SPAG7 deletion causes intrauterine growth restriction, resulting in adulthood obesity and metabolic dysfunction
- Internal Medicine Research Unit, Pfizer Inc, United States
- Center for Research on Reproduction and Women's Health, Perelman School of Medicine, University of Pennsylvania, United States
- Division of Neonatology, Department of Pediatrics, Children’s Hospital of Philadelphia, United States
- Drug Safety Research and Development, Pfizer Inc, United States
- Medicine Design, Pfizer Inc, United States
Figures
Figure 1 with 1 supplement
SPAG7-deficiency causes obesity and insulin resistance.
(A) Graphical representation of an ENU-driven forward genetic screen. (B) Images of WT and SPAG7 KO littermates at 32 weeks of age. (C) WT vs SPAG7 KO body weight over time. n=7. Significance was assessed by Welch’s two sample t-test. (D) WT vs SPAG7 KO lean mass over time. n=7. Significance was assessed by Welch’s two sample t-test. (E) WT vs SPAG7 KO fat mass over time. n=7. Significance was assessed by Welch’s two sample t-test. (F) Body length measured from nose to base-of-tail. n=7. Significance was assessed by Welch’s two sample t-test. (G) Bone mineral density, as determined by DEXA scan. n=7. Significance was assessed by Welch’s two sample t-test. (H) Heart weight. n=7. Significance was assessed by Welch’s two sample t-test. (I) Gastrocnemius muscle weight. n=7. Significance was assessed by Welch’s two sample t-test. (J) Tibialis anterior muscle weight. n=7. Significance was assessed by Welch’s two sample t-test. (K) Kidney weight. n=7. Significance was assessed by Welch’s two sample t-test. (L) Brain weight. n=7. Significance was assessed by Welch’s two sample t-test. (M) Liver weight. N=7. (N) Plasma triglyceride levels following 8 hr fast. n=5. Significance was assessed by Welch’s two sample t-test. (O) Plasma triglyceride levels following 8 hr fast with a 2 hr ad-lib refeed. n=5. Significance was assessed by Welch’s two sample t-test. (P) Plasma total cholesterol levels following 8 hr fast. n=5. Significance was assessed by Welch’s two sample t-test. (Q) Plasma total cholesterol levels following 8 hr fast with a 2 hr refeed. n=5. (R) Plasma NEFA levels following 8 hr fast. n=5. Significance was assessed by Welch’s two sample t-test. (S) Plasma NEFA levels following 8 hr fast with a 2 hr refeed. n=5. Significance was assessed by Welch’s two sample t-test. (T) Blood glucose levels following an oral glucose challenge. n=7. Significance was assessed by Welch’s two sample t-test. (U) Plasma insulin levels following an oral glucose challenge. n=7. Significance was assessed by Welch’s two sample t-test. (V) Blood glucose levels following an IP insulin challenge. n=7. Significance was assessed by Welch’s two sample t-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Figure 1—figure supplement 1
SPAG7 KO adipose tissue and liver.
(A) Gross morphology of WT vs. SPAG7 KO perigonadal (PGAT), intrascapular, subcutaneous inguinal (SQAT), brown (BAT), and mesenteric adipose tissue depots. Scale bars = 10 mm (B) PGAT tissue weights. n=6. Significance was assessed by Welch’s two sample t-test. (C) SQAT tissue weights. n=6. Significance was assessed by Welch’s two sample t-test. (D) BAT tissue weights. n=6. Significance was assessed by Welch’s two sample t-test. (E) Histological sections of WT and SPAG7 KO SQAT stained with hematoxylin and eosin. Scale bars = 200 µm. (F) SQAT average adipocyte area. n=8. Significance was assessed by Welch’s two sample t-test. (G) PGAT average adipocyte area. n=8. Significance was assessed by Welch’s two sample t-test. (H) Number of crown-like structures per mm2 in SQAT. n=8. Significance was assessed by Welch’s two sample t-test. (I) Gross morphology of WT vs SPAG7 KO liver. Scale bars = 10 mm (J) Histological sections of WT vs SPAG7 KO liver tissue. Scale bar = 300 um. (K) Triglyceride content of WT vs SPAG7 KO liver tissue. n=6. Significance was assessed by Welch’s two sample t-test. (L) Circulating ALT levels after a 16 hr fast. N=4. (M) Circulating ALT levels after a 16 hr fast, followed by a 2 hr refeed. N=4. (N) Circulating AST levels following a 16 hr fast. N=4. (O) Circulating AST levels following a 16 hr fast, followed by a 2 hr refeed. n=4. ** p<0.01, *** p<0.001.
Figure 2
SPAG7-deficiency causes decreased locomotor activity and total energy expenditure.
(A) Cumulative food intake as determined by BioDaq Food and Water intake monitoring system. N=7. (B) Hourly energy expenditure as determined by CLAMS metabolic cage system. N=7. (C) Total energy expenditure as determined by CLAMS metabolic cage system. n=7. Significance was assessed by Welch’s two sample t-test. (D) Home cage locomotor activity as determined by CLAMS metabolic cage system. n=7. Significance was assessed by Welch’s two sample t-test. (E) Body weight over time of WT vs SPAG7 KO animals raised at thermoneutrality. n=7. Significance was assessed by Welch’s two sample t-test. (F) Fat mass over time of animals at thermoneutrality. n=7. Significance was assessed by Welch’s two sample t-test. (G) Daily food intake of animals at thermoneutrality. N=7. (H) Total energy expenditure of animals at thermoneutrality as determined by CLAMS. n=7. Significance was assessed by Welch’s two sample t-test. (I) Body weight over time of WT vs SPAG7 KO animals raised on high-fat diet. n=8. Significance was assessed by Welch’s two sample t-test.(J) Fat mass over time of animals fed high-fat diet. n=8. Significance was assessed by Welch’s two sample t-test. (K) Daily food intake of animals fed high-fat diet. N=8. (L) Total energy expenditure of animals fed high-fat diet. n=8. Significance was assessed by Welch’s two sample t-test. (M) Percent body weight difference in SPAG7 KO animals vs WT fed chow diet at room temperature (Chow), fed chow diet at thermoneutrality (TN), or HFD at room temperature (HFD) at 20 weeks of age. n=7. * p<0.05, ** p<0.01.
Figure 3 with 2 supplements
SPAG7-deficiency dampens skeletal muscle function and mitochondrial oxidative capacity.
(A) Distance run until exhaustion for WT vs SPAG7 KO animals in treadmill endurance test. n=7. Significance was assessed by Welch’s two sample t-test. (B) Max VO2 reached during treadmill endurance. n=6. Significance was assessed by Welch’s two sample t-test. (C) In vivo gastrocnemius/soleus complex muscle max force generation. n=6. Significance was assessed by Welch’s two sample t-test. (D) Cross sectional area of myosin heavy chain 2a-expressing fiber in gastrocnemius muscle. n=7. Significance was assessed by Welch’s two sample t-test. (E) Triglyceride content of gastrocnemius muscle. n=6. Significance was assessed by Welch’s two sample t-test. (F) Histological sections of gastrocnemius muscle stained with hematoxylin and eosin. Scale bars = 300 µm. (G) Histological sections of gastrocnemius muscle labeled with antibodies against lectin (red), CD31 (green), and DAPI (blue). Scale bars = 200 µm. (H) Quantification of CD31 +and lectin +capillaries per muscle fiber in gastrocnemius muscle. N=6. (I) Histological sections of gastrocnemius muscle stained for succinate dehydrogenase B activity. Scale bars = 600 µm. (J) Citrate synthase activity of gastrocnemius muscle. n=7. Significance was assessed by Welch’s two sample t-test. (K) Volcano plot of differentially expressed genes in female WT vs SPAG7 KO gastrocnemius muscle following RNAseq. N=7. (L) Gene ontology enrichment pathway analysis of differentially expressed genes in female WT vs SPAG7 KO gastrocnemius muscle following RNAseq. n=7. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Figure 3—figure supplement 1
SPAG7-deficient muscle fiber staining.
(A) Fluorescent microscopy images of WT and SPAG7-deficient gastrocnemius/soleus muscle complex stained for Type 1 Fiber (Orange), Type 2 A Fiber (Green), and Type 2 X Fiber (Red). Scale bars represent 1 mm. (B) SDH staining intensity in WT vs. SPAG7-deficient gastrocnemius muscle. Significance was assessed by Welch’s two sample t-test. (C) Percent SDH-High staining fiber in WT vs. SPAG7-deficient gastrocnemius muscle. Significance was assessed by Welch’s two sample t-test. ** p<0.01.
Figure 3—figure supplement 2
SPAG7-deficient skeletal muscle transcriptomics.
(A) Top 20 genes downregulated in SPAG7 KO gastrocnemius muscle vs WT by log(adjusted p-value). N=7. (B) Top 20 genes upregulated in SPAG7 KO gastrocnemius muscle vs WT by log(adjusted p-value). N=7. (C) Gene expression by qPCR of Spag7 and Hsd17b10 in SPAG7 KO gastrocnemius muscle. n=7. Significance was assessed by Welch’s two sample t-test. (D) Gene expression by qPCR of Spag7 and Hsd17b10 in SPAG7 KO quadriceps muscle. n=7. Significance was assessed by Welch’s two sample t-test. (E) Gene expression by qPCR of Spag7 and Hsd17b10 in SPAG7 KO in perigonadal adipose tissue. n=7. Significance was assessed by Welch’s two sample t-test. (F) Gene expression by qPCR of Spag7 and Hsd17b10 in SPAG7 KO kidney. n=7. Significance was assessed by Welch’s two sample t-test. (G) Gene expression by qPCR of Spag7 and Hsd17b10 in SPAG7 KO liver. n=7. Significance was assessed by Welch’s two sample t-test. (H) Gene expression by qPCR of pag7 and Hsd17b10 in SPAG7 KO brain. n=7. Significance was assessed by Welch’s two sample t-test. * p<0.05, ** p<0.01, *** p<0.001.
Figure 4
Whole-body SPAG7-deficiency induced during adulthood has no effect on systemic metabolism.
(A) Graphic representation of the whole-body inducible SPAG7-deficient mouse model (iSPAG7 KO). (B) Western blot for SPAG7 and bACTIN in liver, brain, kidney, gastrocnemius muscle, and PGAT tissues from iSPAG7 KO animals, 8 weeks following final tamoxifen dose. N=4. (C) iSPAG7 KO body weight over time. N=9. (D) iSPAG7 KO fat mass over time. N=9. (E) iSPAG7 KO lean mass over time. N=9. (F) iSPAG7 KO daily food intake, as measured by hopper weight. Taken 9 weeks after treatment. N=9. (G) Blood glucose levels following an oral glucose bolus. Taken 7 weeks after treatment. N=9. (H) Plasma insulin levels following an oral glucose bolus. Taken 7 weeks after treatment. N=9. (I) Hourly energy expenditure as determined by CLAMS metabolic cage system. Taken 6 weeks after treatment. N=9. (J) Total energy expenditure as determined by CLAMS metabolic cage system. Taken 6 weeks after treatment. N=9. (K) Home cage locomotor activity as determined by CLAMS metabolic cage system. Taken 6 weeks after treatment. N=9. (L) Distance run until exhaustion during treadmill endurance test. Taken 8 weeks after treatment. N=9. (M) In vivo gastrocnemius/soleus complex muscle max force generation. Taken 8 weeks after treatment. n=9.
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Figure 4—source data 1
Uncropped western blot gels.
Raw and annotated WB images. The representative western blot images for 4B are indicated within a blue square.
- https://cdn.elifesciences.org/articles/91114/elife-91114-fig4-data1-v1.zip
Figure 5
SPAG7-deficiency induces intrauterine growth restriction.
(A) Gross morphology of WT, SPAG7 KO Heterozygous, and SPAG7 KO Homozygous pups at p0. Scale bars represent 10 mm. (B) Birth weights of WT, SPAG7 KO Heterozygous, and SPAG7 KO Homozygous pups. Number of WT pups = 25. Number of HET pups = 45. Number of HOM pups = 11. Significance was assessed by one-way ANOVA and Tukey HSD. (C) Mendelian birth genotyping rates expected from Heterozygous x Heterozygous breeding (left), and birth genotyping rates observed from SPAG7 KO Heterozygous x Heterozygous breeding (right). Number of dams = 29. (D) Embryo genotyping rates at e6.5 from SPAG7 KO Heterozygous x Heterozygous breeding. Number of dams = 6. (E) Embryo genotyping rates at e8.5 from SPAG7 KO Heterozygous x Heterozygous breeding. Number of dams = 6. (F) Embryo genotyping rates at e11.5 from SPAG7 KO Heterozygous x Heterozygous breeding. Number of dams = 6. (G) Embryo genotyping rates at e18.5 from SPAG7 KO Heterozygous x Heterozygous breeding. Number of dams = 6. (H) Gross morphology of WT, HET, and SPAG7 KO embryos at e18.5. Scale bars represent 10 mm. (I) Fetus weights at e18.5, comparing WT and SPAG7 KO embryos. n=10. Significance was assessed by one-way ANOVA and Tukey HSD. (J) Placenta weights at e18.5, comparing WT and SPAG7 KO embryos. n=10. Significance was assessed by one-way ANOVA and Tukey HSD. (K) Fetal blood glucose levels at e18.5, comparing WT and SPAG7 KO embryos. n=10. Significance was assessed by one-way ANOVA and Tukey HSD. (L) Fetal plasma insulin levels at e18.5, comparing WT and SPAG7 KO embryos. n=10. Significance was assessed by one-way ANOVA and Tukey HSD. (M) Western blots for bACTIN, IGF1, and IGF2 in WT and SPAG7 fetal liver at e18.5. n=4. (N) Quantification of western blots in Figure 6M. n=4. Significance was assessed by Welch’s two sample t-test. (O) Gene expression levels of igf1 and igf2 in fetal liver at e18.5. n=6. Significance was assessed by Welch’s two sample t-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
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Figure 5—source data 1
Uncropped western blot gels.
Raw and annotated WB images. The representative western blot images for 5 M are indicated within a blue square.
- https://cdn.elifesciences.org/articles/91114/elife-91114-fig5-data1-v1.zip
Figure 6
SPAG7-deficiency induces placental insufficiency.
(A) Histological sections of WT and SPAG7 KO placenta stained with hematoxylin and eosin. Scale bars = 500 um. (B) Histological sections of WT and SPAG7 KO placenta labeled with CD34 antibody. Scale bars = 500 um. (C) Total placenta cross-sectional area of WT and SPAG7 KO placenta at e18.5. n=9. Significance was assessed by Welch’s two sample t-test. (D) Histological sections of WT and SPAG7 KO placenta labyrinth and junctional zones labeled with CD34 antibody. Scale bars = 100 um. (E) Whole placenta gene expression of vascular markers. N=5. (F) Whole placenta mtDNA copy number. n=5. Significance was assessed by Welch’s two sample t-test. (G) Histological sections of WT and SPAG7 KO placenta stained with hematoxylin and eosin. Scale bars = 100 um. (H) Junctional:Labyrinth Zone ratios in WT and SPAG7 KO placenta. n=9. Significance was assessed by Welch’s two sample t-test. (I) Percent Junctional Zone in WT and SPAG7 KO placenta. n=9. Significance was assessed by Welch’s two sample t-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Figure 7
Graphical abstract.
(A) SPAG7-deficiency causes intrauterine growth restriction which leads to obesity and insulin resistance in adulthood.
Author response image 1
Tables
Author response table 1
| SPAG7 KO | ||||
|---|---|---|---|---|
| Body Weight Week 6(g) | 18.76+-0.84 | 18.00+-0.33 | 23.36+-0.74 | 22.47+-1.81 |
| Lean Mass Week 6(g) | 16.76+-0.99 | 15.04+-0.29 | 21.18+-0.34 | 19.55+-1.32 |
| Fat Mass Week 6(g) | 1.54+-0.27 | 2.53+-0.48 | 1.47+-0.24 | 2.42+-0.56 |
| Body Weight Week 20(g) | 22.53+-1.70 | 22.31+-1.72 | 28.6+-2.95 | 30.47+-3.18 |
| Lean Mass Week 20(g) | 21.12+-1.04 | 19.78+-1.04 | 26.25+-1.40 | 24.42+-1.14 |
| Fat Mass Week 20(g) | 3.43+-1.20 | 8.50+-3.87 | 4.15+-1.69 | 6.72+-1.29 |
| Food Intake Week 20(g) | 3.32+-0.77 | 3.01+-0.74 | 3.84+-0.62 | 3.87+-1.00 |
| Total Energy Expenditure Week 11 (kcal) | 31.80+-1.37 | 27.75+-2.76 | 32.02+-1.49 | 26.94+-3.63 |
| oGTT AUC Week 12 | 1227+-94.8 | 1761+-110 | 1139+-92 | 1971+-127 |
Author response table 2
| F WT | F HOM | M WT | M HOM | |
|---|---|---|---|---|
| Body Weight Week 0 (g) | 19.86+-2.84 | 19.55+-1.31 | 25.06+-2.84 | 24.4+-0.75 |
| Body Weight Week 9 (g) | 22.53+-1.70 | 22.31+-1.72 | 28.6+-2.95 | 30.47+-3.18 |
| Lean Mass Week 9 (g) | 19.61+-0.85 | 19.37+-1.02 | 25.07+-2.69 | 25.42+-1.99 |
| Fat Mass Week 9 (g) | 3.31+-0.79 | 2.85+-0.92 | 3.53+-1.23 | 3.70+-0.80 |
| Food Intake Week 9 (g) | 2.12+-0.56 | 2.08+-0.30 | 2.20+-0.44 | 2.18+-0.38 |
| Total Energy Expenditure Week 6 (kcal) | 29.71+-1.28 | 30.72+-1.43 | 31.13+-1.26 | 31.25+-1.2 |
| oGTT AUC Week 7 | 3503+-178 | 3601+-180 | 3217+-145 | 3306+-151 |
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SPAG7 deletion causes intrauterine growth restriction, resulting in adulthood obesity and metabolic dysfunction
eLife 12:RP91114.
https://doi.org/10.7554/eLife.91114.3
