Supplementary Information. Protectin DX alleviates insulin resistance by activating a myokine-liver glucoregulatory axis.

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1 Supplementary Information Protectin DX alleviates insulin resistance by activating a myokine-liver glucoregulatory axis. Phillip J. White, Philippe St-Pierre, Alexandre Charbonneau, Patricia Mitchell, Emmanuelle St-Amand, Bruno Marcotte and André Marette. Supplementary Figures - Nature Medicine: doi:./nm.9

2 Supplementary Figure BLOOD SAMPLE LEGEND ο Pre-infusion sample Pre-clamp sample Clamp samples Post clamp sample H-Glucose Bolus.µCi Kg Insulin Bolus mu Kg - - ο - µg or icle µg or icle Insulin (mu kg min - ) % dextrose variable - - H-Glucose (.µci min ) + RBC (.ml h ) hour intravenous Lipid or Saline infusion (ml kg h - ) Supplementary figure. Lipid-infusion hyperinsulinemic-euglycemic clamp schema. h fasted mice were infused with lipid or saline for h. Prior to the initiation of the infusion a blood sample was drawn to determine pre-infusion glycemia and FFA's. At t= min the tracer stabilization period of the clamp procedure was begun. Immediately prior to the stabilization period a blood sample was taken to determine pre-clamp glycemia and insulinemia. The clamp was begun at t= min with infusion of insulin. Groups were administered µg of or equal volumes of vehicle immediately prior to the infusion and at t=. Nature Medicine: doi:./nm.9

3 Supplementary Figure a Saline Lipid Lipid + µg ml c Percent palmitate Plasma adiponectin Palm Palm + -nm Palm + -nm Nitrite production b pg ml d IL- Palmitate (nm) inos pjnk Thr/Tyr JNK Palm Palm + -nm Palm + -nm IL- ng ml CCL CCL TNF-α IL- Mθ protein (AU).... inos ND Palm Palm + -nm Palm + -nm pjnk/jnk Supplementary figure. dampens lipid-induced inflammation in macrophages. (a) Plasma adiponectin in clamped saline-vehicle, lipid-vehicle and lipid- treated mice. Data are mean ± s.e.m., and are representative of mice per group. (b) Chemokines, cytokines (c) and nitrite in media of J77A. macrophages exposed to vehicle () or palmitate (µm) for h in the presence or absence of. (d) Immunoblots for inos, pjnk Thr/Tyr, and total JNK in palmitate-treated macrophages. Quantitation of densitometry analyses are shown besides the representative gels Data are mean ± s.e.m., and are representative of independent experiments. P<., P.<., P<. calculated using analysis of variance. Nature Medicine: doi:./nm.9

4 Supplementary Figure a b c µg g tissue.... Saline Lipid Lipid + Liver IL- Fold vehicle IL- in media Nor Fold vehicle nm -nm -µm IL- mrna d IL- in media (fold veh) -nm -nm -µm LPS e pampk Thr7 AMPK Mθ protein (AU) - nm -nm -µm - µm min h h pampk/ampk f 9Z E Z E 7Z Z 7(S) (S) CO PD RvD DiHETE 9Z Z E E 7Z Z CO 7(S) (R) PD 7(S) (R) 9Z E Z E 9E Z CO Fold vehicle 7(S) RvD (S) E Z 9E (S) Z CO (S)(S)DiHETE IL- in media Nature Medicine: doi:./nm.9

5 Supplementary figure. selectively induces skeletal muscle IL-. (a) IL- protein in liver from clamped saline-vehicle, lipid-vehicle and lipid- treated mice. Data are mean ± s.e.m., and are representative of mice per group. (b) IL- protein in media of T7i brown adipocytes treated with vehicle (), norepinephrine (Nor, µm) or ( nm) for h. Data are mean ± s.e.m. of independent experiments. (c) IL- mrna (h) and (d) IL- protein in media of J77A. macrophages treated with, or LPS (ng/ml). Data are mean ± s.e.m. of independent experiments. (e) Immunoblots of pampk Thr7 and total AMPK in J77A. macrophages treated with or. Quantification of densitometry analyses are shown below the representative gels. Data are mean ± s.e.m. of independent experiments.(f) IL- in media of CC myotubes treated with or nm of, PD, RvD or (S) (S) DiHETE (DiHETE) for h. Data are mean ± s.e.m. of independent experiments. Chemical structures of, PD, RvD and (S) (S) DiHETE are shown to the left. Structures shared with are colored red while strucutures colored in blue represent a common feature that is the same distance from the methyl end but not the carboxyl terminal of the fatty acid chain due to a difference in chain length. For all figures P<., P<., P<. vs vehicle calculated using analysis of variance. Nature Medicine: doi:./nm.9

6 Supplementary Figure a Glycemia (mm) Glycemia (mm) b GIR (mg kg min ) GIR (mg kg min ) Pre-clamp glycemia c Time (min) d Time (min) Glucose infusion rate Fold increase in Rd % suppression of HGP pg g tissue Peripheral insulin action Hepatic insulin action Muscle IL- e f g pstat Ser77 pampk Thr7 Liver protein (AU) STAT... Relative expression Muscle protein (AU) AMPK pstat/stat Ppargc Pck Gpc pampk/ampk Nature Medicine: doi:./nm.9

7 Supplementary figure. IL- is required for the glucoregulatory actions of. (a) Pre-clamp glycemia (left) and clamp glucose excursion (right), (b) glucose infusion rate (GIR) (left) and mean GIR for the final min of clamp (right) in wild-type () or IL- null () saline-infused (S) (P) or vehicle (V) treated animals. (c) Peripheral insulin action expressed as fold increase in Rd during the clamp (left) and hepatic insulin action expressed as percent suppression of hepatic glucose production (HGP; right), (d) Skeletal muscle IL- content (e) Immunoblots for pstat Ser77 and total STAT and (f) relative mrna expression of Ppargc, Pck and GPc normalized to GAPDH in liver from clamped wild-type () or IL- null () saline-infused (S) (P) or vehicle (V) treated animals. (g) Immunoblots for pampk Thr7 and total AMPK in gastrocnemius muscle from clamped wild-type () or IL- null () saline-infused (S) (P) or vehicle (V) treated animals. Quantification of densitometry analyses for immunoblots is shown below the representative gels. Dotted lines indicate that lanes were run on the same gel but were noncontiguous. -, wild-type saline-infused vehicle, n = mice. -, wild-type saline-infused, n = mice. -, IL- null saline-infused vehicle, n = mice. -, IL- null saline-infused, n= mice. Data are mean ± s.e.m, P<., P<. calculated using analysis of variance. Nature Medicine: doi:./nm.9

8 Supplementary Figure a pakt Ser7 Akt b Fold vehicle -nm -nm -µm IL- in media c pampk Thr7 AMPK CC protein (AU) nm nm pampk/ampk µm -nm -nm -µm d e pstat Ser77 STAT pg ml - - -LV -LP Plasma TNFα Supplementary figure. mediated activation of AMPK is IL- independent. (a) Immunoblot for pakt Ser7 and total Akt in liver from clamped wild-type () or IL- null () saline-infused (S) (P) or vehicle (V) treated mice. Dotted line indicates that lanes were run on the same gel but were noncontiguous. (b) IL- in media and (c) immunoblots for pampk Thr7 and total AMPK in CC myotubes treated with vehicle () or for min. Quantification of densitometry analyses is shown below representative gels. Data are mean ± s.e.m. of independent experiments. (d) Plasma TNF-α from clamped saline (S) or lipid-infused (L) IL- null () mice treated with vehicle (V) or (P). Data are mean ± s.e.m. -, IL- null saline-infused vehicle, n =. -, IL- null saline-infused, n =. -LV, IL- null lipid-infused vehicle, n =. -LP, IL- null lipid-infused, n =. (e) Immunoblot of pstat ser77 and total STAT in liver of vehicle () or acute -treated genetically obese diabetic db/db mice. P<., P<. calculated using analysis of variance. Nature Medicine: doi:./nm.9

9 Supplementary Figure a c e pg mg ewat Glycemia (mm) GIR (mg kg min ) Pre-clamp glycemia Time (min) Glycemia (mm) pg mg ewat,,, GIR (mg kg min ) Time (min) Glucose infusion rate f pg ml b Insulin (ng ml ) d pg µg protein Pre-clamp Post-clamp Muscle IL- pg ml ng ml 9 Plasma IL- Nature Medicine: doi:./nm.9

10 Supplementary figure. Chronic therapy improves insulin sensitivity in db/db mice. (a) Pre-clamp glycemia (left) and clamp glucose excursion (right), (b) Pre and post-clamp insulin and (c) glucose infusion rate (GIR) (left) and mean GIR for the final min of clamp (right) in vehicle (veh) and chronic -treated 7 week old genetically obese diabetic db/db mice. (d) Skeletal muscle (left) and plasma (right) IL- content, (e) Chemokines and cytokines in epididymal white adipose tissue (ewat) and (f) plasma of clamped vehicle (veh) and chronic -treated genetically obese diabetic db/db mice. Data are mean ± s.e.m., and are representative of mice per group. P<. calculated using student's t-test. Nature Medicine: doi:./nm.9