Supplementary Table 1. RT-qPCR primers for CD3, PPARg and CEBP. Assay Forward Primer Reverse Primer 1A CAT TTG TGG CCT TGT GCT CTT TGA TGA GTC ACA GAA AGA ATC AAT TC 1B AGG AAA TGA ACT GAT GAG TCA CAG A ATG TTG GAG CAT TTG ATT GAA AAA T 1C CAT CTC CGA AAG CAA GCT CTT CTA AGG AAA TGA ACT GAT GAG TCA CAG A 1D TAG TCC ATC CAA AAG CAA GG CCA GCT CCT TTG AGA TTT GA 1E CTG TAT AAA TAC TCC TAA GAA GTT AT CAG GAA ATG AAC TGA TGA GTC ACA G 1F GGT TAC AAG CAT GAC TTC TAT TAA AC AAT GAA CTG ATG AGT CAC AGA AAG A Total CD3* GAG ACC TGC TTA TCC AGA ACA CAA T TTC TGT GCC TGT TTT AAC CCA ATT TTT PPARg ATA AAG TCC TTC CCG CTG A CAC CTC TTT GCT CTG CTC CT CEB/Pa AGG GTC TCT AGT TCC ACG CC CAA GGG GAA GCC CAG CCT ATA *Primers specific to exon 3 and exon were used for total CD3 mrna. Supplementary Table. Conserved regulatory elements in the promoters of CD3. Exon 1A 1B 1C Elements within 1kb 5 of the transcription start site MEF, SpI-1/PU.1, TEF1 MEF, PPARα, TEF-1, GR, IFN, NF1, NFK, CK-, PPAR, SRE, SpI-1/PU.1, p3 PPARγ, MEF, PPARα, C/EBP, NFK MEF-Myocyte enhancer factor; SPI-1/PU.1- Macrophage and B cell specific transcriptional activator; TEF1- transcriptional enhancer factor; PPAR - peroxisome proliferator-activated receptor alpha; GR - glucocorticoid response response element; IFN -gamma interferon, NF1- nuclear factor -1; NFK - nuclear factor kappa ; CK- cytokine-specific sequences ; SREsterol-responsive element; C/EBP- CCAAT/enhancer binding protein. 1 American Diabetes Association. Published online at http://care.diabetesjournals.org/lookup/suppl/doi:1.337/dc13-835/-/dc1
Supplementary Table 3. Demographics for subjects genotyped for rs1717 and grouped based on intrahepatic TG content. IHTG <5% IHTG >5% Mean SEM Mean SD Gender (M/F) 7/18 8/ Age (yrs.).. BMI (kg/m ) 3.3.7 37.3.9 FM (kg) 1.7 1.8.5 1.9 FFM (kg) 55.8. 1.,1 IHTG %.8.3.3 1.9 Glucose (mg/dl) 93.7 1.3 95.3 1. Insulin ( U/L) 8.5 1.1 19.9 1.5 Insulin stimulation (%) of glucose Rd a 99.3 3. 18.5 13. Total Cholesterol (mg/dl) 17.3 7. 15. 5. HDL (mg/dl)..1..5 LDL (mg/dl) 13.. 99.5 3.3 Total Triglycerides (mg/dl 1.. 9.9 5. Values are mean ± SEM; FM-fat mass; FFM-fat free mass; IHTG-Intrahepatic triglycerides; a Percent increase in glucose disposal rate during insulin infusion. 1 American Diabetes Association. Published online at http://care.diabetesjournals.org/lookup/suppl/doi:1.337/dc13-835/-/dc1
Supplementary Figure 1. Schematic of the metabolic studies protocol. Subjects were admitted to the Clinical Research Unit (CRU) at Washington University School of Medicine on the evening before the clamp procedure at visit. At 5 h the following morning, after a 1 h overnight fast, a -stage hyperinsulinemic-euglycemic clamp was started and continued for 9 hours. Tissue biopsies were obtained during the basal period. In stage 1, insulin was infused at mu/m body-surface area (3-h) and in stage at 5 mu/m body-surface area (-9h). To determine hepatic, skeletal muscle and adipose tissue insulin sensitivity [,-H] glucose, [,-H]palmitate and % dextrose enriched with labeled glucose were infused. Blood samples were obtained before beginning the tracer infusion to determine background plasma glucose and palmitate tracer-to-tracee ratios (TTRs), and every 1 min during the final 3 min of the basal period and stages 1 and of the clamp procedure to determine glucose, FFA and insulin concentrations and substrate kinetics. 1 American Diabetes Association. Published online at http://care.diabetesjournals.org/lookup/suppl/doi:1.337/dc13-835/-/dc1
Supplementary Figure. Tissue distribution of the minor CD3 alternative transcripts. A) Relative expression of the minor transcripts (1D-F) in commercially available human tissues. B) Transcripts in THP-1 monocytes before and after differentiation into macrophages. C) SGBS human derived preadipocytes before and after differentiation into adipocytes. Bars represent means ± s.e.m. 1 American Diabetes Association. Published online at http://care.diabetesjournals.org/lookup/suppl/doi:1.337/dc13-835/-/dc1
Supplementary Figure 3. Adipose tissue CD3 transcript 1C predicts systemic and tissue insulin sensitivity. Linear regression was used to model the relationship of adipose tissue 1C expression to measures of insulin sensitivity derived from hyperinsulinemic euglycemic clamps. A) Intrahepatic TG, IHTG. B) Hepatic InsulinSensitivity Index (HISI). C) Insulin stimulated muscle glucose disposal, and D) Insulinmediated suppression of Palmitate Ra (n=3). Correlation coefficients and p-values arepresented in Table. A. 5 IHTG (%) 3 1 B.. Log (HISI) 1.5 1..5 C.. 5 Insulin stimulated muscle glucose uptake (%) 3 1 D. Insulin mediated suppression of palmitate(%) 1 8.... Adipose 1C (AU) 1 American Diabetes Association. Published online at http://care.diabetesjournals.org/lookup/suppl/doi:1.337/dc13-835/-/dc1
Supplementary Figure. Adipose Tissue Exons 1C and 1B transcript levels correlate with mrna levels of known transcription regulators, CEBP-A and PPARG.Expression for exons 1B, 1C and total CD3 mrna was related to that of CEBP-Α(A-C) and PPAR-G (D-F) in adipose tissue samples from obese subjects. The data showrelative expression of the transcripts normalized to the human RPLPO gene and arerepresentative of duplicate determinations from at least two experiments. A.. p<.1 D.. p=.3 Adipose Exon 1C...... B. E. 1 p=.1 1 p=. Adipose Exon 1B 8 8 C. p=.3 F. p=.8 Adipose Total CD3.....8 Adipose CEBPA..5 1. 1.5 Adipose PPARG 1 American Diabetes Association. Published online at http://care.diabetesjournals.org/lookup/suppl/doi:1.337/dc13-835/-/dc1