Investigation of 3T3-L1 Cell Differentiation to Adipocyte, Affected by Aqueous Seed Extract of Phoenix Dactylifera L

Background: Obesity, often associated with insulin resistance and type 2 diabetes, is a metabolic disease that can result in dyslipidemia and hyperglycemia. Many reports describe the hypoglycemic and hypolipidemic properties of the Phoenix dactylifera L. seed extract in STZ-induced diabetic rat models, however, its antidiabetic effects in other diabetic models are less characterized in the literature. This study set out to determine the possible effects of the Phoenix dactylifera L. seed extract on adipogenesis and glucose homeostasis. Methods: 3T3-L1 cells were cultured in adipocyte differentiation media with or without varying doses of Phoenix dactylifera L. extract (0.312-1 mg/ml). Assays were performed on days 5, 8, and 12 after induced differentiation. Results: Our results demonstrate that the triglyceride content in treated groups was significantly lower compared to controls. Further, treating 3T3-L1 cells with Phoenix dactylifera L. seed extract reduced adipogenesis through the downregulation of PPAR-γ and CEBP-α, and adipocyte-specific genes involved in fatty acid metabolism including ap2, ACACA, and FAS. Conclusions: Phoenix dactylifera L. seeds have the potential to inhibit adipogenesis and obesity. Overall, this study explored the inhibitory effects of Phoenix dactylifera L. seed extract on adipogenesis in 3T3-L1 cells on the molecular level.


Introduction
Obesity is a universal health problem and its prevalence is on the rise. Adipocyte hypertrophy and dysfunction are associated with various metabolic diseases such as obesity, insulin resistance and inflammation (1). Apart from adipocyte hypertrophy, an increase in adipocyte number (hyperplasia) can also lead to obesity. Moreover, increased adipocyte numbers in obese individuals are correlated more vigorously with severe obesity (2). Within adipose tissue, preadipocytes proliferate and differentiate into mature adipocytes. Adipocytes can emanate from the dedifferentiation of adipocytes to preadipocytes, which then proliferate and redifferentiate into mature adipocytes (1).
The primary function of adipocytes is to maintain energy balance but also provide energy through the storage of triglycerides (TG) (5). Together, fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACACA) are involved in fatty acid synthesis; however, ACACA is also known to regulate fatty acid oxidation in adipocytes. To emphasize their importance in metabolic disease, scientists know now that increased de novo lipogenesis and reduced fatty acid oxidation can lead to both lipid accumulation and obesity (6).
Adipocytokines secreted by adipocytes, such as leptin and adiponectin, are known to regulate insulin sensitivity globally within the body (7). GLUT4, found in skeletal muscle and adipose tissue, is an insulin-regulated glucose transporter that permits the uptake of glucose in cells, and plays a central role in insulin resistance and diabetes type 2 (8). Additionally, Akt2, belonging to the Akt (PKB) subfamily of serine/threonine kinases, mediates signal transduction in adipocytes downstream of insulin, and overall is crucial for maintaining glucose homeostasis (9).
The carrier protein for fatty acids, aP2, is abundantly expressed in mature adipocytes. AP2 is also known as the fatty acid-binding protein 4 (FABP4) and is primarily involved in fatty acid, cholesterol, and phospholipid metabolism. Studies show that high levels of ap2 correlate to insulin resistance and dyslipidemia (10). Thermogenin also referred to as UCP1, is an uncoupling protein, found in the inner mitochondrial membrane (IMM) that generates heat by uncoupling oxidative phosphorylation. Increased expression of PPAR-γ can lead to increased expression of UCP1, browning of white adipose tissue, reduced insulin sensitivity and, in some cases, obesity (11). Expression of brown adipose tissue genes in white adipose tissue is essential for glucose tolerance, energy expenditure, and treating obesity and insulin resistance (12).
Date palm consumption was found to be beneficial for lipid and glucose control in diabetic individuals (13). Phoenix dactylifera L. (P. dactylifera), commonly known as the date palm, belongs to the palm plant genus and is largely found within southwest Asia and North Africa (14). Plants species within this genus are widely used to treat various infectious diseases, and health conditions, such as atherosclerosis, diabetes, hypertension, and cancer (15). Today, decocted Phoenix dactylifera L. seeds are mainly used to heal scorpion stings, cancer, kidney and bladder stones (16). The seeds contain high levels of phenolic compounds, antioxidants, and fibers. Moreover, the phenolic compounds consist of phenolic acids and flavonoids (17). Flavonoids can promote anti-cancer, anti-inflammatory and anti-obesity effects, and act as an antioxidant (18)(19)(20)(21). Both alcoholic and aqueous extracts in P. dactylifera seeds have been shown to decrease blood sugar and lipid levels (22)(23)(24) and protect against diabetic nephropathy (25) and CCl4-induced hepatotoxicity in rats (26).
Further, aqueous extracts from P. dactylifera seeds may produce hypoglycemic effects in diabetic rats when administered with insulin (27). Interestingly, P. dactylifera seeds may also stimulate endogenous insulin secretion in streptozotocin-induced type I diabetic rats (28). Lastly, combining insulin and date seed extracts were found to alleviate the negative consequences of diabetes on the liver and kidneys (29). Despite numerous studies illustrating the anti-glycemic, anti-inflammatory, antioxidant and anti-lipidemic effects of date palm seeds (30)(31), others have denied the hypoglycemic effect of aqueous compounds extracted from the tops of date palm leaves in diabetic rats (32).
Our study aimed to determine whether P. dactylifera seed extracts could produce hypoglycemic and hypolipidemic effects in an in vitro model. We specifically investigated the effects of aqueous extracts from P. dactylifera seeds on adipogenesis and glucose homeostasis, on the molecular level, in a 3T3-L1 cell model that is well-known to differentiate into mature adipocytes.

Materials and methods
All the experiments were approved by the ethical committee of Tehran University of Medical Sciences.

Preparation of aqueous extract of P. dactylifera seed
Seeds obtained from P. dactylifera were washed in water, roasted and crushed. We added distilled water to form a 50 g/L solution. Then, the solution was brought to the boiling point for 90 minutes, and after cooling to room temperature (RT), the solution was filtered and lyophilized. This process yielded ~3.8 g of brown powder that was stored at -20°C for future experiments (29).

Cell culture and maintenance
The 3T3-L1 cell line (preadipocytes derived from mouse embryonic fibroblasts) was purchased from the Iranian Biological Resource Center (IBRC). The cells were grown in Dulbecco's Medium Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin at 37°C in a 5% CO2 incubator.

Cell viability
Cell viability was assessed using an MTT assay. 3T3-L1 cells were seeded to a 96-well plate (15x103cells/well) for 24 h at 37°C in a 5% CO2 incubator. The cells were then treated with various concentrations of extract and incubated for 72 h. MTT stock solution (5 mg/ml, 20 μl) was added to each well and the plate was incubated for 4 h. After discarding the MTT solution, 100 μl of DMSO was added to each well and the plate was left in the dark for 15 minutes with gentle rocking. Finally, the absorbance was measured at 540 nm using a microplate reader (33).

Cell culture and differentiation
Differentiation of 3T3-L1 cells to adipocytes was performed according to a previous protocol with minor modifications (34). 3T3-L1 cells were seeded in 6-well plates (30 × 103 cells/well) or T25 flasks (10 × 104 cells per well). After reaching confluence, the medium was changed, and the cells were incubated for 48 h. To induce differentiation, the cells were incubated for 72 h in DMEM supplemented with a differentiation cocktail (0.5 mM IBMX, 1 µm dexamethasone, 2 µm rosiglitazone, 10 µg/ml insulin) and 10% FBS with or without the extract (day 0). Then, the medium was changed to DMEM containing 10 µg/mL of insulin every other day with or without the extract. It is to be noted that for this study, cell differentiation did not continue past day 12.

Oil-Red O staining
On days 5, 8 and 12 the cultured cells were washed three times with phosphate-buffered saline (PBS) and fixed in 10% formalin for 60 minutes at RT. After washing with deionized water, and an incubation period in 60% isopropanol (10 minutes), the cells were stained with Oil-Red O for 15 minutes at RT (35). To quantify the number of cells, they were washed twice with 60% isopropanol, for 5 minutes between each wash, with gentle rocking. Then, Oil-Red O stain was extracted using 100% isopropanol and the absorbance was measured at 492 nm (33).

Triglyceride assay
On days 5, 8 and 12 the cells in T25 flasks were washed with PBS, harvested and placed in 0.5% NP-40 solution and RIPA buffer separately. Next, cell suspensions were sonicated until a homogenized mixture was formed. NP-40 homogenate was incubated in a water bath at 80°C for 5 minutes to allow TG to precipitate out of solution. TG content was measured using the lipase glycerol kinase enzymatic method and Pars Azmoon kit (Cat. No. 97006). RIPA homogenate was used to determine protein content using the BCA method and Pars Tous kit (Cat. No. A101-251). Finally, TG content was normalized against the total protein concentration. adiponectin and leptin levels (ng/mg) were determined.

Quantitative real-time PCR
Total RNA was extracted from cells on days 5, 8 and 12 following induced differentiation using RiboEx (GeneAll, Cat. No.302-001). cDNA was synthesized using the Primer Script RT Reagent kit (Takara, Cat. No. RR037A) with 1 μg of total RNA. The real-time PCR assay was performed using a Rotor-Gene Q and Quanti Fast SYBR Green PCR kit (QIAGEN, Cat. No. 204054). The relative amount of mRNA, normalized to β-Actin levels, was calculated using the delta-delta method (36). The primer sequences used can be found in Table 1. No. RPN 2232) and exposed to x-ray film. Finally, protein expression was analyzed using ImageJ software and normalized against β-actin.

Statistical Analysis
Statistical differences between groups were determined using the one-way analysis of variance (ANOVA). We used a post-Hoc Dennett's test to analyze multiple comparisons. Data analysis was performed using GraphPad Prism version 8.0, and statistical differences were expressed as the mean ± standard deviation (SD). P values < 0.05 were considered statistically significant.

Effect of extract on adipogenesis
On day 5 we detected the first signs of TG accumulation within the cell cytoplasm as lipid droplets. To visualize lipid droplets, cells were stained with Oil-Red O on days 5, 8 and 12 which are depicted as red areas within the cytoplasm (Fig. 2). On days 8 and 12, we found that both the number and size of lipid droplets in the extract-treated group (0.315, 0.625, and 1.25 mg/mL) were lower compared to controls, despite maintaining a normal phenotype and shape of a mature adipocyte (spherical) (Fig. 3). We performed an Oil-Red O stain to also quantify TG and lipid accumulation, formerly expressed as absorbance values. A higher absorbance indicates increased lipid droplet formation and greater differentiation potential. Next, the relative lipid content (%) was compared to controls (Fig. 4A). Moreover, cytoplasmic TG content was measured on days 5, 8 and 12 and normalized to the total protein concentration (Fig.  4B). Relative lipid and TG content were reduced significantly in treated groups in a dose-dependent manner ( Fig.4A and B). Lipid content in the control group on day 12 was considered 100%. The cytosolic TG content was measured using the lipase glycerol kinase enzymatic method. TG content was normalized to the total protein concentration: (B) on day 12, the control group had a value of one. Relative lipid and TG content were reduced significantly in treated groups compared to controls on days 8 and 12. The results are represented as the mean ± SD of three independent experiments (*p < 0.05, ****p < 0.0001).

Effect of extract on leptin and adiponectin secretion
Both leptin and adiponectin are adipokines secreted by adipocytes. These proteins were measured during 3T3-L1 differentiation in cell culture medium on days 5, 8 and 12. There was a significant decrease in leptin and adiponectin levels in groups treated with extract at the following doses, 0.312, 0.625 and 1.25 mg/ml, compared to controls (Fig. 5A and B). Collectively, our results demonstrate that the presence of aqueous extract at low doses (0.312, 0.625, and 1.25mg/ml) could result in reduced levels of adipokines during differentiation. Thus, by increasing the extract concentration, we see an increase in adiponectin and leptin levels.

Effect of extract on the expression of adipogenic transcription factors and adipocyte-specific genes
PPAR-γ and C/EBP-α are adipogenic TFs involved in the differentiation of preadipocytes to adipocytes. We investigated whether aqueous extract could influence the expression of PPAR-γ and C/EBP-α during adipogenic differentiation. As shown in Fig. 6, the presence of aqueous extract significantly reduced PPAR-γ and C/EBPα expression in a dose-dependent manner, compared to controls. The results strongly indicate that the extract can suppress adipogenic differentiation by decreasing the expression of PPAR-γ and C/EBP-α.
Given the inhibitory effect of the aqueous extract on adipocyte differentiation, we further explored its anti-adipogenic effect through the expression of adipocyte-specific genes including aP2, ACACA, and FAS. Furthermore, we investigated the browning effect of the extract on 3T3-L1 adipocytes by examining UCP1 expression. As shown in Fig.   6, the aqueous extract treatment significantly decreased the mRNA expression of aP2, ACACA, FAS and UCP1 in 3T3-L1 differentiated adipocytes. These results suggest that the extract may trigger a down-stream regulatory pathway that suppresses the expression of adipocyte-specific genes, leading to decreased TG accumulation in adipocytes.

Effects of extract on glucose uptake
We then investigated the effect of the extract on basal glucose uptake of mature 3T3-L1 adipocytes. The results show that there was no significant change in basal glucose uptake in the presence of the extract at the following concentrations, 0.312, 0.625, and 1.25 mg/ml when compared to controls. Furthermore, the extract did not affect insulinstimulated glucose uptake (Fig. 7).

Effect of extract on glucose transportation and insulin signaling pathway
GLUT4 is the insulin-regulated glucose transporter in adipocytes. Akt2 plays a key role in signal transduction downstream of insulin and is activated when phosphorylated. On day 12, we investigated whether aqueous extract (0.312-1.25 mg/ml) could influence GLUT4 and Akt2 (inactive form, dephosphorylated; active form, ser473phosphorylated) expression levels in 3T3-L1 adipocytes. Data analysis demonstrated that the aqueous extract did not affect GLUT4 and Akt2 expression levels in treated groups ( Fig. 8A and B). Results demonstrate no effect of extract (0.312, 0.625, and 1.25 mg/ml) on GLUT4, Akt2 and phospho-Akt2 levels in treated groups (B). Data are represented as the mean ± SD of three independent experiments and were normalized to β-actin levels.

Discussion
Numerous studies have shown that various species of the palm may have hypoglycemic and hypolipidemic properties. For instance, the aqueous extract from the leaves of Europeanpalm, Chamaeropshumilis was shown to reduce plasma glucose, total cholesterol and triglyceride levels in obese rats (37). Moreover, hypoglycemic compounds were found in the roots of American palm, Arocomiaaculeate (38). P. dactylifera, commonly known as the date palm, is reported to be beneficial for lipid and glucose regulation in diabetic individuals (13). In the past, specific parts of the P. dactylifera plant have been used to treat various infectious diseases, and other health conditions including atherosclerosis, diabetes, hypertension, and cancer (15). In streptozotocininduced diabetic rats, palm oil tocotrienol-rich fractions improved dyslipidemia and reduced serum glucose and glycated hemoglobin concentrations (39). Furthermore, ethanol extracts from P. dactylifera leaves were shown to have antioxidant, anti-inflammatory and anti-diabetic effects (40).
Interestingly, phenolic compounds in P. dactylifera seed extracts, including phenolic acids and flavonoids, (17) is known to promote anticancer (18), antioxidants (19), anti-inflammatory (20) and anti-obesity (21) effects. Moreover, alcoholic extracts from P. dactylifera seeds were shown to decrease blood glucose and lipid concentrations in male diabetic rats (24). Another study showed that P. dactylifera aqueous extracts may produce hypoglycemic effects when administered with insulin in diabetic rats (27).
To date, the impact of P. dactylifera seeds on lipid and glucose metabolism has not been fully investigated. In our study, the hypoglycemic and hypolipidemic effects of aqueous extracts were evaluated in vitro using 3T3-L1 cells. Firstly, we evaluated the effect of aqueous extract on TG accumulation. A prominent feature of adipocytes is the presence of lipid droplets in the cellular cytoplasm. We found that lipid droplets began to appear on day 5 following induced differentiation. The results of the Oil-Red O stain on days 8 and 12 revealed that when 3T3-L1 cells were treated with aqueous extract, they maintained a normal adipocyte phenotype (spherical). However, the number and size of lipid droplets in extract-treated groups (0.315-1.25 mg/mL) were lower compared to controls (Fig. 3). Further, quantifying the Oil-Red O stain confirmed a reduction in lipid droplets in treated groups (Fig. 4A). Additionally, the average TG content in treated groups was significantly lower, in a dose-dependent manner, compared to the control group (Fig. 4B). Overall, these results show that the aqueous extract reduced TG accumulation while the 3T3-L1 cells maintained a normal adipocyte phenotype during differentiation.
It is a well-known fact that a decrease in TG accumulation is considered to be a promising therapeutic strategy against obesity, insulin resistance and type 2 diabetes (41). Therefore, we aimed to investigate a possible mechanism to support the anti-adipogenic effect observed in the presence of the aqueous extract. Firstly, we studied two key adipogenic TFs, specifically, PPAR-γ and C/EBP-α. We compared the mRNA levels of both PPAR-γ and C/EBP-α on days 5 (early stage), 8 (intermediate stage) and 12 (final stage) during adipogenic differentiation. We found that the extract inhibited adipogenic differentiation in 3T3-L1 cells by decreasing C/EBP-α and PPAR-γ expression (Fig.6).
The increase in the number and size of differentiated adipocytes from preadipocytes is known to result in obesity (42)(43)(44). Our results show that the extract at high doses (2.5-10 mg/mL) could significantly reduce the viability of 3T3-L1 preadipocytes (Fig.1). The extract (0.315-1.25 mg/mL) also inhibited adipogenic differentiation, in a dose-dependent manner, as shown by fewer mature adipocytes with decreased lipid droplets and reduced levels of accumulated intracellular TG. The reduction in secretory markers in mature adipocytes in treated groups, specifically adiponectin and leptin, demonstrates a lower differentiation potential in extract-treated 3T3-L1 cells ( Fig. 5A and B).
In this study, we found that both C/EBP-α and PPAR-γ induced the expression of adipocytespecific genes, which are key regulators of fatty acid metabolism. These genes include aP2, the fatty acid-binding protein, FAS, a major enzyme responsible for fatty acid synthesis, and ACACA, which regulates the fatty acid synthesis and fatty acid oxidation in adipocytes. Several studies show that high ap2 levels correlate to insulin resistance and dyslipidemia (10). Indeed, in the absence of these important proteins, there is an increase in de novo lipogenesis and reduced fatty acid oxidation resulting in lipid accumulation and obesity (6). Hence, we investigated the effect of aqueous extracts on aP2, FAS and ACACA mRNA levels during differentiation. We found that the extract reduced mRNA expression of FAS and ACACA, proteins involved in the late stage of adipogenesis. Furthermore, aP2 gene expression was also decreased (Fig.6). Taken together, we show that the anti-adipogenic effect of the aqueous extract was mediated by down-regulating PPAR-γ, CEBP-α, aP2, FAS and ACACA expression.
The mechanism and molecular events mediating the hypoglycemic and hypolipidemic effects induced by P. dactylifera seed extracts remain poorly characterized within the literature. An in vivo study proposed that components, such as oleic acid and linoleic acid, found in P. dactylifera alcoholic extracts could produce hypolipidemic effects. Further, the study suggests that elements such as magnesium (Mg) and zinc (Zn) in P. dactylifera seeds can produce a hypoglycemic effect through insulin production and secretion (24).
Another study showed that P. dactylifera aqueous extract combined with exogenous insulin promotes hypoglycemic effects in diabetic rats (27). Interestingly, it has been reported that the extract may stimulate endogenous insulin secretion in streptozotocin-induced type-I diabetic rats (28). The present study explored the hypoglycemic property of the P. dactylifera aqueous extract, and glucose homeostasis on day 12 (late differentiation stage). The results show that the extract had no effect on cellular glucose uptake with or without insulin stimulation (Fig.   7). Furthermore, the extract did not affect GLUT4, Akt2 and p-Akt2 (ser473) protein expression in treated groups ( Fig. 8A and B). Our findings demonstrate that although P. dactylifera seeds produced an anti-adipogenic effect, it had no significant impact on cellular glucose transport and insulin signaling.
To explain why there was no effect on glucose homeostasis, we considered the presence of insulin in the culture medium. As we know, insulin has direct effects on glucose metabolism and transport. We speculate that the extract could not overcome the effects of insulin. However, the extract could play a role in inhibiting the lipogenic activity of insulin in 3T3-L1 cells.
To gain a more accurate picture regarding the relationship between aqueous extract and glucose homeostasis, further investigations should consider in vivo models. This is the first study to illustrate the inhibitory effect of P. dactylifera aqueous extract on adipogenesis in 3T3-L1 cells. Moreover, P. dactylifera seeds demonstrate the potential to reduce adipogenesis through the downregulation of C/EBP-α and PPAR-γ and fatty acid metabolic genes. All things considered, P. dactylifera seed may provide a novel treatment strategy against obesity. The authors declare no conflict of interest.