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

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....

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.......

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 .......

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.