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1 SUPPLEMENTRY INFORMTION doi: /nature11808 NT Phen Met ICR Oligo FCCP pmpk tmpk Supplemental figure Primary hepatocytes were treated with 250 um Phenformin, 1 mm Metformin 250 um ICR, 100 nm Oligomycin, or 500 nm FCCP for 2 hours, and assayed for total and phospho T172 MPK () or treated with 5 nm glucagon for 15 minutes and subjected to assay for cmp (). 1
2 RESERCH SUPPLEMENTRY INFORMTION NT Glucagon C NT Glucagon NT Glucagon D 100 nm Glucagon (min) NT pmpk tmpk Supplemental Figure 2. -C. Primary hepatocytes isolated from fasted mice were plated in M199 media and treated with the indicated concentrations of metformin and pbs or 100 nm glucagon for 18 hours. Metabolites were extracted with perchloric acid and analyzed by HPLC. D. P rim a ry h e p a to cyte s p late d o ve rn ig h t in M m e d ia w e re tre ate d w ith nm g lu ca g o n for th e in d ica te d tim e s a n d e xtra cts w e re p ro b e d b y w e ste rn b lo t fo r th e p h o sp h o ryla tio n sta tu s of M P K. 2
3 SUPPLEMENTRY INFORMTION RESERCH Supplemental figure 3 FRET Ratio (FRET/CFP) Time post-glucagon (10 nm) 500 µm Phenformin No Phenformin CFP Pre-glucagon 200 sec. 600 sec sec. High FRET Low FRET Supplemental figure Primary hepatocytes were isolated and infected with adenovirus expressing the KR3 FRET-based activity probe.cells were treated with the indicated concentrations of phenformin for 2 hours and images were aquired starting 5 minutes before addition of 10 nm glucaogn.. Representative cells from untreated and phenformin treated cells, pseudocolored to reflect the FRET/CFP ratio.. The calculated FRET/CFP ratios from cytoplasmic ROI (one ROI per cell) for untreated, 250 um, and 500 um phenformin treated cells. W W W. N T U R E. C O M / N T U R E 3
4 RESERCH SUPPLEMENTRY INFORMTION MPK α1/α2 lox/lox 0 C Supplemental figure 4.. P rim a ry h e p a tocyte s w e re p la te d o ve rn ig h t in M m e d ia a n d tre ate d w ith p b s or IC R a n d stim u la te d w ith 5 n M g lu ca g o n for 1 5 m in ute s. E xtra cts w e re a ssa ye d fo r P K a ctivity (P K I-se nsitive k e m ptid e p h o sp h oryla tio n ).. P rim a ry h e p ato cyte s iso la te d fro m M P K α1 a n d α2 flo xe d m ice infe cte d w ith V -T G -C re o r V -T G -G F P 1 4 d a ys p rio r w e re tre ate d w ith th e in d ica te d IC R d o se for 2 h o u rs a n d stim u la te d w ith 5 n M g lu ca g o n for 1 5 m in u te s. c M P le ve ls w e re q u a ntifie d b y E L IS. C. P rim ary h e p a to cyte s w e re p la te d o ve rn ig h t in M m e d ia a n d tre a te d w ith µm IC R fo r 2 h o u rs a n d su b seq u en tly stim u la te d w ith 5 n M g lu ca g o n o r 5 0 µm F o rsk o lin for 1 5 m in ute s. c M P le ve ls w e re q u a ntifie d b y E L IS. 4
5 SUPPLEMENTRY INFORMTION RESERCH Supplemental figure 5.. Primary hepatocytes were incubated with 250 and 500 um phenformin for 2 hours and treated with the indicated glucagon concentrations. Western blots were performed, quantified and three representative blots are shown. 5
6 RESERCH SUPPLEMENTRY INFORMTION C PK-DN Phenformin Supplemental figure Primary hepatocytes were isolated, cultured for 18 hours in the presence or absence of 65 um phenformin and treated for 15 minutes with the indicated concentrations of glucagon () or the cell permeable PK agonist SP-8r-cMPS-M (). Western blots were performed for phospho and total PFKF1, CRE, and the IP3R. Three or more western blots were quantified. C. Primary hepatocytes isolated from mice infected with V-TG-PK-DN or V-TG-GFP control virus 7 days earlier were plated and treated for 1 hour with 250 um phenformin or PS and then subjected to a glucose production assay for 2 hours with 10 nm glucagon and 250 um phenformin. Data represents the mean of 3 of these experiments. 6
7 SUPPLEMENTRY INFORMTION RESERCH Supplemental figure 7.. Primary hepatocytes were incubated with 250 um Phenformin for 2 hours, with or without 20 um IMX, 20 um Cilostamide (PDE3i), or 50 um RO (PDE4i) for the final 30 minutes. Cells were treated with 5 nm glucagon for 15 minutes, lysed, and total cellular cmp assayed.. Primary hepatocytes were incubated with 250 um phenformin for 2 hours, treated wiht 5 nm glucagon or 20 um forskolin for 15 minutes, lysed, and total cellular cmp was assayed. 7
8 RESERCH SUPPLEMENTRY INFORMTION C Supplemental figure 8. Membranes from primary hepatocytes were isolated and assayed for adenylyl cyclase activity in the presence of the indicated concentrations of forskolin and glucagon. ll incubations included 100 um GTP.. C assays were performed at low (40 um) and high (500 um) TP concentrations in the presence of 300 um MP. ssays were stopped with PC before or after 10 minutes at 30 C. Nucleotides were quantified by HPLC. C. C assays were perfomed with 500 um TP in the presence or absence of 300 um MP. Nucleotides were separated by TLC and the radioactivity in the TP fraction was quantified in scintillation solution after removal from the TLC plates. 8
9 SUPPLEMENTRY INFORMTION RESERCH Glucagon Metformin ppk Substrates pmpk MPK pcc CC Supplemental figure 9.. Fed mice were fasted for 1 hour and gavaged with water or 500 mg/kg bw of metformin. One hour later mice were injected intraperitoneally with 2 mg/kg glucagon, and liver tissue was collected 5 minutes later. Western blot analysis was performed, examining reactivity to phospho-pk substrate antibodies, phosphorylated and total MPK, and phosphorylated and total CC.. 18 hour fasted mice were gavaged with 50 mg/kg metformin and 1 hour later blood glucose levels were measured. 9
10 RESERCH SUPPLEMENTRY INFORMTION C D Metformin + E pmpk tmpk ppfkf1 tpfkf1 pip3r tip3r pkt 473 tkt Supplemental figure 10.. Mice fed normal chow or high fat diet for 10 weeks were fasted overnight and blood glucose was tested., and the total hepatic MP levels were quantified. C-E. Mice fed high fat diet for 10 weeks were fasted overnight and gavaged with 250 mg/kg metformin. One hour later blood glucose was assayed (C) and livers were collected and assayed by western blots for total and phosphorylated MPK, PFKF1, IP3R, and KT (D). pkt/total KT is quantified (E). 10
11 SUPPLEMENTRY INFORMTION RESERCH Supplemental Figure 11 Proposed Model Metformin enters the cell and acts on the mitochondria, causing increased MP. Elevated cellular MP levels inhibit membrane bound denylyl Cyclase, causing a reduction in cellular cmp levels and decreased PK activation and target phosphorylation. 11
12 RESERCH SUPPLEMENTRY INFORMTION Supplemental Table 1 denine nucleotides in liver M P (m M ) D P (m M ) T P (m M ) M P / T P N C W ate r ± ± ± ±.0 8 N C M e tform in ± ± ± ±.2 4 H F D W ate r ± ± ± ±.0 5 H F D m etform in ± ± ± ±.0 9 N o rm a l ch o w (N C ) a n d hig h fa t d ie t fe d m ice (H F D ) w e re fa ste d 1 8 h o urs a n d g a va g e d w ith w a te r o r m g /kg m etfo rm in. 1 h o ur late r, live rs w e re co lle cte d, a n d a d en in e n u cle o tid e s w e re e xtra cte d a n d q u a ntifie d b y H P L C. M e a su re m e n ts (µm o le s/m g p rote in ) w e re co n ve rte d to co n ce ntratio n s b ase d o n p rote in re co ve ry a n d h e p a to cyte vo lu m e 2 4. de n o te s p< from w a te r co n tro l. 12
13 SUPPLEMENTRY INFORMTION RESERCH Supplemental Table 2 denine nucleotides in primary hepatocytes M P (µm ) D P (m M ) T P (m M ) M P / T P N T ± ± ± ± µm ± ± ± ± µm ± ± ± ± µm ± ± ± ± µm ± ± ± ± µm ± ± ±, ± P rim a ry h e p a tocyte s w e re iso la te d from fe d m ice a n d tre a te d w ith p h e nfo rm in for 2 h o u rs. d e n in e n u cle o tid e s w e re e xtra cte d a n d q u a n tified b y H P L C. C o n ce ntratio n va lu e s w e re ca lcu la te d a s in T a b le 1. T h e M P va lu e s a n d tho se in fig ure 1 d a re d e rive d fro m th e sam e e xp e rim e n t. d e n ote s p < com p a re d to co ntro ls. N T = N o t tre ate d 13
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