Abnormal BAT mitochondrial morphology in Cdkal1 A-KO mice. (A) Electron micrographs of BAT from chow-fed, room temperature housed control (A, C) or A-KO (B, D) mice. (E) Mitochondrial DNA to nuclear DNA ratio in BAT from mice housed at room temperature (RT) (Ctrl, n ¼ 4; A-KO, n ¼ 4) or subjected to 2.5 days of 4 C cold temperature challenge (Ctrl, n ¼ 5; A-KO, n ¼ 6). (F) Western blot for protein levels of mitochondrial electron transport chain complex components (Mitoprofile), UCP1, and Cdkal1 in BAT of 26 week old, chow-fed Ctrl (n ¼ 4) and A-KO (n ¼ 4) male mice. Anti-b-tubulin blot was used to confirm equal protein loading. (G) Coomassie stain of Blue Native gel for mitochondrial lysate from Ctrl and Cdkal1 A-KO BAT. Bands for native mitochondrial electron transport chain complexes are indicated.

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... with adipocyte-specific deletion of Cdkal1 exhibit similar body weight and body composition relative to Cdkal1 flox/flox controls (Figure 1AeC). Neither glucose (Supplementary Figure 3A as identified in this study by co-immunoprecipitation of FLAG-CDKAL1 followed by mass spectrometry (purple arrows). CDKAL1 protein interactors include com- ponents of the cytosolic Fe-S cluster transfer complex (MMS19, FAM96B, and CIAO1) and the mitochondrial protein ANT1. ...

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... Diagram of FLAG-tagged CDKAL1 mutants used in this study. Input (D) and (E) co-immunoprecipitation of FLAG-tagged wild type (Supplementary Figure 3B) was significantly altered in Cdkal1 A-KO mice on a standard chow diet. In addition, adipose-specific deletion of Cdkal1 did not change glucose tolerance (Supplementary Figure 4A, 4B), insulin tolerance (Supplementary Figure 4C), body weight (Supplementary Figure 4D, 4E) or body composition (Supplementary Figure 4F) in mice rendered obese by HFD feeding. ...

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... mitochondrial morphology observed by EM in BAT from Cdkal1 A-KO mice Based on the decreased cellular and mitochondrial rates of respiration, we examined BAT cellular morphology by transmission electron mi- croscopy. In contrast to BAT from control animals ( Figure 3A and C), Cdkal1 A-KO BAT mitochondria exhibit a swollen shape and disordered cristae distribution with a pale matrix (Figure 3B and D). In a medium- power field, dozens of abnormal mitochondria are apparent. ...

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... mitochondrial morphology observed by EM in BAT from Cdkal1 A-KO mice Based on the decreased cellular and mitochondrial rates of respiration, we examined BAT cellular morphology by transmission electron mi- croscopy. In contrast to BAT from control animals ( Figure 3A and C), Cdkal1 A-KO BAT mitochondria exhibit a swollen shape and disordered cristae distribution with a pale matrix (Figure 3B and D). In a medium- power field, dozens of abnormal mitochondria are apparent. ...

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... quantified the ratio of mitochondrial to nuclear DNA in samples from mice housed at room temperature or following a 4 C cold challenge. We observed no differences in mitochondrial DNA content in BAT of Cdkal1 A-KO mice ( Figure 3E). Western blotting for dissociated components of the electron transport chain complexes or the uncoupling protein UCP1 demonstrated no apparent differences in protein levels of these mitochondrial proteins in Cdkal1 A-KO BAT ( Figure 3F). ...

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... observed no differences in mitochondrial DNA content in BAT of Cdkal1 A-KO mice ( Figure 3E). Western blotting for dissociated components of the electron transport chain complexes or the uncoupling protein UCP1 demonstrated no apparent differences in protein levels of these mitochondrial proteins in Cdkal1 A-KO BAT ( Figure 3F). Lastly, we also assessed relative levels of intact, native electron transport chain complexes from control and A-KO BAT mito- chondria using non-denaturing Blue Native gels and Coomassie staining, observing modest decreases in complex I and III ( Figure 3G). ...

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... blotting for dissociated components of the electron transport chain complexes or the uncoupling protein UCP1 demonstrated no apparent differences in protein levels of these mitochondrial proteins in Cdkal1 A-KO BAT ( Figure 3F). Lastly, we also assessed relative levels of intact, native electron transport chain complexes from control and A-KO BAT mito- chondria using non-denaturing Blue Native gels and Coomassie staining, observing modest decreases in complex I and III ( Figure 3G). These findings of altered mitochondrial morphology further support a selective effect of Cdkal1 on mitochondria. ...

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... found that obesity downregulates Cdkal1 mRNA levels in mouse adipose tissue (Supplementary Figure 1D), spurring us to investigate whether adipocyte-specific loss of Cdkal1 can contribute to obesity-related metabolic disorders in mice on a high fat diet. Glucose tolerance and insulin sensitivity were unchanged in mice that lacked Cdkal1 in their adipocytes ( Supplementary Figures 3 and 4). Instead, we observed decreased energy expenditure in Cdkal1 A-KO mice exposed to 4 C cold-temperature challenge, suggesting altered respiration in brown or beige adipocytes in vivo ( Figure 1D and E). ...

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... effects were attributed to loss of modifications on mito- chondrial tRNA species and subsequent mitochondrial translation impairment [40]. We demonstrate several lines of evidence that absence of Cdkal1 in adipocytes can affect mitochondrial morphology (Figure 3AeD) and function ( Figure 2C and D). These adipose mito- chondrial defects occurred independently of differences in body weight or glucose homeostasis ( Supplementary Figures 3 and 4) or mito- chondrial defects due to decreased Cdkal1 levels in other metabolically important tissues. ...

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... demonstrate several lines of evidence that absence of Cdkal1 in adipocytes can affect mitochondrial morphology (Figure 3AeD) and function ( Figure 2C and D). These adipose mito- chondrial defects occurred independently of differences in body weight or glucose homeostasis ( Supplementary Figures 3 and 4) or mito- chondrial defects due to decreased Cdkal1 levels in other metabolically important tissues. Previous mouse studies using whole-body Cdkal1 knockout demonstrated that lowered insulin secretion from islets lacking Cdkal1 coincided with reduced islet mitochondrial ATP pro- duction [20]. ...

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... patients with missense mutations in ANT1 have been shown to develop disorders characterized by mitochondrial dysfunction and progressive weakening of their eye, heart, or skeletal muscles [49,50]. Electron micrographs from human patients with ANT1 mutations reveal mitochondria that display a swollen, disorganized morphology [51], similar to that we observed in Cdkal1 A-KO BAT mitochondria (Figure 3AeD). Patients and mice harboring defective alleles of ANT1 have impaired mitochondrial function and altered mitochondrial morphology [52,53]. ...

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... addition, elevated levels of ANT1 are also observed under pathological conditions including facioscapulohumeral muscular dystrophy [54,55], suggesting that inappropriately low or high levels of this protein can lead to mitochondrial disease patho- genesis. The Cdkal1 A-KO mouse line, which exhibited abnormal mitochondrial morphology (Figure 3) and defective mitochondrial respiration ( Figure 2C and D) in BAT, also exhibited increased levels of ANT1 in white adipose tissue (Figure 5F and G; Supplementary Figure 5). Understanding the mechanisms by which Cdkal1 may affect mitochondrial ANT1 levels could elucidate a novel mechanism by which Cdkal1 can regulate mitochondrial function. ...