The role of insulin in transdifferentiated hepatocyte proliferation and function in serum‐free medium

Gu, Ce; Li, Panpan; Liu, Wei; Zhou, Yan; Tan, Wen‐Song

doi:10.1111/jcmm.14303

Cited by 11 publications

(8 citation statements)

References 42 publications

(54 reference statements)

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“…cDNA was obtained using MLV reverse transcriptase (Promega, USA). Quantitative real-time PCR (qRT-PCR) was performed as previously reported [23]. GAPDH was used as an endogenous control.…”

Section: Rna Isolation and Qrt-pcrmentioning

confidence: 99%

Hypoxia alleviates dexamethasone-induced inhibition of angiogenesis in cocultures of HUVECs and rBMSCs via HIF-1α

Chai

Shen

et al. 2020

Stem Cell Res Ther

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Background and aim: Inadequate vascularization is a challenge in bone tissue engineering because internal cells are prone to necrosis due to a lack of nutrient supply. Rat bone marrow-derived mesenchymal stem cells (rBMSCs) and human umbilical vein endothelial cells (HUVECs) were cocultured to construct prevascularized bone tissue in osteogenic induction medium (OIM) in vitro. The angiogenic capacity of HUVECs was limited in the coculture system. In this study, the effects of the components in the medium on HUVEC angiogenesis were analyzed. Methods: The coculture system was established in OIM. Alizarin red staining and alkaline phosphatase staining were used to assess the osteogenic ability of MSCs. A Matrigel tube assay was used to assess the angiogenic ability of HUVECs in vitro. The proliferation of HUVECs was evaluated by cell counting and CCK-8 assays, and migration was evaluated by the streaked plate assay. The expression levels of angiogenesis-associated genes and proteins in HUVECs were measured by qRT-PCR and Western blotting, respectively. Results: Dexamethasone in the OIM suppressed the proliferation and migration of HUVECs, inhibiting the formation of capillary-like structures. Our research showed that dexamethasone stimulated HUVECs to secrete tissue inhibitor of metalloproteinase (TIMP-3), which competed with vascular endothelial growth factor (VEGF-A) to bind to vascular endothelial growth factor receptor 2 (VEGFR2, KDR). This effect was related to inhibiting the phosphorylation of ERK and AKT, which are two downstream targets of KDR. However, under hypoxia, the enhanced expression of hypoxiainducible factor-1α (HIF-1α) decreased the expression of TIMP-3 and promoted the phosphorylation of KDR, improving HUVEC angiogenesis in the coculture system. Conclusion: Coculture of hypoxia-preconditioned HUVECs and MSCs showed robust angiogenesis and osteogenesis in OIM, which has important implications for prevascularization in bone tissue engineering in the future.

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“…cDNA was obtained using MLV reverse transcriptase (Promega, USA). Quantitative real-time PCR (qRT-PCR) was performed as previously reported [23]. GAPDH was used as an endogenous control.…”

Section: Rna Isolation and Qrt-pcrmentioning

confidence: 99%

Hypoxia alleviates dexamethasone-induced inhibition of angiogenesis in cocultures of HUVECs and rBMSCs via HIF-1α

Chai

Shen

et al. 2020

Stem Cell Res Ther

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“…In previous studies, glucose consumption in normal cells was used as the control [24,25], but we found that insulin has a mitogenic effect on hepatocytes, although there were no changes in proliferation rate within a certain concentration range. This has also been described by other investigators [31,32]. Since cell proliferation affects glucose consumption, we compared cells with similar proliferation rates but selected those with the highest glucose consumption within each group as the control; IR was identified based on a decrease in glucose consumption.…”

Section: Discussionmentioning

confidence: 90%

Dexmedetomidine alleviates insulin resistance in hepatocytes by reducing endoplasmic reticulum stress

Liu

Zhu

et al. 2019

Endocrine

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Purpose Dexmedetomidine (DEX) stabilizes intraoperative blood glucose levels and reduces insulin resistance (IR), a common perioperative complication. However, the molecular mechanisms underlying these effects remain unclear. Since endoplasmic reticulum stress (ERS) is a mechanism of IR, this study sought to examine whether DEX can effectively alleviate IR by reducing ERS. Methods HepG2 and LO2 cells were treated with different concentrations of insulin. The glucose content assay and Cell Counting Kit-8 (CCK-8) were then employed to determine the optimal insulin concentration capable of inducing IR without affecting cell viability. Insulin-resistant hepatocytes were cultured with different concentrations of DEX for 24 h, and the glucose concentration in the supernatant was measured. ERS was assessed by qPCR and western blotting. The latter was also used to quantify the expression of phosphorylated protein kinase B (p-AKT), phosphoenolpyruvate carboxykinase (PEPCK), and glucose 6 phosphatase (G6Pase), which are key proteins involved in the action of insulin. Results After 48-h of culturing with 10 μg/mL insulin, glucose consumption in hepatocytes was found to be reduced. IR hepatocytes cultured with 10, 100, or 1000 ng/ml DEX for 24 h showed a concentration-dependent increase in glucose consumption. Elevated mRNA and protein levels of ERS markers binding immunoglobulin protein (BIP) and ER protein 29 (ERp29), were reversed by DEX treatment. Moreover, reduced p-AKT and increased PEPCK and G6Pase protein levels in IR hepatocytes were also restored following DEX treatment. Conclusion DEX may alleviate IR in hepatocytes by reducing ERS serving to restore insulin action via the IRS-1/PI3K/AKT pathway.

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“…Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot in this study were performed as previously published articles. [14,15] The primer sequences are shown in Table S1; GAPDH was used as the housekeeping gene. The primary antibodies are shown in Table S2, and β-actin served as the internal control.…”

Section: Qrt-pcr and Western Blottingmentioning

confidence: 99%

“…[13,14] Further, a large-scale expansion technique of hiHeps based on serumfree microcarriers has been established to achieve high-density cell culture. [15] However, the expression of the hepatocyte-specific functions of hiHeps is still premature compared to that of primary hepatocytes in vitro, especially for urea synthesis and CYP450 enzyme activity. The expression of some metabolism-related genes and coagulation-related genes has been shown to be significantly improved when hiHeps are incorporated into the liver, suggesting that the liver function of hiHeps can be enhanced by simulating an in vivo microenvironment.…”

Section: Introductionmentioning

confidence: 99%

Human umbilical cord‐derived mesenchymal stem cells affect urea synthesis and the cell apoptosis of human induced hepatocytes by secreting IL‐6 in a serum‐free co‐culture system

Chai

et al. 2021

Biotechnology Journal

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Background: Bioartificial livers (BALs) are emerging as a potential supportive therapy for liver diseases. However, the maintenance of hepatocyte function and viability in vitro is a major challenge. Mesenchymal stem cells (MSCs) have attracted extensive attention for providing trophic support to hepatocytes, but only few studies have explored the interaction between human MSCs and human hepatocytes, and very little is known about the underlying molecular mechanisms whereby MSCs affect hepatocyte function, especially in serum-free medium (SFM). Method and Results:This study aims to explore the effects of human umbilical cordderived MSCs (hUMSCs) on human-induced hepatocytes (hiHeps) function and viability, and know about the underlying molecular mechanism of interaction in SFM.The liver-specific function of hiHeps was evaluated by analysis of albumin secretion, urea synthesis, and metabolic enzyme activity. hiHeps apoptosis was mainly characterized by live/dead staining assay, JC-1 mitochondrial membrane potential assay, reactive oxygen species (ROS) generation, and cell apoptosis detection. The expression of related genes and proteins were measured by qRT-PCR and western blotting.The results indicate that co-culture with hUMSCs improved hiHep urea synthesis and reduced cell apoptosis compared to monoculture in SFM, and this effect was found to be mediated by secreted interleukin-6 (IL-6). Further, studies revealed that IL-6 reduced hiHep apoptosis via the activation of the JAK-Stat3-Ref-1 and JAK-Stat3-Bcl-2/Bax-Caspase3 pathways by binding to the IL-6 receptor. IL-6 also enhanced hiHep urea synthesis through the JAK-Akt-P53-ARG1 pathway. Finally, hiHep-specific functions were further prolonged and increased when co-cultured with hUMSCs on 3D polyethylene terephthalate (PET) fibrous scaffolds. Conclusion:The SFM co-culture strategy showed major advantages in maintaining hiHep function and viability in vitro, which is of great significance for the clinical application of hiHeps in BALs.

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The role of insulin in transdifferentiated hepatocyte proliferation and function in serum‐free medium

Cited by 11 publications

References 42 publications

Hypoxia alleviates dexamethasone-induced inhibition of angiogenesis in cocultures of HUVECs and rBMSCs via HIF-1α

Hypoxia alleviates dexamethasone-induced inhibition of angiogenesis in cocultures of HUVECs and rBMSCs via HIF-1α

Dexmedetomidine alleviates insulin resistance in hepatocytes by reducing endoplasmic reticulum stress

Human umbilical cord‐derived mesenchymal stem cells affect urea synthesis and the cell apoptosis of human induced hepatocytes by secreting IL‐6 in a serum‐free co‐culture system

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