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.

Conflict of interest statement

The authors declare no competing interests.

© 2021. The Author(s).

Figures

Figure 1

Schematic figure showing an overview of interactions between proteins involved in HSP and associated stress-response pathways in response to hypoglycemia. These interactions decide the fate of the downstream signaling pathway. The HSPs and associated proteins interact with the cell surface receptors and/or with each other in response to different stimuli, including accumulated unfolded/misfolded proteins, hormones and cellular/environmental stress (including hypoglycemia) (as indicated by upward red arrows) and regulate different molecules affecting a spectrum of biological functions such as apoptosis, autophagy, cell migration and alterations in the immune response. Schematic created using Biorender (https://biorender.com).

Figure 2

Comparison of circulatory heat shock protein (HSP) related proteins between control and T2D. Proteomic (Somalogic) analysis was undertaken to determine the plasma levels of heat shock proteins [HSPs], Ubiquitin-conjugating enzyme E2 G2 (UBE2G2) (A), MAP kinase-activated protein kinase 5 (MAPKAPK5) (B), E3 ubiquitin-protein ligase CHIP (STUB1) (C), Mothers against decapentaplegic homolog 3 (SMAD3) (D), Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform (PPP3CA) (E), Heat shock protein 90ab (HSP90ab) (F), Ubiquitin-conjugating enzyme E2 N (UBE2N) (G), Stress-induced-phosphoprotein 1 (STIP1) (H), Clusterin (CLU) (I) at baseline (BL), during and after iatrogenic induction of hypoglycemia for control (C) and type 2 diabetes (T2D) subjects. Blood sampling was performed at BL, at hypoglycemia (0 min) and post-hypoglycemia (0.5-h, 1-h, 2-h, 4-h and 24-h) for controls (white circles) and for T2D (black squares). Panels A-C show proteins for which levels differed at baseline between T2D and control subjects. Panels D-I show proteins for which levels differed at hypoglycemia and post-hypoglycemia between control and T2D. *p < 0.05, **p < 0.01, control vs T2D; RFU, relative fluorescent units; BG, blood glucose; Hypo, hypoglycemia. Those HSPs that generally show a trend to be consistently higher in T2D are not shaded; those HSPs that appeared to show no difference in trend between T2D and controls are shaded in blue; for the single HSP (STUB1) that is generally lower in T2D than controls, this graph is shaded in yellow.

Figure 3

Comparison of circulatory HSP related proteins within groups (baseline vs hypoglycemia in Control subjects and subjects with T2D; significance is denoted by the symbol “#” for control and “$” for T2D). Proteomic (Somalogic) analysis was undertaken to determine the plasma levels of HSP related proteins, Heat shock protein beta-1 (HSPB1) (A), Mothers against decapentaplegic homolog 3 (SMAD3) (B), Heat shock 70 kDa protein 1A (HSPA1A) (C), MAP kinase-activated protein kinase 5 (MAPKAPK5) (D), Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform (PPP3CA) (E), Ephrin type-A receptor 2 (EPHA2) (F) at baseline (BL) during and after iatrogenic induction of hypoglycemia for control (C) and type 2 diabetes (T2D) subjects. Blood sampling was performed at BL, at hypoglycemia (0 min) and post-hypoglycemia (0.5-h, 1-h, 2-h, 4-h and 24-h) for controls (white circles) and for T2D (black squares). Panels A-D show proteins for which levels differed between baseline and hypoglycemia in control subjects (significance is denoted by “#”). Panels E–F show proteins for which levels differed between baseline and hypoglycemia in T2D subjects (significance is denoted by “$”). #p < 0.05, ##p < 0.01, $p < 0.05; RFU, relative fluorescent units; BG, blood glucose; Hypo, hypoglycemia. Those HSPs that generally show a trend to be consistently higher in T2D are not shaded; those HSPs that appeared to show no difference in trend between T2D and controls are shaded in blue.

Figure 4

Comparison of circulatory HSP related proteins within groups at post-hypoglycemia (baseline vs 0.5–4 h post-hypoglycemia in control subjects; significance is denoted by the symbol “@”). Proteomic (Somalogic) analysis was undertaken to determine the plasma levels of HSP related proteins, Ubiquitin-conjugating enzyme E2 N (UBE2N) (A), Stress-induced-phosphoprotein 1 (STIP1) (B), Hsp90 co-chaperone Cdc37 (CDC37) (C), Ubiquitin-conjugating enzyme (UBE2L3) (D), Heat shock cognate 71 kDa protein (HSPA8) (E), DnaJ homolog subfamily B member 1 (DNAJB1) (F), Heat shock protein 90ab (HSP90ab) (G), 60 kDa heat shock protein, mitochondrial (HSPD1) (H), Clusterin (CLU) (I) at baseline (BL), during and after iatrogenic induction of hypoglycemia for control (C) and type 2 diabetes (T2D) subjects. Blood sampling was performed at BL, at hypoglycemia (0 min) and post-hypoglycemia (0.5-h, 1-h, 2-h, 4-h and 24-h) for controls (white circles) and for T2D (black squares). All the Panels from A-I show proteins for which levels differed between baseline and 0.5–4 h post-hypoglycemia in control subjects. @p < 0.05, @@p < 0.01, @@@p < 0.001, @@@@p < 0.0001; RFU, relative fluorescent units; BG, blood glucose; Hypo, hypoglycemia. Those HSPs that generally show a trend to be consistently higher in T2D are not shaded; those HSP that appeared to show no difference in trend between T2D and controls are shaded in blue.

Figure 5

Comparison of HSP related proteins within groups at post-hypoglycemia (baseline vs 0.5–4 h post-hypoglycemia in subjects with T2D; significance is denoted by the symbol “%”). Proteomic (Somalogic) analysis was undertaken to determine the plasma levels of HSP related proteins, Mothers against decapentaplegic homolog 3 (SMAD3) (A), Ephrin type-A receptor 2 (EPHA2) (B). Blood sampling was performed at BL, at hypoglycemia (0 min) and post-hypoglycemia (0.5-h, 1-h, 2-h, 4-h and 24-h) for controls (white circles) and for T2D (black squares). Panels A-B show proteins for which levels differed between baseline and 0.5–4 h post-hypoglycemia in subjects with T2D. %%p < 0.01, %%%p < 0.001; RFU, relative fluorescent units; BG, blood glucose; Hypo, hypoglycemia. The HSP that generally shows a trend to be consistently higher in T2D (SMAD3) is not shaded; the HSP that appeared to show no difference in trend between T2D and controls (EPHA2) is shaded in blue.