Inhibition of polyamine biosynthesis preserves β cell function in type 1 diabetes

Abstract

In preclinical models, α-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, delays the onset of type 1 diabetes (T1D) by reducing β cell stress. However, the mechanism of DFMO action and its human tolerability remain unclear. In this study, we show that mice with β cell ODC deletion are protected against toxin-induced diabetes, suggesting a cell-autonomous role of ODC during β cell stress. In a randomized controlled trial (ClinicalTrials.gov: NCT02384889) involving 41 recent-onset T1D subjects (3:1 drug:placebo) over a 3-month treatment period with a 3-month follow-up, DFMO (125-1,000 mg/m²) is shown to meet its primary outcome of safety and tolerability. DFMO dose-dependently reduces urinary putrescine levels and, at higher doses, preserves C-peptide area under the curve without apparent immunomodulation. Transcriptomics and proteomics of DFMO-treated human islets exposed to cytokine stress reveal alterations in mRNA translation, nascent protein transport, and protein secretion. These findings suggest that DFMO may preserve β cell function in T1D through islet cell-autonomous effects.

Keywords: disease modification; islet; ornithine decarboxylase; polyamines; prevention; trial; type 1 diabetes; α-difluoromethylornithine; β cell; β cell stress.

Graphical abstract

Figure 1

The polyamine biosynthetic pathway and the glycemic response of Odc1^Δβ mice to multiple low doses of STZ (A) The polyamine metabolic pathway in mammals. Polyamine production is governed by the biosynthetic enzymes ornithine decarboxylase (ODC), spermidine synthase (SPS), and spermine synthase (SMS) and the catabolic enzymes spermidine/spermine N1-acetyltransferase (SSAT), the flavin-dependent polyamine oxidase (PAOX), and spermine oxidase (SMOX). Spermidine is used as a substrate in the enzymatic modification of Lys50 in the protein eIF5A to generate the hypusine modification. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ODC. (B) A schematic showing the multiple low-dose streptozotocin (STZ) experiments. Male Odc1^Δβ mice and control Cre+ littermates were injected with STZ daily for 5 days. Subsequently, glucose levels and glucose tolerance were monitored. (C) Glucose tolerance test and corresponding AUC analysis on day 10 after the start of STZ injections. (D) Diabetes incidence; mice were considered to have diabetes after two consecutive blood glucose values of 250 mg/dL. (E) Non-fasting blood glucose values over time. (F) Representative immunohistochemistry images of pancreata showing β cells (insulin, brown) and hematoxylin (nuclei, blue) and corresponding β cell mass; scale bar, 200 μm. (G) Representative immunofluorescence images of islets showing ODC (magenta), insulin (green), and nuclei (DAPI, blue) and corresponding percentage of β cells positive for ODC; scale bar, 50 μm. n = 6–8 biological replicates. Data are presented as mean ± SEM. ∗p < 0.05 as indicated.

Figure 2

Key features of study design and findings (A) CONSORT diagram for participants in this trial. (B) Scatterplot showing significant correlation (Pearson r = −0.385; p = 0.02) between DFMO dose (x axis) vs. change in urinary putrescine, the direct downstream product of ODC (y axis). (C and D) Least square means, modeled with ANCOVA using baseline values as a covariate, for (C) mixed-meal C-peptide AUC and (D) fasting proinsulin:C-peptide ratios (PI:C) for participants in the placebo and 1,000 mg/m²/day dosing groups at baseline, after 3 months of treatment, and at the 6-month follow-up visit. Data are presented as least square means ± 95% confidence interval. Sample size for each group was as follows: placebo, n = 10; 125 mg/m², n = 6; 250 mg/m², n = 6; 500 mg/m², n = 6; 750 mg/m², n = 7; 1,000 mg/m², n = 6.

Figure 3

RNA sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics of human islets treated with DFMO and/or proinflammatory cytokines (A) Human islets isolated from 5–6 cadaveric donors were pretreated with either vehicle or 5 mM DFMO, then incubated with or without proinflammatory cytokines (PICs; IL-1β and IFN-γ) for 24 h, followed by either unbiased RNA sequencing or proteomics. (B) Principal-component analysis plot of RNA sequencing data. (C) Volcano plot of differentially expressed genes in control vs. DFMO-treated islets. (D) Volcano plot of differentially expressed genes in PIC vs. PIC plus DFMO-treated islets. (E) Principal-component analysis plot of proteomics data. Circles identify clustering of individual donors. (F) Volcano plot of differentially expressed proteins in control vs. DFMO-treated islets. (G) Volcano plot of differentially expressed proteins in PIC vs. PIC plus DFMO-treated islets. (H) GO biological process pathway analysis of differently expressed proteins of control vs. DFMO-treated islets. (I) GO biological process pathway analysis of differently expressed proteins of PIC vs. PIC plus DFMO-treated islets. (J) Commonly altered genes between transcriptomics and proteomics in PIC vs. PIC plus DFMO-treated islets. n = 5–6 biological replicates.