Anti-TNFα alters the natural history of experimental Crohn's disease in rats when begun early, but not late, in disease.

Anti-TNFα therapy decreases inflammation in Crohn's disease (CD). However, its ability to decrease fibrosis and alter the natural history of CD is not established. Anti-TNF-α prevents inflammation and fibrosis in the peptidoglycan-polysaccharide (PG-PS) model of CD. Here we studied anti-TNF-α in a treatment paradigm. PG-PS or human serum albumin (HSA; control) was injected into bowel wall of anesthetized Lewis rats at laparotomy. Mouse anti-mouse TNF-α or vehicle treatment was begun day (d)1, d7, or d14 postlaparotomy. Rats were euthanized d21-23. Gross abdominal and histologic findings were scored. Cecal levels of relevant mRNAs were measured by quantitative real-time PCR. There was a stepwise loss of responsiveness when anti-TNFα was begun on d7 and d14 compared with d1 that was seen in the percent decrease in the median gross abdominal score and histologic inflammation score in PG-PS-injected rats [as %decrease; gross abdominal score: d1 = 75% (P = 0.003), d7 = 57% (P = 0.18), d14 = no change (P = 0.99); histologic inflammation: d1 = 57% (P = 0.006), d7 = 50% (P = 0.019), d14 = no change (P = 0.99)]. This was also reflected in changes in IL-1ß, IL-6, TNF-α, IGF-I, TGF-ß1, procollagen I, and procollagen III mRNAs that were decreased or trended downward in PG-PS-injected animals given anti-TNF-α beginning d1 or d7 compared with vehicle-treated rats; there was no effect if anti-TNF-α was begun d14. This change in responsiveness to anti-TNFα therapy was coincident with a major shift in the cytokine milieu observed on d14 in the PG-PS injected rats (vehicle treated). Our data are consistent with the clinical observation that improved outcomes occur when anti-TNF-α therapy is initiated early in the course of CD.

Nigrostriatal rAAV-mediated GDNF overexpression induces robust weight loss in a rat model of age-related obesity.

Intraventricular administration of glial cell line-derived neurotrophic factor (GDNF) in primate and humans to study Parkinson's disease (PD) has revealed the potential for GDNF to induce weight loss. Our previous data indicate that bilateral continuous hypothalamic GDNF overexpression via recombinant adeno-associated virus (rAAV) results in significant failure to gain weight in young rats and weight loss in aged rats. Based on these previous results, we hypothesized that because the nigrostriatal tract passes through the lateral hypothalamus, motor hyperactivity mediated by nigrostriatal dopamine (DA) may have been responsible for the previously observed effect on body weight. In this study, we compared bilateral injections of rAAV2/5-GDNF in hypothalamus versus substantia nigra (SN) in aged Brown-Norway X Fisher 344 rats. Nigrostriatal GDNF overexpression resulted in significantly greater weight loss than rats treated in hypothalamus. The nigral or hypothalamic GDNF-induced weight loss was unrelated to motor activity levels of the rats, though some of the weight loss could be attributed to a transient reduction in food intake. Forebrain DA levels did not account for the observed effects on body weight, although GDNF-induced increases in nucleus accumbens DA may have partially contributed to this effect in the hypothalamic GDNF-treated group. However, only nigrostriatal GDNF overexpression induced activation of phosphorylated extracellular signal-regulated kinase (p-ERK) in a small population of corticotrophin-releasing factor [corticotrophin-releasing hormone (CRH)] neurons located specifically in the medial parvocellullar division (MPD) of the paraventricular nucleus of the hypothalamus. Activation of these hypothalamic CRH neurons likely accounted for the observed metabolic effects leading to weight loss in obese rats.

Effect of age on angiotensin II-mediated downregulation of adrenomedullary catecholamine biosynthetic enzymes.

Expression of catecholamine biosynthesizing enzymes, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DbetaH) increase with age in the adrenal medulla, however, the underlying mechanisms are unclear. In the present study, we examined the effect of peripheral angiotensin II (AngII) on the expression of TH and DbetaH, in the adrenal medulla of young (6 mo) and old (23 mo) Fischer-344 rats. Saline or AngII (230 ng/kg/min sc) was infused for 3 days using osmotic minipumps. Adrenomedullary TH and DbetaH mRNA levels increased significantly with age, and while AngII reduced the expression of these enzymes in young animals, it had no such effect in the old animals. Neuropeptide Y (NPY), which is co-released with catecholamines in the adrenal medulla and stimulates the synthesis of TH and DbetaH, was also upregulated with age and downregulated in response to AngII in young rats. However, in the old animals, the already elevated NPY expression remained unchanged following AngII treatment. This data indicate that the hypertensive effect of peripheral AngII is compensated by an inhibition of adrenomedullary catecholamine biosynthesis in young animals, but this mechanism is impaired in senescence, potentially contributing to the age-related increase in catecholamine biosynthesis.

Effects of life-long caloric restriction and voluntary exercise on age-related changes in levels of catecholamine biosynthetic enzymes and angiotensin II receptors in the rat adrenal medulla and hypothalamus.

We examined if life-long mild caloric restriction (CR) alone or with voluntary exercise prevents the age-related changes in catecholamine biosynthetic enzyme levels in the adrenal medulla and hypothalamus. Ten-week-old Fisher-344 rats were assigned to: sedentary; sedentary+8% CR; or 8% CR+wheel running. Rats were euthanized at 6 or 24 months of age. Tyrosine hydroxylase (TH) mRNA expression was 4.4-fold higher in the adrenal medullae and 60% lower in the hypothalamus of old sedentary rats compared to young (p<0.01). Life-long CR reduced the age-related increase in adrenomedullary TH by 50% (p<0.05), and completely reversed the changes in hypothalamic TH. Voluntary exercise, however, had no additional effect over CR. Since angiotensin II is involved in the regulation of catecholamine biosynthesis, we examined the expressions of angiotensin II receptor subtypes in the adrenal medulla. AT(1) protein levels were 2.8-fold higher in the old animals compared to young (p<0.01), and while AT(1) levels were unaffected by CR alone, CR+wheel running decreased AT(1) levels by 50% (p<0.01). AT(2) levels did not change with age, however CR+wheel running increased its level by 42% (p<0.05). These data indicate that a small decrease in daily food intake can avert age-related changes in catecholamine biosynthetic enzyme levels in the adrenal medulla and hypothalamus, possibly through affecting angiotensin II signaling.

Hypothalamic rAAV-mediated GDNF gene delivery ameliorates age-related obesity.

Intraventricular delivery of glial cell line-derived neurotrophic factor (GDNF) results in weight loss. We hypothesized that this effect of GDNF was likely mediated via its effects on dopaminergic neurons in the hypothalamus. Continuous rAAV-mediated GDNF expression in the hypothalamus of young and senescent rats resulted in weight loss compared to controls. However, GDNF-induced weight loss was unrelated to alterations in hypothalamic dopamine levels. The weight loss was associated with decreased food intake and increased energy expenditure, but these effects were not mediated by changes in hypothalamic NPY or POMC expression. Moreover, uncoupling protein 1 levels were unchanged in brown adipose tissue (BAT). The reduction in weight and adiposity were as great or greater in the aged rats even though aged rats are generally resistant to weight loss therapies. In summary, central GDNF gene delivery reduces weight and adiposity in young and aged rats through decreased food intake and increased energy expenditure. Our observations in aged rats suggest that GDNF may be especially effective in reducing obesity in aged obese rats.

Acute pressor effect of central angiotensin II is mediated by NAD(P)H-oxidase-dependent production of superoxide in the hypothalamic cardiovascular regulatory nuclei.

BACKGROUND: Centrally applied angiotensin II (Ang II) increases sympathetic nervous activity and mean arterial blood pressure (MAP), but the mediation of these effects is not fully understood. OBJECTIVE: To test the hypothesis that central effects of Ang II are mediated by reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H]-oxidase-dependent production of superoxide in the hypothalamus. METHODS: Under isoflurane anesthesia, male Sprague-Dawley rats were given an intracerebroventricular infusion of either artificial cerebrospinal fluid or apocynin (4 microg/kg per min), a selective inhibitor for NAD(P)H oxidase, for 30 min, followed by Ang II (20 ng) or carbachol (200 ng), while MAP and heart rate were measured at the femoral artery. At the end of the experiments, hydroethidine, a superoxide-sensitive fluorescent dye, was infused intravenously for 10 min, and superoxide production was assessed in the vasoregulatory hypothalamic nuclei using confocal microscopy. RESULTS: Ang II elicited a rapid 11 +/- 2-mmHg increase in MAP and a 16 +/- 2-beats/min decrease in heart rate. Apocynin abolished these effects of Ang II in a specific manner, as carbachol-induced increases in MAP were unaffected by the inhibition of NAD(P)H oxidase (MAP increased by 9 +/- 2 and 8 +/- 1 mmHg in the absence and presence of apocynin, respectively). In response to Ang II, apocynin-sensitive production of superoxide increased significantly in the nuclei of the anterior hypothalamus, in the subfornical organ, and in the paraventricular nucleus of the hypothalamus. CONCLUSION: These findings demonstrate that acute pressor responses of central Ang II are mediated by NAD(P)H-oxidase-dependent production of superoxide in the hypothalamus.

The age-related discrepancy in the effect of neuropeptide Y on select catecholamine biosynthetic enzymes in the adrenal medulla and hypothalamus in rats.

The elevated levels of circulating catecholamines (CAs) with age may be related to the increased expression of CA biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DbetaH) in the adrenal medulla of senescent compared with younger animals. Neuropeptide Y (NPY) is co-synthesized and co-released with CAs in the adrenal medulla. NPY inhibits the stimulated secretion of CAs, however, its role in regulation of the genes encoding CA biosynthetic enzymes is not clear. We hypothesized that NPY up-regulates TH, DbetaH and NPY expression in the adrenal medullae of young and old Fischer-344 rats. NPY increased mRNA expression of TH, DbetaH, NPY and also enhanced TH protein level in the adrenal medullae of young rats by 50%, 35%, 45% and by 20%, respectively. We also examined the effect of NPY on TH and NPY mRNA in the hypothalamus. Basal expression of TH mRNA was decreased in the hypothalamus with age. DNA binding activities of activator protein-1 and cAMP response element binding protein were also augmented only in the young by 140% and 125%, respectively. We conclude that NPY stimulates the CA biosynthetic pathway in the adrenal medulla and positive auto-regulation of NPY might be involved in this process. The stimulatory effect of NPY on adrenomedullary CA biosynthetic pathway is blunted with age.

Effect of exercise on mRNA expression of select adrenal medullary catecholamine biosynthetic enzymes.

The effect of submaximal endurance training (SET) on sympathoadrenal activity is not clear. We tested the hypothesis that SET (90 min/day, 5 days/wk, for 12 wk) elevates mRNA expression of catecholamine (CA) biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH) in the adrenal medullae of adult, female Sprague-Dawley rats. SET increased TH protein level by 35%, TH activity by 62%, TH mRNA expression by 40%, and DbetaH mRNA expression by 67%. In addition, we examined the effect of SET on Fos-related antigens (FRAs), FRA-2 immunoreactivity, and activator protein (AP)-1 binding activity. SET increased AP-1 binding activity by 78%; however, it did not affect late FRAs and FRA-2 immunoreactivity. Because the regulation of neuropeptide Y (NPY) often parallels that of CAs, we also examined the effect of SET on NPY mRNA expression. Indeed, SET elevated NPY mRNA expression as well. We conclude that 1) SET elicits a pretranslational stimulatory effect on adrenomedullary CA biosynthetic enzymes, 2) another immediate early mRNA product, rather than FRA-2, may contribute to the increase in AP-1 binding activity in response to SET, and 3) SET increases NPY mRNA expression.

Effect of high-fat diet feeding on hypothalamic redox signaling and central blood pressure regulation.

We examined the effect of high-fat (HF) feeding on blood pressure (BP) regulation, including hypothalamic redox signaling, as well as the changes in diurnal patterns and responses to restraint stress. Furthermore, we investigated whether HF feeding affects catecholamine and neuropeptide Y (NPY) biosynthesis in the adrenal medulla. Male obesity-prone Sprague-Dawley rats were fed with standard rat chow or 60% HF diet for 6 months. BP and heart rate (HR) were measured by telemetry, and circadian changes as well as responses to 20 min restraint stress were analyzed. Mean arterial BP was significantly elevated in HF rats both during daytime and nighttime compared with controls, whereas HR was elevated only during the day. BP and HR increased similarly in response to stress in both experimental groups; however, post-stress recovery of BP and HR were significantly delayed in HF animals. Protein levels of angiotensin II type 1 receptor (AT(1)) and NOX2, p67(phox) and p47(phox) subunits of NADPH oxidase, as well as NADPH oxidase activity increased significantly in the hypothalamus with HF feeding, whereas levels of antioxidant enzymes and nitric oxide synthases remained unchanged. In addition, HF diet also elevated the adrenomedullary protein levels of tyrosine hydroxylase and NPY. This study shows that feeding obesity-prone Sprague-Dawley rats with a HF diet results in elevated BP and HR and delayed cardiovascular post-stress recovery, and that these changes are paralleled by increases in the expression and activity of NADPH oxidase in the hypothalamus without a compensatory increase in the antioxidant enzyme levels, possibly leading to superoxide-mediated sympathoexcitation and hypertension.

Central angiotensin II increases biosynthesis of tyrosine hydroxylase in the rat adrenal medulla.

Angiotensin II acting centrally contributes to the regulation of blood pressure and water intake and stimulates the release of catecholamines from the adrenal medulla. We hypothesized that the central angiotensin II is one mediator of biosynthesis of catecholamines in the adrenal medulla. Rats were administered i.c.v. angiotensin II or saline, and TH mRNA and protein levels in adrenal medulla were measured 1 or 3 h later. Angiotensin II did not change TH mRNA or protein 1 h later. However, by 3 h, angiotensin II increased TH mRNA and protein levels. Centrally administered angiotensin II elevates TH mRNA expression and protein levels in the adrenal medulla. In conclusion, one component of central angiotensin II elevation of blood pressure may be the result of increased catecholamine synthesis in the adrenal gland and elevated TH synthesis represents one underlying mechanism.