J. Michael Overton

Glucagon-like peptide-1 receptor stimulation increases blood pressure and heart rate and activates autonomic regulatory neurons

Glucagon-like peptide-1 (GLP-1) released from the gut functions as an incretin that stimulates insulin secretion. GLP-1 is also a brain neuropeptide that controls feeding and drinking behavior and gastric emptying and elicits neuroendocrine responses including development of conditioned taste aversion. Although GLP-1 receptor (GLP-1R) agonists are under development for the treatment of diabetes, GLP-1 administration may increase blood pressure and heart rate in vivo. We report here that centrally and peripherally administered GLP-1R agonists dose-dependently increased blood pressure and heart rate. GLP-1R activation induced c-fos expression in the adrenal medulla and neurons in autonomic control sites in the rat brain, including medullary catecholamine neurons providing input to sympathetic preganglionic neurons. Furthermore, GLP-1R agonists rapidly activated tyrosine hydroxylase transcription in brainstem catecholamine neurons. These findings suggest that the central GLP-1 system represents a regulator of sympathetic outflow leading to downstream activation of cardiovascular responses in vivo.

Effects of central leptin administration on blood pressure in normotensive rats

We tested the hypothesis that intracerebroventricular (i.c.v.) administration of leptin would increase mean arterial pressure (MAP) in ad libitum (AL) fed and food deprived (FD) normotensive rats. Male Sprague-Dawley rats were chronically instrumented with a guide cannula directed at the lateral ventricle and a carotid arterial catheter. Following recovery from surgery, the MAP and heart rate (HR) response to i.c.v. administration of vehicle (5 microl saline over 1 min) or leptin (0.3 microg or 3.0 microg in 5 microl saline) were determined in conscious, unrestrained AL fed (n=7-10) and 48-h FD (n=5-10) rats. Food deprivation significantly reduced MAP (AL=116+/-3; FD=104+/-3 mmHg; P < 0.01) without altering HR. In AL rats, high dose leptin (3.0 microg, i.c.v.) produced a significant increase in MAP when maximal responses were evaluated (9+/-2 mmHg; P < 0.05), but did not significantly alter MAP and HR over time during the 90 min measurement period. In FD rats, low dose leptin (0.3 microg, i.c.v.) produced significant elevations in MAP (7+/-3 mmHg) after a latency of 60 min, while high dose leptin (3.0 microg, i.c.v.) produced an increase in MAP within the first 10 min (10+/-3 mmHg) followed by an additional increase 1 h after injection (6+/-2 mmHg). Leptin administration also produced delayed increases in HR in FD rats (0.3 microg, 34+/-5 b.p.m.; 3.0 microg, 57+/-10 b.p.m). These results indicate that leptin may modulate cardiovascular function through central mechanisms and may do so to a greater extent in food deprived animals.

Estradiol decreases the orexigenic effect of melanin-concentrating hormone in ovariectomized rats

Estradiol exerts an inhibitory effect on food intake via interactions with anorexigenic peptides, like cholecystokinin, that function to decrease meal size. It is currently unknown whether estradiol also interacts with orexigenic compounds implicated in the physiological control of food intake. Thus, the primary goal of this study was to determine whether estradiol decreases the orexigenic effect of melanin-concentrating hormone (MCH), a neuropeptide that, like estradiol, appears to influence food intake by selectively affecting the controls of meal size. Food and water intake were monitored following lateral ventricular (icv) infusions of 5 mug MCH or saline vehicle in oil- and estradiol-treated ovariectomized rats. MCH increased food intake throughout the first 4 h of the dark phase in oil-treated rats, but only for the last 2 h of the same 4-h interval in estradiol-treated rats. As a result, the orexigenic effect of MCH was significantly lower in estradiol-treated rats, relative to oil-treated rats. During this interval of MCH-stimulated feeding, a prandial increase in water intake was not observed in either oil- or estradiol-treated rats. We conclude that estradiol decreases the orexigenic effect of MCH in ovariectomized rats.

Translating Animal Model Research: Does It Matter That Our Rodents Are Cold?

Does it matter that rodents used as preclinical models of human biology are routinely housed below their thermoneutral zone? We compile evidence showing that such rodents are cold-stressed, hypermetabolic, hypertensive, sleep-deprived, obesity-resistant, fever-resistant, aging-resistant, and tumor-prone compared with mice housed at thermoneutrality. The same genotype of mouse has a very different phenotype and response to physiological or pharmacological intervention when raised below or at thermoneutrality.

The effect of food deprivation and experimental diabetes on orexin and NPY mRNA levels

Although exogenous orexin can induce feeding, reports of increased orexin gene expression after caloric manipulations have been inconsistent. We hypothesized that orexin gene expression is increased only by extreme negative energy balance challenges. We measured hypothalamic orexin and NPY mRNA by in situ hybridization and orexin-A immunoreactivity in rats after food deprivation, streptozotocin-induced diabetes, and combined deprivation and diabetes. Neither food deprivation, nor diabetes, nor the combination affected orexin mRNA levels, although orexin-A immunoreactivity was increased by diabetes. NPY mRNA levels were increased by either treatment. These results suggest that increased orexin gene expression is not a consistent correlate of negative energy balance challenges.

Evidence for the role of hindbrain orexin-1 receptors in the control of meal size

Hypothalamic orexin neurons project to the hindbrain, and 4th-ventricle intracerebroventricular (4th-icv) injection of orexin-A treatment increases food intake. We assessed the effects of hindbrain orexin-A and the orexin-1-receptor antagonist SB334867 on meal pattern in rats consuming standard chow. When injected 4th-icv shortly before dark onset, lower doses of orexin-A increased food intake over a 2-h period by increasing the size of the first meal relative to vehicle, whereas the highest dose increased food intake by causing the second meal to be taken sooner. Conversely, hindbrain SB334867 reduced food intake by decreasing the size of the first meal of the dark phase. We also examined the effects of 4th-icv orexin-A and SB334867 on locomotor activity. Only the highest dose of orexin-A increased activity, and SB334867 had no effect. In addition, hindbrain SB334867 induced c-Fos in the nucleus of the solitary tract. These data support the suggestion that endogenous hindbrain orexin-A acts to limit satiation. Both orexin-A and the pancreatic satiation hormone amylin require an intact area postrema to affect food intake, so we asked whether 4th-icv orexin-A impairs the satiating effect of peripheral amylin treatment. Amylin reduced the size of the first meal of the dark cycle when rats were pretreated with 4th-icv saline, yet amylin was ineffective after 4th-icv orexin-A pretreatment. Using double-label immunohistochemistry, we determined that some orexin-A fibers in the area postrema are located in proximity to amylin-responsive neurons. Therefore, hindbrain orexin-A may increase food intake, in part, by reducing the ability of rats to respond to amylin during a meal.

Cardiovascular effects of melanin-concentrating hormone

Melanin-concentrating hormone (MCH) is a cyclic 19-amino acid neuropeptide exclusively synthesized in the lateral hypothalamic area (LHA) and the zona incerta (ZI) that has been implicated in the regulation of energy balance. Despite what is known about the orexigenic effect of MCH, whether MCH has distinct cardiovascular and metabolic effects has yet to be determined. Thus, our goal here was to characterize the concurrent cardiovascular, metabolic, and behavioral responses of male rats to chronic intracerebroventricular (icv) infusion of MCH. Male Long-Evans rats were instrumented with telemetry transmitters for measurement of heart rate (HR) and housed in room calorimeters for assessment of food intake and oxygen consumption (VO(2)) at standard lab ambient temperature (23 degrees C) in order to examine physiological responses to chronic infusion of MCH (8 microg/d and 16 microg/d). Our findings provide the first evidence that chronic administration of MCH induces bradycardia and reduced mean arterial pressure, while it did not affect VO(2). A second experiment was performed in which the physiological responses to an acute icv infusion of MCH were observed. The results of experiment 2 indicate that MCH leads to a low HR that is maintained during the first 2 h post-infusion, the time period during which MCH acutely stimulated feeding. Collectively, these findings confirm that MCH may be an important modulator of sympathetic nervous system activity and thus may play a critical role in coordinating normal responses to negative energy balance.

Increased NPY activity in the PVN contributes to food-restriction induced reductions in blood pressure in aortic coarctation hypertensive rats

We hypothesized that hypothalamic NPYergic mechanisms mediate the blood pressure lowering effect of caloric restriction in hypertensive rats. Aortic coarctation-induced (AC) hypertensive rats (n=25) were assigned to either an ad libitum fed control group (AL) or food restricted group (FR; 60% of AL consumption) for 3 weeks. Rats were instrumented chronically with vascular catheters and bilateral guide cannulae directed at the paraventricular hypothalamic nuclei (PVN). Blood pressure (BP) and heart rate (HR) responses to bilateral PVN microinjection of saline (200 nl) or the putative NPY receptor antagonists [D-Trp32]NPY(1-36) (3.3 micrograms/200 nl) and [D-Tyr27,36 Thr32]NPY(27-36) (D-NPY(27-36); 3.3 micrograms/200 nl) were determined. The FR rats were then refed and cardiovascular responses to PVN injections of NPY receptor antagonists were again determined. FR rats had significantly reduced resting BP (159+/-4 vs. 129+/-4 mmHg) and HR (360+/-11 vs. 326+/-9 bpm) compared to AL controls. Refeeding restored BP and HR of FR rats to levels similar to AL (BP=153+/-4 mmHg, HR=359+/-11 bpm). PVN administration of [D-Trp32]NPY produced foraging behavior and concurrent increases in BP and HR in FR, AL and Re-fed rats. The behavioral activation suggests that [D-Trp32]NPY(1-36) produced activation of NPY receptors. In contrast, D-NPY (27-36) did not produce any behavioral response or affect BP or HR in AL or Re-fed rats. In FR rats, D-NPY (27-36) produced significant increases in BP (peak=15+/-3 mmHg) which partially reversed the effect of FR on BP. Thus, in FR rats with reduced BP, PVN administration of an NPY receptor antagonist increases BP. NPY blockade in the PVN accounted for about 50% of the BP effect of food restriction, thus other mechanisms are likely to be involved. These findings are consistent with the hypothesis that NPYergic mechanisms may contribute to the reduction of BP produced by food restriction.

Attenuated Blood Pressure Responsiveness During Post-Exercise Hypotension

OBJECTIVE To test the hypotheses that acute treadmill exercise would produce post-exercise hypotension (PEH) and that PEH would be associated with reduced mean arterial pressure (MAP) responsiveness to the alpha 1-adrenergic agonist phenylephrine. METHODS Arterial and venous catheters were implanted into exercise-trained female Dahl-salt sensitive rats (n = 9) for measurement of pulsatile blood pressure (BP) and heart rate (HR). The changes in BP following ganglionic blockade (hexamethonium/atropine) and the MAP responses to phenylephrine (PE) injections after ganglionic blockade (GB) were examined on separate days in testing cages (control) and following 40 min of treadmill exercise (post-ex). RESULTS Thirty minutes following graded treadmill exercise (20-40 m/min, 0% grade, 40 min duration) blood pressure was significantly reduced (-9 +/- 1) mmHg compared to control. After exercise, GB produced a 43 +/- 3 mmHg decrease in BP which tended (p = 0.08) to be less than the reduction observed during control studies (51 +/- 2 mmHg). PE-induced increases in BP were significantly lower post-ex than control for each of the dosages tested (0.5, 1.0 and 2.0 micrograms/kg). CONCLUSIONS These results demonstrate that there is sustained reduction in PE-induced MAP responsiveness which accompanies exercise-induced decreases in blood pressure in the Dahl salt-sensitive rat. Thus, decreased alpha-adrenoceptor responsiveness may contribute to the production of PEH.

Long term ablation of protein Kinase a (PKA)-mediated cardiac troponin I phosphorylation leads to excitation-contraction uncoupling and diastolic dysfunction in a knock-in mouse model of hypertrophic cardiomyopathy

Background: The R21C mutation in cardiac troponin I (cTnI) prevents PKA-mediated phosphorylation of serines 23 and 24 of cTnI in vivo. Results: Myofilament function is uncoupled from the intracellular [Ca2+] and delays muscle relaxation. Conclusion: Long term ablation of cTnI phosphorylation leads to hypertrophy, diastolic dysfunction, and dysautonomia in mice. Significance: Restoration of phosphorylated cTnI may prevent hypertrophic cardiomyopathy and diastolic dysfunction. The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca2+ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca2+-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca2+ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca2+ content. This abnormal Ca2+ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na+-Ca2+ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.