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Gastro-Resistant Insulin Receptor-Binding Peptide from Momordica charantia Improved the Glucose Tolerance in StreptozotocinInduced Diabetic Mice via Insulin Receptor Signaling Pathway Hsin-Yi Lo, Chia-Cheng Li, Feng-Yuan Chen, Jaw-Chyun Chen, Chien-Yun Hsiang, and Tin-Yun Ho J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03583 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017

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Journal of Agricultural and Food Chemistry

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Gastro-Resistant Insulin Receptor-Binding Peptide from Momordica charantia Improved

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the Glucose Tolerance in Streptozotocin-Induced Diabetic Mice via Insulin Receptor

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Signaling Pathway

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Hsin-Yi Lo,† Chia-Cheng Li,† Feng-Yuan Chen,† Jaw-Chyun Chen,‡ Chien-Yun Hsiang,§,*,

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Tin-Yun Ho, ǁ,*

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†

Graduate Institute of Chinese Medicine, China Medical University, Taichung, Taiwan

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‡

Department of Medicinal Botany and Healthcare, Da-Yeh University, Changhua, Taiwan

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§

Department of Microbiology, China Medical University, Taichung, Taiwan

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ǁ

Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan

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* Corresponding author. Tin-Yun Ho, Telephone: +886 4 22053366 x 3302. Fax: +886 4

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22032295. E-mail address: [email protected]

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*Corresponding author. Chien-Yun Hsiang, Telephone: +886 4 22053366 x 2163. Fax: +886 4

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22053764. E-mail address: [email protected]

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Short title: Gastro-resistant mcIRBP-9 improved glucose tolerance in diabetic mice

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ACS Paragon Plus Environment


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ABSTRACT

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Momordica charantia is a commonly used food and has been used for the management

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of diabetes. Our previous study has identified an insulin receptor (IR)-binding protein

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(mcIRBP) from Momordica charantia. Here we identified the gastro-resistant

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hypoglycemic bioactive peptides from protease-digested mcIRBP. By in vitro digestion

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and IR kinase activity assay, we found that a 9-amino-acid-residue peptide, mcIRBP-9,

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was a gastro-resistant peptide that enhanced IR kinase activities. mcIRBP-9 activated IR

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signaling transduction pathway, resulting in the phosphorylation of IR, the translocation

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of glucose transporter 4, and the uptake of glucose in cells. Intraperitoneal and oral

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administration of mcIRBP-9 stimulated the glucose clearance by 30.91 ± 0.39% and

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32.09 ± 0.38%, respectively, in streptozotocin-induced diabetic mice. Moreover, a pilot

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study showed that daily ingestion of mcIRBP-9 for 30 days decreased the fasting blood

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glucose levels and glycated hemoglobin (HbA1c) levels by 23.62 ± 6.14% and 24.06 ±

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1.53%, respectively. In conclusion, mcIRBP-9 is a unique gastro-resistant bioactive

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peptide generated after the digestion of mcIRBP. Furthermore, oral administration of

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mcIRBP-9 improves both the glucose tolerance and the HbA1c levels in diabetic mice

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via targeting IR signaling transduction pathway.

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KEYWORDS: diabetes, gastro-resistant peptide, hypoglycemia, insulin receptor,

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Momordica charantia 2


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INTRODUCTION

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Diabetes and its complications are severe global health problems.1 In 2015, there are

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415 million people with diabetes worldwide and five million people die because of

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diabetes. The estimated number of diabetic patients will increase by 55%, reaching

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642 million patients by 2040. Furthermore, the global health cost due to diabetes is

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USD 673 billion worldwide in 2015 and will reach to USD 802 billion by 2040.2

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Diabetes is characterized by hyperglycemia, which is caused by defective insulin

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secretion, resistance to insulin action, or a combination of both.1 Some foods have

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showed functional activities on the regulation of blood glucose. For example,

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jaboticaba (Myrciaria jaboticaba) is rich in polyphenols with anti-oxidative

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activities.3 Intake of jaboticaba peels decreases glucose and insulin levels after the

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second meal, suggesting that jaboticaba improves the insulin sensitivity in healthy

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adults.4 Single oral administration of 1-deoxynojirimycin-enriched mulberry powder

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significantly suppresses the elevation of postprandial blood glucose and the secretion

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of insulin, suggesting that mulberry powder can be used as a dietary supplement for

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the prevention of diabetes in healthy subjects.5 Furthermore, daily administration of

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Ginkgo biloba L. leave dry extract improves the glycemic control in patients with type

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2 diabetes.6 Three-month supplementation of ginger (Zingiber officinale) improves

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glycemic indices in type 2 diabetic patients. Moreover, daily supplementation with 3



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fermented red ginseng lowers postprandial glucose levels in subjects with impaired

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fasting glucose or type 2 diabetes.7 In addition of food extracts, intake of soy proteins

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significantly improves the glucose homeostasis in women with gestational diabetes.8

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Conglutin gamma protein from Lupinus albus seeds has a relevant postprandial

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hypoglycemic effect in humans, suggesting that it could potentially be used to manage

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patients with impaired glucose metabolism.9 Additionally, oral administration of

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insulin-like protein from Costus igneus significantly reduces the blood glucose levels

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in streptozotocin (STZ)-induced diabetic Swiss mice via insulin signaling pathway.10

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These studies therefore promise the beneficial effects of foods and food-derived

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peptides on the regulation of blood glucose.

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Bitter melon (Momordica charantia L.) is a commonly used vegetable and has

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been widely used in traditional medicine for the treatment of diabetes in Asia. Daily

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intake of bitter melon for four weeks shows hypoglycemic effects in patients with

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type 2 diabetes.11 Bitter melon decreases fasting blood glucose levels and lowers 2-h

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plasma glucose levels after oral glucose tolerance test in type 2 diabetic patients.

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However, no patient experiences a symptomatic hypoglycemic event or has a

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documented blood glucose level < 60 mg/dL, suggesting the beneficial effects and

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safety of bitter melon consumption on glucose homeostasis.11 Supplementation of

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bitter melon in the diet decreases the activities of maltase and lactase in the intestines 4


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of type 1 diabetic rats, also suggesting the beneficial role of bitter melon on the

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management of diabetes.12 In previous study, we have identified a hypoglycemic

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polypeptide,

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(mcIRBP).13,14 A 19-amino-acid-residue consensus peptide, mcIRBP-19, has been

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further identified from mcIRBP.15 mcIRBP-19 is a putative IR-binding and blood

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glucose-lowering bioactive peptide motif with a β-hairpin structure. Moreover,

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mcIRBP-19 motif homologs are present in various plants belonging to different

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taxonomic families. Because food proteins are usually cleaved into small peptides in

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gastro-intestinal tracts, we characterized the gastro-resistant mcIRBP-derived peptides

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and investigated the hypoglycemic effects of the identified peptide in STZ-induced

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type 1 diabetic mice. IR kinase activity assay, glucose uptake activity assay, Western

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blot, immunohistochemical staining (IHC), and immunofluorescence (IF) were

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performed to elucidate the hypoglycemic mechanisms of peptides. Fasting blood

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glucose and glucose tolerance test were measured to analyze the blood glucose

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homeostasis and glucose clearance of peptides. Our findings showed that a

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gastro-resistant 9-mer peptide, mcIRBP-9, was a bioactive peptide of Momordica

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charantia that targeted IR and improved the glucose tolerance in type 1 diabetic mice.

Momordica

charantia

insulin

receptor

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94 5


(IR)-binding

protein


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MATERIALS AND METHODS

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Chemicals. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO)

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unless indicated. [γ-32P] ATP, [3H]-2-deoxy-D-glucose, and [125I]-insulin were

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purchased from Perkin Elmer (Boston, MA). Rabbit polyclonal antibodies against IR,

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phospho-phosphoinositide-dependent kinase 1 (PDK1), Akt, phospho-Akt (Thr308),

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phospho-Akt (Ser473), and phospho-glycogen synthase kinase 3β (GSK-3β) was

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purchased from Cell Signaling (Beverly, MA). Rabbit polyclonal antibody against

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glucose transporter 4 (GLUT4) was purchased from Millipore (Temecula, CA).

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Rabbit polyclonal antibody against phospho-IRβ (Tyr1162/Tyr1163) and mouse

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monoclonal antibody against β-actin were purchased from Santa Cruz (Santa Cruz,

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CA). Alexa Fluor 633-conjugated goat anti-rabbit IgG secondary antibody was

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purchased from Invitrogen (Eugene, OR). Insulin (Actrapid®) was purchased from

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Novo Nordisk (Kalundborg, Denmark).

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Cell Lines. 3T3-L1 preadipocytes were purchased from Bioresource Collection

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Research Center (Hsinchu, Taiwan). 3T3-L1 preadipocytes were maintained in

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Dulbecco's modified Eagle medium (Life Technologies, Gaithersburg, MD)

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supplemented with 10% calf serum (Life Technologies, Gaithersburg, MD). Cells

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were incubated at 37°C in a humidified atmosphere containing 5% CO2. 6


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In Vitro Digestion and Peptide Synthesis. All Peptides were synthesized by

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PeptideSynTM technology (LifeTein, Somerset, NJ). Peptides with purities over 95%

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were used in this study. mcIRBP-19 (RVRVWVTERGIVARPPTIG), a consensus

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peptide motif of mcIRBP, is a putative IR-binding and blood glucose-lowering

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peptide with a β-hairpin structure.15 mcIRBP-19 peptide was subjected to in vitro

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digestion to identify the gastro-resistant peptide fragments in this study. In vitro

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digestion was performed as described previously.15 Briefly, mcIRBP-19 was digested

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by pepsin in a simulated gastric fluid (34.2 mM NaCl, 80 mM HCl, pH 3.0) at 37°C

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for 30 min and then digested by pancreatin in a simulated intestinal fluid (100 mM

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KH2PO4, 30 mM NaOH, pH 7.0) at 37°C for 60 min. The digestive peptide fragments

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were identified by liquid chromatography-tandem mass spectrometry using an

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Ultimate capillary LC system (LC Packings, Amsterdam, The Netherlands) coupled to

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a QSTARXL quadrupole-time of flight mass spectrometer (Applied Biosystem/MDS

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Sciex, Foster City, CA).

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IR Kinase Activity Assay and Kinetic Analysis. IR kinase activity assay was

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performed as described previously.15 Briefly, 0.5 µM or various amounts of peptides

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were mixed with IR protein (Sigma-Aldrich, St. Louis, MO) and kinase buffer (25 7



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mM HEPES, pH 7.6, 25 mM MgCl2, 100 µM ATP, 100 µM sodium orthovanadate,

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2.5 mg/mL poly(Glu·Tyr), 25 µCi/mL [γ-32P]ATP). The resulting mixture was

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incubated at 30°C for 10 min, spotted on chromatography papers, and then soaked in

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10% trichloroacetic acid to precipitate poly(Glu·Tyr). The radioactivity incorporated

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into the precipitated poly(Glu·Tyr) was measured by scintillation counter (Beckman

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Coulter, Fullerton, CA). One unit (U) of kinase activity was determined as

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transferring 1 pmol of phosphate from [γ-32P]ATP to poly(Glu·Tyr) per minute at

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30°C. Vmax and KM values were calculated by fitting the data to the Michaelis-Menten

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equation.

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Glucose Uptake Assay. Glucose uptake assay was performed in fully

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differentiated 3T3-L1 adipocytes, prepared as described previously.16 3T3-L1

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adipocytes were cultured in 24-well plates. After a 4.5-h starvation, cells were

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incubated in Krebs-Ringer bicarbonate buffer (118 mM NaCl, 4.7 mM KCl, 1.3 mM

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CaCl2, 1.2 mM MgSO4, 1.2 mM Na2HPO4, 2% bovine serum albumin, 0.5 mM

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glucose, 25 mM NaHCO3, pH 7.4), treated with various amounts of peptides for 30

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min at 37°C, and treated with [3H]-2-deoxy-D-glucose (0.1 µCi/assay) for an

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additional 10 min. Cells were then solubilized in 0.1% sodium dodecyl sulfate (SDS),

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and the radioactivity incorporated into the cells was measured by scintillation counter 8


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(Beckman Coulter, Fullerton, CA). Relative radioactivity was presented as the

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comparison of radioactivity relative to solvent-treated cells. Effective concentration at

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50% (EC50) was determined as the concentration of peptide required to increase

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glucose uptake by 50% relative to solvent-treated cells.

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IR-Binding Assay. IR-binding assay was performed as described previously with

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slight modification.17 Briefly, fully differentiated 3T3-L1 adipocytes were incubated

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with different amounts of mcIRBP-9 for 15 min at 25°C. [125I]-Insulin was added and

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incubated for 90 min at 16°C. Cells were then scraped and centrifuged at 3,000 xg for

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10 min. The supernatant containing unbound ligands was removed. The radioactivity

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of cell pellet containing bound

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counter (Beckman Coulter, Fullerton, CA).

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I-labeled insulin was measured by scintillation

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Western Blot Analysis. Fully differentiated 3T3-L1 adipocytes were treated with

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0.5 µM insulin or various amounts of mcIRBP-9 at 37°C for 30 min. Cells were then

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lyzed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.5% sodium

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deoxycholate, 0.1% SDS, 150 mM sodium chloride, 2 mM EDTA, 50 mM sodium

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fluoride) containing protease/phosphatase

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Technology, Boston, MA). Protein samples (50 µg) were separated by 10%

inhibitor cocktail (Cell Signaling

9



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SDS-polyacrylamide gel electrophoresis and transferred electrophoretically to

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nitrocellulose membranes. Membranes were then probed with primary antibodies and

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the bound antibody was detected with peroxidase-conjugated secondary antibodies

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followed by chemiluminescence (Phototope®-HRP Western detection kit, New

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England Biolabs, Ipswich, MA) and exposed by autoradiography. Bands on the X-ray

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films were measured using Gel-Pro® Analyzer (Media Cybernetics, Silver Spring,

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MD). Quantitative data were normalized by internal control (β-actin) and further

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expressed as fold, which is presented as the comparison with the amount relative to

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mock.

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Animal Experiments. C57BL/6J mice (6-week old, male) were purchased from

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National Laboratory Animal Center (Taipei, Taiwan). Mouse experiments were

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conducted under ethics approval from China Medical University Animal Care and

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Use Committee (Permit No. 104-75-N). Mice were maintained under a 12:12

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light-dark cycle with free access to water and standard diet (#5001, LabDiet, St Louis,

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MO) unless indicated.

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To induce type 1 diabetes, C57BL/6J mice were intraperitoneally injected with 50

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mg/kg STZ for five consecutive days. Two weeks later, fasting blood glucose levels

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and fasting insulin levels were measured using glucometer (Draw-In, Scienco 10


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Technology, Taipei, Taiwan) and insulin enzyme-linked immunosorbent assay kit

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(Millipore, St. Charles, MO), respectively. The fasting insulin levels and the fasting

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blood glucose levels of normal C57BL/6J mice were about 0.8 ng/ml and 75 mg/dL,

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respectively. Mice were considered as type 1 diabetic if their fasting insulin and blood

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glucose levels were about 0.4 ng/ml and ≥250 mg/dL, respectively.18 Intraperitoneal

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glucose tolerance test (IPGTT) was performed as described previously.16 Briefly,

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diabetic mice were fasted for 4 h and then given orally or intraperitoneally with

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various amounts of mcIRBP-9 in phosphate-buffered saline (PBS) (137 mM NaCl,

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1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, pH 7.2) 15 min before

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intraperitoneal administration of 1 g/kg glucose solution. Blood glucose levels were

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measured at indicated time after glucose challenge. Relative blood glucose area (%)

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was presented as the comparison of the area under the curve (AUC) relative to mock.

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Glycated hemoglobin (HbA1c) levels were measured by DCA Vantage Analyzer

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(Siemens Healthcare, Erlangen, Germany). For long-term study, body weight and

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food uptake were measured every five days. Fasting blood glucose levels were

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measured every 14 days. HbA1c levels, IPGTT, and IHC were performed on the 30th

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day.

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Immunohistochemical staining and Immunofluorescence. Parafilm-embedded

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muscle tissue sections were deparaffinized and rehydrated, followed by quenching

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endogenous tissue peroxides. Sections were then incubated with anti-GLUT4

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antibody (1:100 dilution) at 40C overnight. IHC was performed according to

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manufacturer's instructions (Histostain-Plus, Invitrogen, Camarillo, CA). For IF,

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slides were further hybridized with Alexa Fluor 633-conjugated goat anti-rabbit IgG

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secondary antibody (1:500 dilution) for 60 min. Images were captured using an

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inverted confocal microscope (Leica TCS SP2; Leica Microsystems, Wetzlar,

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Germany), with an excitation wavelength of 632 nm. GLUT4-positive area was

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measured using ImageJ (Media Cybernetics, Bethesda, MD). The expression of

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GLUT4 was calculated as (area occupied with brown or red color/area of whole tissue)

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× 100.

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Statistical Analysis. Data were presented as mean ± standard error. Data were

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analyzed by one-way ANOVA and post hoc Bonferroni test using SPSS Statistics

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version 20 (IBM, Armonk, NY). A p-value < 0.05 was considered as statistically

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significant.

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226 12


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RESULTS

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Identification of Gastro-Resistant Peptides within mcIRBP-19. We have

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previously identified a 19-mer peptide (mcIRBP-19) that exhibits hypoglycemic

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effects via affecting IR signaling transduction pathway.15 In vitro digestion was

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further performed to identify the gastro-resistant peptides within mcIRBP-19.

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Digestion of mcIRBP-19 by pepsin and pancreatin released 178 fragments, which

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included 31 kinds of peptides ranging from 5 to 13 amino acid residues in length

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(Figure 1A). Among 178 fragments, mcIRBP-9 (IVARPPTIG) accounted for the

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majority (20.78%) of fragments, followed by mcIRBP-6 (RPPTIG) (11.8%),

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mcIRBP-10 (GIVARPPTIG) (8.99%), and mcIRBP-7 (ARPPTIG) (7.87%).

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Effects of Gastro-Resistant Peptides on IR Kinase Activities and Glucose

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Uptake Activities. To identify whether these gastro-resistant peptides exhibited

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IR-binding abilities, we chemically synthesized the peptides and evaluated their

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IR-binding abilities by IR kinase activity assay and glucose uptake assay. IR is a

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transmembrane protein and intrinsic tyrosine kinase. Upon binding to insulin, IR

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kinase phosphorylates the IR substrate and consequently stimulates the uptake of

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glucose by adipocytes.19 As shown in Figure 1B, IR kinase activities were activated

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by 0.5 µM peptides. Kinase activity of IR reached 62.34 ± 1.64 U/ml in the presence 13



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of mcIRBP-9, followed by mcIRBP-10 (53.17 ± 0.85 U/ml) and mcIRBP-11 (51.77 ±

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0.76 U/ml). By contrast, kinase activity of IR reached 21.71 ± 0.50 U/ml in the

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presence of mcIRBP-6. Glucose uptake assay in 3T3-L1 adipocytes also showed that

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mcIRBP-9 and mcIRBP-10 stimulated the uptake of glucose by 50% at 0.47 ± 0.05

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µM and 8.42 ± 0.87 µM, respectively, while other peptides increased the glucose

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uptake by 50% at more than 20 µM. These findings suggested that mcIRBP-9 was a

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better gastro-resistant peptide that stimulated IR kinase activities and enhanced the

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uptake of glucose in cells.

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Effects of mcIRBP-9 on IR Signaling Transduction Pathways. To further

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analyze the mcIRBP-9-IR interaction and the mcIRBP-9-induced IR signaling

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transduction pathway, we performed IR-binding assay, glucose uptake assay, and

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Western blot in the presence of mcIRBP-9. Fully differentiated 3T3-L1 adipocytes

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were incubated with various amounts of mcIRBP-9 and a fixed amount of 125I-insulin

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and IR. The radioactivity of cell pellets was increased as the amount of mcIRBP-9

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was increased, suggesting that mcIRBP-9 enhanced the binding of insulin to IR

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(Figure 2A). However, the addition of non-radiolabeled insulin decreased the

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radioactivity, suggesting that non-radiolabeled insulin blocked and abolished

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mcIRBP-9-dependent enhancement of insulin binding with specificity. These findings 14


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also suggested that mcIRBP-9 interacted with the sites different from the

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insulin-binding site on IR. Furthermore, fully differentiated 3T3-L1 adipocytes were

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treated with various amounts of mcIRBP-9 and 3H-glucose. The radioactivity in cells

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was gradually increased as the amount of mcIRBP-9 was increased, suggesting that

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mcIRBP-9 stimulated the uptake of glucose in cells (Figure 2B). Moreover, the

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stimulation displayed a dose-dependent manner.

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We further evaluated the IR-binding kinetic parameters of mcIRBP-9 by IR kinase

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activity assay. Upon binding to ligands, the kinase activity of IR was activated. The

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velocity and the affinity of insulin and mcIRBP-9 were therefore calculated on the

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basis of IR kinase activities. As shown in Figure 2C, the radioactivity was increased

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as the amount of mcIRBP-9 or insulin was increased. The radioactivity reached a

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plateau when the concentration of insulin and mcIRBP-9 was ≥ 500 nM and 1 µM,

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respectively. The hyperbolic shape of the radioactivity/concentration curve was

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further expressed by the Michaelis-Menten equation and applied to determine the Vmax

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and KM values of insulin and mcIRBP-9. The KM and Vmax values of insulin on IR

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were 0.053 ± 0.002 nM and 34.86 ± 0.79 U/ml/10 min, respectively, while KM and

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Vmax values of mcIRBP-9 were 0.07 ± 0.02 nM and 20.86 ± 1.16 U/ml/10 min,

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respectively.

15



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Western blot was further performed to elucidate the IR signaling transduction

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affected by mcIRBP-9. Binding of ligands to the extracellular region of IR induces a

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conformational change of IR, resulting in the autophosphorylation of tyrosine residues

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on IR. IR activation subsequently leads to the activation of PDK1 and the

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phosphorylation of Akt. Activated Akt then results in the phosphorylation and

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inactivation of GSK-3β, which promotes glucose storage as glycogen. It also leads to

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the expression and translocation of GLUT4 to the plasma membrane, which promotes

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the uptake of glucose.19 As shown in Figure 3, insulin induced the phosphorylations

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of IR, PDK1, and Akt, resulting in the phosphorylation of GSK-3β and the expression

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of GLUT4. mcIRBP-9 had no effects on the total amounts of IR and Akt. However,

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mcIRBP-9 induced the phosphorylation of IR, which was consistent with the

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aforementioned IR kinase activity data. In addition, mcIRBP-9, like insulin,

295

stimulated the phosphorylation of PDK1, Akt, and GSK-3β, and the expression of

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GLUT4. Moreover, effects of mcIRBP-9 displayed a dose-dependent manner.

297

Therefore, these findings suggested that mcIRBP-9 interacted with IR, activated the

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IR signaling transduction pathway, and induced the uptake of glucose in cells.

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Effects of mcIRBP-9 on the Glucose Clearance in Type 1 Diabetic Mice. The

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glucose clearance ability of mcIRBP-9 was evaluated by IPGTT. In comparison with 16


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other mouse strains, C57BL/6J mice are sensitive to STZ stimulation. Moreover, male

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mice tend to be more susceptible to STZ-induced diabetes.20 Therefore, male

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C57BL/6J mice were chosen in this study for the establishment of type 1 diabetes.

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Type 1 diabetic mice were intraperitoneally injected or orally given with various

306

dosages of mcIRBP-9. Blood glucose levels were determined after glucose challenge.

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In this study, we identified that mcIRBP-9 was a gastro-resistant peptide targeting IR.

308

Therefore, commercial drugs that targeted IR were suitable as positive controls in

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animal experiments. Insulin is the only medication that interacts with IR. Injectable

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insulin was therefore used as a positive control to compare the hypoglycemic effects

311

of mcIRBP-9 given by intraperitoneal injection. As shown in Figure 4A, the amounts

312

of blood glucose reached maximum levels at 60 min after glucose challenge in all

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groups, except insulin. In comparison with mock, all treatments, except 0.5 nmol/kg

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and 1 nmol/kg mcIRBP-9, significantly decreased the blood glucose levels starting

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from 30 min after glucose tolerance. Injection with insulin, as expected, significantly

316

improved the glucose tolerance in type 1 diabetic mice. Injectable insulin at 2.5

317

nmol/kg decreased the blood glucose area by 24.95 ± 0.46%, while injection of

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mcIRBP-9 at 2.5 nmol/kg and 20 nmol/kg decreased the blood glucose area by 13.28

319

± 0.39% and 30.91 ± 0.39%, respectively. No oral insulin is available for the

320

comparison of oral mcIRBP-9. All treatments, except 0.1 µnol/kg mcIRBP-9, 17



321

significantly decreased the blood glucose levels during the experiment period (Figure

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4B). Oral administration of mcIRBP-9 at 20 µmol/kg decreased the blood glucose

323

area by 32.09 ± 0.38%. In comparison with mock, both intraperitoneal and oral

324

administrations of mcIRBP-9 significantly stimulated the clearance of glucose in a

325

dose-dependent manner. However, intraperitoneal injection of mcIRBP-9 at 10

326

nmol/kg and 20 nmol/kg displayed similar hypoglycemic activities to insulin, with no

327

statistical differences. These findings suggested that mcIRBP-9 given by both

328

injection and oral exhibited hypoglycemic effects in type 1 diabetic mice. Moreover,

329

the glucose clearance ability of mcIRBP-9 was comparable to that of insulin.

330

331

Long-Term Effects of mcIRBP-9 on the Regulation of HbA1c and Blood

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Glucose Levels in Type 1 Diabetic Mice. Single administration of mcIRBP-9

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improved the glucose tolerance in type 1 diabetic mice. We wondered whether

334

long-term administration of mcIRBP-9 was capable of regulating the HbA1c levels. A

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pilot study of long-term effects was therefore performed by oral administration of low

336

dose (0.1 µmol/kg) and high dose (1 µmol/kg) of mcIRBP-9 to type 1 diabetic mice

337

for 30 consecutive days. As shown in Figure 5A, the average body weight of mice

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was 19.59 ± 2.11 g/mouse on Day 0 and 20.64 ± 3.29 g/mouse on Day 30. No

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significant change on the body weight was observed during the treatment period. 18


Page 18 of 45

Page 19 of 45


340

Fasting blood glucose levels were increased as the time was increased in both mock

341

group and low-dose mcIRBP-9 group (Figure 5B). However, high dose of mcIRBP-9

342

maintained the fasting blood glucose levels at about 300 mg/dL during the experiment

343

period. IPGTT showed that, in comparison with mock, 1 µmol/kg mcIRBP-9

344

significantly decreased the blood glucose levels starting from 30 min after glucose

345

tolerance (Figure 5C). Oral administration of mcIRBP-9 at 1 µmol/kg significantly

346

decreased the blood glucose area by 23.62 ± 6.14%. The HbA1c level was 7.55 ±

347

0.33% in mock group (Figure 5D). However, mcIRBP-9 significantly decreased the

348

levels of HbA1c by 24.06 ± 1.53%. The HbA1c level dropped to 5.73 ± 0.12% after a

349

30-day administration of high dose of mcIRBP-9. The expression and translocation of

350

GLUT4, a marker of IR signaling pathway, in skeletal muscle tissues was analyzed by

351

IHC and IF. IHC analysis showed that the amount of GLUT4 was increased in mice

352

given with 1 µmol/kg mcIRBP-9 (Figure 5E, middle panel). The GLUT4-positive area

353

was 62.92 ± 3.52% in mcIRBP-9 group and 5.13 ± 1.27% in mock group. Moreover,

354

IF analysis showed that mcIRBP-9 significantly stimulated the translocation of

355

GLUT4 to the plasma membrane (11.65±2.84% vs. 3.03±1.31%) (Fgure 5E, right

356

panel). Therefore, our findings suggested that long-term administration of mcIRBP-9

357

improved both the glucose tolerance and the HbA1c levels in diabetic mice via

358

targeting IR signaling transduction pathway. 19



359

360

361

DISCUSSION

362

In this study, we identified that mcIRBP-9 was a likely gastro-resistant peptide from

363

bitter melon that activated IR signaling transduction pathway and improved the

364

glucose tolerance in type 1 diabetic mice. There are several medications currently

365

available for diabetes. For example, dipeptidyl peptidase 4 (DPP-4) inhibitors prevent

366

the rapid catabolism of endogenous glucagon-like peptide 1 (GLP-1). GLP-1 analogs

367

slow the absorption of intestinal carbohydrate. Insulin secretagogues and peroxisome

368

proliferator-activated receptor (PPAR) agonists enhance the secretion and sensitivity

369

of insulin.21 Some studies have shown that food-derived peptides and molecules can

370

act like these diabetic medications.22 For example, whey proteins reduce postprandial

371

glucose levels and stimulate insulin release in healthy subjects and in subjects with

372

type 2 diabetes by reducing DPP-4 activity in the proximal bowel.23 Short bioactive

373

peptide Ile-Pro-Ala, occurring in hydrolysates of β-lactoglobulin, has been identified

374

as a moderate DPP-4 inhibitor from whey proteins.24 Berries are a rich source of

375

bioactive phenolic compounds that are able to bind and inhibit DPP-4. Anthocyanins,

376

predominantly delphinidin-3-arabinoside, from fermented berry beverages have the

377

potential to modulate DPP-4 and its substrate GLP-1, and to increase insulin secretion 20


Page 20 of 45

Page 21 of 45


378

in pancreatic β-cells.25 Bioactive peptide Ile-Pro, derived from enzymatic proteolysis

379

of defatted rice bran has been identified as an effective DPP-4 inhibitor in vitro.26 Soy

380

peptide (IAVPTGVA) and lupin peptide (LTFPGSAED) have been identified as

381

DPP-4 inhibitors that interact with DPP-4 and inhibit DPP-4 activities.27 Casein

382

glycomacropeptide-derived peptide, IPPKKNQDKTE, increases the glucose uptake in

383

insulin-resistant HepG2 cells and increases the phosphorylation of Akt and GSK-3β,

384

suggesting that casein glycomacropeptide-derived peptide plays a potential role in the

385

prevention and treatment of hepatic insulin resistance and type 2 diabetes.28 Curcumin,

386

a yellow pigment isolated from the rhizomes of Curcuma longa L (turmeric),

387

significantly reduces the number of prediabetic individuals and appears to improve the

388

overall function of β-cells.29 In vitro and in vivo studies indicate that curcumin is a

389

GLP-1 secretagogue that increases the secretion of endogenous GLP-1.30 Furthermore,

390

metformin, the most popular anti-diabetic drug, is developed based on a biguanide

391

compound isolated from French lilac.31

392

Some natural molecules that exhibit insulin-like functions have also been identified

393

from foods or plants. Stevioside, a natural sweetener and diterpene glycoside

394

extracted from Stevia rebaudiana (Bertoni), has been used as an anti-hyperglycemic

395

agent for the treatment of diabetes for decades.32 Stevioside increases the levels of

396

GLUT4 protein and the uptake of glucose in both L6 myotubes and 3T3-L1 21



397

adipocytes.33 Modasima A, an anthraquinone from the roots of Morinda longissima Y.

398

Z. Ruan (Rubiaceae), improves glucose uptake via activation of AMP-activated

399

protein kinase.34 Additionally, roseoside, epigallocatechin gallate, glycyrrhetinic acid,

400

dehydrotrametenolic acid, strictinin, isostrictinin, pedunculagin, epicatechin, and

401

christinin-A from medicinal plants have shown significant insulin-like and

402

antidiabetic activities.35 Few insulin-like proteins or peptides have been identified

403

from foods or medicinal plants. Polypeptide-p or p-insulin is an 11-kDa protein

404

extracted from bitter melon. Polypeptide-p decreases blood glucose levels in gerbils,

405

langurs, and humans when injected subcutaneously.36 Insulin-like protein (ILP) is a

406

5.7-kDa protein extracted from leaves of Costus igneus.37 ILP significantly increases

407

the uptake of glucose, the levels of insulin receptor susbtrate-1, and the translocation

408

of GLUT4 in differentiated L6 myotubes.10 It also decreases the levels of blood

409

glucose when administered orally in type 1 diabetic mice.38 Although these studies

410

suggest that the insulin-like molecules or peptides are present in foods or plants, there

411

are no direct evidences indicating that these natural molecules or peptides interact

412

with IR and activate the autophosphorylation of IR. Moreover, oral stability of these

413

peptides remains to be evaluated. In this study, we identified a gastro-resistant

414

9-amin-acid-residue peptide mcIRBP-9 in bitter melon that targeted IR, activated IR

415

signaling transduction pathway, and stimulated the glucose uptake in cells. 22


Page 22 of 45

Page 23 of 45


416

Additionally, a 30-day oral administration of mcIRBP-9 stimulated the glucose

417

clearance and decreased the HbA1c levels in diabetic mice, suggesting the oral

418

benefits of mcIRBP-9 on the regulation of blood glucose. However, because of the

419

low number of mice in the long-term study, more researches are needed to verify our

420

data.

421

After ingestion, proteins are usually digested to amino acid residues or small

422

peptides by pepsin in the stomach and pancreatin in the small intestine. Our previous

423

study has identified that oral administration of a 68-mer mcIRBP and a 19-mer

424

mcIRBP-19 displayed hypoglycemic effects in mice. It was reasonable to expect that

425

these proteins and peptides would be cleaved by gastrointestinal enzymes after

426

ingestion. Therefore, we performed in vitro digestion using simulated gastric and

427

intestinal fluids in the presence of pepsin and pancreatin, and found that mcIRBP-9

428

was a minimal gastro-resistant bioactive peptide that exhibited hypoglycemic activity

429

in mice when given orally. It raised a question. How could mcIRBP-9 be absorbed by

430

gastrointestinal tract? Small intestine is responsible for the absorption of more than

431

90% of nutrients, such as carbohydrates, proteins, lipids, water, vitamins, and

432

minerals. Endocytosis, phagocytosis, transcytosis, direct penetration, and paracellular

433

transport are responsible for the transport of peptides in the intestinal epithelia.39

434

Small molecules with less than 100-200 daltons can be absorbed through paracellular 23



435

pathways.40 The molecular weight of mcIRBP-9 was approximately 975 daltons made

436

it difficult to be transported by paracellular pathways. Intestinal epithelium is made up

437

of phospholipid bilayer membrane and cholesterol. Upon oral administration, peptides

438

must traverse through this lipoidal membrane before entering into systemic circulation.

439

If peptide is lack of lipophilicity, no passive absorption can be taken place. The amino

440

acid sequence (IVARPPTIG) of mcIRBP-9 consisted of five kinds of hydrophobic

441

amino acid residues, including Ala, Ile, Val, Pro, and Gly. Moreover, the estimated

442

Grand Average of Hydropathy value of mcIRBP was 0.689 using ProtParam Program

443

in ExPASy Server, suggesting the lipophilicity of mcIRBP-9.41 Therefore, passive

444

absorption by intestinal epithelium might be a potent transport mechanism of

445

mcIRBP-9 in the small intestine. The detailed transport mechanism of mcIRBP-9

446

remained to be further elucidated.

447

The kinetic parameters, including Vmax and KM values, were calculated by IR

448

kinase activities. Vmax represents the maximum rate at the saturated concentration of

449

substrate, while the Michaelis constant KM is the substrate concentration at which the

450

reaction rate is half of Vmax. KM is an inverse measure of the affinity of substrate for

451

the enzyme, which means that a small KM value indicates a high affinity for the

452

enzyme.42 In previous study, we have reported that the KM values of mcIRBP and

453

mcIRBP-19 were 0.13 ± 0.02 nM and 0.11 ± 0.01 nM, respectively, and the Vmax 24


Page 24 of 45

Page 25 of 45


454

values were 18.32 ± 0.40 U/ml/10 min and 17.27 ± 0.26 U/ml/10 min, respectively.15

455

In comparison with mcIRBP and mcIRBP-19, the KM value (0.07 ± 0.02 nM) of

456

gastro-resistant mcIRBP-9 was decreased, suggesting that mcIRBP-9 exhibited a high

457

affinity to IR. In addition, mcIRBP-9 displayed similar kinetic parameters with

458

insulin.

459

Some hypoglycemic peptides isolated from foods or plants stimulate the uptake of

460

glucose in vitro or in vivo without the evidences of IR interaction. Here we found that

461

mcIRBP-9 was a unique gastro-resistant 9-mer bioactive peptide of Momordica

462

charantia that targeted IR. Oral administration of mcIRBP-9 stimulated the clearance

463

of blood glucose in type 1 diabetic mice. A pilot study showed that daily ingestion of

464

mcIRBP-9 for 30 days improved the glucose tolerance and decreased the HbA1c

465

levels via IR signaling pathway. In conclusion, our findings might explain the potent

466

blood glucose-modulating effect of Momordica charantia after ingestion. Moreover,

467

mcIRBP-9 might be able to contribute to the anti-diabetic action of bitter melon.

468

469

470

FUNDING SOURCES

471

This work was supported by grants from Ministry of Science and Technology

472

(MOST104-2320-B-039-018-MY3 and MOST105-2320-B-039-017-MY3), China 25



Page 26 of 45

473

Medical University (CMU104-H-01 and CMU104-H-02), and CMU under the Aim

474

for Top University Plan of the Ministry of Education, Taiwan.

475

476

477

NOTES

478

The authors declare no competing financial interest. Patents for mcIRBP-9 have been

479

approved by U.S. (US 8,697,649 B2 and US 9,029,326 B2), Europe (EP 2664622 B1),

480

Japan (JP 5676538 and JP 5947872), Taiwan (I588153), and China Patent Offices (ZL

481

201210164364.4).

482

483

484

ABBREVIATIONS USED

485

AUC, area under the curve; DPP-4, dipeptidyl peptidase 4; EC50, effective

486

concentration at 50%; GLP-1, glucagon-like peptide 1; GLUT4, glucose transporter 4;

487

GSK-3β, glycogen synthase kinase 3β; HbA1c, glycated hemoglobin; IF,

488

immunofluorescence; ILP, insulin-like protein; IPGTT, intraperitoneal glucose

489

tolerance test; IR, insulin receptor; mcIRBP, Momordica charantia insulin receptor

490

(IR)-binding

protein;

PBS,

phosphate-buffered

26


saline;

PDK1,

Page 27 of 45


491

phosphoinositide-dependent kinase 1; PPAR, peroxisome proliferator-activated

492

receptors; SDS, sodium dodecyl sulfate; STZ, streptozotocin; U, unit

27



493

494

495

496

497

Page 28 of 45

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Williams, D. R.; Oh, W. K. Anthraquinones from Morinda longissima and their

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Biomed. 2012, 2, 320-330.

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(36) Krawinkel, M. B.; Keding, G. B. Bitter gourd (Momordica charantia): A dietary approach to hyperglycemia. Nutr. Rev. 2006, 64, 331-337.

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Biochem. Biophy. Res. Commun. 2013, 436, 278-282.

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(38) Hegde, P. K.; Rao, H. A.; Rao, P. N. A review on Insulin plant (Costus igneus Nak). Pharmacogn. Rev. 2014, 8, 67-72. (39) Fosgerau, K.; Hoffmann, T. Peptide therapeutics: current status and future directions. Drug Discov. Today 2015, 20, 122-128.

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(40) Camenisch, G.; Alsenz, J.; van de Waterbeemd, H.; Folkers, G. Estimation of

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permeability by passive diffusion through Caco-2 cell monolayers using the drugs'

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lipophilicity and molecular weight. Eur. J. Pharm. Sci. 1998, 6, 317-324.

617

(41) Gasteiger, E.; Hoogland, C.; Gattiker, A.; Duvaud, S.; Wilkins, M. R.; Appel,

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R. D.; Bairoch, A. Protein identification and analysis tools on the ExPASy server. In

619

The proteomics protocols handbook; Walker J. M., Ed.; Humana Press: New York,

620

NY, 2005, pp. 571-607. 34


Page 34 of 45

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621

622

(42) Grima, R.; Walter, N. G.; Schnell, S. Single-molecule enzymology à la Michaelis-Menten. The FEBS J. 2014, 281, 518-530.

35



Page 36 of 45

623

FIGURE CAPTIONS

624

Figure 1. Identification of gastro-resistant IR-binding peptides within mcIRBP-19. (A)

625

Location and distribution of gastro-resistant peptides. The amino acid sequences of

626

mcIRBP-19 are shown at the top. The relative positions of gastro-resistant peptide

627

fragments are shown by solid lines. The thickness and color of line represent the

628

content (%) of peptide in the mcIRBP-19-digested fragments. Lines in red, blue, green,

629

and purple represent >20%, 6-20%, 3-5%, and 20

mcIRBP-11

ERGIVARPPTIG

>20

mcIRBP-10

ERGIVARPPTIG

8.42 ± 0.87

mcIRBP-9

ERGIVARPPTIG

0.47 ± 0.05

mcIRBP-8

ERGIVARPPTIG

>20

mcIRBP-7

ERGIVARPPTIG

>20

mcIRBP-6

ERGIVARPPTIG

>20

mcIRBP-9-1

ERGIVARPPTIG

>20

mcIRBP-9-2

ERGIVARPPTIG

>20

mcIRBP-9-3

ERGIVARPPTIG

>20

Peptide

40


IR kinase activity (U/ml)

Page 40 of 45

Page 41 of 45


Figure 2 (A)

(B) 2.5

3

2

Relative radioactivity

Relative radioactivity

***

3.5

***

** 1.5

1

0.5

2.5 2 1.5 1 0.5 0

0

0

mcIRBP-9 (µM) Cold insulin

0 ―

1 ―

2.5 ―

5 ―

10 ―

0.5

1

2.5

5

10

20

10 mcIRBP-9 concentration (µM)

＋

8000

8000

7000

7000 Radioactivity (dpm)

Radioactivity (dpm)

(C)

6000 5000 4000 3000 2000 1000

6000 5000 4000 3000 2000 1000

0

0 0

500

1000

1500

2000

2500

0

Insulin concentration (nM)

2000

4000

6000

8000

mcIRBP-9 concentration (nM) 41


10000


Page 42 of 45

Figure 3

mcIRBP-9 (µM) Mock

Insulin

1

2.5

5

IR p-IR p-PDK1 Akt p-Akt (Thr308) p-Akt (Ser473) p-GSK-3β GLUT4 β-Actin

12

Fold relative to mock

10

Insulin

*

mcIRBP-9 1µM mcIRBP-9 2.5µM

8

*

**

*

mcIRBP-9 5µM

** *

6 *

** *

*

4

*

*

2 0 IR

p-IR

p-PDK-1

Akt

p-Akt p-Akt GSK-3β GLUT-4 (Thr308) (Ser473)

42


Page 43 of 45


Figure 4 (A) 110 Relative blood glucose area (%)

1000

Blood glucose (mg/dL)

900 800 700 600 500

Mock Insulin 2.5 nmol/kg mcIRBP-9 0.5 nmol/kg mcIRBP-9 1 nmol/kg mcIRBP-9 2.5 nmol/kg mcIRBP-9 5 nmol/kg mcIRBP-9 10 nmol/kg mcIRBP-9 20 nmol/kg

400 300 200 100

***

**

100 90 80

***

70 60 50

0 0

30

60

90

120

180

Mock Insulin

240

0.5

1

2.5

5

10

20

mcIRBP-9 dosage (nmol/kg)

Time (min)

(B) 110 Relative blood glucose area (%)

1000 900 Blood glucose (mg/dL)

800 700 600 500

Mock mcIRBP-9 0.1 µmol/kg mcIRBP-9 0.5 µmol/kg mcIRBP-9 1 µmol/kg mcIRBP-9 2.5 µmol/kg mcIRBP-9 5 µmol/kg mcIRBP-9 10 µmol/kg mcIRBP-9 20 µmol/kg

400 300 200 100 0 0

30

60

90

100

***

** 90 80 70 60 50

120

180

Mock

240

Time (min)

0.1

0.5

1

2.5

5

mcIRBP dosage (µmol/kg) 43


10

20


Page 44 of 45

Figure 5 (A)

(C)

(D)

(B)

(E)

Mock

5.13±1.27%

3.03±1.31%

mcIRBP-9

62.92±3.52% **

44


11.65±2.84%**

Page 45 of 45


Table of Contents Graphics

mcIRBP

mcIRBP-9 in vitro digestion

45


Gastro-Resistant Insulin Receptor-Binding Peptide from

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