Keloid scarring is a natural human disease model involving chronic inflammation, fibrosis, and tumors, and is characterized by uncontrolled proliferation of fibroblasts and excessive deposition of extracellular matrix [5, 6, 27]. Aerobic glycolysis is considered a hallmark of cancer . Tissue hypoxia is commonly found in most solid tumors and results in metabolic reprogramming. Our previous work demonstrated metabolic reprogramming and the augmented activation of the PI3K/AKT pathway in KFb under hypoxic conditions . However, the role of the PI3K/AKT pathway in modulating glucose metabolism and cell functions in KFb under hypoxia is poorly documented. In the present study, we investigated glycolysis, mitochondrial function, and cell function after PI3K inhibition both under hypoxia and normoxia. In addition, redox homeostasis, along with the relationship between the PI3K-AKT signaling network and HIF-1α were also explored under hypoxia.
The PI3K-AKT signaling network represents the main growth regulatory pathways in mammalian cells, and abnormal activation of this signaling network is considered to be one of the most frequently altered pathways in human cancers . PI3K, which exists as a heterodimer of a catalytic subunit coupled with a regulatory subunit , mainly transfers signals from G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), thereby regulating physiological functions of cells . Besides directly regulating multiple cellular biological processes, including growth, proliferation, and survival, the PI3K signaling network is implicated in metabolic reprogramming. Extensive studies conducted in recent decades have illustrated that the ubiquitous PI3K-AKT pathway exerts a regulatory role in cellular metabolism by either directly modulating metabolic enzymes and nutrient transporters or by controlling transcription factors that regulate the expression levels of critical components of metabolic pathways . Accumulating evidence reveals that PI3K signaling directly regulates glucose uptake and glycolysis in most human cancers. A recent study demonstrated that the PI3K-AKT-mTOR/PFKFB3 pathway promotes aerobic glycolysis in lung fibroblasts and collagen synthesis in lipopolysaccharide-induced pulmonary fibrosis . Keloids exhibit physiological functions similar to those of tumors and fibrotic diseases. However, the regulation of the PI3K-AKT signaling network in glucose metabolism in KFb under hypoxia remains unclear. In our study, the PI3K blockade decreased the protein expression of GLUT1 and LDHA in KFb, suggesting that the activation of the PI3K-AKT pathway promotes glucose intake and enhances the Warburg effect. Additionally, we also detected decreased levels of glycolytic enzymes, including HK2, PFKFB3, PGK1, ENO1, and PKM2, after PI3K inhibition using LY294002 under hypoxic conditions. ECAR indicates real-time changes in glycolytic levels in living cells. When PI3K signaling was inhibited, the results of the glycolytic stress test showed attenuated ECAR, including basic glycolysis, glycolytic capacity, and glycolytic reserve.
The PI3K-AKT signaling pathway is crucial for maintaining mitochondrial function and healthy conditions. Although the regulation of PI3K-AKT signaling in mitochondrial functions has been extensively studied, conflicting findings have been reported. Studies in the last decade have revealed that the PI3K-AKT pathway is negatively related to mitochondrial respiration. Zheng et al. reported that increased phosphorylation in the PI3K-AKT pathway induced by salidroside-inhibited mitochondrial respiratory chain complex I, disturbed phosphorylation coupling, and moderately depolarized the mitochondrial membrane potential in hepatocytes . Zhao et al.  found that blockade of AKT activation by PTEN overexpression suppressed glucose uptake and lactate production and maintained mitochondrial functions, resulting in the transformation of energetic metabolism from glycolysis to oxidative phosphorylation in cultured PTEN-negative human hepatocellular carcinoma (HHCC) cells. In this study, we detected increased mitochondrial mass and MMP in KFb after PI3K inhibition with LY294002. Consistent with previous studies [32, 33], PI3K inactivation enhanced mitochondrial function in our study. Additionally, we also observed mitochondrial ultrastructure, and TEM analysis revealed that the mitochondrial ultrastructure remained previous conditions when KFb was treated with LY294002. We evaluated OCR, representing mitochondrial respiration, using a Seahorse XFp Real-time Extracellular Flux Analyzer in real time. The results demonstrated that KFb treated with PI3K inhibition exhibited increased OCR parameters, including basal respiration, maximal respiration, spare capacity, and ATP production. Hence, our findings collectively indicate that PI3K inhibition suppresses glycolysis and enhances mitochondrial respiration. However, a growing body of evidence has revealed that activation of PI3K is positively associated with mitochondrial function. Activation of PI3K/AKT signaling with the activator IGF-1 resulted in enhanced glycolysis and upregulation of mitochondrial complex I expression and activity in isogenic hepatocyte cell lines . Li et al.  reported that activation of PI3K/AKT due to PTEN loss increases mitochondrial mass and function by regulating estrogen-related receptor α (ERRα) through the AKT/CREB axis in primary hepatocytes.
However, the modulation of the PI3K-AKT pathway in KFb proliferation, migration, invasion, and apoptosis requires further investigation. In this study, KFb was treated with a range of concentrations of LY294002 and incubated under hypoxic or normoxic conditions, following which the functions described above were determined. Interestingly, our findings were consistent with those of previous studies that focused on diverse conditions [36–39]. The results of cell counting showed that PI3K inhibition compromised KFb proliferation in a dose- and time-dependent manner. Furthermore, the inhibition of PI3K on proliferation under hypoxic conditions was more sensitive than that under normoxia. It is generally believed that the PI3K-AKT pathway is closely linked to invasion through the regulation of MMP-2 and MMP-9 expression. The findings of the Transwell chamber suggested that migration and invasion were significantly inhibited in the blockade of the PI3K-AKT signaling pathway. Additionally, the PI3K-AKT signaling network is implicated in mitochondrial apoptosis. We observed that PI3K inhibition markedly promoted KFb apoptosis. Moreover, the pro-apoptotic effect was more distinct under hypoxic conditions. Similar results were observed for keloid fibroblasts. Xin et al. reported that the PI3K/AKT/mTOR pathway activated by CD26 promotes proliferation and invasion in keloid fibroblasts . A recent report by Lv et al.  revealed that inactivation of the PI3K/Akt signaling pathway by circCOL5A1 knockdown in the keloid inhibited KFb proliferation, migration, and enhanced apoptosis. Additionally, we investigated whether the PI3K-AKT pathway regulates KFb proliferation through glycolysis. The ECAR decreased or increased when KFb was treated with a PI3K inhibitor or PI3K activator, respectively. Interestingly, the glycolysis stress test suggested that KFb treated with both PI3K activation and glycolysis inhibition manifested significantly lower ECAR parameters than the control group. We concluded that glycolysis is partly regulated by the PI3K-AKT pathway. It is widely acknowledged that glycolysis and OXPHOS are the two main sources of energy generation. When one pathway is inhibited, cells would tend to depend largely on the other pathway for ATP provision. Hence, KFb exhibited increased OCR when cells were treated with a PI3K inhibitor or PI3K activator coupled with a glycolysis inhibitor. But one point to note is that ATP generation in SC79 and 2-DG group did not increase compared to SC79 group under hypoxia and the special phenomenon needs further study. Furthermore, the proliferative rate of KFb treated with the PI3K activator and glycolysis inhibitor was significantly inhibited compared to that of PI3K activation. These results collectively indicate that the proliferation induced by glycolysis partly depends on the PI3K-AKT pathway. The conclusion that the PI3K-AKT pathway promotes proliferation by enhancing glycolysis is supported by recent studies focusing on tumor metabolism. Hussain et al. reported that inhibition of glycolysis and lipogenesis by the PI3K inhibitor, 3-Dihydro-2-(naphthalene-1-yl) quinazolin-4(1H)-one (DHNQ) represses angiogenesis and decreases proliferation of colon cancer cells . In a recent study by Wang et al., FoxA2 inhibited the proliferation of hepatic progenitor cells by attenuating PI3K-AKT-HK2-mediated glycolysis . Additionally, glycolysis induced by the PI3K-AKT pathway also plays a key role in invasion and apoptosis in other cancer cells, such as breast cancer  and pediatric osteosarcoma . Keloid features with tumor-like physiological functions. Accordingly, we postulated that the PI3K-AKT network can also regulate invasion and apoptosis by glycolysis.
The PI3K-AKT signaling network and HIF-1α are considered the two main pathways for glucose metabolic regulation under hypoxia . However, the relationship between these two pathways remains controversial. An increasing number of studies have demonstrated that activation of PI3K signaling facilitates HIF-1α accumulation in mammalian cells under hypoxic conditions [47–49]. However, the role of HIF-1α in regulating phosphorylation of the PI3K-AKT pathway remains largely unexplored. In the present study, we found that HIF-1α levels decreased when the PI3K-AKT signaling pathway was inhibited by LY294002 in KFb under hypoxia. Moreover, the phosphorylation of the PI3K-AKT pathway was reduced when HIF-1α was inhibited by LW6. This suggests that HIF-1α responds to PI3K signaling in a positive feedback manner. Additionally, Sun et al. reported that HIF1α inhibitor YC-1 inhibits activation of the PI3K/AKT/mTOR pathway during hypoxia in prostate cancer cells . These results collectively suggest that the PI3K-AKT pathway interacts with HIF-1α through a positive feedback mechanism under hypoxia.
We are aware that there are several limitations in the present study. Our understanding of the role of the PI3K-AKT pathway in glucose metabolism in vivo is hindered by the absence of a suitable animal model. Furthermore, further investigation is needed to focus on the regulation of the key metabolic modulator HIF-1α in glycolysis and mitochondrial functions under hypoxia.