2024 Volume 71 Issue 1 Pages 55-63
Visceral fat accumulation is a major determinant of type 2 diabetes mellitus and cardiovascular diseases. Recent studies have reported that glutamate is the most elevated amino acid in the plasma amino acid profile in patients with obesity and/or visceral fat accumulation. Here, we show the relationship between plasma glutamate and the clinical features of patients with type 2 diabetes. The study subjects were 62 (28 men and 34 women) Japanese patients with type 2 diabetes. Blood profiles, including glutamate and adiponectin (APN) levels and estimated visceral fat area (eVFA), were measured. We also evaluated the plasma amino acid levels in mice with or without obesity by GC/MS analysis. In patients with type 2 diabetes, plasma glutamate was positively correlated with BMI, eVFA, and fasting insulin but negatively correlated with APN and duration of diabetes. Additionally, multiple regression analysis revealed that plasma glutamate was a significant determinant of APN. The plasma glutamate level was most significantly increased in obese mice compared to control mice, and it was negatively correlated with APN. These results suggest that the level of plasma glutamate could be a strong indicator of adipocyte dysfunction in patients with type 2 diabetes.
THE NUMBER OF PATIENTS with obesity-related type 2 diabetes mellitus has continued to increase worldwide [1, 2]. Many studies have shown that visceral fat accumulation due to obesity causes not only type 2 diabetes mellitus but also hypertension, dyslipidemia, and atherosclerotic cardiovascular diseases through adipocyte dysfunction and chronic inflammation of adipose tissue [3-5]. Individuals with visceral fat accumulation exhibit dysregulation of adipokines/adipocytokines, such as hypoadiponectinemia [6], and elevated levels of plasma tumor necrosis factor-α (TNF-α) [7] and plasminogen activator inhibitor-1 (PAI-1) [8], which leads to insulin resistance, inflammation, and cardiovascular disease [3].
We and others have shown that adiponectin (APN), an adipose-specific secreted factor, has antidiabetic, antiatherosclerotic, and tissue protective effects [9]. The concentration of circulating adiponectin is negatively correlated with visceral fat area and is an excellent biomarker reflecting adipocyte function [9]. In patients with type 2 diabetes with abdominal obesity, who are presumed to have visceral fat accumulation, the prevalence of hypertension, dyslipidemia and cardiovascular disease is significantly higher than that in patients with type 2 diabetes without abdominal obesity [4]. Therefore, evaluating visceral fat accumulation and the concentration of circulating adiponectin in patients with type 2 diabetes is important for the prevention of atherosclerotic cardiovascular disease.
Alterations in plasma free amino acid concentrations have been reported in various diseases, including fatty liver [10], liver cirrhosis [11], cancer [12] and Alzheimer’s disease [13]. Furthermore, alterations in plasma amino acid concentrations in obesity and visceral fat accumulation have been reported recently [14, 15]. In particular, plasma glutamate was the most elevated amino acid in obese individuals compared to nonobese individuals, and this was also observed in individuals with visceral fat accumulation compared to individuals without visceral fat accumulation [14, 15]. A recent report has shown that glutamate is the most elevated amino acid in the plasma amino acid profile of patients with type 2 diabetes compared to healthy subjects [16]. In addition, we have reported that obesity increases glutamate concentrations in white adipose tissue and causes adipocyte dysfunctions, such as decreased adiponectin secretion and decreased insulin-induced glucose uptake, in animal experiments using mice [17]. We also found that increased glutamate levels within adipocytes in conditions of obesity resulted in significant reductions in glutamate uptake into adipocytes via decreased expression of GLAST, a glutamate transporter. Thus, we speculated that this might be one of the mechanisms underlying the increase in plasma glutamate levels in obesity [17]. Therefore, alterations in glutamate levels in circulation and adipose tissue in conditions of visceral fat accumulation could be associated with adipocyte dysfunctions and some clinical features in patients with type 2 diabetes.
In the present study, we showed relationships between plasma glutamate levels and the clinical features of patients with type 2 diabetes to clarify the clinical importance of plasma glutamate concentrations in type 2 diabetes mellitus. Multiple regression analysis identified plasma glutamate as a significant explanatory factor associated with the serum APN level. The results indicate that the plasma glutamate level could be an excellent parameter reflecting adipocyte dysfunction in patients with type 2 diabetes.
The study subjects were 62 Japanese patients with type 2 diabetes who had been hospitalized for poor glycemic control and/or staging of complications at the Department of Endocrinology and Metabolism, Osaka University Hospital, during the period between April 2012 and March 2014. This study was approved by the human ethics committee of Osaka University (No. 08269-9). Each participant provided written informed consent. Type 2 diabetes was defined according to the 2006 World Health Organization criteria for diabetes [18] and/or treatment for diabetes. The exclusion criteria were as follows: 1) patients who were diagnosed with type 1 diabetes mellitus, 2) patients positive for anti-GAD antibody, and 3) patients for whom blood samples could not be obtained.
Clinical examinationVenous blood samples were collected after overnight fasting. The homeostasis model assessment of insulin resistance (HOMA-IR = fasting plasma glucose (mg/dL) × fasting insulin (μU/mL) /405) was calculated for subjects who were evaluated under fair glycemic control (fasting plasma glucose <140 mg/dL) [19] without insulin treatment. Plasma glutamate levels were analyzed using the Amplex Red Glutamic Acid/Glutamate Oxidase Assay kit (Invitrogen). Serum adiponectin levels were assayed using an adiponectin ELISA kit (Otsuka). The duration of diabetes was determined through medical interviews. Visceral fat accumulation was assessed by the estimated visceral fat area (eVFA) using the bioelectrical impedance analysis method [20]. We previously showed a significant correlation between eVFA and VFA determined by computed tomography [20]. The maximum intima-media thickness (IMT) of the carotid artery and maximum carotid IMT were measured as previously described [4].
Definition of hypertension and dyslipidemiaHypertension and dyslipidemia were defined as previously described [4]. Briefly, hypertension was defined as systolic blood pressure of ≥140 mmHg and/or diastolic blood pressure of ≥90 mmHg. Dyslipidemia was defined as a low-density lipoprotein cholesterol (LDL-C) concentration of >140 mg/dL and/or a triglyceride concentration of >150 mg/dL and/or a high-density lipoprotein cholesterol (HDL-C) concentration of <40 mg/dL. Patients who received antihypertensive and/or anti-dyslipidemic medications were also considered positive for hypertension and/or dyslipidemia, respectively.
Animal studyAt 6 weeks of age, C57BL/6J male mice were randomly divided into six treatment groups and fed a high-fat and high-sucrose (HF/HS) diet (F2HFHSD; Oriental Yeast, Tokyo, Japan) from 6 (16W), 14 (8W), 18 (4W), 20 (2W) or 21 weeks of age (1W) to 22 weeks of age. Mice in the control group were fed a regular diet from 6 to 22 weeks of age (0W). At 22 weeks of age, plasma samples were collected after 4 h of fasting and stored at –80°C until GC/MS measurement. These mice were also used in our previous report [17]. Mice were maintained in rooms set at 22°C with a 12–12 h dark-light cycle (light cycle, 8 a.m. to 8 p.m.). All experimental protocols described in the study were approved by the Ethics Review Committee for Animal Experimentation of Osaka University School of Medicine. The GC/MS analysis for plasma samples of mice was performed based on previous research using GCMS-QP2010 ultra (Shimadzu) [21]. For data analysis, metabolites were identified based on the retention index and mass spectra library. Each peak area was expressed relative to the peak area of the internal standard, ribitol, and described in the text as relative quantity. The absolute quantity of each metabolite was determined using standard curves prepared in the same analytical batch. Plasma glucose and plasma insulin levels were measured by the Glucose CII-Test (Wako Pure Chemical) and the Insulin Measurement ELISA kit (Morinaga), respectively.
Statistical analysisAll values for human subjects are expressed as the mean ± SD (Table 1). The levels of plasma amino acids in mice are expressed as the mean ± SE. In all cases, p values of <0.05 were considered statistically significant. The statistical significance of differences between groups was determined using two-tailed t tests or Dunnett’s test. The Jonckheere-Terpstra trend test was used for the analysis of mouse adiponectin levels. The correlations between plasma glutamate and other parameters were analyzed by simple linear regression analysis and then by multiple regression analysis (Tables 2 and 3). The correlations between APN and other parameters were analyzed by simple linear regression analysis and then by multiple regression analysis (Tables 4 and 5). All analyses were conducted by using JMP version 16.2.0 for Windows (SAS Institute, Cary, NC) or EZR.
Baseline characteristics of the 62 Japanese patients with type 2 diabetes mellitus
| n (males/females) | 62 (28/34) |
| Age (years) | 61.0 ± 14.1 |
| Body mass index (kg/m2) | 27.8 ± 6.1 |
| eVFA (cm2) | 150.0 ± 71.8 |
| Plasma glutamate (μM) | 40.9 ± 15.4 |
| Duration of diabetes mellitus (years) | 13.3 ± 9.8 |
| Fasting plasma glucose (mg/dL) | 152.6 ± 52.4 |
| HbA1c (%) | 8.9 ± 1.7 |
| Fasting insulin (mIU/L) | 8.7 ± 6.3 |
| HOMA-IR (n = 26) | 2.0 ± 1.0 |
| LDL-C (mg/dL) | 114 ± 52.4 |
| HDL-C (mg/dL) | 46.9 ± 13.8 |
| Triglycerides (mg/dL) | 190 ± 251 |
| Uric acid (mg/dL) | 5.7 ± 1.6 |
| Adiponectin (μg/mL) | 5.7 ± 3.7 |
| Hypertension (%) | 82 |
| Dyslipidemia (%) | 87 |
| CCA max IMT (mm) | 1.86 ± 0.82 |
| CCA mean IMT (mm) | 0.99 ± 0.30 |
Data are mean ± SD or number of subjects. eVFA, estimated visceral fat area; HOMA-IR, homeostasis model assessment of insulin resistance; LDL-C, low-density lipoprotein-cholesterol; HDL-C, high-density lipoprotein-cholesterol; CCA, common carotid artery; IMT, intima-media thickness.
Simple regression analyses of the factors associated with plasma glutamate
| Simple linear regression model | ||
|---|---|---|
| r | p-value | |
| gender (male = 1/female = 0) | –0.0146 | 0.9105 |
| Age (years) | –0.1646 | 0.2012 |
| Body mass index (kg/m2) | 0.2885 | 0.023 |
| eVFA (cm2) | 0.3334 | 0.022 |
| Duration of diabetes mellitus (years) | –0.2688 | 0.0362 |
| Fasting plasma glucose (mg/dL) | 0.155 | 0.229 |
| HbA1c (%) | –0.0209 | 0.8718 |
| Fasting insulin (mIU/L) | 0.2991 | 0.046 |
| LDL-C (mg/dL) | 0.0213 | 0.8692 |
| HDL-C (mg/dL) | –0.1112 | 0.3894 |
| Triglycerides (mg/dL) | –0.0351 | 0.7865 |
| Uric acid (mg/dL) | –0.1257 | 0.3305 |
| Adiponectin (μg/mL) | –0.448 | 0.0008 |
| CCA max IMT (mm) | –0.1208 | 0.362 |
| CCA mean IMT (mm) | –0.1619 | 0.2289 |
Pearson correlation coefficient (r) and each probability value (p) are shown. Statistically significant results (p < 0.05) are shown in bold. eVFA, estimated visceral fat area; LDL-C, low-density lipoprotein-cholesterol; HDL-C, high-density lipoprotein-cholesterol; CCA, common carotid artery; IMT, intima-media thickness.
Multiple regression analysis of the factors associated with plasma glutamate
| Multiple regression model | ||
|---|---|---|
| Std β | p-value | |
| Adiponectin (μg/mL) | –0.37059 | 0.0594 |
| eVFA (cm2) | 0.153468 | 0.5235 |
| Duration of diabetes mellitus (years) | 0.010237 | 0.9559 |
| Fasting insulin (mIU/L) | 0.052018 | 0.8142 |
Standardized beta coefficients (Std β) and each probability value (p) are shown. eVFA, estimated visceral fat area.
Simple regression analyses of the factors associated with adiponectin
| Simple linear regression model | ||
|---|---|---|
| r | p-value | |
| gender (male = 1/female = 0) | –0.1442 | 0.303 |
| Age (years) | 0.4207 | 0.0017 |
| Body mass index (kg/m2) | –0.4298 | 0.0013 |
| eVFA (cm2) | –0.4425 | 0.003 |
| Duration of diabetes mellitus (years) | 0.3076 | 0.0251 |
| Fasting plasma glucose (mg/dL) | –0.2698 | 0.0507 |
| HbA1c (%) | –0.1134 | 0.4188 |
| Fasting insulin (mIU/L) | –0.4029 | 0.011 |
| LDL-C (mg/dL) | –0.0519 | 0.7122 |
| HDL-C (mg/dL) | 0.2616 | 0.0585 |
| Triglycerides (mg/dL) | –0.0484 | 0.7308 |
| Uric acid (mg/dL) | –0.2092 | 0.1327 |
| Plasma Glutamate (μM) | –0.448 | 0.0008 |
| CCA max IMT (mm) | 0.2732 | 0.0524 |
| CCA mean IMT (mm) | 0.0133 | 0.9275 |
Pearson correlation coefficient (r) and each probability value (p) are shown. Statistically significant results (p < 0.05) are shown in bold. eVFA, estimated visceral fat area; LDL-C, low-density lipoprotein-cholesterol; HDL-C, high-density lipoprotein-cholesterol; CCA, common carotid artery; IMT, intima-media thickness.
Multiple regression analysis of the factors associated with adiponectin
| Multiple regression model | ||
|---|---|---|
| Std β | p-value | |
| Age (years) | 0.409382 | 0.1007 |
| eVFA (cm2) | 0.073027 | 0.7964 |
| Fasting insulin (mIU/L) | –0.0875 | 0.6597 |
| Plasma Glutamate (μM) | –0.4272 | 0.0177 |
Standardized beta coefficients (Std β) and each probability value (p) are shown. Statistically significant results (p < 0.05) are shown in bold. eVFA, estimated visceral fat area.
To determine the clinical significance of the plasma glutamate level in type 2 diabetes, we investigated the correlation between plasma glutamate levels and clinical parameters in human subjects. The study subjects were 62 Japanese patients with type 2 diabetes (28 men and 34 women; age 61.0 ± 14.1 years [mean ± SD]; BMI 27.8 ± 6.1 kg/m2, eVFA; 150.0 ± 71.8 cm2; duration of type 2 diabetes 13.3 ± 9.8 years; HbA1c 8.9 ± 1.7%) (Table 1). Among these patients, 51 (82%) had hypertension, and 54 (87%) had dyslipidemia.
In the simple linear regression analysis, plasma glutamate was positively correlated with BMI, eVFA and fasting insulin and negatively correlated with APN and duration of diabetes in patients with type 2 diabetes (Table 2). In addition, the plasma glutamate levels were positively correlated with HOMA-IR (Fig. 1A). However, there were no significant correlations between plasma glutamate and HbA1c or plasma glucose and maximum carotid IMT. Interestingly, the absolute value of the correlation coefficient was higher between plasma glutamate and APN (–0.448) than between plasma glutamate and eVFA (0.3334) (Fig. 1B and C). Multiple regression analysis identified that the APN level tended (p = 0.0594) to determine plasma glutamate levels (Table 3).
Correlations between plasma glutamate levels and clinical parameters in 62 Japanese patients with type 2 diabetes mellitus
(A–C) Correlations of plasma glutamate levels with homeostasis model assessment of insulin resistance (HOMA-IR) (A), serum adiponectin (B) and eVFA (C) values in 62 Japanese patients with type 2 diabetes mellitus. In all cases, p values <0.05 were considered statistically significant.
APN is an excellent clinical marker that reflects adipocyte dysfunction, including insulin resistance and dysregulation of adipocytokines caused by visceral fat accumulation [3, 9, 22]. Low serum levels of adiponectin correlate with cardiometabolic diseases [9]. Our previous study showed that elevated levels of glutamate in obese adipocytes did not affect the gene expression level of APN but decreased APN secretion [17]. Furthermore, elevated glutamate levels in obese adipocytes resulted in decreased insulin sensitivity in adipocytes [17]. Based on these findings, we examined the correlation between serum APN and various parameters. In the simple linear regression analysis (Table 4), APN was significantly positively correlated with age and duration of type 2 diabetes. Moreover, APN was significantly negatively correlated with BMI, eVFA, fasting insulin and plasma glutamate. Among them, plasma glutamate (r = –0.448) and eVFA (r = –0.4425) were closely correlated with APN. Multiple regression analysis, which included age, eVFA, fasting insulin and plasma glutamate, identified plasma glutamate as the only significant explanatory factor associated with the levels of APN (Table 5).
Changes in plasma amino acid concentrations in mice with or without obesitySeveral studies have shown that glutamate is the most elevated amino acid in the plasma amino acid profile in patients with obesity and/or visceral fat accumulation [14, 15]. Since backgrounds between individuals can be matched and the degree of obesity can be set from mild to severe, we examined changes in plasma amino acid concentrations due to obesity in mice. At 6 weeks of age, C57BL/6J male mice were randomly divided into six groups and fed a HF/HS diet from 6 (16W), 14 (8W), 18 (4W), 20 (2W) or 21 weeks of age (1W) to 22 weeks of age. Mice in the control group were fed a regular diet from 6 to 22 weeks of age (0W) (Fig. 2). At 22 weeks of age, plasma samples were collected after 4 h of fasting. The body weight of these mice was increased with increasing weeks of the HF/HS diet (Fig. 2A). Plasma glucose levels were significantly increased by the HF/HS diet in all groups (1W, 2W, 4W, 8W and 16W) (Fig. 2B and D). The levels of plasma insulin increased with increasing weeks of the HF/HS diet (Fig. 2C), suggesting the development of diabetes and increasing insulin resistance. In the amino acid profile, the levels of plasma glutamate clearly increased with increasing weeks of the HF/HS diet (Fig. 2D). The fold change in the glutamate level in response to 16 weeks of a HF/HS diet was 1.83. This was the most marked increase of all amino acids, as shown in several previous reports [14-16] of human subjects with obesity and/or visceral fat accumulation. The levels of other amino acids, such as tryptophan, asparagine, aspartate, lysine, serine and threonine were increased by the HF/HS diet. The levels of most of these amino acids increased rapidly after 1 week of the HF/HS diet, and the fold change in these amino acid levels in response to the HF/HS diet was lower than that of glutamate. Glycine was the only amino acid that was decreased by the HF/HS diet. The Fischer ratio, which is the molar ratio of branched-chain amino acids (BCAAs) to aromatic amino acids (AAAs), demonstrated a decreasing trend in the HF/HS diet-fed mice compared to the control mice (0W), but this difference was not statistically significant (Fig. 2E). Taken together, these data from our mouse study indicate that glutamate was the only amino acid whose plasma levels increased linearly with increasing body weight.
Changes in plasma amino acid concentrations in mice with or without diet-induced obesity
At 6 weeks of age, male C57BL/6J mice were randomly divided into six treatment groups (n = 4–6 per group). Mice in the diet-induced obesity groups were fed a high-fat/high-sucrose diet from 6 (16W), 14 (8W), 18 (4W), 20 (2W) or 21 weeks of age (1W) to 22 weeks of age. Mice in the control group were fed a regular diet from 6 to 22 weeks of age (0W). (A) Body weight at 22 weeks of age in these groups. * p < 0.05, ** p < 0.01, *** p < 0.001, control versus diet-induced obesity group(s). Data are presented as the mean ± SEM. The data were modified from our previous report [17]. (B) Plasma glucose levels in the groups. * p < 0.05, ** p < 0.01, control versus diet-induced obesity group(s). Data are presented as the mean ± SEM. (C) Plasma insulin levels in the groups. **p < 0.01, *** p < 0.001, control versus diet-induced obesity group(s). Data are presented as the mean ± SEM. (D) The concentrations of plasma amino acids, lactate and glucose were measured by GC/MS analysis as described in the Methods. All mice were analyzed at the age of 22 weeks after 4 h of fasting. * p < 0.05 versus the control group. Data are presented as the mean ± SEM. (E) The Fischer ratio (BCAA/AAA) of all groups. Data are presented as the mean ± SEM. (F) Plasma adiponectin levels of all groups. Data are presented as the mean ± SEM. (G) The correlation between plasma glutamate levels and adiponectin levels in all mice. p values <0.05 were considered statistically significant.
Measurement of circulating APN levels in all groups showed that the APN levels tended to decrease with increasing duration of the HF/HS diet (p = 0.055 for trend) (Fig. 2F). The APN levels of mice fed the HF/HS diet for 16 weeks demonstrated a decreasing trend compared to that of the control mice (p = 0.057). Moreover, plasma glutamate showed a significant negative correlation with APN in these mice (Fig. 2G), and this correlation was also observed in patients with type 2 diabetes.
Numerous studies from us and others have clinically and fundamentally revealed that obesity and visceral fat accumulation induce glucose intolerance, dyslipidemia and hypertension, which lead to atherosclerotic diseases [3]. In obese adipocytes, adipocyte dysfunction occurs. Such dysfunction includes insulin resistance through the dysregulation of insulin receptor signaling [23, 24] and abnormalities in adipocytokine secretion, such as a decrease in adiponectin secretion. These processes are involved in the pathogenesis of diabetes and atherosclerotic diseases [3]. We have reported that there is no significant difference in the prevalence of microvascular complications between patients with type 2 diabetes with and without visceral fat accumulation [4]. However, the prevalence of hypertension, dyslipidemia and cardiovascular disease was significantly higher in patients with visceral fat accumulation than in patients without visceral fat accumulation [4]. Therefore, it is necessary to properly evaluate patients’ risk of cardiovascular disease when managing type 2 diabetes. It is also important to provide personalized medical care based on factors such as the presence of visceral fat accumulation to improve the prognosis and quality of life of patients.
Recently, we and others have reported that changes not only in metabolites associated with glucose and fatty acid metabolism but also in amino acids and purine metabolites in obesity can modulate adipocyte function [17, 25-27]. Amino acids serve as building blocks of proteins, energy sources, materials for the biosynthesis of nucleic acids and hormones, and intermediate metabolites in protein catabolic processes [28]. In addition, amino acids act as cell signaling molecules and regulators of gene expression and the protein phosphorylation cascade [28-30]. Plasma free amino acids circulate as a medium linking all organs in the human body [31, 32]. Specific abnormalities in amino acid concentrations have been reported in various diseases, including fatty liver [10], liver cirrhosis [11], cancer [12], gout [31], sarcopenia [33] and Alzheimer’s disease [13]. Therefore, plasma free amino acid profiles can be used as reliable markers for monitoring the risks of various diseases in various populations and improving in physiological states [31-33]. For example, the liver is a major organ in amino acid metabolism, and the balance of plasma amino acids is altered in liver diseases. The Fischer ratio, which is the molar ratio of BCAAs to AAAs, is a good indicator of amino acid metabolism dynamics in the liver and is used in clinical practice [34]. Plasma amino acid profiles can be applied for the diagnosis of advanced liver fibrosis in patients with chronic hepatitis [35]. Furthermore, it has been demonstrated that plasma amino acid profiles are altered in various cancers [12, 36, 37]. Additionally, the amino acid profile of each patient can be used to identify those who may benefit from cancer immunotherapy, such as immune checkpoint inhibitors, before treatment [38].
In metabolic research associated with obesity and type 2 diabetes mellitus, specific abnormalities in plasma amino acid concentrations have been reported to be associated with obesity [14], visceral fat accumulation [14, 15] and diabetes status [16]. Among all amino acid levels, glutamate levels are most highly elevated in obesity and visceral fat accumulation compared to healthy subjects [14, 15]. It has also been reported that plasma levels of glutamate increase the most among amino acids in patients with type 2 diabetes compared to healthy subjects [16]. In addition, a recent report showed that the level of plasma glutamate was significantly higher in the low APN group than in the high APN group in a Japanese medical check-up population [39].
Based on the above findings, we hypothesized that changes in plasma glutamate levels may sensitively reflect some pathological conditions in patients with type 2 diabetes mellitus. In the mouse study, glutamate was the only amino acid that increased linearly with increasing body weight, and the rate of increase was the largest among amino acids. Several amino acids, such as tryptophan, threonine, serine and lysine, were increased after a one-week HF/HS diet intervention, possibly due to changes in diet composition rather than weight gain. In patients with type 2 diabetes, glutamate was positively correlated with eVFA, BMI, and insulin levels and negatively correlated with APN. Interestingly, the multiple regression analysis identified plasma glutamate as the only significant explanatory factor associated with the levels of APN. These findings suggested that glutamate concentrations reflect adipocyte dysfunction rather than visceral fat accumulation itself.
We have previously reported, using obese mouse models, that increased glutamate biosynthesis in obese adipose tissue and that elevated glutamate in adipocytes leads to insulin resistance and decreased APN secretion without any alteration in the mRNA levels of APN [17]. In other words, visceral fat accumulation increases not only the glutamate concentration in circulation but also that within adipocytes. This increase in glutamate within adipocytes may be involved in the pathophysiology of visceral fat accumulation. In the same study, we also found that increased glutamate levels within adipocytes in conditions of obesity resulted in significantly reduced glutamate uptake into adipocytes via decreased expression of GLAST, a glutamate transporter in adipocytes, and speculated that this may be one of the mechanisms underlying the increase in plasma glutamate levels in obesity [17]. Another report suggested that changes in the intestinal microbiota during obesity in mice could be linked to the increase in plasma glutamate concentrations [40]. In the present study, multiple regression analysis of the correlations between APN and each parameter revealed that the level of plasma glutamate remained a factor that could explain the serum APN level. The results indicate that glutamate variability may be at least partially involved in the reduction in APN secretion in obesity and visceral fat accumulation. The present study has several limitations. The study was not prospective in design and included a relatively small population limited to Japanese individuals. The study was performed in a single institution. The influence of ethnicity, sex, and unmeasured factors cannot be fully excluded. Further prospective studies with larger populations are needed in the future.
In summary, the present study demonstrated that plasma glutamate was positively correlated with visceral fat accumulation and negatively correlated with serum APN in patients with type 2 diabetes. Multiple regression analysis identified plasma glutamate as the only significant explanatory factor associated with the levels of APN. The plasma glutamate level was significantly increased with increasing weeks of HF/HS diet consumption in obese mice compared to control mice, and it was negatively correlated with APN. These results indicate that the level of plasma glutamate could be a useful parameter reflecting adipocyte dysfunction rather than visceral fat accumulation itself in patients with type 2 diabetes. Elevated plasma glutamate levels may serve as a useful predictor for various pathologies associated with decreased levels of APN. Further investigation will be needed in the future.
This work was supported in part by Grant-in-Aid for Research Activity Start-up number 23K19589 (to H. Nagao), Grant-in-Aid for Scientific Research (C) number 23K08006 (to H. Nishizawa), Gout and Uric Acid Foundation of Japan (to H. Nishizawa), Lotte Research Promotion Grant (to Y.F.), Suzuken Memorial Foundation (to I.S.), and a grant from the “Creation of Innovative Technology for Medical Applications Based on the Global Analyses and Regulation of Disease-Related Metabolites Project” of the Japan Agency for Medical Research and Development (to E.F. and I.S.).
H. Nagao and H. Nishizawa are members of the “Department of Metabolism and Atherosclerosis,” which is a sponsored course endowed by Kowa Co., Ltd. S.K. belongs to the department endowed by Takeda Pharmaceutical Company, Rohto Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., FUJI OIL HOLDINGS INC., and Kobayashi Pharmaceutical Co., Ltd. The funders had no role in the study design, analysis, or preparation of the manuscript.
H. Nagao designed the research, performed the experiments, analyzed the data and wrote the manuscript. H. Nishizawa designed the research and edited the manuscript. S.F., Y.F., S.K. and N.M. interpreted the results and edited the manuscript. T.B. performed metabolomic analysis and analyzed the data. E.F. supervised the metabolomic analysis. I.S. edited the manuscript and supervised the project.
