Increased ghrelin signaling prolongs survival in mouse models of human aging through activation of sirtuin1.

Caloric restriction (CR) is known to retard aging and delay functional decline as well as the onset of diseases in most organisms. Ghrelin is secreted from the stomach in response to CR and regulates energy metabolism. We hypothesized that in CR ghrelin has a role in protecting aging-related diseases. We examined the physiological mechanisms underlying the ghrelin system during the aging process in three mouse strains with different genetic and biochemical backgrounds as animal models of accelerated or normal human aging. The elevated plasma ghrelin concentration was observed in both klotho-deficient and senescence-accelerated mouse prone/8 (SAMP8) mice. Ghrelin treatment failed to stimulate appetite and prolong survival in klotho-deficient mice, suggesting the existence of ghrelin resistance in the process of aging. However, ghrelin antagonist hastened death and ghrelin signaling potentiators rikkunshito and atractylodin ameliorated several age-related diseases with decreased microglial activation in the brain and prolonged survival in klotho-deficient, SAMP8 and aged ICR mice. In vitro experiments, the elevated sirtuin1 (SIRT1) activity and protein expression through the cAMP-CREB pathway was observed after ghrelin and ghrelin potentiator treatment in ghrelin receptor 1a-expressing cells and human umbilical vein endothelial cells. Furthermore, rikkunshito increased hypothalamic SIRT1 activity and SIRT1 protein expression of the heart in the all three mouse models of aging. Pericarditis, myocardial calcification and atrophy of myocardial and muscle fiber were improved by treatment with rikkunshito. Ghrelin signaling may represent one of the mechanisms activated by CR, and potentiating ghrelin signaling may be useful to extend health and lifespan.

Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2.

Subtype 2 of the metabotropic glutamate receptor (mGluR2) is expressed in the presynaptic elements of hippocampal mossy fiber-CA3 synapses. Knockout mice deficient in mGluR2 showed no histological changes and no alterations in basal synaptic transmission, paired-pulse facilitation, or tetanus-induced long-term potentiation (LTP) at the mossy fiber-CA3 synapses. Long-term depression (LTD) induced by low-frequency stimulation, however, was almost fully abolished. The mutant mice performed normally in water maze learning tasks. Thus, the presynaptic mGluR2 is essential for inducing LTD at the mossy fiber-CA3 synapses, but this hippocampal LTD does not seem to be required for spatial learning.

Sympathetic activation of leptin via the ventromedial hypothalamus: leptin-induced increase in catecholamine secretion.

Leptin is an adipocyte-derived blood-borne satiety factor that acts directly on the hypothalamus, thereby regulating food intake and energy expenditure. We have demonstrated that the hypothalamic arcuate nucleus (Arc) is a primary site of the satiety effect of leptin (Neurosci Lett 224:149-152, 1997). To explore the hypothalamic pathway of sympathetic activation of leptin, we examined the effects of a single intravenous or intracerebroventricular injection of recombinant human leptin on catecholamine secretion in rats. We also examined the effects of direct microinjection of leptin into the ventromedial hypothalamus (VMH), Arc, paraventricular nucleus (PVN), and dorsomedial hypothalamus (DMH) in rats. To further assess whether sympathetic activation of leptin is mediated via the VMH, we also examined the effects of a single intravenous injection of leptin in VMH-lesioned rats. A single injection of leptin (0.25-1.0 mg i.v./rat or 0.5-2.0 pg i.c.v./rat) increased plasma norepinephrine (NE) and epinephrine (EPI) concentrations in a dose-dependent manner. Plasma NE and EPI concentrations were increased significantly when leptin was injected directly into the VMH but were unchanged when injected into the Arc, PVN, and DMH. Plasma NE and EPI concentrations were unchanged in VMH-lesioned rats that received a single intravenous injection of leptin. The present study provides evidence that a leptin-induced increase in catecholamine secretion is mediated primarily via the VMH and suggests the presence of distinct hypothalamic pathways mediating the satiety effect and sympathetic activation of leptin.

Involvement of agouti-related protein, an endogenous antagonist of hypothalamic melanocortin receptor, in leptin action.

To understand the role of agouti-related protein (AGRP), an endogenous antagonist of hypothalamic melanocortin receptor, in leptin action, we produced a full-length recombinant AGRP and examined its effect on the satiety effect of leptin. We also studied leptin's regulation of hypothalamic AGRP mRNA expression. A single intracerebroventricular (i.c.v.) injection of AGRP significantly increased cumulative food intake and body weight in a dose-dependent manner in rats. The leptin-induced inhibition of food intake and body weight was reversed by co-injection of AGRP in a dose-dependent manner. Hypothalamic AGRP mRNA expression was upregulated in leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice and downregulated in lethal yellow agouti mice (KKAy mice) with hyperleptinemia. A single i.c.v. injection of leptin reversed the increased AGRP mRNA levels in ob/ob mice but not in db/db mice. In control mice and KKAy mice, AGRP mRNA expression was upregulated during fasting, when plasma leptin concentrations were decreased. No significant increase in AGRP mRNA expression was noted during fasting in control mice and KKAy mice treated with leptin. This study provides the first direct evidence that AGRP is a negative regulator of leptin action, and leptin downregulates hypothalamic AGRP production. Because leptin is shown to increase hypothalamic alpha-melanocyte stimulating hormone (alpha-MSH) production, our data suggest that its action via the hypothalamic melanocortin system is determined by the balance between the levels of its agonist and antagonist, alpha-MSH and AGRP.

Adrenocorticotropin release induced by intracerebroventricular injection of recombinant human interleukin-1 in rats: possible involvement of prostaglandin.

ACTH release induced by iv and intracerebroventricular (icv) injection of recombinant human interleukin-1 beta (IL-1 beta) or alpha (IL-1 alpha) was studied in conscious, unrestrained rats. A dose as small as 3 ng of IL-1 beta injected icv induced a significant rise in plasma ACTH levels, whereas 100 ng/100 g body wt (approximately 300 ng/rat) was needed for a significant ACTH response when injected iv. Intracerebroventricular administration of 30 ng IL-1 alpha tended to increase plasma ACTH levels, but not significantly. Intravenous injection of 1000 ng/100 g IL-1 beta induced a maximal response with a pronounced elevation of plasma ACTH levels at 10 and 30 min after injection, but plasma ACTH levels fell at 60 min post injection. On the other hand, icv injection of 30 ng IL-1 beta raised plasma ACTH levels at 10 min, reaching peak values between 30 and 60 min post injection, and plasma ACTH levels remained elevated for 2-3 h after injection. Pretreatment with indomethacin completely prevented the ACTH response induced by either iv or icv injection of IL-1 beta. Administration of indomethacin did not alter the elevation of plasma ACTH levels induced by immobilization stress, however. On the other hand, vagotomy did not alter the ACTH response to iv administered IL-1 beta. Neither iv nor icv injection of IL-1 beta in a dose which induced a maximal ACTH response altered plasma PRL levels. These findings strongly suggest that the brain is the primary site of action of IL-1 beta, and that IL-1 beta transmits the message of the immune system to the brain and, possibly, CRF neurons. It is also suggested that prostaglandins may be involved in this central action of IL-1 beta.

Interleukin-1 beta increases prostaglandin E2 in rat astrocyte cultures: modulatory effect of neuropeptides.

Recombinant human interleukin-1 beta (IL-1 beta) significantly increased prostaglandin E2 (PGE2) in a dose-dependent manner in rat astrocyte culture. The minimum effective dose of IL-1 beta was 10(-10)M. IL-1 alpha also increased PGE2, but at a higher concentration. The minimum effective dose of IL-1 alpha was 10(-8)M, indicating it to be 100-fold less effective than IL-1 beta. On the other hand neither IL-1 beta nor IL-1 alpha increased PGE2 production by neuron cultures at any concentration tested. PGE2 response to IL-1 beta was suppressed by simultaneous addition of CRH, somatostatin-14 and LHRH, while these neuropeptides alone did not alter the basal PGE2 levels. Substance P, vasoactive intestinal polypeptide and alpha-MSH altered neither basal nor IL-1 beta-induced increase in PGE2 levels. Angiotensin II (AII) alone also increased PGE2 in cultured astrocytes. Combined addition of AII and IL-1 beta induced a synergistic effect in increasing PGE2 levels. The direct action of IL-1 beta on astrocyte culture suggests that astrocytes may be the target cells for IL-1 beta in the central nervous system. In view of the essential role of central PGE2 in IL-1 beta-induced CRH/ACTH release, these findings suggest the presence of a sophisticated regulatory network in the immune-neuroendocrine interaction.

Modulatory role of vasopressin in secretion of atrial natriuretic polypeptide in conscious rats.

To investigate whether vasopressin is involved in the secretory mechanism of atrial natriuretic polypeptide (ANP), effects of arginine-vasopressin (AVP) administered iv on plasma ANP levels were studied in conscious, unrestrained rats. The administration of 100 ng and 1 microgram of AVP caused a dose-dependent increase of the plasma ANP level, which was blocked by a V1-receptor antagonist of AVP, and was attenuated by 5 ml blood volume reduction before the stimulation. The injection of less than 10 ng of AVP induced no significant effects on ANP secretion. However, the administration of 5 ng of AVP significantly enhanced ANP secretion induced by intravascular volume expansion with 3 ml saline infusion. These results suggest the possible physiological significance of AVP as a modulator rather than a direct stimulator of ANP secretion from the heart.

Pathophysiological significance of the obese gene product, leptin, in ventromedial hypothalamus (VMH)-lesioned rats: evidence for loss of its satiety effect in VMH-lesioned rats.

To explore the pathophysiological significance of the obese (ob) gene product, leptin, in ventromedial hypothalamus (VMH)-lesioned rats, we examined the synthesis and secretion of leptin and its satiety effect in VMH-lesioned rats compared with those in sham-operated rats. Northern blot analysis revealed that ob gene expression is markedly augmented in the mesenteric and sc white adipose tissue, but remained unchanged in the epididymal white adipose tissue during the development of obesity in VMH-lesioned rats. Plasma leptin levels were relatively constant in sham-operated rats, but were elevated during the development of obesity in VMH-lesioned rats. In sham-operated rats, a single i.v. (1.0 mg/rat) or intracerebroventricular (2.0 micrograms/rat) injection of recombinant human leptin reduced food intake and body weight gain in sham-operated rats. By contrast, no significant effect on food intake or body weight gain was observed in VMH-lesioned rats. The present study provides evidence that VMH-lesioned rats overproduce leptin and increase its release but cannot respond to it and suggests that the loss of its satiety effect contributes to the development of obesity and the obesity-related phenotypes in VMH-lesioned rats.

Effect of cholecystokinin tetrapeptide amide on the metabolism of 5-hydroxytryptamine in the rat brain.

The effect of cholecystokinin tetrapeptide amide (CCK-4) on the content of 5-hydroxytryptamine (5-HT) and its major metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), was studied in various regions of the brain of rat. After intracerebroventricular administration of CCK-4, the content of 5-HT decreased, while the content of 5-HIAA increased in some areas. These changes were marked 30 min after the injection and relatively small doses (10 and 100 ng) of CCK-4 produced a pronounced effect, but larger doses (1 and 10 micrograms) were less effective. The ratio of 5-HIAA/5-HT increased significantly in most regions of the brain, except in the striatum and the olfactory tubercle. The results indicate that exogenous CCK-4 in small doses stimulates the metabolism of 5-HT in the brain of rat.

Effects of cholecystokinin on apomorphine-induced changes of motility in rats.

Subcutaneous injection of apomorphine in small doses decreased, while larger doses increased motility in rats. The hypomotility was abolished by intracerebroventricular administration of sulphated cholecystokinin octapeptide [CCK-8(s)], but not by non-sulphated CCK-8 [CCK-8(ns)]. In contrast, the hypermotility was not influenced by CCK peptides. An antiserum directed against the C-terminus of CCK did not affect hypermotility induced by apomorphine but suppressed the apomorphine-induced hypomotility further. The results indicate that sulphated CCK-peptides contribute to the control of motility, which can be inhibited by apomorphine.

Impairment of pupillary responses and optokinetic nystagmus in the mGluR6-deficient mouse.

Retinal bipolar cells receive glutamatergic transmission from photoreceptors and mediate a key process in segregating visual signals into ON-center and OFF-center pathways. The segregation of ON responses involves a G protein-coupled metabotropic glutamate receptor (mGluR). The mGluR6 subtype is expressed restrictedly at the postsynaptic site of retinal ON-bipolar cells. Ablation of mGluR6 in the ON-bipolar cells by gene targeting results in a loss of ON responses but unchanged OFF responses in visual transmission. Thus, mGluR6 is essential for inducing ON responses. The aims of this study are analyses of visual responsiveness and possible visual dysfunction in mGluR6-deficient mice. We report here that mGluR6-deficient mice have unaltered locomotor activity in a daily light-dark cycle and exhibit light-stimulated induction of Fos immunoreactivity in the suprachiasmatic nucleus. These findings indicate that mGluR6-deficient mice are capable or responding to light stimulation. The mGluR6 deficiency, however, markedly reduces the sensitivity of pupillary responses to light stimulus and severely impairs the ability to drive optokinetic nystagmus in response to visual contrasts. This study thus demonstrates that mGluR6 contributes to discrimination of visual contrasts.

Interleukin-1 suppresses follicle-stimulating hormone-induced estradiol secretion from cultured ovarian granulosa cells.

The effect of recombinant human interleukin-1 beta (IL-1 beta) on the follicle stimulating hormone-(FSH) induced secretion of estradiol was investigated using cultured granulosa cells obtained from immature rats with diethylstilbestrol implants. Estradiol secretion was significantly reduced by IL-1 beta in cultures containing FSH and either 10(-7) or 10(-8) M androstenedione as a substrate for estradiol synthesis. However, the inhibition of FSH-induced estradiol secretion by IL-1 beta was more apparent in the presence of 10(-8) M as compared to 10(-7) M androstenedione. IL-1 beta suppressed estradiol secretion during a 72 h culture in a dose-dependent manner with a minimum effective dose of 10 ng/ml. The reduction of FSH-stimulated estradiol secretion by IL-1 beta was greatest after a 48 h culture in the presence of 10(-8) M androstenedione. IL-1 beta did not effect estradiol production when cultures were incubated with various doses of androstenedione in the absence of FSH. Finally, IL-1 beta also suppressed the forskolin-induced secretion of estradiol. These results suggest that IL-1 beta may play some role in the multifactorial regulation of aromatase and estrogen secretion in the early developing follicle, and IL-1 beta may exert an effect on the cAMP-adenylate cyclase messenger system in granulosa cells. Taken together with previous studies, these results provide further evidence for the existence of a putative communications network between the immune and reproductive endocrine systems.

Altered responding to cholecystokinins and dopaminergic agonists following 6-hydroxydopamine treatment in rats.

Production of lesions in the brain dopamine (DA) system by intraventricular injection of 6-hydroxydopamine (6-OHDA) resulted in increased responses to apomorphine (0.5 mg/kg, sc) and reduced responses to methamphetamine (0.15 mg/kg, sc). It also made animals increase responding to cholecystokinin octapeptide (CCK-8; 0.5-2 micrograms, intracerebroventricularly [icv]) and reduce responding to cholecystokinin tetrapeptide (CCK-4; 0.5-2 micrograms, icv). Response changes were quantified by measuring the level of general activity. The result indicates that DA dysfunction can affect not only DA receptor sensitivity but also the sensitivity of the CCK system. The response to CCK-8 was partially blocked by a selective CCK-8 antagonist, proglumide (5 micrograms, icv), a result suggesting the involvement of the CCK-8 receptor system. Thus, manipulation of one neuronal system could induce sensitivity changes in another closely related system.

Attenuation of focal cerebral infarct in mice lacking NMDA receptor subunit NR2C.

Neuronal death following cerebral vascular occlusion may be caused in part by the action of glutamate acting through the NMDA receptor. Here we demonstrate that gene disruption of the NR2C subunit of the NMDA receptor attenuates focal cerebral ischemic injury after permanent MCA occlusion, and that a low level of NR2C is expressed and active in the cerebral cortex. NR2C-deficient mice do not show impairment of motor coordination or motor learning. Therefore the development of drugs selectively inhibiting NR2C may prove beneficial in the treatment of stroke and traumatic brain injuries.

Preventive effect of cholecystokinin octapeptide on experimental amnesia in rats.

The effect of intracerebroventricular (ICV) administration of cholecystokinin octapeptide (CCK-8) on electroconvulsive shock (ECS)-induced amnesia in passive avoidance response was studied in rats. In normal rats, CCK-8 in doses from 1 ng to 1 microgram had no effect on the response when injected before the training trials, immediately after foot shock or before the first retention test. However, proglumide, a CCK-8 receptor blocker, induced marked amnesia when injected in doses from 0.1 to 10 micrograms before the training trials and in doses of 1 and 10 micrograms before the first retention test, though not subsequent to foot shock. ECS given immediately after the foot shock caused amnesia in the 24 hr and 48 hr retention tests, which could have been prevented by CCK-8 injected in doses of 10 ng to 1 microgram prior to the training trials, of 10 ng to 1 microgram following ECS and of 0.1 and 1 microgram before the first retention test. In addition, the effects of CCK-8 and proglumide became pronounced following chronic ICV infusion, using an osmotic minipump, for 7 days at a dose of 1 ng/day and 10 ng/day, respectively. The amnesia induced by proglumide was not affected by arginine vasopressin (AVP), while AVP in doses of 10 ng and 100 ng given 30 min before the training trials prevented ECS-induced amnesia. The antiamnesic effect of AVP was abolished by simultaneous administration of proglumide. On the other hand, AVP-antiserum produced marked amnesia which could be antagonized by CCK-8. However, the antiamnesic effect of CCK-8 was not suppressed by AVP-antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)

Passive avoidance deficit following intracerebroventricular administration of cholecystokinin tetrapeptide amide in rats.

The effect of cholecystokinin tetrapeptide amide (CCK-4) injected into the lateral cerebral ventricle on memory processes was examined by a one-trial passive avoidance test in the rat. CCK-4 injection 30 and 60 min before the first retention test caused a shortened latency to response, and its chronic infusion into the lateral ventricle at a rate of 2 micrograms/day shortened the latency of the response to the level of almost complete amnesia. CCK-4 also reduced arginine-vasopressin effect on memory processes when administered simultaneously 30 min before the first retention test, but its amnestic action is short-lasting and antagonized by relatively small amounts of cholecystokinin octapeptide (CCK-8). In addition, the shortened latency to response was admitted to be not always associated with the motility effect of CCK-4.

Fronto-cortical regulation of beta-endorphin actions in the rat.

Ablation of the frontal neocortex markedly enhanced the antinociceptive and cataleptic actions of beta-endorphin injected into the lateral ventricle of rat brain. This enhanced response was not affected by simultaneous administration of cholecystokinin octapeptide (CCK-8). In sham-operated rats, however, CCK-8 suppressed the effects of beta-endorphin in a dose-related manner. Moreover, ablation of a similar amount of occipital neocortex did neither affect beta-endorphin actions nor the interactions of CCK-8.

Effect of vasoactive intestinal peptide on dopaminergic system in the rat brain.

The present study analyzed the effect of vasoactive intestinal peptide (VIP) on the content of dopamine (DA) and its main metabolite, DOPAC, in the rat brain. Intracerebroventricular administration of VIP increased the DA and DOPAC content, causing a dose-dependent increase in the DOPAC/DA ratio in various regions of the brain. The results suggest that VIP facilitates the DA metabolism in the brain.

Activation of CCK-A receptors induces elevation of plasma corticosterone in rats.

Cholecystokinin octapeptide (CCK-8) and ceruletide (1 microgram/kg) produced a pronounced increment of plasma corticosterone levels at 30 min after intraperitoneal administration. The response to these peptides was suppressed by pretreatment with a selective antagonist for CCK-A receptors, (-)L-364,718, in a dose-related manner, but not with an antagonist for CCK-B receptors, (+)L-365,260. However, (-)L-364,718 itself had no effect on basal levels of plasma corticosterone. These results indicate that peripheral administration of CCK-8 and ceruletide stimulates the hypothalamo-pituitary adrenal axis through the activation of CCK-A receptors, but not CCK-B receptors.

Blocking of cholecystokinin octapeptide behavioral effects by proglumide.

An open field apparatus was used to assess the effect of proglumide, a selective antagonist of cholecystokinin octapeptide (CCK-8), to block the behavioral effect of CCK-8 in rats. Intracerebroventricular (ICV) injection of CCK-8 (0.5 to 2 micrograms) was effective in suppressing general exploratory activities and this effect was blocked by proglumide at doses of 2 to 5 micrograms administered ICV or 1 mg/kg administered subcutaneously. The effect of peripherally administered CCK-8 (10 micrograms/kg) was blocked by peripherally administered proglumide at a dose of 2 mg/kg but not by centrally administered proglumide at a dose of 5 micrograms/rat. The behavioral effect of CCK-8 was thus clearly blocked by proglumide.