N-Lactoyl-phenylalanine Improves Cardiac Function in a Mouse Model of Diabetic Cardiomyopathy by Regulating Lipid Metabolism

Summary

This protocol describes in vivo and in vitro approaches to investigate how N-lactoyl-phenylalanine affects diabetic cardiomyopathy, cardiac lipid metabolism, and cholesterol synthesis, with a focus on the adenosine monophosphate-activated protein kinase alpha 1/3-hydroxy-3-methylglutaryl-coenzyme A reductase pathway.

Abstract

Diabetic cardiomyopathy (DCM) is one of the most serious complications of diabetes. N-lactoyl-phenylalanine (Lac-Phe), an endogenous metabolite, can promote weight loss and improve glucose homeostasis. This study sought to investigate the therapeutic effect of Lac-Phe on DCM and further explore the underlying mechanism. Type 2 diabetic mice with DCM were treated with Lac-Phe. Lac-Phe enhanced cardiac contractile function, ameliorated pathological remodeling, and reduced lipid accumulation in the heart. In vitro, the effects of Lac-Phe were investigated in H9c2 cells stimulated with high glucose and palmitic acid. Lac-Phe reduced lipid accumulation by decreasing cholesterol synthesis in H9c2 cells. Mechanistically, Lac-Phe promoted the phosphorylation and activation of adenosine monophosphate-activated protein kinase alpha 1 (AMPKα1). Subsequently, activated AMPKα1 phosphorylated and inhibited its downstream target, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), leading to reduced cholesterol synthesis and ultimately ameliorating lipid metabolism imbalance in H9c2 cells. These findings indicate that Lac-Phe improves lipid metabolism in H9c2 cells in DCM through the AMPKα1/HMGCR pathway, thereby enhancing cardiac contractile function and ameliorating pathological remodeling. Lac-Phe may represent a potential therapeutic approach for diabetic cardiomyopathy.

Introduction

Diabetes is a metabolic disease characterized by persistent hyperglycemia¹. Over the past two decades, diabetes has become one of the top ten causes of death worldwide, with more than six million people dying from diabetes and its complications each year². Diabetic cardiomyopathy (DCM), a cardiac pathological condition resulting from diabetes, represents one of the most severe complications associated with diabetes^3,4. It functions as an independent risk factor for heart failure and is characterized by structural and functional myocardial alterations^5,6, including cardiac fibrosis and cardiomyocyte hypertrophy, which ultimately lead to both diastolic and systolic dysfunction. Although several conventional cardiovascular therapies, such as β-blockers, angiotensin-converting enzyme inhibitors, and angiotensin II type 1 receptor blockers, are currently employed in the management of DCM^7,8, their efficacy remains suboptimal in clinical settings. As the mechanisms underlying DCM have not been thoroughly studied⁹, there is currently no clear treatment strategy targeting this disease^10,11. The clinical manifestations of DCM progress from asymptomatic diastolic dysfunction in the early stage to systolic dysfunction and clinical heart failure in the late stage, and even death^12,13. Therefore, there is an urgent need to explore and develop effective treatment options for this condition.

Imbalanced lipid metabolism is a key characteristic of DCM¹⁴. In DCM, increased levels of free fatty acids (FFAs) in the blood can lead to excessive uptake of fatty acids by cardiomyocytes^15,16. Fatty acid metabolism in cardiomyocytes mainly depends on fatty acid oxidation. Over time, fatty acid oxidation in cardiomyocytes is insufficient to completely metabolize all ingested fatty acids, and excess fatty acids and their intermediate metabolites accumulate in cardiomyocytes¹⁷. This excessive lipid load induces lipotoxicity, disrupts myocardial energy supply, and promotes cardiac oxidative stress and inflammatory responses, ultimately causing severe damage to cardiomyocytes^18,19. Therefore, actively regulating cardiac lipid metabolism may represent a potential therapeutic target for DCM²⁰.

N-lactoyl-phenylalanine (Lac-Phe) is an endogenous metabolite whose concentration rises rapidly after exercise²¹. Mechanistically, Lac-Phe is synthesized by the cytosolic enzyme carnosine dipeptidase 2 (CNDP2) through the condensation of lactate and phenylalanine. This biosynthetic pathway is widely distributed across diverse cell types, including macrophages, epithelial cells, and mesenchymal stem cells, which secrete Lac-Phe into the circulation following physical activity²². Chronic administration of Lac-Phe has been shown to effectively reduce food intake, decrease obesity and body weight, and improve glucose homeostasis²¹. A recent study has shown that Lac-Phe can improve the lipid metabolism capacity of microglia/macrophages following spinal cord injury²³. Interestingly, studies indicate that both acute and chronic administration of metformin, a first-line medication for type 2 diabetes, can elevate Lac-Phe levels and that Lac-Phe mediates the anti-obesity effects of metformin²². Moreover, Sha et al. recently demonstrated that Lac-Phe ameliorates myocardial cell death by alleviating mitochondrial dysfunction, thereby improving transverse aortic constriction (TAC)-induced heart failure²⁴. Prolonged hyperglycemia and obesity are important factors contributing to dysregulation of lipid metabolism in DCM²⁵. Collectively, this evidence suggests that Lac-Phe may have a potential therapeutic effect on DCM. However, to date, the role of Lac-Phe specifically in DCM has not been investigated.

In this study, we found that exogenous administration of Lac-Phe to mice with diabetic cardiomyopathy effectively improved cardiac contractile function and ameliorated cardiac pathological remodeling. We found that Lac-Phe improved cardiac function in mice with DCM through the regulation of lipid metabolism imbalance. Lac-Phe reduced lipid droplet (LD) accumulation in cardiomyocytes of mice with DCM and decreased cholesterol content in the heart. Mechanistically, Lac-Phe promoted the phosphorylation and activation of adenosine monophosphate-activated protein kinase alpha 1 (AMPKα1), thereby promoting the phosphorylation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). HMGCR phosphorylation reduces its activity, resulting in decreased cholesterol production and thereby improving the imbalance of lipid metabolism in cardiomyocytes. These results provide a theoretical foundation for the potential treatment of DCM with Lac-Phe.

Protocol

The animal study protocol was approved by the Institutional Ethics Committee of Nanjing Drum Tower Hospital (Approval Number: 2025AE1065, 26 September 2025) and was conducted in accordance with the guidelines outlined in the Guidelines for the Care and Use of Laboratory Animals (8th Edition), published by the National Institutes of Health of the United States.

Animals

Thirty 5-week-old male C57BL/6J mice were used in this study. After 1 week of adaptive feeding, all mice were housed with access to food and water under an alternating 12 h day-night cycle at a temperature of 22°C ± 2°C and humidity of 40%–60%. An HFD combined with low-dose STZ was used to establish the DCM mouse model (Figure 1A). Four weeks after initiation of high-fat diet (HFD; 60% fat, 20% protein, and 20% carbohydrate) feeding, mice were fasted overnight and subsequently administered streptozotocin (STZ; 8 mg/mL dissolved in citrate buffer, pH 4.5) via intraperitoneal injection at a dosage of 35 mg/kg/day for three consecutive days. Control group mice were fed a control diet (10% fat, 20% protein, and 70% carbohydrate) and received equivalent volumes of citrate buffer. Following 15 additional weeks of high-fat diet maintenance, mice meeting both criteria, random blood glucose of ≥13.3 mmol/L and left ventricular ejection fraction (LVEF) of <55%, were defined as successfully established DCM models. These validated DCM mice were numbered and randomly assigned to the DCM and DCM + Lac-Phe groups using a random number table. Approximately half of the mice failed to develop the model; these animals were euthanized in accordance with animal ethics requirements. In brief, mice were divided into three groups: Control, DCM, and DCM + Lac-Phe (n = 10 for each group). All 10 mice per group underwent echocardiography, body weight measurement, and OGTT. Following the 28-day treatment period, hearts were collected for subsequent analyses. There were 10 mice in each group. Of these, three mice per group were used for transcriptome sequencing, three were used for transmission electron microscopy and histological staining, and three were used for protein expression analysis. The remaining mouse in each group was reserved as a backup. After successful establishment of the DCM model, Lac-Phe was dissolved in normal saline to a final concentration of 10 mg/mL. Mice in the DCM + Lac-Phe group received intraperitoneal Lac-Phe at 50 mg/kg/day in an injection volume of 5 mL/kg, once daily for 28 consecutive days. Mice in the Control and DCM groups were administered an equivalent volume of normal saline as vehicle control.

Figure 1. Lac-Phe improves cardiac function and pathological remodeling in mice with diabetic cardiomyopathy. (A) Schematic of the HFD/STZ-induced DCM mouse model and Lac-Phe treatment protocol. (B) Body weights of mice in the Control, DCM, and DCM + Lac-Phe groups during the 4-week treatment period; n = 10 per group. (C) Oral glucose tolerance test (OGTT) in the indicated groups; n = 10 per group. (D) Representative echocardiographic images at 0, 2, and 4 weeks of treatment. (E,F) Quantification of left ventricular ejection fraction (LVEF) (E) and fractional shortening (FS) (F); n = 10 per group. (G) Representative hematoxylin and eosin (H&E)-stained heart sections. Scale bar = 1 mm. (H,I) Representative Masson’s trichrome staining of heart sections (H) and quantification of fibrotic area (I); n = 3 per group. Scale bar = 50 μm. (J,K) Representative wheat germ agglutinin (WGA) staining of heart sections (J) and quantification of relative cardiomyocyte area (K); n = 3 per group. Scale bar = 20 μm. Data are presented as the mean ± standard deviation. P < 0.05, **P < 0.001. Please click here to view a larger version of this figure.

Cardiac function

Following anesthesia with isoflurane inhalation, mice were secured in a supine position on a temperature-controlled platform (37°C). Left ventricular dimensions and mitral valve blood flow velocity during diastole and systole were measured using an echocardiography machine equipped with a 40 MHz transducer, from which LVEF and shortening fraction (FS) were calculated. Three consecutive cardiac cycles were collected from each mouse.

Cell treatment

H9c2 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Cells were passaged every 2–3 days upon reaching 70%–80% confluence. For experiments, cells were seeded in 10 cm dishes and then divided into three groups: Control, high glucose/palmitic acid (HG/PA), and HG/PA + Lac-Phe. Cells in the HG/PA and HG/PA + Lac-Phe groups were first exposed to 0.25 mM palmitic acid and 50 mM glucose for 24 h. Thereafter, cells in the HG/PA + Lac-Phe group were treated with 10 μg/mL Lac-Phe for an additional 24 h while HG/PA exposure was maintained; cells in the HG/PA group received an equivalent volume of PBS under the same HG/PA conditions. In the p-AMPK inhibition experiments, 10 μM AMPK-IN-3 (dissolved in DMSO) was added simultaneously with Lac-Phe for 24 h, and the corresponding control groups received an equal volume of DMSO. For the atorvastatin experiments, H9c2 cells under HG/PA conditions were treated with 10 μM atorvastatin for 24 h, either alone or in combination with 10 μg/mL Lac-Phe.

Histological staining

After 28 days of Lac-Phe administration, mouse hearts were harvested and immersion-fixed in 4% paraformaldehyde (pH 7.4) for 24 h. Fixed cardiac tissues were paraffin-embedded and sectioned into 6-μm-thick slices. Tissue sections were stained with hematoxylin and eosin, wheat germ agglutinin, and Masson’s trichrome. All staining procedures were performed strictly following the manufacturers’ protocols. Whole-slide imaging was performed at 20× magnification using a high-resolution brightfield microscope equipped with a digital camera system.

Western blot analysis

Protein concentration was determined using the Bradford assay. Tissue lysates were resolved by SDS-PAGE on 10% (w/v) acrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes. Nonspecific binding sites were blocked with 5% bovine serum albumin for 2 h at room temperature. Membranes were then incubated overnight at 4°C with primary antibodies (diluted 1:1,000), followed by six washes with TBST (5 min each) on a shaker. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1:5,000) for 2 h at room temperature, followed by another six washes with TBST (5 min each). Protein bands were visualized using an enhanced chemiluminescence reagent kit. Quantification of western blot results was performed by densitometric analysis using ImageJ software. All antibodies used in this study are listed in the Table of Materials.

Transcriptomics

Total RNA was isolated from cardiac tissue of C57BL/6J mice using TRIzol Reagent and RNeasy mini kits. RNA quality was evaluated using a 2100 bioanalyzer, demonstrating high RNA purity and integrity. RNA-seq libraries were prepared according to the VAHTS Universal V10 RNA-seq Library Prep Kit and sequenced using an Illumina NovaSeq 6000 System (single-read, 150 bp). Differentially expressed genes were identified using DESeq2, with an adjusted P value < 0.05 and an absolute log₂ fold change > 1 considered statistically significant. Gene Ontology (GO) enrichment analysis was conducted on differentially expressed genes, and the significance of enrichment of differentially expressed genes in GO terms was calculated using the hypergeometric distribution algorithm.

Oil Red O staining and LD540 lipid staining

Cardiac tissue was embedded in OCT compound and then cut into 10-μm-thick sections. The frozen sections were equilibrated at room temperature for 30 min and fixed with 4% paraformaldehyde for 10 min. After washing with PBS, the sections were placed in 60% isopropyl alcohol for 15 s and covered with Oil Red O working solution for 30 min. The sections were then treated with 60% isopropyl alcohol for 15 s and washed three times with PBS. For LD540 lipid staining, H9c2 cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were then incubated with 10 μM LD540 for 30 min at room temperature.

Transmission electron microscopy (TEM)

Fresh mouse heart tissues were cut into 1–2 mm³ pieces, fixed in electron microscope fixative at 4°C for 4 h, then washed with double-distilled water and immersed in ethanol solutions of progressively increasing concentrations. The samples were embedded, sectioned, and then observed under a transmission electron microscope.

Total cholesterol (TC) content

Total cholesterol (TC) levels in serum and H9c2 cell samples were measured using the Amplex Red Cholesterol and Cholesteryl Ester Assay Kit according to the manufacturer’s instructions.

Oral Glucose Tolerance Test (OGTT)

Mice were fasted for 14 h (from 19:00 to 9:00) and gavaged with glucose (20% stock, 2 g/kg). Blood glucose levels were determined using blood collected from the tail vein at 0, 15, 30, 60, and 120 min after gavage.

Cell counting kit-8 (CCK-8) measurement

CCK-8 was used to measure cell viability. H9c2 cells were seeded into 96-well plates at an initial density of 3 × 10⁴ cells/well. After treatment with different concentrations of Lac-Phe under HG/PA conditions for 24 h, 10 μL of the kit reagent was added to 100 μL of culture medium per well, followed by incubation at 37°C for 4 h. The absorbance was measured at 450 nm using a multimode microplate reader to obtain the optical density (OD) value.

Molecular docking

The crystal structures of Lac-Phe and AMPKα1 were downloaded from PubChem²⁶. The molecular docking process was carried out using CB-DOCK2²⁷.

Statistical analysis

All echocardiographic measurements, histological staining, and image quantifications were performed by investigators blinded to group assignment. All data analyses were performed using Prism and SPSS 20.0. Each experiment was repeated at least three times, and the data were presented as the mean ± standard deviation. Homogeneity of variance was assessed using Levene’s test. Normality was confirmed by graphical inspection of residuals. Comparisons among multiple groups were conducted using one-way analysis of variance, followed by Tukey's post hoc test for pairwise comparisons. For OGTT data, two-way repeated-measures ANOVA was used, with group as the between-subjects factor and time as the within-subjects factor, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.

Results

Lac-Phe improves cardiac function in diabetic cardiomyopathy mice

We induced DCM in C57BL/6J mice using an HFD combined with STZ injections (Figure 1A) and randomly assigned the successfully established DCM mice to an untreated group (DCM group) and a Lac-Phe treatment group, with control C57BL/6J mice serving as the negative control. According to the study by Li et al.²¹, we selected 50 mg/kg as the therapeutic dose of Lac-Phe. After successful model establishment, the Lac-Phe treatment group received four weeks of exogenous Lac-Phe injections, while the untreated group received injections of an equivalent volume of saline. Compared with the DCM group, mice with diabetic cardiomyopathy treated with Lac-Phe exhibited a significant reduction in body weight after four weeks (Figure 1B). OGTT revealed that, compared with the negative control group, mice in the DCM group had significantly elevated blood glucose levels at all measured time points, indicating impaired glucose tolerance. In contrast, DCM mice treated with Lac-Phe showed improved glucose tolerance (Figure 1C). Echocardiographic analysis revealed that, compared with negative control mice, mice with DCM exhibited significantly reduced LVEF and FS (Figure 1D–F). Treatment with Lac-Phe increased LVEF and FS in DCM mice. Histopathological analysis using H&E staining showed pathological hypertrophy in the cardiac tissue of DCM mice compared with control mice (Figure 1G). Lac-Phe treatment markedly alleviated myocardial hypertrophy. Masson’s trichrome staining further demonstrated that Lac-Phe significantly alleviated myocardial fibrosis in DCM hearts (Figure 1H,I). WGA staining indicated that Lac-Phe significantly reduced the cross-sectional area of cardiomyocytes in DCM hearts (Figure 1J,K). These experiments demonstrate that exogenous administration of Lac-Phe effectively improves cardiac function and ameliorates pathological remodeling in mice with DCM.

Lac-Phe ameliorates dysregulated lipid metabolism in DCM

To further elucidate the mechanism of Lac-Phe in DCM, we performed transcriptomic analysis of mouse cardiac tissues. Following data acquisition, GO and KEGG pathway analyses were conducted. GSEA of the fatty acid metabolic process and KEGG pathway enrichment analysis indicated alterations in pathways related to lipid metabolism (Figure 2A,B). To further investigate how Lac-Phe functions in the hearts of DCM mice, we conducted TEM analysis (Figure 2C,D) to examine whether Lac-Phe treatment altered the ultrastructural morphology of the hearts of DCM mice. TEM imaging revealed a significant increase in LD density within the hearts of DCM mice compared with control mice, whereas Lac-Phe administration effectively reduced this LD density. Given the established significance of lipid metabolism dysregulation in DCM, we further assessed cardiac lipid accumulation using Oil Red O staining (Figure 2E,F). Histological analysis demonstrated markedly increased lipid accumulation in the hearts of DCM mice, whereas Lac-Phe treatment reduced this lipid accumulation. Lac-Phe treatment also reduced the TC level in the serum of DCM mice (Figure 2G). To select an appropriate dose of Lac-Phe for the in vitro experiments, we employed the CCK-8 assay to measure the viability of H9c2 cells treated with various concentrations of Lac-Phe under HG/PA conditions (Figure 2H). H9c2 cells treated with Lac-Phe at a concentration of 10 μg/mL under HG/PA conditions exhibited the highest survival rate among the tested concentrations. Thus, we selected 10 μg/mL Lac-Phe for the subsequent experiments. H9c2 cells exposed to HG/PA showed significant lipid accumulation, as demonstrated by LD540 staining and fluorescence quantification (Figure 2I,J), and TC content was also increased (Figure 2K). Lac-Phe treatment reversed these changes, significantly reducing both lipid accumulation and TC levels in H9c2 cells. Collectively, these experimental findings demonstrate that Lac-Phe effectively attenuates cardiac lipid accumulation and ameliorates lipid metabolism dysregulation in DCM mice and reduces lipid accumulation and TC levels in HG/PA-treated H9c2 cells.

Figure 2. Lac-Phe ameliorates dysregulated lipid metabolism in diabetic cardiomyopathy. (A) Gene set enrichment analysis (GSEA) of the fatty acid metabolic process in the Control and DCM groups. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis comparing the DCM and DCM + Lac-Phe groups. (C) Representative transmission electron microscopy (TEM) images of cardiac tissues, with red arrows indicating lipid droplets (LDs). Scale bar = 2 μm. (D) Quantification of the mean number of LDs per 100 μm²; n = 3 per group. (E,F) Representative Oil Red O staining of cardiac tissues (E) and quantification of the relative Oil Red O-positive area (F); n = 3 per group. Scale bar = 20 μm. (G) Relative total cholesterol (TC) levels in serum from the indicated groups; n = 3 per group. (H) Cell viability measured by the CCK-8 assay in H9c2 cells treated with 0, 1, 5, 10, or 20 μg/mL Lac-Phe under HG/PA conditions. (I,J) Representative LD540 staining of H9c2 cells (I) and quantification of mean LD540 fluorescence intensity (J); n = 3 per group. Scale bars = 200 μm. (K) TC levels in H9c2 cells from the indicated groups; n = 3 per group. Data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Please click here to view a larger version of this figure.

Lac-Phe increases HMGCR phosphorylation in the hearts of DCM mice and H9c2 cells

To identify potential targets through which Lac-Phe improves lipid metabolism, we screened DCM-associated proteins using GeneCards²⁸ and predicted proteins potentially interacting with Lac-Phe using SwissTargetPrediction²⁹ (Figure 3A). By comparing the results, we identified six potential target genes (Figure 3B). Analysis of these six genes revealed that HMGCR is a core regulator of cholesterol metabolism and also plays a central role in DCM (Figure 3C)³⁰. To determine whether Lac-Phe regulates lipid metabolism via HMGCR, we first examined the p-HMGCR/HMGCR ratio in mouse hearts. Western blot analysis revealed that the p-HMGCR/HMGCR ratio was decreased in the hearts of DCM mice, whereas Lac-Phe treatment increased this ratio (Figure 3D,E). These findings are consistent with increased HMGCR phosphorylation and reduced HMGCR activity following Lac-Phe treatment. In vitro experiments showed that, under HG/PA conditions, the p-HMGCR/HMGCR ratio was decreased in H9c2 cells, whereas Lac-Phe treatment increased this ratio (Figure 3F,G). These results demonstrate that Lac-Phe increases HMGCR phosphorylation in the hearts of DCM mice and in HG/PA-treated H9c2 cells, supporting the involvement of HMGCR in the effects of Lac-Phe on lipid metabolism.

Figure 3. Lac-Phe increases HMGCR phosphorylation in the hearts of mice with diabetic cardiomyopathy and in H9c2 cells. (A) Venn diagram showing the overlap between DCM-associated proteins and predicted Lac-Phe-associated proteins. (B) Six overlapping proteins associated with DCM and Lac-Phe. (C) Protein-protein interaction network showing HMGCR within the network of DCM-associated proteins. (D,E) Representative western blot images of p-HMGCR and HMGCR in cardiac tissues from the indicated groups (D) and quantification of the p-HMGCR/HMGCR ratio (E); n = 3 per group. (F,G) Representative western blot images of p-HMGCR and HMGCR in H9c2 cells from the indicated groups (F) and quantification of the p-HMGCR/HMGCR ratio (G); n = 3 per group. β-Tubulin was used as the loading control. Data are presented as the mean ± standard deviation. *P < 0.05. Please click here to view a larger version of this figure.

Lac-Phe combined with atorvastatin improves lipid metabolism in H9c2 cells

To further evaluate the effect of Lac-Phe on lipid metabolism via HMGCR, we used atorvastatin as a control. In vitro experiments showed that, under HG/PA conditions, combined treatment with Lac-Phe and atorvastatin produced a greater reduction in lipid accumulation than atorvastatin treatment alone. LD540 staining and fluorescence quantification demonstrated that combined treatment with Lac-Phe and atorvastatin reduced LD accumulation in H9c2 cells (Figure 4A,B). TC content measurement showed a similar effect (Figure 4C). Western blot analysis showed that combined treatment with Lac-Phe and atorvastatin significantly increased the p-HMGCR/HMGCR ratio (Figure 4D,E). These results indicate that combined Lac-Phe and atorvastatin treatment is associated with reduced lipid accumulation and TC levels and increased HMGCR phosphorylation in H9c2 cells.

Figure 4. Lac-Phe combined with atorvastatin improves lipid metabolism in H9c2 cells. (A,B) Representative LD540 staining of H9c2 cells from the indicated treatment groups (A) and quantification of mean LD540 fluorescence intensity (B); n = 3 per group. Scale bar = 200 μm. (C) Total cholesterol (TC) levels in H9c2 cells from the indicated treatment groups; n = 3 per group. (D,E) Representative western blot images of p-HMGCR and HMGCR in H9c2 cells from the indicated treatment groups (D) and quantification of the p-HMGCR/HMGCR ratio (E); n = 3 per group. β-Tubulin was used as the loading control. Data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, ****P < 0.0001. Please click here to view a larger version of this figure.

Lac-Phe increases AMPKα1 phosphorylation

To further explore the molecular mechanism by which Lac-Phe regulates HMGCR, we reanalyzed the mouse cardiac transcriptome data. Through KEGG analysis of the transcriptomic data, we identified 160 differentially enriched pathways between the DCM and Control groups and 89 differentially enriched pathways between the DCM and DCM + Lac-Phe groups (Figure 5A). Sixty pathways overlapped between these two comparisons. From these 60 overlapping pathways, we selected the AMPK pathway. AMPK plays a critical role in regulating lipid metabolism in DCM³¹. Furthermore, AMPK is upstream of HMGCR (Figure 5B). We hypothesized that Lac-Phe might regulate HMGCR by modulating AMPK, ultimately improving lipid metabolism. To test this hypothesis, we first performed molecular docking of AMPKα1 and Lac-Phe using CB-DOCK2. The molecular docking results showed that the best-ranked predicted binding conformation between Lac-Phe and AMPKα1 had an affinity of −7.6 kcal/mol (Figure 5C). These results suggest a potential interaction between Lac-Phe and AMPKα1 but do not establish direct intracellular binding. To determine whether Lac-Phe affects the AMPK pathway in DCM, we examined AMPKα1 phosphorylation in the hearts of DCM mice. Western blot analysis revealed that the p-AMPKα1/AMPKα1 ratio was decreased in the hearts of DCM mice, whereas Lac-Phe treatment increased this ratio (Figure 5D,E). In vitro experiments showed that, under HG/PA conditions, the p-AMPKα1/AMPKα1 ratio was decreased in H9c2 cells, whereas Lac-Phe treatment increased this ratio (Figure 5F,G). These findings indicate that Lac-Phe increases AMPKα1 phosphorylation in DCM mouse hearts and HG/PA-treated H9c2 cells.

Figure 5. Lac-Phe increases AMPKα1 phosphorylation in the hearts of mice with diabetic cardiomyopathy and in H9c2 cells. (A) Venn diagram showing the overlap between pathway enrichment results for the DCM/Control and DCM + Lac-Phe/DCM comparisons. (B) Schematic illustrating AMPK and HMGCR phosphorylation and the associated inhibition of cholesterol synthesis. (C) Predicted three-dimensional molecular docking interaction between Lac-Phe and AMPKα1; binding affinity = −7.6 kcal/mol. (D,E) Representative western blot images of p-AMPKα1 and AMPKα1 in cardiac tissues from the indicated groups (D) and quantification of the p-AMPKα1/AMPKα1 ratio (E); n = 3 per group. (F,G) Representative western blot images of p-AMPKα1 and AMPKα1 in H9c2 cells from the indicated groups (F) and quantification of the p-AMPKα1/AMPKα1 ratio (G); n = 3 per group. GAPDH was used as the loading control. Data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01. Please click here to view a larger version of this figure.

AMPK inhibition attenuates Lac-Phe-mediated HMGCR phosphorylation and improvement of lipid metabolism in H9c2 cells

We then added a p-AMPK inhibitor to H9c2 cells receiving Lac-Phe treatment. The p-AMPK inhibitor decreased the p-AMPKα1/AMPKα1 ratio (Figure 6A,B) and reduced the p-HMGCR/HMGCR ratio (Figure 6C,D). These findings indicate that inhibition of AMPK phosphorylation is accompanied by reduced HMGCR phosphorylation during Lac-Phe treatment. The p-AMPK inhibitor also diminished the effect of Lac-Phe on lipid metabolism. LD540 staining and fluorescence quantification showed increased lipid accumulation in H9c2 cells treated with the p-AMPK inhibitor compared with cells receiving Lac-Phe without AMPK inhibition (Figure 6E,F). TC levels in H9c2 cells also increased following AMPK inhibition (Figure 6G). These results collectively support the involvement of AMPKα1/HMGCR signaling in the effects of Lac-Phe on lipid metabolism. Lac-Phe increased AMPKα1 phosphorylation, which was associated with increased HMGCR phosphorylation and reduced lipid accumulation and TC levels. Inhibition of AMPK attenuated these effects in H9c2 cells, supporting the hypothesis that Lac-Phe improves lipid metabolism through regulation of the AMPKα1/HMGCR pathway.

Figure 6. AMPK inhibition attenuates the effects of Lac-Phe on HMGCR phosphorylation and lipid accumulation in H9c2 cells. (A,B) Representative western blot images of p-AMPKα1 and AMPKα1 in H9c2 cells treated with HG/PA, Lac-Phe, and/or AMPK-IN-3 as indicated (A) and quantification of the p-AMPKα1/AMPKα1 ratio (B); n = 3 per group. (C,D) Representative western blot images of p-HMGCR and HMGCR in H9c2 cells from the indicated treatment groups (C) and quantification of the p-HMGCR/HMGCR ratio (D); n = 3 per group. GAPDH and β-tubulin were used as loading controls in (A) and (C), respectively. (E,F) Representative LD540 staining of H9c2 cells from the indicated treatment groups (E) and quantification of mean LD540 fluorescence intensity (F); n = 3 per group. Scale bar = 200 μm. (G) Total cholesterol (TC) levels in H9c2 cells from the indicated treatment groups; n = 3 per group. Data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. Please click here to view a larger version of this figure.

Collectively, the findings from the in vivo and in vitro experiments support a proposed mechanism whereby Lac-Phe increases AMPKα1 and HMGCR phosphorylation, which is associated with reduced cholesterol levels and lipid accumulation and improved cardiac function and pathological remodeling in DCM. The proposed AMPKα1/HMGCR pathway underlying the effects of Lac-Phe is summarized schematically in Figure 7.

Figure 7. Proposed mechanism by which Lac-Phe improves lipid metabolism in diabetic cardiomyopathy through the AMPK/HMGCR pathway. Schematic illustrating the proposed mechanism whereby Lac-Phe promotes AMPKα1 phosphorylation, followed by HMGCR phosphorylation, which is associated with reduced lipid droplet accumulation and lipotoxicity in diabetic cardiomyopathy. AMPK, AMP-activated protein kinase; HMGCR, HMG-CoA reductase; LD, lipid droplet. Please click here to view a larger version of this figure.

Data Availability:

The raw data supporting the findings of this study have been deposited in Zenodo and are publicly available at https://zenodo.org/records/21991371 

Discussion

In this study, we provide evidence supporting a therapeutic effect of Lac-Phe on DCM. Lac-Phe improved cardiac remodeling and cardiac dysfunction in DCM while reducing cholesterol production in cardiomyocytes and ameliorating lipid metabolism imbalance. Mechanistically, Lac-Phe increased AMPKα1 phosphorylation. Subsequently, increased AMPKα1 phosphorylation was associated with increased phosphorylation and inhibition of its downstream target HMGCR, leading to a reduction in cholesterol synthesis and ultimately ameliorating lipid metabolism imbalance in cardiomyocytes. In summary, this study demonstrates that Lac-Phe improves cardiac lipid metabolism in DCM through the AMPK/HMGCR signaling pathway. A schematic summary of the main findings and the proposed mechanism is presented in Figure 7.

Lipid metabolism imbalance is one of the key factors contributing to myocardial injury in DCM¹⁵. Excessive lipid accumulation in cardiomyocytes leads to lipotoxicity, resulting in cardiomyocyte dysfunction and pathological cardiac remodeling¹⁶. Long-term hyperglycemia and obesity are important factors leading to lipid metabolism disorders²⁵. Lac-Phe has been shown to be an endogenous substance that can effectively reduce food intake, decrease obesity and body weight, and improve glucose homeostasis²¹. Recent reports have also shown that Lac-Phe can regulate lipid metabolism in microglia/macrophages during spinal cord injury²³, demonstrating that Lac-Phe can regulate lipid metabolism in another disease model. As an endogenous metabolite, the role of Lac-Phe in metabolic diseases has attracted considerable interest. However, the role of Lac-Phe in DCM has not yet been fully investigated. Our study revealed a role for Lac-Phe in cardiomyocyte lipid metabolism in DCM. Through transcriptomic analysis, we found that Lac-Phe treatment was associated with alterations in lipid metabolism-related pathways in the hearts of DCM mice. Oil Red O staining and TEM observations confirmed reduced lipid accumulation in the hearts of Lac-Phe-treated DCM mice. These findings suggest that Lac-Phe exerts cardioprotective effects in DCM by reducing cardiac lipid accumulation and ameliorating pathological remodeling. In vitro experiments also demonstrated that Lac-Phe administration alleviated lipid accumulation in H9c2 cells exposed to HG/PA conditions.

HMGCR is an NADPH-dependent, rate-limiting enzyme in the mevalonate pathway³⁰. HMGCR catalyzes the conversion of HMG-CoA to mevalonate, a critical step in cholesterol biosynthesis^30,31,32. HMGCR represents a therapeutic target relevant to cardiovascular disease and DCM³³. Clinically, inhibition of HMGCR is an established approach for reducing cardiovascular risk³⁴. Our experimental data demonstrate that Lac-Phe increases HMGCR phosphorylation in the hearts of type 2 diabetic mice, consistent with inhibition of HMGCR activity and reduced cholesterol synthesis. In vitro studies further showed that Lac-Phe administration increased HMGCR phosphorylation in H9c2 cells exposed to HG/PA conditions, accompanied by reduced cholesterol levels. Lac-Phe combined with the cholesterol-lowering drug atorvastatin also produced greater effects than atorvastatin alone in the experimental conditions examined. In H9c2 cells treated with atorvastatin, the addition of Lac-Phe increased HMGCR phosphorylation and decreased lipid accumulation. These findings support the feasibility of targeting HMGCR phosphorylation through Lac-Phe to reduce cholesterol production and restore lipid metabolism balance in DCM. However, whether Lac-Phe could provide a safe and effective therapeutic strategy for patients with DCM requires further investigation.

AMPK, a key enzyme in mitochondrial energy metabolism, is central to the study of metabolic diseases such as diabetes^35,36. Through regulation of phosphorylation and dephosphorylation, AMPK plays a pivotal role in cardiac lipid metabolism. Phosphorylated AMPK represents its active form³⁷. p-AMPK promotes fatty acid entry into mitochondria for FAO by regulating CPT-1³⁸. Consistent with previous studies³⁹, our results showed that increased AMPK phosphorylation was associated with increased HMGCR phosphorylation and reduced cholesterol synthesis. In mice with DCM induced by STZ and HFD, persistent hyperglycemia can lead to changes in cardiac p-AMPK levels⁴⁰. In the early stage of diabetes, when cardiac function has not changed, cardiac p-AMPK levels may remain unaffected^10,37,41. In advanced diabetes, with persistent hyperglycemia for more than 8 weeks, pathological cardiac remodeling begins and p-AMPK levels can decrease¹⁰. Maintaining AMPK activation has been proposed as an effective strategy for preventing DCM^10,42. Metformin, a drug widely used to treat type 2 diabetes, has been shown to activate the AMPK pathway but has been reported to have a neutral effect on the heart^43,44. Interestingly, both acute and chronic metformin administration can increase Lac-Phe levels²². These observations raise the possibility that Lac-Phe may contribute to metabolic signaling associated with AMPK activation; however, the present study does not establish that exogenous Lac-Phe produces a more specific or safer effect than metformin.

In our study, AMPKα1 phosphorylation was markedly reduced in the late-stage DCM model, as shown in Figure 5D,E, a finding consistent with previous research⁴². Transcriptomic analysis revealed changes involving the AMPK pathway in the hearts of Lac-Phe-treated type 2 diabetic mice, and western blot analysis showed increased AMPKα1 phosphorylation following Lac-Phe treatment. Molecular docking suggested a potential interaction between Lac-Phe and AMPKα1, which is consistent with a recent study²³. Importantly, molecular docking alone does not establish direct intracellular binding between Lac-Phe and AMPKα1. In vitro experiments showed that treatment with the AMPK inhibitor suppressed AMPKα1 phosphorylation and was accompanied by reduced HMGCR phosphorylation. Following the addition of the AMPK inhibitor, the effects of Lac-Phe were attenuated, coinciding with increased lipid accumulation and elevated cholesterol content in H9c2 cells. Collectively, these results support the involvement of AMPKα1/HMGCR signaling in the effects of Lac-Phe on lipid metabolism. Lac-Phe increased AMPKα1 phosphorylation, which was associated with increased HMGCR phosphorylation, reduced cholesterol levels, and reduced lipid accumulation; inhibition of AMPK attenuated these effects.

There are several limitations to this study. First, all animal experiments were conducted in male mice. Although we demonstrated the therapeutic effect of Lac-Phe on DCM in male C57BL/6J mice, sex is an important factor influencing cardiovascular disease⁴⁵. Therefore, whether Lac-Phe has the same effects in female mice remains unknown. Second, transcriptome sequencing was performed on whole-heart samples, which might have masked cell type-specific changes in the heart following Lac-Phe treatment. Single-cell sequencing could provide deeper insight into the cellular and molecular heterogeneity underlying the observed effects^46,47 and represents an alternative approach for further investigating this hypothesis. Finally, because of limitations of the ultrasound equipment used in this study, we were unable to assess cardiac diastolic function in mice. Further studies are therefore needed to clarify the therapeutic effects of Lac-Phe on DCM, evaluate potential sex-dependent effects, investigate cell type-specific responses, assess diastolic function, and further elucidate the mechanisms underlying the observed effects. In summary, this study provides evidence supporting Lac-Phe as a potential endogenous metabolite of interest for the treatment of DCM. Furthermore, our findings support a proposed mechanism whereby Lac-Phe reduces lipid accumulation in cardiomyocytes during DCM through the AMPKα1/HMGCR pathway. Specifically, Lac-Phe increased AMPKα1 phosphorylation, which was associated with increased HMGCR phosphorylation, reduced cholesterol synthesis and lipid accumulation, and ameliorated pathological remodeling. Further investigation is required to establish the molecular interaction between Lac-Phe and AMPKα1 and to determine the potential translational applications of these findings.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AMPK-IN-3 (p-AMPK inhibitor)	MedChemExpress (MCE)	HY-132921	Used at 10 μM during AMPK inhibition experiments to inhibit AMPK signaling in Lac-Phe-treated cells.
Amplex Red Cholesterol and Cholesteryl Ester Assay Kit	Beyotime	S0211S	Used to determine total cholesterol levels in serum and H9c2 cell samples.
Anti-AMPKα1	Cell Signaling Technology	2532; RRID: AB_330331	Primary antibody used for western blot analysis of AMPKα1.
Anti-GAPDH	Cell Signaling Technology	5174; RRID: AB_10622025	Primary antibody used for western blot analysis of GAPDH.
Anti-HMGCR	Abcam	ab174830; RRID: AB_2749818	Primary antibody used for western blot analysis of HMGCR.
Anti-p-AMPKα1 (Thr172)	Cell Signaling Technology	2535; RRID: AB_331250	Primary antibody used for western blot analysis of phosphorylated AMPKα1.
Anti-p-HMGCR (Ser872)	Abcam	ab124350	Primary antibody used for western blot analysis of phosphorylated HMGCR.
Anti-rabbit IgG, HRP-linked antibody	Cell Signaling Technology	7074; RRID: AB_2099233	Used at 1:5,000 dilution for western blot detection.
Anti-β-tubulin	Cell Signaling Technology	2128; RRID: AB_823664	Primary antibody used for western blot analysis of β-tubulin.
Atorvastatin	MedChemExpress (MCE)	HY-17379	Used alone or in combination with Lac-Phe to assess effects on lipid accumulation, total cholesterol levels, and HMGCR phosphorylation in HG/PA-treated H9c2 cells.
Bovine serum albumin	Sigma-Aldrich	A7906	Used at 5% to block nonspecific binding during western blot analysis.
Bradford assay reagent/kit	Thermo Fisher Scientific	23200	Used to determine protein concentration before western blot analysis.
C57BL/6J mice (5-week-old, male)	Model Animal Research Center of Nanjing University	N/A	Used to establish the HFD/STZ-induced diabetic cardiomyopathy mouse model.
Cell Counting Kit-8 (CCK-8)	Sharebio	SB-CCK8S	Used to measure H9c2 cell viability following treatment.
Citrate buffer	Prepared in-house	N/A	Prepared at pH 4.5 and used to dissolve STZ and as the corresponding vehicle control.
Control diet (10% fat, 20% protein, 70% carbohydrate)	Research Diets, Inc.	D12450J	Used as the diet for control-group mice.
D-(+)-Glucose	Sigma-Aldrich	N/A	Used for HG/PA cell treatment and for the oral glucose tolerance test.
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2650	Used as the solvent for AMPK-IN-3 and as the corresponding vehicle control.
Dulbecco’s modified Eagle’s medium	Gibco, Thermo Fisher Scientific	11965092	Used as the basal culture medium for H9c2 cells.
Echocardiography machine with 40 MHz transducer	FUJIFILM VisualSonics	Vevo 3100 LT; 40 MHz transducer	Used to measure left ventricular dimensions and mitral valve blood flow velocity and to derive LVEF and FS.
Electron microscope fixative	ServiceBio	G1102	Used to fix fresh mouse heart tissue for transmission electron microscopy.
Ethanol	Sigma-Aldrich	E7023	Used in progressively increasing concentrations for dehydration during transmission electron microscopy sample preparation.
Fetal bovine serum	Gibco, Thermo Fisher Scientific	10099141	Used at 10% to supplement H9c2 cell culture medium.
H9c2 cell line	Sunncell	RRID: CVCL_0286	Used as the in vitro cardiac cell model for HG/PA and Lac-Phe treatment experiments.
Hematoxylin and eosin stain	ServiceBio	G1005-1/2	Used for histological assessment of cardiac tissue.
High-fat diet (60 kcal% fat)	Research Diets, Inc.	D12492	Used with low-dose STZ to establish the diabetic cardiomyopathy mouse model.
High-resolution brightfield microscope	Leica	DM2500	Used for whole-slide imaging of stained cardiac tissue at 20× magnification.
ImageJ	National Institutes of Health	N/A	Used for densitometric quantification of western blot results.
Isoflurane	Sigma-Aldrich	792632	Used for anesthesia during mouse echocardiography.
Isopropyl alcohol	Sigma-Aldrich	I9516	Used at 60% during Oil Red O staining of frozen cardiac sections.
LD540 lipid stain	Beyotime	C2050S-1	Used at 10 μM to stain intracellular lipids.
Masson’s trichrome stain	ServiceBio	G1006	Used for histological staining of cardiac tissue.
N-lactoyl-phenylalanine (Lac-Phe)	MedChemExpress (MCE)	HY-146098	Administered to DCM mice and used to treat HG/PA-exposed H9c2 cells.
Oil Red O working solution	ServiceBio	G1015-100ML	Used to stain lipids in frozen cardiac tissue sections.
Omni-ECL Efficient Light Chemiluminescence Kit	EpiZyme	SQ203L	Used for chemiluminescent visualization of protein bands after western blotting.
Palmitic acid	Sigma-Aldrich	P0500	Used at 0.25 mM with high glucose to establish the HG/PA-treated H9c2 cell model.
Paraformaldehyde	Sigma-Aldrich	P6148	Prepared as a 4% solution and used to fix cardiac tissue and cells before histological or lipid staining.
Penicillin-streptomycin	Gibco, Thermo Fisher Scientific	15140122	Used at 1% in H9c2 cell culture medium.
Phosphate-buffered saline (PBS)	Gibco, Thermo Fisher Scientific	10010023	Used for washing during staining procedures and as the corresponding vehicle in cell-treatment experiments.
Prism	GraphPad Software	Version 8.0	Used for statistical analysis of experimental data.
PVDF transfer membrane, 0.45 μm	Thermo Fisher Scientific	88518	Used for electrophoretic transfer of proteins during western blot analysis.
RNeasy Mini Kit	QIAGEN	74104	Used for isolation/purification of total RNA from mouse cardiac tissue.
Sodium chloride solution, 0.9% (normal saline)	Sigma-Aldrich	S8776	Used to dissolve Lac-Phe and as the vehicle control for Control and DCM mice.
SPSS	IBM	Version 20.0	Used for statistical analysis of experimental data.
Streptozotocin (STZ)	Sigma-Aldrich	S0130	Administered with HFD feeding to establish the diabetic cardiomyopathy mouse model.
TBS Tween-20 Buffer (20×)	Thermo Fisher Scientific	28360	Used to wash membranes after primary- and secondary-antibody incubation during western blot analysis.
Tissue-Tek O.C.T. Compound	Sakura Finetek	4583	Used to embed cardiac tissue before preparation of frozen sections for Oil Red O staining.
Transmission electron microscope	HITACHI	HT7800	Used to examine ultrastructural changes in mouse cardiac tissue.
TRIzol Reagent	Invitrogen	15596026	Used for total RNA isolation from mouse cardiac tissue.
VAHTS Universal V10 RNA-seq Library Prep Kit	Vazyme	NR616-01	Used to prepare RNA-seq libraries for transcriptomic analysis.
Wheat germ agglutinin (WGA)	ServiceBio	W7024	Used for histological assessment of cardiac tissue.