Severe hypoglycemia exacerbates myocardial dysfunction and metabolic remodeling in diabetic mice

Journal Pre-proof Severe hypoglycemia exacerbates myocardial dysfunction and metabolic remodeling in diabetic mice Lishan Huang, Yu Zhou, Zhou Chen, Meilian Zhang, Zhidong Zhan, Linxi Wang, Libin Liu PII:

S0303-7207(19)30394-6

DOI:

https://doi.org/10.1016/j.mce.2019.110692

Reference:

MCE 110692

To appear in:

Molecular and Cellular Endocrinology

Received Date: 26 August 2019 Revised Date:

21 December 2019

Accepted Date: 23 December 2019

Please cite this article as: Huang, L., Zhou, Y., Chen, Z., Zhang, M., Zhan, Z., Wang, L., Liu, L., Severe hypoglycemia exacerbates myocardial dysfunction and metabolic remodeling in diabetic mice, Molecular and Cellular Endocrinology (2020), doi: https://doi.org/10.1016/j.mce.2019.110692. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

1

Severe Hypoglycemia Exacerbates Myocardial Dysfunction and Metabolic

2

Remodeling in Diabetic Mice

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Lishan Huang1*, Yu Zhou2*, Zhou Chen2*, Meilian Zhang3, Zhidong Zhan1,

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Linxi Wang1, and Libin Liu1#

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1

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China

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2

Department of Endocrinology, Fujian Medical University Union Hospital, Fuzhou,

Department of Clinical Pharmacy and Pharmacy Administration, School of Pharmacy,

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Fujian Medical University, Fuzhou, China

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3

12

Fuzhou, China

Department of Ultrasound, Fujian Province Hospital for Women and Children,

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*

15

#

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University Union Hospital, Fuzhou 350001, Fujian, People’s Republic of China. Tel:

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+86-591-86218562, Email: [email protected]

These authors contributed equally to this work and share the first authorship. Corresponding author: Libin Liu, Department of Endocrinology, Fujian Medical

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1

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Abstract

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Although several studies have revealed that adverse cardiovascular events in

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diabetic patients are closely associated with severe hypoglycemia (SH)1, the causal

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relationship and related mechanisms remain unclear. This study aims to investigate

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whether SH promotes myocardial injury and further explores the potential

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mechanisms with focus on disturbances in lipid metabolism. SH promoted myocardial

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dysfunction and structural disorders in the diabetic mice but not in the controls. SH

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also enhanced the production of myocardial proinflammatory cytokines and oxidative

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stress. Moreover, myocardial lipid deposition developed in diabetic mice after SH,

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which was closely related to myocardial dysfunction and the inflammatory response.

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We further found that myocardial metabolic remodeling was associated with changes

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in PPAR-β/δ and its target molecules in diabetic mice exposed to SH. These findings

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demonstrate that SH exacerbates myocardial dysfunction and the inflammatory

32

response in diabetic mice, which may be induced by myocardial metabolic

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remodeling via PPAR-β/δ.

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Keywords: severe hypoglycemia; diabetes mellitus; myocardial dysfunction;

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metabolic remodeling

1

severe hypoglycemia

2

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1. Introduction

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Hypoglycemia is a common adverse side effect of hypoglycemic therapy in patients

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with diabetes mellitus (DM). Approximately 30% of type 1 diabetes mellitus (T1DM)

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patients have experienced severe hypoglycemia (SH) (Frier, 2014). Hypoglycemic

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events, particularly when severe, have been linked to subsequent adverse cardiac

42

outcomes and mortality in individuals with diabetes. Although there is suggestive

43

evidence linking hypoglycemia with cardiac disease, there is limited data regarding

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whether this link is causal, predictive of greater vulnerability, or both (Goto, Goto,

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Terauchi et al., 2016,Leong, Berkowitz, Triant et al., 2016,2019). Thus, there is an

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urgency to identify the influences and specific mechanisms linking SH with cardiac

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dysfunction in DM, which could indicate whether measures should be taken to protect

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the myocardium during correction of low blood sugar.

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The heart uses a large amount of fatty acids (FAs) as energy-providing substrates.

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More than 70% of all substrates used for adenosine triphosphate generation are

51

derived from FAs, with the remaining sources being glucose, lactate, ketone bodies,

52

and amino acids (Schulze, Drosatos and Goldberg, 2016). However, some studies

53

have indicated that the excess cardiac lipid content induced by free fatty acids (FFAs)

54

is linked to impaired systolic function and increased left ventricular mass (Carpenter,

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1962,Alpert, 2001). Furthermore, excess cardiac lipids can trigger an inflammatory

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response that is widely considered to be a critical risk factor for cardiovascular disease

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in diabetes with SH (Mani, Puri, Schwartz et al., 2019,Yang, Park and Zhou,

58

2016,Hotamisligil,

59

been demonstrated that both hypoglycemia and hyperglycemia can stimulate the

60

release of FFA (Peterson, Herrero, McGill et al., 2008,Winhofer, Krssak, Wolf et al.,

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2015). However, whether SH can further promote the release of FFA and aggravate

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myocardial lipid deposition in diabetic mice remains unclear.

2017,Joy,

Perkins,

Mikeladze

et

al.,

2016).

It

has

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In the present study, we established a T1DM animal model exposed to SH to

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observe myocardial changes within a short-term time period, which was closely

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related to long-term poor cardiovascular outcomes. We found that myocardial injury,

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metabolic remodeling, and proinflammatory effects occurred in diabetic mice after SH.

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Peroxisome proliferator-activated receptors (PPARs), including PPAR-α, PPAR-β/δ,

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and PPAR-γ, are considered core regulators in myocardium metabolism and are

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associated with cardiovascular disease (Schulze et al., 2016,Puddu, Cravero, Arnone

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et al., 2005,Barger and Kelly, 2000). Furthermore, PPAR-β/δ was identified as a

3

71

potential key regulator that mediates changes in the metabolism of the diabetic

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myocardium following SH.

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2. Materials and Methods

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2.1. Experimental animals

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A total of 60 male C57BL/6J mice (20−25 g) were purchased from the Department

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of Research Animal Center, Shanghai, China. All animals were housed at Fujian

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Medical University under controlled temperature and humidity and a 12/12-h

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dark-light cycle (lights on at 6:00 and off at 18:00), with food and water ad libitum.

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All experiments were approved by the Fujian Animal Research Ethics Committee

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(grant FJMU IACUC 2018-060) and were performed in accordance with the ARRIVE

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guidelines (Animal Research: Reporting In Vivo Experiments guidelines).

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2.2. Experiment grouping and establishment of the SH model in diabetic mice

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All mice were subdivided into the following four test groups (n = 15 per group):

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control group (NC), control + SH (NH), T1DM (DM), and T1DM + SH (DH).

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Diabetic conditions were induced in the DM and DH groups by a single

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intraperitoneal injection of streptozotocin (STZ; S0130; Sigma, St. Louis, MO, USA)

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dissolved in a 1% (w/v) solution of 0.1 M citrate buffer (pH 4.2–4.5) at a dose of 150

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mg/kg. A total of 30 age-matched control mice received an injection of citrate buffer

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alone. The random blood glucose level in mice was measured using a glucometer

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(Freestyle, Abbott, UK) on day 3 following STZ injection to determine whether

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diabetes was successfully induced in mice. Those without diabetes would receive

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retests on day 7. Mice with a random blood glucose level >16.7 mmol/L for three

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consecutive tests as well as behavioral markers of diabetes (increased consumption of

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food and drink, increased urination, and decreased weight) were defined as diabetic

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(Fig. 1A). Animals failing to meet these criteria were administered a second STZ

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injection and retested (Zhou, Huang, Zheng et al., 2018).

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Previous research has revealed that long-term chronic high blood glucose levels can

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mask the effects of hypoglycemia itself (Rezende, Everett, Brooks et al., 2018). To

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minimize the effects of long-term hyperglycemia on the body and investigate the

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effects of hyperglycemia, SH, and their interaction simultaneously, we subsequently

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performed an SH intervention after a successful induction of diabetes. The mice in the

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NH and DH groups were subjected to one episode of SH as previously described, with

4

104

minor modifications (Yu, Zhang, Sun et al., 2017,Wang, Ahmed, Jiang et al., 2017).

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Briefly, after an overnight fast, regular insulin (Wanbang, Jiangsu, China) was

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injected (2 mU/g intraperitoneally [i.p.] for NH mice and 15 mU/g i.p. for DH mice).

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Tail vein glucose was assessed every 30 min to ensure sustained SH levels (<2.0

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mmol/L) for 90 min (Puente, Silverstein, Bree et al., 2010). To terminate

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hypoglycemia, the mice were permitted free access to food or received glucose (1

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mg/kg i.p.). NC and DM mice were administered an equal volume of saline injections

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under the same conditions. Finally, two mice died in the NH group and three died in

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the DH group during SH. The rest of the mice were sacrificed following an

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echocardiographic assessment.

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2.3. Echocardiographic assessment

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Mice (n = 12 per group) were anesthetized with 2% isoflurane at 24 h after the SH

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challenge and placed in the supine position. Two-dimensional and M-mode

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transthoracic echocardiography was performed to evaluate cardiac function by a Vevo

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2100 high-resolution imaging system (Visual Sonics Inc., Toronto, Canada). M-mode

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tracing from the precordium was used to measure the left ventricular end-diastolic

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diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular

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anterior wall end-diastolic depth (LVAWd), and left ventricular posterior wall

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end-diastolic depth (LVPWd) in the short axis view. Left ventricle systolic function

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and mass were determined by calculating the left ventricular ejection fraction (LVEF),

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fractional shortening (FS), and left ventricular mass (LVM) as follows: LV

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end-diastolic (systolic) volume (LVED[S]V) was calculated as [(7.0/(2.4 + LVEDd (s)]

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× LVEDd(s)3. LVEF was calculated as 100 × [(LVEDV – LVESV)/LVEDV], and

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LVFS was calculated as [(LVEDd – LVEDs)/LVEDd] × 100. LVM(g) = 0.8 × 1.053 ×

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[(LVEDd + LVPWDd + LVAWd3 – LVEDd)3 – LVEDd3].

129

2.4. Histologic analyses

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Mice (n = 3 per group) were anesthetized with 5% isoflurane. Heart tissues were

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immediately dissected, fixed in 4% paraformaldehyde for 48 h, and embedded in

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paraffin. A. microtome (RM2016, Leica, Germany) was used to section the

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paraffin-embedded tissue, and 4-µm-thick cross sections were obtained, mounted on

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glass slides, and fixed in 4% formalin. For hematoxylin and eosin (H&E) staining, the

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slides containing heart sections were sequentially stained with hematoxylin, bluing

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solution, and eosin Y by gentle shaking at room temperature. Finally, the structure of

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the heart tissues was displayed with red cardiac fibers and a blue nucleus. To detect

5

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neutral lipids, the sections were stained with oil red O, dyed with hematoxylin, and

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fixed. Lipid droplets appeared as red dots following oil red staining. Heart slices were

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observed under a microscope (Eclipse Ni-U, Nikon Instruments Inc., Tokyo, Japan)

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and quantified using Image J software.

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2.5. Quantitative real-time polymerase chain reaction (PCR)

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Total RNA (n = 8 per group) was extracted from the heart tissue using an EASY

144

spin plus tissue RNA kit (AidLab, Beijing, China). Next, 1 µg total RNA was

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reverse-transcribed with a Prime Script™ RT reagent Kit (TaKaRa, Beijing, China)

146

according to the manufacturer’s instructions. The mRNA expression was quantified

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by quantitative real-time PCR with SYBR® Premix Ex Taq™ II (TaKaRa), including

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atrial natriuretic peptide (ANP); brain natriuretic peptide (BNP); PPARs containing

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PPAR-α, PPAR-β/δ, and PPAR-γ; cluster of differentiation 36 (CD36); fatty acid

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transporter 1 (FATP-1); carnitine palmityl transferase 1 (CPT-1); fatty acyl coenzyme

151

A synthetases (FACS); medium-chain acyl-CoA dehydrogenase (MACD); glucose

152

transporter 4 (GLUT4); and glucose transporter 1 (GLUT1). The complete details of

153

the primer sequences are presented in Table 1. All samples were assessed in triplicate,

154

and β-actin was the control gene for normalization. Data were analyzed using the

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comparative cycle threshold (Ct) method (∆∆Ct). These steps followed the latest

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Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Plain,

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Marsh, Waldron et al., 2014).

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2.6. Immunoblot analysis

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Cardiac protein was extracted using RIPA buffer with protease inhibitors (Beyotime

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Biotechnology, Jiangsu, China). The protein (20 µg) was separated on a 10% (w/v)

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sodium dodecyl sulfate–polyacrylamide gel and transferred onto a polyvinylidene

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difluoride membrane. The nonspecific binding sites on the membrane were blocked

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with a 5% (w/v) nonfat dry milk solution in 0.1% TBS/Tween-20 for 2 h at room

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temperature. The membranes were then incubated overnight at 4°C with the following

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primary antibodies: β-actin (cat. A2103, 1:1000; Sigma), CD36 (cat. DF13262,

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1:2000; Affinity), FATP1 (cat. DF7716, 1:2000; Affinity), CPT-1 (cat. 15184-1-AP,

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1:1000; Proteintech), FACS (cat. 4047s, 1:1000; Cell Signaling), MCAD

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(cat.55210-1-AP, 1:1000; Proteintech), GLUT4 (cat. ab654, 1:1000; Abcam), GLUT1

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(cat. ab652, 1:1000; Abcam), and PPAR-α (cat. ab24509, 1:1000; Abcam), PPAR-β/δ

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(cat. ab23673, 1:1000; Abcam), and PPAR-γ (cat. 2443s, 1:2000; Cell Signaling).

6

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After washing three times in 0.1% TBS/Tween-20, the membranes were incubated

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with a horseradish peroxidase–coupled anti-rabbit secondary antibody (cat. BA1050,

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1:5000; Boster) for 2 h at room temperature. The band density was quantified via

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densitometric analysis.

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2.7. Enzyme-linked immunosorbent assay (ELISA)

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Myocardial tissues were rinsed with ice-cold phosphate-buffered saline (PBS) to

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remove excess blood and then homogenized in PBS (9 mL PBS to 1 mg of tissue

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pieces) with a glass homogenizer on ice. The homogenate was used for detection of

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interleukin 6 (IL-6), interleukin 1β (IL-1β), interleukin 18 (IL-18), tumor necrosis

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factor α (TNF-α), reactive oxygen species (ROS), glutathione (GSH), triglycerides

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(TG), and ceramide. Blood samples were collected using serum separator tubes and

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allowed to clot overnight at 4°C. The serum was extracted, and the detection of FFA,

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cardiac troponin I (cTnI), and insulin was performed. Relevant ELISA (mlbio,

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Shanghai, China) kits were used for all of the above detections, according to the

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respective manufacturer’s instructions.

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Table 1. Summary of the primers used to measure mRNA expression related to

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heart failure and metabolism. Gene

Forward primer sequence (5’-3’)

Reverse primer sequence (5’-3’)

ANP

ACCTGCTAGACCACCTGGAG

CCTTGGCTGTTATCTTCGGTACCGG

BNP

GAGGTCACTCCTATCCTCTGG

GCCATTTCCTCCGACTTTTCTC

PPAR-α

AGAGCCCCATCTGTCCTCTC

ACTGGTAGTCTGCAAAACCAAA

PPAR-β/δ

TCGGGCTTCCACTACGG

ACTGACACTTGTTGCGGTTCT

PPAR-γ

GCCATTGAGTGCCGAGTCTGT

GCATCCGCCCAAACCTGA

CD36

ATGGGCTGTGATCGGAACTG

GTCTTCCCAATAAGCATGTCTCC

FATP1

CTGGGACTTCCGTGGACCT

TCTTGCAGACGATACGCAGAA

FACS

GGAGCTTCGCAGTGGCATC

CCCAGGCTCGACTGTATCTTGT

CPT-1

CTCCGCCTGAGCCATGAAG

CACCAGTGATGATGCCATTCT

MCAD

GATCGCAATGGGTGCTTTTGATAGAA

AGCTGATTGGCAATGTCTCCAGCAAA

GLUT4

CCTTTGCACACGGCTTCCGA

TGTTCAATCACCTTCTGTGGGGCA

GLUT1

GAAGAGGGTCGGCAGATGA

CGAAGATGCTCGTTGAGTAGTAGA

β-actin

GGCTGTATTCCCTCCATCG

CCAGTTGGTAACAATGCCATG

189 190

2.8. Statistical analysis

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Data are presented as mean ± SEM. Statistical analysis was performed using SPSS

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25.0 (Chicago, IL, USA). The effects of hyperglycemia, SH, and their interaction

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were tested by a two-way non–repeated-measures analysis of variance. A least

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significant difference test was used as the post hoc test for multiple group

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comparisons. A P value of <0.05 was considered statistically significant.

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3. Results

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3.1. Characterization of experimental mice

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As expected, compared with the NC group, mice in the DM and DH groups

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presented with significantly higher values of glycemia (P < 0.001; P < 0.001; Fig. 1B)

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and lower serum insulin levels (P < 0.001; Fig. 1C), as well as a significant decrease

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in body weight (P < 0.001; P < 0.001; Fig. 1D), confirming their diabetic status. Fig.

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1E shows that the levels of glucose in mice in the NH and DH groups were

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maintained at less than 2.0 mmol/L during SH.

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Fig. 1. Characterization of experimental mice. (A) Diagram illustrating the

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experimental protocol. (B) Level of glycemia among the four experimental groups. (C)

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Serum insulin levels of the four groups. (D) Body weight measured from four groups.

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(E) Glucose levels among the four groups during the SH episode (<2.0 mmol/L; n =

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15 per group); &&&P < 0.001, DM vs NC; +++P < 0.001, DH vs NC.

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3.2. SH exacerbates cardiac dysfunction in diabetic mice

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Echocardiography was employed to evaluate cardiac function. The representative

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M-mode images showed that compared with the NC group, the DM group exhibited

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decreased cardiac systolic function, which was further reduced following SH (Fig.

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2A). Both LVEF and FS were decreased in the DM group compared with the NC

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group (P < 0.05; Fig. 2B and C) but were decreased in the DH group compared with

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the DM group (P < 0.001; Fig. 2B and C). The LVM of the DH group exhibited

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higher levels than the DM group; however, there were few differences between the

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DM and NC groups (P < 0.05; Fig. 2D). Moreover, the above parameters did not

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show any differences between the NH and NC groups, indicating that SH attenuates

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cardiac function in diabetic mice but not in the control mice.

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The content of serum cTnI, as well as the relative expression of ANP and BNP, was

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further tested to evaluate myocardial injury. Consistent with the echocardiography

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results, cTnI increased in the DM group compared with the NC group (P < 0.001; Fig.

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2E) and was further elevated after the diabetic mice experienced SH (P < 0.01; Fig.

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2E). However, there were no significant changes in the NH group compared with the

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NC group (P > 0.05; Fig. 2E). The mRNA expression results revealed that both ANP

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and BNP were increased in the DM and DH groups compared with the NC group;

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however, there was no significant difference between the DM and DH groups (ANP, P

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< 0.001; BNP, P < 0.001; Fig. 2F and G). This finding indicates that even short-term

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DM was sufficient to impair the myocardial situation of mice, and SH can exacerbate

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such injury based on the diabetic situation.

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Fig. 2. SH exacerbates cardiac dysfunction in diabetic but not control mice. (A)

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Representative M-mode echocardiographic images revealed the LV systolic function

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among the four groups. (B−D) The mean percentage LV, percentage FS, and LVM (n

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≥ 12 per group) as assessed by an echocardiographic analysis. (E) Serum cardiac

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troponin I of the mice tested by ELISA. (F−G) Real-time PCR analyses of ANP and

240

BNP among the four groups. & P < 0.05; && P < 0.01; &&& P < 0.001, DM vs NC; +++ P

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< 0.001, DH vs NC; # P < 0.05; ## P < 0.01; ### P < 0.001, DH vs DM.

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3.3. SH induces myocardial fiber dissolution in diabetic mice

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H&E staining was used to observe the myocardial morphology of the mice. As

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presented in Fig. 3A and B, compared with the NC group, the DM and DH groups

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displayed a significant disorder of the myocardial arrangement with more internal

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loose layers and lower external dense layers. Moreover, multiple dissolutions of the

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myocardial fibers appeared, representing degradation of the cardiac muscle,

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presenting several irregular holes in representative H&E microphotographs, after the

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diabetic mice experienced SH (P < 0.001; Fig. 3C).The study further strengthened the

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evidence of cardiac damage caused by SH on diabetes.

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Fig. 3. Cardiac structural damage was induced by SH in diabetic mice. (A) H&E

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staining of the myocardium was observed under 10× and 200×, respectively, with an

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optical microscope. The blue arrows indicate the sites of cardiac fiber dissolution. (B)

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The relative area of loose layer/dense layers. (C) Quantification of the dissolved area

257

of cardiac fibers. &&& P < 0.001, DM vs NC; ### P < 0.001, DH vs DM.

258 259

3.4. SH promotes myocardial inflammation and oxidative stress

260

The inflammatory reaction was suspected to be one of the most pivotal factors

261

contributing to the cardiovascular events induced by hypoglycemia (Hanefeld, Frier

262

and Pistrosch, 2016). The relative proinflammatory cytokine expression, including

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TNF-α, IL-6, IL-1β, and IL-18, in the hearts of mice was tested. All of the above

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proinflammatory cytokines were higher in the DM group than in the NC group (P <

265

0.05; Fig. 4A− −D). The level of TNF-α, IL-6, and IL-18 were elevated in the NH group

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compared with the NC group (P < 0.05; Fig 4A, B, and D). However, only TNF-α

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and IL-1β were further increased in the DH group compared with the DM group (P <

268

0.05; Fig 4A and C). Because the inflammatory signal is associated with the

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overproduction of cytosolic and mitochondrial ROS (Pashkow, 2011), which further

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enhances the inflammatory effects, we further tested the presence of related molecules

271

containing ROS and GSH. Compared with the NC group, both the NH and DM

272

groups manifested higher oxidative stress, exhibiting higher ROS (P < 0.05; P < 0.001;

273

Fig. 4E) and lower GSH (P < 0.05; P < 0.001; Fig 4 F). The diabetic mice freed more

274

ROS synchronized with reduced GSH after exposure to SH (P < 0.05; Fig. 4E and F).

12

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Fig. 4. SH promotes myocardial inflammation and oxidative stress. (A–D) The

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mean expression of proinflammatory factors, TNF-α, IL-6, IL-1β, and IL-18 tested

278

with an ELISA. (E−F) Related indicators of oxidative stress, ROS and GSH, tested by

279

an ELISA; * P < 0.05; ** P < 0.01; *** P < 0.001, NH vs NC; & P < 0.05; && P < 0.01;

280

&&&

P < 0.001, DM vs NC; # P < 0.05; ## P < 0.01; ### P < 0.001, DH vs DM.

281 282

3.5. SH promotes myocardial lipid deposition in diabetic mice

283

To determine whether SH could induce myocardial lipid metabolism disorder

284

related to heart function and inflammation, oil red staining was used to observe the

285

presence of myocardial lipids. The DH group exhibited higher myocardial lipid

286

deposition compared with the DM group (Fig. 5A and B), consistent with a higher

287

myocardial TG content (P < 0.01; Fig. 5C). In contrast, ceramide, a toxic lipid

13

288

derivative, did not appear to exhibit obvious changes among those groups (Fig. 5D).

289

Interestingly, higher levels of FFA were observed in the NH and DM groups compared

290

with the NC group (P < 0.01; P < 0.001; Fig. 5E), as well as in the DH group

291

compared with the DM group (P > 0.001; Fig. 5E).

292 293

Fig. 5. SH induced myocardial lipid deposition in diabetic mice (A) Representative

294

images of oil red O–stained heart histology. The blue arrowheads indicate significant

14

295

lipid deposition (n = 3–4 per group; 10× and 200×). (B) Quantification of red oil

296

staining. (C−D) The content of myocardial TG and ceramide tested by ELISA (n = 8

297

per group). (E) Free fatty acids derived from the serum (n = 8 per group). * P < 0.05;

298

**

299

NC; # P < 0.05; ## P < 0.01; ### P < 0.001, DH vs DM.

P < 0.01;

***

P < 0.001, NH vs NC; & P < 0.05;

&&

P < 0.01; &&& P < 0.001, DM vs

300 301

3.6. SH inhibited myocardial metabolism related to PPAR-β β /δ δ in diabetic mice.

302

We speculated that SH may reprogram myocardial metabolism in mice to varying

303

degrees, which can be attributed to different basal conditions. To shed light on this

304

presumption, the relative mRNA expression to myocardial FAs uptake and oxidation

305

embracing CD36, FATP1, CPT-1, FACS, and MCAD received further testing.

306

Compared with the NC group, the DM group displayed significantly strengthened

307

myocardial FAs uptake and oxidation (P < 0.001; Fig. 6A). Although the NH group

308

did not reach statistical significance compared with the NC group, all relative mRNA

309

expression appeared to remain on an upward trend (Fig. 6A). However, there was

310

reduced FAs uptake and oxidation, as well as decreased expression of the glucose

311

transporter, GLUT4/GLUT1, in the DH group compared with the DM group (P < 0.05;

312

Fig. 6A). Since the above metabolic indicators served as the downstream target

313

molecules for the PPARs (i.e., PPAR-α, PPAR-β/δ, and PPAR-γ (Lee, Bai, Lee et al.,

314

2017), further examination of PPARs was considered necessary. Not unexpectedly,

315

key transcriptional regulators of FA metabolism, PPAR-α, PPAR-β/δ, and PPAR-γ,

316

were all increased in the DM group compared with the NC group; however, PPAR-β/δ

317

and PPAR-γ were decreased in the DH group after SH delivery (P < 0.001; P < 0.05;

318

Fig. 6B); in particular, PPAR-β/δ was reduced by virtually 32%.

319

The relevant proteins were then detected to further confirm the impact of SH on

320

myocardial metabolism. The immunoblot analysis revealed that except for CPT-1,

321

FACS, GLUT4/GLUT1, and PPAR-α, the corresponding proteins increased in the

322

DM group compared with the NC group (P < 0.05; Fig. 6C− −F). Consistent with gene

323

expression, SH reduced the relative protein expression to myocardial fatty acid uptake

324

and oxidation in diabetic mice (P < 0.01; Fig. 6C− −F). However, different from gene

325

expression, compared with the NC group, the expression of CD36, FATP1, MCAD,

326

PPAR-β/δ, and PPAR-γ proteins was increased in the NH group, with decreased

327

CPT-1 (P < 0.05; Fig. 6C− −F).

15

328 329

Fig. 6. SH inhibited myocardial metabolism in diabetic mice. Real-time PCR

330

analysis of the gene expression involved in myocardial fatty acid transportation,

331

oxidation, and glucose uptake (n = 8 per group). (B) Gene expression of myocardial

332

transcriptional regulator PPARs (n = 8 per group). (C) Representative immunoblot

16

333

images of relative protein expression (n = 6 per group). (D) Quantification of relative

334

protein expression involved in myocardial fatty acid transportation and oxidation. (E)

335

Quantification of the level of glucose transporter protein expression. (F)

336

Quantification of PPARs in the level of proteins; * P < 0.05; ** P < 0.01; *** P < 0.001,

337

NH vs NC; & P < 0.05; && P < 0.01; &&& P < 0.001, DM vs NC; # P < 0.05; ## P < 0.01;

338

###

P < 0.001, DH vs DM.

339 340

4. Discussion

341

In our study, we found that SH was associated with a deterioration in the

342

myocardial function of diabetic mice through an intraperitoneal injection of insulin.

343

SH simultaneously promoted the myocardial inflammatory response and release of

344

FFA in both control and diabetic mice. However, myocardial lipid deposition

345

developed in diabetic mice after SH, which could impair heart function. This led us to

346

further discover that SH exerted myocardial metabolic reprogramming in association

347

with PPAR-β/δ in diabetic mice.

348

There is accumulating evidence showing that hypoglycemia can cause cardiac

349

dysfunction and sudden death (2019), as evidenced by case reports of various cardiac

350

arrhythmias induced by hypoglycemia and studies reporting abnormal cardiac

351

repolarization (Reno, VanderWeele, Bayles et al., 2017,Reno, Daphna-Iken, Chen et

352

al., 2013). However, the effects of hypoglycemia on the heart following the correction

353

of blood glucose tended to be ignored. In this study, we found that SH impaired the

354

cardiac systolic function and structure in diabetic mice but not in control mice. cTnI is

355

a marker of myocardial injury with high sensitivity and specificity (Apple, Sandoval,

356

Jaffe et al., 2017), for which the increase in the DH group revealed that SH induces

357

myocardial injury in diabetic mice. However, it is interesting that the DM group also

358

exhibited cardiac dysfunction, including decreased systolic function and increased

359

short-term cTnI. Compared with the NC group, the myocardial capacity of

360

compensation in the DM group has been reduced. This finding may explain why SH

361

was associated only with a deterioration in myocardial dysfunction in diabetic mice.

362

However, the application of new technology, including echocardiographic strains and

363

strain rate imaging, enables a more reliable and comprehensive assessment of

364

myocardial function (Dandel and Hetzer, 2009). It remains unknown whether SH

365

could affect cardiac function in control mice.

17

366

Inflammatory signaling has been considered to be a critical risk factor for

367

cardiovascular disease in diabetes with SH and usually occurs as an early response to

368

myocardial injury (Fuentes-Antras, Ioan, Tunon et al., 2014). Previous studies found

369

that hypoglycemia can promote the upregulation and release of inflammatory markers

370

(e.g., IL-6, high-sensitivity C-reactive protein, and soluble CD40 ligand) expressed in

371

monocytes in type 1 diabetes (Wright, Newby, Stirling et al., 2010,Gogitidze Joy,

372

Hedrington, Briscoe et al., 2010). These indicators were tested in the blood indirectly

373

obtained from the heart tissue. In our study, we observed that DM significantly

374

stimulated the production of myocardial proinflammatory cytokines and oxidative

375

stress, maintaining levels similar to that observed in previous reports (Kayama, Raaz,

376

Jagger et al., 2015,Bajpai and Tilley, 2018), even in the short term. Although SH

377

could trigger an inflammatory response in the hearts of both the control and diabetic

378

mice, changes in the myocardial inflammatory factors induced by SH varied under

379

different baseline situations. However, because inflammation inordinately exists in the

380

DM group compared with the NC group, it could not be concluded that SH tends to

381

stimulate the myocardial inflammation in control mice compared with that of diabetic

382

mice. Thus, it cannot be excluded that the anti-inflammatory activity of insulin acted

383

on the DM group (Bajpai and Tilley, 2018). ROS is normally produced within the

384

body in limited quantities and constitutes a necessary compound involved in the

385

regulation of processes involving the maintenance of cell homeostasis and

386

functionality (e.g., activation of receptors, signal transduction, and gene expression)

387

(Holmstrom and Finkel, 2014). However, when excess ROS cannot be rapidly cleared,

388

the redox system is imbalanced, which further intensifies the inflammatory response

389

(Hussain, Tan, Yin et al., 2016). Therefore, SH results in myocardial oxidative stress

390

in both control and diabetic mice with increased ROS and decreased GSH. Although

391

the inflammatory response and oxidative stress were increased in the NH group, few

392

changes were found regarding cardiac function with a low expression of cTnI, ANP,

393

and BNP. Aside from the demand for a more sensitive indicator of cardiac function,

394

the heart microstructure or other metabolic functions of the NH group might precede

395

the functional deterioration (Genet, Lee, Baillargeon et al., 2016,Luptak, Qin,

396

Sverdlov et al., 2019). This also implies that appropriately increased inflammation

397

and oxidative stress may be beneficial for self-protection (Holmstrom and Finkel,

398

2014,Medzhitov, 2008).

399

In their study published in 2015, Winhoferÿ et al. found that hypoglycemia could

18

400

promote the release of FFA in patients (Winhofer et al., 2015). Moreover, excess lipid

401

accumulation in the heart can produce “lipotoxicity,” which refers to toxicity arising

402

from the cellular accumulation of lipids and lipid derivatives, leading to an alteration

403

in the morphological structure as well as impaired myocardial performance (Zlobine,

404

Gopal and Ussher, 2016). Lipotoxicity can also promote cardiomyocyte apoptosis via

405

increased ROS production, endoplasmic reticulum stress, and inflammation, leading

406

to the development of heart failure (Ertunc and Hotamisligil, 2016). Our findings

407

identified that FFA was increased in the NH, DM, and DH groups; however, it is

408

important to note that only the DH group developed myocardial deposition.

409

Becausethere is relatively tight coupling between lipid uptake, and oxidation prevents

410

the accumulation of excess lipids in cardiomyocytes (Schulze et al., 2016), it is highly

411

likely that SH might compromise the capacity of cardiac lipid metabolism in diabetic

412

mice. This study revealed that diabetes stimulates fatty acid metabolism of the

413

myocardium, consistent with previous findings (Ritchie, Zerenturk, Prakoso et al.,

414

2017). It is interesting that SH significantly inhibited the lipid metabolism of diabetic

415

mice but stimulated the control mice. This may explain why only the DH group

416

presented with myocardial lipid accumulation, which was not observed in the NH and

417

DM groups, despite the high FFA exhibited in all three groups. It is worth considering

418

that changes in fatty acid metabolism appeared at the protein level but not at the gene

419

level after control mice suffered from SH. This may suggest an important role in

420

protein posttranslational modification (Yang, He, Wang et al., 2017,Yang and Qian,

421

2017,Phillips and Kriwacki, 2019). However, whether this mechanism was weakened

422

in diabetic mice requires further research. GLUT4 is the major isoform that represents

423

approximately 70% of the total glucose transporters involved in myocardial glucose

424

uptake (Mueckler and Thorens, 2013). We found that SH depresses glucose uptake in

425

diabetic mice; however, diabetes did not show a reduction, which appeared to be

426

slightly different from that described in previous studies (Ritchie et al., 2017). PPARs

427

are nuclear hormone receptors and major executors of the modulation of glucose and

428

lipid homeostasis (Barger and Kelly, 2000). There are three PPAR isoforms, including

429

PPAR-α, PPAR-β/δ, and PPAP-γ, which differ in terms of distribution, function, and

430

ligand specificity, and each play a crucial role in cardiovascular disease (Schulze et al.,

431

2016,Puddu et al., 2005). PPAR-α has been found to promote the expression of genes

432

involved in cardiac fatty acid uptake and oxidation pathways and reciprocally

433

suppresses the expression of genes involved in glucose transport and utilization (Finck,

19

434

Lehman, Leone et al., 2002) . However, mice exhibiting heart-specific overexpression

435

of PPAR-α (MHC-PPAR-α) exhibited lipid deposition and myocardial lipid toxicity

436

despite increased myocardial fatty acid metabolism during high-fat feeding (Finck et

437

al., 2002). In contrast to PPAR-α, PPAR-β/δ promotes both the oxidation of fatty acids

438

and the activation of cardiac glucose transport and glycolytic genes (Burkart,

439

Sambandam, Han et al., 2007). Simultaneously, PPAR-β/δ will not increase the uptake

440

of myocardial fatty acids, which is why MHC-PPAR mice do not exhibit lipid

441

deposition and cardiac dysfunction during high-fat feeding compared with

442

MHC-PPAR-α (Burkart et al., 2007). PPAR-γ displays less abundance in the heart,

443

and it could increase the cardiac expression of fatty acid oxidation genes and

444

lipoprotein TG uptake but not influence heart glucose transporter 4 (GLUT4) mRNA

445

expression (Son, Park, Yamashita et al., 2007).

446

In our study, PPAR-β/δ and PPAR-γ expression were increased during the early

447

stages of DM, indicating why the DM group displayed increased metabolism of fatty

448

acids but not decreased glucose uptake. In addition, both PPAR-β/δ and PPAR-γ were

449

downregulated after diabetic mice experienced SH, of which PPAR-β/δ was reduced

450

by approximately 32% at the gene level and 28% at the protein level, leading to the

451

reduction of fatty acid metabolism and glucose uptake. Thus, we considered that the

452

induction of myocardial lipid accumulation in the DH group may be associated with

453

the inhibition of PPAR-β/δ.

454

Compared with the control mice, diabetic mice are associated with some risk

455

factors that may cause cardiac dysfunction, including abnormal lipid metabolism

456

(Peterson et al., 2008), cardiac electrical conduction disorder (Zhang, Wang, Yanni et

457

al., 2019), inflammation (Fuentes-Antras et al., 2014), and blood hypercoagulation

458

(van der Toorn, de Mutsert, Lijfering et al., 2019). Hypoglycemia can also cause such

459

effects (Joy et al., 2016,Winhofer et al., 2015,Reno et al., 2013,Wright et al., 2010,Joy,

460

Tate, Younk et al., 2015) . Therefore, the above factors may be further amplified to

461

cause cardiac dysfunction in diabetic mice following SH. However, compared with

462

diabetic mice, control mice display a stronger compensatory ability. The lack of the

463

above pathological changes may explain why the control mice did not exhibit cardiac

464

dysfunction after SH. In our study, we found that hypoglycemia stimulated the release

465

of FFAs in both control and diabetic mice. However, SH promoted the lipid

466

metabolism of control mice but was inhibited in diabetic mice, which resulted in

20

467

myocardial lipid deposition in diabetic mice following SH, which may impair their

468

heart function.

469

The normal heart tends to prefer FFA as energy substrates and exhibits remarkable

470

fuel flexibility, switching the metabolic substrate when it becomes abundantly

471

available (Schulze et al., 2016). During hypoglycemia, the energy consumption of

472

myocardial contraction and FFAs increase, both of which could assist the control mice

473

in effectively meeting the energy demand under SH via increased cardiac lipid

474

metabolism, so as to improve the resistance of the heart to hypoglycemia (Winhofer et

475

al., 2015,Vileigas, Harman, Freire et al., 2019). However, myocardial lipid

476

metabolism has been in a state of compensatory increase in diabetes (Kolwicz, Purohit

477

and Tian, 2013). With the high oxygen consumption rate of lipid metabolism, the

478

heart tends to produce more inflammatory factors and oxygen free radicals

479

(Evangelista, Nuti, Picchioni et al., 2019). The myocardial lipid metabolism relies

480

greatly on mitochondrial oxidative phosphorylation, but overactive inflammatory

481

signaling and ROS are believed to be strong inhibitors of mitochondrial oxidative

482

phosphorylation, leading to an energy crisis (Lee and Huttemann, 2014). Actually,

483

although the rate of myocardial lipid metabolism is increased in the diabetic state, the

484

efficiency of energy production decreases conversely (Boudina, Sena, Theobald et al.,

485

2007). SH elevates the demand for myocardial energy metabolism in diabetic mice.

486

However, the further load of FFAs, oxidative stress, and the inflammatory response

487

may induce the myocardial compensatory lipid metabolism into the decompensated

488

state, further aggravating the cardiac dysfunction of diabetic mice (Lee and

489

Huttemann, 2014,Wen, Velmurugan, Day et al., 2017).

490

Nevertheless, the present study had several limitations. First, we did not administer

491

a PPAR-β/δ inhibitory agent or genetic intervention, which was insufficient to prove

492

that SH deteriorates the myocardial function of diabetic mice through a lipid

493

metabolism disorder mediated by PPAR-β/δ. Thus, it is necessary to upregulate the

494

expression of PPAR-β/γ and observe the corresponding changes on myocardial

495

function in future studies. Second, we primarily considered the potential damage

496

caused by a disturbance in myocardial lipid metabolism. Because both fatty acid and

497

glucose uptake were decreased in the DH group, there may be a reason to consider

498

that often ignored disorders of the tissue microstructures (e.g., mitochondria) may also

499

be affected. Such effects could also reduce the level of myocardial energy supply,

500

excessive inflammation, and ROS. Finally, whether high doses of STZ could result in

21

501

myocardium toxicity remains unclear.

502 503

5. Conclusion

504

Our study revealed that SH exacerbated myocardial injury and enhanced

505

myocardial inflammation in diabetic mice. Myocardial metabolic remodeling may be

506

mediated by PPAR-β/δ, which can lead to myocardial injury triggered by diabetic

507

hypoglycemia. Implications of the current study could lead to the improvement in

508

treatment strategies that pay careful attention to cardiac function after correcting for

509

blood glucose in hypoglycemia. Moreover, further research into the specific

510

mechanisms of SH aggravating cardiac dysfunction in diabetic mice is required.

511

22

512 513 514

Disclosure statement The authors declare no conflict of interests. Funding

515

This work was supported by the provincial key clinical specialty construction

516

project fund of the Endocrinology Department (Fujian-Wei Medical Policy Letter

517

[2015] 593 hao), the Financial Department Special Funds of Fujian Province

518

(2018B041), and the Startup Fund for scientific research of Fujian Medical University

519

(2018QH2031).

520

Acknowledgements

521

Because it is difficult to list all the participants in this study as authors, I would like

522

to express my heartfelt thanks to the following participants. Thank you to Jinguo

523

Chen, the Director of the Ultrasound Department of Fujian Medical University Union

524

Hospital, and his graduate student, Xiaodong Li, for their technical guidance on a

525

small animal ultrasound. Thank you for the small animal ultrasound imaging platform

526

provided by Fujian University of Traditional Chinese Medicine.

527

23

528

References

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[1] [2]

[3]

[4]

[5] [6] [7] [8]

[9]

[10] [11]

[12]

[13]

[14]

Frier, B.M., 2014. Hypoglycaemia in diabetes mellitus: epidemiology and clinical implications, Nat Rev Endocrinol. 10, 711-22. Goto, A., Goto, M., Terauchi, Y., Yamaguchi, N. and Noda, M., 2016. Association Between Severe Hypoglycemia and Cardiovascular Disease Risk in Japanese Patients With Type 2 Diabetes, J Am Heart Assoc. 5, e002875. Leong, A., Berkowitz, S.A., Triant, V.A., Porneala, B., He, W., Atlas, S.J., Wexler, D.J. and Meigs, J.B., 2016. Hypoglycemia in Diabetes Mellitus as a Coronary Artery Disease Risk Factor in Patients at Elevated Vascular Risk, J Clin Endocrinol Metab. 101, 659-68. 2019. Hypoglycaemia, cardiovascular disease, and mortality in diabetes: epidemiology, pathogenesis, and management, Lancet Diabetes Endocrinol. 7, 385-396. Schulze, P.C., Drosatos, K. and Goldberg, I.J., 2016. Lipid Use and Misuse by the Heart, Circ Res. 118, 1736-51. Carpenter, H.M., 1962. Myocardial fat infiltration, Am Heart J. 63, 491-6. Alpert, M.A., 2001. Obesity cardiomyopathy: pathophysiology and evolution of the clinical syndrome, Am J Med Sci. 321, 225-36. Mani, P., Puri, R., Schwartz, G.G., Nissen, S.E., Shao, M., Kastelein, J.J.P., Menon, V., Lincoff, A.M. and Nicholls, S.J., 2019. Association of Initial and Serial C-Reactive Protein Levels With Adverse Cardiovascular Events and Death After Acute Coronary Syndrome: A Secondary Analysis of the VISTA-16 Trial, JAMA Cardiol. Yang, S.W., Park, K.H. and Zhou, Y.J., 2016. The Impact of Hypoglycemia on the Cardiovascular System: Physiology and Pathophysiology, Angiology. 67, 802-9. Hotamisligil, G.S., 2017. Inflammation, metaflammation and immunometabolic disorders, Nature. 542, 177-185. Joy, N.G., Perkins, J.M., Mikeladze, M., Younk, L., Tate, D.B. and Davis, S.N., 2016. Comparative effects of acute hypoglycemia and hyperglycemia on pro-atherothrombotic biomarkers and endothelial function in non-diabetic humans, J Diabetes Complications. 30, 1275-81. Peterson, L.R., Herrero, P., McGill, J., Schechtman, K.B., Kisrieva-Ware, Z., Lesniak, D. and Gropler, R.J., 2008. Fatty acids and insulin modulate myocardial substrate metabolism in humans with type 1 diabetes, Diabetes. 57, 32-40. Winhofer, Y., Krssak, M., Wolf, P., Anderwald, C.H., Geroldinger, A., Heinze, G., Baumgartner-Parzer, S., Marculescu, R., Stulnig, T., Wolzt, M., Trattnig, S., Luger, A. and Krebs, M., 2015. Free fatty acid availability is closely related to myocardial lipid storage and cardiac function in hypoglycemia counterregulation, Am J Physiol Endocrinol Metab. 308, E631-40. Puddu, G.M., Cravero, E., Arnone, G., Muscari, A. and Puddu, P., 2005. Molecular aspects of atherogenesis: new insights and unsolved questions, J Biomed Sci. 12, 839-53.

24

572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615

[15] [16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

Barger, P.M. and Kelly, D.P., 2000. PPAR signaling in the control of cardiac energy metabolism, Trends Cardiovasc Med. 10, 238-45. Zhou, Y., Huang, L., Zheng, W., An, J., Zhan, Z., Wang, L., Chen, Z. and Liu, L., 2018. Recurrent nonsevere hypoglycemia exacerbates imbalance of mitochondrial homeostasis leading to synapse injury and cognitive deficit in diabetes, Am J Physiol Endocrinol Metab. 315, E973-E986. Rezende, P.C., Everett, B.M., Brooks, M.M., Vlachos, H., Orchard, T.J., Frye, R.L., Bhatt, D.L. and Hlatky, M.A., 2018. Hypoglycemia and Elevated Troponin in Patients With Diabetes and Coronary Artery Disease, J Am Coll Cardiol. 72, 1778-1786. Yu, J., Zhang, Y., Sun, W., Kahkoska, A.R., Wang, J., Buse, J.B. and Gu, Z., 2017. Insulin-Responsive Glucagon Delivery for Prevention of Hypoglycemia, Small. 13. Wang, C.K., Ahmed, M.M., Jiang, Q., Lu, N.N., Tan, C., Gao, Y.P., Mahmood, Q., Chen, D.Y., Fukunaga, K., Li, M., Chen, Z., Wilcox, C.S., Lu, Y.M., Qin, Z.H. and Han, F., 2017. Melatonin ameliorates hypoglycemic stress-induced brain endothelial tight junction injury by inhibiting protein nitration of TP53-induced glycolysis and apoptosis regulator, J Pineal Res. 63. Puente, E.C., Silverstein, J., Bree, A.J., Musikantow, D.R., Wozniak, D.F., Maloney, S., Daphna-Iken, D. and Fisher, S.J., 2010. Recurrent moderate hypoglycemia ameliorates brain damage and cognitive dysfunction induced by severe hypoglycemia, Diabetes. 59, 1055-62. Plain, K.M., Marsh, I.B., Waldron, A.M., Galea, F., Whittington, A.M., Saunders, V.F., Begg, D.J., de Silva, K., Purdie, A.C. and Whittington, R.J., 2014. High-throughput direct fecal PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in sheep and cattle, J Clin Microbiol. 52, 745-57. Hanefeld, M., Frier, B.M. and Pistrosch, F., 2016. Hypoglycemia and Cardiovascular Risk: Is There a Major Link?, Diabetes Care. 39 Suppl 2, S205-9. Pashkow, F.J., 2011. Oxidative Stress and Inflammation in Heart Disease: Do Antioxidants Have a Role in Treatment and/or Prevention?, Int J Inflam. 2011, 514623. Lee, T.W., Bai, K.J., Lee, T.I., Chao, T.F., Kao, Y.H. and Chen, Y.J., 2017. PPARs modulate cardiac metabolism and mitochondrial function in diabetes, J Biomed Sci. 24, 5. Reno, C.M., VanderWeele, J., Bayles, J., Litvin, M., Skinner, A., Jordan, A., Daphna-Iken, D. and Fisher, S.J., 2017. Severe Hypoglycemia-Induced Fatal Cardiac Arrhythmias Are Augmented by Diabetes and Attenuated by Recurrent Hypoglycemia, Diabetes. 66, 3091-3097. Reno, C.M., Daphna-Iken, D., Chen, Y.S., VanderWeele, J., Jethi, K. and Fisher, S.J., 2013. Severe hypoglycemia-induced lethal cardiac arrhythmias are mediated by sympathoadrenal activation, Diabetes. 62, 3570-81. Apple, F.S., Sandoval, Y., Jaffe, A.S. and Ordonez-Llanos, J., 2017. Cardiac

25

616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659

[28] [29]

[30]

[31]

[32]

[33] [34]

[35]

[36]

[37]

[38] [39] [40]

[41]

Troponin Assays: Guide to Understanding Analytical Characteristics and Their Impact on Clinical Care, Clin Chem. 63, 73-81. Dandel, M. and Hetzer, R., 2009. Echocardiographic strain and strain rate imaging--clinical applications, Int J Cardiol. 132, 11-24. Fuentes-Antras, J., Ioan, A.M., Tunon, J., Egido, J. and Lorenzo, O., 2014. Activation of toll-like receptors and inflammasome complexes in the diabetic cardiomyopathy-associated inflammation, Int J Endocrinol. 2014, 847827. Wright, R.J., Newby, D.E., Stirling, D., Ludlam, C.A., Macdonald, I.A. and Frier, B.M., 2010. Effects of acute insulin-induced hypoglycemia on indices of inflammation: putative mechanism for aggravating vascular disease in diabetes, Diabetes Care. 33, 1591-7. Gogitidze Joy, N., Hedrington, M.S., Briscoe, V.J., Tate, D.B., Ertl, A.C. and Davis, S.N., 2010. Effects of acute hypoglycemia on inflammatory and pro-atherothrombotic biomarkers in individuals with type 1 diabetes and healthy individuals, Diabetes Care. 33, 1529-35. Kayama, Y., Raaz, U., Jagger, A., Adam, M., Schellinger, I.N., Sakamoto, M., Suzuki, H., Toyama, K., Spin, J.M. and Tsao, P.S., 2015. Diabetic Cardiovascular Disease Induced by Oxidative Stress, Int J Mol Sci. 16, 25234-63. Bajpai, A. and Tilley, D.G., 2018. The Role of Leukocytes in Diabetic Cardiomyopathy, Front Physiol. 9, 1547. Holmstrom, K.M. and Finkel, T., 2014. Cellular mechanisms and physiological consequences of redox-dependent signalling, Nat Rev Mol Cell Biol. 15, 411-21. Hussain, T., Tan, B., Yin, Y., Blachier, F., Tossou, M.C. and Rahu, N., 2016. Oxidative Stress and Inflammation: What Polyphenols Can Do for Us?, Oxid Med Cell Longev. 2016, 7432797. Genet, M., Lee, L.C., Baillargeon, B., Guccione, J.M. and Kuhl, E., 2016. Modeling Pathologies of Diastolic and Systolic Heart Failure, Ann Biomed Eng. 44, 112-27. Luptak, I., Qin, F., Sverdlov, A.L., Pimentel, D.R., Panagia, M., Croteau, D., Siwik, D.A., Bachschmid, M.M., He, H., Balschi, J.A. and Colucci, W.S., 2019. Energetic Dysfunction Is Mediated by Mitochondrial Reactive Oxygen Species and Precedes Structural Remodeling in Metabolic Heart Disease, Antioxid Redox Signal. Medzhitov, R., 2008. Origin and physiological roles of inflammation, Nature. 454, 428-35. Zlobine, I., Gopal, K. and Ussher, J.R., 2016. Lipotoxicity in obesity and diabetes-related cardiac dysfunction, Biochim Biophys Acta. 1861, 1555-68. Ertunc, M.E. and Hotamisligil, G.S., 2016. Lipid signaling and lipotoxicity in metaflammation: indications for metabolic disease pathogenesis and treatment, J Lipid Res. 57, 2099-2114. Ritchie, R.H., Zerenturk, E.J., Prakoso, D. and Calkin, A.C., 2017. Lipid metabolism and its implications for type 1 diabetes-associated cardiomyopathy,

26

660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703

[42]

[43] [44]

[45] [46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

J Mol Endocrinol. 58, R225-R240. Yang, Y., He, Y., Wang, X., Liang, Z., He, G., Zhang, P., Zhu, H., Xu, N. and Liang, S., 2017. Protein SUMOylation modification and its associations with disease, Open Biol. 7. Yang, X. and Qian, K., 2017. Protein O-GlcNAcylation: emerging mechanisms and functions, Nat Rev Mol Cell Biol. 18, 452-465. Phillips, A.H. and Kriwacki, R.W., 2019. Intrinsic protein disorder and protein modifications in the processing of biological signals, Curr Opin Struct Biol. 60, 1-6. Mueckler, M. and Thorens, B., 2013. The SLC2 (GLUT) family of membrane transporters, Mol Aspects Med. 34, 121-38. Finck, B.N., Lehman, J.J., Leone, T.C., Welch, M.J., Bennett, M.J., Kovacs, A., Han, X., Gross, R.W., Kozak, R., Lopaschuk, G.D. and Kelly, D.P., 2002. The cardiac phenotype induced by PPARalpha overexpression mimics that caused by diabetes mellitus, J Clin Invest. 109, 121-30. Burkart, E.M., Sambandam, N., Han, X., Gross, R.W., Courtois, M., Gierasch, C.M., Shoghi, K., Welch, M.J. and Kelly, D.P., 2007. Nuclear receptors PPARbeta/delta and PPARalpha direct distinct metabolic regulatory programs in the mouse heart, J Clin Invest. 117, 3930-9. Son, N.H., Park, T.S., Yamashita, H., Yokoyama, M., Huggins, L.A., Okajima, K., Homma, S., Szabolcs, M.J., Huang, L.S. and Goldberg, I.J., 2007. Cardiomyocyte expression of PPARgamma leads to cardiac dysfunction in mice, J Clin Invest. 117, 2791-801. Zhang, Y., Wang, Y., Yanni, J., Qureshi, M.A., Logantha, S., Kassab, S., Boyett, M.R., Gardiner, N.J., Sun, H., Howarth, F.C. and Dobrzynski, H., 2019. Electrical Conduction System Remodeling in Streptozotocin-Induced Diabetes Mellitus Rat Heart, Front Physiol. 10, 826. van der Toorn, F.A., de Mutsert, R., Lijfering, W.M., Rosendaal, F.R. and van Hylckama Vlieg, A., 2019. Glucose metabolism affects coagulation factors: The NEO study, J Thromb Haemost. Joy, N.G., Tate, D.B., Younk, L.M. and Davis, S.N., 2015. Effects of Acute and Antecedent Hypoglycemia on Endothelial Function and Markers of Atherothrombotic Balance in Healthy Humans, Diabetes. 64, 2571-80. Vileigas, D.F., Harman, V.M., Freire, P.P., Marciano, C.L.C., Sant'Ana, P.G., de Souza, S.L.B., Mota, G.A.F., da Silva, V.L., Campos, D.H.S., Padovani, C.R., Okoshi, K., Beynon, R.J., Santos, L.D. and Cicogna, A.C., 2019. Landscape of heart proteome changes in a diet-induced obesity model, Sci Rep. 9, 18050. Kolwicz, S.C., Jr., Purohit, S. and Tian, R., 2013. Cardiac metabolism and its interactions with contraction, growth, and survival of cardiomyocytes, Circ Res. 113, 603-16. Evangelista, I., Nuti, R., Picchioni, T., Dotta, F. and Palazzuoli, A., 2019. Molecular Dysfunction and Phenotypic Derangement in Diabetic Cardiomyopathy, Int J Mol Sci. 20.

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[55]

[56]

[57]

Lee, I. and Huttemann, M., 2014. Energy crisis: the role of oxidative phosphorylation in acute inflammation and sepsis, Biochim Biophys Acta. 1842, 1579-86. Boudina, S., Sena, S., Theobald, H., Sheng, X., Wright, J.J., Hu, X.X., Aziz, S., Johnson, J.I., Bugger, H., Zaha, V.G. and Abel, E.D., 2007. Mitochondrial energetics in the heart in obesity-related diabetes: direct evidence for increased uncoupled respiration and activation of uncoupling proteins, Diabetes. 56, 2457-66. Wen, S.Y., Velmurugan, B.K., Day, C.H., Shen, C.Y., Chun, L.C., Tsai, Y.C., Lin, Y.M., Chen, R.J., Kuo, C.H. and Huang, C.Y., 2017. High density lipoprotein (HDL) reverses palmitic acid induced energy metabolism imbalance by switching CD36 and GLUT4 signaling pathways in cardiomyocyte, J Cell Physiol. 232, 3020-3029.

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Highlights Severe hypoglycemia was successfully induced in type 1 diabetic (T1DM) mice. Severe hypoglycemia worsens myocardial dysfunction and structural disorder. Severe hypoglycemia induced PPAR-β/δ–related metabolic remodeling in T1DM mice.