Disruption of mitochondrial fission in the liver protects mice from diet-induced obesity and metabolic deterioration
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Mitochondria and the endoplasmic reticulum (ER) physically interact by close structural juxtaposition, via the mitochondria-associated ER membrane. Inter-organelle communication between the ER and mitochondria has been shown to regulate energy metabolism and to be central to the modulation of various key processes such as ER stress. We aimed to clarify the role of mitochondrial fission in this communication.
We generated mice lacking the mitochondrial fission protein dynamin-related protein 1 (DRP1) in the liver (Drp1LiKO mice).
Drp1LiKO mice showed decreased fat mass and were protected from high-fat diet (HFD)-induced obesity. Analysis of liver gene expression profiles demonstrated marked elevation of ER stress markers. In addition, we observed increased expression of the fibroblast growth factor 21 (FGF21) gene through induction of activating transcription factor 4, master regulator of the integrated stress response.
Disruption of mitochondrial fission in the liver provoked ER stress, while inducing the expression of FGF21 to increase energy expenditure and protect against HFD-induced obesity.
KeywordsDRP1 ER stress FGF 21 Mitochondria–ER juxtaposition structure Mitochondrial dynamics
Activating transcription factor 4
Brown adipose tissue
C/EBP homologous protein
Dynamin-related protein 1
Liver-specific Drp1 knockout
Eukaryotic translation factor 2α
Fibroblast growth factor 21
Intraperitoneal glucose tolerance test
Insulin tolerance test
Mitochondria-associated ER membrane
Mitochondrial fission factor
Normal chow diet
Protein kinase R-like ER kinase
Respiratory exchange ratio
White adipose tissue
Mitochondria are highly dynamic organelles that frequently fuse and divide in response to cellular energy demands, differentiation or pathological conditions [1, 2, 3]. The endoplasmic reticulum (ER) is an extensive, morphologically continuous network of membrane tubules in the eukaryotic cell. It performs a variety of functions in cells including lipid synthesis, intracellular Ca2+ regulation and protein secretion . Recently, it was reported that the ER plays an active role in defining the sites of mitochondrial division . In fact, the two organelles physically interact, forming specialised contacts referred to as mitochondria-associated ER membrane (MAM). MAM has been linked with lipid transfer, apoptotic calcium signalling and mitochondria–ER calcium homeostasis . Furthermore, MAM has recently been shown to regulate mitochondrial shape and motility, energy metabolism and redox status and to be central to the modulation of various key processes such as ER stress, autophagy and inflammasome signalling .
In vertebrates, mitofusin-1 and -2 (MFN1 and MFN2) are involved in mitochondrial fusion and dynamin-related protein 1 (DRP1) and mitochondrial fission factor (MFF) control mitochondrial fission [8, 9]. During the fission cycle, DRP1 first binds to MFF on the surface of mitochondria, followed by entry into a complex that includes ER proteins at the ER–mitochondria interface . Reciprocally, ER-localised inverted formin 2-mediated actin polymerisation is required for efficient mitochondrial fission , suggesting that ER contact is required for initiation of mitochondrial fission. Recently, MFN2 has been shown to be involved in MAM architecture and to regulate insulin signalling and insulin sensitivity in muscle and liver . Furthermore, MAM integrity is required for insulin signalling and is implicated in hepatic insulin resistance . These results led us to hypothesise that DRP1 plays a role in glucose homeostasis through organelle communication between the ER and mitochondria. To investigate such a role of mitochondrial fission, we generated mice with liver-specific deletion of Drp1 (Drp1LiKO). Drp1LiKO mice showed decreased fat mass and were protected from high-fat diet (HFD)-induced obesity. Further analysis revealed that defects of mitochondrial fission in the liver cause ER stress, leading to induction of fibroblast growth factor 21 (FGF21), a metabolic regulator of glucose and lipid homeostasis. In this study, we show that mitochondrial fission in the liver plays an important role in energy homeostasis through induction of FGF21.
Drp1LiKO mice were generated by crossing Drp1 flox/+ mice  with Alb-Cre mice. Mice were fed ad libitum with a normal chow diet (NCD) (5.4% fat, CRF-1; Orient Yeast, Tokyo, Japan) and kept under a 12 h light–dark cycle. For the HFD study, 4-week-old mice were put on an HFD (24% fat, lard fat, 188.28 kJ% fat, D12451; Research Diets, New Brunswick, NJ, USA) for 16–24 weeks. See electronic supplementary material (ESM) Methods for further details.
The in vivo insulin sensitivity was assessed by quantification of Akt phosphorylation in response to exogenous insulin. See ESM Methods for details.
For histochemical and immunohistochemical analyses, adipose tissue and livers were fixed, processed, stained and quantified. The morphologies of mitochondria and ER were analysed by electron microscopy. See ESM Methods for details.
Real-time quantitative RT-PCR was used to determine the relative expression levels of mRNA. Results were normalised to Gapdh expression. Liver mRNA from control and Drp1LiKO mice was subjected to microarray analysis. See ESM Methods for details.
Total liver lipid content was determined by the Folch method . For VLDL secretion assays, mice were fasted for 4 h before being injected with 0.5 g/kg tyloxapol (Triton-1339; Sigma, St Louis, MO, USA). See ESM Methods for details.
All data are expressed as means ± SE and evaluated by Student’s t test using Microsoft Excel software (Mac 2011 version 14.3.5; Microsoft Japan, Tokyo, Japan), two-way ANOVA performed with Tukey’s post hoc test or repeated measures two-way ANOVA performed with the Bonferroni post hoc test using GraphPad Prism 6.0 software (GraphPad; San Diego, CA, USA). To detect an outlier, we performed the Grubbs’ test using the free statistical calculators: QuickCals, http://www.graphpad.com/quickcalcs/. Significance levels were set at p < 0.05, p < 0.01 and p < 0.001.
Ablation of Drp1 protects mice from HFD-induced obesity
When fed an NCD, reduced inflammatory cell infiltration was found in Drp1LiKO mice (Fig. 1i). Reflecting hepatic inflammation, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were 3.17-fold and 2.93-fold higher in the HFD Drp1LiKO mice, respectively, when compared with control mice (Fig. 1j, k).
Hepatic lipid content in Drp1LiKO mice is comparable with that in control mice
Decreased hepatic VLDL secretion in Drp1LiKO mice
The levels of serum triacylglycerol and total cholesterol were significantly lower in Drp1LiKO mice than in control mice after 17 h fasting, while the total serum NEFA level was no different (Fig. 2i–k). Since food intake increases hepatic fatty acid uptake and VLDL secretion, these lipid profiles were also analysed after refeeding for 4 h. The levels of serum triacylglycerol and total cholesterol were significantly lower in Drp1LiKO mice than in control mice. It was noted that the increase in serum triacylglycerol after feeding was completely negated in Drp1LiKO mice (Fig. 2i), suggesting that the defect may also reside in VLDL secretion in Drp1LiKO mice. Serum triacylglycerol values, reflecting hepatic triacylglycerol secretion, were measured after intraperitoneal tyloxapol injection. Drp1LiKO mice showed significantly reduced rates of hepatic triacylglycerol secretion (Fig. 2l). Reduced levels of serum triacylglycerol might be attributed to, in part, defective VLDL secretion.
Drp1LiKO mice display improved glucose metabolism
Loss of Drp1 altered the mitochondria–ER positional relationship
Loss of Drp1 causes ER stress and activates the eukaryotic translation factor 2α–activating transcription factor 4–FGF21 pathway
Top upregulated genes in Drp1LiKO mouse liver
Activating transcription factor 3
Nuclear protein 1
Maternally expressed 3
Insulin-like growth factor binding protein 1
Tribbles homolog 3
FGF21 has recently emerged as a hepatic stress hormone and an effective metabolic regulator of glucose and lipid homeostasis [22, 23, 24]. Furthermore, Fgf21 expression has shown to be induced by the protein kinase R-like ER kinase (PERK)–eIF2α–ATF4 axis [25, 26, 27]. Activation of the elF2α–ATF4 axis was demonstrated in the liver of Drp1LiKO mice (Fig. 5e) so we examined Fgf21 gene expression in Drp1LiKO mice. When mice were fed with NCD, no statistical difference in Fgf21 mRNA expression was found between control and Drp1LiKO mice (Fig. 5g). Feeding with HFD resulted in a significant increase in Fgf21 mRNA expression in Drp1LiKO mice but not in control mice (Fig. 5g). Serum FGF21 concentrations in Drp1LiKO mice were significantly higher than those in control mice on NCD (Fig. 5h); these levels were increased further by an HFD burden. As shown in Fig. 5i, j, mRNA expression of the receptor complex for Fgf21, Fgfr1 and Klb, was increased in WAT of Drp1LiKO mice, suggesting that FGF21 has a possible feedforward effect on its own signalling pathway . The expression of FGF21 was further assessed by immunohistochemistry. There was scattered and faint staining in control liver (Fig. 5k). In contrast, a remarkable increase in FGF21 protein was observed in the cytoplasm of hepatocytes from Drp1LiKO mouse livers (Fig. 5k).
Increased energy expenditure in Drp1LiKO mice
In this study, we showed that lack of DRP1 in mouse hepatocytes caused ER stress, which was dramatically exacerbated by an HFD. In addition, we found that the PERK–eIF2α–ATF4 pathway was dramatically activated in the liver of Drp1LiKO mice (Fig. 5a–e). Loss of DRP1 in the liver had no effect on the inositol-requiring 1α (IRE1α) and ATF6 pathways (data not shown). Although ATF6, PERK and IRE1α share functionally similar luminal sensing domains and are activated in cells treated with ER stress inducers in vitro, they are selectively activated and regulate diverse downstream target genes in vivo . Our results suggest that mitochondrial fission may act upstream to the cascade of the ER stress response and that communication between mitochondria and the ER is indispensable to the maintenance of cellular homeostasis. In this study, we provided direct evidence that loss of DRP1 alters ER architecture. Mitochondrial dynamics, which are changed in response to metabolic burden, may play an important role in maintaining ER morphology and function.
We observed that a defect in mitochondrial fission and subsequent ER stress promoted the expression of FGF21 in the liver and that the increased FGF21 in turn functions as a metabolic regulator, exhibiting anti-obesity and anti-diabetes effects [22, 23, 24]. A recent report by Kim et al demonstrated that autophagy deficiency in muscle leads to protection against obesity and insulin resistance by inducing FGF21 . Interestingly, autophagy deficiency also results in morphologically abnormal, swollen mitochondria, reminiscent of those found in the liver of Drp1LiKO mice on an HFD. These results led us to hypothesise that defects in the inter-organelle communication between mitochondria and ER might depend on FGF21 signalling as a self-defence system to maintain metabolic homeostasis. Recently, Sebastian et al reported that liver-specific Mfn2-knockout mice showed glucose intolerance with hepatic insulin resistance . Loss of MFN2 causes impaired mitochondrial respiration, indicating that MFN2 has multiple roles beyond regulating mitochondrial morphogenesis. There may be different roles for mitochondrial fusion and fission in organelle communication inducing Fgf21. FGF21 increases expression of thermogenic genes in adipose tissue and utilisation of lipids. In addition, FGF21 improves insulin sensitivity by increasing glucose uptake in adipocytes  and FGF21-deficient mice are insulin resistant and hyperinsulinaemic . It is thus conceivable that the favourable whole-body insulin sensitivity in Drp1LiKO mice can be explained, at least in part, by increased FGF21, although they have hepatic insulin resistance.
Energy homeostasis is achieved by multiple mechanisms, including inter-organ communication. As well as humoral factors, recent studies have demonstrated that neuronal pathways play an important role in energy homeostasis as a feedback mechanism, preventing excessive energy intake and enhancing energy expenditure . The interesting possibility that the signal from the liver of Drp1LiKO mice uses neuronal pathways in addition to FGF21 cannot be excluded. Nevertheless, our results suggests that mitochondrial dynamics are relevant to understanding the pathogenesis of human metabolic diseases caused by perturbations resulting from overnutrition.
In conclusion, we provide new insight into the role of mitochondrial fission in energy homeostasis. DRP1 may be a potential target for intervention in metabolic diseases such as diabetes and obesity.
We thank H. Kawate, K. Ashida and K. Ohnaka of the Graduated School of Medical Science, Kyushu University for helpful discussions. We also thank the Research Support Center, Graduate School of Medical Science, Kyushu University, for technical support.
Access to research materials
The microarray data from this publication have been submitted to the GEO database http://www.ncbi.nlm.nih.gov/geo/ and assigned the identifier accession: GSE64222.
This work was supported in part by the Japanese Society for the Promotion of Science (JSPS) KAKENHI (MN, Grant no. 26461383, 12F02426; RT, Grant no. 23390247) and Grants-in-Aid for Research Fellowship for Young Science Foundation and Banyu Science Foundation (LW). The work of MN was supported by a grant from the Medical Research Encouragement Prize of The Japan Medical Association.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
LW and MN designed the project, analysed the data and wrote the manuscript. LW, TI, DS, YH, YT, SS and SY acquired data. YI and KT contributed to the generation of the mice. All authors contributed to analysis and interpretation of the data, revised the manuscript critically for important intellectual content and approved the final version of the paper to be published. MN is the guarantor of the article.