HM-Chromanone, a Major Homoisoflavonoid in Portulaca oleracea L., Improves Palmitate-Induced Insulin Resistance by Regulating Phosphorylation of IRS-1 Residues in L6 Skeletal Muscle Cells

This study investigated the effect of (E)-5-hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone (HM-chromanone) on palmitate-induced insulin resistance and elucidated the underlying mechanism in L6 skeletal muscle cells. Glucose uptake was markedly decreased due to palmitate-induced insulin resistance in these cells; however, 10, 25, and 50 µM HM-chromanone remarkably improved glucose uptake in a concentration-dependent manner. HM-chromanone treatment downregulated protein tyrosine phosphatase 1B (PTP1B) and phosphorylation of c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), which increased because of palmitate mediating the insulin-resistance status in cells. HM-chromanone promoted insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and suppressed palmitate-induced phosphorylation of IRS-1 serine. This activated phosphoinositide 3-kinase (PI3K) and stimulated protein kinase B (AKT) phosphorylation. Phosphorylated AKT promoted the translocation of Glucose transporter type 4 to the plasma membrane and significantly enhanced glucose uptake into muscle cells. Additionally, HM-chromanone increased glycogen synthesis through phosphorylating glycogen synthase kinase 3 alpha/beta (GSK3 α/β) via AKT. Consequently, HM-chromanone may improve insulin resistance by downregulating the phosphorylation of IRS-1 serine through inhibition of negative regulators of insulin signaling and inflammation-activated protein kinases in L6 skeletal muscle cells.


Introduction
Insulin is secreted from pancreatic beta cells after food ingestion, thereby regulating blood glucose to a certain value by increasing glucose uptake into muscle cells or inhibiting the production of glucose in the liver [1]. Intracellular glucose uptake in peripheral tissues is regulated through an insulin signaling pathway and glucose transporter [2]. In particular, skeletal muscles are vital tissues in the processing of glucose, and in response to insulin, they process about 80% or more glucose [3]. However, glucose cannot be taken up into muscle cells when insulin resistance occurs, eventually raising blood glucose levels. Insulin resistance is a condition in which insulin action is lower than normal at physiological insulin concentrations [4].
Insulin resistance is a significantly dangerous constituent in the progression of diabetes, and its prevalence is rapidly increasing [5]. Obesity is a direct cause of insulin resistance development, and fat production by obesity releases free fatty acids (FFAs), causing insulin resistance. Patients with type 2 diabetes (T2D) are characterized by elevated plasma FFA levels [6]. The impairment of glucose utilization and insulin signaling has been revealed in studies with FFA treatment. Among the FFAs present in the blood, the most common FFA is palmitate, which is composed of 16-saturated carbons [7]. Insulin-resistance inducers, such as protein tyrosine phosphatase 1B (PTP1B), c-Jun N-terminal kinase (JNK), and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), are phosphorylated by palmitate and cause changes in the insulin receptor substrate-1 (IRS-1) residue, thereby promoting the insulin resistance indicator IRS-1 serine (IRS-1ser) [8].
The presence of IRS-1ser 307 residue in the vicinity of the phosphotyrosine-binding (PTB) domain by an insulin-resistance inducer decreased the binding force between insulin receptor (IR) and IRS-1 and disassociated the coupling of the IRS-1 signal transduction to phosphoinositide 3-kinase (PI3K). The phosphorylation of IRS-1ser 307 is related to decreased IRS-1-tyrosine (IRS-1tyr), phosphorylation, and insulin resistance [9,10]. As a result, increased phosphorylation of IRS-1ser 307 and dephosphorylation of IRS-1tyr 612 reduced glucose transporter type 4 (GLUT4)-mediated glucose uptake in muscles, resulting in insulin resistance in muscle cells [11].
In addition, defects in glycogen synthesis in muscles have been a significant factor in developing postprandial hyperglycemia and insulin resistance in patients with T2D [12,13]. Phosphorylation (inactivation) of glycogen synthase kinase 3 α/β (GSK3 α/β) by insulin leads to dephosphorylation (activation) of glycogen synthase (GS), thereby synthesizing glycogen. An obstacle in any of these elements can lead to insulin resistance [14].
Portulaca-oleracea L. is a Portulacaceae family that is distributed worldwide. Studies have shown that P. oleracea has a variety of bioactivities: antidiabetic, antibacterial, antioxidant, antiatherosclerotic, renal protection, and immune modulation [15][16][17]. (E)-5hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone (HM-chromanone) from P. oleracea has a 16-carbon-skeleton composed of 2 phenyl rings. Our previous study of this compound revealed its efficacy in increasing glucose uptake in 3T3 adipose cells and muscle cells [15]. Despite previous studies, the mechanism by which HM-chromanone mitigates insulin resistance caused by FFAs remains unclear. Therefore, in this study, insulin resistance was induced by treating L6 skeletal muscle cells with palmitate, and the effect and molecular mechanisms of insulin resistance improvement mediated by HM-chromanone were investigated.

Preparation of Palmitate Stock Solution
The palmitate stock solution was prepared by conjugating palmitate with fatty acidfree bovine serum albumin (BSA) [20]. In brief, palmitate was dissolved in 0.1 N sodium hydroxide (NaOH) and diluted in 9.7% (w/v) of previously warmed BSA solution to yield a stock solution of 8 mM palmitate. The molar ratio of free palmitate to BSA was 6:1.

Cell Culture
Skeletal muscle cell line (L6, Korean cell line bank, KCLB No. 21458) was purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in 90% Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) in 95% air and 5% carbon dioxide (CO 2 ) at 37 • C. For differentiation, the cells were allowed to reach confluence, and the medium was replaced with DMEM containing 1% FBS. After differentiation, myotubes were subjected to various treatments, as described below. L6 skeletal muscle

Preparation of Palmitate Stock Solution
The palmitate stock solution was prepared by conjugating palmitate with fatty acidfree bovine serum albumin (BSA) [20]. In brief, palmitate was dissolved in 0.1 N sodium hydroxide (NaOH) and diluted in 9.7% (w/v) of previously warmed BSA solution to yield a stock solution of 8 mM palmitate. The molar ratio of free palmitate to BSA was 6:1.

Cell Culture
Skeletal muscle cell line (L6, Korean cell line bank, KCLB No. 21458) was purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in 90% Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) in 95% air and 5% carbon dioxide (CO2) at 37 °C. For differentiation, the cells were allowed to reach confluence, and the medium was replaced with DMEM containing 1% FBS. After differentiation,

Glycogen Synthesis
Glycogen synthesis measured the conversion of D-[U-14C] glucose to glycogen in a previous study [21]. To sum it up, cells were incubated with palmitate stock solution in the wells of a 96-well for 16 h before incubating with 10, 25, and 50 µM HM-chromanone for 24 h and then incubated with insulin (100 nM) for 20 min. Cells were rapidly washed using phosphate buffered saline (PBS) and lysed by potassium hydroxide (KOH). Lysed cells were glycogen precipitated with ethanol (EtOH) for 24 h. This glycogen was dissolved in water and transferred to a scintillation vial, and the amount of cell lysate was used for protein determination.

Western Blotting Analysis
Cells were washed using PBS and harvested in a lysis buffer to extract total protein from the cells with insulin resistance. A sample was electrophoresed using 10% sodium dodecyl sulphate (SDS)-polyacrylamide gel. Proteins were transferred to membranes by electrophoresis in skim milk (1 h) and incubated with a primary antibody (24 h, 4 • C). Membranes were incubated with a secondary antibody for 60 min. The antigen-antibody was visualized by an enhanced chemiluminescence (ECL) reagent, and chemiluminescence was detected by a LAS-1000 (FUJIFILM, Tokyo, Japan).

Isolation of Plasma Membranes from L6 Skeletal Muscle Cells
The cells were treated with a medium that contained palmitate (0.75 mM) for 16h and HM-chromanone (10, 25, and 50 µM, 24h) or insulin (100 nM, 20 min). The cells were homogenized using a sonicator and centrifuged to remove non-homogenized cellular debris and nuclei from the homogenate. Another centrifugation was performed at 35,000× g for 1 h, and the resulting pellet was used as the plasma membrane fraction of the cells, whereas the supernatant was used as the cytosolic fraction.

Immunostaining and Microscopy
Cells were incubated with primary antibodies (60 min) and then incubated with secondary antibodies for 60 min. A plasma-membrane filter was prepared using sonication as described previously [22]. The background fluorescence was detected using staining membrane sheets with immunoglobulin G. The image was measured by the DeltaVision system and assayed using SoftWoRx (Applied Precision, Rača, Bratislava, Slovakia).

Statistical Analysis
Statistical analyses were performed using SPSS (26, IBM Corp., Armonk, NY, USA). Differences between the groups were evaluated for significance by a one-way analysis of variance followed by Duncan's post hoc tests.
In Figure 5C, HM-chromanone treatment, like insulin treatment, triggered L6 skeletal muscle cells to display a ring-like distribution of GLUT4, suggesting that more GLUT4 molecules are localized in the plasma membrane or beneath the plasma membrane in these cells. There was a significant reduction in the mean fluorescence intensity on the plasma membranes of palmitate-treated L6 skeletal muscle cells. In contrast, HM-chromanone (10, 25, and 50 µM) appeared markedly different from control cells. Especially, the
In Figure 5C, HM-chromanone treatment, like insulin treatment, triggered L6 skeletal muscle cells to display a ring-like distribution of GLUT4, suggesting that more GLUT4 molecules are localized in the plasma membrane or beneath the plasma membrane in these cells. There was a significant reduction in the mean fluorescence intensity on the plasma membranes of palmitate-treated L6 skeletal muscle cells. In contrast, HM-chromanone (10, 25, and 50 µM) appeared markedly different from control cells. Especially, the effects of 50 µM HM-chromanone were almost comparable to the effect of insulin in recruiting GLUT4 to the plasma membrane. effects of 50 µM HM-chromanone were almost comparable to the effect of insulin in recruiting GLUT4 to the plasma membrane.

Discussion
T2D is characterized by defective insulin secretion and insulin resistance [25], which leads to postprandial and fasting hyperglycemia, dyslipidemia, and hyperinsulinemia [26]. Insulin resistance is generally characterized by elevated plasma FFA levels, and elevated plasma FFA inhibits glucose transport in insulin-dependent cells and leads to hyper-glycemia [27]. Obese patients with T2D often show significantly impaired glucose disposal in skeletal muscles [28].
HM-chromanone, isolated from P. oleracea L, exerted an antidiabetic effect and improved insulin sensitivity in vitro [15]. However, little is known about the effects of HMchromanone on insulin resistance by FFAs released in obesity in skeletal muscle cells. Thus, the potential improvement effect and molecular mechanisms of HM-chromanone on insulin resistance by treating L6 muscle cells with palmitate were investigated in this study.
Our results showed that HM-chromanone improved cell viability and promoted glucose uptake in insulin-resistant cells. Promoting glucose uptake into the cells is essential for alleviating high blood glucose levels, mainly because skeletal muscle cells consume more than 80% of plasma glucose. It has been reported that the promotion of glucose uptake into cells depends on the presence of hydroxyl or methoxyl groups in the compound [29]. HM-chromanone has two hydroxyl groups (C-2 and C-5) and one methoxy group (C-7). According to a study, curcumin, a type of phytopolyphenol, significantly enhanced glucose uptake in insulin resistance-induced skeletal muscle cells. Curcumin consists of two hydroxyl groups and two methoxyl groups, and the enhanced glucose uptake was due to the presence of these functional groups [30].
The factors inducing insulin resistance were investigated to clarify the mechanism of action of HM-chromanone in promoting glucose uptake in insulin-resistant cells. When FFAs are increased in obesity, insulin resistance inducers, such as PTP1B, JNK, and IKKβ, are activated, thereby inducing insulin resistance [31]. PTP1B, a negative regulator of insulin signaling, is widely expressed in insulin-sensitive tissues and acts through dephosphorylation of IRS-1tyr residues [32]. Palmitate induces insulin resistance by enhancing PTP1B expression in insulin target tissues [33].
Additionally, the activities of both IKKβ and JNK are elevated in obesity, and these kinases are essential for the production of inflammatory mediators and in the desensitization of insulin signaling [34]. JNK activation leads to phosphorylation of IRS-1ser; this disrupts insulin signaling, leading to insulin resistance, and ultimately contributing to the pathogenesis of T2D [35]. The phosphorylation of both JNK and IKKβ increased in our study, similar to other studies on palmitate-induced insulin-resistant L6 skeletal muscle cells [36]. However, treatment with HM-chromanone inhibited the phosphorylation of JNK and IKKβ in insulin-resistance-induced L6 skeletal muscle cells. Quercetin, a flavonol from the flavonoid group of polyphenols, attenuated phosphorylation of JNK and IKKβ in insulin-resistance-induced L6 skeletal muscle cells [37].
IRS-1ser phosphorylation (i.e., ser307) leads to an insulin signaling blockade by inhibiting IRS-1tyr phosphorylation induced by insulin. In turn, this action inhibits downstream signaling pathways to promote glucose uptake. Among them, it has been reported that serine phosphorylation of IRS-1, an insulin resistance indicator induced by insulin-resistant inducers such as JNK and IKKβ, is critical to developing insulin resistance [38][39][40]. This study demonstrated that FFA elevation resulted in increased phosphorylation of IRS-1ser 307 in L6 skeletal muscle cells. However, after HM-chromanone treatment, the phosphorylation of IRS-1ser 307 decreased, and the phosphorylation of IRS-1tyr612 increased. The decrease in IRS-1ser 307 phosphorylation seems to be due to the inhibition of insulin resistance inducers such as PTP1B, JNK, and IKKβ by HM-chromanone. It has also been reported that inhibition of several kinases, including JNK, IKKβ, and PTP1B, resulted in decreased phosphorylation of IRS-1ser 307 [41,42].
Deng et al. [30] reported that flavonoids inhibited the phosphorylation of IRS-1ser 307 and increased IRS-1tyr phosphorylation, suggesting an ameliorative effect against FFAinduced insulin resistance in C2C12 muscle cells. Polyphenol compounds have a benzene ring structure substituted with a hydroxyl group and are classified into flavonoids and nonflavonoids according to their chemical structure. Flavonoid compounds (such as flavones, flavonols, isoflavones, and homoisoflavonoids) generally have a typical structure of C 6 -C 3 -C 6 and exist as glycosides bound through a hydroxyl group. One study reported that the presence of multiple hydroxyl groups in flavonoids tends to improve glucose uptake through the IRS pathway by modulating IRS-1ser 307 and IRS-1tyr phosphorylation [43,44]. HM-chromanone is a subclass of flavonoids with two hydroxyl groups and has a heterocyclic C 6 -C 3 -C 6 ring structure. Thus, it improves glucose uptake by regulating the IRS-1 residues in insulin-resistant cells due to its functional groups.
Furthermore, AKT directly phosphorylates AS160 and increases glucose uptake by stimulating the translocation of GLUT4 to the PM in skeletal muscle cells [45]. In agreement with previous research [46], the induction of insulin resistance by palmitate treatment reduced AS160 phosphorylation and reduced the PM-GLUT4 content in this study. However, HM-chromanone treatment restored AS160 phosphorylation and enhanced PM-GLUT4 expression in impaired L6 skeletal muscle cells by palmitate.
Impaired glycogen synthesis and decreased glucose uptake have been found in insulinresistant T2D patients [47]. GSK-3 α/β, an essential enzyme for glycogen synthesis, is a substrate of the AKT signaling pathway, and phosphorylation (activation) of GSK-3 α/β is regulated by AKT-mediated phosphorylation [48]. In this study, treatment with HM-chromanone increased GSK-3 α/β phosphorylation, which resulted in the activation of GS and consequently increased glycogen synthesis. Kaempferol 3-neohesperidoside, the major flavonoid found in Bauhinia forficata leaves, stimulated glycogen synthesis in rat muscle, and its stimulatory effect on glycogen synthesis was due to the activation of AKT-GSK3 [49]. Kaempferol has shown that biological activities are highly dependent on the various functional groups attached to the phenolic groups [50]. HM-chromanone has a hydroxyl group in C-1 and C-2 , corresponding to the B-ring, and like kaempferol, it has a double bond between C-4 and C-5 and a hydroxyl group in C-5. Therefore, we suppose that HM-chromanone increases glycogen synthesis due to this structural feature.

Conclusions
In conclusion, HM-chromanone decreased PTP1B, JNK, and IKKβ and then downregulated the phosphorylation of IRS-1ser and upregulated the phosphorylation of IRS-1tyr. Furthermore, HM-chromanone restored PI3K/AKT pathway activation, and glucose uptake was impaired by palmitate. Additionally, the activation of AKT by HM-chromanone led to the phosphorylation of GSK3 α/β and subsequent activation of GS, resulting in an increase in glycogen synthesis. Taken together, HM-chromanone improved palmitate-induced insulin resistance by decreasing insulin resistance inducers and regulating phosphorylation of IRS-1 residues in L6 skeletal muscle cells (Figure 7). HM-chromanone has the potential to be a glucose homeostasis regulating agent and elucidates the potential antidiabetic effect of a substance isolated from natural products. Further research should be conducted on several aspects. In addition, if HM-chromanone is used clinically for medical purposes, a special permit procedure is required, and research on intake as a functional food should be conducted in the future in vivo.  Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflicts of interest.

Conflicts of Interest:
The authors declare no conflict of interest.