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1.
Chem Commun (Camb) ; 50(38): 4944-6, 2014 May 18.
Article in English | MEDLINE | ID: mdl-24705519

ABSTRACT

Cross-linking of proteins by advanced glycation endproducts (AGEs) causes a host of pathological conditions but their exact roles are unknown. Cross-linking lysyl AGEs were synthesized and incorporated into two types of collagen peptides. The utility of these cross-linked peptides for biochemical investigations was demonstrated by proteolysis studies and circular dichroism.


Subject(s)
Collagen/chemistry , Glycation End Products, Advanced/chemistry , Chromatography, High Pressure Liquid , Circular Dichroism , Collagen/metabolism , Dimerization , Glycation End Products, Advanced/metabolism , Glyoxal/chemistry , Lysine/chemistry , Pyruvaldehyde/chemistry , Trypsin/metabolism
2.
Curr Med Chem ; 19(17): 2828-60, 2012.
Article in English | MEDLINE | ID: mdl-22455587

ABSTRACT

Oxidative stress and mitochondrial dysfunction have been identified by many workers as key pathogenic mechanisms in ageing-related metabolic, cardiovascular and neurodegenerative diseases (for example diabetes mellitus, heart failure and Alzheimer's disease). However, although numerous molecular mechanisms have been advanced to account for these processes, their precise nature remains obscure. This author has previously suggested that, in such diseases, these two mechanisms are likely to occur as manifestations of a single underlying disturbance of copper regulation. Copper is an essential but highly-toxic trace metal that is closely regulated in biological systems. Several rare genetic disorders of copper homeostasis are known in humans: these primarily affect various proteins that mediate intracellular copper transport processes, and can lead either to tissue copper deficiency or overload states. These examples illustrate how impaired regulation of copper transport pathways can cause organ damage and provide important insights into the impact of defects in specific molecular processes, including those catalyzed by the copper-transporting ATPases, ATP7A (mutated in Menkes disease), ATP7B (Wilson's disease), and the copper chaperones such as those for cytochrome c oxidase, SCO1 and SCO2. In diabetes, impaired copper regulation manifests as elevations in urinary CuII excretion, systemic chelatable-CuII and full copper balance, in increased pro-oxidant stress and defective antioxidant defenses, and in progressive damage to the blood vessels, heart, kidneys, retina and nerves. Linkages between dysregulated copper and organ damage can be demonstrated by CuII-selective chelation, which simultaneously prevents/reverses both copper dysregulation and organ damage. Pathogenic structures in blood vessels that contribute to binding and localization of catalytically-active CuII probably include advanced glycation end products (AGEs), as well as atherosclerotic plaque: the latter probably undergoes AGE-modification itself. Defective copper regulation mediates organ damage through two general processes that occur simultaneously in the same individual: elevation of CuII-mediated pro-oxidant stress and impairment of copper-catalyzed antioxidant defence mechanisms. This author has proposed that diabetes-evoked copper dysregulation is an important new target for therapeutic intervention to prevent/reverse organ damage in diabetes, heart failure, and neurodegenerative diseases, and that triethylenetetramine (TETA) is the first in a new class of anti-diabetic molecules, which function by targetting these copper-mediated pathogenic mechanisms. TETA prevents tissue damage and causes organ regeneration by acting as a highly-selective CuII chelator which suppresses copper-mediated oxidative stress and restores anti-oxidant defenses. My group has employed TETA in a comprehensive programme of nonclinical studies and proof-of-principle clinical trials, thereby characterizing copper dysregulation in diabetes and identifying numerous linked cellular and molecular mechanisms though which TETA exerts its therapeutic actions. Many of the results obtained in nonclinical models with respect to the molecular mechanisms of diabetic organ damage have not yet been replicated in patients' tissues so their applicability to the human disease must be considered as inferential until the results of informative clinical studies become available. Based on evidence from the studies reviewed herein, trientine is now proceeding into the later stages of pharmaceutical development for the treatment of heart failure and other diabetic complications.


Subject(s)
Chelating Agents/therapeutic use , Copper/metabolism , Diabetes Mellitus/drug therapy , Animals , Chelating Agents/metabolism , Chelating Agents/pharmacology , Clinical Trials as Topic , Copper/deficiency , Diabetes Complications/drug therapy , Diabetes Complications/metabolism , Diabetes Mellitus/metabolism , Humans , Oxidative Stress/drug effects
3.
Am J Physiol Cell Physiol ; 300(2): C246-55, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21084644

ABSTRACT

In diabetic cardiomyopathy, ventricular dysfunction occurs in the absence of hypertension or atherosclerosis and is accompanied by altered myocardial substrate utilization and depressed mitochondrial respiration. It is not known if mitochondrial function differs across the left ventricular (LV) wall in diabetes. In the healthy heart, the inner subendocardial region demonstrates higher rates of blood flow, oxygen consumption, and ATP turnover compared with the outer subepicardial region, but published transmural respirometric measurements have not demonstrated differences. We aim to measure mitochondrial function in Wistar rat LV to determine the effects of age, streptozotocin-diabetes, and LV layer. High-resolution respirometry measured indexes of respiration in saponin-skinned fibers dissected from the LV subendocardium and subepicardium of 3-mo-old rats after 1 mo of streptozotocin-induced diabetes and 4-mo-old rats following 2 mo of diabetes. Heart rate and heartbeat duration were measured under isoflurane-anesthesia using a fetal-Doppler, and transmission electron microscopy was employed to observe ultrastructural differences. Heart rate decreased with age and diabetes, whereas heartbeat duration increased with diabetes. While there were no transmural respirational differences in young healthy rat hearts, both myocardial layers showed a respiratory depression with age (30-40%). In 1-mo diabetic rat hearts only subepicardial respiration was depressed, whereas after 2 mo diabetes, respiration in subendocardial and subepicardial layers was depressed and showed elevated leak (state 2) respiration. These data provide evidence that mitochondrial dysfunction is first detectable in the subepicardium of diabetic rat LV, whereas there are measureable changes in LV mitochondria after only 4 mo of aging.


Subject(s)
Aging/physiology , Diabetes Mellitus, Experimental/physiopathology , Diabetic Cardiomyopathies/physiopathology , Mitochondria, Heart/physiology , Mitochondrial Diseases/physiopathology , Oxygen Consumption/physiology , Pericardium/physiopathology , Ventricular Dysfunction, Left/physiopathology , Animals , Diabetes Mellitus, Experimental/diagnostic imaging , Diabetic Cardiomyopathies/diagnostic imaging , Echocardiography, Doppler , Heart Rate/physiology , Male , Mitochondria, Heart/diagnostic imaging , Mitochondria, Heart/ultrastructure , Mitochondrial Diseases/diagnostic imaging , Myocardial Contraction , Pericardium/diagnostic imaging , Pericardium/ultrastructure , Rats , Rats, Wistar , Ventricular Dysfunction, Left/diagnostic imaging
4.
Diabetologia ; 53(6): 1217-26, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20221822

ABSTRACT

AIMS/HYPOTHESIS: Treatment with the Cu(II)-selective chelator triethylenetetramine (TETA) improves cardiovascular disease in human patients, and cardiac and vascular/renal disease in rats used as a model of diabetes. Here we tested two hypotheses: first, that TETA elicits greater improvement in organ function than less Cu-selective transition-metal-targeted treatments; second, that the therapeutic actions of TETA are consistent with mediation through suppression of oxidative stress. METHODS: Rats were made diabetic with streptozotocin (55 mg/kg, i. v.) and treated from 8 weeks after disease induction for the following 8 weeks with effective dosages of oral TETA, or one of three less Cu-selective transition-metal-targeted treatments: D-penicillamine, deferiprone or Zn acetate. Treatment effects were measured in ex vivo cardiac and aortic tissues, plasma and urine. RESULTS: Diabetes damaged both cardiac and renal/vascular function by impairing the ability of cardiac output to respond physiologically to rising afterload, and by significantly elevating the urinary albumin/creatinine ratio. Diabetes also lowered total antioxidant potential and heparan sulphate levels in cardiac and arterial tissues, and serum ferroxidase activity, whereas it elevated urinary heparan sulphate excretion. TETA treatment rectified or partially rectified all these defects, whereas the other three experimental treatments were ineffectual. By contrast, none of the four drug treatments lowered diabetes-mediated elevations of plasma glucose or lipid concentrations. CONCLUSIONS/INTERPRETATION: TETA may limit the cardiac and renal/vascular damage inflicted by diabetes through its actions to reinforce antioxidant defence mechanisms, probably acting through selective chelation of 'loosely-bound'/chelatable Cu(II). It may also improve heparan sulphate homeostasis and bolster antioxidant defence by increasing vascular extracellular superoxide dismutase activity. Urinary albumin/creatinine ratio might prove useful for monitoring TETA treatment.


Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Heart/drug effects , Oxidative Stress/drug effects , Trientine/therapeutic use , Adult , Aged , Analysis of Variance , Animals , Aorta/drug effects , Chelating Agents/therapeutic use , Deferiprone , Diabetes Mellitus, Experimental/urine , Heparitin Sulfate/urine , Humans , Kidney/drug effects , Male , Middle Aged , Penicillamine/therapeutic use , Pyridones/therapeutic use , Rats , Rats, Wistar , Zinc Acetate/therapeutic use
6.
Exp Physiol ; 94(8): 914-27, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19429644

ABSTRACT

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited renal diseases. It is associated with the progressive development of renal tubular cysts, which may subsequently lead to renal failure. Studies into the genetic basis of ADPKD have identified two genes, PKD1 and PKD2, that are mutated in ADPKD patients. The PKD1 and PKD2 genes encode for two different proteins, TRPP1 and TRPP2. Previous studies have demonstrated the presence of both TRPP1 and TRPP2 in the renal collecting duct cell line M8. The aim of the following study was to investigate the functional properties of cation currents in these cells and to examine the effect of overexpression of TRPP1 using a transgenic cell model (M7). In M8 cells, initial whole cell currents were low. However, over time there was activation of a flow-sensitive current, which was inhibited by gadolinium (I(Gd)). The I(Gd) was more selective for cations over anions, but did not discriminate between monovalent cations and was Ca2+ permeable. Activation of I(Gd) was dependent on the presence of Ca2+ and also required dephosphorylation. The protein phosphatase 2A inhibitor okadaic acid prevented activation of I(Gd), suggesting that protein phosphatase 2A plays an important role in channel activation. The properties and magnitude of I(Gd) were unaffected in M7 cells, suggesting that overexpression of TRPP1 was without effect. I(Gd) was selectively inhibited by an antibody raised against the C-terminus of TRPP2. However, its selectivity profile was different to TRPP2, suggesting that it is attributable to a TRPP2-like channel or a TRPP2-containing heteromeric channel. In conclusion, these data describe the functional identification of a novel dephosphorylation- and flow-activated TRPP2-related channel in mouse collecting duct cells.


Subject(s)
Kidney Tubules, Collecting/physiopathology , Polycystic Kidney, Autosomal Dominant/physiopathology , TRPP Cation Channels/physiology , Animals , Calcium/metabolism , Cell Line , Electric Conductivity , Gadolinium/pharmacology , Mice , Okadaic Acid/pharmacology , Patch-Clamp Techniques , RNA, Messenger/metabolism , TRPP Cation Channels/immunology
7.
J Dairy Sci ; 92(4): 1543-53, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19307635

ABSTRACT

The production of milk is closely linked to nutritional state in many mammalian species, but the mechanisms by which changes in nutritional state are signaled to the mammary glands are poorly understood. Simultaneous measurements of mammary blood flow and glucose arterio-venous difference were made across the inguinal mammary glands of anesthetized, lactating rats. Blood flow to the mammary glands of previously fed rats was 0.48 mL/min per gram of mammary tissue. Glucose supply was 1.7 mumol/min per gram and 28% was extracted by the mammary glands. After food deprivation for 18 h, mammary blood flow decreased 48%, glucose arterio-venous difference decreased 72%, and hematocrit increased 7%, resulting in a 60% decrease in glucose supply and an 88% decrease in glucose uptake. After 1 h of refeeding, glucose supply had returned to a similar level to that of normally fed animals, but glucose uptake was 60% higher than in the normally fed state. Mammary glucose uptake was not closely linked to either blood flow or glucose supply, suggesting that substrate supply was not the primary determinant of mammary metabolism. Denervation experiments showed that the mammary metabolic response to altered nutritional state was also unlikely to be closely controlled by neural pathways. Severance of the cutaneous branch of the posterior division of the femoral nerve innervating the inguinal mammary glands did not reduce the high glucose uptake by mammary glands of either fed or refed rats, nor did denervation change the low glucose uptake by mammary glands of food-deprived rats. Denervation reduced blood flow in the associated mammary gland, however, indicating that neural pathways may play a role in supporting mammary blood flow when food is available. In in vitro experiments, the rate of glucose uptake was 35% lower in mammary acini from food-deprived rats than in fed rats 2.5 h after tissue removal, indicating some persistence of the food deprivation-induced suppression of mammary metabolism. Administration of insulin increased glucose uptake in acini from both fed and food-deprived rats, indicating that insulin may be involved in signaling the mammary gland of the restoration of nutrient supply when food-deprived rats are refed. The effects of administration of a gut extract in vivo and in vitro are discussed.


Subject(s)
Eating/physiology , Mammary Glands, Animal/blood supply , Mammary Glands, Animal/metabolism , Animals , Blood Glucose/analysis , Denervation/veterinary , Female , Glucose/metabolism , Insulin/blood , Lactation , Mammary Glands, Animal/innervation , Rats , Rats, Sprague-Dawley , Time Factors
8.
Diabetologia ; 52(4): 715-22, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19172243

ABSTRACT

AIMS/HYPOTHESIS: Cu(II)-selective chelation with trientine ameliorates cardiovascular and renal disease in a model of diabetes in rats. Here, we tested the hypothesis that Cu(II)-selective chelation might improve left ventricular hypertrophy (LVH) in type 2 diabetic patients. METHODS: We performed a 12 month randomised placebo-controlled study of the effects of treatment with the Cu(II)-selective chelator trientine (triethylenetetramine dihydrochloride, 600 mg given orally twice daily) on LVH in diabetic patients (n = 15/group at baseline) in an outpatient setting wherein participants, caregivers and those assessing outcomes were blinded to group assignment. Using MRI, we measured left ventricular variables at baseline, and at months 6 and 12. The change from baseline in left ventricular mass indexed to body surface area (LVM(bsa)) was the primary endpoint variable. RESULTS: Diabetic patients had LVH with preserved ejection fraction at baseline. Trientine treatment decreased LVM(bsa) by 5.0 +/- 7.2 g/m(2) (mean +/- SD) at month 6 (when 14 trientine-treated and 14 placebo-treated participants were analysed; p = 0.0056 compared with placebo) and by 10.6 +/- 7.6 g/m(2) at month 12 (when nine trientine-treated and 13 placebo-treated participants were analysed; p = 0.0088), whereas LVM(bsa) was unchanged by placebo treatment. In a multiple-regression model that explained ~75% of variation (R (2) = 0.748, p = 0.001), cumulative urinary Cu excretion over 12 months was positively associated with trientine-evoked decreases in LVM(bsa). CONCLUSIONS/INTERPRETATION: Cu(II)-selective chelation merits further exploration as a potential pharmacotherapy for diabetic heart disease. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN 12609000053224 FUNDING: The Endocore Research Trust; Lottery Health New Zealand; the Maurice and Phyllis Paykel Trust; the Foundation of Research, Science and Technology (New Zealand); the Health Research Council of New Zealand; the Ministry of Education (New Zealand) through the Maurice Wilkins Centre for Molecular Biodiscovery; and the Protemix Corporation.


Subject(s)
Chelating Agents/therapeutic use , Diabetes Mellitus, Type 2/complications , Diabetic Angiopathies/drug therapy , Hypertrophy, Left Ventricular/drug therapy , Trientine/therapeutic use , Adult , Aged , Blood Pressure/drug effects , Body Surface Area , Creatinine/metabolism , Diabetic Angiopathies/physiopathology , Echocardiography , Electrocardiography , Female , Glycated Hemoglobin/metabolism , Heart Ventricles/anatomy & histology , Humans , Hypertrophy, Left Ventricular/physiopathology , Magnetic Resonance Imaging , Male , Middle Aged , Patient Selection , Placebos
9.
Am J Physiol Renal Physiol ; 296(3): F642-8, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19052101

ABSTRACT

Facilitative urea transporters in the mammalian kidney play a vital role in the urinary concentrating mechanism. The urea transporters located in the renal inner medullary collecting duct, namely UT-A1 and UT-A3, are acutely regulated by the antidiuretic hormone vasopressin. In this study, we investigated the vasopressin regulation of the basolateral urea transporter UT-A3 using an MDCK-mUT-A3 cell line. Within 10 min, vasopressin stimulates urea flux through UT-A3 transporters already present at the plasma membrane, via a PKA-dependent process. Within 1 h, vasopressin significantly increases UT-A3 localization at the basolateral membrane, causing a further increase in urea transport. While the basic trafficking of UT-A3 to basolateral membranes involves both protein kinase C and calmodulin, its regulation by vasopressin specifically occurs through a casein kinase II-dependent pathway. In conclusion, this study details the effects of vasopressin on UT-A3 urea transporter function and hence its role in regulating urea permeability within the renal inner medullary collecting duct.


Subject(s)
Casein Kinase II/metabolism , Kidney/metabolism , Membrane Transport Proteins/metabolism , Urea/metabolism , Vasopressins/metabolism , Animals , Cell Line , Dogs , Urea Transporters
10.
Diabetologia ; 51(9): 1741-51, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18636238

ABSTRACT

AIMS/HYPOTHESIS: The selective Cu(II) chelator triethylenetetramine (TETA) extracts systemic Cu(II) into the urine of diabetic humans and rats as a model of diabetes, and in the process also normalises hallmarks of diabetic heart disease. However, the role of Cu and its response to TETA in animals with diabetic nephropathy were previously unknown. Here, we report the effects of TETA treatment on Cu and other essential elements, as well as on indices of renal injury and known pathogenic molecular processes, in kidneys from a rat model of diabetes. METHODS: Rats at 8 weeks after streptozotocin-induction of diabetes were treated with oral TETA (34 mg/day in drinking water) for a further 8 weeks and then compared with untreated diabetic control animals. RESULTS: Renal tissue Cu was substantively elevated by diabetes and normalised by TETA, which also suppressed whole-kidney and glomerular hypertrophy without lowering blood glucose. The urinary albumin: creatinine ratio was significantly elevated in the rat model of diabetes but lowered by TETA. Total collagen was also elevated in diabetic kidneys and significantly improved by TETA. Furthermore, renal cortex levels of TGF-beta1, MAD homologue (SMAD) 4, phosphorylated SMAD2, fibronectin-1, collagen-III, collagen-IV, plasminogen activator inhibitor-1 and semicarbazide-sensitive amine oxidase all tended to be elevated in diabetes and normalised by TETA. CONCLUSIONS/INTERPRETATION: Dysregulation of renal Cu homeostasis may be a key event eliciting development of diabetic nephropathy. Selective Cu(II) chelation can protect against pathogenic mechanisms that lead to or cause diabetic nephropathy and might be clinically useful in the treatment of early-stage diabetic kidney disease.


Subject(s)
Albuminuria/drug therapy , Chelating Agents/therapeutic use , Copper/therapeutic use , Diabetes Mellitus, Experimental/drug therapy , Diabetic Nephropathies/drug therapy , Kidney/pathology , Transforming Growth Factor beta/antagonists & inhibitors , Trientine/therapeutic use , Animals , Disease Models, Animal , Fibrosis , Kidney/drug effects , Kidney Glomerulus/drug effects , Kidney Glomerulus/pathology , Rats
11.
Am J Physiol Endocrinol Metab ; 292(1): E117-22, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16912056

ABSTRACT

Several hormones that regulate nutritional status also impact on bone metabolism. Preptin is a recently isolated 34-amino acid peptide hormone that is cosecreted with insulin and amylin from the pancreatic beta-cells. Preptin corresponds to Asp(69)-Leu(102) of pro-IGF-II. Increased circulating levels of a pro-IGF-II peptide complexed with IGF-binding protein-2 have been implicated in the high bone mass phenotype observed in patients with chronic hepatitis C infection. We have assessed preptin's activities on bone. Preptin dose-dependently stimulated the proliferation (cell number and DNA synthesis) of primary fetal rat osteoblasts and osteoblast-like cell lines at periphysiological concentrations (>10(-11) M). In addition, thymidine incorporation was stimulated in murine neonatal calvarial organ culture, likely reflecting the proliferation of cells from the osteoblast lineage. Preptin did not affect bone resorption in this model. Preptin induced phosphorylation of p42/p44 MAP kinases in osteoblastic cells in a dose-dependent manner (10(-8)-10(-10) M), and its proliferative effects on primary osteoblasts were blocked by MAP kinase kinase inhibitors. Preptin also reduced osteoblast apoptosis induced by serum deprivation, reducing the number of apoptotic cells by >20%. In vivo administration of preptin increased bone area and mineralizing surface in adult mice. These data demonstrate that preptin, which is cosecreted from the pancreatic beta-cell with amylin and insulin, is anabolic to bone and may contribute to the preservation of bone mass observed in hyperinsulinemic states such as obesity.


Subject(s)
Insulin-Like Growth Factor II/pharmacology , Insulin-Secreting Cells/metabolism , Osteogenesis/drug effects , Peptide Fragments/pharmacology , Peptides/pharmacology , Animals , Bone Development/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Humans , Insulin-Like Growth Factor II/metabolism , Male , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoclasts/metabolism , Peptide Fragments/metabolism , Peptides/metabolism , Pertussis Toxin/pharmacology , Rats , Receptors, Islet Amyloid Polypeptide , Receptors, Peptide/antagonists & inhibitors , Swiss 3T3 Cells
12.
J Membr Biol ; 211(3): 173-84, 2006.
Article in English | MEDLINE | ID: mdl-17091215

ABSTRACT

K(+) channels play an important role in renal collecting duct cell function. The current study examined barium (Ba(2+))-sensitive whole-cell K(+) currents (IKBa) in mouse isolated collecting duct principal cells. IKBa demonstrated strong inward rectification and was inhibited by Ba(2+) in a dose- and voltage-dependent fashion, with the K (d) decreasing with hyperpolarization. The electrical distance of block by Ba(2+) was around 8.5%. As expected for voltage-dependent inhibition, the association constant increased with hyperpolarization, suggesting that the rate of Ba(2+) entry was increased at negative potentials. The dissociation constant also increased with hyperpolarization, consistent with the movement of Ba(2+) ions into the intracellular compartment at negative potentials. These properties are not consistent with ROMK but are consistent with the properties of Kir2.3. Kir2.3 is thought to be the dominant basolateral K(+) channel in principal cells. This study provides functional evidence for the expression of Kir2.3 in mouse cortical collecting ducts and confirms the expression of Kir2.3 in this segment of the renal tubule using reverse-transcriptase polymerase chain reaction. The conductance described here is the first report of a macroscopic K(+) conductance in mouse principal cells that shares the biophysical profile of Kir2.3. The properties and dominant nature of the conductance suggest that it plays an important role in K(+) handling in the principal cells of the cortical collecting duct.


Subject(s)
Kidney Cortex/physiology , Kidney Tubules, Collecting/physiology , Potassium Channels, Inwardly Rectifying/physiology , Animals , Barium/pharmacology , Dose-Response Relationship, Drug , Electric Conductivity , Epithelial Sodium Channels/metabolism , Female , Gene Expression , Kidney Cortex/cytology , Male , Membrane Potentials/physiology , Mice , Mice, Inbred C57BL , Mucoproteins/metabolism , Patch-Clamp Techniques , Potassium Channels, Inwardly Rectifying/metabolism , Potassium Chloride/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sodium Chloride/metabolism , Sodium Chloride Symporters/physiology , Uromodulin , gamma-Glutamyltransferase/metabolism
13.
FASEB J ; 20(12): 1974-81, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17012249

ABSTRACT

We report here the application of a previously described method to directly determine the CO2 permeability (P(CO2)) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1-expressing Xenopus laevis oocytes. This method measures the exchange of (18)O between CO2, HCO3(-), and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pH(S)) transients caused by the dominant effect of entry of CO2 vs. HCO3(-) into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces P(CO2) from the normal value of 0.15 +/- 0.08 (SD; n=85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases P(CO2) 2-fold and doubles the alkaline pH(S) gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces P(CO2) of RBCs solely by action on AQP1 as it has no effect in AQP1-deficient RBCs. 3) P(CO2) determinations of RBCs and pH(S) measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS-sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high P(CO2) of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total P(CO2).


Subject(s)
Aquaporin 1/metabolism , Carbon Dioxide/metabolism , Erythrocyte Membrane/metabolism , Animals , Bicarbonates/metabolism , Biological Transport , Cell Membrane Permeability/physiology , Erythrocyte Membrane/physiology , Humans , Hydrogen-Ion Concentration , Oocytes , Oxygen Isotopes/metabolism , Xenopus laevis
14.
Pflugers Arch ; 453(1): 107-16, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16847696

ABSTRACT

TWIK-1, a member of the two-pore domain K(+) channel family, is expressed in brain, kidney, and lung. The aim of this study was to examine the effect of loss of TWIK-1 on the renal cortical collecting duct. Ducts were isolated from wild-type and TWIK-1 knockout mice by enzyme digestion and whole-cell clamp obtained via the basolateral membrane. Current- and voltage-clamp approaches were used to examine K(+) conductances. No difference was observed between intercalated cells from wild-type or knockout ducts. In contrast, knockout principal cells were hyperpolarized compared to wild-type cells and had a reduced membrane conductance. This was a consequence of a fall in a barium-insensitive, quinidine-sensitive conductance (G (Quin)). G (Quin) demonstrated outward rectification and had a relatively low K(+) to Na(+) selectivity ratio. Loss of G (Quin) would be expected to lead to the hyperpolarization observed in knockout ducts by increasing fractional K(+) conductance and Na(+) uptake by the cell. Consistent with this hypothesis, knockout ducts had an increased diameter in comparison to wild-type ducts. These data suggest that G (Quin) contributes to the resting membrane potential in the cortical collecting duct and that a fall in G (Quin) could be an adaptive response in TWIK-1 knockout ducts.


Subject(s)
Kidney Tubules, Collecting/metabolism , Potassium Channels, Tandem Pore Domain/metabolism , Potassium/metabolism , Adaptation, Physiological , Animals , Barium/metabolism , Cations/metabolism , Down-Regulation , Female , In Vitro Techniques , Male , Mice , Mice, Knockout , Patch-Clamp Techniques , Quinidine , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
15.
Eur J Clin Nutr ; 60(1): 77-84, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16118648

ABSTRACT

OBJECTIVE: Ghrelin and leptin play a role in control of food intake and adiposity but mechanisms regulating these hormones in man are poorly defined and evidence that dietary fats may have adverse effects is inconclusive. We investigated whether high-fat meals, which differed in saturated fatty acid (SFA) content acutely modified these hormones. DESIGN: Randomised, double-blind, crossover trial. A high-fat (HF) test meal (59 +/- 4 g fat; 71% of energy as fat) was given for breakfast on two occasions. Meals comprised either high (approximately 70:30) or low (approximately 55:45) saturated:unsaturated fatty acid (SFA:USFA) ratio. Fasting and postprandial measurements of serum total ghrelin (RIA), leptin (enzyme-linked immunosorbent assay (ELISA)) and insulin (RIA) were made over 6 h. Postprandial measurements were also made at 10 and 24 h following a fat-exclusion lunch, snack and dinner. SUBJECTS: A total of 18 lean, healthy men. RESULTS: There was no significant effect of the fatty meal (time, P > 0.05), nor a differential effect of SFA:USFA ratio (treatment*time, P > 0.05) on ghrelin over 6h. Leptin decreased in response to both HF treatments (time, P < 0.001) but increased SFA content did not further inhibit hormone secretion (treatment*time, P > 0.05). There was no significant correlation between ghrelin or leptin and circulating insulin (P>0.05). CONCLUSION: We conclude that HF diets may adversely effect serum leptin, although the circadian decrease may account in part for this response. Increasing dietary SFAs had no deleterious effects on leptin or total ghrelin.


Subject(s)
Dietary Fats, Unsaturated/administration & dosage , Dietary Fats/administration & dosage , Leptin/blood , Peptide Hormones/blood , Adult , Area Under Curve , Circadian Rhythm/physiology , Cross-Over Studies , Dietary Fats/metabolism , Dietary Fats, Unsaturated/metabolism , Double-Blind Method , Fasting , Ghrelin , Humans , Insulin/blood , Male , New Zealand , Postprandial Period
16.
Acta Diabetol ; 42(4): 162-70, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16382303

ABSTRACT

The Zucker diabetic fatty (ZDF) rat is a commonly used animal model of type 2 diabetes yet complete descriptions of insulin resistance in this model are limited. We present a full characterisation of in vivo insulin resistance in obese (fa/fa) animals compared to lean (+/?) littermates. Anaesthetised, ten-week old, obese ZDF rats and their lean littermates underwent a hyperinsulinaemic euglycaemic glucose clamp. Compared with lean littermates, obese ZDF rats required an 89% lower glucose infusion rate to maintain euglycaemia and showed a 35% decrease in peripheral glucose disposal. Insulin-stimulated glucose uptake (R(g')) in obese animals was also significantly less in all skeletal muscles studied. R(g') in cardiac and white adipose tissue was not different between the two groups. Total glycogen content in skeletal and cardiac muscle was significantly less in obese animals, while total glycogen content in the liver was significantly greater than in lean littermates. Glycogen synthesis was also decreased in skeletal muscle of obese animals. Compared with lean animals, total triglyceride content was significantly greater in skeletal muscle, heart and liver of obese ZDF rats. Obese animals also showed significantly increased glucose incorporation into lipid in all of these tissues, indicating an increase in lipogenesis. Collectively, these results provide an integrated characterisation of in vivo insulin resistance in obese ZDF rats and a direct comparison with lean littermates.


Subject(s)
Insulin Resistance , Obesity/genetics , Thinness/genetics , Animals , Blood Glucose/metabolism , Glucose/metabolism , Glucose Clamp Technique , Insulin/blood , Insulin/pharmacology , Kinetics , Male , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Obesity/metabolism , Rats , Rats, Zucker , Thinness/metabolism
17.
Eur J Clin Nutr ; 58(5): 819-27, 2004 May.
Article in English | MEDLINE | ID: mdl-15116086

ABSTRACT

OBJECTIVE: To investigate the effect of moderate changes in dietary fatty acid profile on postprandial risk factors for cardiovascular disease (CVD). DESIGN: Double-blind, randomised, crossover, intervention trial. SETTING: : University of Auckland Human Nutrition Unit, New Zealand. SUBJECTS: A total of 18 lean healthy men. INTERVENTION: A dairy butter fat modified to reduce the saturated:unsaturated fatty acid ratio and a conventional high saturated butter fat were given on two separate occasions as a high-fat test meal (59+/-4 g fat; 71 en% fat) at breakfast. A fat exclusion lunch, dinner and snacks were also given. Blood samples were collected at 0 (baseline), 1, 3, 6, 10 and 24 h. RESULTS: Maximum peak in total triacylglycerol (TAG) occurred 3 h postprandially and was highest on modified treatment (diet, P<0.05) due predominantly to increased TAG within the chylomicron-rich fraction. Transient peaks in total-, LDL- and HDL-cholesterol occurred postprandially, but did not differ between dietary treatments (P>0.05). There were no differential effects of diet on postprandial free fatty acids, apo A, apo B, glucose, insulin, amylin or haemostatic clotting factors (P>0.05). CONCLUSIONS: In a group of healthy young men, replacement of 16% of total saturated fatty acids by mono- and polyunsaturated fats within a dairy lipid did not induce postprandial changes in CVD risk that may be considered beneficial for health. SPONSORSHIP: Fonterra, Wellington; New Zealand.


Subject(s)
Butter , Cardiovascular Diseases/blood , Cholesterol/blood , Dietary Fats, Unsaturated/administration & dosage , Dietary Fats/administration & dosage , Lipids/blood , Cholesterol, HDL/blood , Cholesterol, LDL/blood , Cross-Over Studies , Dietary Fats/metabolism , Dietary Fats, Unsaturated/metabolism , Double-Blind Method , Humans , Male , Postprandial Period , Risk Factors , Triglycerides/blood
18.
Diabetologia ; 46(4): 574-8, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12679865

ABSTRACT

AIMS/HYPOTHESIS: Amylin gene mutations are known to predispose Chinese and Japanese subjects, but not Caucasian subjects, to Type 2 diabetes. New Zealand Maori, who have a high prevalence of Type 2 diabetes, have genetic origins in South East Asia. Amylin gene mutations could therefore predispose New Zealand Maori to Type 2 diabetes. METHODS: The amylin gene was screened for mutations in the proximal promoter region, exons 1 and 2, intron 1, and coding region of exon 3 by polymerase chain reaction amplification and direct sequencing of 131 Type 2 diabetic Maori patients and 258 non-diabetic Maori control subjects. RESULTS: We identified three new amylin gene mutations: two mutations in the promoter region (-215T>G and -132G>A) and a missense mutation in exon 3 (Q10R). The -215T>G mutation was observed in 5.4% of Type 2 Maori diabetic patients and predisposed the carrier to diabetes with a relative risk of 7.23. The -215T>G mutation was inherited with a previously described amylin promoter polymorphism (-230A>C) in 3% of the Maori with Type 2 diabetes, which suggests linkage disequilibrium exists between these two mutations. The -230A>C polymorphism on its own, however, was not associated with Type 2 diabetes in Maori subjects. The -132G>A and Q10R mutations were both observed in 0.76% of Type 2 diabetic patients and were absent in non-diabetic subjects. CONCLUSION/INTERPRETATION: The amylin gene mutations identified in this study are associated with Type 2 diabetes in 7% of Maori. Amylin is likely to be an important susceptibility gene for Type 2 diabetes in Maori people.


Subject(s)
Amyloid/genetics , Diabetes Mellitus, Type 2/ethnology , Diabetes Mellitus, Type 2/genetics , Mutation/genetics , Promoter Regions, Genetic/genetics , Exons/genetics , Female , Gene Frequency/genetics , Humans , Islet Amyloid Polypeptide , Male , Middle Aged , New Zealand/ethnology
19.
Heart ; 89(1): e4, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12482814

ABSTRACT

Coronary artery aneurysms are uncommon and may be complicated by rupture, thromboembolic phenomenon, and more rarely fistulation into one of the cardiac chambers. This case report highlights the difficulty in making a preoperative diagnosis of a coronary artery aneurysm that has fistulated into the right atrium, and lists possible differential diagnoses.


Subject(s)
Aortic Aneurysm/diagnosis , Coronary Aneurysm/diagnosis , Sinus of Valsalva , Vascular Fistula/diagnosis , Adult , Coronary Aneurysm/complications , Coronary Aneurysm/surgery , Diagnosis, Differential , Female , Heart Atria , Humans , Vascular Fistula/complications , Vascular Fistula/surgery
20.
J Cardiovasc Surg (Torino) ; 43(6): 841-2, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12483176

ABSTRACT

Abnormal connections between the ascending aorta and the cardiac chambers are rare. Most are due to ruptured aneurysms of the sinus of Valsalva. Congenital fistulae between the aorta and left atrium are described. We report a case of native tricuspid valve endocarditis associated with a fistula between the right atrium and the aorta.


Subject(s)
Aortic Diseases/diagnosis , Cardiac Surgical Procedures/methods , Endocarditis, Bacterial/diagnosis , Staphylococcal Infections/diagnosis , Vascular Fistula/diagnosis , Angiography , Aortic Diseases/complications , Aortic Diseases/surgery , Cardiac Catheterization , Echocardiography, Transesophageal/methods , Endocarditis, Bacterial/complications , Endocarditis, Bacterial/surgery , Female , Follow-Up Studies , Humans , Middle Aged , Risk Assessment , Sinus of Valsalva , Staphylococcal Infections/complications , Staphylococcal Infections/surgery , Treatment Outcome , Tricuspid Valve , Vascular Fistula/complications , Vascular Fistula/surgery
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