Platycodon grandiflorus Root Extract Attenuates Body Fat Mass, Hepatic Steatosis and Insulin Resistance through the Interplay between the Liver and Adipose Tissue

The Platycodon grandiflorus root, a Korean medicinal food, is well known to have beneficial effects on obesity and diabetes. In this study, we demonstrated the metabolic effects of P. grandiflorus root ethanol extract (PGE), which is rich in platycodins, on diet-induced obesity. C57BL/6J mice (four-week-old males) were fed a normal diet (16.58% of kilocalories from fat), high-fat diet (HFD, 60% of kilocalories from fat), and HFD supplemented with 5% (w/w) PGE. In the HFD-fed mice, PGE markedly suppressed the body weight gain and white fat mass to normal control level, with simultaneous increase in the expression of thermogenic genes (such as SIRT1, PPARα, PGC1α, and UCP1), that accompanied changes in fatty acid oxidation (FAO) and energy expenditure. In addition, PGE improved insulin sensitivity through activation of the PPARγ expression, which upregulates adiponectin while decreasing leptin gene expression in adipocytes. Furthermore, PGE improved hepatic steatosis by suppressing hepatic lipogenesis while increasing expression of FAO-associated genes such as PGC1α. PGE normalized body fat and body weight, which is likely associated with the increased energy expenditure and thermogenic gene expression. PGE can protect from HFD-induced insulin resistance, and hepatic steatosis by controlling lipid and glucose metabolism.


Introduction
Obesity is defined as excessive fat accumulation that may have adverse effects on health, such as type 2 diabetes, insulin resistance, atherosclerosis, dyslipidemia, hepatic steatosis, and cancer [1,2]. Elevated triglycerides, blood pressure, and fasting glucose levels, as well as reduced HDL cholesterol levels, are recognized as major risk factors for these disorders [3]. Obesity-induced inflammation, a

PGE Supplement Lowered Body Weight Gain and Improved Plasma Lipid Profiles and Adipokine Levels in DIO Mice
PGE significantly suppressed the body weight gain from the first week of high-fat feeding and decreased the food efficiency ratio, with no difference in food intake ( Figure 1A). Interestingly, the addition of PGE led to a significant reduction of body weight compared to that in the HFD group, bringing it to the level similar to that in the ND group. PGE significantly decreased not only the total cholesterol (TC) and non-HDL cholesterol levels but also the apolipoprotein (Apo) B levels compared to those in the HFD group. The levels of plasma triglycerides (TG), and FFA were also markedly decreased by the PGE treatment compared to those in the HFD group ( Figure 1B). Similar to the results for plasma lipid profiles, PGE significantly lowered the plasma adipokine levels, such as resistin, leptin, TNF-α, and plasminogen activator inhibitor-1 (PAI-1) levels ( Figure 1C).

PGE Improved Insulin Resistance and Glucose Tolerance by Modulating Activities of Hepatic Glucose-Regulating Enzymes in DIO Mice
The fasting blood glucose concentration was significantly lowered by the PGE treatment after two weeks of high-fat feeding ( Figure 1D). PGE significantly decreased the plasma insulin level as well as the HOMA-IR ( Figure 1E). Moreover, the IPGTT revealed that PGE significantly improved glucose tolerance ( Figure 1F), indicating a decrease in insulin resistance. Hepatic PEPCK and G6Pse activities were suppressed by the PGE treatment ( Figure 1G).

PGE Supplement Decreased Body Fat Mass by Increasing Fatty Acid Oxidation-Related Gene Expression and Energy Expenditure in DIO Mice
The PGE-treated mice showed significantly upregulated mRNA expression levels for a key lipogenic gene, PPARγ, with simultaneous increases in mRNA expression of sterol regulatory element-binding protein 1c (SREBP-1c), stearoyl-CoA desaturase-1 (SCD1), and acetyl-CoA carboxylase 1 (ACC1) in eWAT ( Figure 2D). However, similar to the results for body weight, PGE significantly reduced the weight of all WAT depots (including epididymal, perirenal, retroperitoneal, mesenteric, subcutaneous, and interscapular depots), with a decrease in the epididymal adipocyte size compared with the data obtained for the HFD group of the DIO mice (Figure 2A,B). In addition, whole-body oxygen consumption and energy expenditure of PGE-treat mice were significantly increased relative to those of HFD of the DIO mice ( Figure 2C). Notably, PGE markedly reduced the weight of all adipose tissue depots to the level of that in the ND group. Importantly, the body fat reduction by the PGE treatment was associated with a significant increase in the expression of thermogenic genes, including the SIRT1, PPARα, PGC1α, and UCP1 genes ( Figure 2E). Additionally, the PGE supplementation led to a significant increase in mRNA expression of adiponectin, with a decrease in the TNFα and leptin mRNA expression ( Figure 2F). Western blot analysis revealed that the protein expression of the lipogenic and FA uptake factors, such as PPARγ and CD36, as well as FA oxidation related protein factors PGC1α and CPT2 (carnitine palmitoyltransferase 2) were markedly increased in the PGE-fed mice when compared with their expression in the eWAT of the HFD mice ( Figure 2G). bringing it to the level similar to that in the ND group. PGE significantly decreased not only the total cholesterol (TC) and non-HDL cholesterol levels but also the apolipoprotein (Apo) B levels compared to those in the HFD group. The levels of plasma triglycerides (TG), and FFA were also markedly decreased by the PGE treatment compared to those in the HFD group ( Figure 1B). Similar to the results for plasma lipid profiles, PGE significantly lowered the plasma adipokine levels, such as resistin, leptin, TNF-α, and plasminogen activator inhibitor-1 (PAI-1) levels ( Figure 1C).

PGE Improved Insulin Resistance and Glucose Tolerance by Modulating Activities of Hepatic Glucose-Regulating Enzymes in DIO Mice
The fasting blood glucose concentration was significantly lowered by the PGE treatment after two weeks of high-fat feeding ( Figure 1D). PGE significantly decreased the plasma insulin level as well as the HOMA-IR ( Figure 1E). Moreover, the IPGTT revealed that PGE significantly improved glucose tolerance ( Figure 1F), indicating a decrease in insulin resistance. Hepatic PEPCK and G6Pse activities were suppressed by the PGE treatment ( Figure 1G).

PGE Supplement Decreased Body Fat Mass by Increasing Fatty Acid Oxidation-Related Gene Expression and Energy Expenditure in DIO Mice
The PGE-treated mice showed significantly upregulated mRNA expression levels for a key lipogenic gene, PPARγ, with simultaneous increases in mRNA expression of sterol regulatory element-binding protein 1c (SREBP-1c), stearoyl-CoA desaturase-1 (SCD1), and acetyl-CoA carboxylase 1 (ACC1) in eWAT ( Figure 2D). However, similar to the results for body weight, PGE significantly reduced the weight of all WAT depots (including epididymal, perirenal, retroperitoneal, mesenteric, subcutaneous, and interscapular depots), with a decrease in the epididymal adipocyte size compared with the data obtained for the HFD group of the DIO mice ( Figure 2A,B). In addition, whole-body oxygen consumption and energy expenditure of PGE-treat mice were significantly increased relative to those of HFD of the DIO mice ( Figure 2C). Notably, PGE markedly reduced the weight of all adipose tissue depots to the level of that in the ND group. Importantly, the body fat reduction by the PGE treatment was associated with a significant increase in the expression of thermogenic genes, including the SIRT1, PPARα, PGC1α, and UCP1 genes ( Figure 2E). Additionally, the PGE supplementation led to a significant increase in mRNA expression of adiponectin, with a decrease in the TNFα and leptin mRNA expression ( Figure 2F). Western blot analysis revealed that the protein expression of the lipogenic and FA uptake factors, such as PPARγ and CD36, as well as FA oxidation related protein factors PGC1α and CPT2 (carnitine palmitoyltransferase 2) were markedly increased in the PGE-fed mice when compared with their expression in the eWAT of the HFD mice ( Figure 2G).

PGE Supplement Lowered the Levels of Hepatic Lipids and Lipotoxicity Markers by Altering Hepatic Lipogenic Gene Expression and Enzyme Activities in DIO Mice
The PGE treatment markedly decreased the hepatic cholesterol, TG, and FFA content, as well as glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels in plasma ( Figure 3A,C). In addition, PGE significantly decreased the mRNA expression of the genes and transcription factors involved in lipogenesis and cholesterol synthesis, with a simultaneous increase in FAO in the liver of the DIO mice ( Figure 3D). Tissue morphology analysis also revealed that the accumulation of hepatic lipids was dropped and the cell size was decreased in the PGE group compared with those in the HFD group ( Figure 3B). Furthermore, activities of the hepatic enzymes involved in FA and TG synthesis (FAS, G6PD, ME, and PAP) were significantly decreased by the PGE treatment, with a significant increase in the activity of β-oxidation in the liver ( Figure 3E) compared with that in the HFD group.

PGE Supplement Lowered the Levels of Hepatic Lipids and Lipotoxicity Markers by Altering Hepatic Lipogenic Gene Expression and Enzyme Activities in DIO Mice
The PGE treatment markedly decreased the hepatic cholesterol, TG, and FFA content, as well as glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels in plasma ( Figure 3A,C). In addition, PGE significantly decreased the mRNA expression of the genes and transcription factors involved in lipogenesis and cholesterol synthesis, with a simultaneous increase in FAO in the liver of the DIO mice ( Figure 3D). Tissue morphology analysis also revealed that the accumulation of hepatic lipids was dropped and the cell size was decreased in the PGE group compared with those in the HFD group ( Figure 3B). Furthermore, activities of the hepatic enzymes involved in FA and TG synthesis (FAS, G6PD, ME, and PAP) were significantly decreased by the PGE treatment, with a significant increase in the activity of β-oxidation in the liver ( Figure 3E) compared with that in the HFD group.

PGE Supplement Lowered the Levels of Hepatic Lipids and Lipotoxicity Markers by Altering Hepatic Lipogenic Gene Expression and Enzyme Activities in DIO Mice
The PGE treatment markedly decreased the hepatic cholesterol, TG, and FFA content, as well as glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels in plasma ( Figure 3A,C). In addition, PGE significantly decreased the mRNA expression of the genes and transcription factors involved in lipogenesis and cholesterol synthesis, with a simultaneous increase in FAO in the liver of the DIO mice ( Figure 3D). Tissue morphology analysis also revealed that the accumulation of hepatic lipids was dropped and the cell size was decreased in the PGE group compared with those in the HFD group ( Figure 3B). Furthermore, activities of the hepatic enzymes involved in FA and TG synthesis (FAS, G6PD, ME, and PAP) were significantly decreased by the PGE treatment, with a significant increase in the activity of β-oxidation in the liver ( Figure 3E) compared with that in the HFD group.

PGE Normalized Body Weight Gain and Fat Mass and Increased Expression of Browning Markers in eWAT
Brown adipose tissue (BAT) is a specialized tissue that dissipates energy in the form of heat (nonshivering thermogenesis) by uncoupling FAO from the ATP production via uncoupling protein 1 (UCP1) in mitochondria to protect against obesity [13]. In contrast, WAT does not normally express UCP1 and is the main storage site of excess energy, primarily in the form of triglycerides, via the uptake of lipogenic substrates from the diet and de novo lipogenesis. Abnormal regulation of adipocyte differentiation, as well as lipogenesis, is linked to obesity [14]. Recently, it has been reported that in the obese, white adipocytes, known as "brite/beige" adipocytes (WAT cells that acquire a brown adipose phenotype, i.e., browning), can exhibit brown adipocyte-like characteristics by increasing expression of brown (thermogenic) genes, such as SIRT1, UCP1, PGC1α, and PPARα [7,8]. An increase in the abundance of brite/beige adipocytes in WAT has been linked to the resistance to diet-induced obesity, with improved insulin resistance and increased energy expenditure, similar to the effects provided by the antidiabetic drug thiazolidinedione [15].
P. grandiflorus root (PG) has long been used as a traditional medicine and as a food in Asia. The major bioactivities components of PG are triterpenoid saponins (platycosides), such as platycodin A, D, and E, and platyconic acid, that may act individually, or in synergy to improve human health, including anti-obesity effects, anti-hyperlipidemia, and anti-inflammation [9][10][11].
In this study, the PGE treatment led to elevated lipogenesis through upregulation of PPARγ, thereby increasing the FA synthesis-associated gene expression (SREBP1c, SCD1, and ACC1), as well as increased FA uptake protein CD36 expression in eWAT. Thus, PGE led to increased FA re-esterification into newly synthesized TG by enhancing the adipose FA uptake and SREBP1c, SCD1, and ACC1 genes. Interestingly, despite the activation of the lipogenic genes involved in FA synthesis in eWAT, the PGE supplement markedly decreased the weights of all WAT depots, as well as the body weight. This effect seemed to be associated with the increase in the mRNA expression of SIRT1, which is involved in thermogenesis and in the enhancement of the mRNA expression of FAO-associated genes genes and protein (PPARα and PGC1α). Our data showed the upregulation of SIRT1, PPARα gene expression, as well as increased PGC1α gene/protein expression by the PGE treatment. In addition, PGE supplementation increased expression of fatty acid oxidation related protein in eWAT, such as CPT2, compared to other HFD group. In rodents, activation of SIRT1 in WAT promotes FAO by increasing the levels of PPARα and its coactivator, PGC1α [8,16]. PGC1α is an important transcriptional coactivator for the expression of the UCP1 gene, biogenesis of mitochondria, and energy expenditure in WAT [17]. Downregulation of PGC1α is associated with obesity and an increased risk of diabetes mellitus in the human population [18]. Additionally, obese animals treated with PPARα agonists can benefit from thermogenesis induction, as the PGC1α-dependent myokine irisin which act through PPARα to activate UCP1 [19]. UCP1-mediated thermogenesis in WAT plays an important role in the regulation of energy expenditure [13]. Furthermore, UCP1 is a major determinant of WAT thermogenic activity. Therefore, the increase of energy expenditure in the PGE-treated mice, associated with a significant increase in the mRNA expression of UCP1, can lead to the browning of WAT, which promotes thermogenesis and energy expenditure. In the obese, excessive lipid storage in WAT has been considered a key reason for obesity-associated insulin resistance and hepatosteatosis [20]. Taken together, it is plausible that PGE can contribute to browning of WAT in DIO mice via inducing FOA related genes (SIRT1, PPARα, PGC1α, and UCP1) and proteins (PGC1α, and CPT2). Therefore, PGE can inhibit lipid accumulation via activation of FA oxidation in eWAT, despite increased of lipogenesis.

PGE Lowered Inflammatory Adipokines and Improved Insulin Resistance and Hepatic Steatosis by Alteration of Lipogenic Gene Expression and Glucose Metabolism
Furthermore, activation of PPARγ regulates the expression of adipocyte-secreted transcriptional factors such as adiponectin and leptin in WAT, which act as insulin sensitizers by potentiating insulin signaling in adipocytes [21,22]. Therefore, high adiponectin and/or low leptin levels can enhance insulin sensitivity in WAT and increase FAO in the liver, thus leading to the improvement of diabetes [15].
In our study, the increase in insulin sensitivity by the PGE treatment could also be related to PPARγ activation that led to an increase in adiponectin mRNA expression and a decrease in that of leptin in WAT. Elevation of adiponectin levels was correlated with a reduction of the hepatic fat content [23], similar to the effect of the PGE treatment. The hepatic TG and TC levels were significantly decreased compared with those in the DIO mice, which was accompanied by increased expression of browning genes in eWAT. Furthermore, the cell sizes and weight of the liver in the PGE-treated mice were significantly lower than those in the HFD group owing to the decreased levels of hepatic lipogenic mRNA expression (FAS, SCD1, HMGCR, and ACAT), some enzyme activities (G6PD, FAS, ME, and PAP), and the major transcriptional regulators, SREBP1 and SREBP2. Besides, the PGE treatment enhanced β-oxidation enzyme activity by increasing the expression of PGC1α mRNA, which in turn induced a significant decrease in hepatic LD formation and accumulation. In particular, the excessive release of FAs form dysfunctional and insulin-resistant adipocytes results in lipotoxicity, causing the accumulation of TG-derived toxic metabolites in liver [24,25]. PGE significantly reduced the plasma FFA level with a simultaneous increase in adipocyte FA uptake CD36 protein expression, leading to the reduction of hepatic lipotoxicity via the increased FFA flux to the liver.
In addition, increased accumulation of adipose tissue is accompanied by chronic adipose inflammation, which has been proposed to have an important role in the pathogenesis of obesity-related insulin resistance [4]. PGE significantly increased the adiponectin level, with a simultaneous decrease in the levels of leptin and TNF-α in adipocytes, which enhanced insulin sensitivity. Consistent with these findings, the PGE supplement reduced circulating levels of two inflammatory markers, TNF-α and resistin. Resistin is elevated in obesity and insulin resistance, and its deficiency in the mice led to improved glucose homeostasis. The PGE supplementation led to the normalization of plasma glucose and insulin levels, reflecting improved hepatic insulin sensitivity as evidenced by the reduced HOMA-IR and by the IPGTT data. In addition, PGE significantly decreased activities of hepatic glucose-regulating enzymes (PEPCK and G6Pase).
In recent years, studies have demonstrated that platycosides from the PG showed anti-obesitic, hypolipidemic and anti-diabetic properties that occurred via enhanced insulin sensitivity through decrease of plasma TC, glucose and insulin levels as well as hepatic cholesterol and TG in obese rodents [9][10][11]. This could probably be due to the difference in PGE preparation and composition. Moreover, suppression of body weight gain, body fat reduction, and improvement in insulin sensitivity by PGE supplementation were more potent in PGE in our study. These observations indicated that PGE has the potential to regulate glucose metabolism and hepatic lipid metabolism, thereby ameliorating hepatic steatosis and hyperglycemia in DIO mice. Importantly, an increase in WAT thermogenesis or browning of WAT can be accompanied by improved glucose homeostasis in vivo [26], similar to the effect of the PGE treatment.

Conclusions
This study demonstrated that the PGE treatment normalized the body weight and body fat mass in the HFD-fed mice by increasing FAO. This partly changed the levels of thermogenesis-related genes such as UCP1 as well as PPARα, PGC1α, and SIRT1 and thus could promote BAT-like features in WAT, as well as enhance energy expenditure. Furthermore, reducing the WAT mass by the PGE treatment enhanced the susceptibility to developing hepatic steatosis and insulin resistance.   is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2072-6643/8/9/532/s1, Table S1: Compounds of ethanol extracts of Platycodon grandiflorum. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Conflicts of Interest:
The authors have declared no conflict of interest.