Impact of Drp1 Loss on Organelle Interaction, Metabolism, and Inflammation in Mouse Liver

Abstract

Dynamin-related protein 1 (Drp1) is a crucial player in mitochondrial fission and liver function. The interactions between mitochondria, endoplasmic reticulum (ER), and lipid droplets (LDs) are fundamental for lipid metabolism. This study utilized liver-specific Drp1 knockout (Drp1LiKO) mice to investigate the effects of Drp1 deficiency on organelle interactions, metabolism, and inflammation. Our analysis revealed disrupted interactions between mitochondria and LDs, as well as altered interactions among ER, mitochondria, and LDs in Drp1LiKO mice. Through mass spectrometry and microarray analysis, we identified changes in lipid profiles and perturbed expression of lipid metabolism genes in the livers of Drp1LiKO mice. Further in vitro experiments using primary hepatocytes from Drp1LiKO mice confirmed disturbances in lipid metabolism and increased inflammation. These findings highlight the critical involvement of Drp1 in regulating organelle interactions for efficient lipid metabolism and overall liver health. Targeting Drp1-mediated organelle interactions may offer potential for developing therapies for liver diseases associated with disrupted lipid metabolism.

Keywords: dynamin-related protein 1; lipid metabolism; liver inflammation; organelle interaction.

Figure 1

Increased mitochondria–lipid droplet interactions in Drp1LiKO mice. (A) Transmission electron microscopy images depicting cytochrome c oxidase enzyme activity assay in the livers of control and Drp1LiKO fasted mice. Regions denoted by white dashed squares are magnified in the lower panel. Lipid droplet-associated mitochondria (LDMs) are indicated in green, cytosolic mitochondria (CMs) in red, and lipid droplets (LDs) in yellow. Yellow arrows indicated mitochondria–lipid droplet interaction sites. Scale bar = 1 μm. (B–D) Quantitative analysis of the percentage of lipid droplet-associated mitochondria (n = 23), mitochondria size (n = 105–205), and lipid droplet size (n = 43–198) in control and Drp1LiKO fasted mice. Analysis was performed on a total of 23 images, with 3 mice per group. Values are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Determined by two-way ANOVA with Tukey’s multiple comparisons test. (E) Western blot analysis, corresponding densitometric quantification, and correlation analysis of ACSL1 and ACSL4 expression levels. ACTIN served as the loading control. Technical duplicates labeled as #1 and #2; biological replicates labeled as †1, †2, and †3. Data presented as mean ± SEM (n = 3). * p < 0.05, based on two-way ANOVA with Tukey’s multiple comparisons test. (F) Real-time PCR evaluation of Acsl1 and Acsl4 mRNA expression levels. Gapdh employed as the internal control. Data displayed as mean ± SEM (n = 3–5). * p < 0.05, ** p < 0.01, *** p < 0.001. Based on two-way ANOVA with Tukey’s multiple comparisons test.

Figure 2

Disrupted interactions among mitochondria, the endoplasmic reticulum, and lipid droplets in Drp1LiKO mice. (A–D) Electron microscopy images showing glucose-6-phosphatase enzyme activity assay in the livers of control and Drp1LiKO mice. Mitochondria (Mt) are marked in green, lipid droplets (LDs) in yellow, and endoplasmic reticulum (ER) tubules in magenta. Dashed lines indicate the distances of the interactions among Mt, the ER, and LD. Scale bar = 1 μm in (A) and 100 nm in (B–D). (E–G) Quantitative analysis of the percentage (E) (n = 3) and distances (F–G) (n = 8–53) of interactions among Mt, the ER, and LD in control and Drp1LiKO fasted mice, with 3 mice per group. Values are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Determined by two-way ANOVA with Tukey’s multiple comparisons test.

Figure 3

Aberrant lipid metabolite profiles in Drp1LiKO mice. (A) Schematic representation of the experimental procedures. (B) Body weights of the mice utilized in this study. Values expressed as mean ± SEM. n = 3. * p < 0.05. Determined by two-way ANOVA with Tukey’s multiple comparisons test. (C) Heatmap of lipid profiling. A heatmap was generated from the LC-MS analysis of liver lipid extracts. Biological replicate samples in each condition are numbered as #1, #2, and #3. (D–F) Relative concentrations of specific lipid species in the livers of control and Drp1LiKO mice. The quantities of triacylglycerol and free fatty acids are categorized by total carbon atoms and the number of double bonds. Values expressed as mean ± SEM. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. Determined by two-way ANOVA with Tukey’s multiple comparisons test.

Figure 4

Aberrant gene expression profiles in HFD-Drp1LiKO mice and Drp1LiKO primary hepatocytes. (A,B) KEGG pathway enrichment analysis was conducted on differentially expressed genes in the livers of control and Drp1LiKO mice. The X-axis indicates the fold enrichment, while the Y-axis shows the negative logarithm (base 10) of the p-values. The size of the bubbles corresponds to the number of genes. (C) A heatmap illustrating differentially expressed genes associated with lipid metabolism that were identified through microarray analysis. (D) KEGG pathway enrichment analysis of differentially expressed genes in primary hepatocytes isolated from control and Drp1LiKO mice. (E) Gene ontology analysis of differentially expressed genes was performed on control and Drp1LiKO mouse primary hepatocytes treated with 200 μM palmitate. The X-axis represents fold enrichment, and the Y-axis denotes the negative logarithm (base 10) of p-values. The size of the bubbles corresponds to the gene count. (F) Top ten upregulated genes in control and Drp1LiKO hepatocytes following treatment with 200 μM palmitate, identified through microarray analysis.

Figure 5

Inflammation and hepatocyte death in Drp1LiKO primary hepatocytes and mice. (A) Primary hepatocytes isolated from control and Drp1LiKO mice were treated with PBS (referred to as palmitate-0 μM), palmitate (200 or 800 μM), or oleate (800 μM) for 24 h. Cells and culture supernatants were collected. TNF, IL-6, IL-1β, and IFN-γ levels were determined using a BD cytometric bead array (n = 3). Values are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 determined by two-way ANOVA with Sidak’s post hoc test. (B) Western blot analysis and densitometric quantification of p-P65 NF-κB, P65 NF-κB, and NLRP3 in PBS- or palmitate-treated cells. GAPDH served as the internal control. The technical duplicate samples in each condition were numbered as #1 and #2. Biological samples are numbered from Cont 1-2 and Drp1LiKO 1-2. Values are expressed as mean ± SEM (n = 4). * p < 0.05, ** p < 0.01 determined by two-way ANOVA with Tukey’s multiple comparisons test. (C,D) Control and Drp1LiKO mice were fed either an NCD or HFD for 16–24 weeks. Liver sections were subjected to F4/80 immunostaining and TUNEL assay to evaluate histological changes. (C) Representative images showing liver histology. TUNEL-positive cells indicated by red arrowheads, and necrotic cells indicated by green arrows. Areas indicated with green dashed squares are enlarged and displayed on the bottom left. Scale bar = 50 μm. (D) Quantitative analysis of F4/80-positive cells, TUNEL-positive cells, and necrotic cells performed by counting cells in composite images created by merging 15 high-power fields (20× magnification). Each mouse was analyzed using 3 joint images, with 3–4 mice per experimental group. Values expressed as mean ± SEM. n = 3–4. * p < 0.05, ** p < 0.01, *** p < 0.001. Determined by two-way ANOVA with Tukey’s post hoc test.

Figure 6

Increased lipid droplet formation in Drp1LiKO mouse primary hepatocytes. (A) Primary hepatocytes isolated from control and Drp1LiKO mice were treated with PBS (referred to as palmitate-0 μM), palmitate (200 μM), or oleate (200 μM) for 24 h. Representative images show MitoTracker Red staining for mitochondria and LipidTox Green for lipid droplets. Nuclei are stained with Hoechst 33342 (blue). Differential interference contrast (DIC) imaging enhances contrast in brightfield images. Areas indicated with white dashed squares are enlarged and displayed on the right. Scale bar = 10 μm. (B) Intensity profile analysis of pixels marked by the line shown in panel (A). (C) Quantitative analysis of the total area and average size of lipid droplets in the control and Drp1LiKO primary hepatocytes. Values are expressed as mean ± SEM. n = 9 (3 images from 3 individual experiments). * p < 0.05, ** p < 0.01, *** p < 0.001 determined by two-way ANOVA with Tukey’s post hoc test.

Figure 7

Disrupted lipid metabolism in the liver of Drp1LiKO mice and the potential impact of organelle interactions on lipid levels under varying dietary conditions. In control mice exposed to a high-fat diet (HFD), elevated levels of lysophosphatidic acid (LPA), phosphatidic acid (PA), diacylglycerol (DAG), and triacylglycerol (TAG) were observed. Conversely, NCD-Drp1LiKO mice exhibited significantly higher levels of LPA, PA, DAG, and TAG under normal chow diet (NCD) conditions but significantly lower levels under HFD conditions. The increased mitochondrial–lipid droplet (Mt-LD) interactions observed in NCD-Drp1LiKO mice may play a dual role as both a contributing factor and a consequence of the elevated TAG levels. Upon exposure to a high-fat diet, control mice showed an increase in lipid droplet size along with increased distances between mitochondria and lipid droplets in Mt-ER-LD and Mt-ERs-LD interactions. This spatial arrangement could potentially hinder lipid transfer between mitochondria and lipid droplets. In HFD-Drp1LiKO mice, the absence of increased distances between mitochondria and lipid droplets might be related to the observed reduction in TAG levels.