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At least 505 records · Page 28Linked to original sources
PubMed1998

Binding of enterostatin to the human neuroepithelioma cell line SK-N-MC.

SK-N-MC cells were found to possess binding sites for enterostatin, a peptide with central effects on appetite and sympathetic activation of brown adipose tissue during high-fat feeding. Scatchard analyses of the binding indicated one high-affinity binding (Kd = 0.5-1.5 nM) and one low-affinity binding (Kd = 15-30 nM) for 3H-enterostatin (APGPR). 125I-YGGAPGPR showed similar binding parameters as for the low affinity binding of 3H-APGPR. Met-enkephalin and beta3-casomorphin1-5 were found to displace the binding of 3H-APGPR to the SK-N-MC cells. Affinity purification of solubilized cells revealed an APGPR-binding protein estimated to 53 kDa which may represent a distinct enterostatin receptor. Cross-linking of 125I-YGGAPGPR to intact cells labeled one major protein with the same molecular size. There was no binding of enterostatin to four other human neuroblastoma/neuroepithelioma cell lines, named IMR-92, LAN#5, NB-1 #14 and SH5-SY.

Chromatography, Affinity↗
PubMed1999

On the nature and distribution of enterostatin (Val-Asp-Pro-Asp-Arg)-like immunoreactivity in rat plasma.

Enterostatins, pentapeptides represented at the amino-terminus of the procolipase molecule, are derived following tryptic cleavage of the procolipase molecule in the lumen of the gut. Val-Pro-Asp-Pro-Arg or VPDPR is one such enterostatin. Despite pharmacologic studies suggesting a role for VPDPR in appetite regulation and insulin secretion, the function of this endogenous peptide has been impossible to discern due to the lack of a suitable assay. Using polyclonal antibodies raised against VPDPR and different chromatographic methods, we examined the nature and distribution of enterostatin-like immunoreactivity in rat plasma. The results reported here show for the first time the presence of VPDPR-like immunoreactivity in rat plasma. Further characterization of the plasma VPDPR-like immunoreactivity revealed that a) it is not due to APGPR, VPGPR, or VPDPR but to another peptide similar to VPDPR, and b) plasma VPDPR-like immunoreactivity may circulate bound to large carrier proteins.

Animals↗
PubMed2002

Purification of classical pancreatic lipase from dog pancreas.

The purification of canine classical pancreatic lipase from canine pancreatic juice, but not from pancreatic tissue, has been reported previously. Given the logistic difficulties associated with collection of pancreatic juice in dogs and efforts to minimize experiments in live animals the objective of this project was to purify canine classical pancreatic lipase from dog pancreas. Dog pancreata were collected from research dogs that had been sacrificed for unrelated research projects. Pancreatic tissue was delipidated using organic solvents. The delipidated pancreatic extract was further purified by extracting the enzymes in a Tris-buffer containing two different protease inhibitors, benzamindine and phenylmethylsulfonyl fluoride (PMSF), followed by anion exchange chromatography, gel-filtration, and cation exchange chromatography. The purified protein showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of approximately 50.7. Isoelectric focusing showed isoelectric points ranging from 6.0 to 6.2. N-terminal amino acid sequencing of the first 25 amino acid residues showed the sequence Lys-Glu-Val-X-Phe-Pro-Arg-Leu-Gly-X-Phe-Ser-Asp-Asp-Ser-Pro-Trp-Ala-Gly-Ile-Val-Glu-Arg-Pro-Leu. This sequence showed close homology with classical pancreatic lipase in pigs, horses, and human beings. We conclude that canine classical pancreatic lipase can be successfully purified from canine pancreatic tissue.

Amino Acid Sequence↗
PubMed2002

Enterostatin increases extracellular serotonin and dopamine in the lateral hypothalamic area in rats measured by in vivo microdialysis.

The effect of enterostatin injection into the rat lateral hypothalamic area (LHA) on serotonin and dopamine releases in extracellular space was investigated by in vivo microdialysis technique. The primary focus being to understand whether a small amount of enterostatin crossing the blood-brain barrier correlates with activity changes in serotonergic and dopaminergic nervous systems or not. We found a significant elevation in serotonin release in the LHA. The enterostatin perfusion also induced a smaller but significant increase in dopamine level than serotonin one. This result suggests that enterostatin plays some sort of role in the control of feeding of fat through the control serotonergic and dopaminergic satiety mechanism.

Animals↗
PubMed1993

Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography.

The three-dimensional structure of the lipase-procolipase complex, co-crystallized with mixed micelles of phosphatidylcholine and bile salt, has been determined at 3 A resolution by X-ray crystallography. The lid, a surface helix covering the catalytic triad of lipase, adopts a totally different conformation which allows phospholipid to bind to the enzyme's active site. The open lid is an essential component of the active site and interacts with procolipase. Together they form the lipid-water interface binding site. This reorganization of the lid structure provokes a second drastic conformational change in an active site loop, which in its turn creates the oxyanion hole (induced fit).

Binding Sites↗
PubMed2006

Constant darkness is a circadian metabolic signal in mammals.

Environmental light is the 'zeitgeber' (time-giver) of circadian behaviour. Constant darkness is considered a 'free-running' circadian state. Mammals encounter constant darkness during hibernation. Ablation of the master clock synchronizer, the suprachiasmatic nucleus, abolishes torpor, a hibernation-like state, implicating the circadian clock in this phenomenon. Here we report a mechanism by which constant darkness regulates the gene expression of fat catabolic enzymes in mice. Genes for murine procolipase (mClps) and pancreatic lipase-related protein 2 (mPlrp2) are activated in a circadian manner in peripheral organs during 12 h dark:12 h dark (DD) but not light-dark (LD) cycles. This mechanism is deregulated in circadian-deficient mPer1-/-/mPer2m/m mice. We identified circadian-regulated 5'-AMP, which is elevated in the blood of DD mice, as a key mediator of this response. Synthetic 5'-AMP induced torpor and mClps expression in LD animals. Torpor induced by metabolic stress was associated with elevated 5'-AMP levels in DD mice. Levels of glucose and non-esterified fatty acid in the blood are reversed in DD and LD mice. Induction of mClps expression by 5'-AMP in LD mice was reciprocally linked to blood glucose levels. Our findings uncover a circadian metabolic rhythm in mammals.

Adenosine Monophosphate↗
PubMed2002

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.

Animals↗
PubMed1998

Inhibition of insulin release by enterostatin.

OBJECTIVE: These studies were designed to investigate the mechanism through which enterostatin inhibits insulin secretion from pancreatic islets. DESIGN: A static islet incubation method was used to study the effects of enterostatin on insulin secretion induced by various secretagogues and to investigate the effect of calcium ions and 8-Br-cyclic AMP on the response to enterostatin. Measurements of islet cAMP concentrations in response to enterostatin were also made. RESULTS: Enterostatin (10(-9) to 10(-5) M) inhibited insulin secretion from islets incubated in the presence of 16.7 mM glucose in a dose-dependent manner. Enterostatin also inhibited insulin secretion stimulated by glybenclamide (5.0 and 10 microM), phorbol 12-myristate-13-acetate (TPA) (50 and 100 nM), and the kappa-opioid agonist U50,488 (100 nM). The inhibitory effect of enterostatin on TPA-induced insulin secretion was attenuated but still remained in the absence of extracellular Ca2+. The enterostatin inhibition of insulin secretion was blocked by 8-Br-cAMP (1 mM) independent of extracellular Ca2+. Enterostatin reduced the increase in intracellular cyclic AMP (cAMP) content produced by U50,488 (100 nM) and the changes in cAMP content were parallel with changes in insulin release. CONCLUSION: Enterostatin may suppress insulin secretion through the reduction of cAMP, but other mechanisms may also be possible.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1999

Inhibitory effect of enterostatin on the beta cell response to digestive insulinotropic peptides.

OBJECTIVE AND DESIGN: We have investigated the effect of enterostatin on insulin release by the perfused rat pancreas: (1) under conditions of prolonged fasting and (2) under beta cell stimulation by digestive insulinotropic piptides. RESULTS: In pancreases from 24-h starved rats, the insulin response to glucose was reduced (approximately 75%) as compared to that observed in fed rats. This minimal response was abolished by 100 nM enterostatin. In fed rats, 100 nM enterostatin blocked the insulin output evoked by gastric inhibitory peptide (GIP) (approximately 75%) and by glucagon-like peptide-1 (GLP-1) (approximately 80%). Since both peptides exert their insulinotropic activity by activating the adenylate cyclase/cyclic AMP system, the interference of this pathway by enterostatin may be considered. Enterostatin (100 nM) did not modify the insulin responses to 26-33 fragment of cholecystokinin (CCK-8) and carbachol, substances which activate phosphoinositol turnover within the beta cell. The inhibitory effect of 100 nM enterostatin on glucose-induced insulin release was also observed at 50 nM and 10 nM enterostatin. Finally, des-Arg-enterostatin (100 nM) did not modify glucose-induced insulin output. CONCLUSION: Enterostatin inhibits the insulin response to glucose--both under fed and fasted conditions, and to the main digestive insulinotropic peptides--GIP and GLP-I. The entire enterostatic molecule seems to be necessary for it to exert its beta cell blocking effect. White the physiological role of enterostatin has not as yet been established, our observations allow speculation as to whether this peptide is implicated in the entero-insular axis as an antiincretin agent.

Animals↗
PubMed2000

The specific activities of human digestive lipases measured from the in vivo and in vitro lipolysis of test meals.

BACKGROUND & AIMS: The lipolytic potential of digestive lipases in vivo has always been deduced so far from their in vitro activities under nonphysiologic conditions. In the present study, the specific activities of human gastric lipase (HGL) and pancreatic lipase (HPL) were measured on dietary triglycerides (TGs) during test meal lipolysis. METHODS: Healthy human volunteers ingested a liquid or solid meal. The specific activities of HGL and HPL were estimated from the lipase and free fatty acid (FFA) outputs at the postpyloric and duodenal levels, respectively. Based on the in vivo data, lipolysis was also performed in vitro by mixing the meal either with gastric juice and subsequently with pancreatic juice and bile or with purified HGL and HPL. FFAs were measured by thin-layer chromatography, and the specific activities of HGL and HPL were expressed as micromoles of FFA per minute per milligram of lipase. RESULTS: In vitro, the specific activities on the liquid meal TGs were 32 (gastric juice) and 34 (pure lipase) micromol x min(-1) x mg(-1) with HGL and 47 (pancreatic juice) and 43 (pure lipase) micromol x min(-1). mg(-1) with HPL. The specific activities on the solid meal TGs were 33 (gastric juice) and 32 (pure lipase) micromol x min(-1) x mg(-1) with HGL and 12 (pancreatic juice) and 15 (pure lipase) micromol x min(-1) x mg(-1) with HPL. The in vivo values obtained were in the same range. The secretory lipase outputs were 21.6+/-14.5 mg HGL and 253.5+/-95.5 mg HPL with the liquid test meal and 15.2+/-5.1 mg HGL and 202.9+/-96.1 mg HPL with the solid test meal. CONCLUSIONS: The specific activities of HGL and HPL on meal TGs were much lower than those measured in vitro under optimized assay conditions (1300-8000). However, these low specific activities are enough for the meal TGs to be completely lipolysed, given the amounts of HGL and HPL secreted during a meal.

Adult↗
PubMed2003

The effects of enterostatin intake on food intake and energy expenditure.

Enterostatin (ENT) has been found to inhibit food intake and selectively inhibit fat intake in rats. Both peripheral and central mechanisms have been proposed. It also has been suggested that ENT may increase thermogenesis. The present study investigated the effects of oral ENT administration on food intake, energy expenditure and body weight in subjects with a preference for a high-fat diet. In a double-blind, placebo-controlled, randomized and crossover design, nine female and three male healthy subjects (age 34 (sd 11) years, BMI 24.5 (sd 2.5) kg/m(2)) with a preference for a high-fat diet ingested ENT (3 x 15 mg/d) or placebo (PLA) while consuming a high-fat diet ad libitum for 4 d. Eight subjects ended each intervention with a 36 h stay in the respiration chamber, continuing the diet and treatment. Body-weight loss was significant (ENT 0.8 (se 0.3) kg, P<0.05; PLA 1.3 (se 0.3) kg, P<0.001), but not different between treatments. There was no difference between treatments in total energy intake (ENT 37.1 (se 2.6), PLA 35.9 (se 3.2) MJ), macronutrient composition, hunger, satiety and hedonic scores during the 4 d high-fat diet. Energy expenditure (24 h) (ENT 9.6 (se 0.4), PLA 9.5 (se 0.4) MJ), sleeping and resting metabolic rate, diet-induced thermogenesis, activity-induced energy expenditure and 24 h RQ (ENT 0.77 (se 0.01), PLA 0.77 (se 0.01)) were similar for both treatments. We conclude that oral ENT administration did not affect food intake, energy expenditure or body weight in subjects with a preference for a high-fat diet experiencing a negative energy and fat balance.

Adult↗
PubMed2002

Mitochondrial ATP synthase--a possible target protein in the regulation of energy metabolism in vitro and in vivo.

The increasing prevalence of obesity in the Western world has stimulated an intense search for mechanisms regulating food intake and energy balance. A number of appetite-regulating peptides have been identified, their receptors cloned and the intracellular events characterized. One possible energy-dissipating mechanism is the mitochondrial uncoupling of ATP-synthesis from respiratory chain oxidation through uncoupling proteins, whereby energy derived from food could be dissipated as heat, instead of stored as ATP. The exact role of the uncoupling proteins in energy balance is, however, uncertain. We show here that mitochondrial F1F0-ATP synthase itself is a target protein for an anorectic peptide, enterostatin, demonstrated both after affinity purification of rat brain membranes and through a direct physical interaction between enterostatin and purified F1-ATP synthase. In insulinoma cells (INS-1) enterostatin was found to target F1F0-ATP synthase, causing an inhibition of ATP production, an increased thermogenesis and increased oxygen consumption. The experiments suggest a role of mitochondrial F1F0-ATP synthase in the suppressed insulin secretion induced by enterostatin. It could be speculated that this targeting mechanism is involved in the decreased energy efficiency following enterostatin treatment in rat.

Animals↗