Menthone reduces the hyperglycaemia in 3T3-L1 adipocytes cells by downregulating

the ROS production and increasing antioxidants

M. Revathi 1, K. Padmalochana*2, C. Sankaranarayanan3, S. Nalini1

1
Department of Biochemistry, Shree Ragavendra Arts and Science College,
Keezhamoongiladi , Chidambaram-608102
2
Department of biochemistry, Sri Akilandeswari Womens College,Wandiwash-604 408
3
Department of Biochemistry & Biotechnology, Annamalai University, Annamalai Nagar,
Chidambaram- 608002
Corresponding author: devnathyazhini@gmail.com

Abstract

Background: Diabetes mellitus (DM) is a metabolic disorder characterised by chronic

hyperglycaemia and metabolic consequences caused by insulin defects. Adipose tissue is an
endocrine organ that influences glucose and lipid metabolism by releasing adipokines, pro-
inflammatory factors, and free fatty acids (FFAs), which impair glucose metabolism and
muscle ATP synthesis, promoting the synthesis of toxic lipid metabolites and alter insulin
signalling. Menthone is antioxidative cyclic monoterpenes and is reported to have several
biological activities. The study aimed to evaluate the antioxidative effect of menthone in the
3T3L1cell line.

Methods: The differentiation of 3T3-L1 preadipocytes into mature adipocytes was cultured
and maintained. The effect of menthone on antioxidative parameters of were studied by
Mitochondrial membrane potential (MMP) assay, Reactive oxygen species (ROS), Oil red O
staining, catalase enzymatic activity, MTT assay and Glutathione peroxidase (GP X) assay in
3T3L-1cell line.

Results: The results revealed the treatment (25 mM/L glucose + 0.6 nm/L insulin + 63.22
µM/ml of menthone) with menthone resulted in a significant reduction of lipid accumulation
in the cells. The studies interpreted by MTT assay and viability using 3T3L-1 cell lines found
that treatment increased mitochondrial membrane potential in 3T3L-1 adipocytes, indicating
that menthone exposure reduced mitochondrial damage. In the present study, cell viability
decreased with increased menthone concentration. The IC50 value for menthone was observed
as 70.26 μM/ml. Menthone treatment has significantly suppressed ROS production and
increased
Conclusion: This paper proves that menthone could suppress oxidative stress in 3T3L-1
adipocytic cells.

Introduction

Diabetes, an endocrine disease diagnosed by unusually high blood glucose levels, is

one of the most frequent and fastest-growing diseases globally, with 693 million adults
expected to be affected by 2045 (Cho et al., 2018). According to the World Health
Organization, the incidence of diabetes will be increased up to 422 million worldwide in
2020, with the majority residing in low- and middle-income countries, resulting in 1.5 million
fatalities each year. Preventing or delaying the onset of type 2 diabetes can be accomplished
through a nutritious diet, frequent physical activity, maintaining a healthy body weight, and
avoiding tobacco use. Type I diabetes (insulin-dependent) is caused by the immune system's
destruction of beta cells, resulting in insulin deficiency.

Adipose tissue is an endocrine organ that influences both glucose and lipid
metabolism (Kershaw et al., 2004; Scherer, 2006) by releasing adipokines, pro-inflammatory
factors, and free fatty acids (FFAs), which impair glucose metabolism and muscle ATP
synthesis (Brehm et al., 2006), promote the synthesis of toxic lipid metabolites and alter
insulin signalling (Scherer, 2015; Ravussin et al., 2014) Insulin acts on adipose tissue 1 by
stimulating glucose uptake and triglyceride synthesis and 2) by suppressing triglyceride
hydrolysis and release of FFA and glycerol into the circulation (Saponaro et al., 2015; Boden,
2008). Adipose tissue insulin resistance (Adipo-IR), that is, the impaired suppression of
lipolysis in the presence of high insulin levels, has been associated with glucose intolerance,
and elevated plasma FFA levels have been shown to impair muscle insulin signaling, promote
hepatic gluconeogenesis, and impair glucose-stimulated insulin response (Kashyap et al.,
2003; Ferrannini et al., 1983). Adipose tissue plays an essential role in maintaining lipid and
glucose homeostasis. A key pathogenic mechanism of obesity-related metabolic syndrome is
increased oxidative stress in adipose tissues (Boden, 2008). It has been linked to insulin
resistance and adipocyte hypertrophy, which causes inflammation, altered metabolism, and
adipokine secretion dysregulation (Castro et al., 2016). ROS are also required for adipocytes
differentiation by regulating mitotic clonal expansion during adipogenesis (Han, 2016).

Adipocyte function in obesity and diabetes is extensively studied using the 3T3-L1
adipocyte model. Because diverse cell lines are commonly maintained under these
conditions, these tests are frequently done in the presence of high glucose concentrations (25-
30 mM). Recent research has discovered that extracellular glucose concentrations
significantly impact the assessment of mitochondrial bioenergetics (Valsecchi et al., 2013),
which is important for testing mitochondrial function in diabetic patients. 3T3L1 adipocytes
have been used in many studies for unravelling the pathways of adipogenesis, insulin
signalling and Type 2 diabetes development. Recently, Lavanya et al., (2022) reported the in
vitro activity on glucose uptake of 3T3L-1 adipocytes mediated via PPARγ through in vitro
and molecular docking studies.

The Mentha are of economically important members of Lamiaceae family and have a
long history of usage as a medicinal ingredient. Menthone, menthol, and other compounds are
cyclic monoterpene found in the Mentha genus (Kamatou et al., 2013). Menthone is the
major essential oil that has been proven to have several biological activities such as
antimicrobial, antifungal, anticancer and inflammatory effects (Kamatou et al., 2013). The
cooling minty flavour and smell of plants are due to menthone, iso menthone and other
compounds (Lawrence, 2013). The biological activities of the menthone, on the other hand,
are still barely understood. Hence, we aimed to investigate the hypoglycemic efficacy of
menthone on 3T3-L1 adipocytes in in vitro conditions.

Materials and Methods

Chemical

The Gibco Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS)
and antibiotic solution were from Gibco (USA), Menthone, DCFH-DA, JC-10 and 1x PBS
was procured from Himedia, India. Tris-HCL, EDTA, Glucose, Sodium Chloride, H2O2 were
Purchased from Merk, USA. GSH- Glutathione GSSG- glutathione oxidised form, β-
NADPH- β- Nicotinamide Adenine Dinucleotide Phosphate, EDTA, SodiumeAzide H 2O2
Were Purchased from SRL India. Oil red O powder was obtained from Sigma-Aldrich, USA.

Cell culture and differentiation of 3T3-L1 into mature adipocytes

3T3 L1 adipocytes were purchased from NCCS, Pune. The cells were cultured in
liquid medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 100 µg/ml
penicillin and 100 µg/ml streptomycin and maintained under an atmosphere of 5% CO2 at 37
o
C. The differentiation of 3T3-L1 preadipocytes into mature adipocytes was cultured and
maintained [Green and Kehinde, 1975]. Differentiation medium was prepared 90% DMEM
supplemented with 10% FBS, 1% L-Glutamine, 1% Penicillin+streptomycin antibiotics-
containing DEX (1μM), Insulin (1 μg/mL), and IBMX (0.5 mM). First, 3T3-L1 cells were
seeded preadipocyte expansion medium at 8x104 in a 6-well plate containing 1 mL medium
until they reached confluence. After 48 h for the induction of differentiation 3T3-L1
preadipocytes were cultured with a differentiation medium. After 72 h of the induction of
insulin resistance, the differentiation medium was removed and added by adipocyte
maintenance DMEM supplemented with 10% FBS and insulin (1μg/mL) for another 48 h.
After 5 days of the induction of differentiation, the fresh adipocyte maintenance medium was
replaced every two days until 14 days.

For further experiments the cells were grouped in to four and hyperglycaemic
condition was induced by treating cells with 25 mM/L glucose + 0.6 nm/L insulin. Further
the groups were followed; Group 1: 3T3-L1 preadipocytes + 5 mM/L glucose + 0.6 nm/L
insulin, Group 2: 3T3-L1 mature adipocytes + 25 mM/L glucose + 0.6 nm/L insulin, Group
3: 3T3-L1 mature adipocytes+25 mM/L glucose + 0.6 nm/L insulin + 63.22 µM/ml of
Menthone; Group 4: 3T3-L1 mature adipocytes+25 mM/L glucose + 0.6 nm/L insulin +
63.22 µM/ml of Rosiglitazone.

Determination of cytotoxicity by MTT assay

The toxicity of menthone on 3T3-L1 mature adipocytes cells was determined by MTT
assay. The 3T3 L1 mature adipocytes cells were inoculated (1 x 105 cells/well) in a 96-well
plate and cultivated for 24 hours in a humified environment. Further, the cells were pre-
treated with 25 mM Glucose and administrated with various increasing doses (10-100
μM/ml) of menthone for 24 h at humified incubation. After the incubation period, MTT (20
µL of 5 mg/ml) was added to each well, and the cells were incubated for another 2-4 h until
purple precipitates were visible under an inverted microscope. Then the medium was
aspirated, and the resulting formazan was diluted by 100 µL of DMSO into all wells for 5
min. The absorbance for each well was measured at 570 nm using a microplate reader
(Thermo Fisher Scientific, USA) and the percentage cell viability and IC50 value were
calculated using GraphPad Prism 6.0 software (USA).

Determination of lipid accumulation by Oil red O staining

In brief, the 3T3-L1 mature adipocytes cells were seeded in 6 wells plate (3 x 105) and
cultured in humified condition for 24 h. After the cells were uniformly grown, the group 3
cells were administrated with 63.22 µM/ml of menthone, the group 4 cells were administrated
with 0.1 µM Rosiglitazone for 24 h in CO 2 condition. The group 1 and group 2 cells
remained without treatment. After 24 h of treatment, the cells were washed with PBS and
fixed with the 4%c of formalin in PBS 0.05M, and again washed with the 60% isopropanol
for 2 min and stained with a filtered 0.35% Oil Red O solution in 60% isopropanol for 10 min
at room temperature. Further, the cells were washed with PBS solution and observed under
Olympus light microscope.

Measurement of ROS

The ROS activities induced in 3T3-L1 mature adipocytes cells were ascertained by
treating the cells with DCFH-DA [Wang et al., 1993]. In brief, the cells were seeded in 6
wells plate (3 x 104) and cultured in humified condition for 24 h. After the cells were
uniformly grown, the group 3 cells were administrated with 63.22 µM/ml of menthone, the
group 4 cells were administrated with 0.1 µM Rosiglitazone for 24 h in CO 2 condition. The
group 1 and group 2 cells remained without treatment. Afterwards, the cells were stained with
100 µL of DCFH-DA for 10 min at 37 °C under dark conditions, later, the cells were washed
with PBS and observed under fluorescence microscopy equipped with a digital camera and
appropriate filters.

Measurement of mitochondrial membrane potential

According to the protocol recommended previously, the JC-10 dye was used to stain
the cells to explore the MMP [Johnson et al., 1980]. In brief, the 3T3-L1 mature adipocytes
cells were seeded in 6 wells plate and cultured in humified condition for 24 h. After the cells
were uniformly grown, the group 3 cells were administrated with 63.22 µM/ml of menthone,
and the group 4 cells were administrated with 0.1 µM Rosiglitazone for 24 h in CO 2
condition. The group 1 and group 2 cells remained without treatment. After treatment, the
cells were stained with JC-10 for 30 min. Then the cells were observed for the mitochondrial
membrane alteration under fluorescence microscopy equipped with a digital camera and
appropriate filters.

Estimation of Catalase Activity

The menthone sample was tested for catalase enzymatic activity in 3T3-L1 mature
adipocytes cells. Briefly, the cells were plated at a density of 1×10 5 cells/ml into the 24-well
tissue culture plate in a DMEM medium containing 10 % FBS and 1% antibiotic solution for
24 hours at 37°C. The wells were washed with sterile PBS and treated with test samples
(Group 1 to group 4) in a serum-free DMEM medium. Each sample was replicated three
times, and the cells were incubated at 37°C in a humidified 5% CO 2 incubator for 24 h. After
treatment the cells were lysed using ice-cold homogenate medium (pH 7.4 contain 0.01
mol/L Tris- HCL, 0.0001 mol/l EDTA-2Na, 0.01mol/L Sucrose, 0.8% Sodium chloride
solution) and centrifuged 5000 x g for 10 min at 2-4 OC to obtained supernatant. About 75 µl
of samples were used for assay CAT enzymatic activity was quantified in a
spectrophotometer cuvette. After adding 330 μL H2O2 (30 mM) and adjusting to 1 mL with
PBS, the H2O2 absorbance change was continuously measured at 240 nm every 30 s with a
spectrophotometer (Lambda EZ-150; PerkinElmer Company; Waltham, MA, USA). The
results of enzymatic activity were reported as the U min-1/mg protein.

Catalase enzyme activity was determined using the formula:

Specific activity (units/mg of protein/min) = ΔA 240 nm / (1 min) × 1000/43.6 × mg

protein.

Estimation of GPx activity

The 3T3 L1cells (20,000–50,000 cells/well) were plated to a 24 well plate and
incubated for 24 hr in a DMEM growth medium. After incubation, the plate was washed with
PBS and treated (Group 1 to Group 4) in a serum-free DMEM medium. Again, the plate was
incubated at 37°C in a humidified 5% CO2 incubator for 24 hrs. The samples were lysed in a
lysis medium. Briefly, cells were lysed with 0.9 ml of ice-cold lysis medium (pH 7.4 contain
0.01 mol/L Tris- HCL, 0.0001 mol/l EDTA-2Na, 0.01mol/L Sucrose, 0.8% Sodium chloride
solution) and centrifuged 5000 RPM for 10 min at 2-4 0c to obtained supernatant. 50 µl of
samples were used for the assay.

Result and discussion

Cytotoxicity of the Menthone on mature adipocytes 3T3L-1 cells
The MTT assay was carried out to evaluate the cytotoxic effects of the menthone on
3T3-L1 mature adipocytes cells. As shown in Table 1, the concentration-dependent
cytotoxicity in 3T3L-1 cells was demonstrated by menthone. Briefly, the cells were treated
with different concentrations of menthone for 24h, and the cell viability decreased with an
increase in menthone concentration. The 50% inhibition of cell growth (IC 50) was calculated
as 70.26 μM/ml. The data observed confirmed the effect of menthone on the viability of
3T3L-1 in a dose-dependent manner.
Effect of Menthone on Lipid accumulation by Oil red O staining
The most common use of quantitative oil red O staining is to determine the ability of
cultivated preadipocytes to differentiate under various experimental conditions (Kraus et al.,
2016). Oil red O staining was performed to evaluate the effect of menthone on the lipid
accumulation ability of 3T3-L1 adipocytes. The staining was carried out to distinguish the
lipid droplet in adipocytes treated with menthone at various concentrations for 24 h with the
oil red O kit. The results showed an increased amount of lipid accumulation in mature
adipose cells compared to the preadipose cells. However, the treatment with menthone
resulted in a significant increase of lipid accumulation in the cells. Compared to menthone
treatment, the Rosiglitazone treatment showed a more effective increase in lipid accumulation
in mature adipose cells (Fig. 1).

Effect of Menthone on ROS production

Several metabolic diseases, including T2D, insulin resistance, cardiovascular disease,
and obesity, have been associated with oxidative stress. The ROS production in 3T3L-1 cells
was determined by DCFH-DA staining. In contrast to the control cells (group 1), the depth of
green fluorescence was significantly increased in group 2 cells. However, fluorescence
intensity was significantly reduced when treated with Mentone in insulin-resistant group 3
cells. Despite this, the treatment of Rosiglitazone in insulin-resistant group 4 cells more
effectively reduced the ROS production, as evidenced by decreased fluorescence. The
observation reveals that a decrease in green fluorescence decreased intracellular ROS level
was induced by Menthone and Rosiglitazone in Adipocyte cells (Fig. 2). The increase in
intracellular ROS has been shown to occur during the first phase of adipogenesis (Lee et al.,
2009). In fat accumulation, increased ROS levels induce NADPH oxidase activation via the
high expression levels of NADPH oxidase 4 (Nox4) (Schröder et al., 2009). Elevated ROS
production from accumulated fat also leads to increased oxidative stress in the blood,
negatively affecting the skeletal muscle, aorta, and liver (Furukawa et al., 2004). The
antioxidative property of menthone seems to be effective in inhibiting adipogenesis. In
adipocytes, ROS overproduction has been associated with multiple forms of insulin resistance
(Castro et al., 2016) and changes in the endoplasmic reticulum, mitochondrial functioning,
and cell signaling, all of which contribute to inflammatory conditions (Marimoutou et al.,
2015). The ROS production increased in parallel with fat accumulation. The findings are
following the studies show that accumulation of fat is associated with increased oxidative
stress metabolic syndrome (Furukawa et al., 2004). Hence, uncontrolled ROS generation is a
chief contributor to the development of type 2 diabetes because it contributes to
inflammation, insulin sensitivity, improper functioning of feeding centres in the brain and
imbalanced energy homeostasis.

Effect of Menthone on MMP

Lower membrane potential can be inhibited by lowering the membrane potential. The
results showed that menthone treatment altered the mitochondrial membrane potential in
3T3L-1 adipocytes (Fig. 3). The menthone treatment significantly increased the MMP in
group 3 cells, indicating increased mitochondrial membrane integrity as evidenced by the
reduced fluorescence depth. Moreover, the Rosiglitazone administration significantly
decreased the membrane integrity (group 4). The mitochondrial membrane potential is
directly proportional to ROS formation: the MMP starting at ΔΨm >140 mV, relative oxygen
potential increases rapidly (Liu, 1999). Hyperglycemia in diabetic individuals can cause
mitochondrial superoxide generation, which contributes to the development of problems and
plays a role during normal glycemia periods after hyperglycemia (Brownlee, 2001). Wang et
al., (2015) used rhodamine 123 dye to evaluate the MMP in A549 cells and found a dose-
dependent increase in green fluorescence. The authors found mitochondrial membrane
depolarisation and apoptosis after activating the intrinsic mitochondrial pathway.
Effect of Menthone on Catalase and GPx activity
Cellular antioxidant enzymes, including Gpx, and CAT, play an essential role in the
defence system against oxidative stress. CAT catalyses the dismutation of two molecules of
H2O2 into two molecules of water and one molecule of oxygen. Regarding the catalase
activity, the menthone caused an augmentation in the enzyme activity compared to the group
2 cells (Table 2). An increase in GPX level was observed (0.32±0.02) in menthone
administrated group 3, which is less than the level of GL obtained after treatment with control
(0.21 ±0.01). The GPX level of 0.44±0.02 was observed in Rosiglitazone treated group 4
(Table 3). Wang et al., 2013 reported a dose-dependent reduction in levels of total GL and
reduced GL content on a rat synovial cell line. Baret et al., 2017 reported that GPx in Human
serum albumin-advanced glycation end products (HAS-AGEs) treated 3t3L-1 cells could
originate from the increase in PGC1α (peroxisome proliferator-activated receptor-gamma
coactivator 1-alpha) expression.

Conclusion
 The present study reports the antioxidative properties of menthone as evidenced by
biochemical assays. The menthone has significantly reduced the availability of mature 3T3L-
1 adipocytes in a concentration dependant manner. Moreover, the treatment of menthone
increased mitochondrial membrane potential in 3T3L-1 adipocytes, reduced ROS production,
and increased the catalase and GPx enzyme activity. The results show that the menthone
effectively reduced insulin resistance by enhancing GPx and catalase activity and ROS
reduction in mature 3T3L-1 adipocytes cells.

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Table 1. Cytotoxicity of Menthone on 3T3L-1 adipocytic cells. Values were expressed in

mean ± standard error.

 Tested sample
S. No OD Value at 570 nm Mean value (%)
concentration

1. Control 0.376±0.004 100

2. 100 μM/ml 0.0473±0.003 12.57

3. 90 μM/ml 0.135±0.010 35.97

4. 80 μM/ml 0.198±0.016 52.65

5. 70μM/ml 0.224±0.019 59.68

6. 60 μM/ml 0.265±0.024 70.41

7. 50 μM/ml 0.298±0.014 79.21

8. 40 μM/ml 0.302±0.010 80.45

9. 30 μM/ml 0.323±0.008 86.02

10. 20 μM/ml 0.349±0.006 92.82

11. 10 μM/ml 0.366±0.004 97.25

Table 2. Effect of Menthone on Catalase activity in adipocytic cells. Values were expressed

in mean ± standard error.

Tested sample Units of Catalase

OD Value at 240 nm
concentration (ml) Activity

BLANK 0.03±0.006 0.805±0.11

G1 0.11±0.003 2.52±0.069

G2 0.123±0.01 2.81±0.190

G3 0.130±0.008 2.99±0.1557

G4 0.164±0.01 3.76±0.225
Table 3. Effect of Menthone on GPx activity in adipocytic cells. Values were expressed in

mean ± standard error.

Tested sample Units of GPx Activity Mean value

S. No concentration
(ml) (in triplicates)

1. Blank 0 0 0 0

2. G1 0.199 0.221 0.238 0.219

3. G2 0.207 0.163 0.104 0.158

4. G3 0.295 0.324 0.352 0.324

5. G4 0.407 0.458 0.456 0.440

 Gro
Group 1 ( 5 mM/L glucose + 0.6 nm/L insulin)
up 2 (25 mM/L glucose + 0.6 nm/L insulin)

Gro Gro

up 3 (25 mM/L glucose + 0.6 nm/L insulin + 63.22 up 4 (25 mM/L glucose + 0.6 nm/L insulin + 0.1 µM

µM/ml of Menthone) Rosiglitazone)

Fig. 1. Oil Red O staining shows the effect of menthone on lipid accumulation in mature
preadipocyte 3T3-L1 cells.
Group 1 (5 mM/L glucose + 0.6 nm/L Group 2( 25 mM/L glucose + 0.6 nm/L

insulin) insulin)

Group 3 (25 mM/L glucose + 0.6 nm/L Group 4 (25 mM/L glucose + 0.6 nm/L

insulin + 63.22 µM/ml of Menthone) insulin + 0.1 µM Rosiglitazone

Fig. 2. DCFH-DA staining shows the effect of Menthone on ROS induction in mature
preadipocyte 3T3-L1 cells.
Group 1 (5 mM/L glucose + 0.6 nm/L Group 2( 25 mM/L glucose + 0.6 nm/L

insulin) insulin)

Group 3 (25 mM/L glucose + 0.6 nm/L Group 4 ( 25 mM/L glucose + 0.6 nm/L

insulin + 63.22 µM/ml of Menthone) insulin + 0.1 µM Rosiglitazone)

Fig. 2. JC-10 staining shows the effect of Menthone on MMP in mature preadipocyte
3T3-L1 cells.