High glucose promotes benign prostatic hyperplasia by downregulating PDK4 expression

As men age, a growing number develop benign prostatic hyperplasia (BPH). According to previous research, diabetes may be a risk factor. Pyruvate dehydrogenase kinase 4 (PDK4) is closely related to glucose metabolism and plays a role in the onset and progression of numerous illnesses. This study aimed to determine the direct effects of high glucose environment on prostate epithelial cells, in particular by altering PDK4 expression levels. In this investigation, normal prostatic epithelial cells (RWPE-1) and human benign prostatic hyperplasia epithelial cells (BPH-1) were treated with 50 mM glucose to show the alteration of high glucose in prostate cells. PDK4-target siRNA, PDK4-expression plasmid were used to investigate the effects of PDK4. Rosiglitazone (RG), a PPARγ agonist, with the potential to up-regulate PDK4 expression was also used for treating prostate cells. The expression of PDK4 in human prostate samples was also analyzed. The effects of high glucose therapy on BPH-1 and RWPE-1 cells were demonstrated to enhance proliferation, epithelial-mesenchymal transition (EMT), suppress apoptosis, and down-regulate PDK4 expression. Additionally, diabetes-related BPH patients had reduced PDK4 expression. Following the application of PDK4-target siRNA, a comparable outcome was seen. The PDK4-expression plasmid therapy, however, produced the opposite results. RG with the ability to elevate PDK4 expression might be used to treat BPH. Changes in the metabolism of lipids and glucose may be the cause of these consequences. These findings showed that high glucose treatment might facilitate BPH development, and may be related to the down-regulation of PDK4. PDK4 might be a potential therapeutic target of BPH.


Cell counting Kit-8 (CCK-8) assay
BPH-1 and RWPE-1 cells were seeded in 96-well plates at the density of 3 × 10 3 , 1 × 10 4 cells/well, respectively, for 24 h.After adherence, replaced the medium that contained various concentrations of Rosiglitazone (RG) and cultured for 24 h.An identical volume of DMSO served as the negative control.The cells were then treated with 10% CCK8 reagent (Yeasen Biotech Co., Ltd., Shanghai, China) for 3 h at 37 ºC.The optical density (OD) at 450 nm was then measured.Each concentration was tested in triplicate to ensure accuracy and reproducibility.

Transient transfection
The sequences of the siRNA used to target PDK4 are as follows: sense 5′-CTA CTC GGA TGC TGA TGA A-3′, antisense 5′-TTC ATC AGC ATC CGA GTA G-3′.The siRNA control sequence was provided by Guangzhou RiboBio Co., Ltd.EGFP-tagged PDK4-expression construct, GV208-PDK4-EGFP, and the EGFP-tagged empty vector construct, GV208-EGFP were provided by Shanghai Genechem Co., Ltd.PDK4-expression plasmid or siRNA were transiently transfected into the cells using Lipofectamine 3000.After transfection for 72 h, cells were collected for corresponding experiments.

Lactate assay
Lactate production was measured using a Lactic acid assay kit (Jiancheng Bio, Nanjing, China) according to the manufacturer's protocol.The optical density (OD) was measured at a wavelength of 530 nm and calculated according to specifications to reflect the lactate level.The relative value of the treatment group was computed after giving the control group a value of 1.

Pyruvate assay
Pyruvate was measured using a Pyruvate assay kit (Jiancheng Bio) according to the manufacturer's protocol.The optical density (OD) was measured at a wavelength of 505 nm and calculated according to specifications to reflect the pyruvate level.The relative value of the treatment group was computed after giving the control group a value of 1.

Immunohistochemical staining
The patients with simple BPH and BPH complicated with T2DM were enrolled (n = 3/group).The sections of their prostate transition zone were deparaffinized in gradient xylene.Then, the sections were treated with the 3% hydrogen peroxide (H 2 O 2 ) solution for 10 min to exhaust the endogenous peroxidase activity, and the sections were blocked with 10% normal goat serum for 1 h.Afterward, the sections were incubated with antibodies against PDK4 (1:100; Abclone) at 4 ℃ overnight.The sections were then incubated with biotinylated goat anti-rabbit secondary antibody (1:200; ProteinTech Group) for 30 min and reacted with 3,3-diaminobenzidine (DAB) for 10 min.At last, hematoxylin was used for counterstaining.After staining, the sections were observed under a light microscope (200 × , 400 ×).

Statistical analysis
GraphPad Prism 8 software was used to proceed with statistical analysis.The results were expressed as mean ± standard deviation (SD) from at least three independent experiments.An unpaired student t-test was applied to compare the difference between groups.P < 0.05 was considered statistically significant.

Consent for publication
All authors have read and agreed to publish this manuscript.

High glucose promoted prostatic epithelial cells proliferation, epithelial-mesenchymal transition (EMT), and suppressed apoptosis
BPH-1 and RWPE-1 cells were treated with 50 mM glucose for 24 h to investigate the influence of high glucose on prostatic epithelial cells.The concentration of glucose was referred to others previous work 9,19 .Cell viability was determined using EdU methods.After treatment with high glucose, the proliferation of BPH-1 and RWPE-1 significantly promoted.(Fig. 1A,B) The expressions of epithelial cell markers (E-cad, ZO-1) and mesenchymal cell marker (α-SMA) were detected by western blot to show the influence of high glucose on EMT, and the significant down-regulation of E-cad and ZO-1 indicated that EMT progressed under a high glucose environment.(Fig. 1C-E) Similarly, the decreased expression level of pro-apoptotic genes (BAX, Caspase3) indicated reduced apoptosis incubating with a high glucose medium.(Fig. 1F-H).

Down-regulation of PDK4 might participate in the high glucose regulation effects
We examined the expression of PDK4 in non-diabetes and diabetes BPH patients (n = 3/group) prostate epithelial cells of transition zone by immunohistochemical staining, and we found that compare with non-diabetes patients, the expression of PDK4 lower in diabetes patients.(Fig. 2A,B) In vitro, the expression of PDK4 was also significantly decreased after treatment with high glucose in BPH-1 and RWPE-1 cells (Fig. 2C,D).These results indicated that the down-regulation of PDK4 might participate in the high glucose regulation effects.

Regulated PDK4 expression in prostatic epithelial cells influence the level of proliferation, EMT, and apoptosis
We transfected PDK4-target siRNA into BPH-1 and RWPE-1 cells, and the transfection efficiency was confirmed by western blot after transfecting for 72 h (Fig. 3A,B).Then the proliferation was determined using EdU methods.The results showed that after transfecting for 72 h, the down-regulation of PDK4 significantly promoted the proliferation of BPH-1 and RWPE-1 cells (Fig. 3C,D).Besides proliferation, the influence on EMT and apoptosis were detected by western blot.The siRNA treatment significantly decreased the expression of E-cad, ZO-1, BAX, and Caspase3, which indicated the progression of EMT (Fig. 3E-G) and inhibited apoptosis (Fig. 3H-J).
To further confirm the effects of PDK4 on prostatic epithelial cells, we transfected the PDK4-expression plasmid into BPH-1 cells, and the transfection efficiency was confirmed by western blot after transfected for 72 h (Fig. 4A,B).Then we repeated the experiments mentioned above, and found that overexpression of PDK4 in prostatic hyperplastic epithelial cells could suppress proliferation (Fig. 4C,D), inhibit EMT (Fig. 4E-F), and induce apoptosis (Fig. 4G-H).

RG could increase the expression of PDK4 in prostatic epithelial cells and with the ability to influence the level of proliferation, EMT, and apoptosis
Rosiglitazone (RG), the agonist of PPARγ and a common hypoglycemic drug, has been reported to increase the level of PDK4 20,21 .To determine the impact of RG on the therapy of BPH, we subject BPH-1 and RWPE-1 cells to a variety of RG concentrations and 50 mM Glu.The CCK8 assay results demonstrated that RG could reduce the viability of BPH-1 and RWPE-1 cells after treatment for 24 h, and that this effect was positively linked with concentration (Fig. 5A).Through the application of the western blot, we discovered that RG could enhance PDK4 expression in BPH-1 and RWPE-1 cells (Fig. 5B).The effect of RG on EMT and apoptosis were also demonstrated by western blot.RG treatment significantly decreased the expression of α-SMA in BPH-1 cells and decreased the expression of α-SMA, increased the expression of E-cad and ZO-1 in RWPE-1 cells which indicated that RG Vol:.( 1234567890 www.nature.com/scientificreports/could suppress the progression of EMT (Fig. 5C-D).And the down-regulation of BCL2 in BPH-1 and RWPE-1 cells indicated that RG could enhance apoptosis (Fig. 5E-F).

The glucose and lipid metabolism affected by high glucose and PDK4 regulation in prostatic epithelial cells
To verify the underlying mechanism of high glucose and PDK4 regulation in prostatic epithelial cells, the level of glucose and lipid metabolism were examined.High glucose and PDK4-target siRNA treatment showed decreased pyruvate and lactate content intracellular in BPH-1 and RWPE-1 cells.While the content of citrate increased in BPH-1 cells and decreased in RWPE-1 cells.These changes indicated indirectly that high glucose might downregulate PDK4 expression, consequently, promote cellular aerobic glucose metabolism.The different changes in citrate content might due to the differences in the ability to handle citric acid.However, PDK4-expression plasmid increased pyruvate and lactate content, decreased citrate content intracellular in BPH-1 (Fig. 6A-C).This further confirmed that PDK4 plays a crucial role in the regulation of prostatic epithelial cells glucose metabolism.
We also examined several key enzymes in glucose and lipid metabolism by RT-PCR.Carnitine Octanoyltransferase (CROT) catalyzes the reversible transfer of fatty acyl groups between CoA and carnitine, www.nature.com/scientificreports/palmitoyltransferase II (CPT2) catalyzes the transferring of long-chain fatty acids, they are the key enzymes of β-oxidation of fatty acids.Results showed that the expression of CROT and CPT2 significantly down-regulated after high glucose and siRNA treatment, which suggested that the β-oxidation of fatty acids be inhibited 22,23 .CD36 significantly up-regulated, which indicated the transportation of fatty acids had been facilitated 24 .However, PDK4-overexpression exhibited the opposite trend (Fig. 6D-E).Taken together, these results indicated that PDK4 regulates glucose and lipid metabolism and may contribute to the progression of BPH under high glucose solution.

Discussion
The present study demonstrated that high glucose treatment could induce proliferation and EMT, and suppress apoptosis in prostatic epithelial cells.These effects might relate to the downregulation of PDK4.
BPH, is one of the most common prostate diseases in old-age males, with the annoying lower urinary tract symptoms (LUTS) significantly disturbing patients' quality of life.The precise etiology of BPH needs further exploration, however, the association between BPH and diabetes has been reported by numerous studies 8,25 .However, the direct influence of high glucose in the prostate remains insufficient.Ye et al. 19,26 found that under a high glucose environment, the level of reactive oxygen species (ROS) significantly up-regulated, which induced the proliferation, and suppressed the apoptosis of prostate epithelial cells.In this study, we confirmed that high glucose could affect the proliferation and apoptosis level by EdU and western blot experiment.In the meantime, we demonstrated that high glucose could accelerate the process of EMT, which is in conformity with the results of Yang et al. 9 .
PDK4 is one of the pyruvate dehydrogenase kinase family isoenzymes, involved in glucose metabolism, it inhibits oxidative phosphorylation and affects energy production by phosphorylating pyruvate dehydrogenase complex (PDC) 20 .As the major nutrient of glucose metabolism, the level of glucose could regulate the expression of PDK4 20 .Additionally, PDK4 can affect substance metabolism, and the decrease of PDK4 can promote fatty acid synthesis and amino acid synthesis 27 .The increased expression of PDK4 served as a characteristic of the Warburg effect, and had been found with oncogenic effects in several cancers 28,29 .However, the expression of PDK4 in some cancers, which benefit from a high level of OXPHOS activity is down-regulated [30][31][32] .Particularly,   www.nature.com/scientificreports/consistent with the lacking the Warburg effect, it had been reported that the expression of PDK4 is significantly down-regulated in prostate cancer and is related to tumor recurrence, and drug resistance 16,33 .Beyond that, it had been reported the overexpression of PDK4 could restrain EMT induced by TGFβ 33 .Which had been proven with significant importance in BPH development 34 .Based on these previous researches we surmised that the down-regulation of PDK4 has a role in the development of BPH.
To explore the possible mechanism of how high glucose affects prostate epithelial cells, we examined the expression of PDK4 in non-diabetes, diabetes BPH patients' prostates, and in high glucose treated BPH-1 and RWPE-1 cells.The results indicated that high glucose might decrease the expression of PDK4.This led us to further explore the effects of the downregulation of PDK4 on BPH progression.
As a proliferative disease, abnormal dynamic proliferation and limited apoptosis are the essential characteristics of BPH 35 .By transfection of PDK4-target siRNA into BPH-1, RWPE-1 cells we found that the EdU positive cells increased, which indicated a stronger proliferation ability.Meanwhile, the level of apoptosis was decreased, as the expression of pro-apoptosis genes (BAX, Caspase3) decreased.These results supported that the downregulation of PDK4 could stimulate BPH development.
The decreasing of epithelial cell markers such as E-cad and ZO-1, and the increasing of mesenchymal cell markers such as N-cadherin and α-SMA are generally referred to as EMT 36 .Upon the progression of EMT, the mesenchymal-like cells increased which with stronger proliferation and resisted apoptosis 37 .Recent findings indicated that the change in glycolysis and OXPHOS are intertwined with EMT 38 .PDK4 as a key enzyme of glycolysis, had been reported with the ability to adjust EMT 33 .In this study, we found that the treatment of PDK4-target SiRNA could decrease the expression of E-cad and ZO-1.
To further confirmed the effects of PDK4 regulation on BPH progression and the possible therapeutic effects, we transfected the PDK4 expression plasmid into the BPH-1 cells.The results showed that overexpression of PDK4 in the prostatic hyperplasia cell line could suppress the progression of BPH, manifested as a lower level of proliferation, higher level of apoptosis, and limited EMT.This means drugs target PDK4 might with the potential for the treatment of BPH.And we discovered that RG with the potential to upregulate PDK4 might decrease prostate proliferation and EMT while promoting apoptosis.
Besides the common features of BPH, the content of pyruvic acid, lactic acid, and citric acid was measured tried to have a better understanding of the direct influence of high glucose and PDK4 regulation on BPH development.Our results showed a decreased level of pyruvic acid and lactic acid under high glucose or siRNA treatment, which might indicate a reduction of anaerobic glycolysis and an increase in the TCA cycle.The treatment of PDK4-expression plasmid showed opposite changes, which further confirmed the effects of PDK4 on glucose metabolism.However, changes in the citric acid in the two cell lines are different, which may be due to the different enzymatic activity of the TCA cycle.Since PDK4 is involved in the regulation of fatty metabolism 26 , we also measured the expression of mRNA of key enzymes of lipid metabolism.The PCR showed, the expression of CD36 was significantly up-regulated under high glucose or siRNA treatment, and significantly down-regulated under PDK4-expression plasmid treatment.CD36 is a scavenger receptor expressed in cell membranes that participate in lipid uptake through binds to diverse ligands that could accelerate cells proliferation 39 .These results indicated that the influence of PDK4 in BPH progression might be related to the adjustment of glucose and lipid metabolism.
In this study, we show that the down-regulation of PDK4 might involve in the high glucose effects in BPH development for the first time.However, the specific mechanism had only been briefly discussed, further experiments are needed to show the overall metabolic changes and signal pathways that are related to these effects.

Conclusion
High glucose treatment could accelerate prostate epithelial cells proliferation, EMT, and suppress apoptosis.The down-regulation of PDK4 might be involved in these effects.

Figure 1 .
Figure 1.High glucose promoted prostatic epithelial cells proliferation, epithelial-mesenchymal transition (EMT) and suppressed apoptosis.BPH-1 and RWPE-1 cells were treated with ordinary culture medium or high glucose culture medium (50 mM Glu) for 24 h to evaluate the influence of the high glucose.(A-B) The cell proliferation was measured by EdU assay.(C-E) the protein expression of ZO-1, E-cad, and α-SMA calculated by western blot was used to evaluate the process of EMT.(F-H) the protein expression of BCL2, BAX, and Caspase3 was used to show the level of apoptosis.*P < 0.05; **P < 0.01; ***P < 0.001.NC = normal control, 50 mM Glu = 50 Millimoles per liter of glucose.

Figure 2 .Figure 3 .
Figure 2. High glucose downregulation the expression of PDK4 in prostate epithelial cells.(A) The expression of PDK4 in prostate epithelial cells of non-diabetes and diabetes BPH patients was detected by immunohistochemical staining, then observed by optical microscope under 200 × , 400 × .(B) The level of fasting blood glucose in non-diabetes and diabetes BPH patients.(C-D) Western blot analysis of PDK4 expression in BPH-1, RWPE-1 cells after treating 50mM Glu 24 h.*P < 0.05; ***P < 0.001.NC = normal control, 50 mM Glu = 50 Millimoles per liter of glucose.

Figure 4 .
Figure 4. PDK4-expression plasmid upregulated the expression of PDK4 in BPH-1 cells influencing the level of proliferation, EMT, and apoptosis.(A-B) The expression of PDK4 in BPH-1 cells after treating with negative control or PDK4-expression plasmid for 72 h.(C-D) The influence of PDK4 up-regulation on cellration was measured by EdU assay.(E-F) The protein expression of ZO-1, E-cad, and α-SMA calculated by western blot was used to evaluate the influence of PDK4 up-regulation on EMT.(G-H) the protein expression of BCL2, BAX, and Caspase3 was calculated by western blot to show the influence of PDK4 up-regulation on the level of apoptosis.*P < 0.05; **P < 0.01; ***P < 0.001.NC = negative control of plasmid, OE = overexpression plasmid.

Figure 5 .Figure 6 .
Figure 5. RG could increase the expression of PDK4 in prostatic epithelial cells and with the ability to influence the level of proliferation, EMT, and apoptosis.(A) The value of OD 450 nm in BPH-1 and RWPE-1 cells after treating with 50 mM Glu and different concentrations of RG for 24 h.(B) The expression of PDK4 in BPH-1 and RWPE-1 cells after treating with different concentrations of RG for 24 h.(C-D) The protein expression of ZO-1, E-cad, and α-SMA calculated by western blot was used to evaluate the influence of RG on EMT.(E-F) the protein expression of BCL2, BAX, and Caspase3 was calculated by western blot to show the influence of RG on the level of apoptosis.*P < 0.05; **P < 0.01; ***P < 0.001.RG = Rosiglitazone, 50 mM Glu = 50 Millimoles per liter of glucose.