Role of Intraduodenally Administered Enterostatin in Rats: Inhibition of Food

Central and peripheral administration of enterostatin have been reported to reduce fat or high-fat food intake in rats. Enterostatin is formed in the intestinal lumen by tryptic cleavage of pancreatic procolipase during intraluminal fat digestion. The present experiments were designed to test if enterostatin following intraintestinal infusion would affect food intake in a similar way as intracerebraventricularly or intravenously administered enterostatin. Female Sprague-Dawley rats were fitted with a duodenal catheter and adapted to a feeding schedule for 6 hours each day. After 10 days enterostatin (5.65 and 11.3 nmol/kg/min) or saline were infused into the duodenum and food intake measured. Enterostatin significantly reduced high-fat food intake during the 6 hours of feeding, but had no inhibitory effect on low-fat food intake. Addition of tetracaine to the enterostatin infusates blocked the satiating potency of intestinal enterostatin. These results support the hypothesis of a preabsorptive site of action for enterostatin.     

Effect of enterostatin given intravenously and intracerebroventricularly on high-fat feeding in rats.

The effect of enterostatin, the amino-terminal pentapeptide of pancreatic procolipase, on high-fat food intake has been investigated after intracerebroventricular as well as after intravenous injection. After an overnight fast enterostatin given i.c.v. at doses of 167 pmol and 333 pmol produced a significant and dose-dependent reduction in high-fat food intake, while a higher dose of 667 pmol had no effect. Following intravenous injection of enterostatin the intake of high-fat food was suppressed at doses of 8.3 nmol and 16.7 nmol, while no effect was observed at higher doses. The inhibition of feeding started 3 h after the initiation of feeding and persisted to the end of the test period (6 h). Enterostatin at a dose of 16.7 nmol gave no sign of aversion in an aversion test comparing the effect of enterostatin, lithium chloride and saline on liquid intake. The data suggest that enterostatin may exert its satiety effect on high-fat feeding by being absorbed into the bloodstream.     

Enterostatin inhibition of dietary fat intake is dependent on CCK-A receptors

Enterostatin, a pentapeptide released from the exocrine pancreas and gastrointestinal tract, selectively inhibits fat intake through activation of an afferent vagal signaling pathway. This study investigated if the effects of enterostatin were mediated through a CCK-dependent pathway. The series of in vivo and in vitro experiments included studies of 1) the feeding effect of peripheral enterostatin on Otsuka Long Evans Tokushima Fatty (OLETF) rats lacking CCK-A receptors, 2) the effect of CCK-8S on the intake of a two-choice high-fat (HF)/low-fat (LF) diet, 3) the effects of peripheral or central injection of the CCK-A receptor antagonist lorglumide on the feeding inhibition induced by either central or peripheral enterostatin, and 4) the ability of enterostatin to displace CCK binding in a 3T3 cell line expressing CCK-A receptor gene and in rat brain sections. The results showed that OLTEF rats did not respond to enterostatin (300 microg/kg ip) in contrast to the 23% reduction in intake of HF diet in Long Evans Tokushima Otsuka (LETO) control rats. CCK (1 microg/kg ip) decreased the intake of the HF diet in a two-choice diet regime with a compensatory increase in intake of the LF diet. Peripheral injection of lorglumide (300 microg/kg) blocked the feeding inhibition induced by either near-celiac arterial or intracerebroventricular enterostatin, whereas intracerebroventricular lorglumide (5 nmol icv) only blocked the response to intracerebroventricular enterostatin but not to arterial enterostatin. Enterostatin did not bind on CCK-A receptors because neither enterostatin nor its analogs VPDPR and beta-casomorphin displaced [3H]L-364,718 from CCK-A receptors expressed in 3T3 cells or the binding of 125I-CCK-8S from rat brain sections. The data suggest that both the peripheral and central responses to enterostatin are mediated through or dependent on peripheral and central CCK-A receptors.     

Intragastric beta-casomorphin(1-7) attenuates the suppression of fat intake by enterostatin

The current experiments were designed to compare the feeding response to enterostatin and beta-casomorphin(1-7) injected intragastrically. Sprague-Dawley rats with a gastric cannula were allowed to chose from high-fat diet (HF) or low-fat diet (LF) in separate jars. Enterostatin injected intragastrically into overnight fasted rats caused a U-shaped dose-dependent reduction in the intake of the HF diet for the first two hours after infusion but had no effect on the LF intake. beta-Casomorphin(1-7) stimulated the intake of the HF diet but had no effect on the LF diet. Finally, beta-casomorphin(1-7) blocked the inhibitory effect of enterostatin on HF intake in fasted rats.     

Effect of high-fat diet, surrounding temperature, and enterostatin on uncoupling protein gene expression

Nonshivering thermogenesis induced in brown adipose tissue (BAT) during high-fat feeding is mediated through uncoupling protein 1 (UCP1). UCP2 is a recently identified homologue found in many tissues. To determine the role of UCP1 and UCP2 in thermoregulation and energy balance, we investigated the long-term effect of high-fat feeding on mRNA levels in mice at two different ambient temperatures. We also treated mice with the anorectic peptide enterostatin and compared mRNA levels in BAT, white adipose tissue (WAT), stomach, and duodenum. Here, we report that high-fat feeding at 23 degrees C increased UCP1 and UCP2 levels in BAT four- and threefold, respectively, and increased UCP2 levels fourfold in WAT. However, at 29 degrees C, UCP1 decreased, whereas UCP2 remained unchanged in BAT and increased twofold in WAT. Enterostatin increased UCP1 and decreased UCP2 mRNA in BAT. In stomach and duodenum, high-fat feeding decreased UCP2 mRNA, whereas enterostatin increased it. Our results suggest that the regulation of uncoupling protein mRNA levels by high-fat feeding is dependent on ambient temperature and that enterostatin is able to modulate it.     

Enterostatin inhibition of dietary fat intake is modulated through the melanocortin system

Enterostatin injected into the amygdala selectively reduces dietary fat intake by an action that involves a serotonergic component in the paraventricular nucleus. We have investigated the role of melanocortin signaling in the response to enterostatin by studies in melanocortin 4 receptor (MC4R) knock out mice and by the use of the MC4R and MC3R antagonist SHU9119, and by neurochemical phenotyping of enterostatin activated cells. We also determined the effect of enterostatin in vivo on the expression of AgRP in the hypothalamus and amygdala of rats and in culture on a GT1-7 neuronal cell line. Enterostatin had no effect on food intake in MC4R knock out mice. SHU9119 i.c.v. blocked the feeding response to amygdala enterostatin in rats. Amygdala enterostatin induced fos activation in alpha-melanocyte stimulating hormone (alpha-MSH) neurons in the arcuate nucleus. Enterostatin also reduced the expression of AgRP in the hypothalamus and amygdala and in GT1-7 cells. These data suggest enterostatin inhibits dietary fat intake through a melanocortin signaling pathway.     

Different metabolic responses to central and peripheral injection of enterostatin

Enterostatin, a pentapeptide cleaved from procolipase, suppresses fat intake after peripheral and central administration. Chronic treatment of rats with enterostatin decreases body weight and body fat. The effect was greater than could be accounted by the reduction in food intake alone. Hence, we have investigated the effect of enterostatin on energy metabolism. Male Sprague-Dawley rats adapted to a high-fat diet were implanted with lateral cerebral ventricular or amygdala cannulas. The metabolic effects were determined by indirect calorimetry. After habituation to the test cages, fasted rats were injected with either saline vehicle or enterostatin given either intraperitoneally (100 nmol) or intracerebroventricularly (1 nmol) or into specific brain regions [amygdala (0.01 nmol) or paraventricular nucleus (PVN) (0.1 nmol)]. Respiratory quotient (RQ) and energy expenditure were monitored over 2 h. Intraperitoneal enterostatin reduced RQ (saline: 0.81 +/- 0.02 vs. enterostatin: 0.76 +/- 0.01) and increased energy expenditure by 44%. Intracerebroventricular enterostatin increased the energy expenditure without any effects on RQ, whereas PVN enterostatin increased metabolic rate, while preventing the increase in RQ observed in the control animals. In contrast, neither RQ nor energy expenditure was altered after enterostatin was injected into the amygdala. Enterostatin activated AMP-activated protein kinase in primary cultures of human myocytes in a dose- and time-dependent manner and increased the rate of fatty acid beta-oxidation. These findings suggest that enterostatin regulates energy expenditure and substrate partitioning through both peripheral and central effects.     

Effect of Enterostatin on the Feeding Responses to Galanin and NPY

We have investigated the possibility that enterostatin may inhibit the intake of dietary fat by inhibiting either galanin or NPY-induced feeding pathways. Rats, adapted to either high fat (HF) or low fat-high carbohydrate (HC) diets and fitted with third ventricular cannulas were used to study the effects of intracerebroventricular (icv) enterostatin on icv NPY and galanin induced feeding responses in satiated rats. An equimolar dose of enterostatin (0.1nmoles) inhibited, while a tenfold excess of entersotatin abolished the feeding response to galanin in rats adapted to a HF diet. The galanin stimulation of food intake was reduced in rats adapted to the HC diet and this response was less sensitive to inhibition by enterostatin. Enterostatin had no inhibitory effects on NPY-induced feeding in rats adapted to the HC diet and only a small inhibitory effect, at tenfold molar excess, in rats adapted to the HF diet. The ability of enterostatin to bind to galanin or NPY Y-1 receptors was investigated in lig and binding studies. Enterstatin fialed to dispace ¹²⁵I-galanin or ¹²⁵I-PYY from specific binding sites in rat forebrain homogenates or SK-N-MC cells respectively. The data provide support for the hypothesis that enterostatin specifically inhibits a galanin-responsive fat intake system, but indicate that this effect is not modulated by direct interaction with either galanin or NPY-Y1 receptors.     

Enterostatin decreases postprandial pancreatic UCP2 mRNA levels and increases plasma insulin and amylin

This study investigated the chronic effect of enterostatin on body weight and some of the associated changes in postprandial metabolism. Rats were adapted to 6 h of food access/day and a choice of low-fat and high-fat (HF) food and then given enterostatin or vehicle by an intraperitoneally implanted minipump delivering 160 nmol enterostatin/h continuously over a 5-day infusion period. Enterostatin resulted in a slight but significant reduction of HF intake and body weight. After the last 6-h food access period, enterostatin-treated animals had lower plasma triglyceride and free fatty acid but higher plasma glucose and lactate levels than control animals. Enterostatin infusion resulted in increased uncoupling protein-2 (UCP2) expression in various tissues, including epididymal fat and liver. UCP2 was reduced in the pancreas of enterostatin-treated animals, and this was associated with increased plasma levels of insulin and amylin. Whether these two hormones are involved in the observed decreased food intake due to enterostatin remains to be determined. As lipid metabolism appeared to be altered by enterostatin, we measured peroxisome proliferator-activated receptor (PPAR) expression in tissues and observed that PPARalpha, -beta, -gamma1, and -gamma2 expression were modified by enterostatin in epididymal fat, pancreas, and liver. This further links altered lipid metabolism with body weight loss. Our data suggest that alterations in UCP2 and PPARgamma2 play a role in the control of insulin and amylin release from the pancreas. This implies that enterostatin changes lipid and carbohydrate metabolic pathways in addition to its effects on food intake and energy expenditure.     

Plasma Insulin in Response to Enterostatin and Effect of Adrenalectomy in Rats

Enterostatin has previously been reported to alter serum insulin and corticosterone levels after central administration of the peptide. The purpose of the present study was to investigate the effect of peripheral administration of enterostatin on insulin and corticosterone levels as well as the response of plasma insulin to enterostatin administration in adrena-lectomized rats. Female Sprague-Dawley rats were given a bolus injection intravenously with enterostatin alone or together with glucose. Enterostatin increased basal plasma levels of insulin, but significantly inhibited the increase in plasma insulin stimulated by glucose. Plasma corticosterone levels were not altered after a single intravenous injection of enterostatin. In rats infused chronically with enterostatin, plasma insulin levels were significantly reduced and plasma corticosterone levels were increased. The daily food intake was lower in these rats, but there was no effect on body weight. After adrenalectomy, the responsiveness of plasma insulin to enterostatin infusion was completely abolished. Furthermore, adrenalectomy itself reduced basal plasma levels of insulin and increased plasma levels of endogenous enterostatin. These results suggest that peripheral enterostatin administration produces a similar effect as central infusion of the peptide, and that the glucocorticoid hormones are involved in the regulation of plasma insulin by enterostatin.     

Vagal-Central Nervous System Interactions Modulate the Feeding Response to Peripheral Enterostatin

Enterostatin selectively inhibits the intake of dietary fat after both peripheral and central administration. We have investigated the role of the hepatic vagus nerve in modulating the peripheral response to enterostatin in Sprague-Dawley rats adapted to a high fat (HF) diet. Intraperitoneal (ip) enterostatin reduced intake of HF diet after overnight starvation. This response was abolished by selective vagal hepatic branch transection. Immunohistochemical techniques were used to identify the location of Fos protein in brain nuclei after ip enterostatin. Fos protein was evident in the nucleus tractus solitarius (NTS), parabrachial, paraventricular and supraoptic nuclei. The pattern of expression of Fos-like immunoreactivity differed from that induced by the lipoprivic agent β-mercaptoacetate. Transection of the hepatic vagus blocked the central Fos responses to ip enterostatin. We conclude that afferent hepatic vagal nerve activity is required for the feeding response to peripheral enterostatin.     

Intravenous administration of oxytocin in rats acutely decreases deprivation-induced chow intake,but it fails to affect consumption of palatable solutions

Despite its limited ability to cross the blood-brain barrier, peripherally administered oxytocin (OT) acutely decreases food intake, most likely via the brainstem and hypothalamic mechanisms. Studies performed to date have focused mainly on the effects of subcutaneous or intraperitoneal OT on the consumption of only solid calorie-dense diets (either standard or high-fat), whereas it is unknown whether, similarly to central OT, peripherally administered peptide reduces intake of calorie-dilute and non-caloric palatable solutions. In this project, we established that 0.1 μg/kg intravenous (IV) OT is the lowest anorexigenic dose, decreasing deprivation-induced standard chow intake by ca. 40% in rats and its effect does not stem from aversion. We then used this dose in paradigms in which effects of centrally acting OT ligands on consumption of palatable solutions had been previously reported. We found that IV OT did not change episodic intake of individually presented palatable solutions containing 10% sucrose, 0.1% saccharin, combined 10% sucrose-0.1% saccharin or 4.1%. Intralipid and it failed to affect daily scheduled consumption of a sucrose solution in non-deprived rats. In a two-bottle choice test, IV OT did not shift animals’ preference from sucrose to Intralipid. Finally, OT injected IV prior to the simultaneous presentation chow and a sucrose solution in food-deprived rats significantly decreased chow intake, whereas sugar water consumption remained unchanged. We conclude that IV OT reduces deprivation-induced chow intake without causing aversion, but the dose effective in decreasing energy-driven consumption of high-calorie food fails to affect consumption of palatable calorie-dilute solutions.     

Method of leptin dosing,strain, and group housing influence leptin sensitivity in high-fat-fed weanling mice

High-fat diets are reported to induce resistance to peripherally administered leptin. In an attempt to develop a model of juvenile diet-induced obesity, mice were weaned onto high-fat diet. Male and female, 35-day-old, C57BL/6J high-fat (45% kcal fat) diet-fed mice housed individually on grid floors did not decrease food intake or body weight in response to intraperitoneal (30 microg), lateral ventricle (5 microg), or third ventricle (0.5 microg) injections of leptin. Body weight and fat were significantly reduced by 13-day intraperitoneal infusions of 10 microg leptin/day, which doubled circulating leptin. Leptin infusion also reduced body fat in weanling, high-fat diet-fed NIH Swiss mice. Group housing mice on bedding prevented loss of fat in high-fat diet-fed male and female NIH Swiss and female C57BL/6J mice. These results indicate that peripherally infused leptin reduces fat in part by increasing thermogenesis and that inhibition of food intake in high-fat diet-fed mice requires either chronic activation of central leptin receptors or is independent of receptors that inhibit feeding in response to an acute central injection of leptin.     

Shift of circadian feeding pattern by high-fat diets is coincident with reward deficits in obese mice

Recent studies provide evidence that high-fat diets (HF) trigger both i) a deficit of reward responses linked to a decrease of mesolimbic dopaminergic activity, and ii) a disorganization of circadian feeding behavior that switch from a structured meal-based schedule to a continuous snacking, even during periods normally devoted to rest. This feeding pattern has been shown to be a cause of HF-induced overweight and obesity. Our hypothesis deals with the eventual link between the rewarding properties of food and the circadian distribution of meals. We have investigated the effect of circadian feeding pattern on reward circuits by means of the conditioned-place preference (CPP) paradigm and we have characterized the rewarding properties of natural (food) and artificial (cocaine) reinforcers both in free-feeding ad libitum HF mice and in HF animals submitted to a re-organized feeding schedule based on the standard feeding behavior displayed by mice feeding normal chow ("forced synchronization"). We demonstrate that i) ad libitum HF diet attenuates cocaine and food reward in the CPP protocol, and ii) forced synchronization of feeding prevents this reward deficit. Our study provides further evidence that the rewarding impact of food with low palatability is diminished in mice exposed to a high-fat diet and strongly suggest that the decreased sensitivity to chow as a positive reinforcer triggers a disorganized feeding pattern which might account for metabolic disorders leading to obesity.     

Enterostatin (APGPR) enhances memory consolidation in mice

Enterostatin (APGPR) is a pentapeptide released from its precursor protein, procolipase. We found for the first time that enterostatin has memory-enhancing activity. Enterostatin enhanced memory consolidation after central or oral administration at a dose of 10 nmol/mouse or 300 mg/kg, respectively, in a step-through type passive avoidance test in mice. The memory-enhancing activity of enterostatin was inhibited by pretreatment with lorglumide, an antagonist for cholecystokinin 1 (CCK1) receptor. However, enterostatin had no affinity for CCK receptors. These results suggest that enterostatin improves memory retention through CCK release.     

Inhibiting parabrachial fatty acid amide hydrolase activity selectively increases the intake of palatable food via cannabinoid CB1 receptors

These studies investigated feeding responses to indirect activation of parabrachial cannabinoid CB1 receptors. Arachidonoyl serotonin (AA5HT), an inhibitor of the endocannabinoid degradative enzyme, fatty acid amide hydrolase (FAAH), was infused into the parabrachial nucleus of male Sprague-Dawley rats, and intakes of high-fat/sucrose pellets and standard rodent chow were subsequently evaluated under various feeding schedules. FAAH blockade stimulated the intake of high-fat/sucrose pellets that were presented daily for 4 h during the light period, with compensatory decreases in the consumption of standard chow during the ensuing 20 h. These diet-selective changes were repeated on the next day, indicating a shift in feeding toward the more palatable diet that lasted for 48 h after a single infusion. The cannabinoid CB1 receptor antagonist, AM251, blocked the orexigenic actions of AA5HT, implicating CB1 receptors in mediating the feeding responses to FAAH inactivation. When the feeding schedule was reversed, AA5HT produced nominal increases in the consumption of standard chow for the 4-h access period, but substantial increases in the intake of high-fat/sucrose during the following 20-h interval. When presented with only high-fat/sucrose diet for 24 h, AA5HT increased 24-h food intake. In contrast, when given 24-h access only to standard chow, AA5HT failed to affect intake. Therefore, indirectly activating parabrachial CB1 receptors by blocking the degradation of native ligands selectively stimulates the intake of palatable food, with differential actions on total energy intake depending upon the feeding schedule. Our results support a role for parabrachial cannabinoid mechanisms in providing physiological regulation to neural substrates modulating feeding, energy balance, and behavioral responses for natural reward.     

Enterostatin suppresses food intake in rats after near-celiac and intracarotid arterial injection

Enterostatin (Ent) selectively suppresses the intake of dietary fat after peripheral and central administration. To further investigate the site of action of Ent, we compared the feeding responses to Ent injected intra-arterially near the celiac artery, into the carotid artery, or intravenously in rats adapted to a high-fat diet. After near-celiac arterial injection there was an immediate dose-dependent (0.05-13.5 nmol) inhibition of food intake occurring within 5 min in overnight-fasted rats that lasted up to 20 min. Carotid arterial Ent had a similar, immediate dose-related response, and the inhibitory effect was long lasting. The response to intravenous Ent was only evident at the highest dose (13.5 nmol) and was delayed for at least 120 min. Pretreatment with capsaicin, which causes degeneration of vagal sensory neurons, abolished the inhibitory responses to near-celiac Ent but not to intravenous or intracarotid Ent. These results provide further evidence for both a gastrointestinal site of action for peripheral Ent and a central site of action for intracarotid Ent and suggest that the delayed response to intravenous Ent may reflect either binding or slow uptake of this peptide into the central nervous system.     

Resistance to IP leptin-induced adipose apoptosis caused by high-fat diet in mice

The objectives of this experiment were to determine whether leptin causes adipocyte apoptosis in mice, whether peripheral administration is an effective means of studying leptin-induced adipocyte apoptosis, and whether high-fat feeding results in reduced responsiveness to leptin-induced adipocyte apoptosis. Continuous 13-day intraperitoneal infusion of 10 microg/day leptin significantly increased adipocyte apoptosis in the epididymal/parametrial fat pads of male and female mice, but only male mice developed reduced responsiveness to leptin-induced adipocyte apoptosis after high-fat (45% fat) feeding for 5 or 15 weeks. There was a positive correlation between serum leptin concentration and percent apoptotic adipocytes. These findings demonstrate that leptin administered peripherally is effective in inducing adipocyte apoptosis in mice, thus extending the possibility of studying this effect of leptin in a wider variety of animal models. In addition, high-fat feeding has a gender-specific effect on development of reduced responsiveness to leptin-induced adipocyte apoptosis.     

Nutrient Intake is Modulated By Peripheral Peptide Administration

Many peptides have been shown to modulate nutrient intake. In most cases, these peptides decrease food intake, but in a few cases they have been demonstrated to stimulate feeding. Infusion of insulin peripherally will decrease food intake unless hypoglycemia occurs where the reduced glucose is a stimulus to feeding. Other pancreatic hormones including glucagon, amylin, pancreatic polypeptide, and enterostatin reduce food intake. Of the gastrointestinal hormones, cholecystokinin has been the most widely studied and reduces food intake in a number of species, including human beings. Gastrin-releasing peptide and its relative bombesin have been shown to decrease food intake in experimental animals and man. Somatostatin reduces food intake in experimental animals, but no clinical studies are available. Four pituitary peptides also modify food intake. Vasopressin decreases feeding. In contrast, injections of desacetyl melanocyte stimulating hormone (dMSH), growth hormone, and prolactin are associated with increased food intake. Finally, there are a group of miscellaneous peptides which modulate feeding. β-casomorphin, a hepta peptide produced during the hydrolysis of casein, stimulates food intake in experimental animals. In contrast, the other pep tides in this group including calcitonin, apolipoprotein A-IV, the cyclized form of histidyl-proline, several cytokines, and thyrotropin-releasing hormone decrease food intake. Many of these peptides act on gastrointestinal or hepatic receptors which relay messages to the brain via the afferent vagus nerve. As a group they provide a number of leads for potential drug development.