Proinsulin and heat shock protein 90 as biomarkers of beta-cell stress in the early period after onset of type 1 diabetes

Abstract

Rapid evaluation of therapies designed to preserve β cells in persons with type 1 diabetes (T1D) is hampered by limited availability of sensitive β-cell health biomarkers. In particular, biomarkers elucidating the presence and degree of β-cell stress are needed. We characterized β-cell secretory activity and stress in 29 new-onset T1D subjects (10.6 ± 3.0 years, 55% male) at diagnosis and then 8.2 ± 1.2 weeks later at first clinic follow-up. We did comparisons with 16 matched healthy controls. We evaluated hemoglobin A1c (HbA1c), β-cell function (random C-peptide [C] and proinsulin [PI]), β-cell stress (PI:C ratio), and the β-cell stress marker heat shock protein (HSP)90 and examined these parameters' relationships with clinical and laboratory characteristics at diagnosis. Mean diagnosis HbA1c was 11.3% (100 mmol/mol) and 7.6% (60 mmol/mol) at follow-up. C-peptide was low at diagnosis (P < 0.001 vs controls) and increased at follow-up (P < 0.001) to comparable with controls. PI did not differ from controls at diagnosis but increased at follow-up (P = 0.003) signifying increased release of PI alongside improved insulin secretion. PI:C ratios and HSP90 concentrations were elevated at both time points. Younger subjects had lower C-peptide and greater PI, PI:C, and HSP90. We also examined islets isolated from prediabetic nonobese diabetic mice and found that HSP90 levels were increased ∼4-fold compared with those in islets isolated from matched CD1 controls, further substantiating HSP90 as a marker of β-cell stress in T1D. Our data indicate that β-cell stress can be assessed using PI:C and HSP90. This stress persists after T1D diagnosis. Therapeutic approaches to reduce β-cell stress in new-onset T1D should be considered.

Conflict of interest statement

Conflicts of Interest: No authors on this article have a conflict of interest that is relevant to the subject matter or materials included in this work. No authors on this article have financial or personal relationships to disclose with organizations that could potentially be perceived as influencing the described research. All authors have read the journal’s policy on conflicts of interest and the journal’s authorship agreement.

Copyright © 2016 Elsevier Inc. All rights reserved.

Figures

Fig 1

Expression of HSP90 in islets from CD1 and NOD mice. A representative western blotting of 3 experiments (upper panel) shows that the expression of HSP90 is higher in islets from 10-week-old NOD mice compared with those from the age-matched control (CD1 mice). O.D for HSP90 normalized to the O.D for actin shows that HSP90 increases about 4 times in the islets from NOD relative to those from CD1 (lower panel). *P < 0.05. HSP, heat shock protein; NOD, nonobese diabetic; O.D, optical density.

Fig 2

(A–E): Glycemic control (A), β-cell secretory activity (B, C), and β-cell stress (D, E) quantification. Healthy control and T1D subject sera at diagnosis and honeymoon period were analyzed for C-peptide, PI, and HSP90. (A) Hemoglobin A1c of T1D subjects at diagnosis and at honeymoon. (B) C-peptide levels in control and T1D subjects at diagnosis and at honeymoon. (C) PI levels in control and T1D subjects at diagnosis and at honeymoon. (D) β-cell stress assessed by the ratio of PI to C-peptide (PI:C) in control and T1D subjects at diagnosis and at honeymoon. (E) Stress assessed by serum HSP90 in controls and T1D subjects at diagnosis and at honeymoon. Error bars display standard deviations from the mean. Each circle represents one control subject, each square represents one T1D subject at diagnosis, each triangle represents one T1D subject at honeymoon initiation. *P < 0.05, **P < 0.01, ***P < 0.001 vs control based on independent sample t test. ##P < 0.01, ###P < 0.001 vs T1D subject at time of diagnosis based on paired sample t test. 1For subjects (n = 4) with serum PI below the LLD, a value of one-half the LLD was used. HSP, heat shock protein; LLD, lower limit of detection; PI, proinsulin; T1D, type 1 diabetes.

Fig 3

Pearson correlations of relationship between β-cell secretory activity measures (A and B) and β-cell stress measures (C and D) with time after diagnosis of T1D. (A) A nonsignificant negative correlation is observed with lower C-peptide levels at second time point. (B) Significant positive correlation demonstrating that when second time point was further from diagnosis, C-peptide was higher. (C) Significant positive correlation is observed with subjects returning further from diagnosis having higher levels of PI:C. (D) No correlation was observed between HSP90 and time from diagnosis. Each triangle represents one T1D subject at the time of honeymoon initiation. HSP, heat shock protein; PI, proinsulin; T1D, type 1 diabetes.

Fig 4

Pearson correlation between β-cell secretory activity (A and B) and β-cell stress (C and D) with age at diagnosis. (A) A significant positive correlation is observed with younger subjects having lower levels of C-peptide at honeymoon. (B) No correlation was observed between the age of subject at diagnosis and the absolute change in PI:C levels between diagnosis and honeymoon. (C) A nonsignificant negative correlation is observed between PI at honeymoon and subject age. (D) A significant negative correlation is observed between absolute change in PI levels from diagnosis to honeymoon and subject age. Older subjects were more likely to have a decrease in PI than younger subjects during the honeymoon initiation time point. (E) There was a nonsignificant negative correlation between β-cell stress measured by PI:C ratios and age at diagnosis. (F) A significant negative correlation was observed between the absolute change in PI:C ratio from diagnosis to honeymoon period and subject age at diagnosis. Older subjects were more likely to have a decrease in PI:C ratios at the honeymoon time point. (G) A significant negative correlation was observed between HSP90 levels at honeymoon initiation time point and subject age. Older subjects had lower HSP90 concentrations at honeymoon. (H) A nonsignificant negative correlation was observed between the absolute change in HSP90 between diagnosis and honeymoon. Each triangle represents one T1D subject at the time of honeymoon initiation; each diamond represents the difference between baseline and honeymoon initiation values in one T1D subject. HSP, heat shock protein; PI, proinsulin.