Isoxazole Alters Metabolites and Gene Expression, Decreasing Proliferation and Promoting a Neuroendocrine Phenotype in β-Cells

Abstract

Novel strategies are needed to modulate β-cell differentiation and function as potential β-cell replacement or restorative therapies for diabetes. We previously demonstrated that small molecules based on the isoxazole scaffold drive neuroendocrine phenotypes. The nature of the effects of isoxazole compounds on β-cells was incompletely defined. We find that isoxazole induces genes that support neuroendocrine and β-cell phenotypes and suppresses genes important for proliferation. Isoxazole alters β-cell metabolites and protects glucose-responsive signaling pathways under lipotoxic conditions. Finally, we show that isoxazole improves glycemia in a mouse model of β-cell regeneration. Isoxazole is a prime candidate to alter cell fate in different contexts.

Figure 1

Neuroendocrine gene expression phenotype induced by ISX in cultured human islets and mouse MIN6 β cells. (A) Microarray analysis of long-term cultured human islets treated with DMSO or ISX for 7 days. See Table S1 for human islet donor information. (B–D) qPCR confirmation of selected ISX target genes from long-term cultured human islets. Relative expression is shown for genes that were highly up-regulated (B), moderately upregulated (C), or down-regulated (D). *, P < 0.05. (E) ISX dose–response in INS1E cells shows that ISX slightly decreases proliferation measured indirectly by relative amount of ATP. (F) ISX dose–response in INS1E cells shows no change in viability by lactate dehydrogenase (LDH) activity. (G) ISX dose-dependently lowers cell number in a standard cell counting assay after 72 h. (H) Validation of gene expression altered by ISX in short-term cultured human islets. (I) MIN6 cells were treated with ISX (20 µM) for 2 days, and protein levels of STMN2 were monitored by immunoblotting. *, STMN2 protein increased. (J) MIN6 cells were treated with ISX (20 µM) or DMSO for the indicated times, and gene expression of STMN2 and INSI/II relative to actin were monitored by qPCR. Data are expressed as the ratio of ISX/DMSO for each time point. *, P < 0.05. (K) MIN6 cells were cotreated with ISX (20 µM) and actinomycin D (5 µg/mL) for 6 h, and indicated gene expression was monitored by qPCR. All bar graphs are the average and SE of at least three independent experiments. *, P < 0.05.

Figure 2

MIN6 cell phospho-ERK1/2 response to glucose in the presence of palmitate preserved by ISX. (A) Gene set enrichment analysis indicating a significant normalized enrichment score (NES) for ISX-induced genes in the fatty acyl-CoA biosynthesis pathway. (B) Representative immunoblots for phospho- and total ERK1/2. Below shows a bar graph of the average and SE of three independent experiments. Phospho/total-ERK1/2 ratio was normalized to basal BSA DMSO control.

Figure 3

Metabolomic response to chronic high or low glucose altered by ISX. (A) Relative abundances of metabolites extracted from three independent passages of MIN6 cells. Coloring reflects a log2 scale. (B–E) Volcano plots display the log10 (adjusted p value) on the y axis and the fold-change in relative abundance on the×axis for all metabolites detected in the shown comparisons. Coloring labels fold-change, with blue being down-regulated and yellow being up-regulated.

Figure 4

Metabolites altered by ISX in multiple pathways. Relative amounts of metabolites from LC-MS/MS analyses are shown. (A) Glycolysis and tricarboxylic acid cycle intermediate levels. (B) Metabolites involved in nucleotide biosynthesis are shown. (C) Detected acylcarnitines are shown in order of increasing carbon chain length. (D) Levels of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) are shown. (E) Principle component analysis of metabolomic data shows that replicate samples cluster together. All bar graphs are the average and SE of three independent experiments. *, P < 0.05 DMSO vs ISX, H, P < 0.05 high vs low glucose.

Figure 5

Hyperglycemia in PANIC-ATTAC mice abrogated by ISX treatment during OGTT and whole pancreas insulin content increased. (A) Oral glucose tolerance test (OGTT) in control ISX-treated PANIC-ATTAC mice. (B) Area-under-the-curve (AUC) calculation of the OGTT data from A. A.U., arbitrary units. (C) Pancreata from A were acid-ethanol extracted, and insulin was measured with an ELISA. Data are the average and SE of 6–8 vehicle- and 10–11 ISX-treated mice. *P < 0.05.