BGM, a newly synthesized boron compound,

induces apoptosis and reduces oxidative stress by

inhibiting lipogenesis in 3T3-L1 adipocytes via
PPARγ and CTRP3
Meliha KOLDEMİR-GÜNDÜZ 
(

meliha.koldemirgunduz@ksbu.edu.tr
)
Kutahya Health Sciences University

Research Article

Keywords: 3T3-L1 adipocyte cells, boron glycine mono-ester, CTRP3, PPARy, apoptosis, oxidative stress

Posted Date: April 4th, 2022

DOI: https://doi.org/10.21203/rs.3.rs-1503179/v1

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Abstract
Obesity is a chronic disease associated with increased morbidity and mortality. The rapidly increasing
prevalence of obesity makes it a global health problem, while treatment options remain limited. Given the
potential of boron in the treatment of obesity, the aim of this study is to investigate the anti-adipogenic
activity of the newly synthesized boron glycine monoester compound (BGM) using 3T3-L1 adipocytes by
analysing lipid accumulation, CTRP3 and PPARy gene expression, oxidative stress and apoptotic effects.
3T3-L1 fibroblast cells (ATCC® CL − 173) were transformed into adipocyte cells in vitro. Fat accumulation
in the 3T3-L1 adipocyte cells was detected by oil red O staining. Gene expression levels were determined
with qPCR. Biochemical analyzes were performed using spectrophotometric method (CAT, ALP and ACP)
and ELISA kit (TAS, TOS, NADP-IDH). Apoptosis studies were performed on the Muse Cell Analyzer using
the Muse Annexin V & Dead Cell Assay Kit. When BGM-treated cells were compared to control adipocyte
cells, lipid accumulation decreased in a dose-dependent manner. BGM-treated adipocyte cells had higher
CTRP3 expression levels and lower PPAR-γ gene expression levels compared to control adipocyte cells (p 
< 0.001). While BGM application increased the TAS level, it showed an antioxidant effect by regulating the
activity of oxidative metabolism enzymes (p < 0.001). BGM application increased total apoptosis by 1.5
fold. These results show that BGM is a potential therapeutic agent for obesity by regulating the
expression of genes related to adipogenesis and lipogenesis in adipocyte cells and by affecting the
activity of enzymes of oxidative metabolism and apoptosis.

Introduction
Obesity is a chronic condition in which the quantity of energy consumed by the body exceeds the amount
utilised by the body [1]. Obesity is now recognised as a condition that affects all organs and systems of
the body, particularly the cardiovascular and endocrine systems, and causes a variety of diseases and
even morbidity [2]. According to the World Health Organization, 1.6 billion people are now overweight, and
more than 2.8 million people die each tear as a result of being overweight or obese [3]. Obesity has
evolved into a public health issue, and combating it has become a global concern [4, 5].

As a primary endocrine organ, adipose tissue secretes a number of biologically active chemicals that
circulate in the bloodstream, and are known as adipocytokines. These play a function in
pathophysiological processes as well as regulating metabolism [6]. Adipogenesis is a cell differentiation
process required for adipogenesis and energy homeostasis in the body. Uncontrolled hyperplasia and/or
adipocyte hypertrophy are also thought to play a role in the development of obesity [7]. CCAAT enhancer
binding proteins (C/EBPa) and peroxisome proliferator-activated receptor gamma (PPARγ) are two
transcription factors that control this. In addition, PPARγ is necessary to keep differentiation and fat
deposition mechanisms going [8]. PPARγ regulate gene expression of adipocyte differentiation,
lipogenesis and glucose metabolism and play an important role in obesity. C1q tumour necrosis factor
associated protein 3 (CTRP3) is a C1q tumour necrosis factor-related protein that is a paralogue of
adiponectin. CTRP3 influence glucose, and lipid metabolism, inflammation, proliferation, apoptosis,

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fibrosis, ischemia damage, and vascular calcification metabolism in multiple ways [6]. In adult
adipocytes, PPARγ can bind to the CTRP3 promoter and decrease its transcriptional activity [9].

Boron is a rare element that has biological functions in living organisms [10]. Boron is essential for the
growth of plants, animals, and people [11, 12]. It occurs in the environment in the form of boric acid and
borate salt. The utilisation of numerous compounds associated with life activities at the physiological
level is influenced by metabolism or energy substrates such as boron, glucose, and triglycerides,
nitrogenous substances such as proteins and amino acids, and reactive oxygen derivatives [13]. Boron
influences carbohydrate metabolism. Boron in physiological amounts in the diet has been shown to
increase plasma glucose concentrations [14] and affects insulin metabolism [14]. Boron has been
demonstrated to lower the amount of insulin needed to keep blood glucose levels stable. [15]. Recent
boron research has focused on the study of anticancer activity, oxidative stress, protein biomolecular
interactions, and drug delivery through newly synthesised boron compounds [10, 16]. In animal
experiments, ingested tube is hydrolyzed to boric acid in the small intestine, with the majority of it
eliminated in urine before being digested [10]. Boron is often found in dietary supplements that combine
various nutrients (calcium, magnesium, chondroitin sulphate, glucosamine, curcumin, gelatine, etc.).
However, as recognised types of boron supplementation (boric acid, borax, etc.) tend to accumulate in the
tissues rather than in the bone marrow, they have been incresingly questioned. As a result, as a more
advanced type of boron, boron esters were created. In the literature, little is known about boron esters.
Latest research has focused on addressing this deficit [17–19].

One of the first boron ester compounds to be isolated and structurally characterized from natural plant
sources was calcium fructoborate (Ca-FB) [20]. Efflux across the membranes via passive and energy-
dependent active transport supported by the channel facilitates the transfer of boron (B) into cells in plant
metabolism [21]. A specific boron transporter has not been fully discovered in animal metabolism yet,
although such a transporter has previously been suspected. Previous research has showed that the
putative boron transporter NaBC1 pumps boron [22] and actually transport NH3 and not B [23]. According
to the literature, the transport mechanisms of true boric acid (BA) and boron ester compounds (BEC) in
human and animal cells are not fully understood yet [24]. Since active sugar carriers have a lower
permeability than boron compounds that do not have chelate ester bond, fructoborates and glucoborates,
which are plant BEC complexes, can be transported with them [25]. Compared to BA, the BGM
synthesised by our study group are transported to cells and can be hydrolyzed in aqueous media. Rapid
hydrolysis accelerates the formation of the anion tetrahydroxy borate, resulting a process similar to the
degradation product of boric acid. Therefore, hydrolysis might be considered a crucial factor in the
activation of boron ester compounds [17].

Modelling obesity in vitro, 3T3-L1 adipocytes represent a successful system for investigating adipocytic
lipid metabolism and adiposity. In this study, the cytotoxic effects of BGM [10], synthesised as a novel
boron ester compound, on 3T3-L1 adipocytes were investigated. In addition, analysis of lipid
accumulation, CTRP3 and PPARγ gene expression, oxidative stress and apoptotic effects were
investigated in order to determine the effects of BGM on fat metabolism.
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Material And Method
Synthesis of Boron-Glycine mono Ester 

Glycine (Sigma- Aldrich) was dissolved in a mixture of distilled water and methanol (Sigma-Aldrich). The
solution was stirred for about 1 hour at room temperature on the magnetic stirrer with solid H3BO3
(SigmaAldrich). After some of the solvent was evaporated in the evaporator, the solution was allowed to
crystallise at room temperature until the density of the solution was thick. In a vacuum oven at 50°C, the
finished product was dried. It was then characterised by means of FT-IR spectroscopy, elemental analysis,
thermal analysis, and melting point measurement [10]. Scheme 1 shows the reaction strategies for the
BGM complex compounds prepared.

3T3-L1 Cell Culture

The American Type Culture Collection (ATCC) provided the 3T3L-1 fibroblast cell line used in this research
(Manassas, USA). Cells were cultured in a humidified incubator (37 °C and 5% CO2), in medium
containing DMEM (Dulbecco's modified Eagle's medium) (ATCC, USA) medium + 10% Fetal Bovine Serum
(FBS) (ATCC, USA) + penicillin (100 units / ml) + streptomycin (100 g / ml) (Gibco, USA).

Adipocyte Differentiation from 3T3-L1 Fibroblasts

Differentiation of 3T3-L1 fibroblast cells Miard et al. was performed according to the protocol [26].
Differentiation in the cells was determined morphologically according to Lillie and Ashburn's method
using the oil red o staining method [27].

Cytotoxicity Assay 

The BGM solution was mechanically prepared in the medium. 3T3-L1 adipocytes recieved BGM 250 μM,
500 μM, 1, 5, 10, and 50 mM, and the cells were treated for 48 hours. To the control cells, just culture
media was introducedThe method of Yerlikaya et al. was used to do MTT analysis [28]. The data was
processed and a graph was created using the GraphPad Prism 5.0 program (GraphPad Software, Inc., La
Jolla, CA, USA). Data were standardized using nonlinear regression analysis using GraphPad Prism 5.0
tool to calculate IC50 value. Viability rates of BGM treated cells were compared to cells that had not been
treated. The untreated cells were assumed to be 100% viable, and the percentage viability of the cells was
calculate as follows.

% viability: (Treated cell / untreated cell) X100

Isolation of RNA and q PCR Analysis 

The RNeasy Protect Mini Kit (Qiagen, Germany) was used to isolate total RNA from 3T3-L1 adipoctye
cells according to the manufacturer's recommendations. Total RNA samples were kept at -80°C until they
were needed. For cDNA synthesis, the Transcriptor HiFi cDNA Synthesis Kit was used (Roche). The RT-

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qPCR reaction mixture (BlasTaq 2X qPCR MasterMix (G890, Applied Biological Materials Inc) was
prepared for RT-qPCR. Expression of CTRP3 and PPAR γ gene was determined by qPCR using a ABI
StepOnePlus (Applied Biosystems, Germany) instrument. Measurements were performed at least three
times for each sample analysed. Ct values of 40 were not considered in mathematical calculations.
Expression levels were determined based on the expression of the reference gene (GAPDH). The 2−ΔΔCt 
technique was used to confirm the presence of specific gene products. The primer sequences were:
CTRP3 (forward) 5′-GCT GTT TCC CAG AGG ACT GAC T-3′, (reverse) 5′- AGG GCA AGA TGG CTG TAA
GGT-3′; PPAR γ (forward) 5′-CCC ACC TTC GGA ATC AG-3′ and (reverse) 5′-AAT GCT GAA ATC AAC TGT
GGT A-3′; GAPDH (forward) 5′-AGG GCA AGA TGG CTG TAA GGT-3′ and (reverse) 5′-GGG ACC AAC TTC
GGA ATC AG-3′.

Measurement of Biochemical Analysis in 3T3-L1 Adipocytes

Cells were trypsinised and homogenized with homogenisation buffer after BGM was applied to them. The
work was performed without interrupting the cold chain. After homogenisation, after centrifugation at
10,000 rpm and 4°C for 20 minutes, the pellet was discarded and the supernatant was used for the
experiments.

The total protein concentration was determined by measuring the absorbance of colored solutions
resulting from the binding of the dye Coomassie Brilliant Blue (G-250) to the proteins at 595 nm [29]. The
Aebi [30] technique was used to assess catalase activity (CAT). The determination of the enzyme activity
of Acid phosphatase (ACP) and Alkaline phosphatase (ALP) was evaluated spectrophotometrically
according to Walter method [31].

Total antioxidant status (TAS), total oxidant status (TOS), and NADP+ dependent isocitrate
dehydrogenase enzyme (NADP-IDH) levels were determined using commercial enzyme linked
immunosorbent assay (ELISA) kits (Rel Assay Diagnostics TAS Kit, Turkey; Rel Assay Diagnostics TOS
Kit, Turkey) containing well plates coated with immobilized antibodies specific for the target protein. The
following formula was used to construct the Oxidative Stress Index (OSI) [32]:

Muse® Annexin V & Dead Cell Analysis 

Apoptosis in 3T3-L1 adipocyte cells was determined using the Muse® Annexin V & Dead Cell Kit (Cat. No:
MCH100105, Luminex Corporation). With 250.000 cells per petri dish, all cells were seeded in 60 x 15 mm
petri plates. The experiment consisted of two replicate. Drug treatment was started 24 hours after
seeding. The cells were treated with 500 μM, 2 mM and 8 mM BGM for 8 hours. After drug treatment, cells
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were trypsinised and the commercial kit protocol was applied. Subsequently, all samples were read using
the Muse Cell Analyzer device [33].

Statistical analysis 

Cell viability GraphPad Prism 5 was used to perform the MTT analysis (GraphPad Software, Inc.). Results
are reported as mean + standard deviation. SPSS software was used for statistical analysis (version 23.0;
SPSS Inc., Chicago, IL). When two groups are compared, parametric comparisons are utilized. For
unequal variances, Student's t-test and non-parametric Mann-Whitney U test were utilized, and one-way
ANOVA was used to compare more than two groups; for statistically significant results, Tukey's post hoc
test was performed after ANOVA to determine if there was a significant difference between groups.
Correlation between gene expression levels was determined using Spearman's test. A p-value of less than
0.05 was considered significant.

Results
Cytotoxic Effects of BGM on 3T3-L1 Adipocytes

Adipocyte cells were cultured in regular culture media for 24 hours before being treatment with a boron
compound. 50 mM, 10 mM, 5 mM, 1 mM, 500 μM and 250 μM BGM and glycine as control were added to
the cell culture medium and incubated for 48 hours. The control cells received only the culture media. The
MTT test was accomplished on the cells after the administration of boron chemicals. The GraphPad
Prism 5.0 tool was used for statistical analysis of the MTT test findings. After treating adipocyte cells
with 50 mM, 10 mM, 5 mM, 1 mM, 500 μM, and 250 μM BGM for 48 hours, the IC50 value of BGM was
determined to be 2 mM based on the statistical analysis. This finding indicates that boron compounds
are cytotoxic to adipocyte cells.

To determine the percentage survival rates of 3T3-L1 adipocyte cells after application of different
amounts of compound, 50 mM, 10 mM, 5 mM, 1 mM, 500 μM, and 250 μM BGM were applied to cells
cultured in 96 microplates until they reached logarithmic phase for 48 hours. MTT cytotoxicity testing
was then performed (Figure 1). As a result of applying 50 mM, 10 mM, 5 mM, 1 mM, 500 μM and 250 μM
boron-glycine mono ester, cell viability was determined to be 25%, 33%, 38%, 72%, 77% and 83%,
respectively, when compared to the control. It was determined that 50mM, 10 mM and 5mM BGM had a
lethal effect on 3T3-L1 adipocyte cells. Application of 1 mM and 500 μM BGM was found to have an anti-
proliferative effect on adipocytes. The application of 250 µM BGM had no anti- proliferative effect on
3T3-L1 adipocytes (Figure 1).

Three distinct BGM concentrations (500 µM, 2 mM, 8 mM) were determined based on the MTT results
and used in other experimental procedures.

Effect of BGM Application on Oil Droplet Accumulation

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To visualise the oil droplets after the development of mature adipocyte, oil red-o staining was used.
Different amounts of BGM (0.5, 2 and 8 mM) were administered to the mature adipocytes. A large
amount of oil droplets was observed in the control cells. Depending on the BGM dose, lipid accumulation
in the adipocytes was significantly reduced (Figure 2). These results show that BGM reduces the lipid
content in adipocytes. This shows that BGM can be effective in the treatment of obesity.

Differences in CTRP3 and PPARγ Gene Expression Levels

CTRP3 and PPARγ gene expression levels were analysed after administration of 0.5 mM, 2 mM and 5
mM BGM to adipocyte cells. When 3T3-L1 adipocytes treated with 0.5 mM BGM for 48 hours were
compared with control adipocytes, it was found that CTRP3 and PPARγ gene expression levels decreased
0.41 and 0.47 fold, respectively (p<0.001) (Figure 3.). When comparing 3T3-L1 adipocytes treated with 2
mM BGM for 48 hours and control adipocytes, CTRP3 and PPARγ gene expression levels were found be
increase by 2.87 fold and decrease by 0.61 fold, respectively (p<0.001) (Figure 3). When comparing 3T3-
L1 adipocytes treated with 8 mM BGM for 48 hours and control adipocytes, CTRP3 and PPARγ gene
expression levels were found to be increased 5.94 fold and decreased 0.38 fold, respectively (p<0.001)
(Figure 3).

Correlation of CTRP3 and PPARγ Gene Expressions in BGM-treated 3T3-L1 Adipocytes

A positive correlation was observed between CTRP3 and PPARγ gene expression levels in 3T3-L1
adipocytes after 48 h administiration of 500 μM BGM (rs= 1.00, p=0.01) (Table I). 

Table 1. Correlation of expression levels of CTRP3 and PPARγ genes in response to BGM administration
to 3T3-L1 adipocytes. 

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   CTRP3 Gene Expression PPARγ Gene Expression

  rs p rs p

0.5 mM BGM        

   CTRP3 Gene Expression 1.00 - 1.00 0.01*

   PPARγ Gene Expression 1.00 0.01* 1.00 -

         

2 mM BGM        

   CTRP3 Gene Expression 1.00 - 0.300 0.624

   PPARγ Gene Expression 0.300 0.624 1.00 -

         

8 mM BGM        

   CTRP3 Gene Expression 1.00 - 0.600 0.285

   PPARγ Gene Expression 0.600 0.285 1.00 -

The Spearman correlation coefficient (rs) and statistical significance (p) were used to express the results.
*p=0.01 

Biochemical Analysis in 3T3-L1 Adipocytes 

Effects of BGM on TAS, TOS and OSI in 3T3-L1 Adipocytes

TAS, TOS and OSI analyses were performed to determine the effects of the newly synthesised boron
compound BGM on oxidative stress parameters in adipocyte cells. 

TAS reflects the effect of all antioxidants in body fluids and plasma, while TOS reflects the total effect of
all oxidants. When the statistical significanse between the values of total antioxidant capacity measured
in the study were evaluated; it was found that the increase (p˂0.001) was statistically signifcant in the
cells treated with 2 mM BGM compared to the control group (Fig. 4). When the statistical significance
between the TOS values are evaluated, an increase was found in the cells treated with 2 mM BGM
compared to the control group, p˂0.001) (Fig. 4). OSI is expressed as the percentage ratio of TOS
capacity to TAS capacity. When comparing the OSI value with the adipocyte cells of the control group and
the cells treated with 2 mM BGM, it was found that OSI was lower in the BGM-treated group (p<0.001)
(Figure 4).

Activity of Oxidative Metabolism Enzymes

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The effects of BGM on enzymatic parameters in adipocyte cells were studied using the enzyme activities
CAT, ACP, ALP, and NADP-IDH. The enzyme CAT is an intracellular enzyme involved in the antioxidant
defense mechanism. The activity of enzyme CAT was increased in adipocytes treated with an IC50 dose
of BGM compared to control (p<0.001) (Fig. 5). The enzymes ACP and ALP abundant in nature. This
indicates that these enzymes are important in biochemical reactions. ACP enzyme activity was increased
when 2mM BGM was applied compared to the control (p<0.05) (Fig. 5). The Boron-glycine monoester
group, ALP enzyme activity was statistically significantly increased (p<0.05) (Figure 5). NADP-IDH
enzymes are dependent on oxygen-dependent energy metabolism in mitochondria. The activity of NADP-
IDH was lower (p=0.014), 610.7 (U/L) and, 662.5 (U/L) in the adipocytes treated with 2 mM BGM
compared with the adipocytes in the control group (Figure 5).

BGM promotes apoptosis in 3T3-L1 adipocytes

Flow cytometry was used to determine the apoptotic potential of BGM, allowing the identification of
viable, early apoptotic, late apoptotic, and necrotic cells. To demonstrate the apoptotic effect, cells were
treated with an IC50 concentration. When cells treated with 2mM BGM for 8 hours were compared with
control cells in Figure 6, there was no difference in the percentage of viable cells. However, the application
of BGM increased total apoptosis by 1.5 fold. While early apoptosis was 3.89% with 8-hours of BGM
application, 1.64% was observed in control cells, and early apoptosis increased 2.4 fold compared to
control. While total apoptosis was 7.25% with 2 mM BGM application, it was detected at 4.65% in control
cells. These findings imply that BGM can cause adipocyte cells to apoptosis.

Discussion
The diversity of factors that cause obesity complicates the development of drugs to treat obesity. As a
result, new low-toxicity medications should be created. Boron is a promising substance for treating
obesity, as ashown by the studies on the relationship between boron and obesity [11, 34, 35]. Therefore,
the newly synthesised boron compound BGM was investigated for the development of anti-obesity drugs.

Abdik et al. [34] in their study investigating the effect of boron on adipogenic differentiation and fat
storage, reported that 34, 68, 170, 340, and 510 µM sodium pentaborate pentahydrate (NaB)
administirated from human adipose-derived stem cells (hADSCs) had statistically no toxic effect on cell
viability statistically. In this study, the IC50 value after 48th hours was calculated as 2 mM, according to
the statistical analysis performed as a result of applying BGM doses (50 mM, 10 mM, 5 mM, 1 mM, 500
µM and 250 µM) to 3T3-L1 adipocytes.

To determine the effects of 500 µM, 2 mM and 8 mM BGM concentrations on adipogenesis in adipocyte
cells, Oil Red O staining, CTRP3 and PPARγ mRNA expression levels in cells confirmed adipogenesis and
fat accumulation. In a study investigating the inhibition of adipogenesis, the results of Oil Red O staining
showed a dose-dependent decrease in the size of the staining on adipocytes when NaB was applied [34].

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Similarly, the results of this study, showed that lipid accumulation in adipocytes decreased with
increasing dose of BGM administration. This result shows that BGM reduces fat accumulation.

The expression of CTRP3 and PPARγ genes associated with adipogenesis was investigated in 3T3-L1
adipocytes to evaluated the therapeutic effect of BGM. The effects of NaB on adipogenesis were
investigated in a study, the mRNA level of PPARγ was found to be lower [34]. According to a research
paper that looked into the impacts of boron on the inhibition of adipogenesis, the application of boron
decreased PPARγ gene expression [35]. Similar to the studies in the literature, PPARγ gene expression
level was statistically significantly decreased by the application of BGM compared to the control.
According to this result, BGM can inhibit adipogenesis. CTRP3 gene expression level is lower in obese
individuals compared to lean individuals. In their study, Li et al. [36] investigated the relationship between
CTRP3 gene expression level and adipose tissue inflammation and insulin sensitivity in obese mice and,
they found that CTRP3 gene expression level was lower in obese mice than in healthy control mice. Li et
al. reported that obesity impairs glucose metabolism and insulin sensitivity by reducing CTRP3 [36]. In
this study, CTRP3 gene expression levels increased statistically significantly with BGM application
compared to control. An increased CTRP3 mRNA level could indicate an inhibition of lipogenesis.

One of the factors that cause obesity is oxidative stress. ROS that increase in obesity cause cell damage,
necrosis and apoptosis as a result of oxidation of DNA, proteins and lipids. Increased oxidative stress in
stored fat has been shown in studies to be the a fundamental cause of adipocytokine dysregulation and
the development of the metabolic syndrome [37, 38]. TOS is used to measure the general oxidative status
of the body, TAS to measure the general antioxidant status of the body. OSI is the ratio of TOS to TAS,
which is considered a more accurate index of oxidative stress. In studies conducted in the literature, the
levels of TOS and OSI were found to be high in obese individuals and the levels of TAS were found to be
low [39, 40]. In agreement with the literature, the TAS level was found to be higher in BGM-treated
adipocyte cells than in control adipocyte cells, while the OSI level was low. However, in contrast to the
literature, the TOS level was higher in the BGM group. However, as the antioxidant defence mechanism is
complicated, this issue needs further research and verification. In general, however, the results show that
BGM has an oxidative stress inhibitory effect on adipocyte cells.

CAT is an intracellular enzyme involved in antioxidant defence mechanism [41]. It uses H2O2 formed in
the cells as a substrate and provides H2O2 detoxification by splitting H2O2 into oxygen and water.
Catalese activity has been found to decrease with excessive O2 production [42]. In our study, it was found
that catalase activity increased when BGM was applied to adipocyte cells compared to the control. Our
study showed that the BGM usage activated antioxidant defence systems to reduce oxidative stress in
adipocyte cells. NADP-IDH is involved in the regulation of lipid biosynthesis pathways as well as
protection against oxidative stress [43]. NADP-IDH activity was investigated to determine how BGM-
induced adipocyte cells are affected in lipogenesis and maintenance of redox state. The high activity
measured for NADP-IDH in adipocytes may be due to the cytosolic isoform providing reducing
equivalents for lipid synthesis [44]. Brazhnikova et al. reported in their study investigating the effects of
enzyme of oxidative metabolism on the liver that the activity of the enzyme, NADP-IDH decreased, thus
Page 10/20
reducing oxidative stress [45]. The low NADP-IDH activity in BGM-treated adipocyte cells could indicate a
low need for glutathione regeneration and thus an antioxidative defence.

In their study investigating the effects of boron on the inhibition of adipogenesis, Doğan et al. found that
boron treatment did not induce apoptosis [35]. When the 2 mM BGM application was compared to
apoptosis levels in control adipocyte cells, total apoptosis increased in the BGM-applied group. These
findings imply that BGM can cause adipocytes to apoptosis.

Conclusion
BGM has a suppressive effect on fat accumulation, especially in 3T3-L1 adipocytes. The application of
BGM to adipocyte cells showed a regulatory effect on the function of the antioxidant system and the
activity of the enzymes of oxidative metabolism. Inhibition of BGM during adipogenesis could be useful
in the treatment of obesity. According to the findings of this investigation, other structural modifications
of new boron derivatives that can be used in the treatment of obesity may form promising antiobesity
agents. However, further research is needed to identify how BGM affects metabolism.

Declarations
Funding: This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.

Conflicts of interest/Competing interests: The authors declare that they have no conflict of interest

Ethics approval: Not applicable

Availability of data and material: Not applicable

Code availability: Not applicable

Authors' contributions: Meliha Koldemir-Gündüz: Conceptualization; Data curation; Formal analysis;

Investigation; Methodology; Resources; Supervision; Visualization; Writing - original draft; Writing - review
& editing.

Acknowledgements

I would like to thank Prof. Dr. Dursun Ali KÖSE and the National Boron Institute (BOREN, 2020-30-06-30-
002) for the synthesis of boron glycine mono ester used in the study.

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Scheme
Scheme 1 is available in Supplementary Files section.

Figures

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Figure 1

Different concentrations of BGM have cytotoxic effects in adipocytes. The MTT test was performed after
3T3-L1 adipocytes were treated 50 mM, 10 mM, 5 mM, 1 mM, 500 μM, and 250 μM BGM for 48 hours.

Figure 2

BGM's effects on 3T3-L1 adipocytes. Adipocytes treated with 0.5 mM, 2 mM, and 8 mM BGM were
stained with Oil Red O.

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Figure 3

Relative mRNA expression levels of CTRP3 and PPARγ genes in BGM treated groups and control cells.
Gene expression levels were determined in fully differentiated cells by qPCR and normalised to GAPDH
mRNA levels. 

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Figure 4

Results from TAS, TOS and OSI in 3T3-L1 adipocyte cells treated with IC50 of 48 hours BGM (2 mM).

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Figure 5

IC50 values of BGM (2 mM) for 48 hours were applied in 3T3-L1 adipocyte cells; CAT, ACP enzyme activity,
ALP enzyme activity and NADP-IDH results.

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Figure 6

Apoptotic effect of BGM on adipocytes.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.

scheme1.png

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