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

Abstract

Mild hypoglycemia is common in clinical practice. Severe hypoglycemia results in heat shock protein and associate co-chaperone changes. Whether mild prolonged hypoglycemia elicits a similar response with inflammatory and oxidative-stress responses compared with a severe hypoglycemic event is unclear; therefore, this pilot exploratory study was undertaken. We performed a case-control induced hypoglycemia clamp study, maintaining blood glucose at 2.8 mmol/L (50 mg/dL) for 1 h in 17 subjects (T2D (n = 10); controls (n = 7)). Blood sampling was performed at baseline, hypoglycemia, and 24 h; slow off-rate modified aptamer (SOMA)-scan plasma protein analysis of HSP-related proteins, inflammatory stress markers, and oxidative stress markers was performed. In total, 16 HSPs were analyzed. At baseline, TLR4:MD-2 complex was elevated (p = 0.01), whilst HSPA8 was lower (p < 0.05) in T2D. At hypoglycemia, UBE2N, STIP1, and UBE2L3 increased (all p < 0.05), whilst TLR4:MD-2 and HSPA8 decreased (p < 0.05) in T2D versus baseline. In follow-up after hypoglycemia, HSPs normalized to baseline by 24 h, except UBE2L3 (p < 0.05), which was decreased in controls versus baseline. Correlation of altered inflammatory markers with HSPs revealed the following: at baseline, TLR4:MD-2 correlated with CXCL10 (p < 0.01) and SIGLEC1 (p < 0.05) in controls; HSPA8 negatively correlated with IL5 (p < 0.05) in T2D. A negative correlation between urinary isoprostane 8-iso PGF2α, a marker of oxidative stress, and HSPA1A was seen at 24 h in T2D only (p < 0.05). In conclusion, the HSP changes seen for mild prolonged hypoglycemia were similar to those previously reported for a severe event. However, mild prolonged hypoglycemia appeared to elicit an increased inflammatory response that was associated with heat shock and related proteins.

Trial registration: ClinicalTrials.gov NCT02205996.

Keywords: heat shock proteins; hypoglycemia; inflammatory proteins; oxidative stress; type 2 diabetes.

Conflict of interest statement

None of the authors has any conflict of interest to declare.

Figures

Figure 1

Schematic representing the recognized interactions between heat shock proteins (HSPs) and related proteins that, in response to severe acute hypoglycemia, are differentially expressed. Upward red arrows show proteins that, in T2D, were upregulated in response to hypoglycemia. HSP90 alpha (HSP90AA1, HSP90AB1, HSP90 beta, HSP90 dimer); HSPA1A, heat shock 70 kDa protein 1A; HSPA8, heat shock cognate 71 kDa protein; HSPD1, 60 kDa heat shock protein, mitochondrial; AIMP1, aminoacyl tRNA synthase complex-interacting multifunctional protein 1; CDC37, Hsp90 co-chaperone Cdc37; CLU, clusterin; DNAJB1, DnaJ homolog subfamily B member 1; MAPKAPK2, MAP kinase-activated protein kinase 2; MAPKAPK5, MAP kinase-activated protein kinase 5; PPID, peptidyl-prolyl cis-trans isomerase D; PPP3CA, serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform; STIP1, stress-induced phosphoprotein 1; TLR4, toll-like receptor 4; TLR4:MD-2 complex, toll-like receptor 4 in complex with MD-2; CD274, programmed cell death 1 ligand 1; EPHA2, ephrin type-A receptor 2; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme 2 (UBE2L3, ubiquitin-conjugating enzyme E2 L3; UBE2N, ubiquitin-conjugating enzyme E2 N); UCHL1, ubiquitin carboxyl-terminal hydrolase isozyme L1; E3, ubiquitin ligases; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; AKT, protein kinase B, HSF, heat shock factors; SNO, S-nitrosylation; P38 MAPK, p38 mitogen-activated protein kinases; Bcl-xL, B-cell lymphoma-extra large; LRP1, low-density lipoprotein receptor-related protein 1; IR, insulin receptor; IRS1, insulin receptor substrate 1.

Figure 2

Comparison of plasma levels of the following heat shock proteins: toll-like receptor 4:myeloid differentiation factor-2 (TLR4:MD-2), heat shock protein family A (HSP70) member 8 (HSPA8), and heat shock protein 90 dimer (HSP90a/b) at baseline (BL), at hypoglycemia and 24 h post-hypoglycemia in control and T2D cohorts. Controls (white circles), T2D (black squares). TLR4:MD-2 (A), HSPA8 (B), and HSP90a/b (C). Panels (A,B) show proteins for which levels differed at BL between T2D and control cohorts. Statistics: *, p < 0.05; **, p < 0.01, control vs. T2D; $, p < 0.05, T2D BL vs. Hypo. RFU, relative fluorescent units; Hypo, hypoglycemia.

Figure 3

Comparison of plasma levels of the following heat shock proteins: ubiquitin-conjugating enzyme E2N (UBE2N), stress-induced phosphoprotein 1 (STIP1), and ubiquitin-conjugating enzyme E2L3 (UBE2L3) at baseline (BL), at hypoglycemia, and 24 h post hypoglycemia in control and T2D cohorts. Controls (white circles), T2D (black squares). UBE2N (A), STIP1 (B), and UBE2L3 (C), proteins for which levels differed from baseline to hypoglycemia in T2D and control cohorts. Statistics: $, p < 0.05, T2D BL vs. Hypo; &, p < 0.05, control BL vs. 24 h. RFU, relative fluorescent units; Hypo, hypoglycemia.

Figure 4

Correlations of HSPs that differed at baseline between T2D and control subjects with inflammatory proteins. At baseline, TLR4:MD-2 complex showed a positive correlation with CXCL10 (A) and SIGLEC1 (B) in control subjects. HSPA8 showed a negative correlation with IL5 (C) in T2D subjects. TLR4:MD-2 complex, toll-like receptor 4 in complex with MD-2; HSPA8, heat shock cognate 71 kDa protein; IL5, interleukin 5; RFU, relative fluorescent units.

Figure 5

Correlations of HSPs that changed significantly from baseline to hypoglycemia in T2D with inflammatory markers at hypoglycemia. At hypoglycemia, UBE2N showed a positive correlation with IL5 (A), AZU1 (B), RPS6KA5 (C), TBK1 (D), and PRKCZ (E) in T2D subjects; UBE2N also had a positive correlation with RPS6KA5 (C) in control subjects. STIP 1 showed a positive correlation with IL5 (F), TBK1 (I), and FGF8 (J) in T2D subjects alone and with AZU1 (G) and RPS6KA5 (H) in both T2D and control subjects. UBE2N, ubiquitin-conjugating enzyme E2 N; STIP1, stress-induced-phosphoprotein 1; AZU1, azurocidin 1; RPS6KA5, ribosomal protein S6 kinase 5; TBK1, TANK-binding kinase 1; PRKCZ, protein kinase C zeta type; IL5, interleukin 5; FGF8, fibroblast growth factor 8; RFU, relative fluorescent units.

Figure 6

Correlations of HSPs that changed significantly from baseline to hypoglycemia in T2D with inflammatory markers at hypoglycemia. At hypoglycemia, UBE2L3 showed a positive correlation with IL5 (A), TBK1 (D), PRKCZ (E), and FGF8 (F) in T2D subjects only and with AZU1 (B) and RPS6KA5 (C) in both T2D and control subjects. UBE2L3, ubiquitin-conjugating enzyme E2L 3; IL5, interleukin 5; TBK1, TANK-binding kinase 1; PRKCZ, protein kinase C zeta type; FGF8, fibroblast growth factor 8; AZU1, azurocidin 1; RPS6KA5, ribosomal protein S6 kinase 5; RFU, relative fluorescent units.

Figure 7

Comparison of the correlation of UBE2L3 with inflammatory markers at baseline and at 24 h. In T2D at 24 h, UBE2L3 correlated positively with AZU1 (A), RPS6KA5 (B), HMGB1 (C), and TBK1 (D), and negatively with IL10Rbeta (F). In controls at 24 h, UBE2L3 correlated positively with AZU1 (A), RPS6KA5 (B), IL1beta (E), and FGF8 (G). In comparison with the correlation of those markers at baseline, the same correlations were seen for both T2D and controls for AZU1 (A), RPS6KA5 (B), HMGB1 (C), IL1beta (E), IL10Rbeta (F), and FGF8 (G), and only differed for TBK1, which correlated in T2D at 24 h (D), but not at baseline, and did not correlate with controls at 24 h (D), despite correlating at baseline. UBE2L3, ubiquitin-conjugating enzyme E2L 3; AZU1, azurocidin 1; RPS6KA5, ribosomal protein S6 kinase 5; IL1beta, interleukin 1 beta: FGF8, fibroblast growth factor 8; HMGB1, high mobility group box 1; IL10Rbeta, interleukin 10 receptor beta; TBK1, TANK-binding kinase 1; RFU, relative fluorescent units.

Figure 8

STRING interaction network showing the interactions of heat shock-related proteins. STRING 11.0 (Search Tool for the Retrieval of Interacting Genes) was used to visualize the recognized and predicted protein–protein interactions for the heat shock and inflammatory proteins reported here (https://string-db.org/ (accessed on 1 September 2021)). Network nodes represent proteins, and the lines reflect physical and/or functional interactions of proteins. Empty nodes represent the proteins of unknown three-dimensional structure, and filled nodes represent the proteins with some three-dimensional structure, either known or predicted. Different colored lines between the proteins represent the various types of interaction evidence in STRING (databases, experiments, neighborhood, gene fusion, co-occurrence, text mining, co-expression, and homology): here, known interactions are shown in light blue (from curated databases) and pink (experimentally determined); predicted interactions are shown in dark blue (gene co-occurrence); relationships gleaned from text mining (lime green), co-expression (black), and protein homology (mauve) are also shown. The heat shock proteins and the inflammatory proteins are shown separately (left) and combined (right).