MethodsBackground

Shrimp oil extracted from shrimp waste is a rich source of omega-3 fatty acids and

astaxanthin esters, and possess anti-adipogenic effects in 3T3-L1 preadipocytesIndrayani Phadtare1, Hitesh Vaidya1, Sara Ahmadkelayeh2, Kelly Hawboldt2, Sukhinder K Cheema1

1Department of Biochemistry, 2Faculty of Engineering and Applied Science, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9

Results

Summary

References

Experimental Design

(Adipogenesis)

Hypotheses

Conclusions

Objectives

Statistical Analysis

The province of Newfoundland and Labrador, Canada,

generates over 6000 metric tons of shrimp waste every

year. Shrimp waste is a valuable source of shrimp oil

rich in omega (n)-3 polyunsaturated fatty acids

(PUFA) and astaxanthin (Astx). Astx is a highly potent

antioxidant that exist in either free form or esterified

form (Astx-E) [1]. Previous studies using fish oil (rich

in n-3 PUFA) [2], and free astaxanthin [3], have been

shown to reduce fat accumulation by inhibiting

adipogenesis, thereby protecting against obesity and

insulin resistance. However, the effects of shrimp

extract rich in n-3 PUFA and astaxanthin on

adipogenesis are not known. Furthermore, majority of

the fish oil in the market is suggested to be oxidized

[4]. Astaxanthin is a highly potent antioxidant,

however it is not known whether a combination of fish

oil and astaxanthin prevents the oxidation of fish oil,

and inhibits adipogenesis. We extracted shrimp oil

from shrimp waste, and investigated the effects on

adipogenesis in 3T3-L1 adipocytes. We further

investigated the effects of a combination of fish oil

plus astaxanthin on adipogenesis, compared to fish

oil alone, in 3T3-L1 adipocytes.

Shrimp oil, rich in n-3 PUFA and Astx-E, and a

combination of fish oil plus Astx-E, will reduce fat

accumulation and inhibit adipogenesis to a greater

extent, compared to fish oil or Astx-E alone.

The specific objectives were to:

1. Extract and analyze shrimp oil (SO) from shrimp

waste using the Soxhlet method,

2. Investigate whether Astx-E prevents the oxidation

of fish oil (FO), compared to FO alone using the

peroxide value (PV) assay,

3. Investigate the effects of SO, FO (alone or in

combination with Astx-E) on adipogenesis in 3T3-

L1 adipocytes.

SO extraction and analysis: Shrimp extract (SE) was

obtained from shrimp waste using the Soxhlet method

(hexane: acetone 2:3, v/v) [5]; SO was extracted from

SE using the Folch method [6]. Astx was measured

spectrophotometrically after separation by thin layer

chromatography (TLC) (acetone: hexane (25:75 v/v)

[7]. Total lipids and fatty acids composition was

measured using TLC-flame ionization detection (TLC-

FID) and gas chromatography (GC), respectively.

PV assay: FO alone, or in combination with Astx-E

(100 μg/g) was exposed to 37⁰C forced air; samples

were collected at 0, 4, 8, 12, 24 and 48 hrs. Oxidation

was measured using PV assay [8]; n=3 per group.

Lipid emulsions: Lipid emulsions were prepared [9]

to treat 3T3-L1 cells. Emulsions were analyzed for

particle size using dynamic light scattering method

[10] to confirm uniform distribution of the particles.

Oil Red O staining: Oil Red-O staining and

quantification was performed as per the manufacturer

instructions [11]. Stained cells were viewed using a

Leica DMIL LED Microscope at 400x magnification.

Total RNA extraction and Real time qPCR: Total

RNA was extracted using TRIzol method [12]. Gene

expression analysis was performed using the Bio-Rad

CFX96TM Real-Time System. Expression of target

genes was normalized to RPLP0 as the reference gene

and calculated using ΔΔCt method [13].

Data were analyzed using one-way ANOVA and

Tukey’s post-hoc test using GraphPad Prism 8. Results

are expressed as mean±SD and p<0.05 was

considered significant. Superscripts (a, b, c) represent

significant differences.

Table 2: SO is rich in Astx-E. SE was spotted on pre-coated Silica

gel-G plates, along with free- (Astx) and esterified-astaxanthin (Astx-E)

standards. The spots corresponding to Astx and Astx-E were scraped

and analyzed as mentioned in the methods section, using respective

standard curves.

Figure 1: SO is rich in phospholipids.

Figure 2: Astx-E protects FO from oxidation. FO, alone or in

combination with Astx-E, was exposed to oxidation as described in the

methods section, and PV was measured. Data were assessed using two

tailed paired student’s t-test, P<0.05 was considered significant; n=3.

*represent significant differences at each time point.

Figure 5: SO, SE and Astx-E downregulates, while FO

upregulates the mRNA expression of sterol regulatory element-

binding protein (SREBP)-1c in 3T3-L1 mature adipocytes. The

cells were differentiated to mature adipocytes as explained in the

methods section. Data were analyzed using one-way ANOVA and

Tukey’s post-hoc test. p<0.05 was considered significant. Superscripts

(a, b, c) represent significant differences. FOA=FO plus Astx-E.

Our findings demonstrate that SO from shrimp

waste is rich in phospholipids, n-3 PUFA and

esterified astaxanthin, and has the potential to

reduce fat accumulation. Furthermore, SO and FO

appear to regulate adipogenesis via independent

pathways.

Table 1: SO is rich in n-3 PUFA.

AcknowledgmentsThis research was funded by the Vitamin Research

Fund, Ocean Frontier Institute, Canada.

Fatty acids (% nmol) Shrimp oil

C14:0 0.17±0.00

C16:0 15.73±0.33

C16:1n7 9.58±0.65

C18:0 2.42±0.07

C18:1n9 21.33±1.03

C18:1n7 6.49±1.56

C18:2n6 1.96±0.12

C18:3n3 0.61±0.08

C20:1n9 0.45±0.20

C20:4n6 1.69±0.14

C20:5n3 21.10±0.11

C22:5n3 1.48±0.11

C22:6n3 13.89±0.13

∑ n-3 PUFA 37.09±0.04

∑ n-6 PUFA 3.99±0.11

Control

SE SO

Astx

-E FOFO

A

0.0

0.5

1.0

1.5

2.0

SR

EB

P-1

c m

RN

A e

xpre

ssio

n

(Fol

d C

hang

e)

a

bb

c c c

Control SO

FOAAstx-E FO

SE

Figure 3: SE revealed a decrease in fat accumulation, while FO and

FOA showed an increase in fat accumulation in 3T3-L1 mature

adipocytes. Oil Red O staining and quantification was performed at day 8

of differentiation as explained in the methods section. Data were analyzed

using one-way ANOVA and Tukey’s post-hoc test. p<0.05 was considered

significant. Superscripts (a, b, c) represent significant differences.

Treatments: Dose response effect of treatments was conducted

to establish the dose (0.125-0.5 mg/ml of culture medium).

Preadipocytes were differentiated to mature adipocytes in the

presence or absence of SO or FO emulsions at a final

concentration of 0.25 mg/ml, for 8 days. This concentration of

SO contained 15.9 ng Astx-E; thus, cells were also treated

with 15.9 ng/ml of Astx-E, and FO plus 15.9 ng/ml of Astx-E

(FOA); n=3 per group. NBCS= newborn calf serum, Dex=

dexamethasone, IBMX= 3-isobutyl-1-methylxanthine.

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8. British Pharmacopeia. PV. (2005). IV:2.5.5:129

9. Anez‐Bustillos L. and Dao D.T. (2016). Nutr. Clin Pract. 31(5):609

10. Zhang B. and Cai Q. (2019). Water Research. 149:301

11. Maeda H. and Hosokawa M. (2006). Inter. J. Mol. Med. 18:152

12. Chomczynski P. and Sacchi N. (1987). Anal. Biochem. 162(1):159

13. Livak K.J. and Schmittgen T.D. (2001). Methods. 25:402

Figure 4: SO, SE and Astx-E downregulates, while FO and FOA

upregulates the mRNA expression of peroxisome proliferator-

activated receptor (PPAR)-γ in 3T3-L1 mature adipocytes. The cells

were differentiated to mature adipocytes, for 8 days, as explained in the

methods section. Data were analyzed using one-way ANOVA and

Tukey’s post-hoc test. p<0.05 was considered significant. Superscripts (a,

b, c) represent significant differences. FOA=FO plus Astx-E.

Total RNA extraction

3T3-L1 preadipocytes

70-80% confluent

DMEM+10% NBCS

8 days

Differentiation media +Treatments

(10μg/ml insulin, 1 μM Dex, 0.5mM IBMX)

Oil Red O staining

Mature adipocytes

Harvest Cells

Con

trol

SESO

Astx-

EFO

FOA

0.0

0.5

1.0

1.5

2.0

DG

AT

2 m

RN

A e

xpre

ssio

n

(Fold

Chan

ge)

a

c c

bbb

Figure 6: SO and SE downregulates, while FO upregulates the

mRNA expression of diacylglycerol O-acyltransferase 2

(DGAT2) in 3T3-L1 mature adipocytes. The cells were

differentiated to mature adipocytes as explained in the methods

section. Data were analyzed using one-way ANOVA and Tukey’s

post-hoc test. p<0.05 was considered significant. Superscripts (a, b,

c) represent significant differences. FOA=FO plus Astx-E.

Adipogenesis

DGAT2

Shrimp

waste

SREBP-1c

Lipogenesis

Preadipocytes Mature adipocytes

Shrimp oil

PPAR-γ

Fish oil +

Astx-E

Fish oil

Stery

l Ester

s

Wax

Ester

s

Ethyl

Ester

s

Triacy

lgyc

erol

s

Free Fat

ty A

cids

Alc

ohol

s

Stero

ls

Phosp

holip

ids

0

20

40

60

80

% L

ipid

co

mp

osi

tio

n

Con

trol

SESO

Astx-

EFO

FOA

0.0

0.5

1.0

1.5

2.0

Oil

Red

O (

52

0 n

m) a

a

ab

bcb

c

Control

SE SO

Astx

-E FOFO

A

0.0

0.5

1.0

1.5

2.0

2.5

PP

AR

-γ m

RN

A e

xpre

ssio

n

(Fol

d C

hang

e)

a

a

bc

c c

0 4 8 12 24 48

0

10

20

30

Time (Hours)

PV

(m

Eq

/Kg

)

FO

FO+Astx-E*

*

**

FractionConcentration

(μg/ml SE)

Astx yield, μg/g

shrimp waste

Free Astx 8.24 24.03

Astx-E 64.37 187.76

Publishing Date: May 2020. © 2020. All rights reserved. Copyright rests with the author. No part of this

abstract/poster may be reproduced without written permission from the author.