ALT (U/L) (Relative expression) HDL (mm) (Relative expression) ALT (U/L) (Relative expression)

Transcription

1 a DMT mrna () 8 6 r =.96 P =. DMT mrna () 8 6 r =. P =.6 DMT mrna () 8 6 r =.99 P =.6 DMT mrna () 8 6 r =. P =.9 DMT mrna () BMI (kg/m ) 8 6 r =.7 P =.966 DMT mrna () 8 ALT (U/L) 8 6 r = -.66 P =.76 DMT mrna () AST (U/L) 8 6 r =.7 P =.9 DMT mrna () Triglycerides (mm) 8 6 r =.6 P =.89 DCYTB mrna () 6 8 Cholesterol(mM ) r =.68 P <. DCYTB mrna ()..... HDL (mm) r =.78 P <. DCYTB mrna () 6 LDL (mm) r =.98 P =. DCYTB mrna () Glucose (mm) r =. P =.9 DCYTB mrna () BMI (kg/m ) r =.8 P =. DCYTB mrna () 8 ALT (U/L) r = -. P =. DCYTB mrna () AST (U/L) r =.77 P =.8 DCYTB mrna () Triglycerides (mm) r =.8 P =. PDK mrna () 6 8 Cholesterol(mM ) r =.8 P =.966 PDK mrna ()..... HDL (mm) r = -.8 P =.799 PDK mrna () 6 LDL (mm) r = -.7 P =.78 PDK mrna () Glucose (mm) r = -.7 P =.66 BMI (kg/m ) 8 ALT (U/L) AST (U/L) Triglycerides (mm) PDK mrna () 6 r = -.76 P =.66 PDK mrna () 6 r =. P =.766 PDK mrna () 6 r = -.6 P =.96 PDK mrna () r =.87 P =.999 b 6 8 Cholesterol(mM ) HIFα HIFα β-actin Chow..... HDL (mm) HFD 6 LDL (mm) Chow Glucose (mm) HFD Dmt Dcytb Epo Fpn Pdk Nature Medicine: doi:.8/nm.

2 Supplementary Figure. Increased HIFα signaling in human ileum biopsies is correlated with obesity. (a) Correlative analysis of ileum DMT, DCYTB, and PDK mrna levels with BMI, ALT, AST, triglycerides, cholesterol, HDL, LDL, and glucose. n =. Correlations were assessed by nonparametric Spearman s test. (b) Western blot analysis of HIFα and HIFα protein expression (n = /group) and mrna expression analysis of their target genes in small intestine from chow or HFD-fed mice ( week). n = for chow and n = for HFD. For box plots, the midline represents the median; box represents the interquartile range (IQR) between the first and third quartiles, and whiskers represent the lowest or highest values within. times IQR from the first or third quartiles. P <., P <. versus chow, by two-tailed Student s t-test. Nature Medicine: doi:.8/nm.

3 a Body mass(g) d f 6 Hifα 6 9 Time after HFD (weeks) Duodenum Jejunum b Blood glucose (mg/dl) Ileum Colon Liver Kidney Lung Heart Spleen Time (min) BAT WAT Brain g e HIFα HIFα β-actin h c % Initial blood glucose. 6 9 Time (min) i Body mass(g) 6 9 Time after chow diet (weeks) Liver weight (g).... Liver weight (% body mass) j k l m n Hepatic triglycerides (mg/g of liver) Serum triglyceride (mg/dl) Nature Medicine: doi:.8/nm. Hepatic cholesterol (mg/g of liver) Serum cholesterol (mg/dl) 8 ALT (U/L) 8

4 Supplementary Figure. Lack of intestinal HIFα prevents HFD-induced obesity and improves metabolic homeostasis. (a) Growth curves of HFD-fed and Hifa IE mice. (b) Glucose tolerance testf HFD-fed and Hifa IE mice. (c) Insulin tolerance testf HFD-fed and Hifa IE mice. (d) Hifa mrna expression in different tissues from HFD-fed and Hifa IE mice. (e) Western blot analysis of liver HIFα and HIFα from f HFD-fed and Hifa IE mice (n = /group). (f) Growth curves of chow-fed and Hifa IE mice. (g) Representative H&E staining of liver sections of chow-fed and Hifa IE mice (n =, images/mouse). Scale bars, µm. (h) Liver weights of chow-fed and Hifa IE mice. (i) Liver weight to body weight ratios of chowfed and Hifa IE mice. (j, k) Liver (j) and serum (k) triglyceride content of chow-fed and Hifa IE mice. (l,m) Liver (l) and serum (m) cholesterol content of chow-fed and Hifa IE mice. (n) Serum ALT levels of chow-fed and Hifa IE mice. n = /group. Data are presented as the mean ± sem. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within. times IQR from the first or third quartiles. P <., P <. versus mice, by two-tailed Student s t-test. Nature Medicine: doi:.8/nm.

5 a b Hifa IE M M M M M c e Serum sphingomyelin relative abundance g C6: Hifa C8: C8: C: Dmt C: C: C: Dcytb d f h Serum glucosylceramide relative abundance C6: Sptlc C8: C: C: Sptlc Kdsr Cers Cers C: C: Cers6 Degs Degs..... Smpd Smpd Smpd Smpd Enpp Sgms Sgms Acer Acer Nature Medicine: doi:.8/nm.

6 Supplementary Figure. Loss of HIFα in the intestine affects ceramide metabolism in HFD-fed and Hifa IE mice. (a) Score scatter plot of a PCA model of the serum metabolites between (circle) and Hifa IE (square) mice. (b) S-plot of an OPLS-DA model of the serum metabolites. (c,d) The relative levels of sphingomyelin (c) and glucosylceramide (d) in serum. (e) Expression of intestinal Hifa mrna and HIFα target gene mrnas. (f,g) Intestinal expression of mrnas encoded by ceramide synthesis-related genes, including the de-novo pathway (f) and the sphingomyelinase pathway (g). (h) Intestinal expression of mrnas encoded by ceramide catabolism-related genes. n = 6/group. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within. times IQR from the first or third quartiles. P <., P <. versus mice, by two-tailed Student s t-test. Nature Medicine: doi:.8/nm. 6

7 a Body mass (g) e Activity (Counts) Liver weight (g) Day Night Liver weight (% body mass) Blood glucose (mg/dl) 6 9 Time (min) f EE (kcal/day/kg^.7) % Initial blood glucose 8: 6: : 8: Hepatic triglycerides (mg/g of liver) 8 6 Serum triglyceride (mg/dl) 6 9 Time (min) 6 8: 6: : 8: VO (ml/kg.7 /min) Hepatic cholesterol (mg/g of liver) Cumulative food intake (kcal/day/mouse) Serum cholesterol (mg/dl) VCO (ml/kg.7 /min) i j k l m n o p. b c d g h 8: 6: : 8: ALT (U/L).... Srebpc Cidea Cd6 Fabp Fabp Fabp Fabp Acly Acaca Fasn Scd Elovl6 Dgat Dgat Plin q. r..... Nature Medicine: doi:.8/nm. Acox Acox Cpt Cpt Acsl Acadl Ehhadh Acaaa Tnfa Pai Ccl Ccl Ilb Il6 Il

8 Supplementary Figure. Inhibition of the intestinal HIFα substantially increases the metabolic rate and decreases hepatic steatosis independent of body weight changes in HFD-fed and Hifa IE mice. (a) body weight. (b) Glucose tolerance test. (c) Insulin tolerance test. (d) Cumulative food intake. (e) Activity. (f) Energy expenditure. (g) Oxygen consumption rate. (h) Carbon dioxide production rate. (i) Liver weights. (j) Liver weight to body weight ratios. (k,l) Liver (k) and serum (l) triglyceride content. (m,n) Liver (m) and serum (n) cholesterol content. (o) Serum ALT levels (P =.8). (p) Hepatic expression of mrnas encoding fatty acid transport and lipogenesis. (q) Hepatic expression of mrnas encoding fatty acid oxidation-related enzymes. (r) Hepatic expression of mrnas encoding inflammatory cytokines and chemokines. n = 6 for group and n = for Hifa IE group. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within. times IQR from the first or third quartiles. P <., P <. versus mice, by two-tailed Student s t-test. Nature Medicine: doi:.8/nm. 8

9 a. b. c Hifα Dmt Dcytb d..... Sptlc Sptlc Kdsr Cers Cers Cers6 Degs Degs Smpd Smpd Smpd Smpd Enpp7 e f g Intestinal ceramide (nmol/g) h j Ucp Ucp Ppargca Total C6: Prdm6 Prdm6 Tmem6 Tmem6 C8: C8: C: Hifα fl/fl Hifα ΔIE Elovl Elovl Elovl6 Hifα fl/fl Hifα ΔIE Cox8b C: C: C: i k Serum ceramide (µm) UCP eif.6.. Ucp Ppargca. Neu Total C6: C8: C: Prdm6 Elovl Hifa IE Neu C: C: C: Hifα fl/fl Hifα ΔIE Cox8b UCP/eIF (rel. expression) Neu Portal ceramide (µm) Glb... l Gba Gba Total C6: C8: C: C: C: C: 9 Nature Medicine: doi:.8/nm.

10 Supplementary Figure. Intestinal HIFα deficiency reduces ceramide synthesis in the small intestine independent of body weight changes in HFD-fed and Hifa IE mice. (a) Expression of intestinal Hifa mrna and HIFα target gene mrnas. (b-d) Intestinal expression of mrnas encoded by ceramide synthesis-related genes, including the de-novo pathway (b), the sphingomyelinase pathway (c), and the salvage pathway (d). (e-g) Ceramide levels in the small intestine (e), systematic serum (f), and portal serum (g). (h-j) Thermogenic gene expression in scwat (h), BAT (i), and ewat (j). (k) Western blot analysis of UCP protein expression in scwat (n = /group). Data are presented as the mean ± sem. n = 6 for group and n = for Hifa IE group. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within. times IQR from the first or third quartiles. P <., P <. versus mice, by two-tailed Student s t-test. (l) Representative UCP immunohistochemistry staining of scwat sections (n = images/mice). Scale, µm. Nature Medicine: doi:.8/nm.

11 a c e Vhl mrna () i Pdk mrna ().... Vhl Smpd Vhl/Hifa fl/fl Vhl/Hifa ΔIE Vhl/Hifa ΔIE + PT8 Vhl fl/fl ΔIE Vhl/Hifa fl/fl Vhl fl/fl ΔIE Vhl/Hifa fl/fl Vhl/Hifa ΔIE Vhl/ Vhl/ n.s. n.s. Hifa Vhl/Hifa ΔIE Vhl/ Vhl/ Hifa Vhl/Hifa fl/fl Vhl/Hifa ΔIE Vhl/Hifa ΔIE + PT8 Smpd Smpd Hifa mrna () Neu mrna () n.s. Vhl fl/fl ΔIE Vhl/Hifa fl/fl Vhl fl/fl ΔIE Vhl/Hifa fl/fl n.s. Vhl/Hifa ΔIE Vhl/ Vhl/ n.s. n.s. Vhl/Hifa ΔIE Vhl/ Vhl/ Hifa mrna () Intestinal lactosylceramide C6: relative abundance Supplementary Figure 6. HIFα regulates the ceramide synthesis in the small intestine. (a) Intestinal expression of Vhl, Hifa, Hifa, Dmt and Dcytb mrnas. (b-d) Intestinal expression of mrnas Nature Medicine: doi:.8/nm. f Dmt Smpd Dcytb Enpp7 b d Neu g Vhl/Hifa fl/fl Vhl/Hifa ΔIE Vhl/Hifa ΔIE + PT Neu Neu Sptlc Sptlc Kdsr Cers Cers Cers6 Degs Degs Vhl fl/fl ΔIE Vhl/Hifa fl/fl Vhl/Hifa ΔIE Vhl/ Vhl/ Glb n.s. DMT Gba Normoxia + Normoxia + PT8 Hypoxia (CoCl ) + Hypoxia (CoCl ) + PT8 Dmt mrna () DCYTB Gba 6 Vhl/Hifa fl/fl Vhl/Hifa ΔIE Vhl/Hifa ΔIE + PT8 NEU Vhl fl/fl ΔIE Vhl/Hifa fl/fl Vhl/Hifa ΔIE Vhl/ Vhl/ j k l m h n.s. n.s. Efficiency (%) Control sineu

12 encoded by ceramide synthesis-related genes, including the de-novo pathway (b), the sphingomyelinase pathway (c), and the salvage pathway (d). Male Vhl/Hifa fl/fl and Vhl/ Hifa IE mice fed a chow diet were treated with or without PT8 ( mg/kg) for three consecutive days (n = to 6/group). P <., P <. versus vehicle-treated Vhl/Hifa fl/fl mice, P <., P <. versus vehicle-treated Vhl/Hifa IE mice, by one-way ANOVA with Tukey's correction. (e-j) Intestinal mrna expression levels of Vhl (e), Hifa (f), Hifa (g), Dmt (h), Pdk (i), and Neu (j) in Vhl fl/fl, Vhl IE, Vhl/Hifa fl/fl, Vhl/Hifa IE, Vhl/, and Vhl/Hifa IE mice fed a chow diet (n = to 6/group). (k) The relative levels of lactosylceramide C6: in the small intestine from and Hifa IE mice fed a HFD for weeks (n = 6/group). P <. versus mice, by two-tailed Student s t-test. (l) mrna expression of DMT, DCYTB, and NEU in HCT6 cells treated with vehicle or PT8 and exposed to either vehicle or CoCl (n = /group). P <. versus Normoxia + treatment, P <., P <. versus Hypoxia (CoCl ) + treatment, by one-way ANOVA with Tukey's correction. (m) The knockdown efficiency of sineu in HCT6 cells. P <., by two-tailed Student s t-test. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within. times IQR from the first or third quartiles. Nature Medicine: doi:.8/nm.

13 a b c Intestinal neuraminidase activity (mu/mg protein e g j o Serum ceramide (µm) Hepatic triglycerides (mg/g of liver) DANA Naringin Total Srebpc Cidea C6: DANA Naringin Hepatic neuraminidase activity (mu/mg protein C8: Serum triglyceride (mg/dl) Cd6 Fabp Fabp Fabp Fabp Acly Acaca Fasn Scd Body mass (g) DANA Naringin Nature Medicine: doi:.8/nm C: DANA Naringin White adipose neuraminidase activity (mu/mg protein C: DANA Naringin C: DANA Naringin C: Hepatic cholesterol (mg/g of liver) DANA Naringin DANA Naringin Time after HFD (weeks) DANA Naringin Elovl6 Dgat Intestinal ceramide (nmol/g) DANA Naringin f Dgat Plin 6 Liver weight (g) Serum cholesterol (mg/dl) Total C6:.... DANA Naringin DANA Naringin C8: C8: C: k l m n d h p i Liver weight (% body mass) ALT (U/L) DANA Naringin Tnfa Pai Ccl Ccl Ilb DANA Naringin C: C: C: DANA Naringin DANA Naringin Il6 Il

14 Supplementary Figure 7. NEU inhibitor DANA and naringin treatments protect mice from HFDinduced obesity and hepatic steatosis. (a-c) Neuraminidase activities in intestine (a), liver (b), and white adipose tissue (c). (d,e) Ceramide levels in the small intestine (d) and serum (e). (f) Growth curves. (g) Representative H&E staining of liver sections (n = images/mouse). Scale bars, µm. (h) Liver weights. (i) Liver weight to body weight ratios. (j,k) Liver (j) and serum (k) triglyceride content. (l,m) Liver (l) and serum (m) cholesterol content. (n) Serum ALT levels. (o) Hepatic expression of mrnas encoding fatty acid transport and lipogenesis. (p) Hepatic expression of mrnas encoding inflammatory cytokines and chemokines. n = 6/group. Data are presented as the mean ± sem. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within. times IQR from the first or third quartiles. P <., P <. versus vehicle treatment, by two-tailed Student s t-test. Nature Medicine: doi:.8/nm.

15 a Intestinal ceramide (nmol/g) c e Body mass (g) 6 Total AUC (mg/dl min) C6: C8: C8: Ceramide C: Ceramide C: Time after HFD (weeks) Ceramide C: C: 7 Time (min) Supplementary Figure 8. Administration of ceramide reverses the protective effects of intestinal HIFα inhibition on the HFD-induced obesity and insulin resistance in and Hifa IE. (a,b) Ceramide levels in the small intestine (a) and serum (b). (c) Growth curves. (d,e) Glucose tolerance test (d) and glucose AUC (e). (f) Insulin tolerance test. n = /group. Data are presented as the mean ± sem. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within. times IQR from the first or third quartiles. P <., P <. versus vehicle-treated mice, P <., P <. versus vehicle-treated Hifa IE mice, by one-way ANOVA with Tukey s correction. b d f Serum ceramide (µm) Blood glucose (mg/dl) % Initial blood glucose 6 8 Time (min) 8 Total Ceramide C6: Ceramide C8: C: Ceramide C: C: C: Nature Medicine: doi:.8/nm.

16 a Body mass (g) d or 6 9 Time after HFD (weeks) Blood glucose (mg/dl) Time (min) + + PT8 + + PT8 g h i l Hepatic triglycerides (mg/g of liver) Intestinal ceramide (nmol/g) PT8 + + PT8 + + PT8 Total + + PT8 C6: C8: Serum triglyceride (mg/dl) Nature Medicine: doi:.8/nm. C8: C: b + + PT8 + + PT8 + + PT8 + + PT8 C: C: Hepatic cholesterol (mg/g of liver) C: 6 e or + + PT8 + + PT8 m Liver weight (g) Serum ceramide (µm) Total Serum cholesterol (mg/dl) C6: % Initial blood glucose PT8 + + PT8 j c + + PT8 C8: or 6 8 Time (min) + + PT8 + + PT8 f C: Liver weight (% body mass) + + PT8 + + PT8 k ALT (U/L) + + PT8 C: + + PT8 + + PT8 C: C:

17 Supplementary Figure 9. PT8 prevents mice from HFD-induced obesity and hepatic steatosis through inhibition of the intestinal HIFα-ceramide axis in and Hifa IE mice. (a) Growth curves. (b) Glucose tolerance test. (c) Insulin tolerance test. (d) Representative H&E staining of liver sections (n = images/mouse). Scale bars: µm. (e) Liver weights. (f) Liver weight to body weight ratios. (g,h) Liver (g) and serum (h) triglyceride content. (i,j) Liver (i) and serum (j) cholesterol content. (k) Serum ALT levels. (l) Quantitation of ceramide concentrations in the intestine. (m) Quantitation of ceramide concentrations in serum. n = /group. Data are presented as the mean ± sd. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within. times IQR from the first or third quartiles. P <., P <. versus vehicle-treated mice, by one-way ANOVA with Tukey s correction. Nature Medicine: doi:.8/nm. 7

18 a PT8 + + PT8 b + + PT8 + + PT8 c..... Hifa Dmt + + PT8 + + PT8 Dcytb d Sptlc Sptlc Kdsr Cers Cers Cers6 + + PT8 + + PT8 Degs Degs e f. Srebpc Smpd + + PT8 Cidea Smpd Cd6 Smpd Fabp + + PT8 + + PT8 Fabp Smpd Fabp Enpp7 Fabp Acly Acaca + + PT8 Neu Fasn Scd Neu Elovl6 Neu Dgat Glb Dgat Gba Plin Gba Tnfa Pai Ccl Ccl Ilb Il6 Il 8 Nature Medicine: doi:.8/nm.

19 Supplementary Figure. PT8 inhibits ceramide synthesis in the small intestine and alters fatty acid synthesis, metabolism, and inflammation in the liver dependent on intestinal HIFα in and Hifa IE mice. (a) Expression of Hifa mrna and its target gene mrnas in the intestine. (b-d) Intestinal expression of mrnas encoded by ceramide synthesis-related genes, including the de-novo pathway (b), the sphingomyelinase pathway (c) and the salvage pathway (d). (e) Hepatic expression of mrnas encoding fatty acid transport and lipogenesis. (f) Hepatic expression of mrnas encoding inflammatory cytokines and chemokines. n = /group. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within. times IQR from the first or third quartiles. P <., P <. versus vehicletreated mice, by one-way ANOVA with Tukey s correction. Nature Medicine: doi:.8/nm. 9

20 a Body mass(g) d 6 PT8 Time after HFD (weeks). b Blood glucose (mg/dl) 7 PT8 e PT8 6 9 Time (min) c % Initial blood glucose 6 9 Time (min) PT8 PT8 f..... Dmt Dcytb PT8 g Serum ceramide (µm) Total C6: C8:.6 P=. C: C: PT8 C: C: Sptlc Sptlc Kdsr Cers Cers Cers6 Degs Degs. Smpd Smpd Smpd Smpd Enpp7 h.8 PT8 i PT8..6. Srebpc Cidea Nature Medicine: doi:.8/nm. Cd6 Fabp Fabp Fabp Fabp Acly Acaca Fasn Scd Elovl6 Dgat Dgat Plin Tnfa Pai Ccl Ccl Ilb Il6 Il

21 Supplementary Figure. PT8 reverses metabolic dysfunctions in HFD-induced obese mice. (a) Growth curves. (b) glucose tolerance test. (c) Insulin tolerance test. (d) Expression levels of HIFα target gene mrnas in the intestine. (e) Quantitation of ceramide concentrations in serum. (f,g) Intestinal expression of mrnas encoded by ceramide synthesis-related genes, including the de novo pathway (f) and the sphingomyelinase pathway (g). (h) Hepatic expression of mrna encoding fatty acid transport and lipogenesis-related enzymes. (i) Hepatic expression of mrnas encoding inflammatory cytokines and chemokines. n = for vehicle group, n = for PT8 group. Data are presented as the mean ± sem. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within. times IQR from the first or third quartiles. P <., P <. versus vehicle treatment, by two-tailed Student s t-test. Nature Medicine: doi:.8/nm.

22 a Non-obese HIFα Obese b Chow HIFα HFD 8 weeks Ctrl 7 kd kd kd kd kd 7 kd HIFα kd 7 kd kd kd 7 kd 7 kd β-actin kd β-actin 7 kd kd kd kd kd kd kd HIFα kd kd 7 kd NEU kd 7 kd β-actin kd 7 kd kd Nature Medicine: doi:.8/nm.

23 c Chow HIFα HFD week d HIFα kd kd 9 kd 7 kd kd 7 kd HIFα Ctrl kd kd 7 kd 9 kd 7 kd HIFα β-actin kd kd kd kd 7 kd kd kd 7 kd β-actin kd kd 7 kd kd 7 kd Nature Medicine: doi:.8/nm.

24 e NEU f NEU PT8 kd kd kd kd kd 7 kd kd 7 kd kd kd 7 kd 7 kd kd β-actin kd β-actin kd kd kd 7 kd kd 7 kd kd kd 7 kd 7 kd g UCP KD KD KD eif 7 KD KD KD Nature Medicine: doi:.8/nm.

25 Supplementary Figure. Full western blot gel panels. (a-d) HIFα, HIFα, NEU and β-actin from which the data in Figure b (a), Figure f (b), Supplementary Figure b (c), Figure g (b) and Supplementary Figure e (d) were derived. (e, f) NUE and β-actin from which the data in Figure h (e) and Figure 6k (f) were derived. (g) UCP and EIF from which the data in Supplementary Figure v were derived. Nature Medicine: doi:.8/nm.

26 Supplementary Table. Demographic characteristics of the subjects Characteristics Non-obese Obese Cohort (n = ) Gender, n (%) Male (66.7) (66.7) Female (.) (.) Age, y Mean (sem) 9.7 (.8). (6.) Range Body weight, kg Mean (sem) 9. (.) 76. (.) Range Body mass index, kg/m Mean (sem). (.6) 8. (.8) Range Cohort (n = ) Gender, n (%) Male 9 (.) 7 (.) Female 9 (.) (8.8) Age, y Mean (sem) 7. (.).7 (.) Range Body weight, kg Mean (sem).7 (.) 8. (.) Range Body mass index, kg/m Mean (sem). (.6) 9.8 (.) Range P <. versus Non-obese, by two-tailed Student s t-test. Nature Medicine: doi:.8/nm. 6

27 Supplementary Table. Clinical biochemistry of the subjects Laboratory analytes Non-obese Obese P value Cohort (n = ) Liver enzymes, U/L ALT. (.). (.). AST 7. (.7). (.). Lipids, mm Triglycerides.7 (.).67 (.).7 Cholesterol. (.6).79 (.).69 HDL. (.).99 (.).9 LDL.7 (.).9 (.6).6 Glucose, mm. (.7).9 (.7).9 Cohort (n = ) Liver enzymes, U/L ALT 9.6 (.) 8. (.). AST 9. (.). (.8). Lipids, mm Triglycerides. (.6).79 (.). Cholesterol.6 (.). (.). HDL.9 (.). (.6).6 LDL.99 (.9).67 (.9).7 Glucose, mm. (.8) 6. (.6).798 Data are presented as mean (sem). Two-tailed Student s t-test. Nature Medicine: doi:.8/nm. 7

28 Supplementary Table. Primer list Mouse primers Sequence β-actin FWD '- GGCTGTATTCCCCTCCATCG -' β-actin REV '- CCAGTTGGTAACAATGCCATGT -' Hifa FWD '- ATAGCTTCGCAGAATGCTCAGA -' Hifa REV '- CAGTCACCTGGTTGCTGCAA -' Hifa FWD '- TGAGTTGGCTCATGAGTTGC -' Hifa REV '- TATGTGTCCGAAGGAAGCTG - Dmt FWD '- TGTTTGATTGCATTGGGTCTG - Dmt REV '- CGCTCAGCAGGACTTTCGAG - Dcytb FWD '- CATCCTCGCCATCATCTC - Dcytb REV '- GGCATTGCCTCCATTTAGCTG - Pdk FWD '- TTACTCAGTGGAACACCGCC - Pdk REV '- GTTTATCCCCCGATTCAGGT - Vhl FWD '- ACATCGTCAGGTCACTCTATGA - Vhl REV '- CTCTTGGCTCAGTCGCTGTAT - Sptlc FWD '- CGAGGGTTCTATGGCACATT-' Sptlc REV '- GGTGGAGAAGCCATACGAGT -' Sptlc FWD '- TCACCTCCATGAAGTGCATC -' Sptlc REV '- CAGGCGTCTCCTGAAATACC - Kdsr FWD '- TCCAGTGGCATTGGGAAGTG - Kdsr REV '- CTTCTCTTGTGCCTGCTTTATGA - Degs FWD '- AATGGGTCTACACGGACCAG - Degs REV '- TGGTCAGGTTTCATCAAGGAC - Degs FWD '- AAGCCAATGGACCACAAACT - Degs REV '- TGCTTGGAGAGCCCTTCTAAT - Cers FWD '- AAGTGGGAAACGGAGTAGCG- Cers REV '- ACAGGCAGCCATAGTCGTTC - Cers FWD '- GGATTAGCTGATCTCCGCAC - Cers REV '- CCAGTATGTCTCCTGCCACA - Cers6 FWD '- AAGCCAATGGACCACAAACT - Cers6 REV '- TGCTTGGAGAGCCCTTCTAAT - Smpd FWD '- GTTACCAGCTGATGCCCTTC - Smpd REV '- AGCAGGATCTGTGGAGTTG - Smpd FWD '- AGCAGGATCTGTGGAGTTG - Smpd REV '- CTCCAGCCATGAAGCTCAAC - Smpd FWD '- CCTGACCAGTGCCATTCTTT - Smpd REV '- AGAAACCCGGTCCTCGTACT - Nature Medicine: doi:.8/nm. 8

29 Smpd FWD '- ACCTGGCCCTCAATCCATTTG - Smpd REV '- ATAGGCACAGTCCGAAGTACG - Enpp7 FWD '- AAGCCCAGTATATGACTCCTGC - Enpp7 REV '- ACCGTGCTGGTGGTATTGTAG - Neu FWD '- GGACCGCTGAGCTATTGGG - Neu REV '- CGGGATGCGGAAAGTGTCTA - Neu FWD '- CACAGGCGTCCATGCTTACA - Neu REV '- CTGCGTGCTCATCCGTCTT - Neu FWD '- ATGGAGGCCACATTACCTGG - Neu REV '- TCTGGCACCTCTCAGTAACAT - Glb FWD '- GCACGGCATCTATAATGTCACC - Glb REV '- GTATCGGAATGGCTGTCCATC - Gba FWD '- GCCAGGCTCATCGGATTCTTC - Gba REV '- CACGGGGTCAAGAGAGTCAC - Gba FWD '- GGCTGTGCCGAAAGAGATTC - Gba REV '- ATCCTGGGGTCCACTATCCTC - Galc FWD '- CGCCTACGTGCTAGACGAC - Galc REV '- ACGATAGGGCTCTGGGTAATTT - Srebpc FWD '- GGAGCCATGGATTGCACATT-' Srebpc REV '- GCTTCCAGAGAGGAGGCCAG -' Cidea FWD '- TGACATTCATGGGATTGCAGAC -' Cidea REV '- GGCCAGTTGTGATGACTAAGAC -' Cd6 FWD '- AGATGACGTGGCAAAGAACAG - Cd6 REV '- CCTTGGCTAGATAACGAACTCTG - Fabp FWD '- ATGAACTTCTCCGGCAAGTACC - Fabp REV '- CTGACACCCCCTTGATGTCC - Fabp FWD '- GTGGAAAGTAGACCGGAACGA - Fabp REV '- CCATCCTGTGTGATTGTCAGTT - Fabp FWD '- GTGGAAAGTAGACCGGAACGA - Fabp REV '- CCATCCTGTGTGATTGTCAGTT - Fabp FWD '- AAGGTGAAGAGCATCATAACCCT - Fabp REV '- TCACGCCTTTCATAACACATTCC - Acly FWD '- ACCCTTTCACTGGGGATCACA - Acly REV '- GACAGGGATCAGGATTTCCTTG - Acaca FWD '- ATGGGCGGAATGGTCTCTTTC - Acaca REV '- TGGGGACCTTGTCTTCATCAT - Fasn FWD '- AAGTTGCCCGAGTCAGAGAACC -' Fasn REV '- ATCCATAGAGCCCAGCCTTCCATC -' Scd FWD '- TTCTTGCGATACACTCTGGTGC - 9 Nature Medicine: doi:.8/nm.

30 Scd REV '- CGGGATTGAATGTTCTTGTCGT - Elovl6 FWD '- GAAAAGCAGTTCAACGAGAACG -' Elovl6 REV '- AGATGCCGACCACCAAAGATA -' Dgat FWD '- GACGGCTACTGGGATCTGA -' Dgat REV '- TCACCACACACCAATTCAGG -' Dgat FWD '- CGCAGCGAAAACAAGAATAA -' Dgat REV '- GAAGATGTCTTGGAGGGCTG -' Plin FWD '- GACCTTGTGTCCTCCGCTTAT -' Plin REV '- CAACCGCAATTTGTGGCTC -' Acox FWD '- GGGCACGGCTATTCTCACAG - Acox REV '- CATCAAGAACCTGGCCGTCT - Acox FWD '- ACGGTCCTGAACGCATTTATG - Acox REV '- TTGGCCCCATTTAGCAATCTG - Cpt FWD '- GAACACAAATGTGCAAGCAGC - Cpt REV '- GCCATGACCGGCTTGATCTC - Cpt FWD '- CAGCACAGCATCGTACCCA - Cpt REV '- TCCCAATGCCGTTCTCAAAAT - Acsl FWD '- CGATGGCTGTTGGACTTTGC - Acsl REV '- CACCCAGGCTCGACTGTATC - Acadl FWD '- TCTTTTCCTCGGAGCATGACA - Acadl REV '- GACCTCTCTACTCACTTCTCCAG - Ehhadh FWD '- CGGTCAATGCCATCAGTCCAA - Ehhadh REV '- TGCTCCACAGATCACTATGGC - Acaaa FWD '- AGGCTTCAAGAACACCACCC - Acaaa REV '- GGCTCCTGGCTCAAGAACAT - Tnfa FWD '- AGGGTCTGGGCCATAGAACT - Tnfa REV '- CCACCACGCTCTTCTGTCTAC - Pai FWD '- TTCAGCCCTTGCTTGCCTC - Pai REV '- ACACTTTTACTCCGAAGTCGGT - Ccl FWD '- TTAAAAACCTGGATCGGAACCAA - Ccl REV '- GCATTAGCTTCAGATTTACGGGT - Ccl FWD '- TTCTCTGTACCATGACACTCTGC - Ccl REV '- CGTGGAATCTTCCGGCTGTAG - Ilb FWD '- AAGAGCTTCAGGCAGGCAGTATCA - Ilb REV '- TGCAGCTGTCTAGGAACGTCA - Il6 FWD '- TAGTCCTTCCTACCCCAATTTCC - Il6 REV '- TTGGTCCTTAGCCACTCCTTC - Il FWD '- GCTCTTACTGACTGGCATGAG - Il REV '- CGCAGCTCTAGGAGCATGTG - Nature Medicine: doi:.8/nm.

31 Human primers Sequence GAPDH FWD '- GGAGCGAGATCCCTCCAAAAT -' GAPDH REV '- GGCTGTTGTCATACTTCTCATGG -' HIFA FWD '- GAACGTCGAAAAGAAAAGTCTCG -' HIFA REV '- CCTTATCAAGATGCGAACTCACA -' HIFA FWD '- CGGAGGTGTTCTATGAGCTGG -' HIFA REV '- AGCTTGTGTGTTCGCAGGAA -' DMT FWD '- GCTCTCATACCCATCCTCACATT -' DMT REV '- TCCATTGGCAAAGTCACTCATT -' DCYTB FWD '- GGTGTTTTCGTAAATACGCTTGG -' DCYTB REV '- ATTGCGGTCTGGTGACTATCC -' PDK FWD '- CTGTGATACGGATCAGAAACCG - PDK REV '- TCCACCAAACAATAAAGAGTGCT - NEU FWD '- AAGTGACAACATGCTCCTTCAA - NEU REV '- TCTCCTCGTAGAACGCTTCTC - ChIP primers Sequence NEU FWD '- TGTCAAGAGGGGCCTTTTCC - NEU REV '- ACAATCACACGCAAGGACCA - Construct primers Sequence Neu promoter FWD '- AACTCGAGCACAGACGCTGAAACGAACCC - Neu promoter REV '- AAGGTACCTGCTGCAGTGTGTCAAGAGGG - Neu promoter HRE FWD '- CGCGCAGTCTGTGGAAGGTATCACCGCC - Neu promoter HRE REV '- TTCCACAGACTGCGCGGTGAAGGGGC - Neu promoter HRE FWD '- GAATCCATATCCCATTCTTCCCCACCTAC - Neu promoter HRE REV '- AATGGGATATGGATTCAGTACTTGGTGACAG - Nature Medicine: doi:.8/nm.