Helen M. Frankish

Neuropeptide Y, the hypothalamus, and diabetes: Insights into the central control of metabolism

Neuropeptide Y (NPY), a major brain neurotransmitter, is expressed in neurons of the hypothalamic arcuate nucleus (ARC) that project mainly to the paraventricular nucleus (PVN), an important site of NPY release. NPY synthesis in the ARC is thought to be regulated by several factors, notably insulin, which may exert an inhibitory action. The effects of NPY injected into the PVN and other sites include hyperphagia, reduced energy expenditure and enhanced weight gain, insulin secretion, and stimulation of corticotropin and corticosterone release. The ARC-PVN projection appears to be overactive in insulin-deficient diabetic rats, and could contribute to the compensatory hyperphagia and reduced energy expenditure, and pituitary dysfunction found in these animals; overactivity of these NPY neurons may be due to reduction of insulins normal inhibitory effect. The ARC-PVN projection is also stimulated in rat models of obesity +/- non-insulin diabetes, possibly because the hypothalamus is resistant to inhibition by insulin; in these animals, enhanced activity of ARC NPY neurons could cause hyperphagia, reduced energy expenditure, and obesity, and perhaps contribute to hyperinsulinemia and altered pituitary secretion. Overall, these findings suggest that NPY released in the hypothalamuss, especially from the ARC-PVN projection, plays a key role in the hypothalamic regulation of energy balance and metabolism. NPY is also found in the human hypothalamus. Its roles (if any) in human homeostasis and glucoregulation remain enigmatic, but the animal studies have identified it as a potential target for new drugs to treat obesity and perhaps NIDDM.

Increased neuropeptide Y secretion in the hypothalamic paraventricular nucleus of obese (fa/fa) Zucker rats

NPY is synthesized in the hypothalamic arcuate nucleus (ARC), and NPY injected into the paraventricular nucleus (PVN), the main site of NPY release, induces hyperphagia and reduces energy expenditure. Hypothalamic NPY mRNA and NPY levels are increased in fatty Zucker rats, consistent with increased NPY release. This could explain the hyperphagia and reduced energy expenditure, which lead to obesity in the fatty Zucker rat. We have therefore compared NPY secretion in the PVN of conscious fatty and lean Zucker rats using push-pull sampling. The NPY secretory profile was consistently higher in fatty Zucker rats than in lean rats throughout the 3-h study period (P < 0.01), and mean NPY secretion over the whole 3 h was increased 2-fold in the fatty rats (P < 0.001). We conclude that fatty Zucker rats have increased NPY release in the PVN. This observation further supports the hypothesis that increased activity of the NPYergic ARC-PVN pathway may contribute to obesity in the fatty Zucker syndrome.

The serotonergic agent fluoxetine reduces neuropeptide y levels and neuropeptide y secretion in the hypothalamus of lean and obese rats

Evidence suggests that serotonin and neuropeptide Y neurons in the hypothalamus, which respectively inhibit and stimulate food intake, may interact to control energy homoeostasis. We therefore investigated the effects of fluoxetine, which inhibits serotonin reuptake, on food intake and the activity of the neuropeptide Yergic arcuato-paraventricular projection in lean Wistar and Zucker rats. We also studied its effects in obese Zucker rats, in which obesity is postulated to be due to overactivity of the arcuato-paraventricular projection. Fluoxetine significantly reduced food intake in lean and obese rats, both during continuous subcutaneous infusion and (10 mg/kg/day for seven days) and acutely after a single injection (10 mg/kg). Fluoxetine also significantly reduced neuropeptide Y levels in the paraventricular nucleus, a major site of neuropeptide Y release which is highly sensitive to the appetite-stimulating actions of neuropeptide Y. Push-pull sampling in lean and fatty Zucker rats showed that neuropeptide Y secretion in the paraventricular nucleus was significantly reduced after acute fluoxetine treatment. Furthermore, seven days fluoxetine treatment prevented the significant increases in hypothalamic neuropeptide Y messenger RNA which were induced in lean rats by food restriction which precisely matched the hypophagia induced by the drug. We conclude that fluoxetine inhibits various aspects of the activity of the neuropeptide Yergic arcuato-paraventricular neurons, and suggest that reduced neuropeptide Y release in the paraventricular nucleus may mediate, at least in part, the drugs hypophagic action. We further suggest that serotonin may influence food intake and energy balance by inhibiting the arcuato-paraventricular projection, and that the two neurotransmitters may act together to regulate feeding and energy homoeostasis.

Hyperphagia in cold-exposed rats is accompanied by decreased plasma leptin but unchanged hypothalamic NPY

Chronic cold exposure stimulates sympathetically driven thermogenesis in brown adipose tissue (BAT), resulting in fat mobilization, weight loss, and compensatory hyperphagia. Hypothalamic neuropeptide Y (NPY) neurons are implicated in stimulating food intake in starvation, but may also suppress sympathetic outflow to BAT. This study investigated whether the NPY neurons drive hyperphagia in rats that have lost weight through cold exposure. Rats exposed to 4°C for 21 days weighed 14% less than controls maintained at 22°C ( P < 0.001). Food intake increased after 3 days and remained 10% higher thereafter ( P < 0.001). Increased BAT activity was confirmed by 64, 96, and 335% increases in uncoupling protein-1 mRNA at 2, 8, and 21 days. Plasma leptin decreased during prolonged cold exposure. Cold-exposed rats showed no significant changes in NPY concentrations in any hypothalamic regions or in hypothalamic NPY mRNA at any time. We conclude that the NPY neurons are not activated during cold exposure. This is in contrast with starvation-induced hyperphagia, but is biologically appropriate since enhanced NPY release would inhibit thermogenesis causing potentially lethal hypothermia. Other neuronal pathways must therefore mediate hyperphagia in chronic cold exposure.

Differential effects of the receptor agonist mCPP and the 5-HT1A agonist flesinoxan on hypothalamic neuropeptide Y in the rat: Evidence that NPY may mediate serotonin's effects on food intake

To determine whether NPY and 5-HT interact, we studied the effects on hypothalamic NPY and NPY mRNA levels of acute (10 mg/kg) and 7-day administration (10 mg/kg/day) of mCPP (a 5-HT 1B/2C agonist) and flesinoxan (a 5-HT 1A agonist). After both treatments, mCPP reduced food intake and NPY levels in the PVN (p < 0.01). NPY mRNA levels were only increased by comparable food restriction (p < 0.05). Acute flesinoxan injection increased food intake and NPY levels in the PVN and ARC (p < 0.01). This suggests that food intake changes induced by mCPP and flesinoxan are associated with altered NPYergic activity. Stimulation of different hypothalamic 5-HT1 receptors may alter NPY levels available for release in the PVN, possibly by altering transport along the ARC-PVN projection. Therefore, inhibition of the ARC-PVN projection may at least partly mediate the hypophagic effects of serotonin.

Increased feeding in fatty Zucker rats by the thiazolidinedione BRL 49653 (rosiglitazone) and the possible involvement of leptin and hypothalamic neuropeptide Y.

1 The thiazolidinedione BRL 49653 (rosiglitazone) induces hyperphagia and weight gain in obese, insulin‐resistant fatty Zucker rats but not in lean insulin‐sensitive rats. We investigated whether these responses might involve neuropeptide Y (NPY), leptin and insulin. 2 BRL 49653 (1 mg kg−1 day−1, orally) was given for 7 or 20 days to fatty and lean Zucker and Wistar rats. 3 In lean rats of either strain, BRL 49653 had no effect on food intake, body weight, plasma insulin and corticosterone, NPY or NPY mRNA levels. 4 Fatty rats given BRL 49653 showed a 30% increase in food intake and accelerated body weight gain (both P<0.01) after 7 and 20 days, but without significant changes in regional hypothalamic NPY or NPY mRNA levels. 5 Plasma leptin levels were twice as high in untreated fatty Zucker rats as in lean rats (P<0.01), but were unaffected by BRL 49653 given for 20 days. However, BRL 49653 reduced insulin levels by 42% and increased corticosterone levels by 124% in fatty rats (both P<0.01). 6 Hyperphagia induced in fatty Zucker rats by BRL 49653 does not appear to be mediated by either a fall in circulating leptin levels or increased activity of hypothalamic NPYergic neurones. The fall in plasma insulin and/or rise in corticosterone levels during BRL 49653 treatment may be involved, consistent with the postulated role of these hormones in the control of food intake.

The serotonin (5-HT) antagonist methysergide increases neuropeptide Y (NPY) synthesis and secretion in the hypothalamus of the rat

NPY is synthesized in neurons of the hypothalamic arcuate nucleus (ARC) which project to the paraventricular nucleus (PVN), an important site of NPY release. Serotonin (5-HT) has been suggested to induce satiety and 5-HT fibers contact NPY neurons in the ARC and PVN, suggesting that 5-HT could inhibit the ARC-PVN projection. Methysergide is a 5-HT antagonist which stimulates feeding in rats and increases NPY levels in the hypothalamus. To clarify the effects of methysergide on NPY, we examined its effects on NPY synthesis and on NPY secretion in the PVN using push-pull sampling. Hypothalamic NPY mRNA levels were measured in rats (n = 8/group) given either saline or methysergide (10 mg/kg) and killed after 4 h or after 7 days. Food intake was increased by 33% in the acute study and by 9% in the 7-day study (both P < 0.01). NPY mRNA levels were 80% higher in the 7-day study (P < 0.05) and unchanged in the acute study. NPY secretion was measured over a 3-h period after an i.p. injection of methysergide or saline (10 mg/kg, n = 12) with a flow rate of 15 microliters/min. Mean NPY secretion in the methysergide-injected rats was increased by 34% (P < 0.01). We conclude that methysergide induced feeding is associated with increased activity of the NPY neurons in the ARC-PVN projection. This is consistent with our previous findings suggesting that the NPYergic ARC-PVN projection may mediate, at least in part the effects of 5-HT on feeding and energy balance.

Increased feeding and neuropeptide Y (NPY) but not NPY mRNA levels in the hypothalamus of the rat following central administration of the serotonin synthesis inhibitor p-chlorophenylalanine

Neurons containing serotonin (5-HT), a potent anorexic agent, come into contact with neuropeptide Y-ergic neurons, that project from the arcuate nucleus (ARC) to the paraventricular nucleus (PVN). NPY powerfully stimulates feeding and induces obesity when injected repeatedly into PVN. We hypothesize that 5-HT tonically inhibits the ARC-PVN neurons and that balance between the two systems determines feeding and energy homeostasis. This study aimed to determine whether central injection of the 5-HT synthesis inhibitor p-chlorophenylalanine (pCPA), which increases feeding, increased hypothalamic NPY and NPY mRNA levels. pCPA (10 mg/kg in 3 microliters) was administered into the third ventricle either as a single injection (n = 8) or daily for 7 days (n = 8). Control rats received a similar injection of saline. pCPA significantly increased food intake compared with controls after both single and repeated injections (P < 0.05). NPY levels were measured by radioimmunoassay in microdissected hypothalamic extracts. NPY levels in the acutely treated group were significantly increased in the paraventricular nucleus (PVN; by 41%, P = 0.01), anterior hypothalamic area (AHA; by 34%, P < 0.01) and lateral hypothalamic area (LHA; by 41%, P < 0.02). In the 7-day-treated group, NPY levels were also increased in the same areas, i.e. PVN (by 24%, P < 0.01), AHA (by 30%, P < 0.01) and LHA (by 38%, P = 0.01). There were no significant changes in the ARC or any other region or in hypothalamic NPY mRNA levels. pCPA administration increased NPY levels in several regions notably the PVN. This is a major site of NPY release, where NPY injection induces feeding. We suggest that the hyperphagia induced by pCPA is mediated by increased NPY levels and secretion in the PVN. This is further evidence for interactions between NPY and 5-HT in the control of energy homeostasis.

Role of hypothalamic neuropeptide Y neurons in the defective thermogenic response to acute cold exposure in fatty Zucker rats

The fatty Zucker rat has impaired heat production and fails to mount an adequate thermogenic response to cold exposure, partly because of decreased sympathetic drive to thermogenesis in brown adipose tissue. Neuropeptide Y, synthesized in neurons of the hypothalamic arcuate nucleus and released in the paraventricular nucleus, stimulates feeding and inhibits brown adipose tissue activity. The neuropeptide Y neurons are overactive in fatty Zucker rats and are thought to contribute to hyperphagia, reduced energy expenditure and obesity. We have examined the relationship between thermogenic activity in brown adipose tissue (measured as uncoupling protein messenger RNA levels) and hypothalamic neuropeptide Y and neuropeptide Y messenger RNA levels in response to cold exposure (4 degrees C) for 2.5 and 18 h, in fatty and lean Zucker rats. In lean Zucker rats, cold exposure at 4 degrees C for 2.5 and 18 h significantly increased uncoupling protein messenger RNA levels by 3.5-fold (P<0.01) and 3.3-fold (P<0.01), respectively, compared with warm-maintained controls. Exposure to cold for 18 h also increased neuropeptide Y concentrations in the paraventricular nucleus (P<0.01) and ventromedial nucleus (P<0.001) in lean rats, with no change in neuropeptide Y messenger RNA after either 2.5 or 18 h. By contrast, fatty Zucker rats showed no significant changes in uncoupling protein messenger RNA (P>0.05) at either duration of cold exposure. There were also no significant changes in neuropeptide Y levels in any region nor in neuropeptide Y messenger RNA, with cold exposure for either period (P>0.05). In lean rats, cold exposure therefore stimulates brown fat uncoupling protein messenger RNA and also increases neuropeptide Y concentrations in its hypothalamic sites of release. We suggest that increased brown fat thermogenic capacity induced by cold in lean rats may be mediated, at least in part, by decreased neuropeptide Y release in the paraventricular nucleus, resulting in its accumulation in this site. Defective thermogenic responses in fatty rats may result from central dysregulation of brown adipose tissue due to sustained and non-suppressible overactivity of hypothalamic neuropeptide Y neurons.

Increased hypothalamic neuropeptide Y concentration or hyperphagia in streptozotocin-diabetic rats are not mediated by glucocorticoids

Hypothalamic neuropeptide Y containing neurones are overactive and may mediate hyperphagia in insulin-deficient diabetic rats, but the factors stimulating them remain uncertain. To determine the possible role of glucocorticoids, we investigated the effects of the glucocorticoid receptor blocker mifepristone (RU486) on food intake and regional hypothalamic neuropeptide Y concentrations in streptozotocin-diabetic rats. RU486 (30 mg/kg) or corn oil vehicle control was given orally for 3 weeks to diabetic rats. Food intake and neuropeptide Y levels in the hypothalamic arcuate and paraventricular nuclei were increased in untreated diabetic rat groups (P < 0.01), and though RU486 did increase plasma corticosterone levels (P < 0.01) it did not have any effect on either feeding or neuropeptide Y levels (P = NS). These negative findings suggest that glucocorticoids may not be responsible for increasing hypothalamic neuropeptide Y or for hyperphagia in insulin-deficient diabetes.