AdPLA ablation increases lipolysis and prevents obesity induced by high fat feeding or leptin deficiency

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1 AdPLA AdPLA ablation increases lipolysis and prevents obesity induced by high fat feeding or leptin deficiency Kathy Jaworski, Maryam Ahmadian, Robin E. Duncan, Eszter Sarkadi-Nagy, Krista A. Va rady, Marc K. Hellerstein, Hui-Young Lee, Va rman T. Samuel, Gerald I. Shulman, Kee-Hong Kim, Sarah de Va l, hulho Kang and Hei Sook Sul kb Arm A Arm Arm Targeting vector Neo loxp 1 loxp 2 loxp 3 TK homologous recombination Targeted allele Neo loxp 1 loxp 2 loxp 3 HZ HZ 2.6 kb 2 kb 861 bp 230 bp 7 6' Apa 1 Exon 3 wt allele 6 Apa 1 re recombination loxp mutant allele 6' D Epi WAT Ren WAT AdPLA-372bp ontr-172bp GAPDH 411 bp Apa ' AdPLA ont-172bp Supplemental Fig. 1. Generation of AdPLA null mice. (A) Generation of AdPLA null mice using a re-loxp strategy. The AdPLA targeting vector was constructed using peasy Flox (pef) plasmid. A neo gene with flanking loxp sites was inserted into intron 2 and the thrid loxp site was inserted into intron 3. TK gen was inserted at the 3 end for negative selection. 1-4 designates exons. The final targeting vector was used for homologous recombination in ES cells. ES cells were cultured on gelatin-coated dishes with 1000 units/ml recombinant murine LIF (ESGRO, hemicon) After linearization of the AdPLA-targeting vector with NotI, 50 μg DNA was electroporated into 8 x /SVj E14 ES cells. G418- and ganclicovir-resistant colonies were picked and screened for homologous recombination events occurring between loxp1 and loxp3 by PR using external forward primers upstream to the AdPLA. fragment and reverse primers inside the neomycin gene. Positive colonies were identified, expanded and electroporated with pm-re plasmid. G418 dying colonies were identified and screened for homologous recombination by PR performed with a primer pair flanking exon 3. () Genotyping of mice by PR using primers spanning exon 3., wild type; HZ, heterozygote;, knockout mice. Primer sequences in exon 32 of the mouse fatty acid synthase gene, producing a 172 bp fragment, were used as an internal control. () RT- PR analysis for AdPLA, using RNA form renal fat of and mice. (D) Western blotting showing an absence of AdPLA in epididymal (epi) and renal (ren) WAT of mice. Figure S1

2 P value Red blood cells (x10 6 /mm 3 ) 9.46 ± ± platelets (x10 3 /mm 3 ) 968 ± ± white blood cells (x10 6 /mm 3 ) 10.5 ± ± neutrophil (%) 24.2 ± ± lymphocytes (%) 70.0 ± ± monocyte (%) 4.8 ± ± eosinophil (%) 1.0 ± ± basophil (%) 0 0 na D E lood measure P value Total bilirubin (mg/dl) 0.26 ± ± Aspartate Aminotransferase (U/l) a ± ± Alanine Aminotransferase (U/l) b 45.2 ± ± Alkaline Phosphatase (U/l) 36 ± 1 50 ± Albumin (g/dl) 3.84 ± ± Lactate dehydrogenase (U/l) 1181 ± ± Urea Nitrogen (mg/dl) 25.4 ± ± Sodium (mmol/l) 150 ± ± Potassium (mmol/l) 8.60 ± ± hloride (mmol/l) 83.6 ± ± Phosphorus (mg/dl) 8.90 ± ± a =Aspartate Aminotransferase (AST)= serum glutamic oxaloacetic transaminase (SGOT) b =Alanine Aminotransferase (ALT)= serum glutamic pyruvic transaminase (SGPT) Supplemental Fig. 2. lood profile and markers of inflammation. (A) lood cell counts. () RT-qPR analysis of levels of inflammatory cytokines and macrophage markers in adipose tissue from and AdPLA null () mice. () Serum Il6 and TNF concentrations in and mice. (D) mrna levels of F4/80+ and AdPLA in isolated peritoneal macrophages determined by RT-qPR. (E) Hematological assessment of electrolytes, and markers of liver and renal function. Figure S2

3 Glucose (mg dl -1 ) * ** ** ** D E Liver (ORO) F H P-IRS1-Ser307 IRS-1 total P-IRS1-Ser307 IRS-1 total ontrol ontrol HFD G P-AMPK-Thr172 AMPK total P-AMPK-Thr172 AMPK total ontrol ontrol HFD AO Supplemental Fig. 3. Insulin resistance and ectopic TAG storage in AdPLA null mice. (A) Glucose (GTT) and insulin (ITT) tolerance in 18 wk old male and mice fed a (n=11) () glucose infusion rate (GINF) and () plasma glucose during the hyperinsulinemic euglycemic clamp study in 12 wk old male and mice fed a HFD. (D) Top panel: Livers from16 wk old male mice fed a HFD were stained with hematoxylin and eosin (H&E). ottom panel: cryostat sections of frozen livers from 16 wk old male mice fed a HFD were stained with Oil red O. (E) ryostat sections of frozen soleus muscles from 16 wk old male, HFD fed and mice stained with Oil red O. (F) Immunoblot analysis of total and phosphorylated IRS-1 in gastrocnemius muscle. (G) Top panel: Immunoblot analysis of total AMPK or phosphorylated AMPK in gastrocnemius muscle from 18 wk old male and mice fed a HFD. ottom panel: RT-PR for acyl-oa oxidase (AO) using RNA from gastrocnemius skeletal muscle from 18-wk old male and mice fed a HFD. (H) Oxidation of [U- 14 ]-palmitate in homogenates of skeletal muscle from wild type or AdPLA null (n=4). Results are means ± SEM, *P<0.05. Results are means ± SEM, * P< 0.05, *** P< Figure S3

4 Supplemental Fig. 4. TAG clearance. (A) Serum TAG clearance. We followed serum TAG levels of overnight fasted mice for 5h following oral gavage with 400 l peanut oil (n=6), as described previously (olumbo et al., J. iol. hem. (278) , 2003). () Intestinal TAG absorbance and secretion. Serum TAG levels were measured in mice given a tail vein injection of WR1339 (to inhibit lipoprotein lipase) prior to gavage with 400 l peanut oil (n=6). () Lipoprotein lipase mrna levels in the liver, adipose and skeletal muscle of and mice, normalized to GAPDH, as determined by RT-qPR (n=3). Values are means ± SEM, *P<0.05, **P<0.01. Figure S4

5 Sequence Homology between murine and human AdPLA homologs. Mm 1-LAPIPEPKPGDLIEIFRPMYRHWAIYVGDGYVIHLAPPSEIAGAGAASIMSALTDKAIV H 1-RAPIPEPKPGDLIEIFRPFYRHWAIYVGDGYVVHLAPPSEVAGAGAASVMSALTDKAIV Mm61-KKELLHVAGKDKYQVNNKHDEEYTPLPLSKIIQRAERLVGQEVLYRLTSENEHFVNEL Hs61-KKELLYDVAGKYQVNNKHDDKYSPLPTKIIQRAEELVGQEVLYKLTSENEHFVNEL Mm121-RYGVPRQVRDAVKAVGIAGVGLAALGLVGVMLSRNKKQKQ-162 Hs121-RYGVARQVRDVIIAASVAGMGLAAMSLIGVMFSRNKRQKQ-162 Supplementary Figure 5. Murine and human sequences share 83.3% homology. Figure S5

6 ody weight (g) ± ± 1.02 *** arcass weight (g) ± ± 0.31 *** Water (%) ± ± 2.18 *** Lipid (%) ± ± 1.77 *** Lean Tissue (%) ± ± 0.64 *** Liver weight (g) 1.50 ± ± 0.35 ** Liver TAG content (mg/g) ± ± 4.77 *** Supplemental Table 1. ody composition data for male and mice age 36 wks fed a HFD (n=6). Table S1

7 - - -HFD -HFD ob/ob- Glucose (mg/dl) ± ± ± ± ± * d ± 50.21** Insulin (ng/ml) 0.37 ± 0.10 Triglyceride ± (mg/dl) FFA (mmol/l) 1.03 ± 0.09 Adiponectin ± (µg/ml) 0.69 Leptin (ng/ml) ± ± 0.09* ± ± 0.10* 3.21 ± 0.61*** 2.02 ± 0.77*** 1.76 ± ± 0.60** ± ± * 1.24 ± 0.80 ± ** 9.8 ± ± 0.37** ± 9.29 ± *** 9.37 ± ± ± ± 2.19 _ ± ± ± ± 0.69*** _ Supplemental Table 2. Serum Parameters. Serum parameters were measured in overnight-fasted 18 wk old male and mice fed a (n=11) or a HFD (n=15-21) and 16 wk old ob/ob and d mice fed a (n=8). lood samples collected by cardiac puncture from overnight fasted mice were allowed to clot and centrifuged at 1000 g for 10 min. Fasting glucose was measured by glucometer. Serum triglycerides were analyzed with Infinity Triglyceride reagent. Serum free fatty acids were determined with the NEFA kit (Wako). Serum insulin, leptin and adiponectin levels were determined using enzyme linked immunosorbent assay kits (rystal hem and -ridge). Results are means ± SEM, * P <0.05, ** P<0.01, *** P< omparisons are within diet groups between and mice and between ob/ob and d mice on a. Table S2