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.

D. WT vs SPAG7 KO lean mass over time. n = 7.

E. WT vs SPAG7 KO fat mass over time. n = 7.

F. Body length measured from nose to base-of-tail. n = 7.

G. Bone mineral density, as determined by DEXA scan. n = 7.

H. Heart weight. n = 7.

I. Gastrocnemius muscle weight. n = 7.

J. Tibialis anterior muscle weight. n = 7.

K. Kidney weight. n = 7.

L. Brain weight. n = 7.

M. Liver weight. n = 7.

N. Plasma triglyceride levels following 8-hour fast. n = 5.

O. Plasma triglyceride levels following 8-hour fast with a 2-hour ad-lib refeed. n = 5.

P. Plasma total cholesterol levels following 8-hour fast. n = 5.

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.

S. Plasma NEFA levels following 8-hour fast with a 2-hour refeed. n = 5.

T. Blood glucose levels following an oral glucose challenge. n = 7.

U. Plasma insulin levels following an oral glucose challenge. n = 7.

V. Blood glucose levels following an IP insulin challenge. n = 7. * 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.

D. Home cage locomotor activity as determined by CLAMS metabolic cage system. n = 7.

E. Body weight over time of WT vs SPAG7 KO animals raised at thermoneutrality. n = 7.

F. Fat mass over time of animals at thermoneutrality. n = 7.

G. Daily food intake of animals at thermoneutrality. n = 7.

H. Total energy expenditure of animals at thermoneutrality as determined by CLAMS. N = 7.

I. Body weight over time of WT vs SPAG7 KO animals raised on high-fat diet. n = 8.

J. Fat mass over time of animals fed high-fat diet. n = 8.

K. Daily food intake of animals fed high-fat diet. n = 8.

L. Total energy expenditure of animals fed high-fat diet. n = 8.

M. Percent of body weight increase 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). n = 7. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

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.

B. Max VO2 reached during treadmill endurance. n = 6.

C. In vivo gastrocnemius/soleus complex muscle max force generation. n = 6.

D. Cross sectional area of myosin heavy chain 2a-expressing fiber in gastrocnemius muscle. n = 7.

E. Triglyceride content of gastrocnemius muscle. n = 6.

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.

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. n = 9.

G. Blood glucose levels following an oral glucose bolus. n = 9.

H. Plasma insulin levels following an oral glucose bolus. n = 9.

I. Hourly energy expenditure as determined by CLAMS metabolic cage system. n = 9.

J. Total energy expenditure as determined by CLAMS metabolic cage system. n = 9.

K. Home cage locomotor activity as determined by CLAMS metabolic cage system. n = 9.

L. Distance run until exhaustion during treadmill endurance test. n = 9.

M. In vivo gastrocnemius/soleus complex muscle max force generation. n = 9. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

SPAG7-deficiency induces intrauterine growth restriction.

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

B. Birth weights of WT, SPAG7 KO Heterozygous, and SPAG7 KO Homozygous pups. Number of dams = 11.

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 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.

J. Placenta weights at e18.5, comparing WT and SPAG7 KO embryos. n = 10.

K. Fetal blood glucose levels at e18.5, comparing WT and SPAG7 KO embryos. n = 10.

L. Fetal plasma insulin levels at e18.5, comparing WT and SPAG7 KO embryos. n = 10.

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.

O. Gene expression levels of igf1 and igf2 in fetal liver at e18.5. n = 6. * 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.

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.

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.

I. Percent Junctional Zone in WT and SPAG7 KO placenta. n = 9. * 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.