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-hour fast. n = 5. Significance was assessed by Welch’s two sample t-test.

O. Plasma triglyceride levels following 8-hour fast with a 2-hour ad-lib refeed. n = 5. Significance was assessed by Welch’s two sample t-test.

P. Plasma total cholesterol levels following 8-hour fast. n = 5. Significance was assessed by Welch’s two sample t-test.

Q. Plasma total cholesterol levels following 8-hour fast with a 2-hour refeed. n = 5.

R. Plasma NEFA levels following 8-hour fast. n = 5. Significance was assessed by Welch’s two sample t-test.

S. Plasma NEFA levels following 8-hour fast with a 2-hour 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.

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 = 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 = 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.

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 = 300um.

G. Histological sections of gastrocnemius muscle labeled with antibodies against lectin (red), CD31 (green), and DAPI (blue). Scale bars = 200 um.

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 = 600um.

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.

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.

SPAG7-deficiency induces intrauterine growth restriction

A. Gross morphology of WT, SPAG7 KO Heterozygous, and SPAG7 KO Homozygous pups at p0. Scale bars represent 10mm.

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 10mm.

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 Fig. 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.

SPAG7-deficiency induces placental insufficiency

A. Histological sections of WT and SPAG7 KO placenta stained with hematoxylin and eosin. Scale bars = 500um.

B. Histological sections of WT and SPAG7 KO placenta labeled with CD34 antibody. Scale bars = 500um.

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 = 100um.

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 = 100um.

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.

Graphical abstract

A. SPAG7-deficiency causes intrauterine growth restriction which leads to obesity and insulin resistance in adulthood.