Colipase enhances hydrolysis of dietary triglycerides in the absence of bile salts.

This study explores how dietary lipids are digested when intraduodenal bile salts are low or absent. Long-chain triglycerides emulsified with phosphatidylcholine were found to be hydrolyzed very slowly by pancreatic lipase alone, as if the surface layer of phospholipids enveloping the triglycerides impeded the action of the enzyme. Colipase enhanced triglyceride hydrolysis severalfold, both when added before or after the lipase. Hydrolysis became even more rapid when the emulsion was first incubated with pancreatic phospholipase. Hydrolysis of long-chain triglycerides was also severely impeded when other proteins were added to the system, probably because they adsorbed to the oil-water interface of the emulsion droplets. It was previously known that bile salts can relieve such inhibition, presumably by desorbing the adsorbed proteins. Colipase was found to enhance hydrolysis severalfold in a dose-dependent manner even in the absence of bile salts, i.e., it could partially or completely relieve the inhibition depending upon the amount and the type of inhibitory protein added to the system. Prior exposure of a protein-coated triglyceride emulsion to another lipase also enhanced the rate at which pancreatic lipase could then hydrolyze the lipids. Most dietary triglycerides are probably presented for intestinal digestion in emulsions covered by proteins and/or phospholipids. These emulsions would be hydrolyzed slowly by pancreatic lipase alone. However, through the action of the lipase in stomach contents and of pancreatic phospholipase and through the lipolysis-promoting effects of collipase, these triglycerices can be rather efficiently hydrolyzed, even in the absence of bile salts.

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.

Inhibition of insulin release by intraduodenally infused enterostatin-VPDPR in rats.

Enterostatin, an amino-terminal pentapeptide produced in the intestinal lumen after cleavage of pancreatic procolipase, has been shown to suppress fat intake in rats after intraduodenal infusion. In this study, female Sprague-Dawley rats fitted with a duodenal catheter were intestinally infused with enterostatin (Val-Pro-Asp-Pro-Arg, 11.3 and 22.6 nmol/kg/min) plus 20% Intralipid for 30 min. Plasma insulin levels were significantly reduced, whereas plasma glucose concentrations were not altered by enterostatin-VPDPR. The tripeptide Asp-Pro-Arg was also found to decrease the levels of plasma insulin. However, the pentapeptide with the sequence Val-Pro-Gly-Pro-Arg, des-Arg-enterostatin Val-Pro-Asp-Pro and the tripeptide Pro-Asp-Pro failed to cause the reduction of plasma insulin levels in rats following intestinal infusion of these peptides. Radiolabeled enterostatin ([3H]VPDPR) was identified in plasma by HPLC following intraduodenal infusion of the peptide, indicating that the appearance of an intact enterostatin-VPDPR in blood. It is concluded that intestinally administered enterostatin-VPDPR and its metabolites reduce plasma levels of insulin stimulated by Intralipid.

Intracerebroventricular enterostatin stimulates food intake in non-food-deprived rats.

Previous studies reported that ICV enterostatin reduced high-fat food intake in food-deprived rats. The present study sought to determine if ICV enterostatin would decrease intake of a high-fat food in non-food-deprived rats. Eight doses (0-32 micrograms) were tested. Enterostatin (32 micrograms) significantly stimulated cookie intake at 30 min. Enterostatin did not reduce food intake at any dose. These results conflict with previous reports and suggest that central enterostatin does not play a role in suppressing, but may play a role in stimulating, high-fat food consumption in non-food-deprived rats.

Changes in the microstructure of feeding after administration of enterostatin into the paraventricular nucleus and the amygdala.

The effects of enterostatin (ENT) injected into the paraventricular nucleus (PVN) and the amygdala (AMYG) on the microstructure of feeding was studied by using an automated feeding apparatus. In rats adapted to a 6-h meal feeding regime, ENT reduced the size and duration of the first meal after injection in both the PVN and the AMYG. Similar effects were observed when ENT was given at the beginning of the dark cycle in rats fed ad libitum although the onset of feeding was also delayed in this situation. The number of meals and the size of subsequent meals was unaffected by ENT but the eating rate within the first meal was reduced after ENT injection into the AMYG of meal-fed rats. Enterostatin injected into the AMYG at a dose that suppressed feeding did not produce a conditioned taste aversion. ENT given centrally therefore appears to reduce food intake by delaying the initiation of feeding and/or advancing meal termination suggesting that it affects both appetite and satiation mechanisms.

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 suppresses food intake following injection into the third ventricle of rats.

The effect on food intake of an activation peptide from pancreatic pro-colipase, called enterostatin, has been studied after parenteral or third ventricular administration. The activation peptide (enterostatin = Val-Pro-Asp-Pro-Arg = VPDPR) reduced food intake when given intraperitoneally. Low doses of this peptide also reduced food intake when given into the third ventricle, but high doses were ineffective. Enterostatin did not modify the stimulatory effects on food intake of clonidine, an alpha 2-adrenergic agonist, suggesting that its anorectic effects are not mediated via the alpha 2-adrenergic system. These data suggest that enterostatin, an activation peptide from pro-colipase, may play a role in producing satiety.

Uptake across the blood-brain barrier and tissue distribution of enterostatin after peripheral administration in rats.

Enterostatin, the N-terminal activation pentapeptide of procolipase that is produced by the pancreas, reduces food intake from high-fat diet when injected either peripherally or centrally to rats. We investigated uptake across the blood-brain barrier (BBB) and tissue distribution of enterostatin by giving radioactive-labeled enterostatin (3H-VPDPR) intravenously. Low levels of 3H-VPDPR were detected in many areas of the brain, with greatest radioactivity in the frontal cortex, hippocampus and cerebellum. Radioactivity was found in the plasma and all tissues, with the highest amount detected in the pancreas.

Reduction of feed intake in sheep by enterostatin, the procolipase activation peptide.

Enterostatin, a peptide formed during the activation of pancreatic procolipase in the duodenum, is likely a mediator of satiety in the rat. The objective of this research was to determine whether administration of enterostatin into the lateral cerebral ventricle of sheep unfed for 2 h resulted in reduced feed intake. Bolus doses of 2 micrograms did reduce (P < .03) feed intake during the hour after injection by one- to two-thirds of control. Doses 20 micrograms or greater were not effective, and .2 microgram also did not have any statistically significant effect. This range of effective doses was similar to that observed in the rat, and the effective dose of 2 micrograms was approximately 10-fold greater on a per-animal basis, but less on a brain-size basis, than the amount needed to achieve a similar response in the rat. Therefore, enterostatin can reduce feed intake and thus may also be involved in satiety in sheep.

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.

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 adrenalectomized 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.

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.

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.

Plasma enterostatin: identification and release in rats in response to a meal.

OBJECTIVE: To discover a possible absorption and/or secretion of enterostatin into the circulating blood, as well as to compare the levels of circulating enterostatin after high-fat feeding and low-fat feeding. RESEARCH METHODS AND PROCEDURES: Using a specific enzyme-linked immunosorbent assay, plasma enterostatin levels were determined after feeding a high-fat, a high-fat/-sucrose, or a low-fat meal to Sprague-Dawley rats deprived of food overnight. RESULTS: The enterostatin levels were increased by all diets; the response to the high-fat and the high-fat/-sucrose meals was greater in magnitude and duration than that to the low-fat meal. In addition, enterostatin levels correlated with the intake of dietary fat. Plasma enterostatin levels after high-fat feeding were found to be similar to those after intravenous administration of exogenous enterostatin known to inhibit high-fat food intake. Gel chromatography of pooled postprandial plasma extracts followed by high-performance liquid chromatography analysis showed that plasma enterostatin was identical to synthetic enterostatin. Affinity cross-linking of plasma proteins with 125I-enterostatin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography, revealed a single band with a molecular weight of about 66 kDa, indicating the presence of a potential enterostatin-binding protein in plasma. DISCUSSION: The measurements of plasma enterostatin may be a sensitive indicator for the measurement of fat intake.

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 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.

Chronic ingestion of dietary fat is a prerequisite for inhibition of feeding by enterostatin.

Enterostatin (Ent), the activation pentapeptide from procolipase, inhibits the intake of dietary fat. The selectivity of the response to fat suggests that the rat must recognize a permissive signal related to dietary fat for the Ent biological response. To investigate the nature of this signal, we studied the effects of Ent in rats that were adapted to either a high-fat (HF) or high-carbohydrate/low-fat (HC) diet and then naively exposed to either HF or HC diets. Ent (1 nmol) was injected into the lateral ventricle of overnight-fasted rats, and food intake was measured. Rats adapted to HF diet and tested with HC diet responded to Ent, but rats adapted to HC diet and tested with HF did not respond to Ent. The groups were maintained on their new test diets for up to 21 days and tested again for their response to Ent at 3, 7, 14, and 21 days. Ent response did not appear in HC-adapted rats switched to HF diet before 21 days. Conversely, the HF-adapted rats, which responded to Ent when tested with HC diet for the first time, did not respond at any subsequent testing time. The data suggest that chronic ingestion of dietary fat is required for Ent action and that chronic consumption of fat initiates a postingestion metabolic, endocrine, or neurochemical change that is required for the biological response to Ent.

Intravenous enterostatin does not affect single meal food intake in man.

Enterostatin, a pentapeptide released with colipase from pancreatic procolipase in man, affects eating behaviour in animals. We report the first phase II study of intravenous (i.v.) enterostatin (D3800) in obese but otherwise healthy men. Eighteen men (mean age 37 years, mean body mass index 34.9 kg/m2) completed a double-blind, randomized, crossover placebo controlled trial. After in initial session, each man received i.v. 4 mg D3800, 16 mg D3800 or placebo in random order over three sessions, immediately before a test meal served on a universal eating monitor. No statistically significant effect of i.v. enterostatin on any uptake or rating variable was observed. Several factors may explain the lack of effect, e.g. the inability of i.v. enterostatin to reach a site of action, the time between i.v. administration and eating, and the possibility that the only human responders are those who express particular fat preferences.

Enterostatin--its ability to inhibit insulin secretion and to decrease high-fat food intake.

Enterostatin is a peptide which has been found to decrease food intake with a specificity for the fat contained in the food. In this work we have investigated the effect of enterostatin (Val-Pro-Asp-Pro-Arg) and its proteolytic fragments, des-arg-enterostatin (Val-Pro-Asp-Pro) and the tripeptide Asp-Pro-Arg, on insulin secretion. It was found that enterostatin and desarg-enterostatin inhibited insulin secretion from isolated rat islets by 55.3% (P < 0.05) and 53.6% (P < 0.05) at 1.6 x 10(-4) M concentration, while the tripeptide Asp-Pro-Arg at 1.6 x 10(-4) M concentration had no significant effect and increased insulin secretion by 33.0%. Enterostatin at 200 ng after intraventricular administration was found to inhibit the intake of a high-fat diet by 45.0%, while des-arg-enterostatin (200 ng) had no effect, in agreement with previous findings. The tripeptide Asp-Pro-Arg (200 ng) had no effect on the intake of a high-fat diet compared to saline injection. The ability of enterostatin to inhibit high-fat food intake and decrease insulin secretion may be important for the prevention of obesity and type II diabetes, conditions linked through hyperinsulinemia.

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 beta-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.

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.