Figure 3 |. Protective glucose metabolism directs arginine to the urea cycle away from NO synthesis in islets undergoing inflammation.
a, Histogram of relative abundance of 3160 proteins detected (out of 5399 total, see Supplementary Dataset 1) by LC–MS/MS in 8.7 × 104 purified human ²-cells from n = 3 donors. Green lines indicate gene products related to the urea cycle, argininosuccinate and aspartate metabolism detected at the protein level.
b, Expression levels of genes related to the urea cycle, pyruvate metabolism and arigninosuccinate shunt based on RNAseq analysis of FACS-sorted human β-cells from n=3 human donors, data are in Log2CPM.
c, Co-immunostaining of insulin and individual metabolic enzymes related to the urea cycle in dispersed human islet cells from one donor representing similar results obtained from two additional donors. Representative images are shown (left), scale bar is 10 microns. Mean fluorescence intensity (FI) within the insulin positive region of interest (ROI) was calculated from 5 images per antibody (right). Neg denotes negative control for background Alexa Fluor 488 signal with insulin co-stain. Statistical analyses are student’s t-tests of each enzyme compared to Neg.
Enzyme abbreviations in a–c are ARG2, arginase 2; ASL, argininosuccinate lyase; ASS1, argininosuccinate synthase 1; CPS1, carbamoyl-phosphate synthase 1; DDAH 1, dimethylarginine dimethylaminohydrolase 1; DDAH 2, dimethylarginine dimethylaminohydrolase 2; GOT1, aspartate aminotransferase 1; GOT2, aspartate aminotransferase 2; MDH1, malate dehydrogenase 1; MDH2, malate dehydrogenase 2; NAGS, N-acetyl-glutamate synthase; OAT, ornithine aminotransferase; PC, pyruvate carboxylase; SLC25A12, solute carrier family 25 member 12/calcium-binding mitochondrial carrier protein Aralar 1; SLC25A13, solute carrier family 25 member 13/calcium-binding mitochondrial carrier protein Aralar 2; SLC25A15, solute carrier family 25 member 15/mitochondrial ornithine transporter 1.
d–e, Cytokine-induced changes in the partitioning of 15N2-L-arginine to urea (d) and citrulline/NO (e) synthesis in human islets comparing protective vs non-protective glucose metabolism as modeled by BAD SAHBA SD vs RO0281675 treatment, respectively, n=4 donors.
f, Chemical inhibition of arginase via ABH interferes with the protective effect of BAD SAHBA SD in human islets undergoing inflammation, n=5 donors.
Data are means ± s.e.m. with one-way (d,e) and two-way (f) ANOVA statistical tests with Tukey adjustment for multiple comparisons.