BMI risk SNPs associate with increased CADM1 and CADM2 expression in the cerebellum of human subjects.

Transcription

1 Supplementary Figure 1 BMI risk SNPs associate with increased CADM1 and CADM2 expression in the cerebellum of human subjects. Boxplots show the 25% and 75% quantiles of normalized mrna expression levels (y-axis), solid horizontal lines indicate the median, and whiskers indicate the 10% and 90% quantiles. (a) Elevated expression of CADM1 associates with risk allele (G) of rs in cerebellum. (b) Genotype dependent expression levels of CADM2 for the SNP rs The risk allele (G) is associated with higher expression levels in human cerebellum. Statistical analyses are described in the Methods and Supplementary Table 3.

2 Supplementary Figure 2 Increased Cadm1 expression in multiple brain regions of Lepob/ob mice is reversed upon ketogenic diet feeding. (a) Schematic representation of Cadm1 and Cadm2 engaged in homophilic (homo) and heterophilic (het) interactions. (b) (c) Western blot analysis of Cadm1 and Cadm2 in total and synaptosome-enriched lysates from cerebellum and hippocampus of 4-week-old Lep ob/ob mice and littermate controls. (d) Quantification of western blot analysis of Cadm1 in multiple brain regions of 16-week-old WT and Lep ob/ob mice on chow or ketogenic diet for 45 days. Cortex (ctx), striatum (str), hippocampus (hpc), hypothalamus (hyp), midbrain

3 (mid), hindbrain (hind), and cerebellum (cer) are shown. (e, f) Western blot analysis of Cadm1 in cerebellum and hippocampus of 16- week-old wild-type on normal chow (WT/chow) and Lep ob/ob mice on normal chow (Lep ob/ob /chow) or ketogenic diet (Lep ob/ob /keto). (g) Western blot analysis of Cadm1 from isolated hippocampus of 12-week old C57Bl/6 mice after administration of ketogenic diet for 4 or 8 days. Results are presented as mean ± s.e.m. *P<0.05 and **P<0.01. Boxplots show median, lower and upper quartiles (box), and minimum and maximum (whiskers). Statistical analyses are described in the Methods and Supplementary Table 3.

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5 Supplementary Figure 3 Cadm1 and Cadm2 total knockout mice exhibit decreased body weight, and increased insulin sensitivity and energy expenditure. (a) Body weight curves of Cadm1KO mice (n=10) and littermate controls (n=12). (b) Body weight-to-length ratio in Cadm1KO mice (n=10) and control littermates at age 12-weeks (n=13). (c) Glucose measurements during an insulin tolerance test (ITT) on 12-week old Cadm1KO mice (n=5) and control littermates (n=5). (d) Glucose measurements during an intraperitoneal glucose tolerance test (GTT) on 12-week old Cadm1KO mice (n=5) and control littermates (n=6). (e) Glucose measurements during a pyruvate tolerance test (PTT) on 12-week old Cadm1KO mice (n=5) and control littermates (n=6). (f) Quantification of food intake in 12-week old Cadm1KO (n=5) and littermate controls (n=6) during leptin challenge. Daily food intake and body weight was measured for 5 days prior to leptin administration for base line. Differences in daily food intake were normalized to daily body weight. (g) Quantification of body weight change in 12-week old Cadm1KO (n=5) and littermate controls (n=6) during leptin challenge. Differences in daily body weight were normalized to daily body weight. (h) Quantification of daily food intake in 12-week old Cadm1KO (n=10) and littermate controls (n=10). (i) Quantification of O 2 consumption, CO 2 production, and RER, respectively in 12-week old male Cadm1KO mice (KO) (n=9) and control littermates (WT) (n=11). (j) Quantification of core body temperature (CBT) in 12-week old male Cadm1KO mice (KO) (n=5) and control littermates (WT) (n=5). (k) Quantification of energy expenditure during day and night phases in 12-week old male Cadm1KO mice (n=9) and control littermates (n=11). (l) Locomotor activity measured during day and night phases in 12-week old male Cadm1KO mice (n=8) and control littermates (n=8). (m) Energy expenditure of individual animals plotted against locomotor activity in 12-week old Cadm1KO (n=7) and littermate controls (n=11). (n) Western blot analysis of Cadm2 and Cadm1 in cortex, cerebellum (cereb), hippocampus (hpc), hypothalamus (hyp), liver and lung from Cadm2KO (KO) mice compared to littermate controls (WT). (o) Representative confocal images of primary hippocampal neurons isolated from Cadm2KO and littermate control mice, stained for Cadm2 (green) and MAP2 (red). (p) Body weight curves of Cadm2KO mice (n=8) and littermate controls (n=8) from age post-natal day (q) Glucose measurements during an ITT on 9-week old Cadm2KO mice (n=6) and control littermates (n=6). (r) Glucose measurements during a GTT on Cadm2KO mice (n=3) and littermates (n=8) and quantification of area under the curve (AUC). (s) Quantification of food intake in 10-week old Cadm2KO mice (n=7) and littermate controls (n=9). (t, u) Quantification of O 2 consumption, CO 2 production, and RER, respectively in 10-week old Cadm2KO mice (n=6) and littermate controls (n=8). (v) Quantification of energy expenditure in 10-week old Cadm2KO mice (n=6) and littermate controls (n=8). (w) Quantification of locomotor activity in 10-week old Cadm2KO mice (n=6) and littermate controls (n=8). (x) Energy expenditure of individual animals plotted against lean body mass (LBM) in 10-week old Cadm2KO mice (n=7) and littermate controls (n=8). (y) Energy expenditure of individual animals plotted against locomotor activity in 10-week old Cadm2KO mice (n=7) and littermate controls (n=8). Results in panels a, c, d, e, f, g, p, g, and r are presented as mean ± s.e.m. *P<0.05, and **P<0.01. Boxplots show median, lower and upper quartiles (box), and minimum and maximum (whiskers). Statistical analyses are described in the Methods and Supplementary Table 3.

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7 Loss of Cadm1 reduces inhibitory post-synaptic currents in POMC neurons. (a) Immunostaining of Cadm1 (red), POMC-eGFP (green) and VGLUT2 (magenta) identifies limited Cadm1 and VGLUT2 expression in POMC neurons in the arcuate nucleus region (ARC) of the hypothalamus. High magnification images are outlined by white boxes. (b) Immunostaining quantification of POMC-GFP and Cadm1-positive cells in the ARC (GFP-positive cells: 188±28; GFP- and CADM1- positive cells: 23±5). (c) qrt-pcr analysis of Pomc, Npy and Agrp expression in the ARC region of 12-week old Cadm1KO mice (n=3), Lep ob/ob mice (n=3) and control littermates (WT) (n=4). (d) Summary of miniature IPSC frequency in POMC neurons from Cadm1KO and control littermates at the age of 3 weeks (top left). Example traces of IPSCs recorded from POMC neurons of Cadm1KO or control littermates (top right). Mean amplitude and cumulative probability distribution of IPSC frequencies from POMC neurons showing a significant shift in Cadm1KO mice (n=15) compared to littermate control mice (n=12) (lower left and right). (e) Summary of miniature EPSC frequency in POMC neurons from Cadm1KO and control littermates (top left). Example traces of EPSCs recorded from POMC neurons of Cadm1KO or control littermates (top right). Mean amplitude and cumulative probability distribution of EPSC frequencies from POMC neurons showing no change in Cadm1KO mice (n=7) compared to littermate control mice (n=11) (lower left and right). (f, g) Representative electron micrographs showing perikaryal membranes of egfp-expressing POMC neurons from Cadm1KO (KO) and wild-type littermates (WT) mice identify marked differences in the asymmetrical (excitatory) synaptic inputs contacting POMC neurons (Scale bar, 1 μm). (h) Quantification of asymmetrical, symmetrical and total synapse numbers in in Cadm1KO mice (n=6) and littermate controls (n=8). Results are presented as mean ± s.e.m. *P<0.05, **P<0.01 and ***P< Boxplots show median, lower and upper quartiles (box), and minimum and maximum (whiskers). Statistical analyses are described in the Methods and Supplementary Table 3.

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9 Supplementary Figure 5 Induction of Cadm1 in excitatory neurons increases co-localization with Cadm2. (a) Confocal images revealing partial co-localization of Cadm1 and VGLUT2 in the different mouse brain regions. Cadm1 (green) and VGLUT2 (red) co-localize in hippocampus (Hpc), medial habenula (MHb), and paraventricular hypothalamus (PVH) regions. High magnification images are identified by white boxes. Fluorescent intensity profiles of Cadm1 and VGLUT2 (measured along white lines) quantify the co-localization of Cadm1 and VGLUT2. (b, c) Representative confocal images of Cadm1 and Cadm2 expression in Slc17a6-Cre and (d, e) Slc32a1-Cre-positive primary hippocampal neurons. Immunostaining for Cadm1 or Cadm2 (red), ZsGreen (green), and Map2 (cyan). (f) Representative confocal images of Cadm1 and Cadm2 expression in primary hippocampal neurons of Cadm1KO, Tg-Cadm1, and WT mice. (g) Quantification of O 2 consumption, CO 2 production, RER and energy expenditure (EE), in 14- week old Tg-Cadm1 mice (n=6) and littermate controls (n=8). (h) Representative confocal images of Cadm1, Cadm2, and VGLUT2 expression in primary hippocampal neurons of Cadm1KO, Tg-Cadm1, and WT mice. Immunofluorescence detection of Cadm1 (red), Cadm2 (green), and VGLUT2 (cyan) in dendritic branches identifies areas of co-localization. (i) Fluorescence intensity profiles of Cadm1 and Cadm2 (measured along white dashed lines in (h) quantify areas of co-localization. (j) Pearson correlation indicates increased association between Cadm1 and Cadm2 expression in Tg-Cadm1 (n=9) mice compared to wild-type control littermates (n=10). Results are presented as mean ± s.e.m. *P<0.05. Boxplots show median, lower and upper quartiles (box), and minimum and maximum (whiskers). Statistical analyses are described in the Methods and Supplementary Table 3.

10 Supplementary Figure 6 Loss of Cadm1 in excitatory neurons improves glucose homeostasis and insulin sensitivity. (a) Confocal imaging of coronal brain slice identifying ZsGreen expression in Slc17a6-ires-Cre, lox-zsgreen mice at age 12 weeks. (b) Western blot analysis of Slc17a6-Cre-mediated reduction of Cadm1 expression in hippocampus (hpc), cortex, and olfactory bulb (Olf bulb). (c) Confocal imaging of coronal brain slice identifying egfp expression in Slc32a1-ires-Cre, lox-zsgreen mice at age 12 weeks. (d) Western blot analysis of Slc32a1-Cre-mediated reduction of Cadm1 expression in hypothalamus (Hypoth), striatum (Striat), and olfactory bulb (Olf bulb). (e) Body weight curves in Cadm1 flox/flox mice (n=6) and littermate controls (n=6) from age postnatal day (f) Body weight-to-length ratio in Cadm1 flox/flox mice (n=4) and control littermates (n=8) at age 3-weeks. (g) Body weight curves in Slc32a1-Cre, Cadm1 flox/flox mice (n=8) and littermate controls from 4-12 weeks of age (n=8). (h) Glucose measurements during an ITT on 12-week old Slc32a1-Cre, Cadm1 flox/flox mice (n=4) and control littermates (n=6) and quantification of area under the curve (AUC). (i) Glucose measurements during a GTT on Slc32a1-Cre, Cadm1 flox/flox mice (n=5) and control littermates (n=8). (j) Energy expenditure of individual animals plotted against lean body mass in 12-week old Slc32a1-Cre, Cadm1 flox/flox mice (n=7) and littermate controls (n=8). (k) Energy expenditure of individual animals plotted against locomotor activity in 12-week old

11 Slc32a1-Cre, Cadm1 flox/flox mice (n=7) and littermate controls (n=8). (l) Quantification of locomotor activity and food intake measured in 12-week old Slc32a1-Cre, Cadm1 flox/flox mice (n=8) and littermate controls (n=8). (m) Body weight, blood glucose, and plasma insulin values before initiating hyperinsulinemic-euglycemic clamp studies on 12-week old Cadm1 flox/flox mice (n=6) and control littermates (n=4). (n) Plasma glucose concentrations and glucose infusion rate during hyperinsulinemic-euglycemic clamp studies on 12-week old Cadm1 flox/flox mice (n=6) and control littermates (n=4). (o) Endogenous glucose production in the basal and the clamped state and suppression of hepatic glucose production in Cadm1 flox/flox mice (n=6) and control littermates (n=4). (p) Peripheral glucose uptake, glycolysis rate, glycogen synthesis, plasma insulin levels during the hyperinsulinemic-euglycemic clamp studies in 12-week old Cadm1 flox/flox mice (n=6) and control littermates (n=4). Results in panels e, g, h, I, and n are presented as mean ± s.e.m. *P<0.05, **P<0.01 and ***P< Boxplots show median, lower and upper quartiles (box), and minimum and maximum (whiskers). Statistical analyses are described in the Methods and Supplementary Table 3.

12 Supplementary Figure 7 Loss of Cadm1 in excitatory neurons increases long-term potentiation and long-term depression in hippocampal neurons. (a) Representative electron micrograph images from hippocampus of Cadm1 flox/flox mice and littermate controls. (b) Quantification of excitatory synapse number per volume fraction or per area and (c) Relative post-synaptic densities (PSD) length in hippocampus in Cadm1 flox/flox mice (n=3) and littermate controls (n=3). (d) Western blotting analysis of Cadm1 and PSD95 in total and synaptosome-enriched lysates from hippocampus of 12-week old Cadm1 flox/flox mice (n=2) and control littermates (n=2). (e) Cadm1 glutamatergic deficiency results in increased basal synaptic transmission. Representative fepsps responses at increasing stimulation intensities ( µa, color coded) show higher amplitudes for Cadm1 flox/flox as compared to WT controls. Synaptic transmission was measured as the relationship of fepsp slope vs fiber volley (FV) amplitudes. Two-way RM ANOVA revealed no significant changes for FV amplitudes (P=0.739). Slope of fepsp was significantly facilitated (P=0.029) in Cadm1 flox/flox mice (WT n=18, N=9; Cadm1 flox/flox n=20, N=9). (f) LTP induction in MPP-DG

13 synapses is facilitated in Cadm1 flox/flox mice. Representative traces are the average of 20 fepsps recorded 10 min before (black) and min after LTP induction (grey). Higher LTP values measured min after induction were detected in Slc17a6- Cre, Cadm1 flox/flox as compared to WT control (P=0.021, WT n=8, N=8; Cadm1 flox/flox n=9, N=8). (g) LTD induction in MPP- DG synapses is facilitated in Cadm1 flox/flox mice. Representative traces are the average of 20 fepsps recorded 10 min before (black) and min after LTD induction (grey). Facilitated LTD values measured min after induction were detected in Cadm1 flox/flox as compared to WT control (P=0.032, WT n=9, N=8; Cadm1 flox/flox n=10, N=9). Results are presented as mean ± s.e.m. *P<0.05. Boxplots show median, lower and upper quartiles (box), and minimum and maximum (whiskers). Statistical analyses are described in the Methods and Supplementary Table 3.

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15 Supplementary Figure 8 Tracing analysis identifies VGLUT2-positive afferent inputs to the ARC originating from the PVH, MHb, and Hpc regions. (a) Western blot analysis of Cadm1 expression in total lysates from hippocampus and cortex after delivery of AAV-Cre to floxed Cadm1 mice (flox/flox) and control littermates (+/?). Total hippocampal lysates from Cadm1KO (KO) and Cadm1 flox/flox (Vglut2) serve as positive controls for loss of Cadm1 expression. (b) Quantification of O 2 consumption, CO 2 production, energy expenditure, locomotor activity and RER in 9-week old floxed Cadm1 mice (flox/flox) (n=7) and control littermates (+/?) (n=6). (c) Representative confocal images of coronal sections through the brain of Slc17a6-Cre/POMC-eGFP transgenic mice stereotaxically injected with AAV2/EF1a-DIO-hChR2(H134R)-mCherry into the paraventricular hypothalamus (PVH), habenular nuclei (Hb), and hippocampus (Hpc). Nuclei have been visualized with DAPI staining. High magnification images are identified by white boxes. (d) Representative coronal brain section of Cadm1 floxed mice after stereotaxic raav8-camkiia-mcherry-cre injection to hypothalamic neurons in the paraventricaular hypothalamus (PVH), and ventromedial hypothalamus (VMH) regions to target Cre expression to excitatory neurons in these areas; confocal imaging of mcherry and the neuronal marker NeuN. (e) Representative western blot analysis showing AAV-Cremediated deletion of Cadm1 after stereotaxic injection of raav8-camkiia-mcherry-cre into the PVH region of the hypothalamus. (f) Quantification of locomotor activity, O 2 consumption, CO 2 production, RER, and energy expenditure from 9-week old floxed Cadm1 mice (n=9) and control littermates (n=8) after stereotaxic injection of raav8/camkii-mcherry-cre injection into the hypothalamus. (g) Glucose measurements during an ITT on 9-week floxed Cadm1 mice (n=6) and control littermates (n=6) after stereotaxic injection of raav8/camkii-mcherry-cre injection into the hypothalamus. (h) Representative confocal images of coronal sections through the brain of Slc17a6-Cre/POMC-eGFP transgenic mice stereotaxically injected with AAV2/EF1a-DIO-hChR2(H134R)-mCherry into the hippocampus (Hpc). High magnification image of the PVH is identified by dashed box. (i) Representative confocal images of coronal sections through the brain of Slc17a6-Cre/POMC-eGFP transgenic mice stereotaxically injected with AAV2/EF1a-DIO-hChR2(H134R)- mcherry into the paraventricular hypothalamus (PVH). High magnification image of the hippocampus is identified by dashed box. (j) Confocal image analysis after stereotaxic injection of AAV2/EF1a-DIO-hChR2(H134R)-mCherry to the habenular nuclei (Hb) of Slc17a6-Cre/POMC-eGFP transgenic mice. Axonal varicosities (red) and POMC-positive neurons (green) are visualized within the ARC region of the hypothalamus. Dotted white circles outline egfp-pomc-positive cell body, used for 3D reconstruction analysis. (k) Surface rendering of Amira 3D reconstruction of egfp-pomc-positive cell body receiving afferent inputs from the MHb. Cell is represented in red, while the synaptic input is color-coded, with the cold to warm colors spreading from 0 to 250nm distance between axonal varicosities and the soma (see color-coded horizontal bar for the distance definition). (l) Histogram shows the number of anterograde AAV-mCherry-labelled MHb axonal varicosities found within 250nm distance from the POMC cell body. (m) Double immunostaining of Cadm1 and mcherry in MHb region identify Cadm1-positive anterograde projections to the ARC region of hypothalamus. Results in panel g are presented as mean ± s.e.m. *P<0.05, and **P<0.01. Boxplots show median, lower and upper quartiles (box), and minimum and maximum (whiskers). Statistical analyses are described in the Methods and Supplementary Table 3.

16 Supplementary Figure 9 Lepr-Cre-mediated deletion of Cadm1 impacts insulin sensitivity. (a) Glucose measurements during an ITT on 12-week old Agrp-Cre, Cadm1 flox/flox mice (n=3) and control littermates (n=5). (b) Glucose measurements during an ITT on 12-week old Pomc-Cre, Cadm1 flox/flox mice (n=6) and control littermates (n=6). (c) Glucose measurements during an ITT on 12-week old Sim1-Cre, Cadm1 flox/flox mice (n=6) and control littermates (n=10). (d) Body weight in Lepr- Cre, Cadm1 flox/flox mice (n=4) and littermate controls (n=7). (e) Glucose measurements during an ITT on 12-week old Lepr-Cre, Cadm1 flox/flox mice (n=3) and control littermates (n=5). (f) Correlation of energy expenditure of individual animals plotted against lean body mass in in 12-week old male Lepr-Cre, Cadm1 flox/flox mice (n=7) and littermate controls (n=8). (g) Quantification of locomotor activity and daily food intake in 12-week old Lepr-Cre, Cadm1 flox/flox mice (n=12) and littermate controls (n=12). (h) Body weight in ob/lepr-cre, Cadm1 flox/flox mice (n=3) and Lep ob/ob littermates (n=3). (i) Random-fed and fasted blood glucose and random-fed plasma insulin in 12-week old ob/lepr-cre, Cadm1 flox/flox mice (n=5) and Lep ob/ob littermates (n=7). (j) Glucose measurements during an insulin tolerance test on 12-week old ob/lepr-cre, Cadm1 flox/flox mice (n=5) and Lep ob/ob littermates (n=7). (k) Glucose measurements during a glucose tolerance test on 12-week old ob/lepr-cre, Cadm1 flox/flox mice (n=5) and Lep ob/ob littermates (n=6). (l) Glucose measurements during a pyruvate tolerance test on 12-week old ob/lepr-cre, Cadm1 flox/flox mice (n=3) and Lep ob/ob littermates (n=4). (m) Pancreatic β- cell mass and insulin content measurements in Lepr-Cre, Cadm1 flox/flox (n=5), ob/lepr-cre, Cadm1 flox/flox (n=7), Lep ob/ob (n=5), and wildtype (WT) littermate controls (n=6) from 12 weeks of age. (n) Energy expenditure per individual animals plotted against lean body mass in 12-week old ob/lepr-cre, Cadm1 flox/flox mice (n=6) and Lep ob/ob littermates (n=7). (o) Quantification of locomotor activity and daily food intake in 12-week old ob/lepr-cre, Cadm1 flox/flox mice (n=6) and Lep ob/ob littermates. (n=8). Results in panels a-e, h, and j-l are presented as mean ± s.e.m. *P<0.05, and **P<0.01. Boxplots show median, lower and upper quartiles (box), and minimum and maximum (whiskers). Statistical analyses are described in the Methods and Supplementary Table 3.

17 Supplementary Figure 10 Immunostaining of Cadm1 with Vglut2-positive afferent projections in contact with POMC neurons. (a) Quadruple immunostaining within the ARC region showing Cadm1 and Vglut2-positive afferent projections in contact with POMC neuron. Arrows indicate colocalization of mcherry punctas with Cadm1 and Vglut2. Slc17a6-Cre-positive mice received stereotaxic injection of AAV2-mCherry to the PVH, and were subsequently immunostained for POMC (green), mcherry (red), Cadm1 (magenta) and Vglut2 (cyan). (b) Visualization of Cadm1, mcherry, and Vglut2. Arrows indicate colocalization of mcherry punctas with Cadm1 and Vglut2.

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19 Supplementary Figure 11 Original western blot panels with gel markers for Figures 2a, 2b, and 2f. Complete scanned gels for western blots shown in Figures 2a, 2b, and 2f. Western images are overlaid onto image containing MW markers. White dashed line identifies cropped region shown in respective figure. Replicate experiments are shown at the right and unlabeled lanes contain samples not included in this study. All results are presented as mean ± s.e.m.

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21 Supplementary Figure 12 Original western blot panels with gel markers for Figures 2f and 4e. Complete scanned gels for western blots shown in Figures 2f, and 4e. Western images are overlaid onto image containing MW markers. White dashed line identifies cropped region shown in respective figure. All results are presented as mean ± s.e.m.

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23 Supplementary Figure 13 Original western blot panels with gel markers for Supplementary Figures 2b, 2c, and 2e. Complete scanned gels for western blots shown in Supplementary Figures 2b, 2c, and 2e. Western images are overlaid onto image containing MW markers. White dashed line identifies cropped region shown in respective figure. All results are presented as mean ± s.e.m.

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25 Supplementary Figure 14 Original western blot panels with gel markers for Supplementary Figures 2g, 3n, 6b, and 6d. Complete scanned gels for western blots shown in Supplementary Figures 2g, 3n, 6b, and 6d. Western images are overlaid onto image containing MW markers. White dashed line identifies cropped region shown in respective figure. All results are presented as mean ± s.e.m.

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27 Supplementary Figure 15 Original western blot panels with gel markers for Supplementary Figures 7d, 8a, and 8e. Complete scanned gels for western blots shown in Supplementary Figures 7d, 8a, and 8e. Western images are overlaid onto image containing MW markers. White dashed line identifies cropped region shown in respective figure. All results are presented as mean ± s.e.m.

28 Supplementary Table 1. mice. Plasma measurements from wild-type and Cadm1KO Feeding state Wild-type (WT) Cadm1KO Glucose (mg/dl) Random fed ± 11.58, n= ± 13.22, n=6 Fasted 50.3 ± 2.595, n= ± 3.177, n=6 Insulin (ng/ml) Random fed ± , n= ± , n=6 Fasted ± , n= ± , n=8 Leptin (ng/ml) Random fed 6.38 ± , n= ± , n=6* Adiponectin ( g/ml) Random fed ± 1.235, n= ± 4.429, n=6* Analysis performed on Cadm1KO and littermate controls at 12-weeks of age. Results are presented as mean ± s.e.m. *P<

29 42 43 Supplementary Table 2. Body composition analysis of male Cadm mutant mouse lines and control littermates. Body composition of Cadm1KO mice and control littermates. body mass (g) body fat (%) lean mass (%) body fat (g) lean mass (g) Cadm1 +/? (n=11) ± ± ± ± ± 0.32 Cadm1KO (n=9) ± 0.94 * ± 0.69 * ± 0.58 * ± 0.26 ** ± 0.59 * Data is presented as means ± s.e.m. **P<0.01. Body composition of Cadm2KO mice and control littermates. body mass (g) body fat (%) lean mass (%) body fat (g) lean mass (g) Cadm2 +/? (n=7) ± ± ± ± ± 0.21 Cadm2KO (n=6) ± 0.62 * ± ± ± ± 0.39 * Data is presented as means ± s.e.m. *P<0.05. Body composition of Tg-Cadm1 mice and Slc17a6-Cre + littermates body mass (g) body fat (%) lean mass (%) body fat (g) lean mass (g) Slc17a6-Cre + (n=8) ± ± ± ± ± 0.39 Tg-Cadm1 (n=10) ± 0.48 ** ± 0.61 * ± 0.63 * 7.67 ± 0.20 ** ± 0.40 Data is presented as means ± s.e.m. *P<0.05, ** P<0.01. Body composition of Cadm1 flox/flox mice and Slc17a6-Cre + littermates. body mass (g) body fat (%) lean mass (%) body fat (g) lean mass (g) Slc17a6-Cre + (n=9) ± ± ± ± ± 0.62 Cadm1 flox/flox (n=8) ± 0.53 * ± ± ± 0.19 * ± 0.46 * Data is presented as means ± s.e.m. *P<0.05, **P<0.01. Body composition of ob/mut mice and Lep ob/ob ; Slc17a6-Cre + littermates. body mass (g) body fat (%) lean mass (%) body fat (g) lean mass (g) Lep ob/ob ; Slc17a6- Cre + (n=6) ± ± ± ± ± 0.66 ob/mut (n=5) 36 ± 1.24 * ± 0.70 * 39.5 ± 0.93 * ± 0.78 * 14.2 ± 0.43 * Data is presented as means ± s.e.m. *P<0.05, **P<0.01. Body composition of Slc32a1-Cre, Cadm1 flox/flox mice and Slc32a1-Cre + littermates. body mass (g) body fat (%) lean mass (%) body fat (g) lean mass (g) Slc32a1-Cre + (n=6) ± ± ± ± ± 0.45 Slc32a1-Cre +, Cadm1 flox/flox (n=6) ± ± ± ± ± 0.55 Data is presented as means ± s.e.m. Body composition of ob/lepr-cre, Cadm1 flox/flox mice and Lep ob/ob ; Lepr-Cre + littermates. body mass (g) body fat (%) lean mass (%) body fat (g) lean mass (g) Lep ob/ob ; Lepr-Cre + (n=5) ± ± ± ± ± 0.28 ob/lepr-cre, Cadm1 flox/flox (n=4) ± ± ± ± ± 0.23 Data is presented as means ± s.e.m. *P<

30 44 45 Supplementary Table 3. Summary of statistical analyses Figure Sample size (n) Statistical Test Values 1a AA: n=14 Linear regression t=1.963, P = 0.05 AG: n=44 GG: n=23 1b AA: n=56 Linear regression t=1.984, P = 0.05 AG: n=24 GG: n=1 2c Wild-type (n=4) Cadm1 +/- (n=3) Cadm1KO (n=3) One-way ANOVA 2d 2e 2g 2h 2i 2j 2k 2l 2m 2n 3a 3b 3c 3d Wild-type (n=10), Cadm1KO (n=10) Wild-type (n=12) Cadm1 KO (n=9) Wild-type (n=6) Tg-Cadm1 (n=6) Tg-Cadm1 (n=5) Wild-type (n=10) Tg- Cadm1 (n=7) Tg-Cadm1 (n=7) Wild-type (n=10) Tg- Cadm1 (n=7) Wild-type (n=6), Tg-Cadm1 (n=5) Wild-type (n=7), Tg-Cadm1 (n=5) Tg-Cadm1 (n=7) Wild-type (n=14), Cadm1 fl/fl (n=12) Wild-type (n=6), Cadm1 fl/fl (n=5) Wild-type (n=10), Cadm1 fl/fl (n=9) Wild-type (n=10), Cadm1 fl/fl (n=9) Cadm1: Genotype: F=243.9, P< Cadm2: Genotype: F= 0.229, P=0.801 Multiple comparison: ***P<0.001 Interaction: F=0.2169, P = Time: F=185.3, P < Genotype: F=39.41, P < Multiple comparison: *P<0.05, **P<0.01, ***P<0.001 Linear regression F=6.232, P = Cadm1: t=6.363, P = Cadm2: t= , P = Interaction: F=2.990, P = Time: F=77.91, P < Genotype: F=6.454, P = Multiple comparison: ***P<0.001 Linear regression F=5.559, P = Interaction: F = 5.383, P = Time: F = 83.91, P < Genotype: F = 10.93, P = Multiple comparison: **P<0.01 Linear regression F=0.367, P = Interaction: F=0.6532, P = Time: F=18.79, P < Genotype: F=20.40, P = Multiple comparison: *P<0.05 Interaction: F=2.990, P = Time: F=77.91, P < Genotype3b: F=6.454, P = Multiple comparison: *P<0.05, **P<0.01 t=0.5769, P= Interaction: F=0.3766, P = Time: F=511.9, P < Genotype: F=16.33, P = Multiple comparison: **P<0.01, ***P<0.001 Interaction: F=2.495, P = Time: F=189.4, P < Genotype: F=10.51, P = Multiple comparison: *P<0.05 VO 2: Interaction: F = 3.188, P = Time: F = 130.4, P < Genotype: F = 9.781, P = VCO 2: Interaction: F = 6.209, P = Time: F = 247.8, P < Genotype: F = 7.570, P = Multiple comparison: **P<0.01 RER: Interaction: F = 1.604, P = Time: F = 87.90, P < Genotype: F = 1.669, P = EE: Interaction: F = 4.130, P =

31 3d 3e 3f 3g 3h 3i 3k 4a 4b 4c 4d 4f 4g 4h 4i Wild-type (n=10), Cadm1 fl/fl (n=9) Wild-type (n=10) Slc17a6 -Cre, Cadm1 fl/fl (n=9) Wild-type (n=10) Slc17a6 -Cre, Cadm1 fl/fl (n=9), Cadm1 fl/fl (n=5), Cadm1 fl/fl (n=5), Cadm1 fl/fl (n=5), Cadm1 fl/fl (n=5) Wild-type (n=6), Cadm1 fl/fl (n=10) Wild-type (n=10), Cadm1 fl/fl (n=7) Wild-type (n=10) Slc17a6 -Cre, Cadm1 fl/fl (n=9) Wild-type (n=10) Slc17a6 -Cre, Cadm1 fl/fl (n=9) Lep ob/ob (n=9), ob/mut (n=5) Lep ob/ob (n=8), ob/mut (n=4) Lep ob/ob (n=9), ob/mut (n=5) Lep ob/ob (n=4), ob/mut (n=4) Time: F = 155.7, P < Genotype: F = 8.980, P = Activity: Interaction: F = 6.224, P = Time: F = 64.24, P < Genotype: F = 4.052, P = Multiple comparison: **P<0.01 t=1.670, P= Linear Regression F=15.397, P = Linear Regression F=5.133, P = Interaction: F=2.480, P = Time: F=46.22, P < Genotype: F=7.328, P = Multiple comparison: **P<0.01 Interaction: F=0.1424, P = Time: F=7.334, P = Genotype: F=27.93, P = Multiple comparison: *P<0.05 BAT: t=2.435, P = isat: t=3.125, P = Eye: t=2.998, P = t=0.8458, P = Interaction: F= , P = Time: F=347.7, P < Genotype: F=15.37, P = Multiple comparison: *P<0.05, **P<0.01 Interaction: F=3.658, P = Time: F=60.61, P < Genotype: F=6.238, P = Multiple comparison: **P<0.01 Linear regression F=15.243, P = Linear regression F=5.167, P = Interaction: F=1.432, P = Time: F=77.53, P < Genotype: F=16.44, P = Multiple comparison: **P<0.01 Glucose: t=2.962, P = Insulin: t=4.872, P = Interaction: F=2.883, P = Time: F=12.68, P < Genotype: F=8.184, P = Multiple comparison: *P<0.05, **P<0.01 G6pc: t=2.512, P = Pck1: t=4.731, P = j Lep ob/ob (n=4), t=3.373, P =

32 46 ob/mut (n=3) 4k Lepob/ob (n=6), Linear Regression F=5.600, P = ob/mut (n=5) 4l Lep ob/ob (n=7), t=0.7152, P = ob/mut (n=6) 5c +/? (n=6), Fl/fl (n=6) Interaction: F=8.020, P < Time: F=91.72, P < Genotype: F=5.403, P = Multiple comparison: **P<0.01 5d +/? (n=6), Linear Regression F=7.923, P = Fl/fl (n=7) 5e +/? (n=6), Linear Regression F=0.802, P = Fl/fl (n=7) 5f +/? (n=6), Fl/fl (n=7) t=0.5845, P = m +/? (n=11), Fl/fl (n=9) Interaction: F=3.439, P = Time: F=83.35, P < Genotype: F=9.483, P = Multiple comparison: *P<0.05, **P<0.01 5n +/? (n=9), Linear Regression F=11.57, P = Fl/fl (n=10) 5o +/? (n=9), Linear Regression F=4.433, P = Fl/fl (n=10) 5p +/? (n=9), Fl/fl (n=10) t=0.9483, P = Main Text Wild-type (n=9) Cadm1 KO (n=7) t=7, P < Suppl. Sample size (n) Statistical Test Values Fig. 1a AA: n=19 Linear regression t=2.476, P = AG: n=50 GG: n=34 1b AA: n=74 Linear regression t=2.476, P = AG: n=29 GG: n=0 2d Wild-type (n=3) Lep ob/ob (n=3) Lep ob/ob /Keto (n=3) One-way ANOVA Cortex: F=0.2879, P= Striatum: F=3.963, P= HPF: F=8.276, P= HY: F=0.4978, P= Midbrain: F=3.213, P= Hindbrain: F=3.255, P= Cerebellum: F=12.87, P=< a 3b 3c Wild-type (n=12) Cadm1KO (n=10) Wild-type (n=13) Cadm1KO (n=10) Wild-type (n=5) Cadm1KO (n=5) t=3.761, P= Multiple comparison: *P<0.05, **P<0.01 Interaction: F = 3.626, P = Time: F = 158.2, P < Genotype: F = 27.33, P < Multiple comparison: **P<0.01, ***P<0.001 Interaction: F = 2.126, P = Time: F = 32.14, P < Genotype: F = 6.426, P = Multiple comparison: *P<0.05 3d Wild-type (n=6) Cadm1KO (n=5) Interaction: F = 3.305, P = Time: F = 82.72, P < Genotype: F = 13.97, P = Multiple comparison: *P<0.05, ***P< e Wild-type (n=6) Cadm1KO (n=5) Interaction: F = , P = Time: F = 36.12, P < Genotype: F = 5.651, P = Multiple comparison: *P<0.05 3f Wild-type (n=6) Interaction: F = 3.154, P =

33 3g 3h 3i 3j 3k 3l 3m 3p 3q 3r 3s 3t 3u 3v 3w Cadm1KO (n=5) Wild-type (n=6) Cadm1KO (n=5) Wild-type (n=10) Cadm1KO (n=10) Wild-type (n=11) Cadm1KO (n=9) Wild-type (n=5) Cadm1KO (n=5) Wild-type (n=11) Cadm1KO (n=9) Wild-type (n=11) Cadm1KO (n=9) Wild-type (n=11) Cadm1 KO (n=7) Cadm2KO (n=8) Wild-type (n=5) Cadm2KO (n=3) Wild-type (n=7) Cadm2KO (n=3) Wild-type (n=9) Cadm2KO (n=7) Cadm2KO (n=6) Cadm2KO (n=6) Cadm2KO (n=6) Cadm2KO (n=6) 6 Time: F = 18.25, P < Genotype: F = , P = Multiple comparison: *P<0.05 Interaction: F = 2.171, P = Time: F = 31.02, P < Genotype: F = 3.454, P = Multiple comparison: *P<0.05 t=1.080, P= VO 2: Interaction: F = , P = Time: F = 173.3, P < Genotype: F = 9.411, P = VCO 2: Interaction: F = 2.267, P = Time: F = 357.9, P < Genotype: F = 5.951, P = RER: Interaction: F = , P = Time: F = 146.0, P < Genotype: F = 1.038, P = Multiple comparison: *P<0.05 t=0.8894, P= Linear Regression F=4.508, P = Interaction: F = , P = Time: F = 220.0, P < Genotype: F = 14.31, P = Multiple comparison: *P<0.05 Interaction: F = 1.130, P = Time: F = 147.9, P < Genotype: F = 22.33, P = Multiple comparison: *P<0.05, ***P<0.001 Interaction: F = 3.325, P = Time: F = 227.7, P < Genotype: F = 7.764, P = Multiple comparison: *P<0.05 Interaction: F = , P = Time: F = 24.84, P < Genotype: F = 6.296, P = Interaction: F = Time: F = Genotype: F = Multiple comparison: *P<0.05 t=3.495, P= (AUC) t=0.5274, P= VO 2: Interaction: F = 1.830, P = Time: F = 406.6, P < Genotype: F = 10.83, P = VCO 2: Interaction: F = , P = Time: F = 434.1, P < Genotype: F = 7.411, P = Multiple comparison: *P<0.05, **P<0.01 Interaction: F = 2.580, P = Time: F = 537.1, P < Genotype: F = 1.006, P = Interaction: F = 1.546, P = Time: F = 420.0, P < Genotype: F = 10.12, P = Multiple comparison: **P<0.01 Interaction: F = 5.887, P = Time: F = 149.7, P < Genotype: F = 2.953, P =

34 3x 3y Cadm2 KO (n=7) Cadm2 KO (n=7) Multiple comparison: *P<0.05 Linear Regression F=14.893, P = Linear Regression F=1.884, P = b n=4 t=11.46, P < c Wild-type (n=4) Cadm1KO (n=3) Lep ob/ob (n=3) One-way ANOVA 4d 4e 4h 5g 5j 6e 6f 6g 6h Wild-type (n=12) Cadm1 KO (n=15) Wild-type (n=11) Cadm1 KO (n=7) Cadm1KO (n=6) Tg-Cadm1 (n=6) Wild-type (n=10) Tg-Cadm1 (n=9) Wild-type (n=6) Cadm1 fl/fl (n=6) Cadm1 fl/fl (n=4) Slc32a1-Cre, Cadm1 fl/fl (n=8) Wild-type (n=6) Slc32a1-Cre, Cadm1 fl/fl (n=4) POMC: F = 15.47, P = Npy: F = 6.103, P = AgrP: F = 12.24, P = Multiple comparison: *P<0.05, **P<0.01 Frequency: t=5.54, P< Amplitude: t=1.167, P = Frequency: t=0.3743, P = Amplitude: t=1.179, P = Asymm: P= Symm: P= Total: P= VO 2: Interaction: F = 14.34, P = Time: F = 543.0, P < Genotype: F = 2.360, P = VCO 2: Interaction: F = 14.79, P = Time: F = 761.7, P < Genotype: F = 2.325, P = RER: Interaction: F = 1.431, P = Time: F = 4.515, P = Genotype: F = 2.807, P = EE: Interaction: F = 11.62, P = Time: F = 298.4, P < Genotype: F =3.330, P = Multiple comparison: *P<0.05 t=3.805, P= Interaction: F = 21.24, P < Time: F = 976.2, P < Genotype: F =8.715, P = Multiple comparison: ***P<0.001 t=7.010, P < Interaction: F = Time: F = Genotype: F =3.593 Interaction: F = , P = Time: F = 23.69, P < Genotype: F =6.625, P = AUC: t=2.379, P= i 6j Slc32a1-Cre, Cadm1 fl/fl (n=5) Slc32a1 -Cre, Cadm1 7 Interaction: F = 1.273, P = Time: F = 110.0, P < Genotype: F = , P = Linear Regression F=1.279, P = 0.280

35 6k 6l 6m 6n 6o 6p 7b 7c fl/fl (n=7) Slc32a1 -Cre, Cadm1 fl/fl (n=7) Slc32a1-Cre, Cadm1 fl/fl (n=8) Wild-type (n=4) Cadm1 fl/fl (n=6) Wild-type (n=4) Cadm1 fl/fl (n=6) Wild-type (n=4) Cadm1 fl/fl (n=6) Wild-type (n=4) Cadm1 fl/fl (n=6) Wild-type (n=4) Cadm1 fl/fl (n=4) Wild-type (n=3) Cadm1 fl/fl (n=3) Linear Regression F=0.608, P = Activity: Interaction: F = 2.252, P = Time: F = 33.35, P < Genotype: F = , P = Food: Interaction: F = , P = Time: F = 242.2, P < Genotype: F =1.143, P = Body Weight: t=1.686, P= Blood Glucose: t=0.1078, P= Insulin: t=1.417, P= Glucose: Interaction: F = 1.546, P = Time: F = 1.290, P = Genotype: F =0.5860, P = GIR: Interaction: F = 2.304, P = Time: F = 60.97, P < Genotype: F =6.661, P = Multiple comparison: *P<0.05 t=2.653, P= Basal output: t=1.675, P= Clamped output: t=2.557, P= Clamped suppression: t=5.739, P= Glc Uptake: t=0.7176, P= Glycolysis rate: t=1.236, Glyc. Synth.: t=1.346, P= Insulin: t=0.9284, P= Synapse number per m 2 : n.s. Synapse profiles per m 2 : n.s. t=3.001, P= b +/? (n=6), Fl/fl (n=7) VO 2: Interaction: F = 4.537, P = Time: F = 294.3, P < Genotype: F = 8.264, P = VCO 2: Interaction: F = 4.400, P = Time: F = 443.1, P < Genotype: F = 4.954, P = RER: Interaction: F = , P = Time: F = 453.0, P < Genotype: F =0.1127, P = Activity: Interaction: F = 4.814, P = Time: F = 310.6, P < Genotype: F =7.113, P = EE: Interaction: F = 5.747, P = Time: F = 635.3, P <

36 Genotype: F =4.674, P = Multiple comparison: *P<0.05, **P<0.01 VO 2: Interaction: F = 12.08, P = Time: F = 593.1, P < Genotype: F = 5.415, P = VCO 2: Interaction: F = 12.37, P = Time: F = 626.8, P < Genotype: F = 5.785, P = RER: Interaction: F = , P = Time: F = 97.55, P < Genotype: F =1.203, P = Activity: Interaction: F = 17.10, P = Time: F = 280.2, P < Genotype: F =3.298, P = EE: Interaction: F = 12.98, P = Time: F = 604.9, P < Genotype: F =5.687, P = Multiple comparison: *P<0.05, **P<0.01 Interaction: F = 1.318, P = Time: F = 41.35, P < Genotype: F =0.1742, P = Interaction: F = 2.143, P = Time: F = 32.61, P < Genotype: F =1.151, P = Interaction: F = 1.059, P = Time: F = 46.59, P < Genotype: F =0.9397, P = Interaction: F = , P = Time: F = 76.77, P < Genotype: F =0.1604, P = Interaction: F = , P = Time: F = 168.9, P < Genotype: F =0.2692, P = Interaction: F = , P = Time: F = 26.95, P < Genotype: F =0.3345, P = Linear Regression F=0.002, P = f +/? (n=8), Fl/fl (n=9) 8g +/? (n=6), Fl/fl (n=6) 9a 9b 9c 9d 9e 9f 9g 9h 9i 9j 9k 9l Wild-type (n=5) Agrp-Cre, Cadm1 fl/fl (n=3) Wild-type (n=6) Pomc-Cre, Cadm1 fl/fl (n=6) Wild-type (n=10) Sim1-Cre, Cadm1 fl/fl (n=6) Wild-type (n=7) Lepr-Cre, Cadm1 fl/fl (n=4) Wild-type (n=5) Lepr-Cre, Cadm1 fl/fl (n=3) Lepr -Cre, Cadm1 fl/fl (n=7) Wild-type (n=12) Lepr-Cre, Cadm1 fl/fl (n=12) Lep ob/ob (n=3) ob/lepr-cre, Cadm1 fl/fl (n=3) Lep ob/ob (n=7) ob/lepr-cre, Cadm1 fl/fl (n=5) Lep ob/ob (n=6) ob/lepr-cre, Cadm1 fl/fl (n=5) Lep ob/ob (n=6) ob/lepr-cre, Cadm1 fl/fl (n=5) Lep ob/ob (n=4) ob/lepr-cre, Cadm1 fl/fl (n=3) 9 Activity: t=1.225, P= Food: t=0.2306, P= Interaction: F = , P = Time: F = 312.3, P < Genotype: F = , P = Random fed: t=2.816, P= Fasted: t=2.294, P= Insulin: t=3.353, P= Interaction: F = 5.885, P = Time: F = 29.97, P < Genotype: F =10.40, P = Multiple comparison: ***P<0.001 Interaction: F = 1.571, P = Time: F = 35.12, P < Genotype: F =10.43, P = Multiple comparison: **P<0.01 Interaction: F = , P = Time: F = 19.65, P < Genotype: F =17.04, P = Multiple comparison: *P<0.05, **P<0.01

37 9m 9n 9o Supp Tab. 1 Supp Tab. 1 Supp Tab. 1 Supp Tab. 1 Supp Tab. 2 Supp Tab. 2 Supp Tab. 2 Wild-type (n=6) Lepr-Cre, Cadm1 fl/fl (n=5) Lep ob/ob (n=5) ob/lepr-cre, Cadm1 fl/fl (n=7) Lepr-Cre, Cadm1 fl/fl (n=5) Lep ob/ob (n=6) ob/lepr-cre, Cadm1 fl/fl (n=4) Lep ob/ob (n=6) ob/lepr -Cre, Cadm1 fl/fl (n=7) Lep ob/ob (n=8) ob/lepr-cre, Cadm1 fl/fl (n=6) Wild-type (n=10-12) Cadm1 KO (n=6) Wild-type (n=7-9) Cadm1 KO (n=6-8) Wild-type (n=10) Cadm1 KO (n=6) Cadm1 KO (n=6) Wild-type (n=11) Cadm1 KO (n=9) Wild-type (n=7) Cadm2 KO (n=6) Tg- Cadm1 (n=10) One-way ANOVA 10 Beta cell mass: F= 39.63, P < Multiple comparison: ***P<0.001 Insulin Content: F= 103.5, P= < Multiple comparison: ***P<0.001 Linear Regression F=1.215, P = Activity: t=1.126, P = Food: t= , P = Glucose random fed: t= , P = fasted: t=0.3685, P = Insulin random fed: t=0.5844, P = fasted: t=0.2725, P = Leptin t=2.521, P = Adiponectin t=2.451, P = body mass: t=2.74, P = body fat (%): t=2.752, P = lean mass (%): t=2.175, P = body fat (g): t=3.317, P = lean mass (g): t=2.45, P = body mass: t=2.27, P = body fat (%): t=0.9068, P = lean mass (%): t=0.9418, P = body fat (g): t=0.1477, P = lean mass (g): t=3.067, P = body mass: t=3.023, P = body fat (%): t=2.299, P = lean mass (%): t=2.263, P = body fat (g): t=2.975, P = lean mass (g): t=1.918, P = Supp Tab. Wild-type (n=9), body mass:

38 47 2 Cadm1 fl/fl (n=8) Supp Tab. 2 Supp Tab. 2 Supp Tab. 2 Lepob/ob (n=6), ob/mut (n=5) Wild-type (n=6) Slc32a1 -Cre, Cadm1 fl/fl (n=6) Lep ob/ob (n=5) ob/lepr -Cre, Cadm1 fl/fl (n=4) t=2.81, P = body fat (%): t=1.131, P = lean mass (%): t=0.7095, P = body fat (g): t=2.741, P = lean mass (g): t=2.44, P = body mass: t=2.857, P = body fat (%): t=2.935, P = lean mass (%): t=2.789, P = body fat (g): t=2.79, P = lean mass (g): t=3.011, P = body mass: t=0.167, P = body fat (%): t=0.6027, P = lean mass (%): t=0.5265, P = body fat (g): t=0.568, P = lean mass (g): t= , P = body mass: t=1.131, P = body fat (%): t=1.257, P = lean mass (%): t=1.161, P = body fat (g): t=1.247, P = lean mass (g): t=0.6805, P =