Reversal of dexamethasone induced insulin resistance by Apocynin in L6 myotubes and 3T3L1 adipocytes – a mechanistic approach

doi:10.21203/rs.3.rs-3811238/v1

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Reversal of dexamethasone induced insulin resistance by Apocynin in L6 myotubes and 3T3L1 adipocytes – a mechanistic approach

https://doi.org/10.21203/rs.3.rs-3811238/v1

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Objective The current study aims to decipher the putative interaction between skeletal muscle and adipose tissue in response to apocynin (APO) mediated glucose uptake in Dexamethasone (DXM) induced insulin resistance.

Methods The gene expression of the molecular markers pertaining to insulin signaling pathway including IRTK, IRS-1, PI3K and GLUT-4 along with adipogenic markers like PPARα and adiponectin were studied. The results from the study aided in understanding the molecular effects of apocynin favoring the investigation of the crosstalk among the 3T3-L1 adipocytes and L6 myotubes.

Results After confirming 100 nM DXM was ideal for inducing insulin resistance without much alteration in cell viability. This is taken further for inducing insulin resistance in both the chosen cell lines. The gene expression of IRTK and IRS-1 was found to be increased in APO treated, DXM+APO treated cells and was found to be equivalent to that expressed in 100 nM insulin treated cells. In DXM+APO treated L6 myotubes and 3T3-L1 adipocytes, IRTK showed a 2- fold and 3.7-fold increase compared to control respectively. Whereas IRS-1 showed 2.3-fold and 3.5-fold increase in L6 myotubes and 3T3-L1 adipocytes respectively. In addition to this PI3K exhibited a 2.6-fold increase in L6 myotubes and 3.3-fold change in adipocytes. The maximum fold increase of 7.3 was exhibited by GLUT-4 in adipocytes while it showed a 2.8-fold increase in myotubes. The impact of apocynin on adipogenic markers were also analyzed and it was found that DXM+APO treated cells show 3.6-fold and 3.4-fold decrease in the expression of adiponectin and PPAR-α respectively in 3T3-L1 adipocytes.

Conclusion The results substantiate that APO (1 μM) has both anti-diabetic effect in L6 myotubes along with anti-adipogenic properties in 3T3-L1 adipocytes.

Apocynin

Dexamethasone

Insulin Resistance

type 2 Diabetes Mellitus

Type 2 Diabetes mellitus (T2DM) is the result of malfunctioning in insulin signaling cascade. It is characterized by carbohydrate, protein, lipid metabolism dysfunction and is a result of decreased insulin production and insulin resistance or combined action of both [1]. Insulin, a growth hormone secreted by the beta cell of the pancreas, regulates glucose levels in circulating blood. Insulin influences several metabolic pathways through insulin signaling, especially in muscle and adipocytes. It also regulates the metabolism of glucose in insulin sensitive tissues [2].

Insulin binds to the insulin receptor in healthy cells [3]. This binding event triggers autophosphorylation of the insulin receptor, leading to the activation of its intrinsic tyrosine kinase activity. Subsequently, the activated insulin receptor phosphorylates specific tyrosine residues on IRS (Insulin Receptor Substrate) proteins. These phosphorylated IRS proteins serve as docking sites for PI3K (Phosphoinositide 3-kinase), recruiting and activating it. The activated PI3K enzyme converts phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-trisphosphate (PIP3) within the cell membrane. PIP3 then plays a crucial role in recruiting AKT to the cell membrane, where AKT is phosphorylated and activated by PDK1 (3-phosphoinositide-dependent protein kinase-1) and mTORC2 (mammalian target of rapamycin complex 2). Activated AKT, in turn, phosphorylates various downstream substrates, including AS160 (Akt Substrate of 160 kDa), which acts as a key regulator of GLUT4 translocation. Through phosphorylation of AS160, GLUT4 vesicles are facilitated to translocate from intracellular compartments to the cell membrane, allowing GLUT4 transporters on the cell surface to enable facilitated diffusion of glucose into the targeted cells [4, 5]. Aberrations in the cascades, on the other hand, result in insulin resistance and related metabolic complications. The onset of insulin resistance leads to the failure of glucose transporters located in different cells to translocate to the plasma membrane, resulting in its loss of the ability to recruit glucose from the bloodstream. This leads to an increase in blood glucose levels which aids in developing diabetic complications [4].

Skeletal muscles exhibit the primary defects in T2DM [1]; it has been reported that skeletal muscle is a prominent site for glucose disposal via insulin. Due to decreased phosphorylation of IRT and IRS-1 tyrosine followed by a reduction in PI3K activation, and impaired GLUT-4 translocation, there is a decrease in insulin-stimulated glucose uptake [5. 6, 7, 8]. Phosphorylation of IRS proteins at serine residues by enzymes like JNK (c-Jun N-terminal kinase) and IKK (IκB kinase) disrupts the normal IRS-PI3K-AKT signalling pathway, resulting in decreased AKT activation and hindered GLUT4 translocation. Elevated lipid levels, particularly diacylglycerols (DAG) and ceramides, in muscle cells, can trigger the activation of protein kinase C (PKC) isoforms. These activated PKC isoforms promote the serine phosphorylation of IRS proteins, thus playing a role in the development of insulin resistance. Furthermore, the presence of pro-inflammatory cytokines such as TNF-α (tumour necrosis factor-alpha) and IL-6 (interleukin-6) can stimulate kinases like JNK and IKK, leading to serine phosphorylation of IRS proteins and contribute to insulin resistance. Additionally, impaired mitochondrial function in muscle cells may lead to an increased production of reactive oxygen species (ROS), which in turn can activate stress kinases like JNK and IKK, further contributing to insulin resistance [6, 7, 8].

Insulin resistance in adipocytes is caused by impaired signal transduction in insulin cascade protein, which altered targeted metabolic outcomes such as glucose transport stimulation and lipolysis inhibition [9].

The diabetic population is increasing day by day and the International Diabetes Federation (IDF) has projected that there will be a 2-fold increase in the diabetic population reaching up to 643 million by 2030. In addition to the growing number of diabetics, and the side effects of certain existing anti-diabetic drugs and drug combinations, there is a need for anti-diabetic drugs with minimal to no side effects on both long and short-term consumption. All of this necessitates the development of purely natural or partially natural drugs with minimal side effects and maximum efficacy [10].

Apocynin is an immunomodulatory constituent isolated from the roots of Picrorhiza kurroa, a native and common Ayurvedic medicinal plant found in India, Nepal, Tibet and Pakistan. The antioxidant, anti-inflammatory, and anti-cancer effects of this semi-synthetic drug have also been investigated. Apocynin has anti-diabetic and anti-adipogenic activity in 3T3-L1 adipocytes, according to previous research [11]. The current study looks into the anti-diabetic effects of apocynin on dexamethasone-induced insulin-resistant L6 myotubes and 3T3-L1 adipocytes, as well as the metabolic signaling components involved in one of the potential crosstalk between skeletal muscle and adipose tissue.

Chemicals And Reagents

3T3-L1 preadipocytes and L6 myoblasts were provided by King’s Institute of Preventive Medicine, Chennai and NCCS (National Center for Cell Science), Pune respectively. Dulbecco Modified Eagle Medium (DMEM) with 4.5 g glucose, 4.0 mM L-glutamine and sodium pyruvate were purchased from Himedia Laboratories (India). Fetal Bovine Serum (FBS), 3-(4,5-Dimethylthiazol-2-yl-)- 2,5-Diphenyltetrazolium bromide (MTT), Dexamethasone (DXM), Human Recombinant Insulin, 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-,3-diazol-4-yl)Amino)-2- Deoxyglucose) were acquired from Himedia Laboratories (Chennai, India). 3-Isobutyl-1-Methylxanthine (IBMX) was obtained from Merck India Biosciences (Mumbai, India). Apocynin (APO) was purchased from Sigma Aldrich, India.

Culturing of 3T3-L1 Preadipocytes and L6 Myoblasts

3T3-L1 preadipocytes were cultured in vitro in DMEM (Dulbecco’s Modified Eagle Medium) along with 10% FBS and 1X antibiotic solution that contains penicillin and streptomycin in 5% CO₂ incubator at 37°C [11]. The cells were subjected to passaging when they attained 70% confluency and then taken further for differentiation. In a similar manner, L6 myoblasts were subjected to high glucose - DMEM in addition to 10% FBS, 1X antibiotic-antimycotic solution comprising 10,000 U penicillin, 10 mg streptomycin, and 25 g amphotericin B in saline solution (0.9%) [12]. The 3T3-L1 preadipocytes and L6 myoblasts were maintained in a 5% CO₂ incubator.

Differentiation of 3T3-L1 Preadipocytes and L6 Myoblasts

Differentiation of cells is essential to attain the cell-specific phenotypic characteristics which in turn lead to the production of cell-specific proteins thus characterizing the function of the cell. Adipogenesis is a series of events that occurs when pre-adipocytes (fibroblasts) differentiate into mature adipocytes, which are characterized by lipid drop accumulation visible under an inverted microscope [13]. On the other hand, differentiation of myoblasts to myotubes is characterized by a fusion of cells along with multinucleation visualized under an inverted microscope.

To differentiate the preadipocytes, cells were subjected to 3 different cocktail media - for 3 days namely differentiation medium (DMEM + 10% FBS + 0.5 mM IBMX + 0.25 µM Dexamethasone + 1 mg/L insulin) after which the cells are treated with maintenance media containing DMEM, 10% FBS and 1 mg/L insulin. These media play a role in arresting the growth of the preadipocytes and promoting differentiation. It also allows the transcription factors to access this regulatory response. These steps together lead to the modulation of phenotypes leading to the formation of mature adipocytes. Finally, after 6 days, the cells were cultured with an expansion medium (DMEM + 10% FBS) that aids in differentiated adipocytes.

Confluent myoblasts were then treated with DMEM growth media with 2% FBS for 2 days to aid in differentiation. The formation of myotubes is confirmed by observing the fusion of cells and multinucleation under a phase-contrast microscope. The differentiated cells were maintained in DMEM (high glucose) for further in vitro assessments [14].

Assessment of Cell Viability using MTT assay

The undifferentiated cells were taken for assessment of cell viability upon treatment with dexamethasone (DXM), apocynin (APO) and both DXM + APO (D + A). This step was essential to finalize the optimum concentration of DXM and APO. A 96-well plate with 0.5 x 10⁵ cells per well was seeded, and the cells were then cultured to the subconfluent stage in a 5% CO2 incubator. After this, the cells were subjected to the glucocorticoid (DXM) for 24 h followed by treatment with APO and incubated for 24 h. After a total of 48 hours, 10 µl of MTT (6 mg/ml) was added to the wells followed by 4 hours of incubation with 100 µl of DMSO to dissolve the formazan crystals. DMSO (0.01%) treated cells were used as the solvent control and Triton X-100 (0.01%) served as the positive control. The absorbance was measured using a multi-mode ELISA reader (spectrophotometer) at 570 nm [11, 15].

Glucose Uptake Assessment using 2-NBDG assay

Differentiated cells were co-incubated with drugs (DXM and APO) for 24 h each. The media was completely aspirated and freshly prepared KRPH (Krebs Ringer Phosphate HEPES) buffer (NaCl 118 mM, KCl 5 mM, CaCl₂ 1.2 mM, MgSO4 1.2 mM, KH₂PO₄ 1.2 mM, HEPES 30 mM, and pH 7.2) was added to all wells [11]. Following this, all wells were treated with 100 µM of 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-,3-diazol-4-yl) Amino)-2- Deoxyglucose) in dark. After incubating it for 30 mins, the supernatant was aspirated and SDS was used to get cell lysate. At wavelengths of 485 nm for the excitation and 538 nm for the emission, the fluorescence intensity was measured [16].

Glucose Uptake Activity in the Presence of Inhibitors

Additional research on glucose uptake was done using the inhibitors such as wortmannin, an inhibitor of PI3K and genistein, a IRTK inhibitor respectively. The differentiated myotubes were serum starved for two hours, followed by treatment with DXM and incubation for 24 hours. After 24 h, the cells were introduced to inhibitors, 100 nM of wortmannin, and 50 nM of genistein for 30 mins each [17]. Later, the cells were administered with an optimum concentration of APO and incubated for 24 h in a 5% CO₂ incubator. The effect of APO on glucose uptake in presence of inhibitors was assessed.

Isolation of RNA

The differentiated cells in a 6-well plate were treated with DXM (100 nM) and APO (1 µM) for 24 h each and were incubated in a 5% CO₂ incubator. Following incubation, the cells were subjected to TRIzol for homogenization. Chloroform was added, and after achieving phase separation, the aqueous layer was collected, followed by the addition of isopropanol and centrifuged at 4°C. 75% ethanol was added to the pellet and centrifuged again at 4°C. The obtained pellet was air-dried followed by the addition of DEPC-treated TE buffer. This was then stored for performing further experiments.

RT-PCR Analysis

The extracted RNA was reverse transcribed into cDNA and then used for PCR analysis [18]. 2X KAPA SYBR® FAST master mix of volume 10µl along with specific primers as provided in Table 1 was used for the RT-PCR analysis. The analysis was executed with an initial denaturation at 95°C for 5 mins continued by denaturation at 95°C for 1 min. This was followed by annealing for 1 min at specific annealing temperatures depending on the primers. The extension took place after the annealing for one minute at 72°C. For 40 cycles, the complete procedure—from initial denaturation to extension—was repeated, and it was maintained at 4°C. The -ΔΔct values were determined using GAPDH for normalization, and fold change was reckoned using 2^(−ΔΔct).

Table 1

The primer sequences of key targets of insulin signaling and adipogenesis
Primer	Primer Sequences	Reference
IRTK	Forward Primer: 5’ ATC TGG ATC CCC CTG ATA ACT GTC 3’ Reverse Primer: 5’ ATG TGG GTG TAG GGG ATG TGT TCA 3’	[11]
IRS-1	Forward Primer: 5’ AAG TGG CGG CAC AAG TCG AG 3’ Reverse Primer: 5’ CGG GTG TAG AGA GCC ACC AG 3’
PI3K	Forward Primer: 5’ TGA CGC TTT CAA ACG CTA TC 3’ Reverse Primer: 5’ CAG AGA GTA CTC TTG CAT TC 3’
GLUT-4	Forward Primer: 5’ CGG GAC GTG GAG CTG GCC GAG GAG 3’ Reverse Primer: 5’ CCC CCT CAG CAG CGA GTG A 3’
PPAR-α	Forward Primer: 5’ AGC CCG TTA TCT GAA GAG TTC 3’ Reverse Primer: 5’ CAT CCC GAC AGA AAG CAC TT 3’
Adiponectin	Forward Primer: 5’ CTA CTG TTG CAA GCT CTC C 3’ Reverse Primer: 5’ CTT CAC ATC TTT CAT GTA CAC C 3’
GAPDH	Forward Primer: 5’ CCA CCC ATG GCA AAT TCC ATG GCA 3’ Reverse Primer: 5’ TCT AGA CGG CAG GTC AGG TCC ACC 3’

Statistical Analysis

The experiments were carried out in triplicates (N = 3). The values obtained were represented as mean ± Standard deviation (SD). Depending on whether the data is being compared with a control or positive control, one-way ANOVA was used to evaluate the data, followed by Tukey test, with a statistical significance of p < 0.05. Statistical analyses were performed with the GraphPad Prism version 8.0.1 software.

Cell Viability Assay

Effect of Dexamethasone on Cell Viability

The effect of DXM on L6 myoblasts and 3T3-L1 preadipocytes viability was assessed using MTT assay. Three different concentrations namely 10 nM, 100 nM, and 1µM based on a previous study [19] were taken for finalizing the optimum concentration of DXM for inducing maximum cell viability and least cytotoxicity. The analysis was done by taking untreated cells as control, 0.01% Triton X-100 treated cells as positive control and 0.01% DMSO served as vehicle control. It was found that in 3T3-L1 preadipocytes and L6 myoblasts, there was a dose-dependent reduction in cell viability upon treatment with DXM. In addition, 1µM DXM showed a substantial reduction in cell viability when compared with the control. It in turn also implies that 1 µM DXM has a cytotoxic effect on cells. Therefore, 10 nM and 100 nM DXM were taken for further analysis [Fig. 1A, B].

Based on the results obtained from the inhibitory study, it can be concluded that 100 nM DXM was ideal for inducing insulin resistance without much alteration in cell viability. Thus, this is taken further for inducing insulin resistance in both the chosen cell lines.

Effect of Apocynin on Viability of Dexamethasone Treated Cells

The analysis was performed taking untreated cells as the control and Triton X-100 treated cells as a positive control, and 0.01% DMSO served as vehicle control. A profound difference in cell viability was observed when compared with positive control. Previous study reveals that the preadipocytes showed the least difference in cell viability when compared with the control [11]. In accordance with the previously mentioned study, Fig. 2A, B suggests that apocynin-treated insulin-resistant cells did not show any alteration in the cell viability in both 3T3-L1 preadipocytes and L6 myoblasts except in its highest concentration in Preadipocytes alone.

2-NBDG (Glucose Uptake) Assay

Effect of Dexamethasone on Inhibition of Glucose Uptake

To study the impact of DXM (100 nM) on the inhibition of glucose uptake, 3 x 10³ cells were seeded and cultured in 24-well plates to attain 70% confluency. Post differentiation, dexamethasone was used in different concentrations to treat the cells. A dose-dependent study was carried out to evaluate insulin resistance based on the suppression of glucose absorption after a 24-hour dexamethasone treatment (10nM, 100nM, and 1µM). In comparison to untreated control cells, dexamethasone-treated cells (insulin-resistant model) acquired desensitization of insulin-stimulated glucose absorption to about 50% after 24 hours of treatment (insulin-sensitive model) [Fig. 3] It was evident that 1µM dexamethasone showed the highest glucose uptake inhibition. IC₅₀ was determined using a linear plot based on the percentage inhibition of glucose uptake [Fig. 3]. Dexamethasone was observed to be most effective at inducing insulin resistance in 3T3-L1 adipocytes and L6 myotubes at 100 nM. This concentration can be taken for further experimentation.

Effect of Dexamethasone on Insulin Stimulated Glucose Uptake

Glucose uptake is facilitated by insulin. This objective focused on unraveling whether insulin (100 nM) can revert insulin resistance induced by DXM (100 nM). The optimum concentration of DXM (100 nM) was used to treat both 3T3-L1 adipocytes and L6 myotubes. After 24h of incubation, the cells were incubated with or without insulin (100 nM) for additional 30 mins. Figure 4 showed that there was a significant increase in glucose uptake in the cells treated with insulin when compared to the vehicle control (VC). Also, dexamethasone treated cells exhibited a profound decrease in the glucose uptake on comparison with the positive control (insulin); thereby, suggesting the establishment of insulin resistance.

Insulin treated cells that have been already induced with dexamethasone have shown a significant increase in the glucose uptake comparatively lesser than the positive control, thus instigating that insulin can revert the insulin resistance caused by dexamethasone.

Effect of Inhibitors on the Glucose Uptake of Dexamethasone induced Insulin Resistance

Figure 5 shows the effects of Wortmannin, Genistein, administration on glucose uptake in Dexamethasone-treated insulin resistant cells. Wortmannin is a PI3K (Phosphoinositide 3 kinase) inhibitor and Genistein is an IRTK (Insulin receptor tyrosine kinase) inhibitor. Both of these were used to inhibit the insulin signaling pathway at their specific steps. In Fig. 5, when compared to positive control i.e., insulin (100nM)-treated cells; insulin stimulated cells showed significant decrease in glucose uptake (#p < 0.0001) upon treatment with Wortmannin (100nM) and Genistein (50µM), suggesting that PI3K and IRTK mediated glucose uptake, respectively.

Effect of Apocynin on Dexamethasone Induced Insulin Resistance

The percentage glucose uptake was assessed upon APO treatment in DXM induced insulin resistant cells. From Fig. 6A, B, it is observed that insulin treated cells showed the maximum glucose uptake and served as the positive control for insulin sensitivity. Whereas, dexamethasone treated cells showed a decrease in glucose uptake compared to that of the positive control (insulin) by inducing insulin-resistance. In addition to this, a remarkable dose-dependent increase in glucose uptake was observed upon APO treatment in dexamethasone-pretreated cells which reveals that 1 µM APO showed a substantial rise (p < 0.05) in glucose uptake activity in both insulin resistance-induced 3T3-L1 adipocytes and L6 myotubes and when compared with vehicle control (VC), thereby suggesting the antidiabetic activity of APO in reverting dexamethasone induced insulin resistance. This result was found to be on par with the effect of insulin in reverting insulin resistance as evident by displaying a significant increase in the glucose uptake activity in dexamethasone-pretreated cells. Thus, this concentration along with DXM (100 nM) treated cells was taken ahead to perform gene expression studies.

Gene Expression Study by RT-PCR

The molecular impact of apocynin on glucose uptake targets the various genes in the insulin signaling cascade like IRTK, IRS-1, PI3K, and GLUT-4. It also targets genes pertaining to adipogenesis like PPAR-α and adiponectin. The impact of treatment with APO on IRTK, IRS-1, PI3K, GLUT-4, PPAR-α and adiponectin was analyzed using RT-PCR. The cells administered with 100 nM insulin and 50 µM troglitazone served as positive controls for diabetic and adipogenic markers, respectively.

The effects of apocynin on genes are depicted in Fig. 7. The expression of IRTK and IRS-1 was found to be increased in APO-treated, DXM + APO-treated cells and was found to be equivalent to that expressed in insulin-treated cells. In DXM + APO-treated L6 myotubes and 3T3-L1 adipocytes, IRTK showed a 2- fold and 3.7-fold increase compared to the control. Whereas IRS-1 showed a 2.3-fold and 3.5-fold rise in L6 myotubes and 3T3-L1 adipocytes respectively. In addition to this PI3K exhibited a 2.6-fold rise in L6 myoblast and a 3.3-fold change in adipocytes. The maximum fold increase of 7.3 was exhibited by GLUT-4 in adipocytes while it showed a 2.8-fold increase in myotubes.

The impact of apocynin on adipogenic markers was also analyzed and it was found that DXM + APO treated cells show a 3.6-fold and 3.4-fold drop in the expression of adiponectin and PPAR-α respectively in 3T3-L1 adipocytes suggesting that APO (1 µM) has both anti-diabetic effects in L6 myotubes along with anti-adipogenic effect in 3T3-L1 adipocytes.

The inability of the body to utilize insulin is a characteristic of Type 2 Diabetes Mellitus, which results from aberrations in the insulin signaling cascade. This metabolic disorder not only leads to hyperglycemia and hyperinsulinemia but also leads to chronic and lethal complications. The metabolic aspects behind the cause of the disorder are not fully understood.

Dexamethasone is a glucocorticoid aid in receptor-level blocking of insulin signaling pathways. In L6 skeletal muscle cells, dexamethasone increases IRS-1 phosphorylation at Ser307 while decreasing AMPK and Akt phosphorylation. Dexamethasone also reduces glucose transporter-4 translocation (GLUT4). On the other hand, dexamethasone treatment on adipocytes causes downregulation of IRS-1 phosphorylation and insulin signaling pathways [20].

Apocynin is an immunomodulatory constituent, isolated from the roots of Picrorhiza kurroa. According to previous studies, apocynin is found to have anti-diabetic and anti-adipogenic activity in adipocytes [11] and L6 myotubes. Apocynin is proven as an NADPH oxidase Inhibitor and it promotes antioxidant defense systems which reduce free radical generation [21]. It is anticipated that apocynin, which is known for its anti-diabetic and anti-adipogenic impact on adipocytes in high glucose circumstances, can restore dexamethasone-induced insulin resistance in both adipocytes and skeletal muscle.

The clinical manifestation of insulin resistance has revealed that skeletal muscles and adipocytes are the target cells of insulin [1]. This showcased a hypothesis that there is a crosstalk between muscle and adipocytes that facilitates glucose uptake. Previous research by Sai Bharadhwaj et al., 2021 [11] revealed the antidiabetic and anti-adipogenic effect of apocynin in 3T3L1 adipocytes. Using this study as a base, we aimed to use a more targeted mode of inducing insulin resistance and identify if apocynin could reverse it. Thus, dexamethasone was chosen, which reduces glucose uptake by targeting insulin receptors. The optimum concentration and time of incubation were found to be 100 nM and 24 h respectively which is in par with that of Sangeetha et al., 2013 [19] findings. The crosstalk does not only happen between skeletal muscle and adipocytes but also has a possible connection with endothelial cells which are present in the lining of blood vessels.

Cell viability of skeletal muscle and adipocytes upon treatment with dexamethasone [Fig. 1A, B] and dexamethasone followed by apocynin [Fig. 2A, B] were analyzed using tetrazolium dye assay (MTT). The results indicate that the glucocorticoid (Dexamethasone) and drug i.e., apocynin did not have any effect on cell viability, in turn, indicates that they do not possess any cytotoxicity which is on par with the previous studies [11, 22]. The drug did not show many alterations in cell viability compared to control in endothelial cells as well (unpublished data).

The assay used 2-NBDG, a fluorescent analog that is taken up by cells but will not be metabolized hence serves as a tool to measure and track the extent of glucose uptake upon treatment of drugs [23]. The cells treated with 1 µM apocynin showed optimal glucose uptake by a substantial accumulation of 2-NBDG (significance p < 0.05) in adipocytes and myotubes. A similar effect was observed in endothelial cells (unpublished data).

Along with these findings, the results obtained from RT-PCR analysis revealed that the anti-diabetic and anti-adipogenic properties of apocynin can potentially revert insulin resistance induced by dexamethasone. The insulin signaling cascade is majorly governed by IRTK, IRS-1, PI3K, and GLUT-4. Increase in expression of these markers suggests that apocynin acts upon initial steps in the cascade which in turn facilitates the formation of GLUT-4 vesicles and translocation of the same aids in glucose uptake [24]. The increase in levels of GLUT-4 (7.3-fold change) may not only be because of an increase in other markers but also could be a possible correlating factor connecting skeletal muscle with adipocytes. In addition to this, the RT-PCR analysis also reveals that apocynin decreases the expression of adipogenic markers (PPAR-α and adiponectin) whose upregulation plays a vital role in the activation of PPAR- α, an adipogenic inducer [11].

The possible effects of apocynin in reversing insulin resistance induced by dexamethasone in adipocytes and skeletal muscle suggest that one of the possible mechanisms through which the cells are correlated in terms of glucose uptake lies at the receptor level and might possibly be governed by one or all genes involved at the receptor level. This correlation can be taken further to connect other linked cells like endothelial cells which also have similar effects upon apocynin treatment along with a dose-dependent increase in nitric oxide production (unpublished data).

To summarize, the study examined apocynin's ability to reverse dexamethasone-induced insulin resistance in adipocytes and skeletal muscle. The objective of this research is to determine if apocynin can reverse dexamethasone-induced insulin resistance by investigating cell viability, cytotoxicity, glucose uptake, and gene expression. Insulin's potential for restoring dexamethasone-induced insulin resistance was also explored, which revealed that apocynin mimicked insulin and facilitated glucose uptake in skeletal muscle and adipocytes. Gene expression studies have shown that increased GLUT-4 is one of the possible reasons for the increase in glucose uptake in apocynin-treated cells (7.3-fold change). The increase in GLUT-4 could be attributed to an increase in receptor-based gene expression. Increased expression levels of IRTK, IRS-1, PI3K, and GLUT-4 after apocynin (1µM) treatment supported the theory. Furthermore, 1 µM apocynin reduced adipogenic marker expression (adiponectin and PPAR-α).

All of this suggests that apocynin could be a potential candidate for reversing insulin resistance caused by dexamethasone and hyperglycemia and that it could be tested on endothelial cells to understand the gene expression pertaining to insulin resistance reversal, as well as the possible correlation between clinically manifested cell types (adipocytes, skeletal muscle, and endothelial cells). Understanding this crosstalk may lead to a more precise understanding of the insulin resistance pathway.

Author contributions Material preparation, data collection and analysis were performed by PN, PT, HM, TH, RP and AM. The first draft of the manuscript was written by PN, PT, HM. SS contributed to the study conception and design. All authors read and approved the final manuscript.

Data Availability All the source data are available at the Corresponding Author upon a reasonable request.

Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval and consent to participate Not applicable.

Consent for publication All authors consent to the publication of final manuscript.

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No competing interests reported.

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Reversal of dexamethasone induced insulin resistance by Apocynin in L6 myotubes and 3T3L1 adipocytes – a mechanistic approach

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Abstract

Figures

Introduction

Materials and Methods

Chemicals And Reagents

Culturing of 3T3-L1 Preadipocytes and L6 Myoblasts

Differentiation of 3T3-L1 Preadipocytes and L6 Myoblasts

Assessment of Cell Viability using MTT assay

Glucose Uptake Assessment using 2-NBDG assay

Glucose Uptake Activity in the Presence of Inhibitors

Isolation of RNA

RT-PCR Analysis

Statistical Analysis

Results

Cell Viability Assay

Effect of Dexamethasone on Cell Viability

Effect of Apocynin on Viability of Dexamethasone Treated Cells

2-NBDG (Glucose Uptake) Assay

Effect of Dexamethasone on Inhibition of Glucose Uptake

Effect of Dexamethasone on Insulin Stimulated Glucose Uptake

Effect of Inhibitors on the Glucose Uptake of Dexamethasone induced Insulin Resistance

Effect of Apocynin on Dexamethasone Induced Insulin Resistance

Gene Expression Study by RT-PCR

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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