A mouse model of human mitofusin-2-related lipodystrophy exhibits adipose-specific mitochondrial stress and reduced leptin secretion

Abstract

Mitochondrial dysfunction has been reported in obesity and insulin resistance, but primary genetic mitochondrial dysfunction is generally not associated with these, arguing against a straightforward causal relationship. A rare exception, recently identified in humans, is a syndrome of lower body adipose loss, leptin-deficient severe upper body adipose overgrowth, and insulin resistance caused by the p.Arg707Trp mutation in MFN2, encoding mitofusin 2. How the resulting selective form of mitochondrial dysfunction leads to tissue- and adipose depot-specific growth abnormalities and systemic biochemical perturbation is unknown. To address this, Mfn2^R707W/R707W knock-in mice were generated and phenotyped on chow and high fat diets. Electron microscopy revealed adipose-specific mitochondrial morphological abnormalities. Oxidative phosphorylation measured in isolated mitochondria was unperturbed, but the cellular integrated stress response was activated in adipose tissue. Fat mass and distribution, body weight, and systemic glucose and lipid metabolism were unchanged, however serum leptin and adiponectin concentrations, and their secretion from adipose explants were reduced. Pharmacological induction of the integrated stress response in wild-type adipocytes also reduced secretion of leptin and adiponectin, suggesting an explanation for the in vivo findings. These data suggest that the p.Arg707Trp MFN2 mutation selectively perturbs mitochondrial morphology and activates the integrated stress response in adipose tissue. In mice, this does not disrupt most adipocyte functions or systemic metabolism, whereas in humans it is associated with pathological adipose remodelling and metabolic disease. In both species, disproportionate effects on leptin secretion may relate to cell autonomous induction of the integrated stress response.

Keywords: adipose tissue; cell biology; integrated stress response; leptin; mitochondria; mitofusin; mouse.

Figure 1.. Generation of a Mfn2^R707W knock-in mouse.

(A) Wild-type (WT) nucleotide and amino acid sequence around the Arg 707 codon. The CRISPR/Cas9 nuclease target is indicated in green. Below is part of the ssODN template with mutated nucleotides in blue, including the upstream silent mutation (at codon 704–705) to generate an EcoRV restriction site. (B) Sanger sequencing confirmation of the knock-in (KI) with restriction site in a founder (F0). (C) Illustration of the genotyping strategy: mutant alleles will digest into 327 bp and 196 bp fragments in response to EcoRV digestion. (D) Ear biopsies were digested using chelix and Mfn2 amplified by PCR, then digested using EcoRV. Representative SYBR Safe DNA gel demonstrating genotyping for two WT, heterozygous, and homozygous KI mice. Image is representative of other genotyping gels. (E) Western blot from inguinal and epididymal white adipose tissue (WAT), brown adipose tissue (BAT), liver and skeletal muscle for expression of Mfn1 and Mfn2. Tissues are from WT and homozygous Mfn2^R707W KI mice fed a 45% kcal high fat diet (HFD) for 6 months. Due to variability across tissues, both Gapdh and Beta-tubulin (Tubb) are given as loading controls. The image is representative of at least three biological replicates.

Figure 1—figure supplement 1.. Expression of mitofusins on chow diet.

Western blots showing expression of Mfn2 and Mfn1 in liver (A), heart (B), skeletal muscle (C), BAT (D), inguinal WAT (E), and epididymal WAT (F) in WT and homozygous Mfn2^R707W KI mice fed chow diet for 6 months; and (G) expression of Mfn2 and Mfn1 in BAT in mice fed chow for 4 weeks. Each lane contains samples from a separate animal and blots are representative of at least three replicates. Canx (calnexin), Gapdh, and Tubb (β-tubulin) are loading controls.

Figure 1—figure supplement 2.. Quantification of western blots for mitofusins in tissues of chow-diet-fed mice.

Densitometric quantification of Mfn1 and Mfn2 bands from liver (A), heart (B), skeletal muscle (C), BAT (D), inguinal WAT (E), and epididymal WAT (F) in male mice fed chow diet for 6 months; and (G) BAT in mice fed chow diet for 4 weeks. Data are from n=3–9 animals. Each point represents data from a separate animal. Asterisks indicate p-values from pairwise comparisons (T-tests) that are false-discovery rate (FDR)-adjusted for multiple tests (* p-FDR <.05). WT in blue, homozygous Mfn2^R707W KI in green.

Figure 1—figure supplement 3.. Expression of mitofusins on high-fat diet.

Western blots showing expression of Mfn2 and Mfn1 in liver (A), heart (B), skeletal muscle (C), BAT (D), inguinal WAT (E), and epididymal WAT (F) from WT and homozygous Mfn2^R707W KI mice fed a 45% kcal high fat diet (HFD) for 6 months. Each lane contains samples from a separate animal and blots are representative of at least three replicates. Canx (calnexin), Gapdh, and Tubb (β-tubulin) are given as loading controls.

Figure 1—figure supplement 4.. Quantification of western blots for mitofusins in tissues of high-fat diet-fed mice.

Densitometric quantification of bands for Mfn1 and Mfn2 from male mice fed HFD for 6 months is shown. Data are from liver (A), heart (B), skeletal muscle (C), BAT (D), inguinal WAT (E), and epididymal WAT (F). Data are from n=3–10 animals. Each point represents a separate animal. Asterisks indicate p-values from pairwise comparisons (T-tests) that are FDR-adjusted for multiple tests (* p-FDR <0.05, ** p-FDR <0.01). WT in blue, homozygous Mfn2^R707W KI in green.

Figure 2.. Effect of Mfn2^R707W on mitochondrial structure and function.

(A) Representative transmission electron microscopy (TEM) images of BAT with zoomed-in images of mitochondria (highlighted in yellow) bordering lipid droplets (outlined in red). (B) Quantification of mitochondrial perimeter from TEM on BAT. Each dot represents data from an individual mitochondrial cross section with each diamond showing the separate 6 biological replicates. p.adj gives the FDR-adjusted p-value from across all TEM analyses. (C) Quantification of mitochondrial aspect ratio (length/width) from TEM of BAT. (D) Quantification of mitochondrial-lipid droplet contact from TEM, expressed as proportion (%) of lipid droplet in contact with mitochondrial membrane on BAT. (E) Number of cristae per mitochondrion from TEM of BAT. (F–G) Mitochondrial DNA content in tissues from mice fed chow diet (F) or HFD (G) for 6 months. Each data point represents a separate animal. p-values are from pairwise comparisons (T-tests) between WT and Mfn2^R707W KI that are FDR-adjusted for multiple tests (* p-FDR <0.05). WT in blue, homozygous Mfn2^R707W KI in green. AU, arbitrary units.

Figure 2—figure supplement 1.. Effect of Mfn2^R707W on mitochondrial morphology in white adipose tissue.

(A) Representative TEM images from inguinal WAT with zoomed-in images of mitochondria (highlighted in yellow) bordering lipid droplets (outlined in red). Quantification of mitochondrial perimeter (B) and aspect ratio (length/width) (C) from TEM in inguinal WAT. Each dot represents an individual mitochondrion with diamonds showing biological replicates. p.adj gives the FDR-adjusted p-value from across all TEM analyses. (D) TEM images from epididymal WAT with zoomed-in images of mitochondria (highlighted in yellow) bordering lipid droplets (outlined in red). Quantification of mitochondrial perimeter (E) and aspect ratio (length/width) (F) from TEM on epididymal WAT. FDR-adjusted p-values are from pairwise comparisons (T-tests) between WT and Mfn2^R707W KI. WT in blue, homozygous Mfn2^R707W KI in green.

Figure 2—figure supplement 2.. Effect of Mfn2^R707W on mitochondrial morphology in liver, heart, and muscle.

(A) Representative TEM images from the heart with zoomed-in images of mitochondria (highlighted in yellow). (B) Quantification of mitochondrial perimeter from TEM on heart. Each dot represents an individual mitochondrion with diamonds showing biological replicates. p.adj gives the FDR-adjusted p-value from across all TEM analyses. (C) Quantification of mitochondrial aspect ratio (length/width) from TEM on heart. (D) TEM images from skeletal muscle with zoomed-in images of mitochondria (highlighted in yellow). Quantification of mitochondrial perimeter (E) and aspect ratio (length/width) (F) from TEM on skeletal muscle. (G) TEM images from liver with zoomed-in images of mitochondria (highlighted in yellow). Quantification of mitochondrial perimeter (H) and aspect ratio (length/width) (I) from TEM on liver. FDR-adjusted p-values are from pairwise comparisons (T-tests) between WT and Mfn2^R707W KI. WT in blue, homozygous Mfn2^R707W KI in green.

Figure 2—figure supplement 3.. Altered expression of Oxphos protein subunits in adipose tissue.

Western blots showing expression of Oxphos protein subunits in mice fed chow diet for 6 months. Expression is shown from liver (A), heart (B), inguinal WAT (C), epididymal WAT (D), and BAT (E). Each lane contains samples from a separate animal and blots are representative of at least three replicates. Canx (calnexin) is used as total cellular control and Tomm20 (or citrate synthetase (Cs)) is used as mitochondrial mass loading control.

Figure 2—figure supplement 4.. Quantification of western blots for Oxphos subunits from tissues of chow-diet-fed mice.

Densitometric quantification of bands for mitochondrial Oxphos subunits from male mice fed chow diet for 6 months. Data are from liver (A), heart (B), inguinal WAT (C), epididymal WAT (D), and BAT (E). Data are from n=3–8 animals per genotype. Each point represents a separate animal. Asterisks indicate p-values from pairwise comparisons (T-tests) that are FDR-adjusted for multiple tests (* p-FDR <0.05, ** p-FDR <0.01). WT in blue, homozygous Mfn2^R707W KI in green.

Figure 2—figure supplement 5.. Mfn2^R707W does not impair brown adipose tissue thermogenic capacity.

Ex vivo mitochondrial respirometry in liver (A, n=4) and BAT (B, n=9). Each data point represents data from a separate animal. Eight-week-old chow-diet-fed mice were exposed to cold (10 °C, n=16–18) or thermoneutrality (30 °C, n=13–14) for 4 weeks and then maximum thermogenic capacity was tested using noradrenaline (NA) stimulation under anaesthesia. (C) Trend in energy expenditure (kcal/hr) before and after noradrenaline stimulation for cold (solid line) and thermoneutral (dashed line) housed animals. (D) Quantification of maximum (peak) energy expenditure. P-values represent the difference between groups comparing 10°C and 30°C. (E) Area under the curve analysis using the minimum value at the point of noradrenaline (NA) injection at the base of the curve. (F) Basal energy expenditure prior to NA injection. (G) Difference between peak and baseline energy expenditure under both conditions. WT in blue, homozygous Mfn2^R707W KI in green. Each data point represents results from an individual animal. FDR-adjusted p-values are from pairwise comparisons (T-tests) between groups as indicated.

Figure 3.. No difference in fat mass or glucose homeostasis in Mfn2^R707W mice compared to WT mice on chow or high fat diet.

Seven-week-old mice were fed chow (n=8–12) or 45% kcal HFD (n=13–14) for 6 months. (A) Absolute body mass for mice fed chow (solid line) and HFD (dashed line) over 6 months. (B) Time-domain nuclear magnetic resonance (TD-NMR) measurement of fat and lean mass of mice fed HFD. (C) The ratio of tissue weights versus body mass for multiple tissues, including four WAT depots, from mice fed HFD. (D) Quantification of lipid droplet area from histological specimens of adipose tissue from mice fed HFD for 6 months. (E) Representative histological images from WAT, liver, and BAT from mice fed HFD. Analyses of mouse plasma lactate (F), plasma glucose (G), and insulin (H), homeostatic model of assessment of insulin resistance (HOMA-IR, I) after a 6 hr fast. Change in plasma glucose during intraperitoneal glucose tolerance test (J) and intraperitoneal insulin tolerance test (K). ns, p>0.05 on unpaired T-tests, adjusted for multiple comparisons. WT in blue, homozygous Mfn2^R707W KI in green. Each data point represents an individual animal.

Figure 3—figure supplement 1.. No evidence of altered fat mass or glucose homeostasis in Mfn2^R707W compared to WT mice on chow or high fat diet for 6 months.

Seven-week-old mice were fed chow diet (n=8–12) or 45% kcal HFD (n=13–14) for 6 months. (A) Relative (%) change of body mass for mice fed chow diet (solid line) and HFD (dashed line) over 6 months. (B) TD-NMR measurement of fat and lean mass in mice fed a chow diet for 6 months. (C) Analysis of mouse serum biochemistry after 6 months of diet and a 6 hr fast for total cholesterol. (D) The ratio of tissue weights versus body mass for multiple tissues, including four WAT depots, from mice fed chow diet for 6 months. (E) Serum triglycerides. (F) Quantification of lipid droplet area from histological specimens of adipose tissue from mice fed chow diet for 6 months. (G) Quantification of relative hepatic steatosis from histological images of liver. (H) Serum aspartate aminotransferase. (I) qPCR of genes implicated in the integrated stress response, lipid metabolism or inflammation in liver from animals fed HFD for 6 months. Raw expression values for target genes were normalised to three housekeeping genes (36b4, B2m, and Hprt) and expressed relative to WT liver. ns, p>0.05 on unpaired T-tests, adjusted for multiple comparisons. WT in blue, homozygous Mfn2^R707W in green. Each data point represents results from an individual animal.

Figure 3—figure supplement 2.. Similar phenotype in female Mfn2^R707W knock-in mice fed high fat diet for 6 months.

Seven-week-old female mice were fed 45% kcal HFD (n=5–11) for 6 months. (A) Absolute body mass for over 6 months. (B) Body mass after 12 weeks on HFD. (C) TD-NMR measurement of fat and lean mass of female mice after 12 weeks on HFD. (D–F) Analysis of mouse serum biochemistry after 12 weeks of diet and a 6 hr fast for serum glucose (D), serum insulin (E), and serum adiponectin (F). Analysis of mouse serum leptin at 4 weeks (G), 12 weeks (H), and 24 weeks (I) on HFD after a 6 hr fast. WT in blue, homozygous Mfn2^R707W KI in green. Each data point represents an individual animal. Asterisks indicate p-values from pairwise comparisons (T-tests) between WT and Mfn2^R707W KI that are FDR-adjusted for multiple tests (*** p-FDR <0.001).

Figure 4.. Mfn2^R707W causes an adipose-tissue-specific induction of the integrated stress response.

qPCR of genes involved in the integrated stress response (ISR) for six tissues from animals fed chow diet for 6 months: Atf4 (A), Ddit3 (Chop, B), and Atf5 (C). Each data point represents one animal. Target gene CT values were normalised to three housekeeping genes (36b4, B2m, and Hprt) and expressed relative to WT liver for each gene. WT in blue, homozygous Mfn2^R707W in green. p-values are FDR-adjusted for multiple tests. Western blots from BAT (D), epididymal WAT (E), and liver (F) illustrating Ser51-phosphorylation of eIF2α and expression of Mthfd2 with calnexin (Canx) as loading control. Western blots are representative of at least three biological and technical replicates. (G) Volcano plot from bulk RNA sequencing (n=8 per genotype) of inguinal WAT from mice on HFD. Significantly differentially expressed genes (Log₂ fold change >1.5 and p-FDR <0.001) are highlighted in orange. Pathway analysis using significantly differentially expressed genes for upregulated (H) and downregulated (I) Hallmark gene sets. The X-axis depicts a relative gene set enrichment score. All illustrated gene sets are enriched with p-FDR <0.05. Asterisks indicate p-values from pairwise comparisons (T-tests) between WT and Mfn2^R707W KI that are FDR-adjusted for multiple tests (* p-FDR <0.05, ** p-FDR <0.01, *** p-FDR <0.001, **** p-FDR <0.0001).

Figure 4—figure supplement 1.. Mfn2^R707W causes adipose tissue-specific induction of the integrated stress response with perturbation of mitochondrial gene expression.

qPCR of genes involved in the ISR for six tissues from animals fed a chow diet for 6 months: Gdf15 (A) and Fgf21 (B). Each data point represents data from a separate animal. Target gene CT values were normalised against three housekeeping genes (36b4, B2m, and Hprt) and expressed relative to WT liver for each gene. p-Values are FDR-adjusted for multiple tests. Serum FGF-21 (C) and GDF-15 (D) after 6 months of chow diet or HFD. Western blots from inguinal WAT (E), skeletal muscle (F), and heart (G) illustrating Ser51-phosphorylation of eIF2α and expression of Mthfd2 with calnexin (Canx) as loading control. Western blots are representative of at least three biological replicates. Mthfd2 in heart (G) is the upper band within the box, as indicated by a horizontal line. (H) Heatmap of all genes from the citrate acid (tricarboxylic acid (TCA)) cycle KEGG pathway. Colour illustrates the Log₂ fold change normalised per gene. Those annotated with orange in the leftmost column had significant differential gene expression (DGE) with p-FDR <0.001. (I) Heatmaps comparing mRNA expression of mitochondrial- and nuclear-encoded mitochondrial genes from inguinal WAT from HFD-fed animals. Asterisks indicate p-values from pairwise comparisons (T-tests) between WT and Mfn2^R707W KI that are FDR-adjusted for multiple tests (*** p-FDR <0.001).

Figure 4—figure supplement 2.. Quantification of western blots for components of the integrated stress response in high-fat diet-fed mice.

Densitometric quantification of bands for Mthfd2 and phosphorylated Ser-51 eIF2α from male mice fed HFD for 6 months. (A) BAT, (B) epididymal WAT, (C) liver, (D) inguinal WAT, (E) skeletal muscle, and (F) heart. Data are from three to nine animals. Each point represents a separate animal. Asterisks indicate p-values from pairwise comparisons (T-tests) between WT and Mfn2^R707W KI that are FDR-adjusted for multiple tests (* p-FDR <0.05, ** p-FDR <0.01, *** p-FDR <0.001). WT in blue, homozygous Mfn2^R707W KI in green.

Figure 4—figure supplement 3.. Mfn2^R707W does not affect Oma1 or Opa1 processing in heart, liver, and muscle.

Western blots of heart (A), liver (B), and skeletal muscle (C) for Opa1 and Oma1 are shown. Tissues are from male mice fed chow diet for 6 months. Each lane contains samples from a separate animal, with band intensity quantified densitometrically. Opa1 is quantified as percentage of the short form of Opa1 (bands c and e of S-Opa1) to total Opa1 (long and short forms, bands a-e). ns, p>0.05 on unpaired T-tests, adjusted for multiple comparisons. WT in blue, homozygous Mfn2^R707W KI in green.

Figure 4—figure supplement 4.. Evidence of modest activation of the Oma1-Opa1 pathway in brown adipose tissue from Mfn2^R707W mice.

Western blots of BAT and WAT for Opa1 and Oma1 are shown. Tissues are from male mice fed chow diet. (A) BAT (chow diet for 4 weeks), (B) BAT (chow diet for 6 months), (C) Epididymal WAT (chow diet for 6 months), and inguindal WAT (chow diet for 6 months). Each lane contains samples from a separate animal with band intensity quantified densitometrically. Opa1 is quantified as percentage of the short form of Opa1 (bands c and e of S-Opa1) to total Opa1 (long and short forms, bands a-e). ns, p>0.05 on unpaired T-tests, adjusted for multiple comparisons. WT in blue, homozygous Mfn2^R707W KI in green. * p-FDR <0.05.

Figure 4—figure supplement 5.. Transcriptional evidence of upregulation of the unfolded protein response and mTorc1 pathways in adipose tissue.

(A) Volcano plot from bulk RNA sequencing (n=7 WT and n=6 KI) of inguinal WAT from mice on chow diet, where significantly differentially expressed genes (Log₂ fold change >1.5 and p-FDR <0.001) are highlighted in orange. Pathway analysis using significantly differentially expressed genes for upregulated (B) and downregulated (C) Hallmark gene sets. X-axis depicts a relative gene set enrichment score. (D) Volcano plot from bulk RNA sequencing (n=6 per genotype) of BAT from mice on HFD, where significantly differentially expressed genes (Log₂ fold change >1.5 and p-FDR <0.001) are highlighted in green. Pathway analysis using significantly differentially expressed genes for upregulated (E) and downregulated (F) Hallmark gene sets. X-axis depicts a relative gene set enrichment score. All illustrated gene sets are enriched with p-FDR <0.05. (G) Scatter plot of the top 100 up-/down-regulated genes from bulk RNA sequencing from inguinal WAT. For each gene, the fold change observed under HFD is plotted on the x-axis and fold change under chow diet is shown on the y-axis. P-values for each dietary condition are shown using size (HFD) and color (chow diet).

Figure 5.. Mfn2^R707W decreases adipose secretion of leptin and adiponectin.

Fasting serum leptin (A) and adiponectin (B) from mice after 6 months on chow diet or HFD. Asterisks indicate t-tests comparing WT and Mfn2^R707W KI with p-values adjusted for multiple testing. (C) Relationship between leptin and body weight for animals fed HFD. Each data point represents one measurement of leptin, with multiple measurements per animal. Shaded area represents the 95% confidence interval. Data are from n=13–14 animals. qPCR of Lep (D) and Adipoq (E) from WAT depots. Each data point represents data from a separate animal. Target gene CT values were normalised against three housekeeping genes (36b4, B2m, and Hprt) and expressed relative to WT for each condition. (F–I) mRNA expression of Atf4, Chop, Lep, and Adipoq in primary adipocytes treated with either DMSO control or Thapsigargin (TG, 150 nM) or Tunicamycin (TN, 5 μg/mL) for 6 hr. Each data point represents an individual well from a separate biological experiment (n=5). Asterisks indicate significance on one-way ANOVA with correction for multiple comparisons. (J) Leptin and (K) Adiponectin secretion from primary adipocytes treated with either DMSO control or Thapsigargin (TG, 150 nM) or Tunicamycin (TN, 5 μg/mL) over 6 hr. Asterisks indicate significance on one-way ANOVA with correction for multiple comparisons. (L) Representative western blots of primary adipocytes treated with either DMSO control or Thapsigargin (TG, 150 nM) or Tunicamycin (TN, 5 μg/mL) for 6 hr. (M) Fasting serum adipsin (complement factor D) from mice after 6 months on chow diet or HFD. Asterisks indicate p-values from pairwise comparisons (T-tests) between indicated groups that are FDR-adjusted for multiple tests (* p-FDR <0.05, ** p-FDR <0.01, *** p-FDR <0.001, **** p-FDR <0.0001).

Figure 5—figure supplement 1.. Mfn2^R707W decreases adipose leptin and adiponectin secretion from multiple adipose depots and in different dietary conditions.

(A) Leptin secretion from explants of epididymal WAT in basal conditions and after insulin-dexamethasone stimulation. (B) Adiponectin secretion from epididymal WAT explants. (C) qPCR of Lep and Adipoq from epididymal explants from animals fed a HFD, where each data point represents an explant from a separate animal (n=11–13). (D) Relationship between leptin and body weight for animals on chow diet. Each data point represents one measurement of leptin, with multiple measurements per animal. Shaded area represents the 95% confidence interval. Data are from n=8–12 animals. Asterisks indicate p-values from pairwise comparisons (T-tests) between indicated groups that are FDR-adjusted for multiple tests (* p-FDR <0.05, ** p-FDR <0.01, **** p-FDR <0.0001).

Figure 5—figure supplement 2.. Mfn2^R707W decreases leptin and adiponectin protein expression in white adipose tissue.

Western blots showing expression of leptin and adiponectin in male mice fed 45% kcal HFD for 6 months from epididymal (A) and inguinal (B) WAT. Expression is shown from epididymal and inguinal WAT with Gapdh used as a loading control. Each lane contains samples from a separate animal with band intensity quantified densitometrically. Asterisks indicate p-values from pairwise comparisons (T-tests) between WT and Mfn2^R707W KI that are FDR-adjusted for multiple tests (** p-FDR <0.01, **** p-FDR <0.0001). WT in blue, homozygous Mfn2^R707W KI in green.

Author response image 1.. Representative images from transmission electron microscopy on liver and brown adipose tissue from wild-type and Mfn2-R707W knock-in mice.

Endoplasmic reticulum (ER) could be clearly identified and mitochondrial-ER contacts quantified in liver (top), with no difference by genotype. In white and brown adipose tissue there was poor preservation of ER with very infrequent mitochondrial-ER contacts, such that a formal assessment was not possible. Data representative from n=4 (liver) and n=8 (adipose tissue) mice.