxygen species (ROS) in PRCP(gt/gt) mice as detected by dihydroethidium or Amplex Red fluorescence or lucigenin luminescence. The importance of ROS is evidenced by the fact that treatment of PRCP(gt/gt) mice with antioxidants (mitoTEMPO, apocynin, Tempol) abrogates the hypertensive, prothrombotic phenotype. Mechanistically, our studies reveal that PRCP(gt/gt) aortas express reduced levels of Kruppel-like factors 2 and 4, thrombomodulin, and eNOS mRNA, suggesting endothelial cell dysfunction. Further, PRCP siRNA treatment of endothelial cells shows increased ROS and uncoupled eNOS and decreased protein C activation because of thrombomodulin inactivation. Collectively, our studies identify PRCP as a novel regulator of vascular ROS and homeostasis.

Molecular mechanisms of pancreatic dysfunction induced by protein malnutrition.

BACKGROUND & AIMS: Dietary protein deficiency results in diminished capacity of the pancreas to secrete enzymes needed for macronutrient digestion. Previous work has suggested that modulation of the mammalian target of rapamycin (mTOR) pathway by the hormone cholecystokinin (CCK) plays an important role in normal digestive enzyme synthesis after feeding. The purpose of this study was to elucidate the role of mTOR in protein deficiency-induced pancreatic dysfunction. METHODS: Wild-type and CCK-null mice were fed protein-deficient chow for 4 days and then allowed to recover on control chow in the presence or absence of the mTOR inhibitor rapamycin. RESULTS: The size and secretory capacity of the pancreas rapidly decreased after feeding protein-deficient chow. Refeeding protein-replete chow reversed these changes in both wild-type and CCK-null mice. Changes in the size of the pancreas were paralleled by changes in the content and secretion of digestive enzymes, as well as the phosphorylation of downstream targets of mTOR. Administration of the mTOR inhibitor rapamycin decreased regrowth of the pancreas but did not affect digestive enzyme content or secretory capacity. CONCLUSIONS: These studies demonstrate that dietary protein modulates pancreatic growth, but not digestive enzyme synthesis, via CCK-independent activation of the mTOR pathway.

Vascular smooth muscle cell-selective peroxisome proliferator-activated receptor-gamma deletion leads to hypotension.

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists are commonly used to treat diabetes, although their PPARgamma-dependent effects transcend their role as insulin sensitizers. Thiazolidinediones lower blood pressure (BP) in diabetic patients, whereas results from conventional/tissue-specific PPARgamma experimental models suggest an important pleiotropic role for PPARgamma in BP control. Little evidence is available on the molecular mechanisms underlying the role of vascular smooth muscle cell-specific PPARgamma in basal vascular tone. METHODS AND RESULTS: We show that vascular smooth muscle cell-selective deletion of PPARgamma impairs vasoactivity with an overall reduction in BP. Aortic contraction in response to norepinephrine is reduced and vasorelaxation is enhanced in response to beta-adrenergic receptor (beta-AdR) agonists in vitro. Similarly, vascular smooth muscle cell-selective PPARgamma knockout mice display a biphasic response to norepinephrine in BP, reversible on administration of beta-AdR blocker, and enhanced BP reduction on treatment with beta-AdR agonists. Consistent with enhanced beta2-AdR responsiveness, we found that the absence of PPARgamma in vascular smooth muscle cells increased beta2-AdR expression, possibly leading to the hypotensive phenotype during the rest phase. CONCLUSIONS: These data uncovered the beta2-AdR as a novel target of PPARgamma transcriptional repression in vascular smooth muscle cells and indicate that PPARgamma regulation of beta2-adrenergic signaling is important in the modulation of BP.

Single histidine button in cardiac troponin I sustains heart performance in response to severe hypercapnic respiratory acidosis in vivo.

Intracellular acidosis is a profound negative regulator of myocardial performance. We hypothesized that titrating myofilament calcium sensitivity by a single histidine substituted cardiac troponin I (A164H) would protect the whole animal physiological response to acidosis in vivo. To experimentally induce severe hypercapnic acidosis, mice were exposed to a 40% CO(2) challenge. By echocardiography, it was found that systolic function and ventricular geometry were maintained in cTnI A164H transgenic (Tg) mice. By contrast, non-Tg (Ntg) littermates experienced rapid and marked cardiac decompensation during this same challenge. For detailed hemodymanic assessment, Millar pressure-conductance catheterization was performed while animals were treated with a beta-blocker, esmolol, during a severe hypercapnic acidosis challenge. Survival and load-independent measures of contractility were significantly greater in Tg vs. Ntg mice. This assay showed that Ntg mice had 100% mortality within 5 min of acidosis. By contrast, systolic and diastolic function were protected in Tg mice during acidosis, and they had 100% survival. This study shows that, independent of any beta-adrenergic compensation, myofilament-based molecular manipulation of inotropy by histidine-modified troponin I maintains cardiac inotropic and lusitropic performance and markedly improves survival during severe acidosis in vivo.

Blood content of asymmetric dimethylarginine: new insights into its dysregulation in renal disease.

BACKGROUND: Plasma asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is significantly elevated in patients with kidney disease and is a potential risk factor for cardiovascular disease. Here, we tested whether human whole blood (WB), as in rodent blood, can accumulate free ADMA and whether this accumulation is a function of disease burden. METHODS: In 16 healthy control subjects (CO), 18 patients with ESRD and 18 matched hypertensive patients with normal renal function (HTN), we compared using high-pressure liquid chromatography baseline plasma and WB supernatant (WBSUP) ADMA and symmetrical dimethylarginine (SDMA) concentrations and accumulation during a 5-h incubation. We measured protein turnover in incubated WBSUP to determine if proteolytic processes drive ADMA accumulation. RESULTS: Elevated plasma ADMA was confirmed in ESRD and HTN populations while basal WBSUP ADMA was significantly higher in ESRD subjects than controls (P = 0.05 versus CO; P = 0.02 versus HTN). Plasma SDMA followed a similar pattern. Incubation of WBSUP resulted in ADMA release from protein-incorporated stores while SDMA was unaffected. ADMA accumulation in ESRD samples was significantly greater than that in HTN (P = 0.03). CO and HTN men showed significantly greater ADMA accumulation than women (P = 0.01 and P = 0.003, respectively) but no gender difference was observed in the ESRD group (P = 0.26). ADMA accumulation correlated with ex vivo protein turnover (R = 0.76, P < 0.0001). CONCLUSIONS: Human blood is capable of releasing physiologically significant quantities of ADMA via proteolytic pathways. Dysregulated ADMA release from WB reservoirs may contribute to the distinctly high plasma ADMA levels in ESRD populations.

Vascular dysfunction in the alpha-galactosidase A-knockout mouse is an endothelial cell-, plasma membrane-based defect.

Fabry disease results from an X-linked mutation in the lysosomal alpha-galactosidase A (Gla) gene. Defective Gla results in multi-organ accumulation of neutral glycosphingolipids (GSLs), especially in the vascular endothelium, with the major GSL accumulated being globotriaosylceramide (Gb3). Excessive endothelial Gb3 accumulation is associated with increased thrombosis, atherogenesis and endothelial dysfunction. However, the mechanism(s) by which endothelial dysfunction occurs is unclear. The purpose of the present study was to further characterize the vasculopathy associated with a murine model of Fabry disease. Vascular reactivity was performed in vessels from wild-type (Gla(+/0)) and Gla-knockout (Gla(-/0)) mice. Conscious blood pressure and heart rate were measured in Gla(+/0) and Gla(-/0) mice by telemetry. The present study demonstrates that vascular smooth muscle (VSM) contractions to phenylephrine and serotonin, but not to U46619, were blunted in Gla(-/0) mice. Endothelium-dependent contraction and receptor-mediated endothelium-dependent relaxation to acetylcholine were significantly attenuated in vessels from Gla(-/0) mice. However, receptor-independent endothelium-dependent relaxation to the calcium ionophore ionomycin remained intact in vessels from Gla(-/0) mice. Furthermore, VSM reactivity was normal in aortas from Gla(-/0) mice in the absence of endothelium. These changes in vascular function were observed without changes in whole-animal blood pressure or heart rate. These results suggest that the vasculopathy associated with Fabry disease is localized to the endothelium, despite the accumulation of GSLs throughout the vasculature.

Double oxygen-sensing vector system for robust hypoxia/ischemia-regulated gene induction in cardiac muscle in vitro and in vivo.

High-fidelity genetically encoded bio-sensors that respond to changes in cellular environmental milieu in disease offer great potential in a range of patho-physiological settings. Here a unique hypoxia-regulated vector-based system with double oxygen-sensing transcriptional elements was developed for rapid and robust hypoxia-regulated gene expression in the heart. Hypoxia-responsive cis elements were used in tandem with a single proline-modified oxygen-dependent degradation (ODD) domain of hypoxia-inducible factor-1alpha to form a double oxygen-sensing vector system (DOSVS). In adult cardiac myocytes in vitro, the DOSVS demonstrated a low background expression not different from baseline control in normoxia, and with 100% efficiency, robust, 1,000-fold induction upon hypoxia. In the heart in vivo, hypoxic and ischemic challenges elicited rapid 700-fold induction in living animals, exceeding that obtained by a high-fidelity constitutive cytomegalovirus (CMV) viral promoter. DOSVS also showed high temporal resolution in the heart in response to cyclical bouts of hypoxia in vivo. We propose that DOSVS will be valuable for a range of applications, including bio-sensing and therapeutic gene expression in the heart and other organ systems that are confronted by chronic or episodic hypoxic/ischemic stresses in vivo.

CCK-induced pancreatic growth is not limited by mitogenic capacity in mice.

In mice fed trypsin inhibitor (camostat) to elevate endogenous CCK, pancreatic growth plateaus by 7 days. It is unknown whether this represents the maximum growth capacity of the pancreas. To test the ability of CCK to drive further growth, mice were fed chow containing camostat (0.1%) for 1 wk, then fed standard chow for 1 wk, and finally returned to the camostat diet for a week. Pancreatic mass increased to 245% of initial value (iv) following 1 wk of camostat feeding, decreased to 147% iv following a 1 wk return to normal chow, and increased to 257% iv with subsequent camostat feeding. Camostat feeding was associated with significant increases in circulating CCK and changes in pancreatic mass were paralleled by changes in protein and DNA content. Moreover, regression of the pancreas following camostat feeding was associated with changes in the expression of the autophagosome marker LC3. Pancreatic protein synthetic rates were 130% of control after 2 days on camostat but were equivalent to control after 7 days. Changes in the phosphorylation of 4E-BP1 and S6, downstream effectors of mammalian target of rapamycin (mTOR), paralleled changes in protein synthetic rates. Cellular content of Akt, an upstream activating kinase of mTOR, decreased after 7 days of camostat feeding whereas expression of the E3 ubiquitin-ligases and the cell cycle inhibitor p21 increased after 2 days. These results indicate that CCK-stimulated growth of the pancreas is not limited by acinar cell mitogenic capacity but is due, at least in part, to inhibition of promitogenic Akt signaling.

Direct monitoring pressure overload predicts cardiac hypertrophy in mice.

Pressure overload (POL) is a classical model for studying cardiac hypertrophy, but there has been no direct measure of hemodynamics in a conscious ambulatory mouse model of POL. We used abdominal aortic constriction to produce POL and radiotelemetry to measure the blood pressure and heart rate for three weeks. The cardiac size correlated with the systolic pressure in the last week is better than other hemodynamic parameters. Cardiac fibrosis was more correlated to the cardiac size than to the systolic pressure. The expression of the cardiac genes that are typically associated with cardiac hypertrophy was correlated with both cardiac size and systolic pressure. In conclusion, the systolic pressure is the major determinant of cardiac hypertrophy in the murine POL model. In contrast, cardiac fibrosis shows the influence of other factors besides systolic pressure. The combination of the POL model with continuous direct measurements of hemodynamics represents a significant technological advance and will lead to an extended usefulness of POL methodologically.