J. M. Overton

Kv1.3 Channel Gene-Targeted Deletion Produces “Super-Smeller Mice” with Altered Glomeruli, Interacting Scaffolding Proteins, and Biophysics

Mice with gene-targeted deletion of the Kv1.3 channel were generated to study its role in olfactory function. Potassium currents in olfactory bulb mitral cells from Kv1.3 null mice have slow inactivation kinetics, a modified voltage dependence, and a dampened C-type inactivation and fail to be modulated by activators of receptor tyrosine signaling cascades. Kv1.3 deletion increases expression of scaffolding proteins that normally regulate the channel through protein-protein interactions. Kv1.3-/- mice have a 1,000- to 10,000-fold lower threshold for detection of odors and an increased ability to discriminate between odorants. In accordance with this heightened sense of smell, Kv1.3-/- mice have glomeruli or olfactory coding units that are smaller and more numerous than those of wild-type mice. These data suggest that Kv1.3 plays a far more reaching role in signal transduction, development, and olfactory coding than that of the classically defined role of a potassium channel-to shape excitability by influencing membrane potential.

Behavioral and physiologic responses to caloric restriction in mice

The purpose of the review is to highlight the influences of ambient temperature (T(a)) and caloric restriction (CR) on metabolism, cardiovascular function and behavior in mice. Standard vivarium ambient temperatures (T(a)?23 degrees C) are a mild cold stress for mice requiring elevated metabolic rate and food intake. Increasing T(a) into the zone of thermoneutrality (TMN?29-33 degrees C) markedly reduces food intake, metabolic rate, heart rate (HR) and blood pressure in mice. Mice are members of a diverse, yet unique group of homeothermic animals that respond to thermal and energetic challenges by allowing body temperature (T(b)) to fall to less than 31 degrees C, a condition known as torpor. In mice housed at standard T(a), torpor is induced by a single night of fasting or a few days of CR. The mechanisms responsible for initiating torpor are related to reduced caloric availability, but do not require leptin. Mice housed at TMN and subjected to CR exhibit physiologic reductions in metabolic rate and HR, but do not appear to enter torpor. Finally, mice exhibit differential locomotor activity responses during CR that depends on T(a). At standard T(a), mice display increased light-phase home-cage activity with CR. This response is virtually eliminated when CR is performed at TMN. We suggest that researchers using mice to investigate energy homeostasis and cardiovascular physiology carefully consider the influence of T(a) on physiology and behavior.

Phenotyping small animals as models for the human metabolic syndrome: thermoneutrality matters

It is standard practice in preclinical biomedical research to house mammalian model organisms at an ambient temperature substantially below the thermoneutral zone. These experimental studies are performed using animals that are chronically challenged by mild cold stress. This condition increases food intake, metabolic rate, sympathetic activity, blood pressure and heart rate. Furthermore, this condition alters the behavioral and physiological responses to drug administration, energy restriction and overfeeding. This paper will review these observations, which must be understood in the context of phenotyping small mammals to enhance our understanding of the biology of human disease.

Diet-induced obesity and cardiovascular regulation in C57BL/6J mice.

1. In the present study, we determined the effect of diet‐induced obesity on cardiovascular and metabolic regulation in mice at standard laboratory temperatures (ambient temperature (Ta) = 22°C) and during exposure to thermoneutrality (Ta = 30°C).

Kv1.3 gene-targeted deletion alters longevity and reduces adiposity by increasing locomotion and metabolism in melanocortin-4 receptor-null mice

Objective:Gene-targeted deletion of the voltage-gated potassium channel, Kv1.3, results in ‘super-smeller’ mice that have altered firing patterns of mitral cells in the olfactory bulb, modified axonal targeting to glomerular synaptic units, and behaviorally have an increased ability to detect and discriminate odors. Moreover, the Kv1.3-null mice weighed less than their wild-type counterparts, have modified ingestive behaviors, and are resistant to fat deposition following a moderately high-fat dietary regime. In this study, we investigate whether or not gene-targeted deletion of Kv1.3 (Shaker family member) can abrogate weight gain in a genetic model of obesity, the melanocortin-4 receptor-null mouse (MC4R-null).Design:Mice with double gene-targeted deletions of Kv1.3 and MC4R were generated by interbreeding Kv1.3 (Kv)- and MC4R-null mouse lines to homozygosity. Developmental weights, nose to anus length, fat pad weight, fasting serum chemistry, oxygen consumption, carbon dioxide respiration, locomotor activity and caloric intake were monitored in control, Kv-null, MC4R-null and Kv/MC4R-null mice. Physiological and metabolic profiles were acquired at postnatal day 60 (P60) in order to explore changes linked to body weight at the reported onset of obesity in the MC4R-null model.Results:Gene-targeted deletion of Kv1.3 in MC4R-null mice reduces body weight by decreasing fat deposition and subsequent fasting leptin levels, without changing the overall growth, fasting blood glucose or serum insulin. Gene-targeted deletion of Kv1.3 in MC4R-null mice significantly extended lifespan and increased reproductive success. Basal or light-phase mass-specific metabolic rate and locomotor activity were not affected by genetic deletion of Kv1.3 in MC4R-null mice but dark-phase locomotor activity and mass-specific metabolism were significantly increased resulting in increased total energy expenditure.Conclusions:Gene-targeted deletion of Kv1.3 can reduce adiposity and total body weight in a genetic model of obesity by increasing both locomotor activity and mass-specific metabolism.

Central Leptin Infusion Attenuates the Cardiovascular and Metabolic Effects of Fasting in Rats

The role of reduced leptin signaling in the regulation of cardiovascular responses to negative energy balance is not known. We tested the hypothesis that central infusion of leptin would attenuate the cardiovascular and metabolic responses to fasting. Male Sprague-Dawley rats, instrumented with telemetry devices and intracerebroventricular cannulas, were housed in metabolic chambers for continuous (24 hours) measurement of dark-phase (active) and light-phase (inactive) mean arterial pressure, heart rate, oxygen consumption, and respiratory quotient. Rats received central infusions of either saline (0.5 &mgr;L/h) or leptin (42 ng/h) for 6 days through osmotic pumps and were either fed ad libitum or were fasted for 48 hours followed by refeeding for 4 days. In ad lib animals, continuous intracerebroventricular leptin infusion significantly reduced caloric intake, body weight, and respiratory quotient compared with saline controls while having no effect on mean arterial pressure or heart rate. Fasting reduced mean arterial pressure, heart rate, oxygen consumption, and respiratory quotient in rats receiving saline infusions. Fasting-induced reductions in mean arterial pressure were specific to the active phase and were not attenuated by central leptin infusion. In contrast, intracerebroventricular leptin, at a dose that had no cardiovascular effects in ad lib control animals, completely prevented fasting-induced decreases in light-phase heart rate and oxygen consumption and blunted fasting-induced reductions in dark-phase heart rate and oxygen consumption. The results are consistent with the hypothesis that reductions in central leptin signaling contribute to the integrated cardiovascular and metabolic responses to acute caloric deprivation.

Cardiovascular and metabolic responses to fasting and thermoneutrality in Ay mice.

Several lines of evidence support a role for reduced melanocortin signaling in the regulation of metabolic rate and cardiovascular function during negative energy balance. We tested the hypothesis that agouti yellow (B6.Cg-A(y)) mice would exhibit blunted physiologic responses to fasting and thermoneutrality. Male B6.Cg-A(y) mice (A(y); n=11, 34+/-2 g) and lean B6 littermates (B6; n=7, 26+/-2 g) were implanted with telemetry devices and housed in metabolic chambers (T(a)=23 degrees C) to determine the effects of a 24-h fasting and exposure to thermoneutrality (T(a)=30 degrees C) on mean arterial pressure (MAP), heart rate (HR), AP and HR variability (time and frequency domain), oxygen consumption (VO(2)), and locomotor activity. A(y) mice exhibited elevated baseline light-period MAP (A(y): 113+/-4; B6: 99+/-3 mm Hg) and VO(2) (A(y): 1.82+/-0.08 vs. B6: 1.45+/-0.13 ml/min) with no difference in HR (A(y): 530+/-12 vs. B6: 548+/-19 bpm). At 12-24 h after food removal, A(y) mice displayed normal fasting-induced bradycardia (A(y): -106+/-12; B6: -117+/-19 bpm) and reduction in VO(2) (A(y): -0.19+/-0.04 vs. B6: -0.28+/-0.05 ml/min), but with augmented hypotension (A(y): -9+/-2 vs. B6: -0.5+/-2 mm Hg) and blunted hyperactivity (A(y): 27+/-23 vs. B6: 122+/-42 m/11 h). Fasting was associated with increased HR variability in both time and frequency domain in B6 but not A(y) mice. Exposure to thermoneutrality produced comparable reductions in MAP, HR, and VO(2) in both strains. We conclude that inhibition of melanocortin signaling is not requisite for, but participates in, the metabolic and cardiovascular responses to negative energy balance.

Combined amylin-leptin treatment lowers blood pressure and adiposity in lean and obese rats.

Objective:To examine the cardiovascular effects of combined amylin (AMN) and leptin (LEP) treatment in lean and obese rats.Research design:Rats were instrumented for telemetry and given LEP (300 μg kg–1 day–1), AMN (100 μg kg–1 day–1), AMN+LEP or vehicle (VEH; 0.9% normal saline) via a subcutaneous mini-osmotic pump for 7 days. The VEH group was subdivided into ad libitum fed and pair-fed to the amount of food AMN+LEP animals ate daily. Rats were housed in metabolic chambers for analysis of cardiovascular physiology and metabolism.Subjects:Male Fisher 344 × Brown Norway (FBNF1; Harlan; age=3–5 months; n=72) rats were placed on standard rodent chow (LEAN, n=41) or moderately high-fat diet (OBESE; n=31) to produce obesity.Results:AMN+LEP potently reduced food intake (LEAN: 57% OBESE: 59%) and abdominal fat mass (LEAN: 56% OBESE: 41%). Pair-fed rats displayed bradycardia and metabolic suppression. In contrast, AMN+LEP increased heart rate and oxygen consumption above levels in LEP or AMN-treated rats. LEP reduced blood pressure in both lean and obese rats but AMN had no effect. LEP-induced reductions in blood pressure were not altered by AMN+LEP treatment. Thus, AMN+LEP treatment decreased food intake, body fat and blood pressure in lean and obese rats.Conclusion:We conclude that the potent anti-adiposity actions of AMN+LEP are due in part to prevention of the bradycardia and metabolic suppression typically observed with negative energy balance. Furthermore, the hypotensive actions of peripheral LEP treatment are observable in spite of the potent AMN+LEP activation of anorexic and thermogenic mechanisms in the central nervous system.

Electrophysiological and behavioral phenotype of insulin receptor defective mice

The olfactory bulb expresses one of the highest levels of insulin found in the brain. A high level of expression of the concomitant insulin receptor (IR) kinase is also retained in this brain region, even in the adult. We have previously demonstrated in a heterologous system that insulin modulates the voltage-dependent potassium channel, Kv1.3, through tyrosine phosphorylation of three key residues in the amino and carboxyl terminus of the channel protein. Phosphorylation also induces current suppression of the Kv1.3-contributed current in cultured olfactory bulb neurons (OBNs) of rodents. In order to explore the behavioral importance of this kinase-induced modulation of the channel for the olfactory ability of the animal, mice with a targeted-gene deletion of the insulin receptor were electrophysiologically and behaviorally characterized. Mice heterozygous for the insulin receptor kinase (IR+/-) gene performed the same as wild-type (+/+) mice when challenged with a traditional, non-learning-based task to test gross anosmia. There was also no significant difference across the two genotypes in tests designed to measure exploratory behavior or in a battery of systems physiology experiments designed to assess metabolic energy usage (locomotion, ingestive behaviors, weight, oxygen consumption, and respiratory quotient). Object memory recognition tests suggest that IR+/- mice have an impairment in recognition of familiarized objects; IR+/- mice demonstrate poor performance for both short-term (1 h) and long-term (24 h) memory tests in comparison to that of wild-type mice. Electrophysiological experiments indicate that mitral cell neurons cultured from both heterozygous and homozygous-null mice (IR+/- and IR-/-) have an decreased peak current amplitude compared with that recorded for wild-type (+/+) animals matched for days in vitro (DIV). These data indicate that the loss of one allele of the IR kinase gene modifies the electrical phenotype of the mitral cell neurons in the olfactory bulb without a change in gross olfactory ability. Given our findings that there are no significant changes in metabolic balance of the IR (+/-) mice but some impairment in memory retention, future experiments testing for specific olfactory behaviors or functional deficits in IR-/+ mice models of diabetes will need to either be tasks that do not require learning or will require a different model (such as diet-induced diabetes) that may evoke a stronger phenotype.

Antihypertensive effects of food-intake restriction in aortic coarctation hypertension

Objective To test the hypothesis that reductions in mean arterial pressure (MAP) induced by food-intake restriction in aortic coarctation hypertension are the result of a reduction of the sympathetic support of the MAP. We also wanted to determine whether the baroreflex control of the heart rate, and α- and β-adrenergic responsivenesses were influenced by chronic food-intake restriction. Methods Four days after aortic coarctation, female Sprague–Dawley rats were assigned to a group that had access ad libitum to food (CON; n = 19) or to a food-intake-restricted group (FRG; n = 17) that was allowed 60% of the CON groups food intake per rat. After 3 weeks, carotid and jugular catheters were implanted for measurement of the MAP and infusion of drugs into conscious rats. The sympathetic contribution to the blood pressure was assessed by measuring the depressor response to ganglionic blockade by hexamethonium plus atropine (30.0 and 0.1 mg/kg intravenously). The baroreflex control of the heart rate was assessed by administering alternating bolus doses of phenylephrine and nitroprusside. The α-adrenergic sensitivity was assessed by measuring the response of the MAP to phenylephrine in areflexive rats (after ganglionic blockade), and the β-adrenergic sensitivity was assessed by measuring the responses of the MAP and heart rate to isoproterenol administration both in reflexive and in areflexive rats. Results Four days after catheterization, both the MAP (CON 150 ± 5 mmHg, FRG 116 ± 4 mmHg) and the heart rate (CON 414 ± 8 beats/min, FRG 365 ± 11 beats/min) were significantly lower in rats of the FRG. That the sympathetic support of the MAP had diminished in FRG rats was evidenced by an attenuated depressor response to ganglionic blockade (40 ± 3 versus 65 ± 3 mmHg). FRG rats exhibited significantly greater reflex bradycardia in response to phenylephrine (slope −1.44 ± 0.07 versus −0.54 ± 0.05 beats/min per mmHg), whereas their reflex tachycardia was not altered (slope − 1.58 ± 0.08 versus − 1.53 ± 0.13 beats/min per mmHg). FRG rats also displayed blunted responses of the heart rate and MAP to isoproterenol administration. Conclusion Food-intake restriction attenuates the rise in MAP which occurs after aortic coarctation significantly. The antihypertensive effect of food-intake restriction may be mediated via a reduction in sympathetic tone.