Artigo 7

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Review Article
Mechanism of GLP-1 Receptor Agonists-Mediated Attenuation of
Palmitic Acid-Induced Lipotoxicity in L6 Myoblasts
Mo-wei Kong ,1 Yu Gao ,1 Yu-yu Xie,2 En-hong Xing,1 Li-xin Sun,1 Hui-juan Ma ,3
and Han-ying Xing3
1Department of Endocrinology, Affiliated Hospital of Chengde Medical University, 36 Nanyingzi Street, Shuangqiao District,
Chengde City, Hebei Province, China
2Department of Dermatology, Chengdu Fifth People’s Hospital, 33 Ma Shi Street, Wenjiang District, Chengdu City,
Sichuan Province, China
3Department of Endocrinology, Hebei Provincial People’s Hospitall, No. 348, Heping West Road, Xinhua District, Shijiazhuang City,
Hebei Province, China
Correspondence should be addressed to Yu Gao; yugao815@163.com
Received 16 June 2022; Revised 8 October 2022; Accepted 2 December 2022; Published 28 December 2022
Academic Editor: Richard K.D. Ephraim
Copyright © 2022 Mo-wei Kong et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Object. L6 cells were cultured to explore the possible mechanism underlying the improvement of insulin resistance by Liraglutide
(LR). Methods. Cells were divided into 5 groups—control, high-fat, 10 nmol/L LR + 0:6mmol/L palmitic acid (PA) (10LR),
100 nmol/L LR + 0:6mmol/L PA (100LR), and 1000 nmol/L LR + 0:6mmol/L PA (1000LR). CCK-8 method to detect cell
viability, GPO-PAP enzymatic method to detect intracellular triglyceride content, and reverse transcription quantitative real-
time polymerase chain reaction (RT-qPCR) and western blotting methods to detect fatty acid translocase CD36 (FAT/CD36)
and fatty acid binding protein 4 (FABP4) in L6 cells, glucose-regulated protein 78 (GRP78), glucose transporter 4 (GLUT4)
expression at the mRNA and protein levels, respectively, were performed. Results. We found that after PA intervention for
24 h, the cell viability decreased significantly; the cell viability of the LR group was higher than that of the high-fat group
(Pto a density of 9 × 104 cells/mL and
transferred into wells of 96-well plates, 100μL per well,
and incubated at 37°C for 48 h. Following interventions,
10μL of CCK-8 reagent was added to each well, and the
plates were placed at 37°C for 1 h. The optical density
(OD) was measured at 450nm using a microplate reader
(BD680 type automatic microplate reader Bio Rad company,
USA). Stimulation index SI = ðODvalue of each stimulated
well −ODvalue of mediumÞ/ðODvalue of unstimulated well
−ODvalue of mediumÞ.
2.6. Lipid Deposition. Lipid deposition was measured using
an Oil Red O Staining Kit (Solarbio Life Sciences; Beijing,
China). Cells were washed twice with PBS, fixed with 4%
paraformaldehyde for 15min; washed twice with PBS, pre-
treated with 60% isopropanol for 20 s, and washed twice
with PBS after 30min shading, and separated with 60% iso-
propanol for 5 s, and then washed twice with PBS. After the
staining area was calibrated and normalized using Image J
image processing software, the data were counted. Stained
cells were photographed with an inverted microscope (Tu
Ming Company, Shanghai, China) within 2 h of staining.
2.7. Triglyceride Content. Intracellular triglyceride content
was quantified using a Triglyceride (TG) Content Assay
Kit (Solarbio Life Sciences; Beijing, China) according to the
manufacturer’s protocol.
2.8. mRNA Expression. Total RNA was extracted from
treated cells using kit (TIANGEN Biochemical Technology
Company, Beijing, China) according to the manufacturer’s
protocol. The gene expression levels were measured by
reverse transcription quantitative real-time PCR using Load-
ing Buffer PCR Master Mix (TIANGEN Biochemical Tech-
nology Company). Primers were synthesized by
GeneCopoeia (Rockville MD, USA; Table 1). Cycling condi-
tions were as follows: 95°C for 10min; 40 cycles of 60°C for
20 s, and 70°C for 10 s. Amplification was carried out on a
Cobasz 480 (Roche Company, Switzerland) to confirm the
amplification curve, dissolution curve, and Ct value using
2-ΔΔCt to calculate the mRNA expression of the target gene.
2.9. Protein Expression. Protein expression was quantified
using western blotting with GAPDH as the internal refer-
ence. Protein samples were separated by polyacrylamide gel
2 BioMed Research International
electrophoresis, transferred to a nitrocellulose membrane,
and the membranes blocked with milk for 1 h and probed
overnight at 4°C with primary antibodies. The antibodies
used were as follows:
The immunoreactive bands were separated by polyacryl-
amide gel electrophoresis, transferred to a nitrocellulose
membrane, and blocking repeated. Membranes were
washed, probed overnight at 4°C with sheep anti-rabbit
(GRP78, FAT/CD36, FABP4, and GLUT4) or sheep anti-
rat (GAPDH) secondary antibodies. Immunoreactive bands
were detected using chemiluminescence reagent (TIANGEN
Biochemical Technology Company) according to the manu-
facturer’s instructions. Protein expression was quantified
using an enzyme labeling instrument (BD 680 automatic
microplate reader) at 570nm.
2.10. Statistical Analyses. All data are expressed as means ±
standard deviation (n = 3). Statistical significance was
assessed using Fisher LSD t-tests, and single factor ANOVA;
correlations were determined using linear correlation. SPSS
20.0 (IBM Corp; Armonk NY, USA) was used for statistical
analysis, and GraphPad Prism 6.01 (GraphPad Software; La
Jolla CA, USA) was used for graphic analysis. Statistical sig-
nificance was set at P 0:05), but
both the 100LR (average SI = 2:64) and 1000LR (average SI
= 2:50) groups had higher cell viability (PGroup GRP78 mRNA FABP4 mRNA FAT/CD36 mRNA GLUT4 mRNA
Contol group 1 ± 0:00 1 ± 0:00 1 ± 0:00 1 ± 0:00
High fat group 8:15 ± 0:35a 2:46 ± 0:08a 8:29 ± 0:52a 0:34 ± 0:03a
10LR group 6:83 ± 0:11b 2:28 ± 0:06 3:23 ± 0:11b 0:47 ± 0:06b
100LR group 5:52 ± 0:48bc 1:24 ± 0:06bc 2:42 ± 0:11b 0:50 ± 0:04b
1000LR group 4:36 ± 0:32bd 1:00 ± 0:04bd 2:88 ± 0:55b 0:75 ± 0:04b
aPsummary, LR reduced the ERS response and intracel-
lular lipid deposition, improved lipid metabolism, and
downregulated the expression of GRP78, FAT/CD36, and
FABP4 mRNA and protein in muscle cells with PA-
induced lipotoxicity. In addition, LR improved the state of
IR by regulating the expression of GLUT4 in the insulin
transduction pathway. This study gives insight into how
LR improves IR and provides theoretical support for its clin-
ical application.
5. Limitation
There are also some limitations in our experiments. Some
experiments can be added as proof of the result: (1) cell via-
bility assays have been performed but apoptosis assays like
Annexin V assay or Hoechst assay can be performed to visu-
alize the percent of cells undergoing apoptosis with palmitic
acid and liraglutide treatment [31–33]. (2) ORO staining has
been performed to measure the lipid deposition but semi-
quantification of the lipid droplets in ORO staining or
counting the ORO positive cells in each group can be per-
formed to determine the differences between each group
[34]. (3) Lipid deposition could be verified at mRNA and
protein levels using perilipin as a marker to provide addi-
tional support to the results [35].
Data Availability
All data are fully available without restriction.
Conflicts of Interest
The authors declare that there are no conflicts of interest.
Authors’ Contributions
This article involves cooperation between multiple depart-
ments. Mo-wei KONG wrote the manuscript; each of the
remaining authors reviewed and revised the manuscript.
Acknowledgments
This work was supported by the Hebei Provincial Depart-
ment of Bureau Science and Technology of “Technology
Innovation Guidance Project-Science and Technology Work
Conference” project (2020-2022), the Hebei Province Basic
Research Project (Natural Science Foundation)
(C2022406011), and the Basic Research Project of Chengde
Science and Technology Bureau (202205B074).
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	Mechanism of GLP-1 Receptor Agonists-Mediated Attenuation of Palmitic Acid-Induced Lipotoxicity in L6 Myoblasts
	1. Introduction
	2. Materials and Methods
	2.1. Materials
	2.2. Cell Culture
	2.3. Palmitic Acid-Supplemented Culture Medium
	2.4. Interventions
	2.5. Cell Viability
	2.6. Lipid Deposition
	2.7. Triglyceride Content
	2.8. mRNA Expression
	2.9. Protein Expression
	2.10. Statistical Analyses
	3. Results
	3.1. Cell Viability
	3.2. Triglyceride Content
	3.3. Lipid Deposition
	3.4. mRNA Expression
	3.5. Protein Expression
	3.6. Correlations
	4. Discussion
	5. Limitation
	Data Availability
	Conflicts of Interest
	Authors’ Contributions
	Acknowledgments