Abstract
Objective
The main purpose of this study was to evaluate the associations between circulating angiopoietin-like protein 6 (ANGPTL6) levels and various diabetes- and atherosclerosis-related variables in patients with type 2 diabetes.
Methods
Serum ANGPTL6 levels in patients with type 2 diabetes hospitalized for glycemic control and/or diabetic education were measured using a chemiluminescent immunoassay.
Results
Most patients had elevated hemoglobin (Hb) A1c levels; 85.7% and 71.4% of patients had HbA1c levels ≥8%, and ≥9%, respectively. ANGPTL6 levels were significantly higher in patients with type 2 diabetes than in non-diabetic controls. In patients with type 2 diabetes, ANGPTL6 was significantly and positively correlated with the duration of diabetes, systolic blood pressure, gamma-glutamyl transpeptidase, C-reactive protein (CRP), and the intimal medial complex thickness of the carotid artery (IMT), and inversely correlated with HbA1c. In the multiple regression analysis, ANGPTL6 had a significant positive association with triglyceride in one of the models in which it was included as a variable. Furthermore, ANGPTL6 also showed significant positive associations with CRP and IMT in models in which they were included as variables.
Conclusion
The current study suggests that circulating levels of ANGPTL6 may be negatively associated with poor glycemic control and positively associated with the degree of atherosclerosis, as reflected by IMT, in patients with type 2 diabetes, most of whom had elevated HbA1c levels.
Keywords: ANGPTL6, type 2 diabetes, IMT
Introduction
Angiopoietin-like proteins (ANGPTLs) are a family of secreted glycoproteins that are structurally similar to angiopoietins and are involved in angiogenesis (1,2). At present, 8 types of ANGPTL have been identified (ANGPTL1-8) and these proteins each contain an N-terminal coil domain, linker peptide regions, and a C-terminal fibrinogen-like domain (1,2), with the exception of ANGPTL8 (also called betatrophin), which does not possess a C-terminal fibrinogen-like domain (1,2). Although the main role of most ANGPTLs is considered to be the regulation of angiogenesis, as well as angiopoietins (1-4), importantly, ANGPTLs do not bind to the well-known angiopoietin binding receptor, that is, tyrosine kinase with immunoglobulin-like and EGF-like domain 1 (Tie1) and the endothelial-specific receptor kinase (Tie2), which are largely expressed in endothelial and early hematopoietic cells (1-3,5,6). This suggests that the mechanisms of angiogenesis by ANGPTLs may differ from those of angiopoietins (1,3,4).
Among these ANGPTLs, ANGPTL2, 3, 4, 6, and 8 are easily found in circulation (3,7,8), and ANGPTL3, 4, and 8 can regulate and play an important role in lipid metabolism mainly by their inhibition of lipoprotein lipase.
ANGPTL2 is mainly expressed in visceral adipose tissues (1,9-11) and is associated with vascular inflammation (11,12) and increased oxidative stress (13). Furthermore, ANGPTL2 causes insulin resistance related to inflammation in adipose tissues and can therefore be involved in glucose metabolism (9-11). In clinical studies, circulating levels of ANGPTL2 have been reported to be associated with new-onset type 2 diabetes (14), and cardiovascular events or mortality in patients with type 2 diabetes (15).
On the other hand, ANGPTL6 [also called angiopoietinrelated growth factor (AGF)] is mainly expressed in the liver (1,3). This protein can also be involved in improving glucose metabolism, unlike ANGPTL2 (16), in addition to its effects on epidermal proliferation and wound healing (17,18). Animal studies have shown that ANGPTL6 increases energy expenditure and insulin sensitivity and counteracts obesity (3,16). In clinical studies, serum levels of ANGPTL6 have been shown to be elevated in patients with diabetes (19) or metabolic syndrome (20) in comparison to healthy subjects, probably through compensatory mechanisms. Interestingly, a recent clinical study demonstrated that elevated levels of circulating ANGPTL6 are related to a low incidence of diabetes (21), in contrast to the results for ANGPTL2 (14). In contrast, high circulating levels of ANGPTL6 are an independent predictor of metabolic syndrome (22). Although these findings suggest that ANGPTL6 may have a potential protective effect on glucose homeostasis and on factors related to metabolic syndrome, the effect appears to be somewhat complex. Furthermore, the association between ANGPTL6 and factors related to diabetes and atherosclerosis has not been fully described, particularly in patients with poorly controlled type 2 diabetes. In this background, we investigated these associations in patients with type 2 diabetes, most of whom had elevated levels of hemoglobin (Hb) A1c.
Materials and Methods
Patients
Patients (n=70; 45 men and 25 women) and non-diabetic control subjects (n=9; 5 men and 4 women) were included in our previous study [registered as UMIN000025767 (23)], in which ANGPTL2 measurements were performed (24). In these subjects, ANGPTL6 levels were measured using samples frozen at -80°C.
The key inclusion and exclusion criteria for patients with type 2 diabetes, which have also been described in a clinical trial registry (23), were as follows: 1) patients (male or female) of ≥20 years of age at enrollment; 2) patients diagnosed with type 2 diabetes (based on the criteria from the Japan Diabetes Society 2010); 3) patients who were hospitalized for glycemic control and/or diabetes education for type 2 diabetes; and 4) patients who provided their written consent for participation in this study. The key exclusion criteria were as follows: 1) patients with type 1 diabetes, pancreatic diabetes, or secondary diabetes (Cushing syndrome, acromegaly, etc.); 2) patients who were pregnant, possibly pregnant, or lactating; and 3) patients who were judged as inadequate for participation by a medical doctor. For control subjects, the inclusion criterion was that the subjects (male or female) were ≥20 years of age at recruitment. Individuals with a prior diagnosis of diabetes during a regular medical checkup were excluded from the control group. Control subjects were not admitted to the hospital.
In a previous study (24), concrete definitions for the degrees of glycemic control, such as “normal,” “moderate,” “poor,” were not decided in advance, and the patients, who were hospitalized for the treatment for glycemic control and/or diabetic education, were enrolled consecutively and prospectively during the study period.
The detailed characteristics and laboratory data of the patients and non-diabetic control subjects are presented in Table 1.
Table 1.
Clinical Features and Laboratory Data in Patients with Type 2 Diabetes and Control Subjects.
| All patients | Control subjects | p | |
|---|---|---|---|
| No. (male/female) | 70 (45/25) | 9 (5/4) | |
| Age (year) | 60 (48.3, 72.8) | 61 (50.0, 63.0) | 0.3503 |
| Mean duration of diabetes (year) | 7 (2, 11.8) | ||
| BMI (kg/m2) | 24.5±4.5 | 24.8±2.7 | 0.8438 |
| FPG (mg/dL) | 174 (143.3, 257.8) | 106 (97, 111) | <0.0001 |
| HbA1c (%) | 9.8 (8.8, 11.7) | 5.7 (5.6, 5.8) | <0.0001 |
| eGFR (mL/min/1.73 m2) | 79.7 (64.2, 96.1) | 76.1 (65.8, 88.8) | 0.6769 |
| SBP (mmHg) | 121 (114.3, 134.8) | ||
| DBP (mmHg) | 73.0±11.7 | ||
| TG (mg/dL) (69) | 127 (99, 177) | ||
| HDL-C (mg/dL) (69) | 47 (41, 63) | ||
| LDL-C (mg/dL) (69) | 123.6±45.0 | ||
| CRP (mg/dL) | 0.256 (0.063, 0.355) | ||
| Fibrinogen (mg/dL) | 349.6±98.2 | ||
| AST (U/L) | 18 (15,23) | ||
| ALT (U/L) | 18 (13, 26.5) | ||
| GGT (U/L) (69) | 30 (20, 39) | ||
| IMT (mm) (58) | 0.95 (0.8, 1.1) | ||
| UAE (mg/g·Cr) (69) | 17 (6.5, 75.5) | ||
| Diabetic therapy | |||
| Insulin (-) | 35 | ||
| Metformin (+) | 24 | ||
| D/ D+A/D+P/D+Sg/D+Gn/ | 7/3/2/2/1/ | ||
| D+P+Sg/D+P+Sg+Gp/D+A+Sg/ | 1/1/2/ | ||
| D+A+Su/Gp/Gp+P+Sg | 1/2/2 | ||
| Metformin (-) | 11 | ||
| D/D+A/D+A+Su/D+Su/ | 2/1/1/1/ | ||
| P+A+D/P+Gp/Gp+Gn/N | 1/1/1/3 | ||
| Insulin (+) | 35 | ||
| Metformin (+) | 16 | ||
| D/D+A/D+P/D+Sg/D+Gn/ | 4/1/2/1/1/ | ||
| D+P+Sg/D+P+Sg+Gp/D+A+Sg/ | 1/0/0/ | ||
| D+A+Su/M+Gp/Gp+P+Sg/ | 0/0/0/ | ||
| D+Su/D+P+A/M/A/A+Su | 1/1/2/1/1 | ||
| Metformin (-) | 19 | ||
| D/D+A/D+A+Su/D+Su/ | 5/2/0/0/ | ||
| P+A+D/P+Gp/Gp+Gn/N/ | 0/0/0/3/ | ||
| P/D+P/D+Gn/A | 3/4/1/1 | ||
| Anti-hypertensive drugs | 31 | ||
| Ar/C/A+C/Ar+C+B/ | 4/6/16/1/ | ||
| Ar+C+Ab/Ar+C+T/C+T/C+B+Ab | 1/1/1/1 | ||
| Anti-hyperlipidemic drugs | |||
| Statins (+) | 20 | ||
| S/S+E/S+C | 18/2/0 | ||
| Statins (-) | 50 | ||
| E/C/N | 2/1/47 | ||
| Smoking history | 29 |
Data are expressed as median (with 25th and 75th interquartile range) or mean±standard deviation. For the variables which were not measured in all diabetic patients, the number of patients is described in brackets.
Diabetic therapy: the number of the patients with respective diabetic therapies, M: metformin, D: dipeptidyl peptidase 4 inhibitors, A: α glucosidase inhibitor, P: pioglitazone, Sg: sodium glucose cotransporter 2 inhibitors, Gn: glinides, Gp: glucagon-like peptide-1 receptor agonists, Su: sulfonylureas, N: no antidiabetic drugs, Anti-hypertensive drugs: the number of the patients with respective anti-hypertensive drugs, Ar: angiotensin-II receptor blockers, C: calcium channel blockers, T: thiazides, B: beta adrenalin receptor blockers, Ab: α adrenalin receptor blockers, Anti-lipids drugs: S: statins, E: ezetimibe, C: colestimide
The HbA1c range among the patients was 6.8-18%, and the numbers of patients with HbA1c levels of ＜6%, ＜7%, 7-7.9%, 8-8.9%, 9-9.9%, and ≥10% were 0, 1, 9, 10, 16, and 34, respectively. Overall, 1.4%, 85.7%, and 71.4% of patients had HbA1c levels ＜7%, ≥8%, and ≥9%, respectively.
Methods
This cross-sectional study targeted patients with type 2 diabetes who were hospitalized for glycemic control and/or diabetic education.
Blood samples were obtained in the morning after fasting overnight for at least 10 hours the day after hospital admission. Body weight (BW) and blood pressure were measured during hospitalization. The methods for the measurement of HbA1c, fasting plasma glucose (FPG), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), C-reactive protein (CRP), fibrinogen, estimated glomerular filtration rate (eGFR), ANGPTL2, and the evaluation method for diabetic retinopathy have been previously described (24).
ANGPTL6 measurement Serum ANGPTL6 levels were measured in this hospital by a chemiluminescent immunoassay using an ANGPTL6 kit (MBS2532115, MY BioSource, San Diego, USA). Based on the manufacturer's information, the intra- and inter-assay coefficients of variation were 9.01-10.56% and 8.26-11.45%, respectively (25).
The IMT was measured on the right side using echography (Aplico i900 and AplicoXV; Toshiba, Tokyo, Japan). The maximum of 3 suitable consecutive points on the carotid artery, at 1 cm intervals, was estimated as the intima-media thickness. The plaque score was estimated as the total IMT value thickened by ＞1.1 mm in the section with a range (6.0 cm) from the base point (branch point of the internal and external artery) to the peripheral side (1.5 cm) and to the proximal (heart direction) side (4.5 cm). The carotid artery stenosis rate was evaluated using the area stenosis rate.
Urinary albumin excretion (UAE) UAE was measured by once daily collection of urine during hospitalization using an immunoturbidimetric method with the TAC-2 test albumin U reagent kit (Medical & Biological Laboratories, Nagoya, Japan). Albumin values were corrected for urinary creatinine levels.
Urinary C-peptide (U-CPR) U-CPR measurement was performed once in a 24-hour urine specimen during admission by a chemiluminescent enzyme assay, based on a 2-step sandwich method using a Lumipulse-presto C-peptide reagent kit (Fujirebio, Tokyo, Japan).
Ethical considerations All subjects provided their written informed consent for inclusion in the previous study, which was registered as UMIN000025767. In the current study, the frozen blood samples were approved, and all patients approved the reuse of the samples in future studies. The previous and current studies were approved by the Local Ethics Committee at the Dokkyo Medical University Saitama Medical Center (approval numbers: 1654, 1/20/2017 and 2077, 9/17/2020, respectively). This study was conducted in accordance with the guidelines of the Declaration of Helsinki.
Statistical methods The normality of data for each variable was examined using a Shapiro-Wilk test and/or a Kolmogorov-Smirnov test. ANGPTL6 showed skewed distribution. Furthermore, in 3 of 70 patients with type 2 diabetes and in 5 of 9 healthy subjects, ANGPTL6 could not be detected due to very low circulating levels of ANGPTL6. We defined these values as 0, and for the correlations of ANGPTL6 levels with other variables and the comparisons between or among subgroups, nonparametric methods were chosen. The correlations were examined using Spearman correlation. However, in the multiple regression analysis, we decided to use a log10-transformation for ANGPTL6 as a dependent variable. Before log-transforming ANGPTL6, we decided to use the minimum value of ANGPTL6 that could be detected in this study instead of the 3 missing values. ANGPTL6 exhibited a normal distribution after log-transformation. In this analysis, we created 4 models; the independent variables were chosen based on the results of previous reports in which the correlations between ANGPTL6 and other variables were investigated (19-21,24-26). For the independent variables, we performed a log-transformation to bring these variables as close as possible to a normal distribution. After log-transformation, the body mass index (BMI), HbA1c, TG, CRP, and eGFR showed normal distributions. Although the log-transformed IMT and duration did not show normal distributions in the 2 above-described analyses for normality, the log-transformed IMT and duration exhibited normal distributions, as confirmed by D'Agostino's Skewness, Kurtosis and Omnibus tests, and the D'Agostino's Skewness test, respectively. On the other hand, although log-transformed age, gamma-glutamyl transpeptidase (GGT), and systolic blood pressure (SBP) were not normally distributed, even by all of the above-described analyses, we decided to include these independent variables in the analysis because we considered that they may be important as confounding factors. Furthermore, for sex, we applied dummy variables of 1 for men and 0 for women. Comparisons of ANGPTL6 between the 2 subgroups of patients with type 2 diabetes were performed using the Mann-Whitney U test. Comparison of ANGPTL6 among the 3 groups was performed using the Kruskal-Wallis test with a post hoc Steel-Dwass test. All statistical analyses were performed using Bell Curve for Excel (Social Survey Research Information, Tokyo, Japan). p values of ＜0.05 were considered to indicate statistical significance.
Results
The median (25th and 75th percentile) levels of ANGPTL6 in type 2 diabetic patients and non-diabetic control subjects, after exclusion of the subjects in whom ANGPTL6 were not detected, were 0.346 (0.134, 0.517) ng/mL and 0.158 (0.115, 0.212) ng/mL, respectively. ANGPTL6 levels in diabetic patients were significantly higher than those in healthy subjects (p＜0.001). There were significant positive correlations between ANGPTL6 and the duration of diabetes, SBP, CRP, GGT, and IMT, while a significant negative correlation was found between ANGPTL6 and HbA1c. The correlations between ANGPTL6 and all examined variables are summarized in Table 2. The results of the multiple regression analysis with log-transformed ANGPTL6 as a dependent variable are summarized in Table 3.
Table 2.
The Correlation of ANGPTL6 with Multiple Variables.
| ρ | p | ||
|---|---|---|---|
| Age (year) | (70) | 0.0496 | 0.6835 |
| Duration (year) | (70) | 0.2770 | 0.0203* |
| BMI (kg/m2) | (70) | 0.0061 | 0.9599 |
| FPG (mg/dL) | (70) | -0.1419 | 0.2412 |
| HbA1c (%) | (70) | -0.2614 | 0.0288* |
| SBP (mmHg) | (70) | 0.2632 | 0.0277* |
| DBP (mmHg) | (70) | 0.1049 | 0.3873 |
| TG (mg/dL) | (69) | 0.2052 | 0.0908 |
| HDL-C (mg/dL) | (69) | 0.0149 | 0.9036 |
| LDL-C (mg/dL) | (69) | -0.1511 | 0.2152 |
| CRP (mg/L) | (70) | 0.2547 | 0.0334* |
| Fibrinogen (mg/dL) | (70) | 0.1536 | 0.2044 |
| AST (U/L) | (70) | 0.1471 | 0.2242 |
| ALT (U/L) | (70) | 0.1513 | 0.2113 |
| GGT (U/L) | (69) | 0.2600 | 0.0310* |
| eGFR (mL/min/1.73 m2) | (70) | -0.1197 | 0.3236 |
| UAE (mg/g·Cr) | (69) | 0.1556 | 0.2017 |
| IMT (mm) | (58) | 0.4532 | <0.001* |
| Plaque score | (58) | 0.3533 | 0.0065* |
| Area stenosis rate | (58) | 0.4065 | 0.0015* |
| U-CPR (μg/day) | (69) | -0.0548 | 0.6549 |
| ANGPTL2 (ng/mL) | (70) | -0.0524 | 0.6668 |
ρ: Spearman’s correlation coefficient, p: p value, p<0.05 is defined as statistical significance (*). Parentheses means the number of the patients.
Table 3.
Multiple Regression Analysis with ANGPTL6 as the Dependent Variable.
| Model 1 | β | p | (R2: 0.1971) |
| Age (year) | 0.0037 | 0.9793 | |
| Gender (m:1, f:0) | -0.0938 | 0.4277 | |
| Duration (year) | 0.2124 | 0.1184 | |
| BMI (kg/m2) | -0.0668 | 0.6157 | |
| HbA1c (%) | -0.2438 | 0.0540 | |
| TG (mg/dL) | 0.2559 | 0.0417* | |
| Model 2 | β | p | (R2: 0.2414) |
| Age (year) | 0.0002 | 0.9990 | |
| Gender (m:1, f:0) | -0.1183 | 0.3082 | |
| Duration (year) | 0.2357 | 0.0754 | |
| HbA1c (%) | -0.2303 | 0.0598 | |
| TG (mg/dL) | 0.1746 | 0.1556 | |
| GGT (U/L) | 0.2184 | 0.0682 | |
| Model 3 | β | p | (R2: 0.3895) |
| Age (year) | -0.0438 | 0.7587 | |
| Gender (m:1, f:0) | -0.1304 | 0,2800 | |
| HbA1c (%) | -0.2234 | 0.0648 | |
| SBP (mmHg) | 0.1345 | 0.2670 | |
| CRP (mg/dL) | 0.3069 | 0.0103* | |
| IMT (mm) | 0.3958 | 0.0053* | |
| Model 4 | β | p | (R2: 0.3793) |
| Age (year) | -0.0035 | 0.9820 | |
| Gender (m:1, f:0) | -0.1828 | 0.1100 | |
| HbA1c (%) | -0.2396 | 0.0533 | |
| eGFR (mL/min/1.73 m2) | 0.0907 | 0.5239 | |
| CRP (mg/dL) | 0.3159 | 0.0090* | |
| IMT (mm) | 0.4228 | 0.0039* |
β: standard partial regression, R2: coefficient of determination, p: p value, p<0.05 is defined as statistical significance (*).
In the independent variables in the models presented in Table 3, when age, GGT, and SBP were deleted due to the apparent lack of normal distribution (i.e., re-analysis), ANGPTL6 exhibited significant associations with HbA1c (negative, p=0.0465) and TG (positive, p=0.0397) in model 1, TG (positive, p=0.0450) in model 2, CRP (positive, p=0.0058) and IMT (positive, p=0.0010) in model 3, and HbA1c (negative, p=0.0483), CRP (positive, p=0.0067), and IMT (positive, p=0.0011) in model 4.
There were no sex-related differences in ANGPTL6, and no significant differences were observed between subgroups of patients treated with and without insulin, metformin, ARBs, or statins. No significant differences were observed in ANGPTL6 levels in patients with or without a history of smoking, or in patients with no diabetic retinopathy (NDR), simple diabetic retinopathy (SDR) or proliferative diabetic retinopathy (PDR).
Discussion
In the current study, the median ANGPTL6 concentration in patients with diabetes was 0.346 ng/mL. Some studies have reported higher ANGPTL6 concentrations than those in our study (19-22,26,27), while the values measured in patients with type 2 diabetes in a study by Sharma et al. (28) were similar to those observed in this study. These findings appear to suggest that absolute circulating values of this protein can vary considerably, likely due to differences in race, glycemic control status, or the kit used for to measure ANGPTL6.
The circulating ANGPTL6 levels in patients with type 2 diabetes were significantly higher than those in non-diabetic control subjects, consistent with the findings of previous studies (19,21,26,28). Given the fact that ANGPTL6 improves insulin sensitivity by increasing energy expenditure, at least partially, and probably by inhibiting gluconeogenesis in the liver (1,3,16), the difference in circulating ANGPTL6 levels between diabetic and non-diabetic subjects may be due to compensatory mechanisms that counteract hyperglycemia, as proposed previously (3,22).
In this study, ANGPTL6 also showed a significant positive correlation with diabetes duration. This may be due to compensatory mechanisms of ANGPTL6. However, because this association disappeared in the multiple regression analysis, a more detailed examination is needed.
Interestingly, a significant positive correlation was observed between ANGPTL6 levels and the IMT of the carotid artery, which reflects systemic atherosclerosis; this was also evident in the multiple regression analysis that included this variable. To our knowledge, this is the first study to examine this correlation. Although ANGPTL6, as well as angiopoietins and most other ANGPTLs, can induce angiogenesis, it is not yet clear whether ANGPTL6 can directly influence the thickening of the intima or atherosclerosis in large arteries. Thus, it would be interesting to investigate the potential direct effect of ANGPTL6 on arteries, despite the fact that ANGPTL6 remains an orphan ligand (1,2). Furthermore, ANGPTL6 also exhibited a significant positive correlation with SBP, partially supporting a previous report that circulating ANGPTL6 levels were elevated in women who subsequently developed pregnancy-induced hypertension (29). Therefore, the involvement of blood pressure as a confounding factor in the association between ANGPTL6 and IMT should be considered. However, ANGPTL6 also showed a strong association with IMT in a multiple regression analysis in a model that included SBP. Therefore, this association appeared to be independent of hypertension.
A positive association was observed between ANGPTL6 and CRP in our study. In a previous study (19), Ebert et al. observed a similar correlation in patients undergoing chronic hemodialysis but not in healthy subjects. Therefore, ANGPTL6 may be associated with the degree of inflammation, as reflected by CRP, likely only under specific conditions (e.g., chronic hemodialysis or a glycemic state, reflected by elevated HbA1c levels). However, it remains unknown whether ANGPTL6 can be directly related to the increase in CRP levels due to its orphan status. Although its clinical significance is not evident, if ANGPTL6 can directly influence inflammation in large arteries, this may also partially explain the positive association between ANGPTL6 and IMT as described above.
ANGPTL6 also correlated positively with GGT. This may be because ANGPTL6 is mainly expressed in the liver. A similar correlation was reported in patients with nonalcoholic fatty liver disease (27). Taken together, these findings suggest that circulating ANGPTL6 levels may be associated with liver dysfunction.
In the current study, we investigated the correlation between circulating levels of ANGPTL6 and ANGPTL2. While ANGPTL6 improves insulin resistance (1,3,16), ANGPTL2 enhances insulin resistance due to adipose inflammation, at least in part (9-11). Additionally, circulating levels of ANGPTL2 have been associated with new-onset type 2 diabetes (14), while circulating levels of ANGPTL6 have been associated with a low incidence of diabetes (21). Therefore, we expected to observe negative associations between these proteins in circulation. However, contrary to our expectations, no significant correlation was found between ANGPTL6 and ANGPTL2, which was consistent with the results of a previous study in which this correlation was investigated in patients with nonalcoholic fatty liver disease (27). Therefore, our results and those of a previous study suggest that the involvement of ANGPTL6 and ANGPTL2 in insulin resistance is complex and that there is no direct association between circulating levels of these proteins, at least in these patients.
The present study was associated with several limitations. First, this was a cross-sectional observational study and the number of patients was relatively small. Second, there is an important issue attributed to glycemic control among the patients included in this study. In this study, patients who were hospitalized for glycemic control and/or diabetic education for type 2 diabetes were consecutively and prospectively enrolled, as noted in the inclusion criteria, and in these criteria, concrete definitions for the degree of glycemic control (e.g., “well”, “normal”, “poor”) was not decided in advance. We did not estimate the proportion of patients enrolled relative to all diabetic outpatients during the study period. However, we considered that it was likely ＜10% because only a proportion of the patients eligible for enrollment agreed to hospitalization. For the above reason we could not evaluate glycemic control among all patients, we instead investigated the data from our recent study of outpatients with diabetes who were managed in our hospital (30), who likely exhibited glycemic control similar to the patients in this study. In that study, the median HbA1c level was 7.3% (6.6% of the 25th percentile, 8% of the 75th percentile; mean value: 7.4±1.1%), and the proportions of patients with HbA1c levels ＜6%, ＜7%, ≥8%, and ≥9% were 5.8%, 35.5%, 27%, and 7%, respectively, while the levels in patients enrolled in this study were 0%, 1.4%, 85.7%, and 71.4%, respectively, as previously described. Therefore, we speculate that HbA1c levels among the patients enrolled in this study tended to be higher than those among all outpatients during the study period. Thus, the results of this study were only reflected in patients with type 2 diabetes, most of whom exhibited elevated HbA1c levels.
In conclusion, circulating ANGPTL6 levels in patients with type 2 diabetes were significantly higher than those in non-diabetic subjects. ANGPTL6 levels were positively correlated with diabetes duration, SBP, GGT, CRP, and IMT, and negatively correlated with HbA1c. With respect to the association between ANGPTL6 and atherosclerosis, as reflected by IMT, a more detailed analysis in a prospective study is warranted.
The authors state that they have no Conflict of Interest (COI).
Financial Support
This work was supported in part by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) [grant Numbers 19K09018 (Koshi Hashimoto) and 20K11394 (Kenji Hara)].
References
- 1.Santulli G. Angiopoietin-like proteins: a comprehensive look. Front Endocrinol (Lausanne) 5: 4, 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Carbone C, Piro G, Merz V, et al. Angiopoietin-like proteins in angiogenesis, inflammation and cancer. Int J Mol Sci 1: 431, 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Oike Y, Tabata M. Angiopoietin-like proteins - potential therapeutic targets for metabolic syndrome and cardiovascular disease. Circ J 73: 2192-2197, 2019. [DOI] [PubMed] [Google Scholar]
- 4.Oike Y, Yasunaga K, Suda T. Angiopoietin-related/angiopoietin-like proteins regulate angiogenesis. Int J Hematol 80: 21-28, 2014. [DOI] [PubMed] [Google Scholar]
- 5.Kim I, Kwak HJ, Ahn JE, et al. Molecular cloning and characterization of a novel angiopoietin family protein, angiopoietin-3. FEBS Lett 443: 353-356, 1999. [DOI] [PubMed] [Google Scholar]
- 6.Ward NL, Dumont DJ. The angiopoietins and Tie2/Tek: adding to the complexity of cardiovascular development. Semin Cell Dev Biol 13: 19-27, 2002. [DOI] [PubMed] [Google Scholar]
- 7.Abu-Farha M, Abubaker J, Tuomilehto J. ANGPTL8 (betatrophin) role in diabetes and metabolic diseases. Diabetes Metab Res Rev 33: 2017. [DOI] [PubMed] [Google Scholar]
- 8.Takebayashi K, Hara K, Terasawa T, et al. Serum betatrophin levels and clinical features in patients with poorly-controlled Type 2 diabetes. J Clin Med Res 9: 782-787, 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Kadomatsu T, Tabata M, Oike Y. Angiopoietin-like proteins: emerging targets for treatment of obesity and related metabolic diseases. FEBS J 278: 559-664, 2011. [DOI] [PubMed] [Google Scholar]
- 10.Thorin-Trescases N, Thorin E. High circulating levels of ANGPTL2: beyond a clinical marker of systemic inflammation. Oxid Med Cell Longev 2017: 1096385, 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Tabata M, Kadomatsu T, Fukuhara S, et al. Angiopoietin-like protein 2 promotes chronic adipose tissue inflammation and obesity-related systemic insulin resistance. Cell Metab 10: 178-188, 2009. [DOI] [PubMed] [Google Scholar]
- 12.Farhat N, Thorin-Trescases N, Mamarbachi M, et al. Angiopoietin-like 2 promotes atherogenesis in mice. J Am Heart Assoc 10: 2, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Aoi J, Endo M, Kadomatsu T, et al. Angiopoietin-like protein 2 accelerates carcinogenesis by activating chronic inflammation and oxidative stress. Mol Cancer Res 12: 239-249, 2014. [DOI] [PubMed] [Google Scholar]
- 14.Doi Y, Ninomiya T, Hirakawa Y, et al. Angiopoietin-like protein 2 and risk of type 2 diabetes in a general Japanese population: the Hisayama study. Diabetes Care 36: 98-100, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Gellen B, Thorin-Trescases N, Sosner P. ANGPTL2 is associated with an increased risk of cardiovascular events and death in diabetic patients. Diabetologia 59: 2321-2330, 2016. [DOI] [PubMed] [Google Scholar]
- 16.Oike Y, Akao M, Yasunaga K, et al. Angiopoietin-related growth factor antagonizes obesity and insulin resistance. Nat Med 11: 400-408, 2005. [DOI] [PubMed] [Google Scholar]
- 17.Okazaki H, Hirakawa S, Shudou M, et al. Targeted overexpression of Angptl6/angiopoietin-related growth factor in the skin promotes angiogenesis and lymphatic vessel enlargement in response to ultraviolet B. J Dermatol 39: 366-374, 2012. [DOI] [PubMed] [Google Scholar]
- 18.Zhang Y, Hu X, Tian R, et al. Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins. Biochem Biophys Res Commun 347: 100-108, 2006. [DOI] [PubMed] [Google Scholar]
- 19.Ebert T, Bachmann A, Lössner U, et al. Serum levels of angiopoietin-related growth factor in diabetes mellitus and chronic hemodialysis. Metabolism 58: 547-551, 2009. [DOI] [PubMed] [Google Scholar]
- 20.Namkung J, Koh SB, Kong ID, Choi JW, Yeh BI. Serum levels of angiopoietin-related growth factor are increased in metabolic syndrome. Metabolism 60: 564-568, 2011. [DOI] [PubMed] [Google Scholar]
- 21.Fan KC, Wu HT, Wei JN, et al. Serum angiopoietin-like protein 6, risk of type 2 diabetes, and response to hyperglycemia: a prospective cohort study. J Clin Endocrinol Metab 105: dgaa103, 2020. [DOI] [PubMed] [Google Scholar]
- 22.Namkung J, Sohn JH, Chang JS, et al. Increased serum angiopoietin-like 6 ahead of metabolic syndrome in a prospective cohort study. Diabetes Metab J 43: 521-529, 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.UMIN-CTR clinical trial [Internet]. https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000029642.
- 24.Suzuki T, Takebayashi K, Hara K, Tsuchiya T, Inukai T. Association between angiopoietin-like protein 2 and lectin-like oxidized low-density lipoprotein receptor 1 ligand containing apolipoprotein B in patients with type 2 diabetes. J Int Med Res 46: 4167-4180, 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.In: MY BioSource, ANGPTL6 kit (MBS2532115). Product insert. [Google Scholar]
- 26.Qaddoumi MG, Alanbaei M, Hammad MM, et al. Investigating the role of myeloperoxidase and angiopoietin-like protein 6 in obesity and diabetes. Sci Rep 10: 6170, 2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Erkan G, Muratoglu S, Ercin U, Bilgihan A. Angiopoietin-like protein 2 and angiopoietin-like protein 6 levels in patients with nonalcoholic fatty liver disease. Arch Med Sci 14: 781-787, 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Sharma V, Pangtey GS, Gupta R, Rehan HS, Gupta LK. Correlation of long-term glycemic control as measured by glycated hemoglobin with serum angiopoietin-like 6 protein levels in type 2 diabetes mellitus patients. Indian J Pharmacol 49: 250-253, 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Tuuri AL, Jauhiainen MS, Ehnholm CP, Tikkanen MJ, Nicholls MG, Kaaja RJ. Elevated serum angiopoietin-like protein 6 in women with subsequent pregnancy-induced hypertension: a preliminary study. Hypertens Pregnancy 32: 203-213, 2013. [DOI] [PubMed] [Google Scholar]
- 30.Takebayashi K, Yamauchi M, Hara K, Tsuchiya T, Hashimoto K. Seasonal variations and the influence of COVID-19 pandemic on hemoglobin A1c, glycoalbumin, and low-density lipoprotein cholesterol. Diabetol Int 13: 599-605, 2022. [DOI] [PMC free article] [PubMed] [Google Scholar]