Weight cycling promotes fat gain and altered clock gene expression in adipose tissue in C57BL/6J mice.

Repeated attempts to lose weight by temporary dieting may result in weight cycling, eventually further gain of body fat, and possible metabolic adaptation. We tested this with a controlled experiment in C57BL/6J mice subjected to four weight cycles (WC), continuous hypercaloric feeding (HF), or low-fat feeding (LF). To search for genes involved in an adaptive mechanism to former weight cycling and avoid acute effects of the last cycle, the last hypercaloric feeding period was prolonged by an additional 2 wk before euthanization. Total energy intake was identical in WC and HF. However, compared with HF, the WC mice gained significantly more total body mass and fat mass and showed increased levels of circulating leptin and lipids in liver. Both the HF and WC groups showed increased adipocyte size and insulin resistance. Despite these effects, we also observed an interesting maintenance of circulating adiponectin and free fatty acid levels after WC, whereas changes in these parameters were observed in HF mice. Global gene expression was analyzed by microarrays. Weight-cycled mice were characterized by a downregulation of several clock genes (Dbp, Tef, Per1, Per2, Per3, and Nr1d2) in adipose tissues, which was confirmed by quantitative PCR. In 3T3-L1 cells, we found reduced expression of Dbp and Tef early in adipogenic differentiation, which was mediated via cAMP-dependent signaling. Our data suggest that clock genes in adipose tissue may play a role in metabolic adaptation to weight cycling.

The distribution of NPY-containing nerves and the catecholamine contents of canine enteric nerve plexuses.

Our previous study demonstrated that PYY was a major neuropeptide in the canine enteric nervous system, the present study defines the locations of NPY-containing enteric neurons. NPY-positive nerve cell bodies and fibers were numerous in gastric and pyloric myenteric plexuses as were positive nerve fibers in antral and pyloric muscle layers, pyloric sphincter muscle layers, and surrounding blood vessels. In contrast to findings for PYY, there were considerably fewer NPY-positive nerve cell bodies and fibers in the canine ileum and colon. Noradrenaline was the predominant catecholamine in all plexuses, the rank order of its contents being: deep muscular > submucous > myenteric plexus. The dopamine/noradrenaline ratio was constant in all plexuses; adrenaline was present in minor amounts. PYY is the more abundant neuropeptide in ileum and colon, and NPY has a greater presence in the gastric antrum, pylorus, and surrounding blood vessels. PYY and NPY may play different functional roles in the GI tract.

Human insulin-mediated enhancement of vascular beta-adrenergic responsiveness.

Insulin may play an important role in the physiological and/or pathophysiological regulation of the cardiovascular system. Defects in insulin secretion and insulin receptor responsiveness have been associated with increased peripheral resistance and hypertension. The mechanisms linking these events remain unclear. To assess the effect of insulin on beta-adrenergic-mediated vasodilation, we examined aortic ring segments obtained from normotensive male Wistar and spontaneously hypertensive rats. Vessels were maximally preconstricted with phenylephrine (3 mumol/L). Relaxation was induced by either isoproterenol (10 mumol/L) or sodium nitroprusside (10 nmol/L), and the relaxant response was followed for 20 minutes. Insulin exposure did not alter phenylephrine-mediated constriction. However, insulin mediated a dose-dependent increase in isoproterenol-induced relaxation, to a maximum of 120 +/- 4% of baseline isoproterenol-mediated relaxation, with an EC50 for insulin of 32 pmol/L in aortic rings from Wistar rats. Insulin exposure also did not alter nitroprusside-mediated relaxation. In contrast to the results obtained in rings from Wistar rats, insulin did not enhance isoproterenol-mediated responses in rings from spontaneously hypertensive rats. Thus, insulin mediates a selective enhancement of vascular beta-adrenergic responsiveness in aortas from normotensive but not hypertensive animals.

Vascular beta-adrenoceptor-mediated responses in hypertension and ageing in rats.

1. In normotensive Wistar (W) and spontaneously hypertensive (SHR) rats between 5 and 20 weeks of age, there was an age-related increase in blood pressure (r2 = 0.770 and r2 = 0.801 respectively). Except for adrenaline (r2 = 0.979) in SHRs, plasma catecholamines and age were unrelated. 2. In ring segments of thoracic aorta from 5, 10, 15 and 20 week old rats, the respective EC80s (-log M) for phenylephrine (PE)-induced contractions were 8.20 +/- 0.37, 7.96 +/- 0.10, 7.15 +/- 0.12 and 7.12 +/- 0.21 in W and 7.73 +/- 0.13, 7.72 +/- 0.16, 7.37 +/- 0.08 and 7.40 +/- 0.03 in SHR tissues (means +/- SEM; n = 5-7). 3. In PE-preconstricted rings, the respective EC50s (-log M) for isoprenaline (IPNA)-induced relaxation were 7.97 +/- 0.11, 7.74 +/- 0.10, 6.96 +/- 0.19 and 6.57 +/- 0.26 in W and 8.03 +/- 0.24, 7.62 +/- 0.08, 6.88 +/- 0.13 and 6.73 +/- 0.14 in SHR tissues (n = 5-7). 4. In PE-preconstricted rings from 5 and 20 week old rats, a single concentration of IPNA (approximating the respective IPNA EC50s) induced relaxation which was sustained over 2 h in W but not SHR tissues. The SHR:W ratios of the net relaxant responses, at 5 and 20 weeks, were 0.6461 and 0.6167 respectively. 5. Thus, W rats exhibit an age-related loss in both vascular alpha- and beta-adrenoceptor responsiveness which appears unrelated to plasma catecholamines. SHRs also exhibit an age-related loss in vasodilator beta-adrenoceptor responsiveness, which may involve adrenaline-induced desensitization, but appear to maintain vasoconstrictor alpha-adrenoceptor responsiveness. It is proposed that an age-related decline in beta-adrenoceptor responsiveness coupled to maintenance of alpha-adrenoceptor responsiveness may lead to chronic blood pressure elevation, as observed in SHRs, while a parallel decline in both alpha- and beta-adrenoceptor responsiveness, as observed in W rats, may preclude hypertension development.

Combined effect of neonatal sympathectomy and adrenal demedullation on blood pressure and vascular changes in spontaneously hypertensive rats.

Neonatal sympathectomy using a combined treatment with antiserum to nerve growth factor and guanethidine during the first 4 weeks after birth was carried out in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Bilateral adrenal demedullation was performed in 4-week-old sympathectomized SHR and WKY rats. The development of hypertension in SHR was prevented by sympathectomy, but the blood pressure (BP) was still higher than in age-matched WKY rats. Demedullation reduced the BP of sympathectomized SHR to the same level as that of WKY rats. Heart rates of SHR and WKY rats were not affected by the treatments. Morphometric measurements of the mesenteric arteries showed that sympathectomy significantly reduced the medial mass in the mesenteric arteries of SHR, mainly through a reduction in the number of smooth muscle cell layers. In sympathectomized SHR, demedullation increased the lumen size of muscular arteries under maximally relaxed conditions, which might explain the further reduction in BP in these animals. Demedullation in sympathectomized SHR and WKY rats caused a decrease in smooth muscle cell layers in the superior mesenteric artery, but the same treatment resulted in a slight increase in the number of smooth muscle cell layers in the large and small mesenteric arteries of SHR and WKY rats. Adventitial area was increased in some mesenteric arteries of SHR and WKY rats by sympathectomy, and demedullation caused a further increase in the size of adventitia in WKY rats. Heart weight in SHR was normalized to the level found in WKY rats by sympathectomy and demedullation. We conclude that in sympathectomized SHR, the elevated BP was maintained by the adrenal medulla.

Genetic aspects of variability in superficial vein responsiveness to norepinephrine.

Venoconstriction of the dorsal hand vein by local norepinephrine infusion was measured by the linear variable differential transformer method in 15 healthy unrelated subjects and eight pairs of monozygotic and six pairs of dizygotic twins. Incremental norepinephrine infusion produced dose-related venoconstriction. In unrelated subjects the doses of norepinephrine constricting basal vein diameter by 50% (ED50) ranged from 3.9 to 120.5 ng/min. There was a positive linear relationship between doses of norepinephrine infused and local steady-state plasma concentrations of norepinephrine achieved in each subject. The reciprocals of the slopes of these dose-concentration relationships, which reflect local norepinephrine clearance (disposition) in the vein, ranged from 0.47 to 1.86 ml/min. Plasma concentrations of norepinephrine associated with reduction of basal vein diameter by 50% (EC50) ranged from 1.4 to 110.2 ng/ml, with variability similar to that of ED50. There was a very high level of concordance in ED50, EC50, and clearance of norepinephrine within pairs of monozygotic twins but not within dizygotic twins. Differences in pharmacokinetics of infused norepinephrine exert a minor impact on overall intersubject variability. Genetic aspects of "tissue responsiveness" (i.e., vascular alpha-adrenoceptor response, smooth muscle contractility, and endothelial function) appear to be largely responsible for the wide intersubject variability in venoconstrictor responsiveness to norepinephrine.

Effect of adrenal demedullation and adrenaline on hypertension development and vascular reactivity in young spontaneously hypertensive rats.

1. The effects of adrenal demedullation and subcutaneous depots of adrenaline, on hypertension development and vascular reactivity, were determined in young spontaneously hypertensive rats (SHRs). 2. Plasma adrenaline, but not noradrenaline, was significantly lower in all 16-week-old demedullated SHRs, irrespective of the time of demedullation. However, hypertension development was attenuated only in SHRs demedullated at 6 weeks of age or younger. 3. Adrenaline depots restored hypertension development in SHRs demedullated at 4 weeks, irrespective of the time of depot implantation, but were without effect in sham-operated rats. 4. Pressor responses to exogenous noradrenaline, in isolated perfused mesenteric arteries from 16-week-old demedullated and sham-operated SHRs and those treated with adrenaline depots, did not differ significantly. Maximal neurogenic pressor responses were, however, reduced in mesenteries from all demedullated rats, including those treated with depot adrenaline. Adding adrenaline to the perfusate facilitated neurogenic responses only. 5. Thus, the adrenal medulla appears to be involved in modulating sympathetic neurogenic vasoconstriction. The nature of this sympathoadrenal interaction and its role in the early development of hypertension in the SHR is unclear and is not explicable simply in terms of a facilitation of sympathetic neurogenic responses by adrenaline.

Interaction between sympathetic nervous system and adrenal medulla in the control of cardiovascular changes in hypertension.

The role of the sympathetic nervous system and adrenal medulla in the development of cardiovascular changes and hypertension was studied in spontaneously hypertensive rats (SHRs), and the results compared with age-matched normotensive Wistar-Kyoto (WKY) rats. Sympathectomy was initiated in newborn rats through daily injection with antiserum to nerve growth factor for 1 week, followed by daily injection with guanethidine for 3 weeks. Removal of the adrenal medulla was carried out in 4-week-old rats after the last guanethidine injection. Such a combination treatment was effective in permanently preventing the development of hypertension in the SHRs, and the blood pressure was maintained at the level of WKY rats. The heart rate of the SHRs and WKY rats was not affected by such treatment. Hypertrophy of the heart and of the vessel wall in the mesenteric arteries was also prevented by such treatment. We conclude that in the SHR, the sympathetic nervous system and the adrenal medulla are essential for the development of cardiovascular changes and hypertension.

Hemodynamic and endocrine effects of mental stress in untreated borderline hypertension.

Mental arithmetic and mirror tracing were compared in 40 untreated patients with borderline hypertension, tested in random sequence in standardized protocols. Both tasks significantly increased systolic and diastolic blood pressure, heart rate, cardiac index, plasma renin, and decreased peripheral resistance. Mental arithmetic also increased cholesterol, triglycerides and HDL; plasma catecholamines were not changed significantly. Lipid changes were correlated with blood pressure changes. These methods will be useful in exploring the relationships between hemodynamic reactivity to stress, and the presence and progression of atherosclerosis, as well as testing the effects of antihypertensive drugs on stress-induced changes that may influence atherosclerotic complications of hypertension.

Presynaptic receptors in hypertension.

The physiological status of presynaptic receptors, regulating sympathetic neurotransmitter release, remains subject to debate. Nevertheless, pharmacological techniques have shown presynaptic alpha-adrenoceptors, mediating a negative feedback inhibition of neuronal noradrenaline (NA) release, and presynaptic beta-adrenoceptors mediating a positive feedback facilitation. Decreased presynaptic alpha- or increased beta-adrenoceptor responsiveness might be expected to result in enhanced per-pulse release of NA and may contribute to hypertension development and maintenance. A potential role in hypertension development, but not its maintenance, has been established for presynaptic beta-adrenoceptors. Attempts to identify altered presynaptic adrenoceptor responsiveness in hypertension have, however, been inconclusive.

Studies on the adrenomedullary dependence of kappa-opioid agonist-induced diuresis in conscious rats.

1. The dependence of kappa-opioid agonist-induced diuresis, upon an intact and functional adrenal medulla in conscious rats, was investigated in order to test the hypothesis that the diuresis is mediated by a blood-borne 'diuretic factor', of adrenomedullary origin, released by kappa-opioid receptor stimulation. 2. Confirming previous observations, adrenal demedullation significantly attenuated diuretic responses to the kappa-opioid agonists U50488H, ethylketocyclazocine (EKC) and tifluadom, but did not affect basal urine output, furosemide-induced diuresis or the antidiuretic response to the mu-opioid agonist, buprenorphine. Naloxone abolished U50488H-induced diuresis, confirming an involvement of opioid receptors. 3. Transfusion studies established that blood, from intact rats treated with U50488H, induced diuresis in intact and demedullated recipient rats, whether or not the recipients had been pretreated with naloxone. However, blood from demedullated rats treated with U50448H was unable to induce diuresis when administered to intact or demedullated recipients. 4. It is concluded that kappa-opioid agonist-induced diuresis is dependent upon an intact and functional adrenal medulla and appears to be mediated by a blood-borne 'diuretic factor' of adrenomedullary origin.

Kappa-opioid agonist induced diuresis. Is it mediated by a blood-borne factor of adrenomedullary origin?

Intravenous administration of the kappa-opioid agonists U50488H, tifluadom, and ethylketocyclazocine induced a characteristic diuresis in conscious, intact, saline-loaded rats. Naloxone pretreatment antagonized U50488H-induced diuresis. The diuretic response to the kappa-opioid agonists was significantly attenuated in adrenal demedullated rats. However, basal urine output, the diuretic response to furosemide, and the antidiuretic response to the mu-opioid agonist buprenorphine were unaffected. Transfusion studies established that 1 mL of blood, from intact donor rats treated with U50488H, induced a diuretic response when administered to intact or demedullated recipient rats, whether or not the recipients had been pretreated with naloxone. However, blood from demedullated rats treated with U50488H was unable to induce diuresis in intact or demedullated recipients. The results indicate that kappa-opioid agonist induced diuresis appears to be mediated by a nonopioid blood-borne "diuretic factor" of adrenomedullary origin and that this factor might be responsible for the dependence of the diuretic response upon an intact and functional adrenal medulla in conscious rats.

Epinephrine facilitates neurogenic responses in isolated segments of dog mesenteric arteries.

Epinephrine (EPI) bitartrate (10(-9)-10(-8) M) significantly enhanced tension development in response to electrical field stimulation in isolated segments of dog mesenteric arteries. Responses to exogenous norepinephrine (NE) were generally unaffected, indicating that the EPI-induced increases in response to field stimulation are not explicable in terms of changes in postjunctional sensitivity. The facilitatory effects of EPI (5 X 10(-9) M) were unaffected by atenolol (10(-6) M) but were completely abolished by timolol (2 X 10(-7) M), suggesting an involvement of beta 2-adrenoreceptors in mediating the EPI-induced facilitation of neurogenic responses. Responses to exogenous NE were usually unaffected by either beta-adrenoreceptor antagonist. The results suggest that release of endogenous sympathetic neurotransmitter, measured in terms of postjunctional effects on vascular smooth muscle tone, appears to be modulated by prejunctional facilitatory beta 2-adrenoreceptors and that these receptors may be a physiologic site of action of EPI.

On the mechanisms of kappa-opioid-induced diuresis.

In conscious saline loaded rats, the kappa-opioid agonists tifluadom, U50488, and ethylketocyclazocine, given subcutaneously, induced a characteristic diuresis which could be antagonized by naloxone. Bilateral adrenal demedullation significantly reduced adrenal gland catecholamine content and plasma adrenaline levels, but did not significantly affect plasma corticosterone levels, indicating that the adrenal cortex remained both intact and functional. Seven days following bilateral adrenal demedullation, the subcutaneous administration of the kappa-agonists no longer induced diuresis. However, demedullation did not affect the diuretic response to frusemide or clonidine, nor did it affect the antidiuretic response induced by the mu-opioid agonists morphine and buprenorphine. Adrenal catecholamines do not appear to be involved in kappa-opioid-induced diuresis, since pretreatment with propranolol, prazosin and idazoxan did not affect the diuretic response in intact animals. The results indicate a link between the adrenal medulla and kappa-opioid-induced diuresis and suggest that a peripheral mechanism may also be involved in mediating this effect.

The effect of adrenal demedullation on cardiovascular responses to environmental stimulation in conscious rats.

Circulating plasma adrenaline has been implicated in the facilitation of neurogenic pressor responses and development of hypertension. Bilateral adrenal demedullation in rats did not affect body weight, urine output, urinary electrolyte (Na+, K+ and Cl-) excretion, nor plasma corticosterone concentration, indicating the selective nature of the demedullation procedure. Adrenal demedullation did induce significant reductions in adrenal catecholamine content, plasma adrenaline levels, resting blood pressure and heart rate in conscious rats, but did not affect alerting-induced increases in blood pressure. The adrenal medulla and circulating plasma adrenaline appear to contribute to the maintenance of resting cardiovascular parameters, but would not appear to be involved in nor facilitate the cardiovascular responses to environmental stimulation.

Adrenaline and the development of spontaneous hypertension in rats.

The effects of chronic alterations in plasma adrenaline levels, on the development of a raised blood pressure in young spontaneously hypertensive (SHR) rats, have been investigated. Bilateral adrenal demedullation (at 4 weeks) significantly reduced plasma adrenaline levels and attenuated the development of hypertension. Pressor responses to phenylephrine (0.3-10 micrograms, i.v.), measured in the pithed animals 11 weeks after demedullation, were unaffected although neurogenic pressor responses were significantly reduced. Subcutaneous implants of procaterol and salbutamol (0.005 and 0.165 mumol/kg, respectively) restored hypertension development in demedullated rats and significantly enhanced neurogenic pressor responses, while responses to i.v. phenylephrine remained unaltered, in the pithed rats. Implants of adrenaline (0.165 and 0.5 mumol/kg, s.c.) also restored hypertension development. However, pressor responses to phenylephrine were reduced and neurogenic responses only slightly enhanced when compared to those obtained in untreated pithed demedullated rats. The pro-hypertensive effect of adrenaline (0.5 mumol/kg, s.c.) was abolished by treatment with the beta 2-adrenoreceptor selective antagonist ICI 118551 (25 mg/kg/day, p.o.). In the subsequently pithed rats, neurogenic pressor responses were slightly reduced when compared to those in animals treated with adrenaline alone. In control demedullated rats, ICI 118551 had no effect on blood pressure nor on the neurogenic and phenylephrine-induced pressor responses. However, in sham-operated animals, ICI 118551 attenuated the development of hypertension and significantly reduced neurogenic pressor responses in the subsequently pithed rats. Responses to phenylephrine remained unaltered. The results support the suggestion that a beta 2-adrenoreceptor-mediated facilitation of sympathetic neurotransmission may be involved in mediating the pro-hypertensive effects of circulating adrenaline in the SHR rat.

Epinephrine enhances neurogenic vasoconstriction in the rat perfused kidney.

Epinephrine has been implicated in the genesis of some forms of hypertension. We have investigated the effects of epinephrine on vasoconstrictor responses evoked by adrenergic stimuli in the isolated perfused rat kidney. Low concentrations of epinephrine (2.5 - 5 X 10(-9) M) increased the amplitude of vasoconstrictor responses evoked by electrical stimulation of the renal adrenergic nerves. These concentrations of epinephrine had no effect on the basal perfusion pressure of the kidney or on the amplitude of vasoconstrictor responses evoked by exogenous norepinephrine. The potentiating effect of epinephrine persisted after infusion of the amine had ceased. Kidneys that had been perfused with 3H-epinephrine accumulated radioactivity, which could then be released by renal nerve stimulation. Cocaine (3 X 10(-5) M) reduced the renal accumulation of 3H-epinephrine and abolished both the persistent potentiating effect of the amine and the release of radioactivity evoked by subsequent nerve stimulation. The potentiating effect of epinephrine infusion was abolished by the beta 2-selective adrenergic receptor antagonist ICI 118,551 (3 X 10(-8) M), but not by the beta 1-selective adrenergic receptor antagonist atenolol (10(-6) M). These results indicate that concentrations of epinephrine that can be achieved during acute stress can enhance the amplitude of neurogenic vasoconstrictor responses. This effect appears to be mediated via a prejunctional beta 2-adrenergic receptor. The persistent nature of this effect may be due to the neuronal accumulation and subsequent release of epinephrine.

Adrenaline and the development of genetic hypertension.

The effects, on the development of a raised blood pressure in spontaneously hypertensive (SHR) rats, of bilateral adrenal demedullation and subcutaneous slow-release implants containing adrenaline, salbutamol and procaterol were studied. Systolic blood pressure (SBP) measurements were made, using an indirect tail-cuff method in groups of male SHR rats aged from three to 14 weeks, which were sham-operated or bilaterally adrenal demedullated under ether anaesthesia at four weeks of age. Bilateral adrenal demedullation attenuated the development of a raised blood pressure, while slow-release implants of adrenaline restored the development of hypertension in adrenal demedullated rats and this effect was abolished by concomitant treatment with the beta 2-adrenoceptor antagonist [C] 118551. Implants of salbutamol and procaterol were also effective in restoring hypertension development in adrenal demedullated rats. It is suggested that adrenaline exerts its pro-hypertensive effects via an action at beta 2-adrenoceptors; possibly those located prejunctionally which are involved in the facilitation of sympathetic neurotransmission.