Electroacupuncture modulates myocardial insulin signaling and inflammatory markers in a rat model of type 2 diabetes

Abstract

Objective:

To evaluate the effects of electroacupuncture (EA) on myocardial insulin resistance (IR) in Zucker diabetic fatty (ZDF) rats, an established model for type 2 diabetes mellitus (T2DM).

Methods:

Twenty-four ZDF-Lepr^fa/fa rats were randomized to: (1) ZDF group (n = 8); (2) ZDF + PIO (pioglitazone) group (n = 8); and (3) ZDF + EA group (n = 8). An additional control group of eight healthy ZDF^+/fa rats was included (Lean group). We examined protein and mRNA expression levels of critical insulin signaling pathway intermediates including insulin receptor substrate (IRS)-1, phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), adenosine monophosphate (AMP)-activated protein kinase (AMPK), ribosomal protein S6 kinase (p70S6K), glycogen synthase kinase 3β (GSK3β) and glucose transporter type 4 (GLUT4), as well as serum levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and C-reactive protein. Echocardiography and cardiac histology were performed.

Results:

Significant improvements in glucose metabolism, reflected in reduced fasting insulin levels and fasting blood glucose levels, were demonstrated after EA and PIO treatment. EA treatment also led to a notable decrease in inflammatory cytokine levels. In addition, there were marked improvements in myocardial structural integrity, as evidenced by histological analyses. Moreover, increased GLUT4 expression in myocardial tissue suggested improved insulin signaling, further supported by reductions in markers of myocardial injury such as serum troponin T type 2 (TNNT2) and B-type natriuretic peptide (BNP).

Conclusion:

EA ameliorated myocardial IR in a rat model of T2DM and positively impacted TNNT2 and BNP levels, as well as phosphorylation status and mRNA expression of several genes involved in the insulin signaling pathway. Our findings underscore the potential of EA to modulate multiple therapeutic targets in the treatment of myocardial IR. If these effects can be replicated clinically, EA may represent a promising non-pharmacological option for the management of cardiometabolic risks associated with diabetes.

Keywords

acupuncture complementary medicine diabetes diabetes and endocrinology electroacupuncture endocrinology internal medicine

Introduction

Diabetes mellitus (DM) is a multifaceted metabolic disorder that is characterized by the presence of hyperglycemia due to impaired insulin secretion and/or insulin action. Globally there were 529 million people suffering from DM in 2021, with type 2 diabetes mellitus (T2DM) accounting for 96%.¹ Insulin resistance (IR) is a major defect in T2DM and significantly increases the risk of cardiovascular disease (CVD), which is the leading cause of morbidity and mortality among diabetic populations.^1–3 While hypotheses such as hyperglycemia, inflammation, obesity and genetic susceptibility have linked DM to CVD, the underlying pathophysiology remains incompletely elucidated.⁴ In patients with T2DM, even in the absence of coronary artery disease, insulin-mediated glucose uptake in normally contracting myocardium is significantly reduced, indicating that T2DM itself is an independent and significant factor affecting myocardial IR.^3,4 Consequently, conventional treatments may not directly address myocardial IR. Furthermore, certain mainstream insulin sensitizers used to treat T2DM, such as thiazolidinediones (e.g. pioglitazone, rosiglitazone and troglitazone), have been associated with a higher risk of heart failure.^5–8 Thus, exploring therapeutic targets for myocardial IR and seeking efficacious therapies without significant side-effects are of practical significance for reducing the healthcare burden and improving patients’ quality of life.

Under physiological conditions, insulin regulates cardiac substrate utilization by stimulating glucose uptake and oxidation.⁹ However, under a state of IR there is a reduced capacity for glucose utilization, which impairs cardiac adaptability to fluctuations in energy demand.^10,11 T2DM is associated with obesity, which is often associated with elevated circulating levels of inflammatory cytokines, such as interleukin-6 (IL)-6 and tumor necrosis factor (TNF)-α. Thus, inflammation within the heart may be a contributing factor to the development of myocardial IR.¹² In support of this assertion, an animal study reported that a high-fat-diet exacerbated cardiac inflammation in obese mice, evidenced by increased infiltration of macrophages and cytokines into the heart, mediated by IL-6.¹³

When insulin binds to the insulin receptor, the receptor undergoes auto-phosphorylation, thereby initiating a signaling cascade involving tyrosine phosphorylation of insulin receptor substrate (IRS). This cascade subsequently phosphorylates phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt), which leads to translocation of glucose transporter type 4 (GLUT4) to the membrane, facilitating glucose uptake into cells.^3,14 In the heart, contraction-mediated GLUT4 translocation may represent the predominant mechanism regulating glucose entry.¹⁵ In addition to glucose uptake, insulin-mediated activation of PI3K and Akt stimulates other intracellular signaling intermediates, such as mechanistic target of ribosomal protein S6 kinase (p70S6K) and glycogen synthase kinase (GSK)3β.¹⁶ Adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) serves as a critical mediator of energy balance in various tissues, including the heart.¹⁷ AMPK can directly enhance endothelial insulin signaling, thus preventing IR.¹⁸

Acupuncture—a traditional Chinese medical therapy—has been demonstrated to effectively enhance insulin sensitivity and mitigate obesity in patients with IR.^19–21 In animal studies, electroacupuncture (EA)—a technique that involves electrical stimulation of needles inserted into the body—has been demonstrated to exert a multi-organ, multi-target regulatory effect. The mechanisms involved include amelioration of inflammation, enhancement of energy metabolism and mitigation of oxidative stress. A recent study reported that EA ameliorated abnormal energy metabolism by attenuating oxidative stress, reducing ectopic fat deposition and modulating metabolic fluxes via the gut-liver axis.²² Acupuncture is internationally recognized as an alternative or complementary therapy and is usually administered at specific traditional acupuncture point locations, which can have organ-specific effects (e.g. on the heart and stomach) depending on their anatomic location and their segmental innervation.²³

The aim of this research was to observe the effects of EA at ST36 (Zusanli) and SP6 (Sanyinjiao) on myocardial IR in a spontaneously insulin-resistant animal model—the Zucker diabetic fatty (ZDF) rat—and to elucidate the underlying mechanisms.

Methods

Animals and experimental design

Twenty-four ZDF-Lepr^fa/fa rats (homozygous recessive for the fatty allele (fa) mutation in the leptin receptor gene) and eight Zucker lean littermates (ZDF^+/fa)—all male and 7 weeks old—were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All rats were housed in a specific pathogen-free (SPF) chamber under controlled conditions of temperature (22–24°C) and relative humidity (40–60%) and a constant 12 h light/dark cycle. A special diet (Purina 5008) and sterile water were provided ad libitum. All experimental procedures were approved by the Laboratory Animal Center Ethics Committee of Guangzhou University of Chinese Medicine and conducted according to the Chinese Guidelines for Laboratory Animals (GB14925-2001 and MOST 2006a).

ZDF-Lepr^fa/fa rats were randomly assigned based on their fasting blood glucose (FBG) levels and fasting body weight (FBW) into three groups: (1) the ZDF group (n = 8); (2) the ZDF + PIO (pioglitazone) group (n = 8); and (3) the ZDF + EA group (n = 8). The eight healthy ZDF^+/fa rats were assigned to the normal control group (Lean group).

Rats in the ZDF + EA group underwent acupuncture at bilateral SP6 and ST36. Stainless steel acupuncture needles (diameter 0.32 mm, length 25 mm, GB2024-1994; Huatuo, Suzhou, China) were inserted to a depth of 5–8 mm and connected to an EA apparatus (model no. SDZ-V, Suzhou Medical Appliance Factory, China). The electrical stimulation was set at a current intensity of 2 mA with a dense-disperse wave frequency of 2/100 Hz (pulse width 0.2 ms, phase switching every 5 s). The treatment was administered for 20 min every other day for a duration of 4 weeks. PIO was administered via oral gavage as a positive pharmacologic control to rats in the ZDF + PIO group at a dose of 10 mg/kg once daily for a duration of 4 weeks. At the same time, the rats in the Lean, ZDF and ZDF + EA groups were subjected to gastric lavage using the same volume of saline as the ZDF + PIO group.

Euthanasia and tissue sampling

Prior to tissue collection, all groups of rats were fasted for 10 h but retained access to water ad libitum. The rats were weighed to accurately calculate the required dose of pentobarbital sodium for deep anesthesia, which was administered by intraperitoneal (i.p.) injection at a dose of 50 mg/kg body weight. Once a deep plane of anesthesia was established and confirmed by a lack of response to toe-pinch reflex and muscle flaccidity, the abdomen was surgically opened to expose the abdominal aorta. A total of 0.5–1 mL of whole blood was collected into pre-warmed clot-activating tubes (without any anticoagulant) using a 23G needle, gently inverted 5–6 times, and allowed to clot at room temperature for 60 min. Immediately thereafter, 0.5–1 mL of blood was drawn into heparinized tubes (pre-coated with 10 U lithium heparin per mL of blood), inverted 8 times to ensure anticoagulation, and kept on ice. Death was confirmed by cessation of respiration and absence of corneal reflexes. Subsequently, proximal ligation of the abdominal aorta was performed to facilitate hemostasis and target organs were swiftly harvested to ensure tissue integrity. The animal carcass was then disposed of according to institutional guidelines.

Cardiac tissues were harvested with precise dissection of the left ventricle (LV) anterior wall (5 mm × 5 mm × 2 mm), oriented with the epicardial surface facing upward. Tissues for histology were fixed in 10% neutral buffered formalin (Solarbio, G2240, Beijing, China) for 16 h at room temperature to avoid over-hardening. Following fixation, tissues were rinsed three times with 0.1 M phosphate-buffered saline (PBS) (10 min each), then dehydrated through a graded ethanol series starting with 50% ethanol (1 h) to minimize shrinkage, followed by 70%, 85%, 95% and 100% ethanol (1 h each). Tissues were cleared in xylene for 15 min (single step) to prevent excessive brittleness, then embedded in paraffin at 56°C–58°C using a Leica EG1150H embedder (Wetzlar, Germany) after being oriented to facilitate transverse sectioning. Liver was sampled for reverse transcription-polymerase chain reaction (RT-PCR).

Plasma and serum assays

For serum separation, clotted samples of whole blood were centrifuged at 1400 g for 15 min at 4°C. The supernatant serum was carefully aspirated with a 200-μL pipette, maintaining a 2-mm distance from the cell pellet to prevent hemolysis and then aliquoted into cryotubes for storage at −80°C. For plasma preparation, heparinized blood was centrifuged at 700 g for 10 min at 4°C. To remove residual cells, the plasma was subjected to a second centrifugation step at 1000 g for 5 min. The clarified plasma was then transferred into sterile tubes, avoiding the platelet layer, and stored at −80°C until analyzed.

FBG levels were determined in plasma using the glucose oxidase method. Enzyme-linked immunosorbent assay (ELISA) was employed to determine the concentrations of C-peptide (630-24149; FUJIFILM Wako, Japan), C-reactive protein (CRP) (70-EK394-96; Multisciences, Hangzhou, China), IL-6 (70-EK306HS-96; Multisciences), IL-1β (70-EK301BHS-96; Multisciences), insulin (633-07279; FUJIFILM Wako), monocyte chemoattractant protein-1 (MCP-1) (BMS631INST; Invitrogen, Carlsbad, CA, USA), TNF-α (70-EK382HS-96; Multisciences), troponin T type 2 (TNNT2) (E-EL-R0151; Elabscience, Wuhan, China) and B-type natriuretic peptide (BNP) (ab108815; Abcam, Cambridge, UK) in plasma.

Measurement of cardiac function by echocardiography

At the end of treatment, rats underwent a standard transthoracic echocardiographic examination to assess cardiac function and morphology. Echocardiography was performed using an Vivid q system (GE Vingmed, Horten, Norway). Two-dimensional B-mode and M-mode were utilized to measure left atrial diameter (LA), interventricular septal thickness (IVST), posterior wall thickness (PWT), left ventricular end-diastolic diameter (LVEDd) and left ventricular end-systolic diameter (LVESd). Heart rate (HR) was recorded in beats per minute and ejection fraction (EF) was calculated as a percentage. Pulsed wave Doppler was used to evaluate the mitral inflow velocity and calculate the early peak/artial peak (E/A) ratio. Systolic blood pressure (SBP) was measured using the tail-cuff plethysmography technique with a CODA non-invasive blood pressure system (Kent Scientific, Torrington, CT, USA). The final value was taken as the average of three consecutive valid readings.

Masson’s trichrome staining of myocardial tissues

Myocardial sections of 4- to 5-µm-thickness sections were cut, deparaffinized in xylene, rehydrated in graded ethanol (100%, 95% and 70%, respectively) and rinsed in distilled water. The sections were stained with Weigert’s iron hematoxylin solution for 5–10 min to visualize the nuclei, then rinsed in running tap water. Masson’s trichrome stain solution (consisting of acid fuchsin, phosphomolybdic acid and aniline blue) was applied for 30 min. After a brief rinse in distilled water, sections were differentiated in 1% acetic acid for 1–2 min, then dehydrated in graded ethanol and cleared in xylene. Finally, the sections were mounted using glycerin jelly (Sigma-Aldrich, St Louis, MO, USA) and allowed to dry completely before observation under an Eclipse E600 light microscope (Nikon, Tokyo, Japan) at 200× magnification. Myocardial structural integrity was assessed by examining collagen fibers (which appeared blue), muscle fibers (which appeared red) and nuclei (which appeared dark blue or black).

Myocardial ultrastructural examination by transmission electron microscopy

Cardiac tissue samples were cut into small pieces (1 mm³) and fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.4) at 4°C overnight. After dehydration in graded ethanol, the samples were embedded in Epon812 and sectioned into thin slices (50–100 nm thickness). Then, the samples were observed and photographed under transmission electron microscopy (TEM; HT-7800, Hitachi, Japan), with the following imaging parameters of the lens mode: zoom-1 HC1; acceleration voltage 80.0 kV; spot size 8 μm; and ×7.0k magnification.

Myocardial evaluation through hematoxylin–eosin staining

Fixed cardiac tissue was sliced to obtain sections of 4–6 μm thickness. Then, the sections were stained with hematoxylin staining solution (Sigma-Aldrich) for 3–5 min until they turned blue-purple, and then rinsed in running water. Next, the sections were stained with eosin staining solution (Sigma-Aldrich) for 1–2 min until they appeared pink, followed by another rinse in running water. Finally, the stained cardiac tissue sections were mounted on slides with a transparent mounting medium and observed under an Eclipse E600 light microscope to evaluate their structure and cellular morphology.

Western blotting

Proteins were extracted from myocardial tissue and quantified using a bicinchoninic acid (BCA) assay kit (23225; Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (20–30 μg) were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer of proteins from the gel onto polyvinylidene fluoride (PVDF) membranes. Subsequently, nonspecific sites were blocked using 5% skim milk or bovine serum albumin (BSA). Next, the membranes were incubated with rabbit primary antibodies against IRS-1 (1:1000, 2382S; Cell Signaling Technology (CST), Danvers, MA, USA), Akt (1:1000, 9272S; CST), mammalian target of rapamycin (mTOR) (1:1000, 2972S; CST), GSK-3β (1:1000, 9315S; CST), p70S6K (1:1000, 9202S; CST), AMPK (1:1000, 2532S; CST), TNNT2 (1:2000, A4914; ABclonal, Wuhan, China) and GLUT4 (1:500, ab313775; Abcam) overnight at 4°C. Following this, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig)G secondary antibody (1:8000, SA00001-2; Proteintech, Chicago, IL, USA) for 1 h at room temperature, and specific protein signals were detected using chemiluminescence, followed by signal capture and quantitative analysis using a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA).

Real-time polymerase chain reaction

Total RNA was extracted from cardiac tissues using Trizol reagent (Invitrogen), according to the manufacturer’s instructions. Prime Script™ RT reagent kits with gDNA Eraser (Takara, Japan) were used for reverse transcription. Real-time PCR was performed utilizing Fast Start Universal SYBR Green master mix (Roche Diagnostics, Indianapolis, IN, USA) and an ABI 7500/7500 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The 2^−ΔΔCt method was used to calculate relative gene expression, with beta-actin (Actb) as the internal control. The primers used in the experiment were as follows: Actb forward 5′-ACAACCTTCTTGCAGCTCCTC-3′ reverse 5′-CTGACCCATACCCACCATCAC-3′; Pik3ca (phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha) forward 5′-GAGAAACACTCCGCTTGATA-3′ reverse 5′-ATGTAACCCTGATGACTGAC-3′; Prkaa2 (protein kinase AMP-activated catalytic subunit alpha 2) forward 5′-TGACAGGCCATAAAGTGGCAG-3′ reverse 5′-TCGAACAATTCACCTCCAGACA-3′; Rps6kb1 (ribosomal protein S6 kinase B1) forward 5′-TTCAGCGCCACTTCCAATCC-3′ reverse 5′-CCTCACACATGCCCTTCCAG-3′; Gsk3b forward 5′-ACACACCTGCCCTCTTCAAC-3′ reverse 5′-GAAGCGGCGTTATTGGTCTG-3′; Irs1 forward 5′-CTGCATCGGACTCTACCAGG-3′ reverse 5′-AGGGAAAGGCAGTGGGTCTA-3′; Akt2 forward 5′-GTAGCCAACAGTCTGAAGCA-3′ reverse 5′-TTGCCGAGGAGTTTGAGATA-3′; and Slc2a4 (solute carrier family 2 member 4) forward 5′-TCCTTCTATTTGCCGTCCTC-3′ reverse 5′-GGGTTTCACCTCCTGCTCTA-3′.

Immunofluorescence double staining of TNF-α and GLUT4 in myocardial tissue

Dual immunofluorescence staining for TNF-α and GLUT4 was performed on myocardial tissue sections. First, tissue sections were deparaffinized in xylene and rehydrated through graded ethanol solutions. Antigen retrieval was conducted by heating the sections in citrate buffer (pH 6.0, C301552; Aladdin, Shanghai, China) at 95°C for 10 min. After cooling to room temperature, the sections were blocked with 5% BSA for 30 min to prevent nonspecific binding. The sections were then incubated overnight at 4°C with polyclonal rabbit primary antibodies against TNF-α (1:100, GB11188; Servicebio, Wuhan, China) and GLUT4 (1:200, GB11052; Servicebio). Following the primary antibody incubation, the sections were washed with PBS and incubated with a mixture of Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1:200, ZF-0511; ZSGB-BIO, Beijing, China) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200, ZF-0516; ZSGB-BIO) in 5% normal donkey serum/PBS at room temperature for 1 h in the dark. After thorough washing, the slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Finally, the stained sections were mounted using an anti-fade mounting medium and observed under a fluorescence microscope (Eclipse C1, Nikon) at 200× magnification. Fluorescence signals were analyzed and captured for TNF-α (red), GLUT4 (green) and nuclei (blue).

Statistical analysis

Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) 25.0 (IBM Corp., Armonk, NY, USA). Normality of data distribution was assessed using the Shapiro–Wilk test. Normally distributed data were presented as mean ± standard deviation and homogeneity of variance was confirmed. Groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni or Tamhane’s T2 post hoc tests. P < 0.05 was considered statistically significant and all tests performed were two-tailed.

Results

Effects of EA on general condition and inflammatory cytokines of ZDF rats

As shown in Table 1, in the ZDF group, rats exhibited a significant increase in FBW (p < 0.001) compared to the Lean group. FBW in ZDF + EA versus ZDF group did not significantly differ; however, it was significantly higher (p < 0.01) in the ZDF + PIO group compared to the Lean group. Regarding FBG, the ZDF group showed a dramatic increase in glucose levels compared to the Lean group (p < 0.001). The ZDF + EA group exhibited significantly reduced FBG levels (p < 0.001) in comparison with the ZDF group. Similarly, the ZDF + PIO group exhibited a substantial reduction in FBG (p < 0.001). In terms of fasting insulin (FINS), the ZDF group displayed a marked increase in insulin levels (p < 0.001) compared to the Lean group. The ZDF + EA group exhibited significantly reduced insulin levels (p < 0.05) and a further reduction was observed in the ZDF + PIO group (p < 0.01) compared to the ZDF group. CRP was elevated in the ZDF group as compared to the Lean group (p < 0.05) and both the EA and PIO interventions appeared to significantly alter CRP levels (p < 0.01). It was observed that C-peptide levels were significantly higher in the ZDF group than in the Lean group (p < 0.05) and were reduced by both EA and PIO treatment (p < 0.05). Further evidence of inflammation was provided by the elevated levels of IL-6 in the ZDF group, in which a sharp contrast with the Lean group was observed (p < 0.001). The application of EA and PIO were both associated with a reduction in IL-6 levels (p < 0.05). By contrast, EA treatment did not significantly reduce IL-1β concentration, whereas PIO did, when compared to no treatment (p < 0.05). TNF-α was also significantly increased in the ZDF group compared to the Lean group (p < 0.001). This increase points to the multifaceted nature of inflammation in ZDF rats, which—in a similar pattern to the CRP levels—was significantly attenuated by both EA and PIO (p < 0.001). Finally, MCP-1 levels were raised in the ZDF group (p < 0.05) and were notably decreased following EA treatment (p < 0.05).

Table 1.

Basic parameters and inflammatory markers.

Variable	Groups
Variable	Lean	ZDF	ZDF + EA	ZDF + PIO
FBW	318.13 ± 11.47	357.38 ± 13.85^###	346.38 ± 16.14	388.75 ± 13.85**
FBG	6.90 ± 0.70	24.26 ± 1.40^###	19.29 ± 1.82***	16.71 ± 1.91***
FINS	0.10 ± 0.04	1.06 ± 0.33^###	0.65 ± 0.18*	0.55 ± 0.24**
C-peptide	0.65 ± 0.31	1.30 ± 0.33^#	0.52 ± 0.37**	0.59 ± 0.28*
CRP	1.80 ± 0.11	2.72 ± 0.30^###	2.19 ± 0.27**	2.08 ± 0.32**
IL-6	9.67 ± 2.85	22.51 ± 3.59^##	14.82 ± 6.45*	12.02 ± 5.47*
IL-1β	0.90 ± 0.10	1.82 ± 0.28^###	1.42 ± 0.27	1.48 ± 0.27*
TNF-α	38.74 ± 8.38	201.08 ± 15.17^###	152.42 ± 11.33***	144.09 ± 8.52***
MCP-1	3.80 ± 1.01	6.47 ± 0.96^#	4.47 ± 1.47*	5.38 ± 1.58

FBW, fasting body weight; FBG, fasting blood glucose; FINS, fasting insulin; CRP, C-reactive protein; IL, interleukin; TNF, tumor necrosis factor; MCP, monocyte chemoattractant protein; ZDF, Zucker diabetic fatty; EA, electroacupuncture; PIO, pioglitazone. Data are mean ± standard deviation (n = 5-8 per group).

p < 0.05; ^##p < 0.01; ^###p < 0.001 versus Lean group; *p < 0.05; **p < 0.01; ***p < 0.001 versus ZDF group.

Effects of EA on hemodynamic parameters and cardiac function in ZDF rats

Hemodynamic and echocardiography parameters are shown in Table 2. No significant differences were observed in HR across groups. The ZDF group exhibited a reduced EF compared to the Lean group (p < 0.01). EF did not significantly differ between ZDF + EA and (untreated) ZDF groups, but was significantly increased in ZDF + PIO versus ZDF groups (p < 0.05). SBP was significantly elevated in the ZDF group compared to the Lean group (p < 0.01) and reduced by PIO (p < 0.05) but not EA. LA dimensions and LVEDd did not significantly differ between groups. LVESd was significantly increased in ZDF versus Lean groups (p < 0.05) but not significantly impacted by the EA or PIO interventions. PWT and IVST were significantly increased in the ZDF group compared to the Lean group (p < 0.05). Both EA and PIO treatments reduced IVST (p < 0.05). The E/A ratio was decreased in ZDF versus Lean groups (p < 0.01) and not significantly impacted by EA or PIO.

Table 2.

Hemodynamic indices and echocardiography study for heart function.

Variable	Groups
Variable	Lean	ZDF	ZDF + EA	ZDF + PIO
HR (bpm)	321 ± 4	326 ± 5	323 ± 3	322 ± 5
EF (%)	76.9 ± 1.21	71.7 ± 2.41^##	74.7 ± 2.26	75.0 ± 1.87
SBP (mm Hg)	113.6 ± 2.18	127.8 ± 7.00^#	117.2 ± 7.42	115.7 ± 3.59*
LA (mm)	5.48 ± 0.36	5.63 ± 0.19	5.56 ± 0.40	5.51 ± 0.19
LVEDd (mm)	8.18 ± 0.17	8.34 ± 0.19	8.22 ± 0.19	8.21 ± 0.19
LVESd (mm)	4.09 ± 0.15	4.49 ± 0.25^#	4.21 ± 0.22	4.25 ± 0.21
PWT (mm)	1.47 ± 0.06	1.61 ± 0.05^#	1.51 ± 0.08	1.52 ± 0.09
IVST (mm)	1.47 ± 0.06	1.61 ± 0.04^##	1.52 ± 0.06*	1.51 ± 0.05*
E/A	1.73 ± 0.05	1.62 ± 0.07^##	1.65 ± 0.03	1.67 ± 0.03

HR, heart rate; bpm, beats per minute; EF, ejection fraction; SBP, systolic blood pressure; LVEDd, left ventricular end-diastolic diameter; LVESd, left ventricular end-systolic diameter; PWT, posterior wall thickness; IVST, interventricular septal thickness; E/A, early peak/artial peak ratio; ZDF, Zucker diabetic fatty; EA, electroacupuncture; PIO, pioglitazone. Data are mean ± standard deviation (n = 5 per group).

p < 0.05; ^##p < 0.01 versus Lean group. *p < 0.05 versus ZDF group.

Effects of EA on TNNT2 protein expression and serum levels of TNNT2 and BNP in ZDF rats

As shown in Figure 1(a), there was a significant increase in TNNT2 protein expression in the ZDF group compared to the Lean group (p < 0.001) and both the ZDF + EA and ZDF + PIO groups showed significantly reduced TNNT2 expression compared to the ZDF group (p < 0.001). Serum TNNT2 levels (Figure 1(b)) were significantly elevated in the ZDF group compared to the Lean group (p < 0.001) and both EA and PIO treatment significantly lowered TNNT2 levels (p < 0.01). Similarly, serum BNP levels (Figure 1(c)) were markedly increased in ZDF compared to Lean groups (p < 0.001) and both EA and PIO treatments resulted in significant decreases in BNP levels compared to no treatment (p < 0.05).

Figure 1.

Effects of electroacupuncture (EA) and pioglitazone (PIO) on troponin T type 2 (TNNT2) protein expression and serum levels of TNNT2 and B-type natriuretic peptide (BNP) in Zucker diabetic fatty (ZDF) rats. (a) Representative Western blots of myocardial TNNT2 levels and densitometric semi-quantitative determination of protein expression levels. (b–c) Serum TNNT2 and BNP levels, measured by enzyme-linked immunosorbent assay. Data are mean ± standard deviation (n = 5–6 per group).

Effects of EA on histological and ultrastructural changes in myocardial tissue of ZDF rats

As shown in Figure 2(a), the histological evaluation of myocardial tissue using hematoxylin–eosin staining in ZDF rats revealed distinct variations across the different treatment groups. The Lean group displayed a regular pattern of striations and normal cardiac muscle morphology, with no signs of fibrosis or inflammatory cell infiltration. The cardiac tissues of rats in the ZDF group exhibited signs of structural disarray, with increased interstitial spacing. By contrast, the ZDF + EA group showed a marked improvement in tissue architecture. The myocardial fibers appeared more organized and closely resembled the Lean group, with reduced interstitial spacing. Similarly, the ZDF + PIO group demonstrated subjective improvements in cardiac muscle histology. The ultrastructural assessment of myocardial tissue in ZDF rats was conducted using TEM, in order to examine subcellular effects of EA/PIO treatment on myocardial IR in this rat model. In the Lean group, the myocardial ultrastructure was well-preserved with clear and organized sarcomeres, intact mitochondria and minimal intracellular edema (Figure 2(b)). The ZDF group, however, demonstrated significant ultrastructural derangements. These included disrupted myofibrillar patterns, swollen mitochondria with distorted cristae and increased presence of lipid droplets. Treatment with EA appeared to mitigate some of the myocardial ultrastructural damage observed in the untreated ZDF rats. Myofibrils showed improved alignment and organization, the mitochondria appeared less swollen and there was a noticeable decrease in lipid accumulation. The ZDF + PIO group also displayed improvements in myocardial ultrastructure, with better-preserved myofibril organization and mitochondrial morphology. Masson staining (Figure 2(c)) further confirmed these observations, with increased collagen deposition in the ZDF group, which was reduced following EA and PIO treatments, indicating a reduction in fibrosis.

Figure 2.

Electroacupuncture (EA) and pioglitazone (PIO) improved histological and ultrastructural changes and alleviated myocardial fibrosis in Zucker diabetic fatty (ZDF) rats. Representative images of myocardial tissue using: (a) hematoxylin-eosin staining (scale bar: 50 μm); (b) Masson staining (scale bar: 80 μm); and (c) transmission electron microscopy. Black arrows indicate mitochondria and black bold circles indicate lipid droplets (scale bar: 8 μm).

Effects of EA on protein phosphorylation and gene expression in the myocardium of ZDF rats

Protein expression ratios of phosphorylated to total IRS-1, Akt, AMPK, p70S6K and GSK-3β and total protein expression of PI3K and GLUT4 were assessed, as well as mRNA expression.

In the ZDF group, PI3K expression was significantly reduced compared to the Lean group (p < 0.001; Figure 3(a)). Both EA and PIO treatments restored PI3K levels (p < 0.01). The phosphorylation ratio of p-Akt (Ser473)/Akt decreased in the ZDF group relative to the Lean group (p < 0.001) and was significantly increased by both EA and PIO treatments (p < 0.05). Both treatments also significantly reduced the phosphorylation ratio of p-IRS-1(Ser1101)/IRS-1 (p < 0.01; Figure 3(b)). EA treatment resulted in a significant increase in the phosphorylation ratio of p-GSK-3β(Ser9)/GSK-3β compared to the ZDF group (p < 0.01), whereas PIO treatment had no statistically significant impact on this ratio (P > 0.05). The phosphorylation ratio of p-AMPK(Thr172)/AMPK was significantly increased in the ZDF + PIO group compared to the untreated ZDF group (p < 0.05; Figure 3(c)). However, no significant changes were observed in the phosphorylation ratio of p-p70S6K(Thr389)/p70S6K following either EA or PIO treatment (p > 0.05). For GLUT4, there was a pronounced decrease in the ZDF group relative to the Lean group (p < 0.001) and both EA and PIO treatments notably increased GLUT4 expression compared to no treatment in ZDF rats (p < 0.001).

Figure 3.

Electroacupuncture (EA) and pioglitazone (PIO) regulate the expression of key proteins in the myocardial tissue of Zucker diabetic fatty (ZDF) rats. (a) Phosphoinositide 3-kinase (PI3K), protein kinase B (Akt) and phosphorylated (p)-Akt. (b) Insulin receptor substrate (IRS)-1, p-IRS-1, glycogen synthase kinase (GSK)-3β and p-GSK-3β. (c) Ribosomal protein S6 kinase (p70S6K), p-p70S6K, adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK), p-AMPK and glucose transporter (GLUT)4 alongside their corresponding phosphorylation statuses. (d) Relative mRNA expression levels of phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha (Pik3ca), Akt1, Irs1, Gsk3b, ribosomal protein S6 kinase B1 (Rps6kb1), protein kinase AMP-activated catalytic subunit alpha 2 (Prkaa1) and solute carrier family 2 member 4 (Slc2a4), assessed by reverse transcription-polymerase chain reaction. Data are mean ± standard deviation (n = 4 per group).

mRNA expression of various genes involved in insulin signaling pathways was assessed using RT-PCR. As shown in Figure 3(d), Irs1, Prkaa2, Akt2, Pik3ca, Gsk3b and Slc2a4 were significantly downregulated in the ZDF group compared to the Lean group (p < 0.05), indicating a disruption in these key signaling pathways. However, treatment with EA or PIO partially restored their expression levels. In the ZDF + EA group, Irs1, Prkaa2, Akt2 and Pik3ca mRNA levels were significantly upregulated compared to the ZDF group (p < 0.05). The ZDF + PIO group showed a similar trend, with significant upregulation of Irs1, Prkaa2, Akt2 and Pik3ca mRNA levels compared to the ZDF group (p < 0.05). For Gsk3b and Slc2a4, no significant differences were observed between the ZDF group and either treatment group (ZDF + EA or ZDF + PIO).

Effects of EA on TNF-α and GLUT4 expression in myocardial tissue of ZDF rats

As shown in Figure 4(a), moderate expression of GLUT4 and low expression of TNF-α were observed in the Lean group, with minimal overlap between the two markers. In the ZDF group, there was a marked increase in TNF-α expression along with a reduction in GLUT4 expression, indicating an inflammatory state and impaired insulin signaling. The ZDF + EA group showed reduced TNF-α levels and improved GLUT4 expression compared to the ZDF group, suggesting alleviation of inflammation and restored insulin sensitivity following EA. The ZDF + PIO group also displayed decreased TNF-α and elevated GLUT4 levels. Quantitative analysis of fluorescence intensity further validated these histological observations (Figure 4(b)).

Figure 4.

Electroacupuncture (EA) and pioglitazone (PIO) alleviate myocardial inflammation and enhance insulin signaling in Zucker diabetic fatty (ZDF) rats. (a) Representative immunofluorescence images of myocardial tissue, showing the expression of tumor necrosis factor (TNF)-α (red), glucose transporter (GLUT)4 (green) and nuclei stained with 4’,6-diamidino-2-phenylindole (blue). (b) Quantitative analysis of fluorescence intensity (scale bar: 100 μm). Data are mean ± standard deviation (n = 5 per group).

Discussion

Myocardial IR is increasingly recognized as a central player in the pathophysiology of cardiometabolic diseases. This study clearly demonstrated significant effects of EA on myocardial IR in ZDF rats. Following EA treatment, an improvement in glucose metabolism was observed, as reflected in both FBG and FINS levels. This finding resonates with recent research suggesting that EA can enhance insulin sensitivity.²⁴ Scientific consensus indicates that IR is closely associated with an elevated inflammatory state, which contributes to alterations in insulin signaling pathways within cardiac tissue, and it appears that EA can exert an anti-inflammatory effect.²⁵ In this study, EA ameliorated damage to myocardial tissue architecture and cellular ultrastructure, and diminished ectopic lipid deposition, potentially in association with the mechanisms underlying the improvement of myocardial IR. Moreover, by modulating the activity of key nodes in the insulin signaling pathway, such as the phosphorylation levels of IRS-1, Akt and GSK3β, EA may directly influence insulin signaling at a molecular level. These results offer new insights into how EA ameliorates myocardial IR.

In our study, ZDF rats exhibited significant myocardial IR, accompanied by elevated levels of CRP and C-peptide. Following EA or PIO treatments, both markers significantly decreased, suggesting that EA and PIO might have similar regulatory effects on these pathways and potentially alleviate myocardial IR by suppressing inflammation and improving pancreatic β-cell function.²⁶ IL-6 and IL-1β, which are key cytokines in inflammatory responses, are elevated under various inflammatory conditions and can exacerbate IR by promoting inflammatory reactions within the insulin signaling pathway.²⁷ Our results showed that EA significantly reduced IL-6 levels but had no significant effect on IL-1β, suggesting that EA might selectively target inflammatory pathways more relevant to myocardial IR. TNF-α and MCP-1 also play roles in the development of IR, which is closely related to the inflammatory state of adipose tissue, as well as insulin signaling and cardiovascular pathology.²⁸ EA treatment was significantly effective at reducing TNF-α levels and, while the reduction in MCP-1 was not statistically significant, it remains possible that the ameliorative effects of EA on myocardial IR might involve multiple inflammatory factors and their corresponding signaling pathways.

TNNT2 and BNP are essential biomarkers in the evaluation of cardiac function and damage, particularly in conditions such as diabetic cardiomyopathy.²⁹ Elevated levels of TNNT2 in the bloodstream are indicative of myocardial injury, as damaged or necrotic cardiomyocytes release TNNT2 into the circulation.³⁰ By contrast, BNP is a hormone that is primarily secreted by ventricular myocytes in response to increased wall stress, such as that caused by volume overload or heart failure.³¹ BNP functions to reduce blood volume and pressure, thereby serving as a compensatory mechanism in heart failure.³¹ Nevertheless, elevated BNP levels over an extended period have been linked to unfavorable cardiac outcomes. In this study, the ZDF group exhibited a decline in left ventricular ejection fraction (LVEF) and other key parameters, indicating impaired systolic function. These findings corresponded with elevated serum TNNT2 and BNP levels, suggesting both structural and functional damage. BNP is particularly important here, as its elevation is associated with left ventricular dysfunction and increased ventricular pressure, both of which are critical factors leading to heart failure.³² However, EA-induced improvements in BNP and TNNT2 levels were not accompanied by statistically significant changes in echocardiographic parameters, with the single exception of IVST. Nevertheless, given that TNNT2 reflects myocardial damage and BNP is a marker of heart failure and ventricular pressure, decreased TNNT2 and BNP levels following EA suggest decreased cellular injury and an alleviation of the compensatory pressure response in the heart, respectively.

Our immunofluorescence results further demonstrated a regulatory effect of EA on myocardial IR. GLUT4 is a key glucose transporter in cardiomyocytes and its reduction is typically associated with IR and impaired glucose metabolism.³³ TNF-α (a pro-inflammatory cytokine) is known to inhibit insulin signaling when overexpressed, which can exacerbate IR. In the ZDF group, there was a significant reduction in GLUT4 expression and a significant increase in TNF-α expression, indicating pronounced inflammation and IR in the myocardial tissue of diabetic rats. By contrast, the EA-treated group showed a marked increase in GLUT4 expression and a decrease in TNF-α levels, suggesting that EA may restore myocardial insulin signaling by reducing inflammation. This aligns with the reduction in serum TNNT2 and BNP levels in the ZDF + EA group, providing further support that EA improves myocardial metabolic function and structural integrity by modulating inflammation and insulin signaling pathways. The reduction in TNF-α likely contributed to decreased myocardial injury, as reflected by the lower TNNT2 levels, which in turn may have alleviated the compensatory stress on the heart, resulting in lower BNP levels. Moreover, the increased GLUT4 expression following EA treatment highlights its potential role in enhancing glucose uptake in cardiomyocytes, which is arguably crucial for improving insulin sensitivity and energy metabolism in the diabetic heart. By potentially mitigating the detrimental effects of TNF-α and restoring GLUT4 function, EA may play a critical role in preventing further deterioration of cardiac function in diabetic cardiomyopathy.

An intricate network of insulin signaling pathways, which includes key molecular players such as IRS-1, Akt, AMPK, p70S6K and GSK3β, is pivotal to the regulation of myocardial insulin sensitivity.³⁴ It is evident from our results that the phosphorylation states of IRS-1 and Akt, which are crucial for insulin signaling and glucose homeostasis, were significantly modulated by EA treatment. The phosphorylated to total protein ratios of IRS-1 and Akt in the EA-treated group showed marked improvement, suggesting that EA promotes the activation of these signaling pathways. This is consistent with the notion that IRS-1 is an essential mediator of insulin action and that Akt activation is linked to enhanced glucose uptake and improved metabolic profiles.³⁴ It is noteworthy that the ratio of phosphorylated to total p70S6K did not significantly change post-intervention, which could either be indicative of a time-dependent response that was not captured in our study timeframe or a potential difference in the sensitivity of this particular pathway to the interventions.^35,36 PIO treatment significantly increased the phosphorylation of AMPK, which may have contributed to improved metabolic outcomes, such as increased insulin sensitivity and glucose homeostasis in myocardial tissue. While EA modulated other critical pathways involved in insulin signaling, its effects on AMPK activation might be limited or require additional stimuli to achieve significance. On the other hand, the phosphorylation status of GSK-3β, a negative regulator of insulin signaling, was significantly reduced by EA and PIO, further affirming their roles in promoting insulin sensitivity.³⁷ Furthermore, mRNA expression levels of these proteins were also altered following treatment, reflecting the transcriptional changes induced by EA and PIO, which could have long-term implications for protein expression and the functional status of these signaling molecules. Collectively, these findings suggest that EA may exert its therapeutic effects through a multi-targeted approach, influencing both the phosphorylation state and mRNA expression of key insulin signaling molecules.

While our study presents promising preclinical evidence regarding the efficacy of EA as a potential therapeutic approach for myocardial IR, there are several limitations that must be acknowledged. First, the research was conducted on a specific animal model—the ZDF rat—which, while valuable, does not completely recapitulate the complex pathophysiology of human T2DM and its cardiovascular manifestations. This species-specific response underscores the need for cautious extrapolation of the results to the human condition and practice of acupuncture. Another limitation is the scope of the molecular analysis. Although we investigated key proteins involved in insulin signaling, the network governing myocardial IR is far more extensive. Looking to the future, it would be prudent to design studies that delve deeper into the mechanistic aspects of EA in terms of its effects on myocardial IR. Such studies should aim to map out the entire signaling network and identify all the relevant mediators and modulators.

In summary, EA ameliorated myocardial IR in a rat model of T2DM and positively impacted TNNT2 and BNP levels, as well as phosphorylation status and mRNA expression of several genes involved in the insulin signaling pathway. Our findings underscore the potential of EA to modulate multiple therapeutic targets in the treatment of myocardial IR. If effects can be replicated clinically, EA may represent a promising non-pharmacological option for the management of cardiometabolic risks associated with diabetes.

Footnotes

Acknowledgements

The authors would like to thank the South China Research Center for Acupuncture and Moxibustion for supplying experimental equipment and the research environment, as well as Dr Yifan Zhang for his advice and guidance.

Contributors

Z-X.L., X-X.L., J.S. and M.L. conceived and designed the experiments. X-X.L., J-Y.Q., L.Z. and L-L.N. performed the experiments. H-H.Z., X-X.L. and X-Z.C. analyzed the data. X-X.L. wrote the paper. All authors read and approved the final version of the manuscript accepted for publication.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This work was supported by Science, Technology and Innovation Commission of Shenzhen Municipality (grant no. JCYJ20230807094802005) and National Natural Science Foundation of China (grant nos 81804167 and 81774394).

Ethical approval

All experimental procedures were approved by the Laboratory Animal Center Ethics Committee of Guangzhou University of Chinese Medicine and conducted in accordance with the Chinese Guidelines for Laboratory Animals (GB14925-2001 and MOST 2006a).

ORCID iDs

Xiao-Xiao Liu

Jian Sun

Data sharing statement

The authors are willing to share the data generated by this study. Please contact Xiao-Xiao Liu (330408076@qq.com).

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