KR101063895B1 - Peptides having AMPK activity - Google Patents

Abstract

The present invention relates to a peptide having AMPK activity, and more particularly, to a peptide containing Pro-Gly as an active ingredient and having AMPK activity, and more particularly to a peptide having an anti-obesity and anti-diabetic effect.

The peptide having an amino acid sequence comprising Pro-Gly according to the present invention may have anti-obesity and anti-diabetic effects by promoting energy metabolism of AMPK activity of skeletal muscle cells. The physiological and pharmacological effects of the peptide having an amino acid sequence including Pro-Gly can be developed as a dietary supplement for treating obesity and diabetes as well as for treating obesity and diabetes.

Description

Peptide having the AMPK activity

The present invention relates to a peptide having AMPK activity, and more particularly to a peptide containing Pro-Gly as an active ingredient and having a AMPK activity, to peptides having an effect of promoting energy metabolism, inhibiting fat synthesis, and lowering blood sugar It is about.

In general, diabetes is a disease in which glucose obtained by digesting foods due to lack of insulin secretion or decreased cellular reactivity to insulin is not properly used in our body and blood sugar is high, and overeating, lack of exercise, and increased stress due to socioeconomic development Due to the increase in diabetes population. Currently, about 4.8 million people, or 10% of the nation's 48 million people, are estimated to be diabetic. If blood glucose is not properly controlled, cardiovascular disease, cerebrovascular disease, kidney disease, retinal disease, and other peripheral vascular neuropathy There is a problem that can cause complications.

Therefore, the treatment of diabetes has a goal of glycemic control and oral hypoglycemic agents and insulin have been used for this purpose. Oral hypoglycemic agents are classified into sulfonurea agents, biguanides, and alpha glucosidase inhibitors. The representative mechanism of action of metformin, a major biguanide agent, is that of AMP-activated protein kinase (AMPK). Since it has been found to be associated with increased activity, research on new antidiabetic agents through the regulation of AMPK activity has been ongoing (Fedele D, Tiengo A, Nodadini R, Marchiori E, Briani G, Garotti MC, and Muggeo M. ( (1976) Diabetes Metab 2: 127-133) (Hermann LS. (1979) Diabtes Metab 5: 233-245) (Davidson MB, and Peters AL. (1997) Am J Med 102: 99-102) (Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyl-Melody J, Wu M, Ventre J, Doebber T, Fujji N, Musi N, Hirshman MF, Goodyear LJ, and Moller DE. (2001) J Clin Invest 108: 1167 -1174).

The AMPK is an enzyme that acts as an energy sensor for intracellular fat and glucose metabolism. It is activated when ATP decreases in the cell and AMP increases to increase the enzymatic activity of the ATP production process and the enzymatic activity of the ATP consuming process irrelevant to cell survival. (Hardie DG. (2003) Endocrinology 144: 5179-5183). The AMPK is widely expressed in human organs and tissues such as heart, skeletal muscle, liver, and fat, and exhibits the above-described action. Increased cardiac AMPK activity increases glucose uptake into cells (Russell RR 3rd, Bergeron R, Shulman GO, and Young LH. (1999) Am J Physiol. 277: H643-9), which activates glycolysis and fatty acid oxidation. Is promoted (Makinde AO, Gamble J, and Lopaschuk GD. (1997) Circ Res. 80 (4): 482-9), and in skeletal muscle only increases glucose influx and increases ATP production due to glycolysis and fat oxidation. (Ai H, Ihelemann J, Hellsten Y, Lauritzen HP, Hardie DG, Galbo H, and Ploug T. (2002) Am J Physiol 282: E1291-1300), and also promote the development of mitochondria (Zong H, Ren JM, Young LH, Pypaert M, Mu J, Birnbaum MJ, and Shulman GI. (2002) Proc Natl Acad Sci US A. 99 (25): 15983-7), increased expression of uncoupling protein (UCP). (Zhou M, Lin BZ, Coughlin S, Vallea G, and Pilch PF. (2000) Am J Physiol Endocrinol Metab 279: E622-E629) (Putman CT, Kiricsi M, Pearcey J, MacLean IM, Bamford JA, Murdoch GK, Dixon WT, and Pette D. (2003) J Physiol 551: 169-178) (Pedersen SB, Lund S, Buhl ES, and Richelson B. (2001) Biochem Biophys Res Commun 283: 19-25). Hepatic AMPK also inhibits fatty acid and cholesterol synthesis (Henin N, Vincent MF, Gruber HE, and Van den Berghe G. (1995) FASEB J 9: 541-546), and inhibits your production (Vincent MF, Marangos PJ). , Gruber HE, and Van den Berghe G. (1991) Diabetes 40: 1259-1266), which are known to inhibit fatty acid synthesis and promote lipolysis in fats (Yin W, Mu J, and Bimbaum MJ. (2003) J Biol Chem. 278: 43074-43080).

After the hypoglycemic action of metformin was mediated by AMPK, studies on the anti-diabetic and anti-obesity effects of AMPK continued, revealing that AMPK-mediated action of leptin, a representative anti-obesity hormone, was also mediated by AMPK. Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, and Kahn BB. (2002) Nature 415: 339-343). Leptin inhibits fatty acid synthesis and promotes fatty acid oxidation in muscle cells. When leptin activates AMPK, AMPK converts acetyl CoA to malonyl-CoA (which speeds up fatty acid synthesis). Determination step) Fatty acid synthesis is inhibited by inhibiting the activity of acetyl-CoA carboxylase (hereinafter referred to as 'ACC'). In addition, malonyl-CoA inhibits carnitine palmitoyltransferase (hereinafter referred to as "CPT"), which introduces fatty acids into the mitochondria (steps to determine the rate of fatty acid oxidation) (Paulson DJ, Ward KM and Shug AL. (1984) FEBS Lett. 176: 381-384), When AMPK inhibits the activity of ACC, the amount of malonyl-CoA decreases and CPT is activated to increase fatty acid oxidation in mitochondria (McGarry). JD and Brown NF. (1997) Eur J Biochem 244: 1-14) (Alam N and Saggerson ED (1998) Biochem J 334: 233-241) (Winder WW and Hardie DG. (1999) Am J Physiol 277: E1 -E10). In addition to leptin, another anti-obesity hormone, adiponectin, is known to be mediated by AMPK for fat oxidation and glucose influx (Yamauchi T, Kmon J, Minokoshi Y, Ito Y, Waki H, Ychida S, Yamashita). S, Noda M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P, Fougelle F, Ferre P, Carling D, Kimura S, Nagai R, Kahn BB, and Kadowaki T. (2002) Nat Med 8: 1288 -1295).

As mentioned above, there are many studies that are effective in anti-obesity and anti-diabetes, but there is an urgent need for development of more effective anti-diabetic and anti-obesity agents for those still suffering from obesity and diabetes. To this end, AMPK activity in various prior studies (Korean Patent Application No. 10-2006-0067833, Korean Patent Application No. 10-2006-0136534, Korean Patent Application No. 10-2007-0125701, Korean Patent Application No. 10-2007-0133156, etc.) Peptides and complexes thereof have been reported. However, the characteristics of peptides with AMPK activity have not yet been clarified.

Accordingly, the present invention seeks to provide a method for developing an AMPK active material more effectively by finding an amino acid sequence characteristic of a peptide having AMPK activity and a complex thereof. In other words, by verifying the activity of the AMPK of the peptide of various amino acid sequences to provide a novel peptide material having an energy metabolism promoting effect on the peptide having a specific amino acid sequence.

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In another aspect, the present invention provides a peptide having an AMP-activated protein kinase (AMPK) activity containing the amino acid sequence of PG, PGV, PPG, PPGV or IPPGVPY as an active ingredient.

The present invention also provides an anti-obesity or anti-diabetic composition comprising the amino acid sequence of PG, PGV, PPG, PPGV or IPPGVPY as an active ingredient.

As described above, the peptide having an amino acid sequence comprising Pro-Gly according to the present invention may have anti-obesity and anti-diabetic effects by promoting energy metabolism of AMPK activity of skeletal muscle cells. The physiological and pharmacological effects of the peptide having an amino acid sequence including Pro-Gly can be developed as a dietary supplement for treating obesity and diabetes as well as for treating obesity and diabetes.

In order to solve the above problems, the present invention measures and analyzes AMPK (5'-AMP activated protein kinase) activation of peptides of various amino acid sequences, so that the peptide containing Pro-Gly has an excellent effect on AMPK activation. Invented the show.

More details will be described through the following examples. It is obvious that the following examples do not limit the scope of the present invention.

Example 1 Selection and Synthesis of Peptides

Among the prior arts mentioned in the background art, the AMPK activity of the Cheonggukjang peptide (The-Pro-Gly-Lys-Phe) showing the AMPK activity of Korean Patent Application No. 10-2007-0125701 and the Korean Patent Application No. 10-2007-0133156 Focusing on the fact that PG is commonly included in the black soybean peptide (Ile-Pro-Pro-Gly-Val-Pro-Tyr), it was studied whether the peptide containing PG generally had excellent AMPK activity.

Peptides used in the present invention, such as Pro-Gly and peptides having the amino acid sequence, were synthesized and used in Peptron Co., Ltd. AMPK activity measurement is described through Example 2 below.

Example 2 AMPK Activity Verification of Peptides

1) Cell culture

The C2C12 cell line, known as rat muscle differentiation cell, was referred to as DMEM (DMEM) containing 10% fetal bovine serum (FBS) in a 6 well plate. (Life Technologies) was cultured in the medium. When the cell density was about 85-90%, the cells were replaced with DMEM medium containing 2% horse serum and differentiated into muscle cells for 6 days.

2) sample treatment

After 16 hours of incubation in serum-free DMEM (serum free DMEM), positive control (2 mM AICAR) and peptide were treated for 1 hour.

3) protein lysis

After removing the medium, the differentiated cells were washed once with Phosphate Buffered Saline (hereinafter referred to as 'PBS') and then lysis buffer (50 mM Tris-HCl, pH 7.3, 50 mM NaF). , 30 mM glycol phosphate, 250 mM sucrose, 1 mM sodium metavanadate, and 0.4 mg / ml) were added to dissolve the cell membrane to obtain cytoplasmic protein and ultracentrifugation. (13,000rpm, 10min, 4 ℃) to obtain a supernatant and the protein concentration was determined using the Bradford Protein Assay (Bradford Protein Assay (Bio-Rad)).

4) Identification of activated AMPK through immunoassay

Phosphor-AMPK-α-antibody as primary antibody and anti-rabbit IgG and anti-rabbit IgG as secondary antibody (HRP-linked) The amount of AMPK activated with the antibody was confirmed by western blotting.

Example 3 AMPK Activity of Di-peptide

 Among the di-peptides consisting of two amino acids, Pro-Gly of the present invention (hereinafter referred to as 'PG') and Gly-Pro (hereinafter referred to as 'GP') which changed the order of the sequence and di-peptides having molecular weight similar to PG AMPK activity of Ser-Ala (hereinafter referred to as 'SA') was measured by the method of Example 2 described above.

As a result, it is shown in FIG. It shows AMPK activity when C2C12 cells were treated with PG and peptides including the same. C (-) is AMPK activity when not treated with a negative control, based on 1.0. AMPK activity expressed pAMPK / b-actin of treated cells relative to control. In other words,

AICAR determines that pAMPK / b-actin is greater than 1.0 whether the experiment proceeded normally when treated with a positive control.

As shown in FIG. 1, PG showed 2.3 times higher activity than control, whereas GP or SA showed low activity below 1.0.

Example 4 AMPK Activity of Peptides Containing PG

Among the peptides, peptides IPPGVPY, PPGV, PGV, peptides containing PG, and peptides IPVPY and VPPQ not containing PG were measured according to Example 2. The results are shown in Table 1.

Table 1 Comparison of AMPK Activity of PG-containing Peptides and PG-Free Peptides

Peptide AMPK activity Peptide AMPK activity IPPGVPY 1.9 PPG 2.5 PPGV 2.4 IPVPY 1.4 PGV 2.7 VPPQE 1.1

As shown in Table 1, the peptide containing PG, such as IPPGVPY showed a relatively high activity of 1.9 to 2.7 compared to the control, whereas the peptide containing no PG, such as IPVPY showed a low activity of 1.4 or less.

Therefore, PG-containing peptides activate AMPK, which activates glycolysis by activated AMPK, promotes fatty acid oxidation and degradation, increases ATP production, and increases the expression of uncoupling proteins, thereby promoting energy consumption. do. Therefore, it can be seen that the peptide containing PG has an antidiabetic effect and an obesity inhibitory effect.

Figure 1 shows the AMPK activity when the C2C12 cells treated with PG and the peptide containing the same.

Claims (3)

delete A composition having an AMP-activated protein kinase (AMPK) activity comprising a peptide represented by the following amino acid sequence as an active ingredient: PG, PGV, PPG, PPGV or IPPGVPY. An anti-obesity or anti-diabetic composition comprising a peptide represented by the following amino acid sequence as an active ingredient. PG, PGV, PPG, PPGV or IPPGVPY.

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