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. Author manuscript; available in PMC: 2019 Aug 28.
Published in final edited form as: Cell Rep. 2019 Jul 16;28(3):759–772.e10. doi: 10.1016/j.celrep.2019.06.058

Figure 6. Expression of hSCS-GTP Modifies Mitochondrial Morphology and Metabolic Regulation of Insulin Secretion.

Figure 6.

(A) Representative TEM images of indicated constitutive expression cell lines grown under normal culture conditions. Scale bar, 1 μm.

(B) Average size of mitochondrion for each cell line.

(C) Average aspect ratio of mitochondria analyzed via TEM imaging in each cell line.

(D) Form factor values for the cell lines.

(E) Mitochondrial oxygen consumption rate (OCR) (antimycin and rotenone subtracted) measured using the Agilent Seahorse ‘‘mito stress test’’ protocol following glucose stimulation (16.7 mM glucose), oligomycin (oligo), and FCCP in indicated constitutive overexpression cell lines. ATP = (OCRglucose – OCRantimycin/rotenone) – OCRoligomycin; Leak = OCRoligomycin – OCRantimycin/rotenone.

(F–J) Simultaneous monitoring of insulin secretion (F), OCR (G), NAD(P)H (H), Ca2+ (I), and extracellular lactate (J) from perifusion of hSCS-ATP and hSCS-GTP cells at basal and upon exposure to high glucose (16.7 mM) or amino acids (10 mM L-leucine, 4 mM L-glutamine) (n = 5).

(K) Insulin secretion in response to 2.5 mM glucose (G2.5) and indicated stimuli (BCH, 2- aminobicyclo[2,2,1]heptane-2-carboxylic acid; SAME, succinate methyl ester, propionate) (n = 6 per group).

(L) Insulin secretion in response to incubations with G2.5, 9 mM glucose (G9.0), and G9.0 + pharmacological agents (TBT, tolbutamide; Ex-4, exendin-4; GKa, glucokinase activator).

(M) Insulin secretion in response to 2.5 mM glucose (G2.5) 10 mM L-arginine (L-Arg), 10 mM L-leucine (L-Leu), 10 mM L-pyruvate (L-Pyr), and 10 mM ketoiso-caproate (α-KIC) (n = 6 per group).

The data are means ± SEMs (for B: control n = 299, hSCS-ATP n = 322, hSCS-GTP n = 232; for C: control n = 448, hSCS-ATP n = 440, hSCS-GTP n = 324; for D: control n = 43, hSCS-ATP n = 29, hSCS-GTP n = 35; for E: control and hSCS-GTP n = 8 per group, hSCS- ATP n = 7 per group; for F and H–J: n = 3 separate experiments; for G: n = 5 per group; for K–M: n = 6 per group; technical replicates for all panels). Statistical significance is *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test (B–D), two-way ANOVA with Tukey’s multiple comparisons test (E), multiple two-tailed t tests with the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (F–H), Holm-Sidak’s multiple two-tailed t tests (I and J), and two-way ANOVA with Dunnett’s multiple comparisons test (K–M).