Lesley L. Walker

Mice lacking angiotensin-converting enzyme have increased energy expenditure, with reduced fat mass and improved glucose clearance

In addition to its role in the storage of fat, adipose tissue acts as an endocrine organ, and it contains a functional renin-angiotensin system (RAS). Angiotensin-converting enzyme (ACE) plays a key role in the RAS by converting angiotensin I to the bioactive peptide angiotensin II (Ang II). In the present study, the effect of targeting the RAS in body energy homeostasis and glucose tolerance was determined in homozygous mice in which the gene for ACE had been deleted (ACE−/−) and compared with wild-type littermates. Compared with wild-type littermates, ACE−/− mice had lower body weight and a lower proportion of body fat, especially in the abdomen. ACE−/− mice had greater fed-state total energy expenditure (TEE) and resting energy expenditure (REE) than wild-type littermates. There were pronounced increases in gene expression of enzymes related to lipolysis and fatty acid oxidation (lipoprotein lipase, carnitine palmitoyl transferase, long-chain acetyl CoA dehydrogenase) in the liver of ACE−/− mice and also lower plasma leptin. In contrast, no differences were detected in daily food intake, activity, fed-state plasma lipids, or proportion of fat excreted in fecal matter. In conclusion, the reduction in ACE activity is associated with a decreased accumulation of body fat, especially in abdominal fat depots. The decreased body fat in ACE−/− mice is independent of food intake and appears to be due to a high energy expenditure related to increased metabolism of fatty acids in the liver, with the additional effect of increased glucose tolerance.

Change in Salt Intake Affects Blood Pressure of Chimpanzees. Implications for Human Populations

Background— Addition of up to 15.0 g/d salt to the diet of chimpanzees caused large rises in blood pressure, which reversed when the added salt was removed. Effects of more modest alterations to sodium intakes in chimpanzees, akin to current efforts to lower sodium intakes in the human population, are unknown. Methods and Results— Sodium intakes were altered among 17 chimpanzees in Franceville, Gabon, and 110 chimpanzees in Bastrop, Tex. In Gabon, chimpanzees had a biscuit diet of constant nutrient composition except that the sodium content was changed episodically over 3 years from 75 to 35 to 120 mmol/d. In Bastrop, animals were divided into 2 groups; 1 group continued on the standard diet of 250 mmol/d sodium for 2 years, and sodium intake was halved for the other group. Lower sodium intake was associated with lower systolic, diastolic, and mean arterial blood pressures in Gabon (2-tailed P<0.001, unadjusted and adjusted for age, sex, and baseline weight) and Bastrop (P<0.01, unadjusted; P=0.08 to 0.10, adjusted), with no threshold down to 35 mmol/d sodium. For systolic pressure, estimates were −12.7 mm Hg (95% confidence interval, −16.9 to −8.5, adjusted) per 100 mmol/d lower sodium in Gabon and −10.9 mm Hg (95% confidence interval, −18.9 to −2.9, unadjusted) and −5.7 mm Hg (95% confidence interval, −12.2 to 0.7, adjusted) for sodium intake lower by 122 mmol/d in Bastrop. Baseline systolic pressures higher by 10 mm Hg were associated with larger falls in systolic pressure by 4.3/2.9 mm Hg in Gabon/Bastrop per 100 mmol/d lower sodium. Conclusions— These findings from an essentially single-variable experiment in the species closest to Homo sapiens with high intakes of calcium and potassium support intensified public health efforts to lower sodium intake in the human population.

The relaxin gene-knockout mouse : A model of progressive fibrosis

Abstract: Relaxin is well known for its actions on collagen remodeling. To improve our understanding of the physiologic role(s) of relaxin, the relaxin gene‐knockout (RLX‐KO) mouse was established by our group and subsequently phenotyped. Pregnant RLX‐KO mice underwent inadequate development of the pubic symphysis as well as the mammary glands and nipples compared to wild‐type mice, thus preventing lactation. Later studies showed that these deficiencies were associated with increased collagen, primarily in the nipple and vagina. Analysis of male RLX‐KO mice also demonstrated inadequate reproductive tract development. The testis, epididymis, and prostate of RLX‐KO mice showed delayed tissue maturation and growth associated with increased collagen deposition. In nonreproductive tissues, an age‐related increase in interstitial collagen (fibrosis) was also detected in the lung, heart, and kidneys of RLX‐KO mice and was associated with organ dysfunction. From 6‐9 months of age and onwards, all organs of RLX‐KO mice, particularly male mice, underwent progressive increases in tissue weight and collagen content (all P <.05) compared with wild‐type animals. The increased fibrosis contributed to bronchiole epithelium thickening and alveolar congestion (lung), atrial hypertrophy and increased ventricular chamber stiffness (heart) in addition to glomerulosclerosis (kidney). Treatment of RLX‐KO mice with recombinant human relaxin in early and developed stages of fibrosis caused the reversal of collagen deposition in the lung, heart, and kidneys. Together, these findings suggest that relaxin is a naturally occurring inhibitor of collagen deposition during normal development, aging, and pregnancy and can be used to prevent the progression of fibrosis.

Relation of addiction genes to hypothalamic gene changes subserving genesis and gratification of a classic instinct, sodium appetite

Sodium appetite is an instinct that involves avid specific intention. It is elicited by sodium deficiency, stress-evoked adrenocorticotropic hormone (ACTH), and reproduction. Genome-wide microarrays in sodium-deficient mice or after ACTH infusion showed up-regulation of hypothalamic genes, including dopamine- and cAMP-regulated neuronal phosphoprotein 32 kDa (DARPP-32), dopamine receptors-1 and -2, α-2C- adrenoceptor, and striatally enriched protein tyrosine phosphatase (STEP). Both DARPP-32 and neural plasticity regulator activity-regulated cytoskeleton associated protein (ARC) were up-regulated in lateral hypothalamic orexinergic neurons by sodium deficiency. Administration of dopamine D1 (SCH23390) and D2 receptor (raclopride) antagonists reduced gratification of sodium appetite triggered by sodium deficiency. SCH23390 was specific, having no effect on osmotic-induced water drinking, whereas raclopride also reduced water intake. D1 receptor KO mice had normal sodium appetite, indicating compensatory regulation. Appetite was insensitive to SCH23390, confirming the absence of off-target effects. Bilateral microinjection of SCH23390 (100 nM in 200 nL) into rats’ lateral hypothalamus greatly reduced sodium appetite. Gene set enrichment analysis in hypothalami of mice with sodium appetite showed significant enrichment of gene sets previously linked to addiction (opiates and cocaine). This finding of concerted gene regulation was attenuated on gratification with perplexingly rapid kinetics of only 10 min, anteceding significant absorption of salt from the gut. Salt appetite and hedonic liking of salt taste have evolved over >100 million y (e.g., being present in Metatheria). Drugs causing pleasure and addiction are comparatively recent and likely reflect usurping of evolutionary ancient systems with high survival value by the gratification of contemporary hedonic indulgences. Our findings outline a molecular logic for instinctive behavior encoded by the brain with possible important translational–medical implications.

Regulation of renal tubular sodium transport by angiotensin II and atrial natriuretic factor.

1 The effects of angiotensin II (AngII) on water and electrolyte transport are biphasic and dose‐dependent, such that low concentrations (10−12 to 10−9 mol/L) stimulate reabsorption and high concentrations (10−7 to 10−6 mol/L) inhibit reabsorption. Similar dose‐response relationships have been obtained for luminal and peritubular addition of AngII. 2 The cellular responses to AngII are mediated via AT1 receptors coupled via G‐regulatory proteins to several possible signal transduction pathways. These include the inhibition of adenylyl cyclase, activation of phospholipases A2, C or D and Ca2+ release in response to inositol‐1,4,5,‐triphosphate or following Ca2+ channel opening induced by the arachidonic acid metabolite 5,6,‐epoxy‐eicosatrienoic acid. In the brush border membrane, transduction of the AngII signal involves phospholipase A2, but does not require second messengers. 3 Angiotensin II affects transepithelial sodium transport by modulation of Na+/H+ exchange at the luminal membrane and Na+/HCO3 cotransport, Na+/K+‐ATPase activity and K+ conductance at the basolateral membrane. 4 Atrial natriuretic factor (ANF) does not appear to affect proximal tubular sodium transport directly, but acts via specific receptors on the basolateral and brush border membranes to raise intracellular cGMP levels and inhibit AngII‐stimulated transport. 5 It is concluded that there is a receptor‐mediated action of ANF on proximal tubule reabsorption acting via elevation of cGMP to inhibit AngII‐stimulated sodium transport. This effect is exerted by peptides delivered at both luminal and peritubular sides of the epithelium and provides a basis for the modulation by ANF of proximal glomerulotubular balance. The evidence reviewed supports the concept that in the proximal tubule, AngII and ANF act antagonistically in their roles as regulators of extracellular fluid volume.

Angiotensinogen and angiotensin-converting enzyme gene copy number and angiotensin and bradykinin peptide levels in mice.

Objective To test the hypothesis that changes in gene expression that may accompany angiotensinogen (AGT) and angiotensin-converting enzyme (ACE) gene polymorphism cause alteration in angiotensin and bradykinin peptide levels. Design Mice with one or two genes for AGT and ACE allow assessment of the effects of modest alteration in AGT and ACE gene expression on angiotensin and bradykinin peptide levels. Methods Angiotensin and bradykinin peptides were measured in the blood, kidney, heart, lung, adrenal, brain, and aorta of mice that were either wild-type (+/+), heterozygous (+/−) or null (−/−) for either the AGT or ACE gene. Results Angiotensin I and angiotensin II were not detectable in blood or tissues of AGT −/− mice, which had increased bradykinin levels in kidney and lung. ACE −/− mice had markedly reduced angiotensin II levels and increased bradykinin levels in blood and tissues. However, despite reduced AGT and ACE gene expression, angiotensin and bradykinin peptide levels in AGT and ACE +/− mice were no different from the levels in wild-type mice. Conclusion Although the AGT and ACE genes are fundamental determinants of angiotensin and bradykinin peptide levels, compensatory mechanisms attenuate the effect of modest change in AGT and ACE gene expression on the levels of these peptides. Identification of these compensatory mechanisms may provide new candidate genes for investigation in humans.

The effects of angiotensin II on blood perfusion in the rat renal papilla

1 Systemic infusion of angiotensin II (AII) increased papillary blood perfusion (PBP) measured by laser‐Doppler flowmetry in rats, aged about 5 weeks. 2 The mechanisms involved in this response were determined by infusion of AII in the presence of systemic doses of losartan (a type 1 AII receptor antagonist), HOE‐140 (a bradykinin B2 receptor antagonist), and an inhibitor of NO production ‐ Nω ‐nitro‐L‐arginine (NOLA). 3 Mean arterial blood pressure (MAP) and PBP increased in a dose‐dependent manner in response to intravenous infusions of AII. Infusion of losartan abolished these responses to AII but HOE‐140 was without effect. Infusion of NOLA abolished the increase in PBP but did not affect the pressor response to AII. Systemic infusion of sodium nitroprusside restored the response to AII in experiments with NOLA infusion. 4 The results indicate that the increase in PBP caused by AII is mediated via angiotensin AT1 receptors and does not involve bradykinin B2 receptors. The AII‐induced increase in PBP is dependent upon the presence of NO, thus providing a mechanism for maintenance of papillary perfusion in the face of generalized renal vasoconstriction due to AII.

Osmoregulatory fluid intake but not hypovolemic thirst is intact in mice lacking angiotensin

Water intakes in response to hypertonic, hypovolemic, and dehydrational stimuli were investigated in mice lacking angiotensin II as a result of deletion of the angiotensinogen gene (Agt-/- mice), and in C57BL6 wild-type (WT) mice. Baseline daily water intake in Agt-/- mice was approximately threefold that of WT mice because of a renal developmental disorder of the urinary concentrating mechanisms in Agt-/- mice. Intraperitoneal injection of hypertonic saline (0.4 and 0.8 mol/l NaCl) caused a similar dose-dependent increase in water intake in both Agt-/- and WT mice during the hour following injection. As well, Agt-/- mice drank appropriate volumes of water following water deprivation for 7 h. However, Agt-/- mice did not increase water or 0.3 mol/l NaCl intake in the 8 h following administration of a hypovolemic stimulus (30% polyethylene glycol sc), whereas WT mice increased intakes of both solutions during this time. Osmoregulatory regions of the brain [hypothalamic paraventricular and supraoptic nuclei, median preoptic nucleus, organum vasculosum of the lamina terminalis (OVLT), and subfornical organ] showed an increased number of neurons exhibiting Fos-immunoreactivity in response to intraperitoneal hypertonic NaCl in both Agt-/- mice and WT mice. Polyethylene glycol treatment increased Fos-immunoreactivity in the subfornical organ, OVLT, and supraoptic nuclei in WT mice but only increased Fos-immunoreactivity in the supraoptic nucleus in Agt-/- mice. These data show that brain angiotensin is not essential for the adequate functioning of neural pathways mediating osmoregulatory thirst. However, angiotensin II of either peripheral or central origin is probably necessary for thirst and salt appetite that results from hypovolemia.

The effect of urocortin on ingestive behaviours and brain Fos immunoreactivity in mice

The influence of urocortin (UCN) on ingestive behaviours and brain neural activity, as measured immunohistochemically by the presence of Fos protein, was determined in mice. Rat UCN was administered by continuous intracerebroventricular (ICV) or subcutaneous (SC) infusion. ICV infusion of UCN (100 ng/h, 14 days) transiently reduced daily food and water intakes (days 1–4) but body weight was reduced from day 2 into the post‐infusion period. Sodium intake was reduced from day 3 to the end of infusion. SC infusion of UCN caused similar but smaller reductions in food and water intakes and body weight, without change in sodium intake. In separate experiments, Fos immunoreactivity was increased in several brain nuclei known to be involved in the control of body fluid and energy homeostasis, e.g. central nucleus of the amygdala, median preoptic nucleus, bed nucleus of the stria terminalis and arcuate nucleus. Increased Fos expression was similar for ICV and SC infusions when measured on days 2–3 or 6–7 of infusion. In conclusion, increases of brain activity by UCN may be associated with stimulation of adrenocorticotrophic hormone release and sympathetic nervous activity, but increases may also indicate suppression of ingestive behaviours by stimulating central inhibitory mechanisms located in areas known to control body fluid and energy homeostasis.

Angiotensin-converting enzyme (ACE) interacts with dopaminergic mechanisms in the brain to modulate prepulse inhibition in mice

A renin-angiotensin system, separate to that in the periphery, has been found in the brain. Angiotensin-converting enzyme (ACE) is crucial in the synthesis of angiotensin II, breakdown of bradykinin and the hydrolysis of several other neuropeptides such as enkephalin, substance P, dynorphin and neurotensin. Changes in the levels of ACE have been found in brains of schizophrenia patients, suggesting an involvement of ACE in the illness which awaits further investigation. Prepulse inhibition (PPI) has been suggested to be an operational measure of sensorimotor gating and is disrupted in patients with schizophrenia. We found that ACE knockout mice have increased startle responses but no differences in baseline PPI compared to wildtype controls. Treatment with the dopamine receptor agonist, apomorphine, or the dopamine-releasing drug, amphetamine, produced significant disruption of PPI in control mice but not in ACE knockout mice. Pretreatment with the ACE inhibitor, captopril, which itself did not affect PPI, caused a reduction in the effect of apomorphine on PPI, similar to that seen in the ACE knockout mice. These data suggest an important role of ACE substrates in modulating dopaminergic mechanisms involved in PPI. Further studies are needed to ascertain if angiotensin or other neuropeptides are involved in these interactions and to investigate the neurochemical mechanism behind these effects.