Impact of severe hypoglycemia on the heat shock and related protein response

Abstract

Heat shock proteins contribute to diabetes-induced complications and are affected by glycemic control. Our hypothesis was that hypoglycemia-induced heat shock and related protein changes would be amplified in type 2 diabetes (T2D). This prospective, case–control study enrolled 23 T2D patients and 23 control subjects who underwent hyperinsulinemic-induced hypoglycemia (≤ 2.0 mmol/L (36 mg/dl)) with blood sampling at baseline, at hypoglycemia and after a 24-h post-hypoglycemia follow-up period. Proteomic analysis of heat shock-related and pro-inflammatory proteins was performed. At baseline, MAPKAPK5 (p = 0.02) and UBE2G2 (p = 0.003) were elevated and STUB1 decreased (p = 0.007) in T2D. At hypoglycemia: PPP3CA (p < 0.03) was increased and EPHA2 (p = 0.01) reduced in T2D; by contrast, three proteins were reduced in controls [HSPA1A (p = 0.007), HSPB1 (p < 0.02), SMAD3 (p = 0.005)] while only MAPKAPK5 was elevated (p = 0.02). In the post-hypoglycemia follow-up period, most proteins normalized to baseline by 24-h; however, STIP1 (p = 0.003), UBE2N (p = 0.004) and UBE2L3 (p < 0.04) were decreased in controls at 24-h. No protein differed from baseline at 24-h in T2D. Pro-inflammatory interleukin-6 increased at 4-h post-hypoglycemia in controls and T2D (p < 0.05 and p < 0.003, respectively) and correlated with HSPA1A; anti-inflammatory IL-10 decreased 2-h post-hypoglycemia in T2D only. Other pro-inflammatory proteins, IL-1α, IFN-γ and TNF-α, were unchanged. Heat shock and related proteins differed at baseline between T2D and controls, with an exaggerated response of heat shock and related proteins to hypoglycemia that returned to baseline, though with changes at 24-h in controls alone. An increase in pro-inflammatory IL-6, with a decrease in anti-inflammatory IL-10, suggests that the HSP system is overactivated due to underlying inflammation in T2D.Trial registration: ClinicalTrials.gov NCT03102801.

Heat Shock-Related Protein Responses and Inflammatory Protein Changes Are Associated with Mild Prolonged Hypoglycemia

“…We have previously reported that inflammatory regulators are increased at the time of hypoglycemia, are exaggerated in T2D, and that all apart from C-reactive protein return to normal at 24 h [21]. These data are in accordance with the HSP response to transient severe hypoglycemia reported previously, although here there was a greater association between the HSP and the inflammatory responses [25].…”

“…The UPS is coordinated sequentially by three enzymes: ubiquitinactivating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). In accord with the findings in severe hypoglycemia, there were significant increases in UBE2N, STIP1, and UBE2L3 seen in hypoglycemia only in the T2D cohort that returned to baseline by 24 h. UBE2L3 showed differences between controls and T2D at 24 h that reflected those seen in severe hypoglycemia [25]. UBE2N, a ubiquitin-conjugating enzyme E2; STIP1, an E3 ubiquitin ligase; and UBE2L3, also a ubiquitin ligase, are all key players in the ubiquitin pathway and critical for misfolded protein degradation [41]; our results suggest that, in T2D, hypoglycemia may be predisposed to increased protein misfolding, therefore activating this pathway.…”

“…The findings for HSPA8 and HSP90 were similar between severe [25] and mild hypoglycemia. The level of HSPA8 was lower in T2D at baseline, in accordance with those reporting lower HSP70 levels in diabetes [31], and decreased following hypoglycemia compared with controls.…”

“…Details of the response of the TLR4:MD-2 complex have not been reported before in hypoglycemia because, whilst the proteomic technology was the same [25], the proteomic panels differed slightly. TLR4:MD-2 complex was elevated in T2D and fell at hypoglycemia, although it did not return to baseline levels by 24-h. TLR4, a member of the TLR family, has an important role in modulating innate immunity [35]; activation of TLRs, by either exogenous or endogenous molecules, leads to a signaling cascade, resulting in cytokine production and induction of an adaptive immune response [36].…”

“…Glucose modulation is associated with changes in HSPs [24], and severe hypoglycemia has been associated with HSP changes at baseline that exhibit an exaggerated response to hypoglycemia associated with changes in inflammatory markers [25]. We hypothesized that mild, although prolonged, hypoglycemia would have less effect than severe transient hypoglycemia on the HSP and related protein responses and the associated inflammatory and oxidative changes; hence, this pilot exploratory study was performed.…”