Protein folding in the endoplasmic reticulum (ER) requires a high ratio of oxidized to reduced glutathione (GSSG/rGSH). Since the GSSG/rGSH depends on total glutathione (tGSH = GSSG + rGSH) levels, we hypothesized that limiting GSH biosynthesis will ameliorate protein misfolding by enhancing the ER oxidative milieu. As a proof-of-concept, we used DL-buthionine-(S,R)-sulfoximine (BSO) to inhibit GSH biosynthesis in Akita mice, which are prone to proinsulin misfolding. We conducted a 2-week intervention to investigate if BSO was safe and a 6-week intervention to find its effect on glucose intolerance. In both cohorts, male heterozygous Akita (AK) and wild-type (WT) mice were continuously administered 15 mM BSO. No adverse effects were observed on body weight, food intake, and water intake in either cohort. Unaltered levels of plasma aspartate and alanine aminotransferases, and cystatin-C, indicate that BSO was safe. BSO-induced decreases in tGSH were tissue-dependent with maximal effects in the kidneys, where it altered the expression of genes associated with GSH biosynthesis, redox status, and proteostasis. BSO treatment decreased random blood glucose levels to 80% and 67% of levels in untreated mice in short-term and long-term cohorts, respectively, and 6-h fasting blood glucose to 82% and 74% of levels in untreated mice, respectively. BSO also improved glucose tolerance by 37% in AK mice in the long-term cohort, without affecting insulin tolerance. Neither glucose tolerance nor insulin tolerance were affected in WT. Data indicate that BSO might treat misfolded proinsulin-induced glucose intolerance. Future studies should investigate the effect of BSO on proinsulin misfolding and if it improves glucose intolerance in individuals with Mutant Insulin Diabetes of Youth.
Graphical abstract
1) Male heterozygous C57BL/6-Ins2Akita/J (AK) mice suffer from misfolded proinsulin-induced glucose intolerance.
(a) Proinsulin misfolding occurs due to a genetic mutation in Ins2 gene that substitutes Cys with Tyr, (b) Due to heterozygosity, AK mice produce both wild-type and mutated proinsulin, (c) Mutated proinsulin forms aggregates with itself and with the bystander native proinsulin, (d) Proinsulin aggregation results in lower functional insulin, and (e) AK mice suffer from impaired glucose tolerance.
2) Treating mice with BSO improved glucose tolerance.
(a) Mice were treated with continuous administration of 15 mM DL -buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione biosynthesis (b), BSO treatment increased the renal mRNA quantity of several genes involved in glutathione biosynthesis, glutathione redox status, and proteostasis, (c) we hypothesize that BSO-induced changes in cellular redox status and gene expression ameliorates proinsulin aggregation and increases the functional insulin levels in β-cells, and (d) BSO treatment significantly improved glucose intolerance in AK mice. Note: AUC - Area under the curve, GCL -γ-g-glutamylcysteine ligase, GS - Gluatthione synthetase.
