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. 2021 Feb 6;24(3):102149. doi: 10.1016/j.isci.2021.102149

GSK3 inhibitor ameliorates steatosis through the modulation of mitochondrial dysfunction in hepatocytes of obese patients

Yaqiong Li 1, Yi Lin 2, Xueya Han 1, Weihong Li 1, Wenmao Yan 2, Yuejiao Ma 1, Xin Lu 1, Xiaowu Huang 3, Rixing Bai 2,4,∗, Haiyan Zhang 1,4,5,∗∗
PMCID: PMC7900441  PMID: 33665568

Summary

Obesity is an important risk factor and a potential treatment target for hepatic steatosis. The maladaptation of hepatic mitochondrial flexibility plays a key role in the hepatic steatosis. Herein, we found that hepatocyte-like cells derived from human adipose stem cell of obese patients exhibited the characteristics of hepatic steatosis and accompanied with lower expression of the subunits of mitochondrial complex I and lower oxidative phosphorylation levels. The GSK3 inhibitor CHIR-99021 promoted the expression of NDUFB8, NDUFB9, the subunits of mitochondrial complex I, the basal oxygen consumption rate, and the fatty acid oxidation of the hepatocytes of obese patients by upregulating the expression of the transcription factor PGC-1α, TFAM, and NRF1 involved in mitochondrial biogenesis. Moreover, CHIR-99021 decreased the lipid droplets size and the triglyceride levels in hepatocytes of obese patients. The results demonstrate that GSK3 inhibition ameliorates hepatic steatosis by elevating the mitochondrial function in hepatocytes of obese patients.

Subject areas: human metabolism, molecular biology

Graphical abstract

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Highlights

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    Obese patients’ adipose-stem-cell-derived hepatocytes reveal hepatic steatosis

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    Hepatic steatosis is accompanied the mitochondrial dysfunction

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    The mitochondrial dysfunction is governed by the low expression PGC-1α, TFAM, and NRF1

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    GSK3 inhibitor ameliorates hepatic steatosis via mitochondrial dysfunction modulation

human metabolism; molecular biology

Introduction

Fatty liver disease (FLD) has emerged as the most prevalent chronic liver disease and greatest risk factor for liver-related morbidity and mortality (Estes et al., 2018; Fan et al., 2017). Hepatic steatosis, which describes a range of conditions caused by triglyceride deposition within hepatocytes, is a major driver of FLD, which can progress to more advanced stages, including steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (Engin, 2017; Friedman et al., 2018; Singh et al., 2015). Given that the worldwide increase in FLD prevalence parallels the evolution of the obesity epidemic, obesity is considered the most important risk factor and a potential target for the treatment of hepatic steatosis and FLD (Cusi, 2012; Eslam et al., 2018; Fan et al., 2017; Grohmann et al., 2018; Samuel and Shulman, 2018). Indeed, the high levels of fatty acids entering, high levels of triglyceride synthesis, and lower capacity of beta-oxidation in mitochondria lead to steatosis in the liver (Cusi, 2012; Engin, 2017). Notably, the continuous maladaptation of hepatic mitochondrial flexibility in obese individuals plays a key role in the pathogenesis of hepatic steatosis (Koliaki et al., 2015; Sunny et al., 2017). However, validation of predictive biomarkers of inadequate mitochondrial adaptation and response to therapy is lacking and is urgently needed. Moreover, an obese individual's susceptibility to substrate overload is highly variable, because of many different properties such as sedentary lifestyle, diet, intrauterine environment, and aging (Fan et al., 2017). Thus, understanding of the hepatic mitochondrial flexibilities and their maladaptation responses in hepatic steatosis will be helpful to offer new precision molecular targets for treatment of FLD and associated diseases.

Accumulating evidence indicates that human adipose stem cell (hASC)-derived hepatocyte-like cells (HLCs) offer an attractive tools for establishing an alternative therapy for liver dysfunction (Aurich et al., 2009; Banas et al., 2007; Hu et al., 2019; Li et al., 2014, 2018; Seo et al., 2005; Yuan et al., 2015). Recent studies showed that the environment or niche of obesity-related adipose tissue may have latent effects on immunophenotypic profile and adipogenic differentiation (Louwen et al., 2018; Oñate et al., 2013; Pachon-Pena et al., 2016), immune properties (Serena et al., 2016, 2017), and primary cilia function (Ritter et al., 2018). However, evidence showing the hepatic differentiation capability of obese patient hASCs is currently lacking.

Here, we found that hASC-HLCs from obese subjects exhibited the characterization of hepatic steatosis associated with maladaptation of mitochondrial oxidative activity, as indicated by lower expression of the subunits of mitochondrial complex I, the basal oxygen consumption rate (OCR), and fatty acid oxidation. Moreover, we found that the GSK3 pharmacological inhibitor CHIR-99021 modified the mitochondrial dysfunction of the hASC-HLCs of obese patients by upregulating the expression of the transcription factor PGC-1α, TFAM, and NRF1 involved in mitochondrial biogenesis and respiratory function. Meantime, CHIR-99021 treatment ameliorates hepatic steatosis in hASC-HLCs of obese patients.

Results

hASCs from obese patients possess the potential to differentiate into hepatocytes

We initially isolated and evaluated the phenotypes of the ASCs from the visceral adipose tissue of eighteen obese patients undergoing bariatric surgery and eight lean control donors undergoing selective caesarean section at term (Table 1). The majority of the cells from the both lean control and obese donors exhibited a typical uniform spindle-shaped appearance of morphogenic fibroblasts at the 6th passage (Figure S1). The cultured hASCs from both lean controls and obese donors exhibited the same expression of these typical surface markers, with more than 96% of the samples positive for CD73, CD90, and CD105 and almost all the samples negative for CD34 (Figure S2). The data confirmed that the hASCs from the obese patients maintained a similar morphology and phenotype as those from the control donors.

Table 1.

Characteristics of the patients

Lean (n = 8) Obese (n = 18) p Value
Age (years) 35.88 ± 5.239 32.67 ± 1.464 0.4373
Gender (male/female) 8 (2/6) 18 (10/8) /
Body mass index (kg/m2) 22.21 ± 0.8169 42.44 ± 2.015 <0.0001
Glucose (mmol/L) 4.715 ± 0.2111 7.314 ± 0.7909 0.0417
Fasting serum insulin (uU/mL) / 20.24 ± 2.333 /
HOMA-IR / 2.690 ± 0.3271 /
Triglyceride (mmol/L) 1.104 ± 0.1726 2.894 ± 0.8006 0.1551
Total cholesterol (mmol/L) 4.429 ± 0.2426 4.536 ± 0.2674 0.8073
HDL-cholesterol (mmol/L) 1.245 ± 0.07741 0.9650 ± 0.04861 0.0045
LDL-cholesterol (mmol/L) 2.644 ± 0.1993 2.810 ± 0.2761 0.7079
HbA1c (%) / 7.667 ± 0.4658 /
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HOMA-IR, homeostasis model assessment of insulin resistance; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

Data are expressed as the means ± SEM.

To assess whether hASCs derived from obese patients possess the potential to differentiate into HLCs, hASCs from lean control donors and obese donors were differentiated using a three-stage differentiation protocol as previously described (Li et al., 2014). The properties of the differentiated cells in the two groups were analyzed on day 5, day 10, and day 20 during the process of hepatic differentiation of hASCs. Immunofluorescence staining data verified that, on day 5, the cells in the two groups were positive for GATA-binding protein 4 (GATA4) (Figure 1A), which is a marker of definitive endoderm cells; on day 10, the cells were positive for α-fetoprotein (AFP) (Figure 1B), which is a marker of hepatic progenitor cells; and on day 20, they were positive for glutathione S-transferase alpha 2 (GSTA2), which is a marker of hepatocytes (Figure 1C). These data indicated that hASCs derived from obese donors can be differentiated into HLCs that exhibit hepatocyte-specific proteins by mimicking liver embryogenesis and maturation induction. Therefore, we named the HLCs from the two groups: lean HLCs (HLCs derived from ASCs of lean donors) and obese HLCs (HLCs derived from ASCs of obese patients).

Figure 1.

Figure 1

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hASCs from obese donors possess the potential to differentiate into hepatocyte-like cells

Immunofluorescence analysis of differentiated hASCs in the two groups was used to determine the expression of GATA4 (A), AFP (B), and GSTA2 (C). Scale bars, 50 μm and 75 μm. Obese, obese donor; lean, lean donor; 1, 2, and 3 represent donor 1, donor 2, and donor 3, respectively. (D) Relative mRNA levels of hepatic functional markers, namely, HNF4α, ALB, CYP3A4, ABCC2, PCK2, and CPS1, in the lean HLCs and obese HLCs were determined by real-time RT-PCR. n = 6 different donors per group. Significance compared with the lean HLCs, as analyzed by unpaired two-tailed Student's t test. Data are presented as the means ± SEM. N.S., not significant. hASCs, human adipose stem cells; lean HLCs, hepatocyte-like cells derived from adipose stem cell of lean donors; obese HLCs, hepatocyte-like cells derived from adipose stem cell of obese patients; GATA4, GATA-binding protein 4; AFP, α-fetoprotein; GSTA2, glutathione S-transferase alpha 2; HNF4α, hepatocyte nuclear factor 4 alpha; ALB, albumin; CYP3A4, cytochrome P450 family 3 subfamily A member 4; ABCC2, ATP-binding cassette, sub-family C (CFTR/MRP), member 2; PCK2, phosphoenolpyruvate carboxykinase 2; CPS1, carbamoyl-phosphate synthase 1. See also Figures S1–S3.

To further characterize the obese HLCs, typical hepatic markers were determined using real-time RT-PCR. Quantitative comparisons of gene expression revealed that the expression of ATP-binding cassette, subfamily C (CFTR/MRP), member 2 (ABCC2), and phosphoenolpyruvate carboxykinase 2 (PCK2) in the cells of the two groups did not differ. However, the mRNA levels of hepatocyte nuclear factor 4 alpha (HNF4α), albumin (ALB), cytochrome P450 family 3 subfamily A member 4 (CYP3A4), and carbamoyl-phosphate synthase 1 (CPS1) in the cells derived from obese donors were relatively lower than were those in the cells derived from the control lean donors (Figures 1D and S3). These data indicated differential gene expression of the obese HLCs and lean HLCs.

Differential gene expression in the lean HLCs and obese HLCs

To obtain an initial perspective on global gene expression changes, RNA sequencing was performed to compare the gene expression in the lean HLCs and obese HLCs. The results revealed that 48 genes were significantly upregulated and 49 were significantly downregulated in the obese HLCs (Figure 2A). A functional gene annotation analysis of these differentially expressed genes was performed according to gene subset to explore the functions of these regulated genes in the lean HLCs and obese HLCs. Analysis of the significantly upregulated genes in the obese HLCs led to the identification of functional groups, such as “bleb assembly,” “ossification,” “cellular response to extracellular stimulus”, “cytolysis,” “cell morphogenesis,” and “glycosaminoglycan catabolic process” (Figure 2B). Analysis of the significantly downregulated genes found in the obese HLCs led to the identification of functional groups, such as “renal absorption,” “regulation of cell proliferation,” “positive regulation of protein phosphorylation,” “cell chemotaxis,” “blood coagulation,” “positive regulation of peptidyl-tyrosine phosphorylation,” “positive regulation of leukocyte chemotaxis,” and “positive regulation of reactive oxygen species metabolic process” (Figure 2C).

Figure 2.

Figure 2

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Comparative analysis of differentially expressed genes in the lean HLCs and obese HLCs based on RNA sequencing

(A) Heatmap generated by the common genes shared between the two groups based on different gene subsets using MeV v4.8 (http://www.tm4.org/mev/). In the heatmap, high expression is depicted in red, and low expression is depicted in green. The columns are log10 plots of the normalized intensity of triplicate samples in each group.

(B) Results of GO analysis of biological process of the differentially expressed upregulated genes in the obese HLCs.

(C) Results of GO analysis for the biological process of the differentially expressed downregulated genes in the obese HLCs.

(D) GSEA for the KEGG pathways and GO analysis of the differentially expressed upregulated genes in the obese HLCs.

(E) GSEA for KEGG pathways and GO analysis of the differentially expressed downregulated genes in the obese HLCs. n = 3 different donors per group. Lean HLCs, hepatocyte-like cells derived from adipose stem cell of lean donors; obese HLCs, hepatocyte-like cells derived from adipose stem cell of obese patients; GO, gene ontology; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes.

See also Figures S4–S6.

GSEA analyses revealed that the pathways related to “glycerolipid metabolism (HSA00561),” “steroid biosynthesis (HSA00100),” “sphingolipid signaling (HSA04071),” “cholesterol biosynthetic process (GO0006695),” and “the positive regulation of interleukin 6 production (GO0032755)” were significantly upregulated (Figure 2D). Genes involved in these pathways are listed in the Supplemental information, Figure S4. However, the pathways related to “alanine aspartate and glutamate metabolism (HSA00250),” “glycosylphosphatidylinositol anchor (00563),” “ABC transporters (HSA02010),” “liver regeneration (GO0097421),” and “histone deacetylase binding (GO0042826)” were significantly downregulated (Figure 2E). Genes involved in these pathways are listed in the Supplemental information, Figure S5.

To validate the accuracy of the gene indexes calculated from RNA sequencing, five upregulated expression genes, including glycerol kinase (GK) in the glycerolipid metabolism pathway, sterol-C5-desaturase (SC5D) in the steroid biosynthesis pathway, Rac family small GTPase 3 (RAC3) in the sphingolipid signaling pathway, insulin-induced gene 1 (INSIG1) in the cholesterol biosynthetic process, and lipopolysaccharide binding protein (LBP) in the positive regulation of interleukin-6 production were confirmed by real-time RT-PCR (Figure S6A). In addition, five downregulated genes, including glutamine-fructose-6-phosphate transaminase 2 (GFPT2) in the alanine aspartate and glutamate metabolism pathways, post-GPI attachment to protein inositol deacylase 1 (PGAP1) in the glycosylphosphatidylinositol anchor pathway, transporter 2, ATP-binding cassette subfamily B member (TAP2) in the ABC transporter pathway, proliferating cell nuclear antigen (PCNA) in liver regeneration, and DNA topoisomerase II beta (TOP2B) in histone deacetylase binding, were also confirmed by real-time RT-PCR (Figure S6B). These data suggest that obese HLCs exhibit the characteristics of abnormally elevated lipid metabolism and reduced liver regeneration compared with the metabolism and regeneration in the lean HLCs.

Obese HLCs exhibit the characteristics of hepatic steatosis

Obesity-related hepatic steatosis represents imbalances of the processes that maintain the normal homeostasis of lipid metabolism in the hepatocyte can lead to hepatic dysfunction (Gluchowski et al., 2017). To investigate whether obese HLCs exhibit the characteristics of hepatic steatosis, the expression levels of genes involved in lipid synthesis and lipolysis in the lean HLCs and obese HLCs were quantitatively compared using real-time RT-PCR. As shown in Figure 3, the results indicated that genes related to fatty acid synthesis, including stearoyl-CoA desaturase (SCD) and fatty acid desaturase 1 (FADS1), and genes involved in the rate-limiting enzyme step of cholesterol synthesis, including 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), were significantly upregulated in the obese HLCs (Figure 3A). Genes related to the fatty acid beta-oxidation pathway, such as peroxisome proliferator-activated receptor alpha (PPARα), acyl-CoA oxidase 1 (ACOX1), phospholipase A2 group IVA (PLA2G4A), and apolipoprotein L3 (APOL3), were significantly downregulated in the obese HLCs (Figure 3B) compared with the levels in the lean HLCs. Fatty acid synthase (FASN), which is related to fatty acid synthesis, carboxylesterase 1 (CES1), which participate in hepatic lipid metabolism, and carnitine palmitoyltransferase 1A (CPT1A) in the fatty acid beta-oxidation pathway were not significantly different in the obese HLCs and the lean HLCs (Figure 3A and 3B).

Figure 3.

Figure 3

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Comparisons of the lipid metabolic properties in the lean HLCs and obese HLCs

(A and B) Relative mRNA levels of lipid-metabolism-related genes in the lean HLCs and obese HLCs were determined by real-time RT-PCR. (A) Lipid synthesis genes, namely, SCD, FASN, FADS1, HMGCS1, and HMGCR; (B) genes related to the fatty acid β-oxidation pathway, namely, PPARα, CPT1A, ACOX1, CES1, PLA2G4A, and APOL3. n = 6 different donors per group.

(C) LDs in the lean HLCs and obese HLCs were determined by HCS LipidTOX green neutral lipid staining. Scale bars, 25 μm. Total LD area in each cell was analyzed using ImageJ software. n = 3 different donors per group; the number of cells was 100 in each donor respectively.

(D) The size of LD in the lean HLCs and obese HLCs was determined using TEM. The red asterisk indicates an LD. Scale bars, 2 μm. The average LD size in the cells was determined using ImageJ software. n = 3 different donors per group.

(E) TG levels of HLCs from lean donors and obese donors were determined by using a triglyceride assay kit. Significance compared with the lean HLCs, as analyzed by unpaired two-tailed Student's t test. N.S., not significant. Data are presented as the means ± SEM. n = 3 different donors per group. Lean HLCs, hepatocyte-like cells derived from adipose stem cell of lean donors; obese HLCs, hepatocyte-like cells derived from adipose stem cell of obese patients; SCD, stearoyl-CoA desaturase; FASN, fatty acid synthase; FADS1, fatty acid desaturase 1; HMGCS1, 3-hydroxy-3-methylglutaryl-CoA synthase 1; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; PPARα, peroxisome-proliferator-activated receptor alpha; CPT1A, carnitine palmitoyltransferase 1A; ACOX1, acyl-CoA oxidase 1; CES1, carboxylesterase 1; PLA2G4A, phospholipase A2 group IVA; APOL3, apolipoprotein L3; LDs, lipid droplets; TEM, transmission electron microscopy.

See also Figures S7 and S8.

The cytoplasmic lipid droplets (LDs) are lipid storage organelles in hepatocytes; the aberrant accumulation of LDs is the hallmark of steatosis, a key pathological feature of obesity, FLD, and metabolic syndrome (Gluchowski et al., 2017). To demonstrate changes in lipid accumulation, neutral lipids in the HLCs were quantitatively compared using HCS LipidTOX green neutral lipid stain. The results revealed that LDs were present both in the lean HLCs and in the obese HLCs. Quantitative analysis of the total area of LDs in the cells derived from three lean donors and obese donors revealed that the total area of LDs was significantly higher in the obese HLCs compared with that in the lean HLCs (Figures 3C and S7A). TEM analysis also demonstrated that the sizes of LDs in the obese HLCs were significantly larger than those in the lean HLCs (Figures 3D and S7B).

Free fatty acids (FAs) are taken up by hepatocytes and converted into triglycerides (TGs) for storage with cholesterol in LDs or imported to mitochondria for β-oxidation. TG levels were quantitatively compared in the lean HLCs and obese HLCs. Consistent with the increased LDs areas, the TG levels in the obese HLCs were higher than the lean HLCs (Figure 3E). By visualizing FAs in live cells, the labeled FA in LDs were accumulated in the obese HLCs than those in the lean HLCs (Figures S8A and S8B). However, the labeled FA localized with mitochondria were decreased in the obese HLCs than those in the lean HLCs (Figures S8C and S8D). These data suggest that the metabolic imbalances between the synthesis or storage and degradation of lipid triggered increased TG and LD accumulation in obese HLCs. These characteristics are consistent with those of hepatocyte steatosis.

Obese HLCs exhibit altered mitochondrial structure and oxidative phosphorylation function

Recent evidence suggests that continuous maladaptation of mitochondrial energetics, gene expression, morphology, and content plays a key role in the pathogenesis of simple steatosis (Sunny et al., 2017). To investigate changes in the mitochondria in the obese HLCs, the mitochondrial structure and function were first evaluated. The results revealed that the mitochondrial mass in the obese HLCs was significantly lower than that in the lean HLCs (Figures 4A and S9A). TEM analysis also demonstrated that the mitochondria in the obese HLCs contained immature mitochondria with a globular shape and poorly developed cristae indicative of a less-active mitochondrial state, whereas the lean HLCs possessed a complex morphology with developed cristae, a dense matrix, and an elongated appearance (Figures 4B and S9B). The mtDNA content in the obese HLCs was significantly decreased compared with that in the lean HLCs (Figure 4C). The mitochondrial membrane potential in the obese HLCs was also significantly lower than that in the lean HLCs (Figures 4D and S10).

Figure 4.

Figure 4

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Comparisons of the mitochondrial mass and oxidative phosphorylated activity levels in the lean HLCs and obese HLCs

(A) Mitochondrial mass in the lean HLCs and obese HLCs was determined by MitoTracker Green FM. Scale bars, 25 μm. The fluorescence intensity of MitoTracker Green in each cell was analyzed using ImageJ software. n = 3 different donors per group; the number of cells was 100 in each donor respectively.

(B) Mitochondrial morphology in the lean HLCs and obese HLCs was determined by TEM. Scale bars, 500 nm. The relative length of mitochondria in the cells was determined using ImageJ software. n = 3 different donors per group.

(C) Relative mitochondrial DNA content (measured as mitochondrial tRNA and 16S-rRNA) in the lean HLCs and obese HLCs was determined by real-time PCR. n = 3 different donors per group.

(D) Mitochondrial potential in the lean HLCs and obese HLCs was determined by TMRM. Scale bars, 25 μm. The fluorescence intensity of TMRM in each cell was analyzed using ImageJ software. n = 3 different donors per group; the number of cells was 100 in each donor respectively.

(E) ATP production rate as determined using the Seahorse XF Real-Time ATP rate assay and the mitoATP and glycoATP production rates in the lean HLCs and obese HLCs were calculated. Obese HLCs, n = 3 different donors.

(F) The basal OCR and OCR/ECAR were calculated in the lean HLCs and obese HLCs. n = 3 different donors per group.

(G) The production levels of mitochondrial ROS in the lean HLCs and obese HLCs, as labeled by MitoSox and immediately measured using flow cytometry. n = 3 different donors per group. Significance compared with the lean HLCs, as analyzed by unpaired two-tailed Student's t test. Data are presented as the means ± SEM. Lean HLCs, hepatocyte-like cells derived from adipose stem cell of lean donors; obese HLCs, hepatocyte-like cells derived from adipose stem cell of obese patients; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; mitoATP, mitochondrial oxidative phosphorylation pathway contributing to the ATP; glycoATP, glycolysis pathway contributing to the ATP; ROS, reactive oxygen species; TEM, transmission electron microscopy.

See also Figures S9 and S10.

Adenosine triphosphate (ATP) production rates measured using Agilent Seahorse XF technology demonstrated that the proportion of the mitochondrial oxidative phosphorylation pathway contributing to the ATP (mitoATP) production rate in the lean-HLCs was ~37%, whereas the proportional mitoATP production rate in the obese HLCs was only ~21% ± 6% (Figure 4E). The proportion of the basal OCR and ECAR in the obese HLCs was significantly lower than that in the lean HLCs (Figure 4F).

Mitochondrial superoxide is generated as a by-product of oxidative phosphorylation. The production of ROS derived from mitochondria was determined using MitoSOX red mitochondrial superoxide indicator, a fluorogenic dye for the highly selective detection of superoxide in the mitochondria of live cells. The results revealed that the source of mitochondrial ROS in the obese HLCs was lower than that in the lean HLCs (Figure 4G). These data indicated that the obese HLCs exhibit an altered mitochondrial structure and lower oxidative phosphorylation activities than were exhibited by the lean HLC controls.

Obese HLCs exhibit lower expression of the subunits of mitochondrial complex I

The mitochondrial respiratory chain complexes respond by converting energy produced during metabolism into synthesized ATP during oxidative phosphorylation. To investigate the expression of the mitochondrial electron transport chain complex in the obese HLCs, the mRNA levels of the electron transport chain complex components were analyzed using RNA sequencing according to gene subset. Interestingly, the significantly downregulated genes were focused on the accessory or supernumerary subunits of respiratory chain complex I (NADH:ubiquinone oxidoreductase), which included NADH:ubiquinone oxidoreductase subunit (NDUF)A8, NDUFA9, NDUFB6, NDUFB8, NDUFB9, and NDUFS8 (Figure S11). Real-time RT-PCR analysis showed that the expression of NDUFA8, NDUFB6, NDUFB8, NDUFB9, and NDUFS8 was significantly decreased in the obese HLCs compared with the levels in the lean HLCs (Figure 5A). Western blot analysis confirmed that the protein levels of NDUFB8 and NDUFB9 were significantly decreased in the obese HLCs compared with their levels in the lean HLCs (Figure 5B). These data suggested that the lower expression of the subunits of mitochondrial complex I may be attributed to lower oxidative phosphorylation activities in the obese HLCs.

Figure 5.

Figure 5

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Comparative analysis of the expression of the complex I subunits of mitochondria in the lean HLCs and obese HLCs

(A) Relative mRNA levels of NDUFB6, NDUFA8, NDUFB8, NDUFS8, NDUFA9, and NDUFB9 in the lean HLCs and obese HLCs were determined by real-time RT-PCR. n = 6 different donors per group.

(B) Protein levels of NDUFB8 and NDUFB9 in the lean HLCs and obese HLCs were determined by western blotting. Relative band densities of NDUFB8 and NDUFB9 were normalized to that of GAPDH and analyzed using ImageJ software. n = 3 different donors per group. Significance compared with the lean HLCs, as analyzed by unpaired two-tailed Student's t test. Data are presented as the means ± SEM. N.S., not significant. Lean HLCs, hepatocyte-like cells derived from adipose stem cell of lean donors; obese HLCs, hepatocyte-like cells derived from adipose stem cell of obese patients; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NDUFB6, NADH: ubiquinone oxidoreductase subunit B6; NDUFA8, NADH: ubiquinone oxidoreductase subunit A8; NDUFB8, NADH: ubiquinone oxidoreductase subunit B8; NDUFS8, NADH: ubiquinone oxidoreductase subunit S8; NDUFA9, NADH: ubiquinone oxidoreductase subunit A9; NDUFB9, NADH: ubiquinone oxidoreductase subunit B9.

See also Figure S11.

CHIR-99021 promotes mitochondrial remodeling in the obese HLCs

Previously, we found that a pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3), CHIR-99021, promoted mitochondrial remodeling and oxidative phosphorylation activity during definitive endodermal differentiation of hASCs by upregulating the expression of transcription factors, involved in mitochondrial biogenesis (Ma et al., 2019). To assess the effect of CHIR-99021 on remodeling in the obese HLCs, the obese HLCs were treated with 2 μM CHIR-99021 or vehicle control (DMSO) for 24 h. The mitochondrial contents and function in the obese HLCs were determined in the CHIR-99021-treated obese HLCs and compared with their expression in the vehicle-control-treated obese HLCs.

The results revealed that the mitochondrial contents including mitochondrial mass, the mtDNA content, and the protein levels of NDUFB8 and NDUFB9 were significantly increased in the CHIR-99021-treated obese HLCs compared with the levels in the vehicle-control-treated obese HLCs (Figures 6A–6C and S12). The mitochondrial mass and the protein levels of NDUFB8 and NDUFB9 were also significantly increased in the CHIR-99021-treated lean HLCs compared with the levels in the vehicle-control-treated lean HLCs (Figure S13).

Figure 6.

Figure 6

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CHIR-99021 promotes mitochondrial biogenesis and function in the obese HLCs

(A) Mitochondrial mass in the CHIR-99021-treated and vehicle-control-treated obese HLCs was determined by MitoTracker Green FM. Scale bars, 25 μm. The fluorescence intensity of MitoTracker Green in each cell was analyzed using ImageJ software. n = 3 different donors; the number of cells was 100 in each group respectively.

(B) Relative mitochondrial DNA content level (measured as mitochondrial tRNA and 16S-rRNA) in the CHIR-99021-treated and vehicle-control-treated obese HLCs was determined by real-time PCR.

(C) Protein levels of NDUFB8 and NDUFB9 in the CHIR-99021-treated and vehicle-control-treated obese HLCs were determined by western blotting. Relative band densities of NDUFB8 and NDUFB9 were normalized to that of GAPDH and analyzed using ImageJ software. n = 3 different donors.

(D) Mitochondrial membrane potential in the CHIR-99021-treated and vehicle-control-treated obese HLCs was determined by TMRM. Scale bars, 50 μm. The fluorescence intensity of TMRM in each cell was analyzed using ImageJ software. n = 3 different donors; the number of cells was 100 in each group respectively.

(E) Kinetic profile of the OCR was measured in the CHIR-99021-treated and vehicle-control-treated obese HLCs using a Seahorse XF Cell Mito Stress test. Black arrows show times of treatment with Oligo, FCCP, Rot, and AA. The basal respiration, ATP production, proton leak, maximal respiration, and non-mitochondrial respiration were calculated. n = 2 different donors.

(F) The CHIR-99021-treated and vehicle-control-treated obese HLCs were incubated with Red C12. Mitochondrial were labeled using MitoTracker Green FM. Fluorescent co-localization of fatty acids and mitochondria represents fatty acid oxidation. Scale bars, 25 μm. The fluorescent co-localization between Red C12 signal and mitochondria in the experiment was quantified by Pearson's coefficient analysis using ImageJ software. n = 3 different donors; the number of cells was 45 in each group respectively.

(G) Protein levels of CPT1A in the CHIR-99021-treated and vehicle-control-treated obese HLCs were determined by western blotting. Relative band densities of CPT1A were normalized to that of GAPDH and analyzed using ImageJ software. n = 3 different donors. Significance compared with the vehicle control, as analyzed by unpaired two-tailed Student's t test. Data are presented as the means ± SEM. Obese HLCs, hepatocyte-like cells derived from adipose stem cell of obese patient; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NDUFB8, NADH: ubiquinone oxidoreductase subunit B8; NDUFB9, NADH: ubiquinone oxidoreductase subunit B9; OCR, oxygen consumption rate; AA, antimycin A; Oligo, oligomycin; Rot: rotenone; CPT1A, carnitine palmitoyltransferase 1A. See also Figures S12–S17.

The mitochondrial membrane potential and oxidative phosphorylation activities, which include the level of basal OCR values and ATP production, were significantly increased in the CHIR-99021-treated obese HLCs compared with that in the vehicle-control-treated obese cells (Figures 6D, 6E, and S14). The mitochondrial membrane potential and oxidative phosphorylation activities were also significantly increased in the CHIR-99021-treated lean HLCs compared with the levels in the vehicle-control-treated lean HLCs (Figure S15).

Mitochondria represent the primary site for fatty acid (FA) β-oxidation where FAs are enzymatically broken down to sustain cellular lipid dynamic regulation (Rambold et al., 2015). Using a pulse-chase labeling method to visualize movement of FAs in live cells, we demonstrate that CHIR-99021 promote the FAs flux into mitochondria in the obese HLCs (Figures 6F and S16). The protein levels of carnitine palmitoyl transferase 1 A (CPT1A), which allows the entry of FAs into mitochondria, were significantly increased in the CHIR-99021-treated obese HLCs compared with the levels in the vehicle-control-treated obese HLCs (Figures 6G and S17). These results suggest that CHIR-99021 may promote mitochondrial structural and functional remodeling in the obese HLCs.

CHIR-99021 upregulated the expression of transcription factors involved in mitochondrial biogenesis

In an attempt to understand the mechanism by which CHIR-99021 induces mitochondrial remodeling, we next examined the expression of transcription factors, including peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α (PGC-1α), the master regulator of mitochondrial biogenesis and oxidative phosphorylation activity; mitochondrial transcription factor A (TFAM), a direct regulator of mitochondrial DNA replication and/or transcription; and nuclear respiratory factor 1 (NRF1), which controls all ten nucleus-encoded cytochrome oxidase subunits in the lean HLCs and obese HLCs. Real-time RT-PCR analyses showed that the mRNA levels of PGC-1α, TFAM, and NRF1 in the obese HLCs were significantly lower than those in the lean HLCs (Figure 7A). Western blotting analyses showed that the protein level of PGC-1α in the obese HLCs was lower than that in the lean HLCs (Figure 7B). Immunocytochemistry analyses showed that the relative fluorescence intensity for TFAM and NRF1 in the obese HLCs was also lower than it was in the lean HLCs (Figures 7C, 7D and S18).

Figure 7.

Figure 7

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CHIR-99021 upregulates the expression of transcription factors involved in mitochondrial biogenesis in the obese HLCs

(A) Relative mRNA levels of PGC-1α, TFAM, and NRF1 in the lean HLCs and obese HLCs were determined by real-time RT-PCR. n = 6 different donors per group.

(B–D) (B) Protein levels of PGC-1α in the lean HLCs and obese HLCs were determined by western blotting. The relative band density of PGC-1α was normalized to that of GAPDH and analyzed using ImageJ software. n = 3 different donors per group. The relative protein levels of TFAM (C) and NRF1 (D) in the lean HLCs and obese HLCs were determined by immunofluorescence staining. Relative immunofluorescence staining intensity of TFAM and NRF1 in each cell were analyzed using ImageJ software. Scale bars, 50 μm. n = 3 different donors per group; the number of cells was 100 in each donor respectively. See also Figure S18.

(E) Relative mRNA levels of PGC-1α, TFAM, and NRF1 in the CHIR-99021-treated and vehicle-control-treated obese HLCs were determined by real-time RT-PCR.

(F–H) (F) Protein levels of PGC-1α in the CHIR-99021-treated and vehicle-control-treated obese HLCs were determined by western blotting. The relative band density of PGC-1α was normalized to that of GAPDH and was analyzed using ImageJ software. n = 3 different donors. The relative protein levels of NRF1 (G) and TFAM (H) in the CHIR-99021-treated and vehicle-control-treated obese HLCs were determined by immunofluorescence staining. Relative immunofluorescence staining intensity of TFAM and NRF1 in each cell were analyzed using ImageJ software. Scale bars, 50 μm. n = 3 different donors; the number of cells was 100 in each group respectively. Significance compared with the lean HLCs or the vehicle control, as analyzed by unpaired two-tailed Student's t test. Data are presented as the means ± SEM. N.S., not significant. Lean HLCs, hepatocyte-like cells derived from adipose stem cell of lean donors; obese HLCs, hepatocyte-like cells derived from adipose stem cell of obese patients; PGC-1α, peroxisome-proliferator-activated receptor gamma (PPARγ) coactivator 1 alpha; TFAM, mitochondrial transcription factor A; NRF1, nuclear respiratory factor 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

See also Figures S18–S20.

Furthermore, the mRNA levels of PGC-1α, TFAM, and NRF1 in the CHIR-99021-treated obese HLCs were significantly increased compared with those in the vehicle-control-treated cells (Figure 7E). Western blot analyses showed that the protein levels of PGC-1α in CHIR-99021-treated obese HLCs were significantly increased compared with those of the vehicle-control-treated cells, but not in lean HLCs (Figures 7F and S19). Immunocytochemistry analyses showed that the relative fluorescence intensities of NRF1 and TFAM were significantly increased in the CHIR-99021-treated obese HLCs or CHIR-99021-treated lean HLCs compared with those of the vehicle-control-treated cells (Figures 7G, 7H, and S20). The changes of protein levels of TFAM between the CHIR-99021-treated HLCs and the vehicle-control-treated cells had inter-individual variability (Figure S20). These findings indicated that CHIR-99021 promoted mitochondrial adaptation in the obese HLCs by upregulating the expression of the key transcription factors involved in mitochondrial biogenesis.

CHIR-99021 ameliorates hepatic steatosis in obese HLCs

Evidence suggests LD size itself represents a fundamental physical parameter dictating the mechanistic processes used for cellular triacylglycerol catabolism (Gluchowski et al., 2017). The presence of smaller LDs in hepatocyte is intimately linked with a dedicated cytoplasmic repository for the sequestration of toxic-free fatty acids via esterification into triacylglycerol (Rutkowski et al., 2015). Then, the LD size was evaluated in obese HLCs after treatment with CHIR-99021. The results showed that the LD size in the CHIR-99021-treated obese HLCs for 24 h was significant smaller than those in the vehicle-control-treated cells (Figures 8A and S21). Meanwhile, the TG levels were dramatically decreased in the CHIR-99021-treated HLCs for 24 h and 72 h compared with those of the vehicle-control-treated cells (Figures 8B and S22). These findings suggest that CHIR-99021 may ameliorate hepatic steatosis of obese patients.

Figure 8.

Figure 8

Open in a new tab

CHIR-99021 ameliorates hepatic steatosis in the obese HLCs

(A) The LDs in the CHIR-99021-treated and vehicle-control-treated obese HLCs were determined by HCS LipidTOX green neutral lipid staining. Scale bars, 50 μm and 25 μm. The average size of LD in each cell was analyzed using ImageJ software. The number of cells was 15 in each group. n = 3 different donors.

(B) TG levels of the CHIR-99021-treated and vehicle-control-treated obese HLCs for 24 h and 72 h were determined by using a triglyceride assay kit. n = 3 different donors. Significance compared with the vehicle control, as analyzed by unpaired two-tailed Student's t test. Data are presented as the means ± SEM. obese HLCs, hepatocyte-like cells derived from adipose stem cell of obese patients; LDs, lipid droplets.

See also Figures S21 and S22.

Taken together, these results suggest that hASCs derived from obese patients can differentiate into HLCs. Obese HLCs exhibit the characteristics of hepatic steatosis. The steatosis of obese HLCs can alter the numerous metabolic pathways regulating hepatic lipids and mitochondrial adaption. CHIR-99021 may ameliorate hepatic steatosis in obese HLCs through the modulation of mitochondrial biogenesis, oxidative function, and fatty acid oxidation by upregulating the expression of the key transcription factors PGC-1α, TFAM, and NRF1 involved in mitochondrial biogenesis and respiratory function. In our opinion, obese HLCs represent an important bridge connecting obesity and FLD and are important to the search for FLD treatments. GSK3 inhibitors may provide a novel treatment strategy for fatty liver disease.

Discussion

Excessive fat deposition in visceral adipose tissue is intimately related to the development of hepatic steatosis in obese individuals and is thought to be a key contributor to the development of FLD and FLD-related cirrhosis and hepatocellular carcinoma (Friedman et al., 2018; Polyzos et al., 2019; Samuel and Shulman, 2018). Therefore, understanding of the processes in obesity that contribute to hepatic steatosis and the search for treatments has increased exponentially (Polyzos et al., 2019; Saltiel and Olefsky, 2017). Given the high variability and complexity of biological, behavioral, and environmental factors among individuals with obesity, studying hepatocytes from individual obese patients is a more valid method for dissecting disease-promoting molecular pathways (Bluher, 2019; Graffmann et al., 2016; Ouchi et al., 2019; Parafati et al., 2018).

Cumulative evidence indicates that HLCs derived from human stem cells may serve as platforms for disease modeling and drug discovery (Corbett and Duncan, 2019; Graffmann et al., 2016; Ouchi et al., 2019; Parafati et al., 2018). Nevertheless, it was not clear whether HLCs from obese individual hASCs have the features of hepatic steatosis. In the present study, we found that HLCs derived from obese patient hASCs exhibit markers typical of hepatocytes and similar to the HLCs from lean donor hASCs. Obese HLCs exhibit the characteristics of hepatic steatosis. More importantly, the upregulated gene expression involved in glycerolipid metabolism, steroid biosynthesis, sphingolipid signaling, cholesterol biosynthetic processes, the positive regulation of interleukin-6 production, and the accumulation of fat droplets identified in the obese HLCs indicated the impairment of intracellular lipid metabolism, which was closely related to the observation in the parenchymal cells of the livers in obese and FLD patients (Iozzo et al., 2010; Latorre et al., 2017). These results suggested that obese HLCs may be influenced by changes in the systemic metabolism status.

Oxidative stress and particularly alterations in mitochondrial function are thought to play significant roles in the simple steatosis to FLD transition (Garcia-Ruiz and Fernandez-Checa, 2018; Mansouri et al., 2018; Sunny et al., 2017). However, the features of maladaptation of mitochondrial oxidative flux are intricate, as documented in patients and experimental models (Koliaki et al., 2015; Rector et al., 2010; Satapati et al., 2015; Yamada et al., 2018). We found that, compared with the lean HLCs, the obese HLCs had altered mitochondrial structure; lower expression of the subunits of mitochondrial complex I, such as NDUFB8 and NDUFB9 (Fiedorczuk and Sazanov, 2018; Piekutowska-Abramczuk et al., 2018); and lower levels of mtDNA; oxidative phosphorylation function (Arruda et al., 2014; Hammerschmidt et al., 2019); mitochondrial ROS (Mansouri et al., 2018), and fatty acid oxidation (Sunny et al., 2017). These indicated that the mitochondrial dysfunction is closely associated with the steatosis in obese HLCs.

The PGC-1 family of regulated coactivators, consisting of PGC-1α, PGC-1β, and PRC, plays a central role in the regulatory network governing the transcriptional control of mitochondrial biogenesis and respiratory functions under cellular stress (Scarpulla, 2011; Wenz, 2013). We found that the mRNA and protein levels of PGC-1α were lower in the obese HLCs than they were in the lean HLCs. Moreover, the expression of NRF1 (also known as NFE2 L1) (Huss and Kelly, 2004), which controls all ten nucleus-encoded cytochrome oxidase subunits, and TFAM (Campbell et al., 2012), a direct regulator of mitochondrial DNA replication and/or transcription, was lower in the obese HLCs than it was in the lean HLCs. These results suggested that mitochondrial dysfunction in the obese HLCs may be influenced by the lower expression of transcription factors PGC-1α, NRF1, and TFAM (Handa et al., 2014; Koliaki et al., 2015).

Previously, we reported that the pharmacological inhibition of GSK-3α and GSK-3β with the specific inhibitor CHIR-99021 promoted mitochondrial biogenesis and oxidative phosphorylation activity during the definitive endodermal differentiation of hASCs (Ma et al., 2019). In this study, we found that CHIR-99021 modified the mitochondrial oxidative phosphorylation activity by upregulating the expression of PGC-1α, NRF1, and TFAM. It has been reported that GSK3β regulates mitochondrial energy metabolism in neurons and glia by altering PGC-1α protein stability, localization, and activity (Martin et al., 2018). Inactivation of GSK-3β enhances skeletal muscle oxidative metabolism (Theeuwes et al., 2017). GSK3β inhibition suppresses hepatic lipid accumulation (Hinds et al., 2016). GSK-3α promotes fatty acid uptake and lipotoxic cardiomyopathy in the context of obesity (Nakamura et al., 2019). These results suggested that mitochondrial dysfunction and steatosis in the obese HLCs may be influenced by the lower expression of these transcription factors, which involved in mitochondrial biogenesis and respiratory function.

Metabolic disorders such as obesity are accompanied by the synthesis and degradation of LDs and can result in the accumulation of significant amounts of lipid deposition, a characteristic feature of hepatocytes in patients with FLD. Maladaptation of mitochondrial oxidative flux is a central feature of hepatic steatosis to FLD transition. Agents targeting mitochondrial dysfunction may provide a novel treatment strategy for hepatic steatosis and NAFLD (Sunny et al., 2017). Herein, we showed that modulating the dysfunction of mitochondrial fatty acid oxidation by CHIR-99021 or GSK3 inhibitors provides an attractive therapeutic strategy for FLD and obesity associated metabolic disease.

Limitations of the study

The mechanism by which CHIR-99021 alleviates steatosis in hepatocytes of obese patients still need to be further explored.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Prof. Haiyan Zhang (culture@ccmu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The datasets generated and/or analyzed during the current study are available in the GEO repository (GSE151760/https://www.ncbi.nlm.nih.gov/geo).

All original, unprocessed images of western blot in the paper were deposited on Mendeley at [https://doi.org/10.17632/54rxxvrwxr.1].

Methods

All methods can be found in the accompanying Transparent methods supplemental file.

Acknowledgments

This research was supported by The National Natural Science Foundation of China (81770616) and the Beijing Natural Science Foundation (5172009). The authors thank Chenguang Zhang, Ping Wang, Xiao Han, Jun Deng, and Hua Wei for advice in performing these experiments.

Author contributions

YL was responsible for conception and design, collection and assembly of data, and data analysis and writing the manuscript. YL was responsible for collecting the information of patients and data analysis. XH was responsible for perform data analysis of flow cytometry. WL was responsible for administrative support and collection and assembly of data. WY was responsible for collecting the information of patients and data analysis. YM was responsible for performing QPCR assays for mtDNA. XL was responsible for collecting data and histochemistry technical support. XH was responsible for collecting the information of patients and data analysis. RB was responsible for conception and design, data analysis and interpretation, and final approval of the manuscript. HZ was responsible for conception and design, data analysis and interpretation, writing the manuscript, funding acquisition, and final approval of the manuscript. All authors read and approved the final manuscript.

Declaration of interests

The authors declare that they have no competing interests.

Published: March 19, 2021

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.102149.

Contributor Information

Rixing Bai, Email: brx5168@163.com.

Haiyan Zhang, Email: culture@ccmu.edu.cn.

Supplemental information

Document S1. Transparent methods, Figures S1–S22, and Tables S1 and S2
mmc1.pdf (3.7MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent methods, Figures S1–S22, and Tables S1 and S2
mmc1.pdf (3.7MB, pdf)

Data Availability Statement

The datasets generated and/or analyzed during the current study are available in the GEO repository (GSE151760/https://www.ncbi.nlm.nih.gov/geo).

All original, unprocessed images of western blot in the paper were deposited on Mendeley at [https://doi.org/10.17632/54rxxvrwxr.1].

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