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Enterostatin, human, mouse, rat是可以减少脂肪摄入量的五肽。
Enterostatin, human, mouse, rat is a pentapeptide that reduces fat intake.

Definition

Enterostatin is a pentapeptide generated by the action of trypsin on procolipase in the intestinal lumen. Pharmacologic studies have suggested a role for these peptides in appetite regulation and insulin secretion 1.

Related Peptides

Enterostatins belong to a family of peptides (e.g., Val-Pro-Asp-Pro-Arg, VPDPR; Ala-Pro-Gly-Pro-Arg, APGPR; and Val-Pro-Gly-Pro-Arg, VPGPR) derived from the tryptic cleavage of amino-terminal pentapeptide from procolipase 1.

Discovery

In 1993, Mei et al., identified the peptapeptide obtained from the tryptic cleavage of the N-terminal of procolipase and named it enterostatin 2.

Structural Characteristics

Enterostatin structure is highly conserved in evolution, with an amino acid sequence of XPXPR. Three enterostatin sequences, Val-Pro-Asp-Pro-Arg (VPDPR), Val-Pro-Gly-Pro-Arg (VPGPR), and Ala-Pro-Gly-Pro-Arg (APGPR), have been studied extensively and shown to be almost equally effective in their ability to decrease dietary fat preference 3. Modifications in the N-terminus of VPDPR abrogate biological function, other modifications result in retention of biological activity. The importance of the N-terminal residue of enterostatin was investigated by replacing the L-valine with (i) D-valine, (ii) L-tyrosine or (iii) addition of an L-tyrosine to generate the hexapeptide, YVPDPR. Replacement of the N-terminal L-valine residue with D-valine abrogated the insulin inhibitory activity of the peptide. This demonstrates that the N-terminal valine may be necessary for biological function and is consistent with a previous report showing a lack of effect of D-VPDPR on high-fat food consumption 4.

Mode of Action

A high-fat diet easily promotes hyperphagia giving an impression of an uncontrolled process. Fat digestion itself however provides control of fat intake through the digestion, carried out by pancreatic lipase and its protein cofactor colipase, and through enterostatin, released from procolipase during fat digestion 5. Neural mechanisms arising from the intestine involving vagal afferent nerves may be important for the early satiety mechanism induced by enterostatin during a meal 6. Enterostatin acts through an opioid pathway, rather as a µ-antagonist acting antagonistic to ß-casomorphin. In its mechanism of action enterostatin gives an exothermic reaction, while the opiate has an endothermic reaction. The decreased thermogenesis by opiates could explain an increased appetite and body weight, while an increased thermogenesis by enterostatin could explain a reduction in appetite and loss of body weight. The hypocholesterolemic effects of APGPR and VPDPR are mediated by a CCK1 receptor-dependent mechanism 7.

Functions

Enterostatin released from the exocrine pancreas and gastrointestinal tract, selectively inhibits fat intake through activation of an afferent vagal signaling pathway 8. Recent studies indicate that enterostatin modulates insulin release in response to a variety of agents that affect first phase as well as the second phase of insulin release 4.

References

1. Imamura M, Sumar N, Hermon-Taylor J, Robertson HJ, Prasad C (1998). Distribution and characterization of enterostatin-like immunoreactivity in human cerebrospinal fluid. Peptides, 19(8):1385-1391.
2. Mei J, Bowyer RC, Jehanli AMT, Patel G, Erlanson-Albertsson C (1993). Identification of enterostatin, the pancreatic procolipase activation peptide in the intestine of rat. Pancreas, 8:488-493.
3. Prasad C, Imamura M, Debata C, Svec F, Sumar N, Hermon-Taylor J (1999). Hyper- enterostatinemia in Premenopausal Obese Women. J Clin Endocrinol Metab., 84(3):937-941.
4. Tadayyon M, Liou S, Briscoe CP, Badman G, Eggleston DS, Arch JRS, York DA (2002). Structure-function studies on enterostatin inhibition of insulin release. Int J Diabetes & Metabolism., 10:14-21.
5. Berger K, Winzell MS, Mei J, Erlanson-Albertsson C (2004). Enterostatin and its target mechanisms during regulation of fat intake. Physiol Behav., 83(4):623-630.
6. Mei J, Sörhede-Winzell M, Erlanson-Albertsson C (2002). Plasma Enterostatin: Identification and Release in Rats in Response to a Meal. Obes Res., 10(7):688–694.
7. Takenaka Y, Shimano T, Mori T, Hou IC, Ohinata K, Yoshikawa M. (2008). Enterostatin reduces serum cholesterol levels by way of a CCK1 receptor-dependent mechanism. Peptides.,29(12):2175-2178.
8. Lin L, Thomas SR, Kilroy G, Schwartz GJ, York DA (2003). Enterostatin inhibition of dietary fat intake is dependent on CCK-A receptors Am J Physiol Regul Integr Comp Physiol., 285(2):321-328.

多肽H2N-Ala-Pro-Gly-Pro-Arg-COOH的合成步骤：

1、合成CTC树脂：称取2.81g CTC Resin（如初始取代度约为0.4mmol/g）和1.35mmol Fmoc-Arg(Pbf)-OH于反应器中，加入适量DCM溶解氨基酸（需要注意，此时CTC树脂体积会增大好几倍，避免DCM溶液过少），再加入3.37mmol DIPEA（Mw：129.1,d：0.740g/ml），反应2-3小时后，可不抽滤溶液，直接加入1ml的HPLC级甲醇，封端半小时。依次用DMF洗涤2次，甲醇洗涤1次，DCM洗涤一次，甲醇洗涤一次，DCM洗涤一次，DMF洗涤2次（这里使用甲醇和DCM交替洗涤，是为了更好地去除其他溶质，有利于后续反应）。得到  Fmoc-Arg(Pbf)-CTC Resin。结构图如下：

2、脱Fmoc：加3倍树脂体积的20%Pip/DMF溶液，鼓氮气30分钟，然后2倍树脂体积的DMF 洗涤5次。得到 H2N-Arg(Pbf)-CTC Resin 。（此步骤脱除Fmoc基团，茚三酮检测为蓝色，Pip为哌啶）。结构图如下：

3、缩合：取3.37mmol Fmoc-Pro-OH 氨基酸，加入到上述树脂里，加适当DMF溶解氨基酸，再依次加入6.74mmol DIPEA，3.2mmol HBTU。反应30分钟后，取小样洗涤，茚三酮检测为无色。用2倍树脂体积的DMF 洗涤3次树脂。（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。得到Fmoc-Pro-Arg(Pbf)-CTC Resin。氨基酸：DIPEA：HBTU：树脂=3：6：2.85：1（摩尔比）。结构图如下：

4、依次循环步骤二、步骤三，依次得到

H2N-Pro-Arg(Pbf)-CTC Resin

Fmoc-Gly-Pro-Arg(Pbf)-CTC Resin

H2N-Gly-Pro-Arg(Pbf)-CTC Resin

Fmoc-Pro-Gly-Pro-Arg(Pbf)-CTC Resin

H2N-Pro-Gly-Pro-Arg(Pbf)-CTC Resin

Fmoc-Ala-Pro-Gly-Pro-Arg(Pbf)-CTC Resin

以上中间结构，均可在专肽生物多肽计算器-多肽结构计算器中，一键画出。

最后再经过步骤二得到 H2N-Ala-Pro-Gly-Pro-Arg(Pbf)-CTC Resin，结构如下：

5、切割：6倍树脂体积的切割液（或每1g树脂加8ml左右的切割液），摇床摇晃 2小时，过滤掉树脂，用冰无水乙醚沉淀滤液，并用冰无水乙醚洗涤沉淀物3次，最后将沉淀物放真空干燥釜中，常温干燥24小试，得到粗品H2N-Ala-Pro-Gly-Pro-Arg-COOH。结构图见产品结构图。

切割液选择：1）TFA：H2O=95%：5%、TFA：H2O=97.5%：2.5%

2）TFA：H2O：TIS=95%：2.5%：2.5%

3）三氟乙酸：茴香硫醚：1，2-乙二硫醇：苯酚：水=87.5%：5%：2.5%：2.5%：2.5%

（前两种适合没有容易氧化的氨基酸，例如Trp、Cys、Met。第三种适合几乎所有的序列。）

6、纯化冻干：使用液相色谱纯化，收集目标峰液体，进行冻干，获得蓬松的粉末状固体多肽。不过这时要取小样复测下纯度 是否目标纯度。

7、最后总结：

杭州专肽生物技术有限公司（ALLPEPTIDE https://www.allpeptide.com）主营定制多肽合成业务，提供各类长肽，短肽，环肽，提供各类修饰肽，如：荧光标记修饰（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基团修饰肽（叠氮、炔基、DBCO、DOTA、NOTA等），同位素标记肽（N15、C13），订书肽（Stapled Peptide）,脂肪酸修饰肽（Pal、Myr、Ste），磷酸化修饰肽（P-Ser、P-Thr、P-Tyr），环肽（酰胺键环肽、一对或者多对二硫键环），生物素标记肽，PEG修饰肽，甲基化修饰肽

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