Potential Therapeutic Effects of Empagliflozin Through Elevated Plasma Citrulline Levels in Heart Failure With Preserved Ejection Fraction

Yoshiro Tsuruta; Shuichi Kitada; Yu Kawada; Yasuhiro Shintani; Tatsuya Mizoguchi; Masashi Yokoi; Sayuri Yamabe; Kento Mori; Shohei Kikuchi; Tsuyoshi Ito; Toshihiko Goto; Yoshihiro Seo

doi:10.1253/circj.CJ-24-0953

Abstract

Background: In large clinical trials, sodium-glucose cotransporter 2 (SGLT2) inhibitors have improved prognosis in heart failure with preserved ejection fraction (HFpEF). Although several beneficial pharmacological effects of SGLT2 inhibitors for HFpEF have been suggested, their presumed metabolic pathways remain insufficiently proven.

Methods and Results: We compared the metabolomic profile, determined using liquid chromatography–mass spectrometry, of 16 patients with HFpEF before and after empagliflozin therapy. Only citrulline levels (expressed as a ratio to methionine sulfone levels) were significantly elevated after therapy (3.57±1.88 vs. 6.47±3.78; P=0.006).

Conclusions: Empagliflozin significantly increased citrulline levels in HFpEF patients. Although further studies are needed, it would be intriguing if this metabolite change were related to the cardiovascular protective effects of empagliflozin.

The prevalence of heart failure (HF) increases with age and, particularly for older people, HF is a life-threatening critical issue.¹ Older patients with HF usually present with HF with preserved ejection fraction (HFpEF). Although appropriate treatment approaches for HFpEF that improve prognosis are limited, large clinical trials have shown that sodium-glucose cotransporter 2 (SGLT2) inhibitors can improve prognosis in HFpEF, and these drugs now play an essential role in HFpEF treatment.²^,³ Several pharmacological effects of SGLT2 inhibitors have been suggested to be responsible for the improvement in prognosis in HF.⁴ Metabolic impairment is essential to HF pathogenesis and progression. In patients with HF, cardiac metabolic flexibility is impaired, requiring a change from glucose or fatty acid metabolism to metabolism in which ketone bodies and branched-chain amino acids (BCAAs) are preferentially used. SGLT2 inhibitors increase ketone bodies and are thought to provide beneficial pharmacological effects by shifting to energy-efficient myocardial metabolism using increased ketone bodies.⁵ However, a recent report on the metabolomic profiling of patients with HFpEF who received SGLT2 inhibitor therapy did not fully support this hypothesis, and the presumed beneficial metabolic pathways for HF induced by SGLT2 inhibitors remain insufficiently proven.⁶ Therefore, the aim of the present study was to investigate changes in metabolic pathways associated with SGLT2 inhibitor treatment using metabolomic profiling of patients with HFpEF.

Methods

Study Patients

This study prospectively enrolled 16 patients diagnosed with HFpEF who were SGLT2 inhibitor naïve. Patients underwent metabolomic profiling twice: once before and once after empagliflozin therapy (10 mg daily). The diagnosis of HFpEF was based on a history of hospitalization for acute decompensated HF or the HFA-PEFF score, as recommended by the Heart Failure Association of the European Society of Cardiology.⁷ Patients were required to have a left ventricular ejection fraction ≥50% and no concomitant ischemic heart disease assessed by coronary angiography or infiltrative cardiomyopathy (e.g., cardiac amyloidosis) on ^{99 m}Tc-pyrophosphate myocardial scintigraphy and/or cardiac magnetic resonance imaging. Patients were excluded from this study if they had severe renal dysfunction (defined as an estimated glomerular filtration rate <30 mL/min/1.73 m² or requiring hemodialysis), significant valvular disease requiring cardiac surgery, right ventricular dysfunction, pulmonary arterial hypertension, and/or were receiving pacing therapy via a pacemaker or implantable cardiac defibrillator.

This prospective study was conducted in accordance with the Declaration of Helsinki. The study was approved by the Institutional Review Boards and Ethics Committees of Nagoya City University Graduate School of Medical Sciences, Japan. All patients received an explanation about the study and provided informed consent before enrollment.

Metabolomic Profiling

Targeted metabolomic profiling of 141 metabolites was performed, namely 9 glycolysis and tricarboxylic acid cycle metabolites, 4 urea cycle metabolites, 4 mevalonic acid and methylerythritol phosphate pathway metabolites, 5 shikimic acid pathway metabolites, 10 methionine and transsulfuration pathway metabolites, 24 nucleic acid-related compounds, 4 catecholamines, 24 amino acids, 17 organic acids, 3 vitamins, 5 coenzymes, 3 alkaloids, and other 29 metabolites (Table). Metabolic profiling was performed twice: once before and 14 weeks after empagliflozin therapy (allowance period was 2 weeks before and after the therapy). Blood samples were taken after patients had fasted for at least 10 h. Blood samples were collected in tubes containing EDTA and incubated on ice until centrifugation at 3,000 rpm for 15 min at 4℃. Plasma was stored at −80℃ until subsequent analysis.

Table.

Concentrations of 141 Primary Metabolites, Expressed as a Ratio to Methionine Sulfone Levels, Before and After Empagliflozin Therapy

Metabolic pathway (no. components)	Metabolite	Before empagliflozin	After empagliflozin	P value
Glycolysis (n=2)	Lactic acid	1.90±7.42E⁻¹	2.10±5.95E⁻¹	0.320
Glycolysis (n=2)	Pyruvic acid	3.21E⁻²±1.22E⁻²	2.71E⁻²±8.25E⁻³	0.209
TCA cycle (n=7)	α-Ketoglutaric acid	9.79E⁻³±6.47E⁻³	6.46E⁻³±2.62E⁻³	0.081
	Aconitic acid	3.58E⁻¹±1.03E⁻¹	4.02E⁻¹±1.15E⁻¹	0.122
	Citric acid	8.67±1.65	9.25±1.60	0.171
	Fumaric acid*
	Isocitric acid	7.90±1.49	8.46±1.42	0.152
	Malic acid	3.78E⁻²±1.14E⁻²	4.29E⁻²±1.55E⁻²	0.103
	Succinic acid*
Urea cycle (n=4)	Arginine	32.12±8.61	30.00±9.66	0.334
	Argininosuccinic acid*
	Citrulline	3.57±1.88	6.47±3.78	0.006
	Ornithine	6.73±1.29	7.08±2.72	0.437
Mevalonic acid/MEP pathway (n=4)	Malic acid	3.78E⁻²±1.14E⁻²	4.29E⁻²±1.55E⁻²	0.103
	Phosphomevalonic acid	5.58E⁻³±1.36E⁻³	5.80E⁻³±1.92E⁻³	0.707
	1-Deoxy-D-xylulose-5-phosphate*
	2C-methyl-D-erythritol-4-phosphate*
Shikimic acid pathway (n=5)	3-Dehydroquinic acid*
	3-Dehydroshikimic acid*
	Chorismic acid*
	Shikimic acid*
	Shikimic acid 3-phosphate	6.11E⁻⁴±4.29E⁻⁴	9.04E⁻⁴±3.92E⁻⁴	0.053
Methionine/transsulfuration pathway (n=10)	5-Glutamylcysteine*
	Cystathionine	6.10E⁻²±3.51E⁻²	7.06E⁻²±3.55E⁻²	0.433
	Cysteine*
	Cystine	5.88±2.68	7.55±3.03	0.074
	Glutathione*
	Homocysteine*
	Methionine	1.07±3.93E⁻¹	8.74E⁻¹±3.40E⁻¹	0.162
	Oxidized glutathione*
	S-Adenosylhomocysteine*
	S-Adenosylmethionine*
Nucleic acid-related components (n=24)	Adenine*
	Adenosine*
	Adenosine 3′,5′-cyclic monophosphate	1.47E⁻²±8.73E⁻³	1.15E⁻²±6.49E⁻³	0.258
	Adenosine monophosphate	5.37E⁻²±4.85E⁻²	9.65E⁻²±9.95E⁻²	0.163
	Adenylsuccinic acid*
	5-aminoimidazole-4-carboxamide ribonucleotide*
	Allantoin	1.46E⁻³±8.92E⁻⁴	2.10E⁻³±1.10E⁻³	0.069
	Cytidine	3.64E⁻³±3.03E⁻³	4.44E⁻³±4.14E⁻³	0.424
	Cytidine 3′,5′-cyclic monophosphate*
	Cytidine monophosphate	8.06E⁻³±5.18E⁻³	8.23E⁻³±2.98E⁻³	0.918
	Cytosine	8.90E⁻³±4.44E⁻³	1.09E⁻²±4.97E⁻³	0.296
	Guanine*
	Guanosine	8.99E⁻³±8.54E⁻³	7.67E⁻³±4.38E⁻³	0.497
	Guanosine 3′,5′-cyclic monophosphate*
	Guanosine monophosphate	7.63E⁻³±5.88E⁻³	7.55E⁻³±4.49E⁻³	0.966
	Hypoxanthine*
	Inosine	2.54E⁻²±5.92E⁻²	9.87E⁻³±8.32E⁻³	0.310
	Thymidine*
	Thymidine monophosphate*
	Thymine*
	Uracil	9.46E⁻³±3.87E⁻³	8.42E⁻³±3.85E⁻³	0.414
	Uric acid	9.29E⁻¹±1.34E⁻¹	8.67E⁻¹±1.43E⁻¹	0.095
	Uridine	8.29E⁻²±3.99E⁻²	6.16E⁻²±3.73E⁻²	0.171
	Xanthine	8.02E⁻³±1.06E⁻²	6.07E⁻³±6.27E⁻³	0.219
Catecholamines (n=4)	Dopamine*
	Epinephrine	4.28E⁻²±5.98E⁻³	4.08E⁻²±7.01E⁻³	0.427
	Norepinephrine*
	Serotonin	1.84E⁻²±3.06E⁻²	1.40E⁻²±1.18E⁻²	0.628
Amino acids (n=24)	4-Hydroxyproline	1.51±5.00E⁻¹	1.55±4.49E⁻¹	0.793
	Alanine	20.27±3.85	22.23±5.16	0.215
	Anthranilic acid*
	Asparagine	1.41±3.08E⁻¹	1.47±2.59E⁻¹	0.447
	Aspartic acid	3.21E⁻¹±7.21E⁻²	3.43E⁻¹±1.04E⁻¹	0.447
	Asymmetric dimethylarginine	7.38E⁻¹±1.48E⁻¹	6.80E⁻¹±9.57E⁻²	0.169
	Dimethylglycine	5.56E⁻²±2.45E⁻²	6.16E⁻²±1.77E⁻²	0.427
	Glutamic acid	5.60±1.36	5.42±1.46	0.652
	Glutamine	31.48±3.38	31.87±3.40	0.698
	Glycine	1.21±5.67E⁻¹	1.03±3.77E⁻¹	0.142
	Histidine	33.86±3.68	33.43±3.28	0.748
	Homocysteine *
	Isoleucine	44.04±6.69	40.21±11.06	0.158
	Leucine	41.77±6.12	40.37±11.45	0.663
	Lysine	57.80±7.32	53.59±16.35	0.374
	Methionine sulfoxide	7.68E⁻²±1.01E⁻¹	7.35E⁻²±4.66E⁻²	0.909
	Phenylalanine	70.96±7.27	70.10±10.14	0.774
	Proline	3.03±1.10	3.59±1.54	0.175
	Serine	7.60±1.75	7.40±1.73	0.669
	Symmetric dimethylarginine	9.46E⁻¹±2.97E⁻¹	9.72E⁻¹±2.63E⁻¹	0.633
	Threonine	5.39±1.22	5.14±1.14	0.346
	Tryptophan	20.86±4.23	21.58±3.69	0.566
	Tyrosine	15.19±4.27	14.44±3.63	0.556
	Valine	4.22±8.27E⁻¹	4.06±8.08E⁻¹	0.566
Organic acids (n=17)	2-Aminobutyric acid	6.01E⁻¹±1.68E⁻¹	7.30E⁻¹±7.56E⁻¹	0.496
	4-Aminobenzoic acid*
	4-Aminobutyric acid	1.79E⁻²±2.36E⁻³	9.30E⁻²±2.84E⁻¹	0.306
	Caffeic acid*
	Cholic acid*
	Creatine	1.77±1.03	1.93±1.26	0.531
	Ferulic acid*
	Glycolic acid*
	Glyoxylic acid*
	Ophthalmic acid*
	Orotic acid*
	p-Coumaric acid*
	Phenyllactic acid	5.73E⁻³±4.00E⁻³	8.83E⁻³±1.19E⁻²	0.178
	Phenylpyruvic acid*
	Taurocholic acid*
	Urocanic acid	9.16E⁻³±1.17E⁻²	6.12E⁻³±5.91E⁻³	0.371
	Vanillic acid*
Vitamins (n=3)	Folic acid*
	Pantothenic acid	1.21E⁻²±2.08E⁻²	7.42E⁻³±6.84E⁻³	0.224
	Pyridoxal phosphate*
Coenzymes (n=5)	Flavin adenine dinucleotide*
	Flavin mononucleotide*
	Nicotinamide adenine dinucleotide*
	Niacinamide	7.59E⁻²±3.65E⁻²	6.72E⁻²±2.71E⁻²	0.474
	Nicotinic acid	3.23E⁻²±1.75E⁻²	2.74E⁻²±6.60E⁻³	0.292
Alkaloids (n=3)	Higenamine*
	Reticuline*
	Tetrahydropalmatine*
Others (n=29)	4-Aminophenylalanine*
	4-Aminophenylpyruvic acid*
	4-Hydroxybenzoic acid	2.42E⁻²±5.71E⁻²	5.83E⁻¹±1.58	0.181
	Acetylcarnitine	24.00±9.37	23.51±4.99	0.857
	Acetylcholine	2.13E⁻²±4.21E⁻³	2.25E⁻²±5.98E⁻³	0.401
	Carnitine	14.89±2.25	13.92±4.37	0.417
	Carnosine*
	Catechol*
	Choline	2.52±4.20E⁻¹	2.57±3.15E⁻¹	0.726
	Citicoline	3.08E⁻³±4.86E⁻³	2.25E⁻³±1.95E⁻³	0.556
	Creatinine	12.15±3.09	12.22±3.04	0.915
	Cysteamine*
	Dihydroxyphenylacetaldehyde*
	Dihydroxyphenylacetic acid*
	Dihydroxyphenylalanine*
	Ergothioneine	7.86E⁻¹±8.28E⁻¹	8.00E⁻¹±9.82E⁻¹	0.869
	Histamine*
	Histidinol*
	Hydroxytyrosol*
	Indole*
	Kynurenine	5.89E⁻¹±2.67E⁻¹	5.96E⁻¹±1.74E⁻¹	0.887
	Methyl-dihydroxyphenylalanine*
	Protocatechuic acid*
	Protocatechuic aldehyde*
	Resveratrol*
	Salicylic acid	2.37E⁻²±5.51E⁻²	5.50E⁻¹±1.49	0.179
	Sinapic acid*
	Tyramine*
	Vanillin*

*Metabolites for which the signal-to-noise ratio was <10, and so were considered undetectable. Unless indicated otherwise, data are given as the mean±SD. MEP, methylerythritol phosphate; TCA, tricarboxylic acid cycle.

The metabolomic profile was quantified using a Nexera X3 UHPLC system (Shimadzu, Kyoto, Japan) coupled with an LCMS-8050 triple quadrupole mass spectrometer (Shimadzu). The following parameters were used for ionization: electrospray ionization voltage, +4.0 kV (positive) and −3.0 kV (negative); heat block temperature, 400℃; desolvation line temperature, 250℃; interface temperature, 300℃; nebulizer gas, 3 L/min; heading gas, 10 L/min; and drying gas, 10 L/min. A Shim-pack GIST PFPP (2.1×150 mm, 3.0 μm; Shimadzu) analytical column was used for chromatographic separation. The mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), and the flow rate was 0.25 mL/min (0.5 mL/min from 17 to 19 min). The following gradient elution program was used: 0–2.0 min, 0% Solvent B; 2.0–5.0 min, 25% Solvent B; 5.0–11.0 min, 35% Solvent B; 11.0–16.0 min, 50% Solvent B; 16.0–19.0 min, 95% Solvent B; 19.0–30.0 min, 0% Solvent B. Data were acquired in multiple reaction monitoring mode with negative electrospray ionization for the 141 metabolites.

Statistical Analysis

Metabolite concentrations are expressed as a ratio to methionine sulfone levels. Continuous data are presented as the mean±SD, and metabolite concentrations before and after empagliflozin therapy were compared using paired t-tests. Two-tailed P<0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 29.0 (SPSS Japan Inc., Tokyo, Japan).

Results

In all 16 patients, metabolic profiling was performed twice, once before and once after empagliflozin therapy. The mean period from the start of therapy to the second profiling was 14.5±1.9 weeks. We measured the concentrations of the 141 primary metabolites listed in the Table. When measuring the intensity of individual metabolite ions separated by their mass-to-charge ratio, we considered 70 metabolites with a signal-to-noise ratio <10 as undetectable (no before and after values in the Table) and compared concentrations before and after therapy for the remaining 71 metabolites. Comparisons of metabolite concentrations before and after empagliflozin therapy revealed that only citrulline levels increased significantly after therapy (3.57±1.88 vs. 6.47±3.78; P=0.006). There were no significant changes in the concentrations of the other 70 metabolites, including ornithine, aspartic acid, arginine, asymmetric dimethylarginine, and symmetric dimethylarginine, from before to after empagliflozin therapy (Table; Figure).

Figure.

Changes in the mean (±SD) concentrations of main metabolites from before to after empagliflozin therapy, expressed as a ratio to methionine sulfone levels. Comparisons of 71 measurable metabolites before and after empagliflozin therapy revealed that only citrulline levels increased significantly after treatment (from 3.57±1.88 to 6.47±3.78; P=0.006). No significant changes were seen for the other 70 metabolites, including ornithine, aspartic acid, arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA).

Discussion

The present comparison of the metabolomic profiles of HFpEF patients before and after empagliflozin therapy demonstrated a significant increase in citrulline levels. No significant increase in ornithine, aspartic acid, arginine, asymmetric dimethylarginine, or symmetric dimethylarginine were found. These findings included the possibility that promoting the conversion pathway from arginine to citrulline, a concomitant that releases nitric oxide (NO) into the systemic circulation. In contrast, there was no evidence of changes in metabolites from ketone-related pathways indicating a transition to energy-efficient myocardial metabolism using ketone bodies.

Some previous studies have reported that SGLT2 inhibitor therapy significantly increases plasma citrulline levels.⁸^,⁹ For example, in an experimental model of left ventricular pressure overload by transverse aortic constriction, metabolomic studies demonstrated that empagliflozin administration increased citrulline levels in the myocardium and reduced arginine levels.⁸ AKT is one of the major signaling molecules in the insulin signaling pathway and is known to upregulate endothelial NO synthase (eNOS) activity. In another study, an SGLT2 inhibitor was reported to increase levels of phosphorylated AKT in cardiac tissues of an ischemia-reperfusion injury model and to activate AKT/eNOS signaling in human umbilical vein endothelial cells.¹⁰ In that study, transcriptome analysis showed that empagliflozin administration enhanced the insulin/AKT pathway and activated NO production through phosphorylation of eNOS, resulting in increased metabolism from arginine to citrulline.¹⁰

In humans, some studies have reported increases in citrulline levels in patients with type 2 diabetes following administration of an SGLT2 inhibitor. Although plasma citrulline levels are known to be reduced in patients with type 2 diabetes,¹¹ Jojima et al.⁹ found that empagliflozin therapy (10 mg/day, 12 weeks) significantly increased in plasma citrulline levels in patients with type 2 diabetes. Similarly, in the present study, we have shown, for the first time, that empagliflozin therapy increases plasma citrulline levels in HFpEF patients. In addition to the direct effect of activating the insulin/AKT pathway, several indirect effects that reflect the multifaceted actions of SGLT2 inhibitors may be involved in the increase in citrulline associated with empagliflozin therapy. Previous studies have reported that plasma citrulline levels are lower in individuals with visceral obesity. However, weight loss following bariatric surgery, such as gastric bypass or sleeve gastrectomy, was associated with an increase in plasma citrulline levels.¹² The weight loss induced by empagliflozin therapy may contribute to the increase in citrulline levels seen in the present study. Another possibility is that improvements in liver dysfunction after empagliflozin administration may increase citrulline levels. Because citrulline is produced in the liver via the urea cycle, the improved liver function after empagliflozin therapy may contribute to increased citrulline production.¹³ Furthermore, empagliflozin therapy may enhance endothelial function, potentially leading to greater citrulline synthesis in endothelial cells.¹⁴ Finally, the increase in citrulline levels may be related to an anti-inflammatory effect of empagliflozin. Although the underlying mechanisms remain unclear, a significant decrease in inflammatory mediators following SGLT2 inhibitor therapy has been noted in animal models and patients with type 2 diabetes.¹⁵ Although various potential mechanisms have been proposed to account for the increase in citrulline levels associated with empagliflozin therapy, the exact mechanisms remain unclear.

The pathophysiology of HFpEF is based on increased vascular and LV stiffness with impaired relaxation and endothelial dysfunction. These impairments are associated with an imbalance of NO metabolism triggered by coronary microvascular endothelial inflammation and oxidative stress.¹⁶ Therefore, increasing citrulline levels and promoting NO release in the vascular and myocardium may become potential therapeutic targets for HFpEF.

In clinical practice, oral citrulline supplementation increases the plasma concentration of arginine, a substrate for NO synthase, and subsequently augments NO production, improving endothelial function.¹⁷ Furthermore, oral citrulline administration to HFpEF patients was associated with improvements in vascular endothelial function as assessed by flow-mediated dilation, reductions in pulmonary artery pressure as assessed by echocardiography, and increases in the 6-min walk distance.¹⁸ Conversely, a treatment that produces NO endogenously rather than via oral citrulline intake has also been proposed for HFpEF. An oral soluble guanylate cyclase (sGC) stimulator directly generates cGMP and restores the sensitivity of sGC to endogenous NO.¹⁹^,²⁰ However, 2 clinical trials investigating the therapeutic effects of the sGC stimulator in HFpEF did not show improvements in physical activity or quality of life.²¹^,²² One possibility is that impaired NO-sGC-cGMP signaling may not be a major pathophysiological factor in patients with HFpEF. Another possibility is the influence of patient selection in these trials. The 2 trials that failed to show improvements in physical activity or quality of life with an sGC stimulator in HFpEF included more patients with severe HFpEF who had a history of HF hospitalization than trials that demonstrated the therapeutic efficacy of SGLT2 inhibitors for HFpEF. In the Empagliflozin Outcome Trial in Patients with Chronic Heart Failure with Preserved Ejection Fraction (EMPEROR-Preserved), approximately 20% of the trial population had no hospitalization for HFpEF during the 12 months before enrollment.² In addition, the Dapagliflozin Evaluation to Improve the Lives of Patients with Preserved Ejection Fraction Heart Failure (DELIVER) trial included patients without a history of hospitalization for HFpEF, who accounted for approximately 40% of the trial population.³ Enrolling a less severely ill population with no history of hospitalization for HFpEF in outpatient clinics may have produced different results.

At present, treatment options for HFpEF promising to improve prognosis are limited. However, in large clinical trials, empagliflozin therapy significantly improved prognosis in HFpEF.²^,³ Because the SGLT2 inhibitor has been reported to have several beneficial pharmacological effects in HF,⁴ it is unclear whether the metabolic pathway related to increased citrulline levels contributed significantly to the improvement in prognosis in HFpEF. In addition, because the present study was a single-arm trial without a control group, it is difficult to conclusively prove that 14 weeks of empagliflozin administration truly increased citrulline levels. Although the present study has several limitations, if empagliflozin enhances myocardial NO synthase and provides cardiovascular protection; increased citrulline levels after empagliflozin therapy in HFpEF would be interesting.

Conclusions

The current comparison of metabolomic profiling of patients with HFpEF before and after empagliflozin therapy demonstrated a significant increase in citrulline levels. Although further studies are needed, it would be intriguing if this metabolite change were related to the cardiovascular protective effects of empagliflozin.

Sources of Funding

This study did not receive any specific funding.

Disclosures

Y. Seo is a member of Circulation Journal’s Editorial Team.

IRB Information

This study was approved by the Institutional Review Board and Ethics Committee of Nagoya City University Graduate School of Medical Sciences, Japan (Reference no. 60-24-0014).

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