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.     

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.     

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.     

Effects of Enterostatin on Consumption of Optional Foods in Non-Food-Deprived Rats

Enterostatin, the activation peptide of procolipase, has been reported to reduce high-fat food consumption in rats. This reduction has been reliably demonstrated using procedures in which the test diet was also the maintenance diet of the animals. Other reports, though, have shown that peripherally administered enterostatin had no effect on the consumption of oil provided as an option to the diet, and that centrally administered enterostatin had no effect on the consumption of an optional high-fat mixed food. However, the effects of peripherally administered enterostatin on the consumption of an optional high-fat mixed food have not been examined. This experiment, then, examined the effects of peripherally administered enterostatin on the consumption of optional, mixed foods (no-fat and high-fat cookies) provided in addition to a standard diet under choice and nonchoice conditions. Four experiments were conducted. In experiment I, the effect of enterostatin in a two-choice feeding paradigm was assessed. In experiment II, the effect of enterostatin in a nonchoice feeding paradigm was assessed. In experiment III, the effect of enterostatin administered at five different pretreatment times in a non-choice feeding paradigm was assessed. Enterostatin had no effect on cookie intake in any of these experiments. Finally, experiment IV was undertaken to verify the activity of the peptide. Enterostatin significantly reduced the consumption of a standard diet in overnight food-deprived rats, thus confirming the activity of the peptide used in experiments I to III. Enterostatin may not play a major role in the regulation of food intake that is stimulated by optional foods that are periodically provided in addition to a standard well-balanced diet.     

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.     

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.     

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.     

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 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 reduces serum cholesterol levels by way of a CCK(1) receptor-dependent mechanism

Enterostatin (APGPR), an anorectic pentapeptide derived from the amino terminus of procolipase, significantly reduced serum cholesterol levels after oral administration at a dose of 100 mg/kg for 3 days in mice fed a high-cholesterol-cholic acid diet. The hypocholesterolemic effect of APGPR was inhibited by pretreatment with lorglumide, an antagonist for cholecystokinin 1 (CCK(1)) receptor, even though APGPR does not have any affinity for CCK(1) receptors. Similarly, the hypocholesterolemic activity of VPDPR, an APGPR analogue, was blocked by lorglumide. These results suggest that the hypocholesterolemic effects of APGPR and VPDPR are mediated by a CCK(1) receptor-dependent mechanism.     

Enterostatin (APGPR) suppresses the analgesic activity of morphine by a CCK-dependent mechanism

Enterostatin (APGPR) found in the gastrointestinal tract and brain is an anorectic pentapeptide. We found that APGPR inhibited morphine-induced analgesia after intracerebroventricular administration in mice at a dose of 10nmol/mouse. The anti-analgesic effect of APGPR was inhibited by pretreatment with lorglumide and LY225910, antagonists for cholecystokinin 1 (CCK1) and cholecystokinin 2 (CCK2) receptors, respectively. The anti-analgesic effect of APGPR may be mediated by CCK release, since APGPR does not have affinity for CCK receptors.     

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.     

Comparative study of enterostatin sequence in five rat strains and enterostatin binding proteins in rat and chicken serum

Enterostatin, a pentapeptide derived from the precursor protein procolipase has been shown to inhibit dietary fat intake and to reduce body fat after chronic administration in rats. We repeat that the enterostatin amino acid sequence from the genomic DNA of 5 different rat strains is APGPR. 125I-APGPR bound to three proteins (300, 205 and 60 kDa) in rat serum and one 60 kDa protein in chicken serum. These serum binding proteins were also eluted by APGPR affinity chromatography. Western blot analysis of serum protein identified enterostatin-like immunoreactivity associated with the same molecular weight bands. Our results demonstrate the enterostatin sequence in rat is APGPR and suggest the presence of enterostatin binding proteins in rat and chicken serum.     

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.     

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.     

beta-Lactotensin, a neurotensin agonist peptide derived from bovine beta-lactoglobulin, enhances memory consolidation in mice

beta-Lactotensin (His-Ile-Arg-Leu) is an ileum-contracting tetrapeptide isolated from bovine beta-lactoglobulin. We previously reported that a neurotensin agonist beta-lactotensin shows antinociceptive effect through neurotensin NT(2) receptor. We found that centrally or orally administered beta-lactotensin at a dose of 60nmol/mouse or 300-500mg/kg, respectively, increased memory consolidation in the step-through-type inhibitory avoidance test in mice. The memory-enhancing activity of beta-lactotensin was inhibited by the dopamine D(2) receptor antagonist raclopride but not the D(1) receptor antagonist SCH23390. Taken together, beta-lactotensin might improve memory consolidation through activating the dopamine D(2) receptor.     

The F1-ATPase beta-subunit is the putative enterostatin receptor

It has been suggested that the F₁-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin_1–7, in the absence and presence of cold enterostatin. ¹²⁵I-β-casomorphin_1–7 weakly binds to the rat F₁-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin_1–7 binding to the F₁-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin_1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin_1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F₁-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F₁-ATPase complex. Western blot analysis showed the F₁-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F₁-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin_1–7 bind to distinct sites on the protein.     

Methanolic Extract of Piper nigrum Fruits Improves Memory Impairment by Decreasing Brain Oxidative Stress in Amyloid Beta(1–42) Rat Model of Alzheimer’s Disease

The present study analyzed the possible memory-enhancing and antioxidant proprieties of the methanolic extract of Piper nigrum L. fruits (50 and 100 mg/kg, orally, for 21 days) in amyloid beta(1–42) rat model of Alzheimer’s disease. The memory-enhancing effects of the plant extract were studied by means of in vivo (Y-maze and radial arm-maze tasks) approaches. Also, the antioxidant activity in the hippocampus was assessed using superoxide dismutase-, catalase-, glutathione peroxidase-specific activities and the total content of reduced glutathione, malondialdehyde, and protein carbonyl levels. The amyloid beta(1–42)-treated rats exhibited the following: decrease of spontaneous alternations percentage within Y-maze task and increase of working memory and reference memory errors within radial arm-maze task. Administration of the plant extract significantly improved memory performance and exhibited antioxidant potential. Our results suggest that the plant extract ameliorates amyloid beta(1–42)-induced spatial memory impairment by attenuation of the oxidative stress in the rat hippocampus.     

Evidence for a functional role of cholecystokinin receptors in the rat thyroid gland.

The aim of the present study was to examine the role of cholecystokinin (CCK) and/or cholecystokinin receptors subtypes (CCK1R and CCK2R) in the regulation of the thyroid gland structure and function. Animals were autopsied after 6 days of treatment with CCK or CCK receptor-specific antagonists (CCK1a--PD 140,548 or CCK2a--PD 135,158) solely or in combination with CCK. Results suggest that CCK exerts a stimulatory effect on follicular thyroid cells manifested by increased epithelium/colloid volume fraction ratio (E/C). Application of selective antagonists of CCK receptor subtypes has demonstrated that CCK acts through the CCK1 receptor subtype at the level of pituitary TSH. The model of endogenous hormone action reveals that thyroid CCK1 is responsible for the thyroid growth. It can be concluded that the physiological activity of CCK1 receptor plays a significant role in a complex interrelationship between TSH, vagal system and CCK1-dependent function of the thyroid gland.