Dynamic Changes of BVRA Protein Levels Occur in Response to Insulin: A Pilot Study in Humans

Abstract

Biliverdin reductase-A (BVRA) is involved in the regulation of insulin signaling and the maintenance of glucose homeostasis. Previous research showed that BVRA alterations are associated with the aberrant activation of insulin signaling in dysmetabolic conditions. However, whether BVRA protein levels change dynamically within the cells in response to insulin and/or glucose remains an open question. To this aim, we evaluated changes of intracellular BVRA levels in peripheral blood mononuclear cells (PBMC) collected during the oral glucose tolerance test (OGTT) in a group of subjects with different levels of insulin sensitivity. Furthermore, we looked for significant correlations with clinical measures. Our data show that BVRA levels change dynamically during the OGTT in response to insulin, and greater BVRA variations occur in those subjects with lower insulin sensitivity. Changes of BVRA significantly correlate with indexes of increased insulin resistance and insulin secretion (HOMA-IR, HOMA-β, and insulinogenic index). At the multivariate regression analysis, the insulinogenic index independently predicted increased BVRA area under curve (AUC) during the OGTT. This pilot study showed, for the first time, that intracellular BVRA protein levels change in response to insulin during OGTT and are greater in subjects with lower insulin sensitivity, supporting the role of BVR-A in the dynamic regulation of the insulin signaling pathway.

Keywords: biliverdin reductase-A; diabetes; insulin signaling; metabolism; obesity.

Figure 1

Changes of (A) glycemia (mg/dL) and (B) insulin (μU/mL) circulating levels along with (C) BVRA protein levels evaluated in PBMCs isolated from subjects with different levels of insulin sensitivity (n = 12) underwent an oral glucose tolerance test (OGTT). Data are presented as mean ± SEM. * p < 0.05, *** p < 0.01 (paired Student’s t-test).

Figure 2

Significant correlations (A–M) found among BVRA protein levels in PBMCs, circulating insulin levels, and surrogate markers of insulin resistance (Pearson’s coefficient) in our cohort (N = 12).

Figure 3

Changes of (A) BVRA protein levels (ng/μg of proteins) evaluated in PBMCs along with mean changes of (B) insulin (μU/mL) and (C) glycemia (mg/dL) circulating levels in the subjects of our cohort categorized according to the HOMA-IR value < 2.5 (n = 6) and >2.5 (n = 6). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 (paired Student’s t-test).

Figure 4

BVRA protein levels along with the activation of main proteins of the insulin signaling pathway were evaluated in HEK293 cells treated with 100 nM insulin for 15, 30, 60, and 120 min. (A,B) Representative Western blot and total load images and densitometric evaluation of (C) BVRA protein levels; (D) IRS1 activation evaluated as a ratio between the IRS1 active form (IRS1Y632) and the IRS1 total protein levels (pIRS1Y632/IRS1); (E) AKT activation evaluated as a ratio between the AKT active form (AKTS473) and the AKT total protein levels (pAKTS473/AKT); and (F) MAPK activation evaluated as a ratio between the MAPK active forms (pMAPK42/44 T202/Y204) and the MAPK total protein levels (pMAPK/MAPK). Data are shown as mean ± SEM (n = 4–5 independent cultures/group) One-way ANOVA with Bonferroni post hoc test: * p < 0.05, ** p < 0.01.

Figure 5

Proposed mechanism through which insulin promotes BVRA protein levels changes depending on the regulatory role for BVRA within the insulin signaling pathway. Upstream in the pathway (red oval): Under physiological conditions, the insulin receptor (IR) activation promotes the phosphorylation of its direct substrate (IRS1) on specific tyrosine (Y) residues. In parallel, IR phosphorylates BVRA on specific Y residues and activates BVRA to function as S/T/Y kinase. Then, as part of a regulatory loop, BVRA phosphorylates IRS1 on inhibitory serine (S) residues (e.g., S307) to avoid IRS1 aberrant activation in response to IR. In subjects with reduced insulin sensitivity, a greater decrease in BVRA protein levels can be observed in response to insulin to favor IRS1 activation. Downstream in the pathway (blue oval): Once activated, IRS1 works as a scaffold protein, driving the activation of the two main arms of the insulin signaling: (1) the Ras/Raf/MAPK pathway (ERK1/2) mainly involved in gene transcription and (2) the PI3K/Akt axis that is critical for glucose uptake as well as for protein and lipid metabolism. Within both axes, BVRA works as a kinase or as a scaffold protein facilitating (1) the ERK1/2 phosphorylation and the subsequent translocation in the nucleus; (2) the PDK1-mediated activation of AKT; and (3) the PDK1-mediated activation of the atypical PKCζ; both these latter events are needed for GLUT4 translocation to the plasma membrane to increase glucose uptake. See text for more details. In subjects with reduced insulin sensitivity, a greater increase in BVRA protein levels can be observed in response to insulin to favor the activation of MAPK and AKT axes. Green arrows refer to BVRA protein levels; blue arrows refer to the molecular events regulated by BVRA.