This study is a single-arm pilot study without a control group, where the baseline clinical characteristics collected from all participants were compared before and after treatment. 18 patients diagnosed with MetS were enrolled from the Endocrinology Department of Xiamen Traditional Chinese Medicine Hospital from June 2023 to June 2024. All participants were vetted to ensure they satisfied the predefined inclusion and exclusion criteria. This study was meticulously conducted in adherence to the ethical principles enshrined in the Helsinki Declaration, and the ethical approval was reviewed and approved by the Medical Ethics Committee of Xiamen Traditional Chinese Medicine Hospital (Protocol Number: 2023-K029-01). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from all individual participants.

The following criteria were established for the inclusion of participants in the study: (i) all participants between the age of 18 to 65 years; (ii) central obesity (waist circumference of ≥90 cm in males or ≥80 cm in females, and at least two of the following factors: elevated triglyceride (TG) ≥1.7 mmol/L, high-density lipoprotein (HDL) <1.03 mmol/L for males and <1.29 mmol/L for females, systolic blood pressure (SBP) ≥130 mmHg or diastolic blood pressure (DBP) of ≥85 mmHg, fasting blood glucose (FBG) ≥5.6 mmol/L; (iii) the duration of both diabetes and hypertension should not exceed 5 years; (iv) all participants were required to provide informed consent and to confirm their voluntary participation in the study (17).

The study applied the following exclusion criteria to ensure the selection of appropriate participants: (i) females who were pregnant or lactating; (ii) acute and chronic diabetes complications; (iii) participants with severe liver or kidney dysfunction, significant cardiovascular diseases, chronic infections, thyroid dysfunction, malignant tumor or other serious health issues; (iv) individuals with neurogenic anorexia or malnutrition; (v) neurological and Psychiatric Conditions.

All participants were instructed to discontinue the use of hypoglycemic, lipid-lowering, and antihypertensive medications for one week. The subjects underwent a 9-day VLCD regimen. This regimen consisted of three phases: a 2-day buffer period at 600 kcal/day, a 5-day intensive phase (days 3-7) at 300 kcal/day, and a 2-day recovery phase (days 8-9) at 600 kcal/day. The diet maintained a consistent macronutrient distribution of 55% carbohydrates, 20% protein, and 25% fat throughout all phases (18). During the intervention, participants were advised to restrict physical activity to basic daily tasks and light exercise.

Among the 18 participants included in this study, there were 10 males (55.6%) and 8 females (44.4%). Among the 18 participants, due to personal willingness and case dropout, serum metabolomics profiling was ultimately conducted on 14 participants, comprising 7 males (50%) and 7 females (50%). Before and after the short-term VLCD intervention, a comprehensive array of baseline clinical characteristics was assessed in the patients. These baseline clinical characteristics included anthropometric measurements such as body weight (BW), waist circumference (WC), BMI, body fat mass, body fat percentage, and blood pressure.

Fasting peripheral venous blood samples were collected after an 8–10 hour fast. Levels of fasting blood glucose (FBG) and fasting insulin (FINS) were quantified using a fully automated biochemical analyzer and a chemiluminescence immunoassay analyzer, respectively. The homeostasis model assessment of insulin resistance (HOMA-IR) was then calculated from FBG and FINS to evaluate insulin resistance. Lipid profiles, including total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL), were also analyzed. Additionally, the safety index of liver and kidney function was assessed.

Following an 8–10 hour fasting period, 3mL of peripheral venous blood was collected from each participant. The samples were centrifuged at 3000×g for 10min to isolate serum. A 400μL serum was then mixed with 200μL phosphate buffer solution and subjected to high-speed centrifugation at 13,000×g for 20min at 4°C. Subsequently, 500μL of the resulting supernatant was transferred to a 5mm NMR tube for analysis. Finally, all processed samples were detected by a ¹H-NMR spectrometer (Bruker AVANCE-III 600MHz, Germany).

The ¹H-NMR spectra of all samples underwent rigorous phase and baseline corrections using MestReNova v9.0.1 software. Subsequently, the data were imported into SIMCA-P 14.0 software. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) was then conducted to differentiate between the metabolic profiles of the samples. Finally, differential metabolites with VIP>1 and P<0.05 were pointed out for before and after treatment.

SPSS software (version 26, IBM, United States) was applied for statistical analyses of clinical variables. The data were performed using paired t-tests for normally distributed parameters, while the Wilcoxon signed-rank test was employed for variables that deviated from normality. P < 0.05 was considered statistically significant. All statistical analyses were conducted using SPSS software(IBM, United States).

Before and after the treatment of a short-term VLCD, the baseline clinical characteristics were observed. The results showed that levels of BW, WC, BMI, SBP, DBP, FBG, FINS, HOMA-IR, HDL, and TG were decreased significantly after treatment (P < 0.05). However, there was no significant alteration in the levels of body fat mass, body fat percentage, LDL and TC after treatment by short-term VLCD (P>0.05). Moreover, albumin (ALB), alanine aminotransferase (ALT), and creatinine (CR), which were related to the function of the liver and kidney, were not altered (P>0.05) (Table 1). Notably, AST was significantly increased after treatment by short-term VLCD (P < 0.05), but it remained in the normal range.

Although a total of 18 participants were enrolled in this study, there were only 14 participants who agreed to be assayed for serum metabolites. ¹H-NMR was utilized to identify the characteristic metabolites in the serum. Meanwhile, the differential metabolites were fingered in the ¹H-NMR spectra (Figure 1).

To point out biomarkers that reflect changes in metabolite profiles before and after the short-term VLCD, an OPLS-DA model was employed for a more in-depth analysis. Obviously, there was a significant discrete between the before and after treatment by short-term VLCD (Figure 2). The results shown that the samples from the two groups did not exhibit any crossover or significant overlap, indicating a clear separation and good dispersion (P < 0.05). This finding was crucial as it points to specific metabolites that may serve as biomarkers for the metabolic changes induced by the short-term VLCD treatment.

Serum metabolomic profiling identified 20 differential metabolites that were significantly altered following the short-term VLCD intervention in patients with MetS (P<0.05). All identified differential metabolites were including acetone, alanine, betaine, diethyl methylmalonate, α-glucose, β-glucose, glycerophosphocholine, glutamate, glutamine, glycerol, glycine, lactic acid, low-density lipoprotein, methyl histidine, phosphatidylcholine, very low-density lipoprotein, leucine, serine, threonine, and valine, were significantly decreased after treatment (Table 2, Figure 3). Subsequently, KEGG enrichment analysis of these metabolites revealed 13 major metabolic pathways that were significantly enriched, based on a threshold of -log(p)>1 (Figure 4).

A short-term VLCD treatment is recognized as an effective approach for weight reduction through caloric restriction, potentially enhancing energy metabolism. Implementation of a short-term VLCD treatment results in decreased blood glucose levels, thereby reducing the dependency on glucose as a primary energy source. Moreover, this dietary intervention may enhance insulin sensitivity and diminish insulin resistance, facilitating more efficient insulin utilization and promoting glucose uptake into cells. Lactic acid can be integrated into the tricarboxylic acid (TCA) cycle either via gluconeogenesis or through the enzymatic conversion of lactate to pyruvate by lactate dehydrogenase and pyruvate dehydrogenase, thereby contributing to ATP synthesis (21). Similarly, acetone and glycerol can be metabolized to generate energy (22, 23). Leucine and valine not only serve as energy substrates but also enter the TCA cycle, providing additional energy for physiological processes (24, 25). Threonine participates in protein synthesis and gluconeogenesis, aiding in the maintenance of energy equilibrium (26). Metabolic disturbances are widely acknowledged as a hallmark feature of patients with MetS (17). Notably, short-term VLCD treatment can modulate key aspects of energy metabolism, including glucose, lactate, acetone, and glycerol pathways. The findings of this study demonstrate that short-term VLCD treatment facilitates effective weight management, enhances insulin sensitivity, and reduces insulin resistance which are pivotal in the therapeutic management of MetS.

LDL and VLDL are the principal lipid carriers in the bloodstream. VLDL is synthesized in the liver and plays a crucial role in transporting triglycerides to peripheral tissues. In the context of obesity, the apolipoprotein C-II (Apo C-II) present on the VLDL surface activates lipoprotein lipase, promoting the hydrolysis of triglycerides. Subsequently, a significant proportion of VLDL is converted into LDL. An excessive accumulation of LDL, particularly oxidized LDL, predisposes to induce arteriosclerosis (27). A short-term VLCD effectively modulates the metabolism of LDL and VLDL, thereby contributing to the management of MetS and reducing cardiovascular risk. Additionally, glycerol serves as a critical intermediate in fatty acid metabolism. By regulating glycerol metabolism and reducing fat accumulation, a short-term VLCD treatment offers an effective therapeutic approach for addressing MetS.

Leucine and valine are metabolized within muscle tissue, where they not only generate energy and promote muscle mass augmentation but also stimulate insulin secretion. Nevertheless, their metabolic intermediates carry the potential to induce insulin resistance (25, 28). It has been demonstrated that regulating branched-chain amino acid (BCAA) metabolism can alleviate insulin resistance (29). Glycine enhances insulin secretion by activating receptors on pancreatic β-cells (30), while serine modulates insulin sensitivity in the liver (31). Glutamine facilitates the release of glucagon-like peptide-1 and boosts insulin secretion (32), whereas glutamate can elevate glucagon secretion and worsen insulin resistance (33, 34). A short-term VLCD treatment significantly modulates the levels of these relevant metabolites. By optimizing their metabolic profiles, this dietary intervention may enhance insulin sensitivity and mitigate insulin resistance in patients with MetS.

Glycine mitigates the levels of pro-inflammatory cytokines through the regulation of NF-κB, while methylhistidine exerts an inhibitory effect on pro-inflammatory cytokines (35, 36). Glutamine influences glycolytic processes and the subsequent inflammatory response (37), and glutamate modulates inflammatory signaling pathways through its specific receptors (38). Betaine participates in methylation reactions, thereby reducing the production of inflammatory mediators (39). Glutathione, a tripeptide composed of glutamic acid, cysteine, and glycine, exhibits potent antioxidant properties (40). Betaine enhances non-enzymatic antioxidant defenses, further contributing to cellular protection (41). MetS is intricately associated with chronic inflammation and oxidative stress (42–44). The short-term VLCD treatment significantly modulates the levels of these pertinent metabolites, indicating its potential anti-inflammatory and antioxidant effects. This observation is consistent with previous studies demonstrating that caloric restriction diets reduce inflammatory cytokine levels and mitigate oxidative damage. Collectively, these findings underscore the multiple metabolic pathways through which a short-term VLCD treatment contributes to the amelioration of MetS (45, 46).

Glutamate functions as the predominant excitatory neurotransmitter in the central nervous system, significantly influencing the release of neurohormones (47). Glutamine plays a pivotal role in the synthesis and transmission of glutamate, ensuring its availability for neurotransmission. Betaine can modulate the methylation processes of neurohormone receptors, thereby affecting their function and expression (48). MetS is closely associated with dysregulation of neurohormones, which are crucial for maintaining energy balance, regulating appetite, and orchestrating other physiological processes (49). The administration of a short-term VLCD treatment effectively modulates the levels of these relevant metabolites. By optimizing neurohormone signaling pathways, this dietary intervention exerts a beneficial effect on the management and alleviation of MetS.

In conclusion, the metabolic profiles associated with MetS underwent significant alterations, with short-term VLCD treatment effectively regulating characteristic metabolites involved in energy metabolism, lipid metabolism, insulin sensitivity, inflammation, oxidative stress, cell signaling, and neurotransmitter-related pathways. Furthermore, short-term VLCD treatment may improve anthropometric parameters, blood pressure, and lipid metabolism indices in MetS patients by modulating multiple metabolites.

In this study, the observed alterations cannot be solely ascribed to the short-term VLCD treatment for MetS, since factors such as time effects, regression to the mean, or medication withdrawal may also play a role. It should be noted that the sample size in this study was relatively limited. In the future, a 9-month follow-up observation will be established to assess the durability of therapeutic efficacy and its long-term effects on the cardiovascular system. Moreover, 50 to 100 participants will be enrolled, and targeted quantitative techniques applied to further validate the results of this study.