Supplemental Table 1. List of primers used for real time PCR. Primer Sequence Primer Sequence Mouse Pcsk9-F TTGCAGCAGCTGGGAACTT Mouse Scd1-F CATCATTCTCATGGTCCTGCT Mouse Pcsk9-R CCGACTGTGATGACCTCTGGA Mouse Scd1-R CCCAGTCGTACACGTCATTTT Mouse Ldlr-F GAGGAGCAGCCACATGGTAT Mouse Fgb-F TCTTCAGCACGTACGACAGG Mouse Ldlr-R GCTCGTCCTCTGTGGTCTTC Mouse Fgb-R TGTAAAGGCCACCCCAGTAG Mouse Idol-F ACACTGTGTGCTGCGAGAG Mouse Fdft1-F CCAACTCAATGGGTCTGTTCCT Mouse Idol-R ACATGGCTTGTCCATCGAGT Mouse Fdft1-R TGGCTTAGCAAAGTCTGTTCCT Mouse Srebp-1c-F GGCCCGGGAAGTCACTGT Mouse Hnf4α-F GCATGGATATGGCCGACTAC Mouse Srebp-1c-R GGAGCCATGGATTGCACATT Mouse Hnf4α-R CTCGAGGCTCCGTAGTGTTT Mouse Fasn-F GCTGCGGAAACTTCAGGAAAT Mouse Apoa1-F TATGTGGATGCGGTCAAAGA Mouse Fasn-R AGAGACGTGTCACTCCTGGACTT Mouse Apoa1-R CAGAAGTCCCGAGTCAATGG Mouse Srebp-2-F GCGTTCTGGAGACCATGGA Mouse Hnf1α-F TTCTAAAGCTGAGCCAGCTGCAGACG Mouse Srebp-2-R ACAAAGTTGCTCTGAAAACAAATCA Mouse Hnf1α-R GCTGAGGTTCTCCGGCTCTTTCAGA Mouse Hmgcr-F CTTGTGGAATGCCTTGTGATTG Mouse Acc-F TGACAGACTGATCGCAGAGAAAG Mouse Hmgcr-R AGCCGAAGCAGCACATGAT Mouse Acc-R TGGAGAGCCCCACACACA Mouse Fdps-F ATGGAGATGGGCGAGTTCTTC Mouse Serpina1-F CAGGATAGGGGGCATAGACA Mouse Fdps-R CCGACCTTTCCCGTCACA Mouse Serpina1-R GACCTCTCCGGAATCACAGA Mouse Tbp-F ACCCTTCACCAATGACTCCTATG Mouse Albumin-F GCTGAGACCTCCACCTTCCA Mouse Tbp-R TGACTGCAGCAAATCGCTTGG Mouse Albumin-R CTTGTGCTTCACCAGCTCAG Mouse 18s-F GTAACCCGTTGAACCCCATT Mouse Fasn-Chip-F1 CCAAGCTGTCAGCCCATGT Mouse 18s-R CCATCCAATCGGTAGTAGCG Mouse Fasn-Chip-R1 CGTCTCTCTGGCTCCCTCTA Mouse Pcsk9-Chip CAGAAGGCTTCCACCTTCAC Mouse Pcsk9-Chip GCTCAGTCCTCTAGCCTCAGAA
Supplemental Table 2. Laboratory reference ranges for measured patient parameters, as per the NIH Clinical Center laboratory. Parameter Reference Range Body Mass Index (kg/m 2 ) 18.5 24.9 (ng/ml) 3.3-18.3 (BMI=22) Fasting Plasma Glucose (mg/dl) 74 100 Fasting Plasma Insulin (µu/ml) 2.6 24.9 2-hr OGTT Plasma Glucose (mg/dl) < 140 Hemoglobin A1C (%) 4.0 6.0 Total Cholesterol (mg/dl) <200 LDL Cholesterol (mg/dl) 60-130 HDL Cholesterol (mg/dl) 40-60 Triglycerides (mg/dl) <150
Supplemental Table 3. Correlation between change in PCSK9 and metabolic parameters upon leptin treatment. Change in Clinical Variable Correlation with Change in ln(pcsk9) P value Body Mass Index, kg/m 2 9 0.12, ng/ml 6 0.25 Fasting Plasma Glucose, mg/dl -0.02 0.97 Fasting Plasma Insulin, µu/ml 2 0.23 2-hr OGTT Plasma Glucose, mg/dl 0.33 0.47 Hemoglobin A1C, % -0.37 0.48 OGIS, ml/min/m 2-0.25 8 Total Cholesterol, mg/dl 0.41 0.31 LDL Cholesterol, mg/dl 0.75 0.03 HDL Cholesterol, mg/dl 8 0.17 Triglycerides, mg/dl -0.17 0.72 Pearson correlation coefficient. Testing for zero correlation. Bold denotes statistical significance.
% of Total Plasma Cholesterol 100% 80% 60% Big HDL HDL LDL VLDL 40% 20% 0% Supplemental Figure 1. Effects of leptin on the distribution of cholesterol among the different lipoprotein fractions in male ob/ob mice. Equal volumes of non-fasted plasma from leptin- or vehicle-treated mice were pooled (n=4-5/group) and were subjected to size-exclusion liquid chromatography. The proportion of cholesterol in each of the fractions was calculated. Similar results were obtained in a second cohort.
Relative Enrichment Relative Enrichment A. Pcsk9 promoter B. Fasn promoter 8 15 6 4 2 10 5 0 IgG Pol II 0 IgG Pol II Supplemental Figure 2. Effects of leptin on PolII occupancy of the Pcsk9 promoter in male ob/ob mice. Male ob/ob mice were treated with leptin or vehicle as described in Fig. 1. Equal aliquots of liver from two mice per group were pooled and subjected to chromatin immunoprecipitation with antibodies against Pol II or control IgG, and the relative enrichment compared to input was calculated. Analysis of the Pcsk9 (A) and Fasn (B) promoters is shown. Data are presented as mean + SEM of two independent experiments. P value 0.05 compared to vehicle-treated mice, Student s t-test.
Relative mrna 1.0 0.0 Supplemental Figure 3. Effects of leptin on mrna of Hnf1α and its targets in male ob/ob mice. Mice were treated as detailed in Figure 1. Gene expression was measured by real time PCR, normalized to Tbp, and expressed relative to vehicle-treated mice. Data represent mean + SEM, and are representative of two independent experiments. P value 0.05 compared to vehicle treatment, Student s t-test; n=4-5/group.
Relative mrna Relative mrna Relative mrna A. Pcsk9 mrna B. 1 0 1.0 0.0 Srebp1-c Acc Fasn Scd1 C. 1.0 0.0 Srebp-2 Hmgcr Squalene Fdft1 Synthase Supplemental Figure 4. Effects of leptin on Pcsk9 mrna and SREBP target genes in female ob/ob mice. Hepatic gene expression was measured by real time PCR, normalized to 18s, and expressed relative to vehicle-treated mice. Data are pooled from two independent experiments; mean and SEM are shown. P value 0.05 versus vehicle-treated mice; n = 12/group.