Milk-Derived Tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro

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Bioactive Constituents, Metabolites, and Functions

Milk-derived Tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) Enhance Insulin Sensitivity and Prevent Insulin Resistance in 3T3-F442A Pre-adipocytes Subhadeep Chakrabarti, Forough Jahandideh, Sandra T. Davidge, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02051 • Publication Date (Web): 30 Aug 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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Journal of Agricultural and Food Chemistry

Milk-derived Tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) Enhance Insulin Sensitivity and Prevent Insulin Resistance in 3T3-F442A Pre-adipocytes

Subhadeep Chakrabarti †,‡,||, Forough Jahandideh †,‡,||, Sandra T Davidge ‡,⸸ ,§,¥ and Jianping Wu †,‡,*

Short Title: IPP and VPP improve insulin functions

†Dept.

of Agricultural, Food and Nutritional Science, ‡Cardiovascular Research Centre, ⸸ Dept. of

Obstetrics and Gynecology, §Dept. of Physiology, and ¥Women and Children’s Health Research Institute, University of Alberta, Edmonton, AB, Canada

Address for correspondence: Dr. Jianping Wu 4-10 Ag/For Centre, Department of Agricultural, Food & Nutritional Science, University of Alberta, Edmonton, AB, Canada, T6G 2P5 (telephone 1-780- 492-6885; fax 1-780- 492-4265; email: [email protected]) ||

Both authors contributed equally to this work

1 ACS Paragon Plus Environment


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Abstract

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There is great interest in developing naturally-derived compounds especially bioactive peptides

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with potential insulin sensitizing effects and/or preventing insulin resistance. Previously, we

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showed adipogenic and insulin mimetic actions of IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro), the

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milk-derived tripeptides on cultured pre-adipocytes, in addition to their previously characterized

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anti-hypertensive and anti-inflammatory functions. However, the effect of these peptides on

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insulin signaling is not known. Therefore, we examined IPP and VPP effects on insulin signaling

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in pre-adipocytes, a well-established model for studying insulin signaling. Our results suggested

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both peptides enhanced insulin signaling and contributed towards the prevention of insulin

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resistance in the presence of tumor necrosis factor (TNF). Inhibition of inflammatory mediator

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NF-kB under TNF stimulation was a likely contributor to the prevention of insulin resistance.

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VPP further enhanced the expression of glucose transporter 4 (GLUT4) in adipocytes and

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restored glucose uptake in TNF-treated adipocytes. Our data suggested the potential of these

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peptides in the management of conditions associated with impairments in insulin signaling.

15 16

Keywords: IPP, VPP, lactotripeptides, insulin resistance, adipocyte


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Introduction

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Metabolic syndrome and its associated complications such as atherosclerosis and type II diabetes

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are growing health problems worldwide.

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underlying pathologies common to hypertension and insulin resistance has gained a great deal of

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interest in recent decades.

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pathophysiological changes affecting both hypertension and insulin resistance.

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surprisingly, there has been scientific interest in evaluating anti-hypertensive therapies for their

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potential effects on insulin signaling. Such therapies may provide a dual protection against the

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main complications of metabolic syndrome.

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converting enzyme (ACE) inhibitors and angiotensin II receptor blockers may suppress

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inflammation and exert insulin mimetic and/or sensitizing functions.

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known side effects of many of these pharmaceutical drugs, there are doubts regarding their

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suitability for long-term usage in metabolic syndrome sufferers.

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Natural health products such as food-derived proteins and peptides have gained substantial

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scientific interest as therapeutic compounds given their wide availability, usage in food sources

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and perceived lack of serious adverse effects. 10, 11 Milk is a prominent source of dietary proteins

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and bioactive peptides.

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form the enzymatic digestion of casein, the main protein fraction in cow’s milk. IPP and VPP,

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identified as ACE-inhibitors, have anti-hypertensive effects as shown in several animal as well as

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clinical trials in human subjects.

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also enhance vascular relaxation, and exert anti-inflammatory and antioxidant properties.

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Previously, we demonstrated adipogenic and anti-inflammatory effects of IPP and VPP in 3T3-

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2, 3

1

Developing novel therapies that may target the

Adipose tissues have been identified as a major site of

5-7

4

Not

Indeed, recent studies suggests that angiotensin

8, 9

However, due to well-

IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) are the tripeptides generated

13-15

In addition to their ACE-inhibitory effect, IPP and VPP


16-18


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F442A pre-adipocytes similar to insulin, highlighting their potential benefits in controlling the

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complications of metabolic syndrome. 19

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Insulin exerts its mitogenic and metabolic effects on pre-adipocyte differentiation and glucose

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uptake through distinct signaling pathways which are known to be perturbed in insulin

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resistance. Indeed, inflamed adipocytes which fail to respond normally to insulin become insulin

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resistant. Despite the potential insulin-like and anti-inflammatory effects of IPP and VPP in

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adipocytes, the role of these peptides, if any, on improving insulin functions and/or modulating

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insulin resistance remained unknown. Given the critical importance of adipocytes in metabolic

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syndrome and the insulin-mimetic and anti-inflammatory profiles of these lactotripeptides, we

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investigated their potential to enhance insulin signaling and prevent insulin resistance in cultured

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pre-adipocytes for this study.


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Materials and Methods

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Bioactive Peptides

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IPP (purity = 99.9%) and VPP (purity = 98.6%) were purchased from Genscript (Piscataway, NJ,

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USA). Peptides’ purity was determined and reported by the manufacturer using HPLC. After

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dissolving in 1X phosphate buffered saline (PBS), peptides were aliquoted and stored at -20οC

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for cell culture experiments.

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Reagents

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Dulbecco’s phosphate buffered saline (PBS) and dithiothreitol (DTT) from Sigma Aldrich (St

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Louis, MO, USA), dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS)

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from Gibco/ Invitrogen (Carlsbad, CA, USA), murine tumor necrosis factor (TNF) from

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Peprotech (Rocky Hill, NJ, USA), type 1 Collagenase from Worthington Biochemical

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Corporation (Lakewood, NJ, USA), and triton-X-100 from VWR International (West Chester,

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PA, USA) were used in this study.

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Cell culture

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The murine pre-adipocyte cell line 3T3-F442A (Sigma Aldrich) obtained in passage 8 was used

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for all experiments. Cells were thawed cultured using DMEM supplemented with heat-

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inactivated FBS at 10% v/v and antibiotics. The cells were grown in T-25 flasks to confluence

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prior to sub-culture in gelatin-coated 48 well plates for the actual experiments. For glucose

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transporter 4 (GLUT4) expression and translocation studies, cells were grown in T-25 flasks.

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Cells were used in passages 10–18 for all experiments.

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The confluent monolayers of 3T3-F442A pre-adipocytes were treated with 50 µM tripeptides

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prior to addition of 10 µg/mL insulin to study the effects of IPP and VPP on insulin signaling. 5 ACS Paragon Plus Environment


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To induce insulin resistance and inflammation in adipocytes, murine TNF (10 ng/mL) was added

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to the medium for 23 h after pre-treating cells with the tripeptides for 1 h. Signaling events were

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studied by 30 min treatment with insulin (10 µg/mL).

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For glucose uptake and GLUT4 translocation experiments, confluent monolayers of adipocytes

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were first stimulated with 10 µg/mL insulin for 48 h to induce differentiation. Cells were washed

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and TNF (10 ng/mL) was added to the medium with/without IPP and VPP (50 µM each) for

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another 24 h.

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Glucose Uptake Assay

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Glucose uptake in adipocytes was determined according to the method described elsewhere

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with some modifications. The differentiated adipocytes incubated with TNF with/without

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tripeptides were washed and incubated in Krebs-Henseleit (KHH) buffer (0.1 % bovine serum

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albumin (BSA), 10 mM Hepes, and 2 mM sodium pyruvate, PH 7.4) for 2 h. The media was then

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changed with the buffer containing 11 mM glucose for another 4 h. A Glucose CII-Test kit

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(Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used afterwards to determine the

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glucose concentration in KHH buffer. The differences in glucose concentrations in the KHH

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buffer before and after culture determined the amount of glucose uptake by cells.

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GLUT4 translocation

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For translocation experiments, after incubating differentiated adipocytes in KHH buffer for 2 h,

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cells were incubated in KHH buffer containing glucose in the presence of insulin (10 µg/mL) for

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30 min. Membrane and cytosolic proteins were separated from cells using a Mem‐PER Plus

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Membrane Protein Extraction Kit (Thermo Fisher Scientific, IL USA) following the kit’s


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instructions. In brief, cells were washed twice with cell wash solution and the pellet was

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incubated with permeabilization buffer to separate cytosolic proteins. After centrifugation

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(16 000 × g, 15 min) and separation of the supernatant containing cytosolic proteins, the pellet

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was incubated with solubilization buffer. The solubilized membrane and membrane‐associated

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proteins were collected after centrifugation (16 000 × g, 15 min) and used for western blotting

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experiments.

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Western Blotting

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The samples for western blotting experiments were prepared by lysing cells in hot Laemmle’s

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buffer containing 50 µM dithiothreitol and 0.2% Triton-X-100 as described previously.

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running the SDS-PAGE and transferring proteins to nitrocellulose membranes, they were

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immunoblotted with antibodies against phospho-Akt and phospho-ERK1/2 (rabbit polyclonal

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antibody from Cell Signaling Technology, ON, Canada), Akt (mouse monoclonal antibody from

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Santa Cruz, CA, USA), ERK1/2 and phospho-IRbeta (mouse monoclonal antibody from Cell

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Signaling Technology), IRbeta (rabbit monoclonal antibody from Cell Signaling Technology),

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GLUT4 (rabbit monoclonal antibody from Abcam), COX-2 and the loading control α-tubulin

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(rabbit polyclonal antibody from Abcam). The concentration of primary antibodies used in this

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study was between 0.4 and 1 µg/mL. Goat anti-rabbit and Donkey anti-mouse fluorochrome-

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conjugated secondary antibodies were from Licor Biosciences (Lincoln, NB, USA). A Licor

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Odyssey BioImager was used to detect the protein bands which then quantified by densitometry

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using corresponding software (Licor Biosciences, Lincoln, NB, USA). COX-2 bands were

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normalized to their corresponding α-tubulin band while phospho-AKT, phospho-ERK1/2 and

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phospho-IRbeta bands were normalized to their corresponding total AKT, ERK1/2, and IRbeta


21

After


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bands respectively. For comparison purposes, untreated cell lysates were loaded onto gels in all

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the experiments.

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Statistical Analysis

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All data are presented as mean ± SEM from 3-7 independent experiments. Data were tested for

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normal distribution using Shapiro-Wilk normality test. For normally distributed data one-way

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analysis of variance (ANOVA) with the appropriate post-hoc test (Dunnett’s test for comparison

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to control group and Tukey’s test for multiple comparisons) were used for determination of

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statistical significance. Kruksal-Wallis with Dunn’s post-hoc test was used in non-normally

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distributed data. The PRISM 7 statistical software (Graph Pad Software, San Diego, CA) was

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used for the analyses. P0.05). While insulin increased

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Akt phosphorylation in control cells more than 5 times (529.4 ± 105.7 vs 100), pre-treatment

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with IPP and VPP significantly enhanced this response (907.7 ± 205.4 and 773.4 ± 135.1 for IPP

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and VPP respectively) in pre-adipocytes. This is an indication for the insulin sensitizing actions

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of IPP and VPP in adipocytes. The insulin sensitizing effect of an egg white hydrolysate on Akt

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phosphorylation in adipocytes has also been reported recently.

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exerted its insulin sensitizing effect potentially downstream to insulin receptor. We also

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evaluated the effects of these peptides on the other key downstream signaling target of insulin,

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mitogen activated protein kinase ERK1/2, in adipocytes. Phosphorylation of ERK1/2 is mainly

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involved in the mitogenic responses of insulin in adipocytes.

22

In our previous study, we showed insulin-like effects of IPP and VPP in 3T3-

22

19

Insulin

Akt activation is involved in mediating insulin effects on glucose 23

As shown in Fig 1A, IPP and VPP alone had no


25

24

The egg white hydrolysate

In a similar vein, IPP and VPP


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alone had no significant effects on ERK1/2 phosphorylation (P>0.05), while VPP pretreated cells

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enhanced insulin effects on ERK1/2 phosphorylation significantly compared to control cells

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treated with exogenous insulin (Fig 1B, 230.9 ± 23.3 vs 171.2 ± 9.5). ERK1/2 activation in

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response to insulin in IPP-treated cells was comparable to that observed in control cells treated

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with insulin (Fig 1B, 188.0 ± 10.4 vs 171.2 ± 9.5).

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IPP and VPP restore insulin signaling in insulin-resistant adipocytes

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Chronic inflammation, aging, and stress are believed to play a key role in the pathogenesis of

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insulin resistance and metabolic syndrome.

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harmful adipokines with adverse effects on adipose tissue and other insulin sensitive organs in

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the body which eventually results in the occurrence of systematic insulin resistance.

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one of the major pro-inflammatory cytokines involved in the pathogenesis of insulin resistance

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through interfering with insulin signaling cascade. Indeed, TNF is known to impair insulin

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signaling in both adipose and muscle tissues by inhibiting tyrosine kinase activity of insulin

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receptor and inducing serine phosphorylation of insulin receptor substrate proteins 26, 28 which in

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fact inactivates insulin receptor proteins leading to inactivation of insulin signaling cascade. TNF

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is a potential predictor of cardiovascular events and diabetes with occurrence of the metabolic

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syndrome. 29 Since we showed that IPP and VPP exerted insulin sensitizing effects in adipocytes,

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next, we explored the potential effects of these tripeptides on insulin signaling in insulin resistant

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adipocytes. TNF was used to induce insulin resistance in adipocytes. Indeed, insulin effects on

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Akt and ERK1/2 phosphorylation were lost when adipocytes were chronically treated with TNF,

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which is an indication of development of insulin resistance in adipocytes. Interestingly, pre-

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treatment with IPP and VPP restored insulin induced Akt and ERK1/2 phosphorylation in insulin

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resistant adipocytes comparable to untreated cells stimulated with insulin in the absence of TNF

26

Inflammation in adipocytes leads to the release of


27

TNF is

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(Fig 2A & 2B). Furthermore, in insulin resistant adipocytes the increase in Akt and ERK1/2

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protein phosphorylation upon IPP and VPP pre-treatment was significantly higher than the

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insulin treated cells (Fig 2A & 2B), which clearly shows the insulin sensitizing effects of IPP and

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VPP in insulin resistant adipocytes. Consistent with our finding, VPP has been reported to

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enhance insulin sensitivity in obese mice. 30

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Insulin sensitizing effect of IPP and VPP appears to be independent from insulin receptor

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in adipocytes

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We also studied the involvement of insulin receptor in the observed insulin sensitizing effects of

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IPP and VPP in adipocytes. The insulin receptor is composed of two alpha-subunits linked by

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disulphide bonds to two beta-subunits.

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activates beta-subunit with tyrosine kinase activity which phosphorylates receptor substrate

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proteins at tyrosine residues and the association with downstream signaling mediating metabolic

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and mitogenic responses of insulin.

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phosphorylation of IRbeta (Fig 3A). Only insulin enhanced IRbeta phosphorylation and pre-

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treatment with IPP or VPP did not further enhance phosphorylation of IRbeta (Fig 3A). These

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data indicate that insulin sensitizing effects of IPP and VPP occurs potentially downstream of

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IRbeta. Furthermore, in insulin resistant state where adipocytes were pretreated chronically with

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TNF, IRbeta phosphorylation was also not adversely affected (Fig 3B). Although we did not

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observe any change in IRbeta phosphorylation in 3T3-F442A adipocytes upon chronic treatment

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with TNF, this cytokine was reported to reduce tyrosine phosphorylation of insulin receptor in

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3T3-L1 adipocytes in a dose dependent manner.

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phosphorylation was observed mostly at 25 and 50 ng/mL of TNF. 33 Our data show that insulin

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effect on IRbeta phosphorylation was independent from TNF or tripeptide pre-treatment (Fig

31

32

Binding of insulin to insulin receptor alpha-subunit

IPP and VPP treatments alone did not enhance

33

The inhibitory effect on IRbeta



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3B). This indicates that effects of TNF on inducing insulin resistance as well as the protective

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effects of tripeptides on insulin signaling in insulin resistant cells occurs downstream of IRbeta

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phosphorylation in 3T3-F442A adipocytes.

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IPP and VPP reduce inflammatory response in insulin resistant adipocytes

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Inflamed and abnormally differentiated adipocytes release harmful molecules which aggravate

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insulin resistance and diabetes.

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eicosapentaenoic acid, palmitoylethanolamide, beta-mercaptoethanol, and St John’s wort extracts

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have been reported to suppress the adverse effects of pro-inflammatory stimuli on adipocytes. 37-

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40

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involved in mediating inflammatory process. 41 COX-2, the inducible form of COX, is markedly

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upregulated in response to inflammation. Indeed, COX-2 plays a crucial role in adipose tissue

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inflammation and systemic insulin resistance.

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reported to significantly reverse adipose tissue inflammation and improve glucose homeostasis

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and insulin sensitivity in obese rodent models.

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COX-2 levels in 3T3-F442A cells, while a one-hour pre-treatment with IPP or VPP abolished

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this response (Fig 4). This inhibition in COX-2 protein expression indicates the beneficial anti-

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inflammatory capabilities of these tripeptides in adipocytes. Activation of adipocyte COX-2 has

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been reported to increase the production of proinflammatory adipokines and decrease

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adiponectin production mainly via the activation of the NF-kB pathway.

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previous study we showed that IPP and VPP also prevented TNF mediated loss of adiponectin

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release from 3T3-F442A cells. The anti-inflammatory effect of IPP and VPP was mediated

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through NF-κB pathway by inhibiting p65 phosphorylation. 19

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VPP restores glucose uptake in TNF-treated adipocytes

34-36

Several compounds with anti-inflammatory effects such as

Cyclooxygenase (COX) is an enzyme preventing the production of prostaglandins which are

42

COX-2 inhibition on the other hand, has been

43

Treatment with TNF for 24 h upregulated


43

Interestingly, in our

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Insulin has several roles in the body. In addition to its anti-inflammatory and adipogenic roles,

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insulin mediates glucose uptake and utilization through Akt phosphorylation in various tissues. 32

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Since we showed that IPP and VPP were capable of restoring Akt phosphorylation in TNF-

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mediated insulin resistant adipocytes, the effects of these peptides on glucose uptake was also

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tested in these cells. While glucose uptake was significantly increased in differentiated

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adipocytes (stimulated with insulin), chronic TNF treatment abolished this effect considerably as

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shown in Fig 5. Interestingly, VPP restored glucose uptake in insulin resistant adipocytes while

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IPP did not affect glucose uptake in these cells (Fig 5). Previous studies by our group also

225

showed a similar dichotomy among related tripeptides (IRW vs. IQW) as shown on anti-

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inflammatory activities in endothelial cells. Although both IRW and IQW inhibited TNF-induced

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up-regulation of intercellular cell adhesion molecule-I (ICAM-1), the upregulation of the

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vascular cell adhesion molecule-I (VCAM-1) was only inhibited by IRW. Moreover, the nuclear

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factor-κB pathway was also differentially regulated by IRW and IQW.

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supported the significance of peptide sequence in determining its function.

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VPP increased GLUT4 expression without affecting translocation in TNF-treated

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adipocytes

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GLUT1 is an insulin-independent glucose transport protein widely distributed in different tissues

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whereas GLUT4 is an insulin-dependent glucose transporter protein responsible for the majority

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of glucose uptake in adipose tissue. Both the GLUT1 and GLUT4 facilitate glucose transport

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into cells. ,

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adipocytes. As shown in Fig 6A, both insulin and VPP enhanced GLUT4 expression

238

significantly in adipocytes whereas, IPP did not induce such an effect. GLUT4 is translocated

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from storage vesicles to the plasma membrane via an exocytic pathway following insulin

45

44

Our study further

We evaluated the potential effects of IPP and VPP on GLUT4 expression in



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240

stimulation enabling the transport of circulating glucose into the cell.

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translocation to the plasma membrane was enhanced in differentiated adipocytes (incubated with

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insulin) as shown in Fig 6B. However, chronic TNF treatment abolished this effect considerably

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and neither IPP nor VPP restored GLUT4 translocation in TNF-treated adipocytes. This implies

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that, the effect of VPP on enhanced glucose uptake in TNF-treated adipocytes is potentially

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mediated through insulin-independent mechanism(s). Further research is warranted to elucidate

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the mechanism by which VPP enhances glucose uptake in insulin resistant adipocytes.

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To summarize, we found that both IPP and VPP enhanced insulin sensitivity that was associated

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with upregulation of Akt phosphorylation in normal and TNF-exposed adipocytes (i.e. insulin-

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resistant) and reduced TNF-mediated COX-2 expression in these cells. VPP further enhanced

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GLUT4 expression in adipocytes and restored glucose uptake in TNF-treated adipocytes, which

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is an indication of additional benefits of this peptide on exerting anti-diabetic effects. IPP and

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VPP are well established for their antihypertensive activities.

253

recent research by others

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tissue including enhancing adipocyte differentiation, reducing inflammation, enhanced glucose

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uptake, and insulin signaling. Therefore, IPP and VPP may be considered as novel alternatives to

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reduce several complications of the metabolic syndrome.

16, 30

17, 47-49

As expected, GLUT4

Results from our study and

highlights the potential effects of IPP and/or VPP on adipose


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Figure 1. IPP and VPP on rapid insulin signaling in adipocytes. Confluent monolayers of

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3T3-F442A pre-adipocytes were pre-treated for 24 h with IPP or VPP (50 µM each) prior to 30 21 ACS Paragon Plus Environment


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min stimulation with insulin (10 µg/mL). Cell lysates were prepared and then immunoblotted for

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total and phosphorylated forms of Akt (A) and ERK1/2 (B). A representative set of images is

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shown. Data are presented as mean±SEM from 6-7 independent experiments. *, *** and ****

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indicate p

Milk-Derived Tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro

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