Preventing Adipogenesis and Preserving Mitochondria and GLUT-4 Functions by Extracts and Isolated Compounds of Australian Acacia saligna

Acacia saligna’s secondary metabolites show promise in treating type 2 diabetes mellitus and its related conditions. We previously discovered that methanolic extracts, isolated flavonoids, and cyclitols effectively preserve mitochondria in 3T3-L1 adipocytes. In this current work, quantification of lipid droplet levels with Oil Red O assay showed a noticeable decrease in lipogenesis in 3T3-L1 cells. Methanolic leaf and bark extracts and isolated compounds, (−)-epicatechin 6 and myricitrin 8, reduced cellular lipid levels by 21.15% to 25.28%, respectively. mRNA levels of key regulators of mitochondrial biogenesis, such as adiponectin, PGC-1α, and mtTFA, were increased. Methanolic flower extract (FL-MeOH) and its chemical components, naringenin 1 and D-(+)-pinitol 5a, increased these gene levels from 10% to 29% at the higher dose. Our study found that FL-MeOH slightly reduced pro-inflammatory cytokines TNF-α and IL-6, attributed to two phytochemicals, naringenin-7-O-α-L-arabinofuranoside 2 and D-(+)-pinitol 5a. Western blot analysis also showed that adipocytes treated with MeOH extracts had higher GLUT-4 expression levels than untreated adipocytes. Overall, A. saligna extracts and their isolated compounds demonstrated anti-lipogenesis activity during 3T3-L1 cell differentiation, modulation of transcriptional levels of adiponectin, PGC-1α, and mtTFA, reducing TNF-α and IL-6 mRNA levels, promoting mitochondrial biogenesis, and enhancing GLUT-4 expression.


Introduction
Type 2 diabetes (T2D) is often associated with obesity [1][2][3][4].In the study by Gao et al. [5], alteration in the mitochondria was observed in 3T3-L1 adipocytes (Figure 1) and in an in vitro model of white adipose tissue.The changes included the overproduction of mitochondrial reactive oxygen species (mt-ROS) and a decrease in mitochondrial membrane potential (MMP).Such changes were linked to oxidative stress and reduced cellular glucose uptake.However, our previous study showed that treatment with Australian Acacia saligna extracts and isolated compounds (as listed in Table 1) [6] improved cellular glucose uptake by activating adenosine 5 -monophosphate-activated protein kinase (AMPK) phosphorylation.This treatment also helped restore mitochondrial homeostasis by reducing mt-ROS and improving MMP.However, the biological mechanism for this protection of mitochondria remains unclear.Therefore, this study was conducted to determine the possible mechanism involved in the positive effects of A. saligna treatment.
Studies on mitochondrial dysfunction in cell lines [5], mice models [7], and T2D patients [8][9][10] found that levels of peroxisome proliferator-activated receptor-γ coactivators-1 (PGCs-1) were lower than healthy controls.This reduction in PGC-1α can impact mitochondrial transcription factor A (mtTFA), which is essential in regulating the transcription of mitochondrial genes and mitochondrial DNA replication in adipocytes.AMPK has been confirmed as the key regulator of PGCs activation and expression [11].Furthermore, the activation of PGC-1α requires adiponectin-induced AMPK activation [12].As A. saligna treatment can increase the expression of AMPK, we hypothesised that A. saligna extracts and isolated compounds could enhance the expression of adiponectin, PGC-1α, and mtTFA.Therefore, this study evaluated the effects of methanolic extracts and their constituents on transcriptional levels of these vital genes involved in mitochondrial biogenesis using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis.
coactivators-1 (PGCs-1) were lower than healthy controls.This reduction in PGC-1α can impact mitochondrial transcription factor A (mtTFA), which is essential in regulating the transcription of mitochondrial genes and mitochondrial DNA replication in adipocytes.AMPK has been confirmed as the key regulator of PGCs activation and expression [11].Furthermore, the activation of PGC-1α requires adiponectin-induced AMPK activation [12].As A. saligna treatment can increase the expression of AMPK, we hypothesised that A. saligna extracts and isolated compounds could enhance the expression of adiponectin, PGC-1α, and mtTFA.Therefore, this study evaluated the effects of methanolic extracts and their constituents on transcriptional levels of these vital genes involved in mitochondrial biogenesis using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis.
Figure 1.A proposed link between mitochondrial dysfunction and insulin resistance in an adipocyte model.The increased intracellular ROS triggered by obesity and metabolic challenges (e.g., excess nutrient intake) leads to mt-ROS overproduction, exacerbating the total ROS generation and decreasing MMP and ATP production [13].The impaired mitochondria reflected by the collapse of mt-DNA content and depletion of mitochondrial biogenesis alter adiponectin level, resulting in decreased OXPHOS caused by reduced activation of AMPK and PGC-1α as its regulator [14].In addition, the compromised mitochondria function causes the accumulation of lipid metabolites because of the reduced β-oxidation and the increase in pro-inflammatory cytokines leading to the induction of PKC isoforms and Ser/Thr kinases [15,16].Consequently, the inhibition of the insulin signalling cascade, including the disruption of GLUT-4 transduction, leads to the defect of the insulin-dependent glucose uptake pathway.ROS: reactive oxygen species; mt-ROS: mitochondrial reactive oxygen species; MMP: mitochondrial membrane potential; ATP: adenosine 5′-triphosphate; mt-DNA: mitochondrial DNA; OXPHOS: oxidative phosphorylation; AMPK: adenosine 5′-monophosphate-activated protein kinase; PGC-1α: peroxisome proliferator-activated response-γ coactivator-1α; PKC: protein kinase C. A proposed link between mitochondrial dysfunction and insulin resistance in an adipocyte model.The increased intracellular ROS triggered by obesity and metabolic challenges (e.g., excess nutrient intake) leads to mt-ROS overproduction, exacerbating the total ROS generation and decreasing MMP and ATP production [13].The impaired mitochondria reflected by the collapse of mt-DNA content and depletion of mitochondrial biogenesis alter adiponectin level, resulting in decreased OXPHOS caused by reduced activation of AMPK and PGC-1α as its regulator [14].In addition, the compromised mitochondria function causes the accumulation of lipid metabolites because of the reduced β-oxidation and the increase in pro-inflammatory cytokines leading to the induction of PKC isoforms and Ser/Thr kinases [15,16].Consequently, the inhibition of the insulin signalling cascade, including the disruption of GLUT-4 transduction, leads to the defect of the insulin-dependent glucose uptake pathway.ROS: reactive oxygen species; mt-ROS: mitochondrial reactive oxygen species; MMP: mitochondrial membrane potential; ATP: adenosine 5 -triphosphate; mt-DNA: mitochondrial DNA; OXPHOS: oxidative phosphorylation; AMPK: adenosine 5 -monophosphate-activated protein kinase; PGC-1α: peroxisome proliferator-activated response-γ coactivator-1α; PKC: protein kinase C.  Interestingly, when mitochondrial regulators are downregulated, there is a significant decrease in phosphorylated Akt and glucose transporter 4 (GLUT-4) levels, along with an increase in the production of pro-inflammatory markers that impede glucose uptake in adipocytes [15,17].Additionally, along with GLUT-4 oxidation and carbonylation, the excessive triglyceride deposits in adipocytes can promote the pro-inflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6), resulting in decreased GLUT-4 expression [18].The levels of cytokines increase proportionally to adiposity and insulin resistance [19].Our previous study showed pronounced glucose uptake modulation [20], which could be attributed to improved mitochondrial function that protects GLUT-4.However, the study has not provided empirical information yet to support the hypothesis.Thus, this study also assesses the protein level of GLUT-4 in the adipocytes after treatment with A. saligna by conducting an immunoblot analysis.To further ensure the anti-inflammatory beneficial effect of the extract and isolated compounds, the TNF-α and IL-6 mRNA levels were also measured by RT-qPCR.Interestingly, when mitochondrial regulators are downregulated, there is a significant decrease in phosphorylated Akt and glucose transporter 4 (GLUT-4) levels, along with an increase in the production of pro-inflammatory markers that impede glucose uptake in adipocytes [15,17].Additionally, along with GLUT-4 oxidation and carbonylation, the excessive triglyceride deposits in adipocytes can promote the pro-inflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6), resulting in decreased GLUT-4 expression [18].The levels of cytokines increase proportionally to adiposity and insulin resistance [19].Our previous study showed pronounced glucose uptake modulation [20], which could be attributed to improved mitochondrial function that protects GLUT-4.However, the study has not provided empirical information yet to support the hypothesis.Thus, this study also assesses the protein level of GLUT-4 in the adipocytes after treatment with A. saligna by conducting an immunoblot analysis.To further ensure the anti-inflammatory beneficial effect of the extract and isolated compounds, the TNF-α and IL-6 mRNA levels were also measured by RT-qPCR.

LF-MeOH/8
Molecules 2023, 28, x FOR PEER REVIEW 3 of 14 Interestingly, when mitochondrial regulators are downregulated, there is a significant decrease in phosphorylated Akt and glucose transporter 4 (GLUT-4) levels, along with an increase in the production of pro-inflammatory markers that impede glucose uptake in adipocytes [15,17].Additionally, along with GLUT-4 oxidation and carbonylation, the excessive triglyceride deposits in adipocytes can promote the pro-inflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6), resulting in decreased GLUT-4 expression [18].The levels of cytokines increase proportionally to adiposity and insulin resistance [19].Our previous study showed pronounced glucose uptake modulation [20], which could be attributed to improved mitochondrial function that protects GLUT-4.However, the study has not provided empirical information yet to support the hypothesis.Thus, this study also assesses the protein level of GLUT-4 in the adipocytes after treatment with A. saligna by conducting an immunoblot analysis.To further ensure the anti-inflammatory beneficial effect of the extract and isolated compounds, the TNF-α and IL-6 mRNA levels were also measured by RT-qPCR.

FL-MeOH/2.58
Molecules 2023, 28, x FOR PEER REVIEW 3 of 14 Interestingly, when mitochondrial regulators are downregulated, there is a significant decrease in phosphorylated Akt and glucose transporter 4 (GLUT-4) levels, along with an increase in the production of pro-inflammatory markers that impede glucose uptake in adipocytes [15,17].Additionally, along with GLUT-4 oxidation and carbonylation, the excessive triglyceride deposits in adipocytes can promote the pro-inflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6), resulting in decreased GLUT-4 expression [18].The levels of cytokines increase proportionally to adiposity and insulin resistance [19].Our previous study showed pronounced glucose uptake modulation [20], which could be attributed to improved mitochondrial function that protects GLUT-4.However, the study has not provided empirical information yet to support the hypothesis.Thus, this study also assesses the protein level of GLUT-4 in the adipocytes after treatment with A. saligna by conducting an immunoblot analysis.To further ensure the anti-inflammatory beneficial effect of the extract and isolated compounds, the TNF-α and IL-6 mRNA levels were also measured by RT-qPCR.Interestingly, when mitochondrial regulators are downregulated, there is a significant decrease in phosphorylated Akt and glucose transporter 4 (GLUT-4) levels, along with an increase in the production of pro-inflammatory markers that impede glucose uptake in adipocytes [15,17].Additionally, along with GLUT-4 oxidation and carbonylation, the excessive triglyceride deposits in adipocytes can promote the pro-inflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6), resulting in decreased GLUT-4 expression [18].The levels of cytokines increase proportionally to adiposity and insulin resistance [19].Our previous study showed pronounced glucose uptake modulation [20], which could be attributed to improved mitochondrial function that protects GLUT-4.However, the study has not provided empirical information yet to support the hypothesis.Thus, this study also assesses the protein level of GLUT-4 in the adipocytes after treatment with A. saligna by conducting an immunoblot analysis.To further ensure the anti-inflammatory beneficial effect of the extract and isolated compounds, the TNF-α and IL-6 mRNA levels were also measured by RT-qPCR.Interestingly, when mitochondrial regulators are downregulated, there is a significant decrease in phosphorylated Akt and glucose transporter 4 (GLUT-4) levels, along with an increase in the production of pro-inflammatory markers that impede glucose uptake in adipocytes [15,17].Additionally, along with GLUT-4 oxidation and carbonylation, the excessive triglyceride deposits in adipocytes can promote the pro-inflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6), resulting in decreased GLUT-4 expression [18].The levels of cytokines increase proportionally to adiposity and insulin resistance [19].Our previous study showed pronounced glucose uptake modulation [20], which could be attributed to improved mitochondrial function that protects GLUT-4.However, the study has not provided empirical information yet to support the hypothesis.Thus, this study also assesses the protein level of GLUT-4 in the adipocytes after treatment with A. saligna by conducting an immunoblot analysis.To further ensure the anti-inflammatory beneficial effect of the extract and isolated compounds, the TNF-α and IL-6 mRNA levels were also measured by RT-qPCR.

LF-MeOH/1
Molecules 2023, 28, x FOR PEER REVIEW 3 of 14  Interestingly, when mitochondrial regulators are downregulated, there is a significant decrease in phosphorylated Akt and glucose transporter 4 (GLUT-4) levels, along with an increase in the production of pro-inflammatory markers that impede glucose uptake in adipocytes [15,17].Additionally, along with GLUT-4 oxidation and carbonylation, the excessive triglyceride deposits in adipocytes can promote the pro-inflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6), resulting in decreased GLUT-4 expression [18].The levels of cytokines increase proportionally to adiposity and insulin resistance [19].Our previous study showed pronounced glucose uptake modulation [20], which could be attributed to improved mitochondrial function that protects GLUT-4.However, the study has not provided empirical information yet to support the hypothesis.Thus, this study also assesses the protein level of GLUT-4 in the adipocytes after treatment with A. saligna by conducting an immunoblot analysis.To further ensure the anti-inflammatory beneficial effect of the extract and isolated compounds, the TNF-α and IL-6 mRNA levels were also measured by RT-qPCR.Interestingly, when mitochondrial regulators are downregulated, there is a significant decrease in phosphorylated Akt and glucose transporter 4 (GLUT-4) levels, along with an increase in the production of pro-inflammatory markers that impede glucose uptake in adipocytes [15,17].Additionally, along with GLUT-4 oxidation and carbonylation, the excessive triglyceride deposits in adipocytes can promote the pro-inflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6), resulting in decreased GLUT-4 expression [18].The levels of cytokines increase proportionally to adiposity and insulin resistance [19].Our previous study showed pronounced glucose uptake modulation [20], which could be attributed to improved mitochondrial function that protects GLUT-4.However, the study has not provided empirical information yet to support the hypothesis.Thus, this study also assesses the protein level of GLUT-4 in the adipocytes after treatment with A. saligna by conducting an immunoblot analysis.To further ensure the anti-inflammatory beneficial effect of the extract and isolated compounds, the TNF-α and IL-6 mRNA levels were also measured by RT-qPCR.

LF-MeOH/5
Molecules 2023, 28, x FOR PEER REVIEW 3 of 14  Interestingly, when mitochondrial regulators are downregulated, there is a significant decrease in phosphorylated Akt and glucose transporter 4 (GLUT-4) levels, along with an increase in the production of pro-inflammatory markers that impede glucose uptake in adipocytes [15,17].Additionally, along with GLUT-4 oxidation and carbonylation, the excessive triglyceride deposits in adipocytes can promote the pro-inflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6), resulting in decreased GLUT-4 expression [18].The levels of cytokines increase proportionally to adiposity and insulin resistance [19].Our previous study showed pronounced glucose uptake modulation [20], which could be attributed to improved mitochondrial function that protects GLUT-4.However, the study has not provided empirical information yet to support the hypothesis.Thus, this study also assesses the protein level of GLUT-4 in the adipocytes after treatment with A. saligna by conducting an immunoblot analysis.To further ensure the anti-inflammatory beneficial effect of the extract and isolated compounds, the TNF-α and IL-6 mRNA levels were also measured by RT-qPCR.Interestingly, when mitochondrial regulators are downregulated, there is a significant decrease in phosphorylated Akt and glucose transporter 4 (GLUT-4) levels, along with an increase in the production of pro-inflammatory markers that impede glucose uptake in adipocytes [15,17].Additionally, along with GLUT-4 oxidation and carbonylation, the excessive triglyceride deposits in adipocytes can promote the pro-inflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6), resulting in decreased GLUT-4 expression [18].The levels of cytokines increase proportionally to adiposity and insulin resistance [19].Our previous study showed pronounced glucose uptake modulation [20], which could be attributed to improved mitochondrial function that protects GLUT-4.However, the study has not provided empirical information yet to support the hypothesis.Thus, this study also assesses the protein level of GLUT-4 in the adipocytes after treatment with A. saligna by conducting an immunoblot analysis.To further ensure the anti-inflammatory beneficial effect of the extract and isolated compounds, the TNF-α and IL-6 mRNA levels were also measured by RT-qPCR.Interestingly, when mitochondrial regulators are downregulated, there is a significant decrease in phosphorylated Akt and glucose transporter 4 (GLUT-4) levels, along with an increase in the production of pro-inflammatory markers that impede glucose uptake in adipocytes [15,17].Additionally, along with GLUT-4 oxidation and carbonylation, the excessive triglyceride deposits in adipocytes can promote the pro-inflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6), resulting in decreased GLUT-4 expression [18].The levels of cytokines increase proportionally to adiposity and insulin resistance [19].Our previous study showed pronounced glucose uptake modulation [20], which could be attributed to improved mitochondrial function that protects GLUT-4.However, the study has not provided empirical information yet to support the hypothesis.Thus, this study also assesses the protein level of GLUT-4 in the adipocytes after treatment with A. saligna by conducting an immunoblot analysis.To further ensure the anti-inflammatory beneficial effect of the extract and isolated compounds, the TNF-α and IL-6 mRNA levels were also measured by RT-qPCR.

Lipid Accumulation in 3T3-L1 Adipocytes Treated with Extracts and Isolated Compounds
Based on the data presented in Figure 2, there were varying amounts of lipid droplets in the adipocytes treated with extracts compared to the control group.The LF-MeOHtreated adipocytes at 50 µg/mL showed a significant decrease in lipid droplets by 28.68% compared to the control, indicating antiadipogenic activity.A similar effect was observed in the positive control treatment with N-acetyl cysteine (NAC) at 10 mM for 29.55%.A slight decrease in lipid droplets was observed from treatment with hexane leaf extract (LFhex) 50 µg/mL, BK-MeOH 50 µg/mL, LF-MeOH 12.5 µg/mL, FL-H 2 O 12.5 µg/mL, and NAC 5 mM by 10.18, 11.9, 15.35, 16.81, and 22.14%, respectively.Conversely, 12.5 µg/mL of BK-MeOH, 50 µg/mL of dichloromethane leaf extract (LF-DCM), and 12.5 µg/mL of dichloromethane bark extract (BK-DCM) significantly increased lipid droplets by 30.4%, 40.9%, and 45.9%, respectively.

Expression of mRNAs Related to the Mitochondrial Biogenesis of Adipocytes
Our initial study has revealed that active extracts and compounds found in A. saligna can enhance glucose uptake by activating AMPK-α in adipocytes and improving mitochondrial function [20].Since activation of AMPK is also linked to the biogenesis of mitochondria, we believe that these extracts and phytochemicals may also increase the expression of mtTFA by upregulating PGC-α, which is known to be linked to the biogenesis of mitochondria [12].To determine the effect of methanolic extracts and isolated compounds on the transcriptional levels of key genes involved in mitochondrial biogenesis, such as adiponectin, PGC-1α, and mtTFA, we conducted a quantitative reverse transcription-polymerase chain reaction (RT-qPCR) study.The expression of target mRNAs, adiponectin, PGC-1α, and mtTFA, increased with the treatment of the three methanolic extracts of A. saligna.All three MeOH extracts at 12.5 µg/mL slightly increased the expression of adiponectin by approximately 7% compared to the control group.At 50 µg/mL, the expression of adiponectin improved, and the FL-MeOH extract showed a significant increase in expression, which was 28.92% higher than the control.Furthermore, at 12.5 µg/mL, BK-MeOH showed small increases in the expression of PGC-1α and mtTFA by 9.9 and 10.7%, respectively.Moreover, the expression of PGC-1α was slightly more upregulated when treating FL-, LF-, and BK-MeOH at 50 µg/mL by 11.4, 13.4, and 10.7%, respectively.On the other hand, only adipocytes treated with FLand BK-MeOH extracts at 50 µg/mL showed noticeable increases in mtTFA expression by 11.5 and 12.6%, respectively.Figure 4 and Table S4 demonstrate that all three extracts consistently affected target gene expression.Our results align with our previous study [20], showing a twofold increase in mitochondrial membrane potential (MMP) from the FL-MeOH treatment at 50 µg/mL.These findings reinforce the beneficial impact of methanolic extracts on adipocyte mitochondrial biogenesis, as evidenced by the reduction in mt-ROS and enhancement of MMP status.This improvement, therefore, was brought about by the transcriptional expression of adiponectin, PGC-1α, and mtTFA.

Expression of mRNAs Related to the Mitochondrial Biogenesis of Adipocytes
Our initial study has revealed that active extracts and compounds found in A. saligna can enhance glucose uptake by activating AMPK-α in adipocytes and improving mitochondrial function [20].Since activation of AMPK is also linked to the biogenesis of mitochondria, we believe that these extracts and phytochemicals may also increase the expression of mtTFA by upregulating PGC-α, which is known to be linked to the biogenesis of mitochondria [12].To determine the effect of methanolic extracts and isolated compounds on the transcriptional levels of key genes involved in mitochondrial biogenesis, such as adiponectin, PGC-1α, and mtTFA, we conducted a quantitative reverse transcriptionpolymerase chain reaction (RT-qPCR) study.The expression of target mRNAs, adiponectin, PGC-1α, and mtTFA, increased with the treatment of the three methanolic extracts of A. saligna.All three MeOH extracts at 12.5 µg/mL slightly increased the expression of adiponectin by approximately 7% compared to the control group.At 50 µg/mL, the expression of adiponectin improved, and the FL-MeOH extract showed a significant increase in expression, which was 28.92% higher than the control.Furthermore, at 12.5 µg/mL, BK-MeOH showed small increases in the expression of PGC-1α and mtTFA by 9.9 and 10.7%, respectively.Moreover, the expression of PGC-1α was slightly more upregulated when treating FL-, LF-, and BK-MeOH at 50 µg/mL by 11.4, 13.4, and 10.7%, respectively.On the other hand, only adipocytes treated with FLand BK-MeOH extracts at 50 µg/mL showed noticeable increases in mtTFA expression by 11.5 and 12.6%, respectively.Figure 4 and Table S4 demonstrate that all three extracts consistently affected target gene expression.Our results align with our previous study [20], showing a twofold increase in mitochondrial membrane potential (MMP) from the FL-MeOH treatment at 50 µg/mL.These findings reinforce the beneficial impact of methanolic extracts on adipocyte mitochondrial biogenesis, as evidenced by the reduction in mt-ROS and enhancement of MMP status.This improvement, therefore, was brought about by the transcriptional expression of adiponectin, PGC-1α, and mtTFA.
At a concentration of 0.5 µM, all isolated compounds caused a slight increase in transcriptional levels of adiponectin, PGC-1α, and mtTFA mRNA, as shown in Figure 5 and Table S5.However, when incubated at 10 µM, naringenin 1 resulted in a 14.42% increase in adiponectin expression, while (+)-pinitol 5a led to a 12.1% increase in PGC-1α expression.At a concentration of 0.5 µM, all isolated compounds caused a slight increase in transcriptional levels of adiponectin, PGC-1α, and mtTFA mRNA, as shown in Figure 5 and Table S5.However, when incubated at 10 µM, naringenin 1 resulted in a 14.42% increase in adiponectin expression, while (+)-pinitol 5a led to a 12.1% increase in PGC-1α expression.
The findings demonstrate a correlation between the observed increases in MMP by 1.67-and 2-fold when treated with naringenin 1 and D-(+)-pinitol 5a in the adipocytes [20].Notably, naringenin 1 and D-(+)-pinitol 5a were found in FL-MeOH extract with percentages (w/w) of 1.75 and 2.5%, respectively [20].The presence of these two active compounds suggests the noticeable effect of FL-MeOH extract on the increase in gene expression.Previous studies have shown that naringenin 1 can modulate mRNA expression of adiponectin and PGC-1α in human white adipocyte cultures, leading to increased energy expenditure and insulin sensitivity [21].Additionally, in obese mice, D-(+)-pinitol 5a stimulated PGC-1α mRNA expression by upregulating the cAMP response element-binding protein (CREB) [22].

Expression of mRNAs of Inflammatory Markers of Adipocytes
Our previous study found that methanolic extracts from A. saligna and isolated compounds effectively reduced cellular ROS and mt-ROS levels in 3T3-L1 adipocytes [20].This inhibition of mt-ROS production can potentially prevent the inflammatory response that leads to impaired glucose homeostasis [16].Cytokine levels are directly proportional The findings demonstrate a correlation between the observed increases in MMP by 1.67-and 2-fold when treated with naringenin 1 and D-(+)-pinitol 5a in the adipocytes [20].Notably, naringenin 1 and D-(+)-pinitol 5a were found in FL-MeOH extract with percentages (w/w) of 1.75 and 2.5%, respectively [20].The presence of these two active compounds suggests the noticeable effect of FL-MeOH extract on the increase in gene expression.Previous studies have shown that naringenin 1 can modulate mRNA expression of adiponectin and PGC-1α in human white adipocyte cultures, leading to increased energy expenditure and insulin sensitivity [21].Additionally, in obese mice, D-(+)-pinitol 5a stimulated PGC-1α mRNA expression by upregulating the cAMP response element-binding protein (CREB) [22].

Expression of mRNAs of Inflammatory Markers of Adipocytes
Our previous study found that methanolic extracts from A. saligna and isolated compounds effectively reduced cellular ROS and mt-ROS levels in 3T3-L1 adipocytes [20].This inhibition of mt-ROS production can potentially prevent the inflammatory response that leads to impaired glucose homeostasis [16].Cytokine levels are directly proportional to adiposity and insulin resistance [18], and thus, the levels of TNF-α and IL-6 mRNA were measured to assess the inhibitory effects of the extracts and isolated compounds on inflammatory markers.This helps explain the mechanism of reduced mt-ROS levels and enhanced glucose uptake in 3T3-L1 adipocytes.
The transcriptional levels of TNF-α and IL-6 were altered in the A. saligna MeOHextract-treated adipocytes compared to the vehicle control group (Figure 6 and Supplementary Table S4).The FL-MeOH-treated adipocytes at 50 µg/mL showed slight decreases in TNF-α and IL-6 expression by approximately 15.9% and 10.5%, respectively.At the same concentration, the LF-MeOH had a slightly lower effect than the methanolic extract of flowers, resulting in 11.6% and 10.4% decreases in TNF-α and IL-6 expression, respectively.On the other hand, the BK-MeOH extract showed a 12% reduction in TNF-α expression and no effect on IL-6 expression.These findings suggest that these extracts have a weak inhibitory effect on pro-inflammatory gene expression in adipocytes.The isolated compounds reduced the mRNA expression of two pro-inflamma adipokines, as shown in Figure 7 and Supplementary Table S5.When treated naringenin-7-O-α-L-arabinofuranoside 2 and D-(+)-pinitol 5a at 10 µM, there were s decreases in TNF-α expression by 14.2 and 19.6%, respectively.Additionally, treatm with naringenin 1 and quercitrin 4 at the same dose decreased TNF-α expression b and 12.7%, respectively.However, there was only a minimal inhibitory effect on th pression of IL-6 mRNA when treated with naringenin 1, quercitrin 4, D-(+)-pinitol 5a naringenin-7-O-α-L-arabinofuranoside 2 at the same concentration.These results gested that FL-MeOH inhibited TNF-α expression mainly through naringenin 1, q citrin 4, and compounds 2 and 5a.Inhibition of IL-6 expression may be due to other The isolated compounds reduced the mRNA expression of two pro-inflammatory adipokines, as shown in Figure 7 and Supplementary Table S5.When treated with naringenin-7-O-α-L-arabinofuranoside 2 and D-(+)-pinitol 5a at 10 µM, there were slight decreases in TNF-α expression by 14.2 and 19.6%, respectively.Additionally, treatment with naringenin 1 and quercitrin 4 at the same dose decreased TNF-α expression by 11 and 12.7%, respectively.However, there was only a minimal inhibitory effect on the expression of IL-6 mRNA when treated with naringenin 1, quercitrin 4, D-(+)-pinitol 5a, and naringenin-7-O-α-L-arabinofuranoside 2 at the same concentration.These results suggested that FL-MeOH inhibited TNF-α expression mainly through naringenin 1, quercitrin 4, and compounds 2 and 5a.Inhibition of IL-6 expression may be due to other unisolated compounds in the extract.Our findings suggest that extracts and phytoconstituents from A. saligna can potentially protect adipocyte mitochondria against the harmful effects of pro-inflammatory cytokines, such as TNF-α and IL-6, which have been known to hamper the adiponectin pathway and mitochondrial biogenesis [23,24].
The isolated compounds reduced the mRNA expression of two pro-inflammatory adipokines, as shown in Figure 7 and Supplementary Table S5.When treated with naringenin-7-O-α-L-arabinofuranoside 2 and D-(+)-pinitol 5a at 10 µM, there were slight decreases in TNF-α expression by 14.2 and 19.6%, respectively.Additionally, treatment with naringenin 1 and quercitrin 4 at the same dose decreased TNF-α expression by 11 and 12.7%, respectively.However, there was only a minimal inhibitory effect on the expression of IL-6 mRNA when treated with naringenin 1, quercitrin 4, D-(+)-pinitol 5a, and naringenin-7-O-α-L-arabinofuranoside 2 at the same concentration.These results suggested that FL-MeOH inhibited TNF-α expression mainly through naringenin 1, quercitrin 4, and compounds 2 and 5a.Inhibition of IL-6 expression may be due to other unisolated compounds in the extract.Our findings suggest that extracts and phytoconstituents from A. saligna can potentially protect adipocyte mitochondria against the harmful effects of pro-inflammatory cytokines, such as TNF-α and IL-6, which have been known to hamper the adiponectin pathway and mitochondrial biogenesis [23,24].

Expression of GLUT-4 of 3T3-L1 Adipocytes Treated with MeOH Extracts
Our prior research demonstrated the potential of methanolic extracts and isolated compounds to improve glucose uptake in 3T3-L1 adipocytes [20].We hypothesised that this effect was attained by activating AMPK and increasing GLUT-4.To validate this pathway, we examined the expression of GLUT-4 in adipocytes.The methanolic extracts of A. saligna were found to increase the level of GLUT-4 protein in adipocytes after treatment.This finding is supported by Figure 8 and Supplementary Table S3.The increases in GLUT-4 at a concentration of 12.5 µg/mL were 46.76%, 37.27%, and 18.84% for FL-, LF-, and BK-MeOH, respectively, in comparison to the vehicle control.
Additionally, at a concentration of 50 µg/mL, FL-MeOH treatment significantly increased the GLUT-4 expression by 61.9%, while this effect was weaker for LF-and BK-MeOH treatments, which increased the protein expression by 50.4% and 28.2%, respectively.In other words, treatment with FL-MeOH extract produced the best effect on the increased expression of GLUT-4.This finding supports our earlier report that the adipocytes exposed to FL-MeOH extract had significantly improved glucose uptake by 41.5% and 85.3% at 12.5 and 50 µg/mL, respectively [20].The study demonstrated a connection between the higher expression of GLUT-4 and the enhanced absorption of glucose in adipocytes.Though the Western blot analysis was repeated twice, additional research with more replications is necessary to establish the accuracy of this initial observation.We previously found that all methanolic extracts could inhibit free radicals [6] by reducing cellular ROS and mt-ROS levels in 3T3-L1 adipocytes [20].The FL-MeOH extract was particularly effective, with its ability to reduce cellular ROS and mt-ROS by 6.48% to 32%, respectively, in a dose-dependent manner [20].This reduction in ROS levels has been shown to protect the function of glucose transporters.For example, in H9c2 cells, glucose-induced oxidative stress caused a decrease in GLUT-4 mRNA expression.However, treatment with Trolox restored the expression of the glucose transporter in a concentration-and time-dependent manner [25].Similarly, in another study, high glucose treatment in HepG2 cells caused a decrease in GLUT-4 protein, which was Molecules 2023, 28, 6677 9 of 14 preserved after incubation with flavonoids such as apigenin and luteolin, which are also natural ROS inhibitors [26].
lar ROS and mt-ROS levels in 3T3-L1 adipocytes [20].The FL-MeOH extract was particularly effective, with its ability to reduce cellular ROS and mt-ROS by 6.48% to 32%, respectively, in a dose-dependent manner [20].This reduction in ROS levels has been shown to protect the function of glucose transporters.For example, in H9c2 cells, glucose-induced oxidative stress caused a decrease in GLUT-4 mRNA expression.However, treatment with Trolox restored the expression of the glucose transporter in a concentration-and time-dependent manner [25].Similarly, in another study, high glucose treatment in HepG2 cells caused a decrease in GLUT-4 protein, which was preserved after incubation with flavonoids such as apigenin and luteolin, which are also natural ROS inhibitors [26].FL-MeOH showed consistent effects on mitochondrial regulators, pro-inflammatory markers, and glucose transporters.Specifically, when treated with 50 µg/mL of FL-MeOH, there were significant increases in adiponectin, PGC-1α, and mtTFA levels (around onethird, one-tenth, and one-tenth increases, respectively), as well as GLUT-4 expression (three-fifths higher than the vehicle control).Additionally, FL-MeOH treatment resulted in a ~10% reduction in TNF-α and IL-6 mRNA levels.These findings are consistent with previous studies showing that obese mice with increased mitochondrial regulators [27] and reduced cytokines [28] have higher levels of GLUT-4 protein compared to control groups.According to the findings, extracts from A. saligna may positively impact mitochondrial homeostasis in adipocytes.This occurred in synergism with the restoration of the GLUT-4 level.Mitochondrial dysfunction can worsen cellular ROS production, inflammatory response, and carbonylation of cellular components [29].Activating the mitochondrial key regulators with A. saligna methanolic extracts can help maintain the adipocytic redox balance.This is shown by a reduction in mt-ROS and MMP values [20], which help preserve GLUT-4 protein from the damaging effects of excessive ROS.
The following materials (Table 2) for the RT-qPCR experiments were supplied by Promega (Madison, WI, USA): nuclease-free water, dNTP mix (10 mM), reverse transcription kits of cDNA first-strand synthesis, and random primers.The following target primers (TaqMan™ GeneExpressionAssay (FAM)) were purchased from Thermo-Fisher Scientific (Waltham, MA, USA).The other materials were a Trizol reagent for RNA isolation (Sigma-Aldrich, St. Louis, MO, USA), a 96-well PCR plate and adhesive seals (Bio-Rad, Hercules, CA, USA), solvents including isopropanol, chloroform, and ethanol (Point of Care Diagnostics, North Rocks, NSW, Australia), and a master mix for RT-PCR (Meridian Bioscience, Cincinnati, OH, USA).

Estimation of Lipid Accumulation Using Oil Red O (ORO) Assay
This assay protocol was adapted from Kraus et al. [30].On the day of induction of differentiation (day 0), the confluent preadipocytes were exposed to the differentiation medium of induction in the presence of extracts (12.5 and 50 µg/mL) or isolated compounds (0.5 and 10 µM) followed by 48 h incubation.The cells were fed with fresh basal media containing insulin in the presence of the extracts every other 2 days until day 8 of the induction.On the day of staining (day 8), the treated cells and control were washed with PBS and then fixed with 10% formalin solution (dissolved in PBS, 100 µL/well) for 30 min at room temperature.The cells were washed with 60% isopropanol and then stained with 60% Oil Red O (0.7 g of ORO/200 mL of isopropanol, 100 µL/well) for 1 h at room temperature.After liquid removal, the stained cells were washed with water and eluted with 100% isopropanol.The plate was incubated for 10 min at room temperature with a shaker.The absorbance was measured at 510 nm through a microplate reader.The lipid level was expressed by: Fold change of adipogenesis = Absorbance of treated adipocytes Absorbance of control × 100% 3.4.Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-qPCR) Total RNA was extracted using Trizol reagent, followed by precipitation with isopropyl alcohol, three times washing with 75% EtOH in nuclease-free water, solvent evaporation, and dissolving in nuclease-free water.The concentration of RNA was quantified using a Nanodrop 2000 spectrometer (Thermo-Fisher Scientific, Waltham, MA, USA).The firststrand cDNA was synthesised from 1 µg of the isolated RNA according to the protocol of the manufacturer.The reaction was conducted in a Bio-Rad T100 Thermal Cycler (Hercules, CA, USA).The concentration of cDNA product used as a template for amplification of RT-qPCR was estimated using the nanodrop apparatus.The qPCR was carried out in a Bio-Rad CFX96 Real-Time System (Hercules, CA, USA) following the instructions of the kit.The reaction was conducted in the following stages: polymerase activation (95 • C, 2 min), denaturation (95 • C, 10 s), and the annealing step (60 • C, 30 s) for 50 cycles.

Western Blot Analysis
The Western blot protocol was adapted with modifications from Montt-Guevara et al. [31].After treatment, the medium of adipocytes was discarded, followed by washing out with pre-chilled PBS, then incubation with RIPA containing protease and phosphatase inhibitor cocktail buffer for 30 min, and moved into microtubes before being centrifuged at 12,000 rpm for 20 min at 4 • C. Subsequently, the supernatant was collected into new microtubes, and the protein concentration was quantified using a BCA protein assay kit.Before separation, the protein supernatant of 35 µg was mixed with loading buffer and boiled at 95 • C on a heating block for 10 min.The electrophoresis was carried out by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for 1 h and 30 min using a Mini Gel Tank (Thermo-Fisher Scientific, Waltham, MA, USA).The separated proteins were transferred into PVDF membranes using an iBlot Western blot transfer system (Thermo-Fisher Scientific, Waltham, MA, USA), followed by blocking with 5% skim milk solution in Tris-buffered saline with Tween (TBST) buffer for an hour.After washing three times with TBST (10 min per each at room temperature), the membranes were incubated with the targeted antibodies solution (1:1000) overnight at 4 • C.After washing with TBST three times, the membranes were incubated with the secondary antibody solution (1:10,000) at room temperature for an hour.Following this, they were rewashed three times with TBST.Finally, the protein strips were detected by the ECL reagents and captured by a Chemidoc system (Amersham Image-quant, Marlborough, MA, USA).The protein expression bands were quantified using Image J (National Institute of Health, Bethesda, MD, USA).The experiment was independently replicated twice.

Statistical Analysis
A descriptive analysis using GraphPad Prism 9 (San Diego, CA, USA) was conducted to determine the mean ± standard error mean (SEM) from the three independent experiments (n = 3), except the Western blot analysis (n = 2).The difference between the two means of each sample and the vehicle control optical densities was calculated using a one-way ANOVA followed by Tukey's post hoc tests, where p < 0.05 was considered significant.

Conclusions
Treatment with LF-MeOH extract inhibited adipogenesis of 3T3-L1 cells, as demonstrated by the ORO staining assay.LF-MeOH's effectiveness is reflected in the significant decrease in lipid accumulation caused by (−)-epicatechin 6 and myricitrin 8.The methanolic extracts and phytochemicals also increased mRNA levels of key regulators of mitochon-drial biogenesis, including adiponectin, PGC-1α, and mtTFA.FL-MeOH treatment showed a consistent increase in PGC-1α and mtTFA levels among the three methanolic extracts.The effect of FL-MeOH on PGC-1α and mtTFA expression is supported by the ability of its isolated compounds, namely naringenin 1 and D-(+)-pinitol 5a, to increase the mRNA expression.All three methanolic extracts slightly reduced the expression of TNF-α and IL-6 at around 10%.However, FL-MeOH was more effective at reducing the expression of IL-6 compared to LF-MeOH and BK-MeOH.The results indicate that these extracts have a weak inhibitory effect on adipocyte pro-inflammatory gene expression.Naringenin 1, compound 2, quercitrin 4, and D-(+)-pinitol 5a are the isolated compounds of FL-MeOH.When tested at a concentration of 10 µM, these compounds showed a slight reduction in TNF-α expression but did not affect IL-6 expression.This suggests that these compounds are responsible for inhibiting TNF-α expression.However, other unisolated compounds in the extract may be responsible for inhibiting IL-6 expression by FL-MeOH.These findings align with the immunoblot analysis outcome, showing that treatment with MeOH extracts increased the expression of GLUT-4 protein in the adipocytes.This suggests that A. saligna extracts and isolated compounds can reduce lipid accumulation, promote adipocytes' mitochondria health, and reduce inflammation, resulting in increased GLUT-4 expression.

Supplementary Materials:
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules28186677/s1:Experimental procedure: Extraction and compound isolation and identification from the flowers, leaves, and bark of Acacia saligna; Table S1: Estimated lipid content from adipogenesis assay with ORO staining agent on the 3T3-L1 adipocytes treated with extracts; Table S2: Estimated lipid content from adipogenesis assay with ORO staining agent on the 3T3-L1 adipocytes treated with isolated compounds within the differentiation process (day-0 to day-8); Table S3: Estimated expression of GLUT-4 normalised by α-tubulin of the 3T3-L1 adipocytes treated with extracts; Figure S1: Original membrane images of Western blot analysis for GLUT-4; Figure S2: Original membrane images of Western blot analysis for α-tubulin; Table S4: Quantitative data of the expression of the target mRNA normalised by β-actin from the RT-qPCR of adipocytes treated with MeOH extracts; Table S5: Quantitative data of the expression of the target mRNA normalised by β-actin from the RT-qPCR of adipocytes treated with MeOH-isolated compounds.

Figure 1 .
Figure1.A proposed link between mitochondrial dysfunction and insulin resistance in an adipocyte model.The increased intracellular ROS triggered by obesity and metabolic challenges (e.g., excess nutrient intake) leads to mt-ROS overproduction, exacerbating the total ROS generation and decreasing MMP and ATP production[13].The impaired mitochondria reflected by the collapse of mt-DNA content and depletion of mitochondrial biogenesis alter adiponectin level, resulting in decreased OXPHOS caused by reduced activation of AMPK and PGC-1α as its regulator[14].In addition, the compromised mitochondria function causes the accumulation of lipid metabolites because of the reduced β-oxidation and the increase in pro-inflammatory cytokines leading to the induction of PKC isoforms and Ser/Thr kinases[15,16].Consequently, the inhibition of the insulin signalling cascade, including the disruption of GLUT-4 transduction, leads to the defect of the insulin-dependent glucose uptake pathway.ROS: reactive oxygen species; mt-ROS: mitochondrial reactive oxygen species; MMP: mitochondrial membrane potential; ATP: adenosine 5 -triphosphate; mt-DNA: mitochondrial DNA; OXPHOS: oxidative phosphorylation; AMPK: adenosine 5 -monophosphate-activated protein kinase; PGC-1α: peroxisome proliferator-activated response-γ coactivator-1α; PKC: protein kinase C.

Table 2 .
List of target Taqman primers used for the RT-qPCR experiments.