Impact of Severe Hypoglycemia on the Heat Shock and Related Response

Alexander S. Atkin1@, Abu Saleh Md Moin, PhD2@, Manjula Nandakumar PhD2, Ahmed Al-

Qaissi MD3,4, Thozhukat Sathyapalan MD3, Stephen L. Atkin MD5*, Alexandra E. Butler MD2*

1Trinity College, Cambridge University, UK

2 Diabetes Research Center (DRC), Qatar Biomedical Research Institute (QBRI), Hamad Bin

Khalifa University (HBKU), Qatar Foundation (QF), PO Box 34110, Doha, Qatar

3 Academic Endocrinology, Diabetes and Metabolism, Hull York Medical School, Hull, UK.

4 Leeds Medical School, Leeds, UK

5 Royal College of Surgeons of Ireland, Bahrain

@ joint first authors *joint senior authors Supplementary table 1. Demographic and clinical characteristics of the study participants. Data are presented as mean ± SD.

Baseline Type 2 Controls p-value Diabetes (n=23) (n=23) Age (years) 64±8 60±10 <0.0001 Sex (M/F) 12/11 11/12 0.77 Weight (kg) 90.9±11.1 79.5±8.8 <0.0001 Height (cm) 167±14 169±5 0.64 BMI (kg/m2) 32±4 28±3 <0.0001 Systolic BP (mmHg) 132±8 122±8 0.001 Diastolic BP (mmHg) 81±7 75±6 0.003 Duration of diabetes 4.5±2.2 N/A (years) HbA1c (mmol/mol) 51.2±11.4 37.2±2.2 <0.0001 HbA1c (%) 6.8±1.0 5.6±0.2 <0.0001 Total cholesterol (mmol/l) 4.2±1.0 4.8±0.77 0.014 Triglyceride (mmol/l) 1.7±0.7 1.34±0.6 0.055 HDL-cholesterol (mmol/l) 1.1±0.3 1.5±0.4 0.001 LDL-cholesterol (mmol/l) 2.23±0.8 2.7±0.87 0.051 CRP (mg/l) 3.0±2.7 5.1±10.3 0.33

BMI: Body mass index, BP: Blood pressure, HDL-cholesterol: High density lipoprotein cholesterol, LDL-cholesterol: Low density lipoprotein cholesterol, CRP: C-reactive protein.

HbA1c: Hemoglobin A1c Supplementary table 2. Student’s t-test values comparing protein levels for baseline versus hypoglycaemia and baseline versus 24-hours post- hypoglycemia in type 2 diabetes (T2D) and control subjects for the twenty-six included in the analysis. HSP = .

T2D vs Control Abbreviation (Baseline) Baseline vs Hypoglycaemia Baseline vs 24 hours T2D p- T2D p Control p-value Control p value value value HSP90AA1 alpha 0.44 0.88 0.95 0.72 0.85 0.5 HSP90AB1 HSP90 beta 0.26 0.59 0.12 0.65 Heat shock 70 kDa protein 0.27 HSPA1A 0.02 0.07 0.91 0.32 1A Heat shock cognate 71 kDa 0.20 HSPA8 0.06 0.78 0.51 0.51 protein 0.37 HSPB1 Heat shock protein beta-1 0.015 0.10 0.48 0.9

60 kDa heat shock protein, 0.74 HSPD1 0.72 0.33 0.50 0.40 mitochondrial Aminoacyl tRNA synthase 0.10 AIMP1 complex-interacting 0.07 0.60 0.96 0.09 multifunctional protein 1 0.36 CDC37 Hsp90 co- Cdc37 0.09 0.58 0.11 0.34 0.16 CLU 0.88 0.76 0.69 0.72 DnaJ homolog subfamily B 0.80 DNAJB1 0.08 0.23 0.38 0.60 member 1 MAP kinase-activated 0.65 MAPKAPK2 0.37 0.64 0.67 0.50 protein kinase 2 MAP kinase-activated 0.04 MAPKAPK5 0.04 0.27 0.83 0.07 protein kinase 5 Peptidyl-prolyl cis-trans 0.24 PPID 0.17 0.70 0.94 0.94 isomerase D Serine/threonine-protein 0.28 PPP3CA phosphatase 2B catalytic 0.72 0.056 0.25 0.27 subunit alpha isoform Stress-induced- 0.57 STIP1 0.10 0.66 0.006 0.09 phosphoprotein 1 E3 -protein ligase 0.01 STUB1 0.78 0.60 0.48 0.57 CHIP TLR4 Toll-like receptor 4 0.30 0.76 0.33 0.82 0.40 0.48 TLR4:MD-2 Toll-like receptor 4 in 0.05 0.72 0.52 0.19 complex complex with MD-2

HSP 90a/b HSP90 dimer 0.20 0.70 0.57 0.44 0.35 Programmed cell death 1 0.54 CD274 0.97 0.88 0.98 0.90 ligand 1 0.82 EPHA2 Ephrin type-A receptor 2 0.32 0.03 0.50 0.07

Mothers against 0.88 SMAD3 0.008 0.75 0.29 0.39 decapentaplegic homolog 3 Ubiquitin-conjugating 0.006 UBE2G2 0.83 0.90 0.76 0.50 enzyme E2 G2 Ubiquitin-conjugating 0.66 UBE2L3 0.09 0.37 0.04 0.27 enzyme E2L 3 Ubiquitin-conjugating 0.80 UBE2N 0.07 0.21 0.006 0.09 enzyme E2 N 0.46 Ubiquitin carboxyl-terminal UCHL1 0.37 0.73 0.69 0.30 hydrolase isozyme L1

Supplementary Figure 1 Hyperinsulinemic -e- Control Euglycemic clamp -It- T2D 12.5 :::::- **** 0 10.5 E .._..E 8.5 * Q) � Cl) 0 () 6.5 :J Ol 4.5 � 0 0 ca 2.5 0.5

BL 0 0.5 1 2 4 24 Time (h) 1Htpol

Supplementary Figure 2 Supplementary figure 3. Proteins that did not differ either between T2D and control subjects or during the post hypoglycemia time course.

Basal �ood collect�n Basal �oodcollection Basal bloodcollect�n Basal �ood collection BS(mM): 7.5 ± 0.4 (T2D) BS (mM): 7.5 ± 0.4 (T2D) BS (mM): 7.5 ± 0.4 (T2D) BS(mM): 7.5 ± 0.4 (T2D) 5.0±0.1(C) 5.0±0.1(C) 5.0 ±0.1 (C) 5.0±0.1(C)

Hypogycemia starts Hypoglycemiastarts Hypoglycemia starts Hypogycemiastarts BS (mM): 2.0± 0.03 (T2D) BS (mM): 2.0 ±O.Q3 (T2D) BS(mM): 2.0 ± 0.03 (T2D) BS (mM): 2.0 ± 0.03 (T2D) 1.8± 0.05 (C) 1.8±0.05(C) 1.8±0.05(C) 1.8 ± 0.05(C) A B C D 340 -e- Control 1300 -e- Control 200 -e- Control 2400 .e, Control • T2D ... T2D • Diabetic • T2D o::J�� Ol u. S' ::J S' ·-C Cl'.'.� u. u. Ql - [ ...... [ 0 'I"' [ 'I"' �I 270 1000 160 � � 1600 ....J ::t!oa.� I'- a:: I u Ol N ....J O 0 I- u Cl. ::J �(/) u ... I Cll-..... I 200 700 120 800 f------r f------r f------r f------r BL 0 0.5 2 4 24 BL 0 0.5 2 4 24 BL 0 0.5 2 4 24 BL 0 0.5 2 4 24 Time(h) Time(h) Time(h) Time(h) 1H!po1 � 1H!po1 1H!po1 Supplementary figure 4

Basal bloodcollection BS (mM): 7.5, 0.4 (T2D) Basal blood collection Basal blood collection 5.0,0.1 (C) BS (mM): 7.5, 0.4 (T2D) BS (mM): 7.5, 0.4 (T2D) A B 5.0,0.1 (C) C 5.0,0.1 (C) Hypoglycemia starts BS (mM): 2.0, 0.03 (T2D) Hypoglycemia starts Hypoglycemia starts -e- Control BS (mM): 2.0, 0.03 (T2D) 300 1.8, 0.05 (C) @ 400 BS (mM): 2.0, 0.03 (T2D) 1.8, 0.05 (C) T2D 1.8, 0.05 (C) ... 1500

5'250 5'300 LL LL i:2 1000 � � 0 .,..... � :=!

Basal bloodcollection Basal bloodcollection Basal blood collection D BS (mM): 7.5, 0.4 (T2D) E BS (mM): 7.5, 0.4 (T2D) BS (mM): 7.5, 0.4 (T2D) 5.0,0.1 (C) 5.0,0.1 (C) F 5.0,0.1 (C) -e- Control Hypoglycemia starts Hypoglycemia starts -e- Control Hypoglycemia starts 400 BS (mM): 2.0, 0.03 (T2D) BS (mM): 2.0, 0.03 (T2D) 500 BS (mM): 2.0, 0.03 (T2D) T2D 1.8, 0.05 (C) 1.8, 0.05 (C) 1.8, 0.05 (C) ... -e- Control 2000 ...T2D T2D ... 5' 5' LL 450 5'300 1500 LL � � � Ctl Ctl :§.400 N 1000 � .,..... � Ctl :=! 200 Ctl LL Z 350 z 500 I- LL =l 100 300 0 I--- I--- BL 0.5 1 2 4 24 I--- BL 0.5 1 2 4 24 Time [hours] BL 0.5 1 2 4 24 IHyp� IHyp� Time [hours] IHyp� Time [hours] G H - Control r=0.47, p=0.03 • Control 500 500 • T2D ....., • T20 ..... :::> 400 • ::, 400 • • LL • • LL • • • � 300 � 300 • .c • • .c N 200 • N 200 • � (0 • 100 ...J 100 .,;..' 0 0

0 2500 5000 7500 10000 0 100 200 300 400 500 2h [RFU] CDC37 2h [RFU] Supplementary figure legends

Supplementary Figure 1. Schematic figure showing an overview of interactions between

HSP and associated proteins that are differentially expressed in response to hypoglycemia. These interactions decide the fate of the downstream signaling pathway. The

HSP 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 and regulate different molecules affecting a spectrum of biological functions such as apoptosis, autophagy, cell migration and alterations in the immune response. The pathways depicted in the figure are the general pathways of

HSP related signaling and are not restricted to a certain tissue or cell type.

Schematic created using Biorender (https://biorender.com)

HSP90 alpha (HSP90AA1, HSP90AB1, HSP90 beta, HSP 90a/b HSP90 dimer);

HSPA1A, Heat shock 70 kDa protein 1A; HSPA8 Heat shock cognate 71 kDa protein ;

HSPB1 Heat shock protein beta-1; 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; SMAD3, Mothers against decapentaplegic homolog 3; E1,

Ubiquitin activating enzyme; E2, Ubiquitin conjugating enzymes 2 (UBE2G2, Ubiquitin- conjugating enzyme E2 G2; UBE2L3, Ubiquitin-conjugating enzyme; UBE2N,Ubiquitin- conjugating enzyme E2 N); UCHL1, Ubiquitin carboxyl-terminal hydrolase isozyme L1;

E3, Ubiquitin ligases; STUB1, E3 ubiquitin-protein ligase CHIP; NFB, 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; TGFR, Transforming growth factor beta receptors; IR,

Insulin receptor; IRS1, Insulin receptor substrate 1.

Supplementary Figure 2. The comparison of blood glucose levels at baseline, at hypoglycaemia and post-hypoglycaemia up to 24 h. Blood sampling was performed at baseline (BL), at hypoglycaemia (0 min) and post-hypoglycaemia (0.5, 1, 2, 4 and 24 h) for controls (white circles) and for type 2 diabetes (T2D) (black squares). At BL, blood sugar (BS) was 7.5 ± 0.4 mmol/l (for T2D) and 5.0 ± 0.1 mmol/l (for control, C). Insulin was infused (at a rate of 2 mU/ml/Kg body weight) by hyperinsulinemic euglycemic clamp. At hypoglycemia,

BS was 2.0 ± 0.03 mmol/l (for T2D) and 1.8 ± 0.05 mmol/l (for controls).

Supplementary Figure 3. Circulatory HSP and related proteins that did not differ with hypoglycemia or between T2D and controls. Proteomic (Somalogic) analysis was undertaken to determine the plasma levels of HSP related proteins, Toll-like receptor 4 (TLR4)

(A), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) (B), Heat shock protein

90AA1 (HSP90A1A) (C), Programmed cell death 1 ligand 1 (CD274) (D) 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-hour, 1-hour, 2-hours, 4-hours and 24-hours) for controls (white circles) and for T2D (black squares).

Supplementary Figure 4. Changes of circulatory pro-inflammatory and anti- inflammatory cytokines in response to hypoglycemia in control subjects and subjects with T2D. Proteomic (Somalogic) analysis was undertaken to determine the plasma levels of HSP related proteins, Interleukin 6 (IL-6) (A), Interleukin 10 (IL-10) (B), Interleukin 1A (IL-1A)

(C), Interleukin 12 (IL-12) (D), Interferon gamma (IFN gamma) (E), TNF alpha (TNF alpha)

(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-hour, 1-hour, 2-hours, 4-hours and 24-hours) for controls

(white circles) and for T2D (black squares). Correlations of plasma levels of IL-6 at 2-hours post-hypoglycemia with HSP70 (G) and CDC37 (H).