#METABOLOMICS WORKBENCH manikana_20251010_114021 DATATRACK_ID:6551 STUDY_ID:ST004286 ANALYSIS_ID:AN007126 PROJECT_ID:PR002703 VERSION 1 CREATED_ON October 13, 2025, 2:19 pm #PROJECT PR:PROJECT_TITLE Limitations in PPAR⍺-dependent mitochondrial programming restrain the PR:PROJECT_TITLE differentiation of human stem cell-derived β cells PR:PROJECT_SUMMARY Pluripotent stem cell (SC)-derived islets offer hope as a renewable source for PR:PROJECT_SUMMARY β cell replacement for type 1 diabetes (T1D), yet functional and metabolic PR:PROJECT_SUMMARY immaturity may limit their long-term therapeutic potential. Here, we show that PR:PROJECT_SUMMARY limitations in mitochondrial transcriptional programming impede the formation of PR:PROJECT_SUMMARY SC-derived β (SC-β) cells. Utilizing transcriptomic profiling, assessments of PR:PROJECT_SUMMARY chromatin accessibility, mitochondrial phenotyping, and lipidomics analyses, we PR:PROJECT_SUMMARY observed that SC-β cells exhibit reduced oxidative and mitochondrial fatty acid PR:PROJECT_SUMMARY metabolism compared to primary human islets that are related to limitations in PR:PROJECT_SUMMARY key mitochondrial transcriptional networks. Surprisingly, we find that PR:PROJECT_SUMMARY reductions in glucose-stimulated mitochondrial respiration in SC-islets were not PR:PROJECT_SUMMARY associated with alterations in mitochondrial mass, structure, or genome PR:PROJECT_SUMMARY integrity. In contrast, SC-islets show limited expression of targets of PPAR⍺, PR:PROJECT_SUMMARY which regulate mitochondrial programming, yet whose functions in β cell PR:PROJECT_SUMMARY differentiation are unknown. Importantly, treatment with WY14643, a potent PR:PROJECT_SUMMARY PPAR⍺ agonist, induced expression of mitochondrial targets, improved insulin PR:PROJECT_SUMMARY secretion, and increased the formation of SC-β cells both in vitro and PR:PROJECT_SUMMARY following transplantation. Thus, PPAR⍺-dependent mitochondrial programming PR:PROJECT_SUMMARY promotes the differentiation of SC-β cells and may be a promising target to PR:PROJECT_SUMMARY improve β cell replacement efforts for T1D. PR:INSTITUTE University of Michigan PR:DEPARTMENT Division of Metabolism, Endocrinology and Diabetes and Department of Internal PR:DEPARTMENT Medicine PR:LABORATORY Scott A. Soleimanpour PR:LAST_NAME Arnipalli PR:FIRST_NAME Manikanta PR:ADDRESS 3815-301B Green Brier apt, Ann Arbor, Michigan, 48105, USA PR:EMAIL manikana@umich.edu PR:PHONE 734-272-8779 #STUDY ST:STUDY_TITLE Limitations in PPAR⍺-dependent mitochondrial programming restrain the ST:STUDY_TITLE differentiation of human stem cell-derived β cells ST:STUDY_SUMMARY Pluripotent stem cell (SC)-derived islets offer hope as a renewable source for ST:STUDY_SUMMARY β cell replacement for type 1 diabetes (T1D), yet functional and metabolic ST:STUDY_SUMMARY immaturity may limit their long-term therapeutic potential. Here, we show that ST:STUDY_SUMMARY limitations in mitochondrial transcriptional programming impede the formation of ST:STUDY_SUMMARY SC-derived β (SC-β) cells. Utilizing transcriptomic profiling, assessments of ST:STUDY_SUMMARY chromatin accessibility, mitochondrial phenotyping, and lipidomics analyses, we ST:STUDY_SUMMARY observed that SC-β cells exhibit reduced oxidative and mitochondrial fatty acid ST:STUDY_SUMMARY metabolism compared to primary human islets that are related to limitations in ST:STUDY_SUMMARY key mitochondrial transcriptional networks. Surprisingly, we find that ST:STUDY_SUMMARY reductions in glucose-stimulated mitochondrial respiration in SC-islets were not ST:STUDY_SUMMARY associated with alterations in mitochondrial mass, structure, or genome ST:STUDY_SUMMARY integrity. In contrast, SC-islets show limited expression of targets of PPAR⍺, ST:STUDY_SUMMARY which regulate mitochondrial programming, yet whose functions in β cell ST:STUDY_SUMMARY differentiation are unknown. Importantly, treatment with WY14643, a potent ST:STUDY_SUMMARY PPAR⍺ agonist, induced expression of mitochondrial targets, improved insulin ST:STUDY_SUMMARY secretion, and increased the formation of SC-β cells both in vitro and ST:STUDY_SUMMARY following transplantation. Thus, PPAR⍺-dependent mitochondrial programming ST:STUDY_SUMMARY promotes the differentiation of SC-β cells and may be a promising target to ST:STUDY_SUMMARY improve β cell replacement efforts for T1D. ST:INSTITUTE University of Michigan ST:DEPARTMENT Division of Metabolism, Endocrinology and Diabetes and Department of Internal ST:DEPARTMENT Medicine ST:LABORATORY Scott A. Soleimanpour ST:LAST_NAME Arnipalli ST:FIRST_NAME Manikanta ST:ADDRESS 3815-301B Green Brier apt, Ann Arbor, Michigan, 48105, USA ST:EMAIL manikana@umich.edu ST:PHONE 734-272-8779 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - S1 11_16_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S1 11_16_22.d SUBJECT_SAMPLE_FACTORS - S1 9_14_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S1 9_14_22.d SUBJECT_SAMPLE_FACTORS - S1 12_17_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S1 12_17_22.d SUBJECT_SAMPLE_FACTORS - S2 11_18_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S2 11_18_22.d SUBJECT_SAMPLE_FACTORS - S2 9_16_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S2 9_16_22.d SUBJECT_SAMPLE_FACTORS - S2 12_19_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S2 12_19_22.d SUBJECT_SAMPLE_FACTORS - S3 11_20_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S3 11_20_22.d SUBJECT_SAMPLE_FACTORS - S3 9_14_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S3 9_14_22.d SUBJECT_SAMPLE_FACTORS - S3 9_18_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S3 9_18_22.d SUBJECT_SAMPLE_FACTORS - S4 11_24_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S4 11_24_22.d SUBJECT_SAMPLE_FACTORS - S4 9_18_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S4 9_18_22.d SUBJECT_SAMPLE_FACTORS - S4 9_22_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S4 9_22_22.d SUBJECT_SAMPLE_FACTORS - S5 12_01_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S5 12_01_22.d SUBJECT_SAMPLE_FACTORS - S5 9_25_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S5 9_25_22.d SUBJECT_SAMPLE_FACTORS - S5 9_29_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S5 9_29_22.d SUBJECT_SAMPLE_FACTORS - S6 10_04_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S6 10_04_22.d SUBJECT_SAMPLE_FACTORS - S6 10_08_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S6 10_08_22.d SUBJECT_SAMPLE_FACTORS - S6 12_10_22 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=S6 12_10_22.d SUBJECT_SAMPLE_FACTORS - 10_05_22 HI_1 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=10_05_22 HI_1.d SUBJECT_SAMPLE_FACTORS - 10_07_22 HI_2 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=10_07_22 HI_2.d SUBJECT_SAMPLE_FACTORS - 10_15_22 HI_3 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=10_15_22 HI_3.d SUBJECT_SAMPLE_FACTORS - 10_28_22 HI_4 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=10_28_22 HI_4.d SUBJECT_SAMPLE_FACTORS - 10_28_22 HI_5 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=10_28_22 HI_5.d SUBJECT_SAMPLE_FACTORS - Vehicle 10_3_24 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=Vehicle 10_3_24.d SUBJECT_SAMPLE_FACTORS - WY 10_3_24 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=WY 10_3_24.d SUBJECT_SAMPLE_FACTORS - WY 11_5_24 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=WY 11_5_24.d SUBJECT_SAMPLE_FACTORS - Vehicle 10_31_24 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=Vehicle 10_31_24.d SUBJECT_SAMPLE_FACTORS - Vehicle 11_5_24 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=Vehicle 11_5_24.d SUBJECT_SAMPLE_FACTORS - WY 10_31_24 Sample type:Human | Sample source:Pancreas RAW_FILE_NAME(Raw file name)=WY 10_31_24.d #COLLECTION CO:COLLECTION_SUMMARY Cells were cultured according to Hogrebe et al4. On the day of sample CO:COLLECTION_SUMMARY collection, samples were incubated in MCDB 131 with 10.5 g BSA, 5.2 mL GlutaMAX, CO:COLLECTION_SUMMARY 5.2 mL P/S, 5 mg heparin, 5.2 mL MEM nonessential amino acids (Corning, CO:COLLECTION_SUMMARY 20–025-CI), 84 μg ZnSO4 (MilliporeSigma, 10883), 523 μL Trace Elements A CO:COLLECTION_SUMMARY (Corning, 25–021-CI), and 523 μL Trace Elements B (Corning, 25–022-CI) and CO:COLLECTION_SUMMARY either 5.5 mM or 20 mM glucose for 3 hr. CO:SAMPLE_TYPE Pancreas #TREATMENT TR:TREATMENT_SUMMARY Treatment not applied. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Media were removed, and Acyl carnitines were extracted with 500 μL solvent SP:SAMPLEPREP_SUMMARY (8:1:1 ratio of methanol/Chloroform/water) containing stable isotope-labeled SP:SAMPLEPREP_SUMMARY internal standards. The resulting homogenate was sonicated on ice for 10 sec. SP:SAMPLEPREP_SUMMARY Samples were centrifuged at 15,000 rpm for 10 mins and 4°C. Supernatant is SP:SAMPLEPREP_SUMMARY removed. Dry down at 45°C. Samples were reconstituted in 50 μL of 5 mM SP:SAMPLEPREP_SUMMARY Ammonium Acetate, filtered and 4 μL were injected for analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY an Agilent 6410 triple quadrupole MS system equipped with an Agilent 1200 LC CH:CHROMATOGRAPHY_SUMMARY System and an ESI source was utilized. Metabolite separation was achieved using CH:CHROMATOGRAPHY_SUMMARY gradient elution on a reverse phase XBridge C18 Column (50 mm × 2.1 mm, 2.5 CH:CHROMATOGRAPHY_SUMMARY μm, Waters, Milford, MA, USA) with the corresponding guard column (5 mm x 2.1 CH:CHROMATOGRAPHY_SUMMARY mm, 1.7 μm) maintained at 40°C. LC vials were maintained at 4°C in a CH:CHROMATOGRAPHY_SUMMARY thermostatic autosampler, and the injection volume was set at 4 µL. The mobile CH:CHROMATOGRAPHY_SUMMARY phase consisted of solvent A, 5 mM Ammonium Acetate, and solvent B, CH:CHROMATOGRAPHY_SUMMARY Acetonitrile. The flow rate was 0.25 mL/min. The gradient elution program was as CH:CHROMATOGRAPHY_SUMMARY follows: 0–8.50 min, 2% B; 1.5–9 min, linear gradient from 2% to 50% B; CH:CHROMATOGRAPHY_SUMMARY 9–14 min, linear gradient from 50% to 95% B; hold at 95% B for 3 min. The flow CH:CHROMATOGRAPHY_SUMMARY rate was 300 μL/min. Acylcarnitine species were each detected by their CH:CHROMATOGRAPHY_SUMMARY characteristic LC retention time in the MRM mode following ESI and comparing CH:CHROMATOGRAPHY_SUMMARY relative areas with those of corresponding standards. Concentrations of CH:CHROMATOGRAPHY_SUMMARY carnitine, acetylcarnitine (C2), propionylcarnitine (C3), butyrylcarnitine (C4), CH:CHROMATOGRAPHY_SUMMARY isovalerylcarnitine (C5), hexanoylcarnitine (C6), octanoylcarnitine (C8), CH:CHROMATOGRAPHY_SUMMARY myristoylcarnitine (C14), palmitoylcarnitine (C16), and oleoylcarnitine (C18) CH:CHROMATOGRAPHY_SUMMARY were calculated by ratios of peak areas of known concentrations of stable CH:CHROMATOGRAPHY_SUMMARY isotopically-labeled analogs. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 6490 CH:COLUMN_NAME Waters XBridge C18 (50 x 2.1 mm, 3 μm) CH:SOLVENT_A 100% Water; 5 mM Ammonium Acetate CH:SOLVENT_B 100% Acetonitrile CH:FLOW_GRADIENT The gradient elution program was as follows: 0–8.50 min, 2% B; 1.5–9 min, CH:FLOW_GRADIENT linear gradient from 2% to 50% B; 9–14 min, linear gradient from 50% to 95% B; CH:FLOW_GRADIENT hold at 95% B for 3 min. CH:FLOW_RATE 300 μL/min CH:COLUMN_TEMPERATURE 40°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6490 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The gas temperature was set at 200°C, the drying gas flow at 14 L/min, the MS:MS_COMMENTS nebulizer at 20 psi, the sheath gas temperature at 250°C, and the sheath gas MS:MS_COMMENTS flow at 11 L/min. The VCap at 3,000 V. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples S1 11_16_22 S1 9_14_22 S1 12_17_22 S2 11_18_22 S2 9_16_22 S2 12_19_22 S3 11_20_22 S3 9_14_22 S3 9_18_22 S4 11_24_22 S4 9_18_22 S4 9_22_22 S5 12_01_22 S5 9_25_22 S5 9_29_22 S6 10_04_22 S6 10_08_22 S6 12_10_22 10_05_22 HI_1 10_07_22 HI_2 10_15_22 HI_3 10_28_22 HI_4 10_28_22 HI_5 Vehicle 10_3_24 WY 10_3_24 WY 11_5_24 Vehicle 10_31_24 Vehicle 11_5_24 WY 10_31_24 Factors Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas Sample type:Human | Sample source:Pancreas L-CARNITINE TRIMETHYL-D9 (C0:0) 9184238 7262518 13377580 14239401 7290058 7531950 22169776 19499335 14622127 8711102 17041923 11461470 12827873 13207282 11064924 10539379 2770984 15112118 1353115 2510805 2625352 8656093 817706 ND ND ND ND ND ND L-Carnitine (C0:0) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 809137 150452 294353 134285 983339 ND ND ND ND ND ND Acetylcarnitine (C2:0) 102854 200576 128055 104449 112126 294667 111079 173980 100125 106125 100588 30154 150285 210033 136819 3331725 583926 740680 213637142 42076656 63519918 9517296 173259392 440086.00 434255.00 490733.00 340817.00 454980.00 228479.00 O-ACETYL (N-METHYL-D3) (C2:0) 201041302 187353638 205128073 210383424 190000986 185707014 197999992 197845838 193453865 200561279 188676084 71086990 190053701 192029290 190244237 190251616 153048681 194524456 162779993 183594198 185994404 192831727 146254924 71472003.00 68853172.00 75802058.00 89204317.00 86879084.00 93219017.00 O-PROPIONYL (N-METHYL-D3) (C3:0) 212151535 187821879 222499761 216806650 191160993 213664537 203219074 201859131 192959172 208447118 190883394 30952890 189416709 194147910 188823230 184859123 139876016 196197955 189117143 191690262 187107445 189796273 165524049 115788842.00 111514804.00 115348719.00 96771205.00 109421535.00 104685657.00 Propionylcarnitine (C3:0) 40782 74625 50831 29239 47457 43129 42925 66161 47481 57997 51278 11702 238157 95952 39672 1473661 192105 1175693 65200180 3984916 8043064 3729562 42226292 671572.00 486130.00 947024.00 308203.00 535611.00 289403.00 Butyrylcarnitine (C4:0) 36109 38853 42359 40800 37165 27443 66918 63577 54131 124689 39654 57705 70711 86153 31311 768035 99682 277504 21319616 861731 751595 302625 10641211 1598573.00 1443917.00 3557413.00 1300372.00 2065626.00 1229131.00 O-BUTYRYL (N-METHYL-D3) (C4:0) 71940696 69669228 79711757 71817474 69747825 74422286 70486402 72870799 67658083 72126058 70719054 17823379 71334926 69747946 69071537 66030851 58074568 68167003 64694275 69179027 68689988 69504299 62652542 94754626.00 91026746.00 90446918.00 88664151.00 93846491.00 91745331.00 Isovalerylcarnitine (C5:0) 62306 75665 125570 36953 51281 54034 25391 276950 82344 97296 74576 41663 154651 122805 34123 607731 99328 680393 10588480 709426 1315899 286284 2723097 755709.00 722500.00 3199759.00 1474238.00 2137833.00 1061967.00 O-ISOVALERYL (N,N,N-TRIMETHYL-D9) (C5:0) 83553816 76210840 82309952 84297374 77849997 82644660 80110958 79632142 77647067 79511719 75489471 19918327 76622281 77584231 76379450 73829469 62292321 80104758 76431026 76702442 74384077 77548516 70725330 80523731.00 79734798.00 86946860.00 84694051.00 90289706.00 91137737.00 Palmitoylcarnitine (C16:0) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 55494 ND ND 18208773 231383 166071 45022 995421 ND ND ND ND ND ND O-PALMITOYL (N-METHYL-D3) (C16:0) 45654962 4513667 564311 3769256 13822966 144243 302442 665156 373347 1273261 8107003 57552 15035085 1478992 300893 7750767 53848 2979921 24890801 7894500 6033438 6443710 20312423 ND ND ND ND ND ND Oleoylcarnitine (C18:1) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 14096245 129063 144119 70699 976077 ND ND ND ND ND ND Stearoylcarnitine (C18:0) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 9932350 71874 61543 ND 216874 ND ND ND ND ND ND MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name RT KEGG_ID L-CARNITINE TRIMETHYL-D9 (C0:0) 1.4 L-Carnitine (C0:0) 1.4 C00318  Acetylcarnitine (C2:0) 1.5 O-ACETYL (N-METHYL-D3) (C2:0) 1.5 O-PROPIONYL (N-METHYL-D3) (C3:0) 2.66 Propionylcarnitine (C3:0) 2.66 C03017 Butyrylcarnitine (C4:0) 3.63 C02862 O-BUTYRYL (N-METHYL-D3) (C4:0) 3.64 Isovalerylcarnitine (C5:0) 4.36 O-ISOVALERYL (N,N,N-TRIMETHYL-D9) (C5:0) 4.38 Palmitoylcarnitine (C16:0) 11.08 C02990 O-PALMITOYL (N-METHYL-D3) (C16:0) 11.07 Oleoylcarnitine (C18:1) 11.35 Stearoylcarnitine (C18:0) 12.35 METABOLITES_END #END