Circulating Angiopoietin-like Protein 6 Levels and Clinical Features in Patients with Type 2 Diabetes.

Aims 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 (CLIA). Results Most patients had elevated HbA1c levels; 85.7% and 71.4% of patients had HbA1c levels of (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 (SBP), gamma-glutamyl transpeptidase (GGT), C-reactive protein (CRP), and the intimal medial complex thickness of the carotid artery (IMT), and inversely correlated with hemoglobin A1c (HbA1c). In the multiple regression analysis, ANGPTL6 had a significant positive association with triglyceride (TG) 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.

Angiopoietin-like growth factor-derived peptides as biological activators of adipose-derived mesenchymal stromal cells.

Adipose-derived mesenchymal stromal cells (AD-MSCs) are an essential issue in modern medicine. Extensive preclinical and clinical studies have shown that mesenchymal stromal/stem cells, including AD-MSCs, have specific properties (ability to differentiate into other cells, recruitment to the site of injury) of particular importance in the regenerative process. Ongoing research aims to elucidate factors supporting AD-MSC culture and differentiation in vitro. Angiopoietin-like proteins (ANGPTLs), known for their pleiotropic effects in lipid and glucose metabolism, may play a significant role in this context. Regeneration is a complex and dynamic process controlled by many factors. ANGPTL6 (Angiopoietin-related growth factor, AGF), among many activities modulated the biological activity of stem cells. This study examined the influence of synthesized AGF-derived peptides, designated as AGF9 and AGF27, on AD-MSCs. AGF9 and AGF27 enhanced the viability and migration of AD-MSCs and acted as a chemotactic factor for these cells. AGF9 stimulated chondrogenesis and lipid synthesis during AD-MSCs differentiation, influenced AD-MSCs cytokine secretion and modulated transcriptome for such basic cell activities as migration, transport of molecules, and apoptosis. The ability of AGF9 to modulate the biological activity of AD-MSCs warrants the consideration of this peptide a noteworthy therapeutic agent that deserves further investigation for applications in regenerative medicine.

Screening for Lipid-Metabolism-Related Genes and Identifying the Diagnostic Potential of ANGPTL6 for HBV-Related Early-Stage Hepatocellular Carcinoma.

Lipid metabolic reprogramming is one of the hallmarks of hepatocarcinogenesis and development. Therefore, lipid-metabolism-related genes may be used as potential biomarkers for hepatocellular carcinoma (HCC). This study aimed to screen for genes with dysregulated expression related to lipid metabolism in HCC and explored the clinical value of these genes. We screened differentially expressed proteins between tumorous and adjacent nontumorous tissues of hepatitis B virus (HBV)-related HCC patients using a Nanoscale Liquid Chromatography-Tandem Mass Spectrometry platform and combined it with transcriptomic data of lipid-metabolism-related genes from the GEO and HPA databases to identify dysregulated genes that may be involved in lipid metabolic processes. The potential clinical values of these genes were explored by bioinformatics online analysis tools (GEPIA, cBioPortal, SurvivalMeth, and TIMER). The expression levels of the secreted protein (angiopoietin-like protein 6, ANGPTL6) in serum were analyzed by ELISA. The ability of serum ANGPTL6 to diagnose early HCC was assessed by ROC curves. The results showed that serum ANGPTL6 could effectively differentiate between HBV-related early HCC patients with normal serum alpha-fetoprotein (AFP) levels and the noncancer group (healthy participants and chronic hepatitis B patients) (AUC = 0.717, 95% CI: from 0.614 to 0.805). Serum ANGPTL6 can be used as a potential second-line biomarker to supplement serum AFP in the early diagnosis of HBV-related HCC.

Energy Homeostasis Gene Nucleotide Variants and Survival of Hemodialysis Patients-A Genetic Cohort Study.

BACKGROUND: Patients undergoing hemodialysis (HD) therapy have an increased risk of death compared to the general population. We investigated whether selected single nucleotide variants (SNVs) involved in glucose and lipid metabolism are associated with mortality risk in HD patients. METHODS: The study included 805 HD patients tested for 11 SNVs in FOXO3, IGFBP3, FABP1, PCSK9, ANGPTL6, and DOCK6 using HRM analysis and TaqMan assays. FOXO3, IGFBP3, L-FABP, PCSK9, ANGPTL6, and ANGPTL8 plasma concentrations were measured by ELISA in 86 individuals. The Kaplan-Meier method and Cox proportional hazards models were used for survival analyses. RESULTS: We found out that the carriers of a C allele in ANGPTL6 rs8112063 had an increased risk of all-cause, cardiovascular, and cardiac mortality. In addition, the C allele of DOCK6 rs737337 was associated with all-cause and cardiac mortality. The G allele of DOCK6 rs17699089 was correlated with the mortality risk of patients initiating HD therapy. The T allele of FOXO3 rs4946936 was negatively associated with cardiac and cardiovascular mortality in HD patients. We observed no association between the tested proteins' circulating levels and the survival of HD patients. CONCLUSIONS: The ANGPTL6 rs8112063, FOXO3 rs4946936, DOCK6 rs737337, and rs17699089 nucleotide variants are predictors of survival in patients undergoing HD.

Relationship of ANGPTL6 With Neonatal Glucose Homeostasis and Fat Mass Is Disrupted in Gestational Diabetic Pregnancies.

CONTEXT: Angiopoietin-like protein 6 (ANGPTL6) is a hepatokine, which, in animal studies, improves insulin sensitivity and increases energy expenditure to counteract insulin resistance. OBJECTIVE: Evaluate in a human population, the role of serum ANGPTL6 in gestational diabetes mellitus (GDM) or its presence in fetal circulation. RESEARCH DESIGN AND METHODS: A total of 190 women (115 controls and 75 GDM) and their offspring were studied. Insulin, glucose, ANGPTL6, retinol binding protein 4 (RBP4), and retinol, as well as leptin and adiponectin, were determined in maternal serum obtained at term and from umbilical artery blood at delivery. RESULTS: At term, pregnant women with GDM showed higher serum concentrations of ANGPTL6, insulin, homeostatic model assessment, and apo-RBP4 (free RBP4) than controls but not of glucose, which remained similar in both groups. Also, in arterial cord serum, ANGPTL6 concentration was increased in GDM neonates with respect to the control group (201 ±â€…12 ng/mL vs 119 ±â€…8 ng/mL, respectively). No effect of maternal insulin treatment of some GDM mothers in neonates of either sex on ANGPTL6 levels was observed. In GDM, circulating ANGPTL6 showed no correlation with glucose or insulin concentration or with neonatal adiposity. However, in control pregnancies, the variation in glucose concentration was positively correlated with ANGPTL6 concentration, both in maternal and in cord samples, and cord ANGPTL6 was negatively correlated with neonatal fat mass. Furthermore, in control pregnant women, serum concentrations of ANGPTL6 and apo-RBP4 were negatively correlated. CONCLUSION: Serum ANGPTL6 levels are associated with maternal glucose homeostasis and fetal adiposity in normal pregnancy. ANGPTL6 levels in maternal and cord serum GDM pregnancy at term are increased, although its mechanism and physiological role are unknown yet.

Use of proteomics to identify mechanisms of hepatocellular carcinoma with the CYP2D6*10 polymorphism and identification of ANGPTL6 as a new diagnostic and prognostic biomarker.

BACKGROUND: Although an association between the cytochrome P4502D6 (CYP2D6) *10 (100C>T) polymorphism and hepatocellular carcinoma (HCC) is known, the mechanism remains unclear. Here we aimed to explore mechanisms of CYP2D6*10 (100C>T) polymorphism conferring to HCC, and screen markers for HCC. METHODS: Label-free global proteome profiling with 34 normal livers and peritumor tissue from 61 HCC patients was performed, and angiopoietin-like protein-6 (ANGPTL6) was evaluated in 2 liver samples validation cohorts and 2 blood specimens validation cohorts. RESULTS: We found a significantly decreased frequency of TT in HCC patients which reduced HCC susceptibility by 69.2% and was accompanied by lowered enzymatic activity for CYP2D6. Proteomic analysis revealed 1342 differentially expressed proteins (DEPs) that were associated with HCC and 88 DEPs were identified as 100 TT-related proteins, likely underlying the susceptibility to HCC. Twenty-two upregulated DEPs and 66 downregulated DEPs were mainly related to lipid metabolism and the extracellular matrix, respectively. High ANGPTL6 was associated with a higher risk to HCC and worse prognosis. ANGPTL6 was both an independent risk factor and an independent prognostic factor for HCC and exhibited strong potential for predicting HCC occurrence, with comparable AUC values and higher sensitivity compared with alpha-fetoprotein. CONCLUSIONS: The TT genotype-associated decreased risk of HCC appears to be related to lowered CYP2D6 activity and altered protein expression in the tumor microenvironment, and ANGPTL6 is a promising new diagnostic and prognostic biomarker for HCC. Our findings reveal new mechanistic insights for polymorphisms related to HCC risk and provide avenues for screening for HCC.

Weight-loss-independent benefits of exercise on liver steatosis and stiffness in Japanese men with NAFLD.

BACKGROUND & AIMS: A weight-loss-independent beneficial effect of exercise on non-alcoholic fatty liver disease (NAFLD) management has been reported, but the underlying mechanism is unknown. To help determine this mechanism, the effects of exercise on individual tissues (liver, adipose tissue, and skeletal muscle) were retrospectively studied. METHODS: Data from Japanese obese men with NAFLD in a 3-month exercise regimen were analysed and compared with those in a 3-month dietary restriction program designed to achieve weight loss. The underlying mechanism was studied in a smaller subcohort. RESULTS: Independent of the effect of weight loss, the exercise regimen reduced liver steatosis by 9.5% and liver stiffness by 6.8% per 1% weight loss, and resulted in a 16.4% reduction in FibroScan-AST score. Improvements in these hepatic parameters were closely associated with anthropometric changes (reduction in adipose tissue and preservation of muscle mass), increases in muscle strength (+11.6%), reductions in inflammation and oxidative stress (ferritin: -22.3% and thiobarbituric acid: -12.3%), and changes in organokine concentrations (selenoprotein-P: -11.2%, follistatin: +17.1%, adiponectin: +8.9%, and myostatin: -21.6%) during the exercise regimen. Moreover, the expression of target genes of the transcription factor Nrf2, an oxidative stress sensor, was higher in monocytes, suggesting that Nrf2 is activated. Large amounts of high-intensity exercise were effective at further reducing liver steatosis and potentiating improvements in pathophysiological parameters (liver enzyme activities and organokine profiles). CONCLUSIONS: The weight-loss-independent benefits of exercise include anti-steatotic and anti-stiffness effects in the livers of patients with NAFLD. These benefits seem to be acquired through the modification of inter-organ crosstalk, which is characterised by improvements in organokine imbalance and reductions in inflammation and oxidative stress. LAY SUMMARY: We investigated the effects of exercise on non-alcoholic fatty liver disease (NAFLD) that were not related to weight loss. We found that exercise had considerable weight-loss-independent benefits for the liver through a number of mechanisms. This suggests that exercise is important for NAFLD patients, regardless of whether they lose weight.

The association between dietary antioxidants and adipokines level among obese women.

AIM: Adipokines are associated with several oxidative stress-related diseases and pathologic conditions. We aimed to assess the association between antioxidants and adipokines in obese adults. METHODS AND MATERIALS: In this cross-sectional study, a total of 160 obese women were included. Body composition and anthropometric characteristics were measured. Dietary intakes were assessed by 3-day, 24-h dietary recall. Blood samples were obtained following an overnight fasting. Serum concentrations of adipokines including progranulin, retinol binding protein 4 (RBP4) and Angiopoietin-related growth factor 6 (ANGPTL6) was measured using an enzyme-linked immunosorbent assay. ANCOVA and the linear regression model analysis was performed to assess the relationship between Progranulin, RBP4, AnGPTL6, and antioxidants. RESULTS: Mean age of included women was 39.31 ±â€¯12.10. Mean and standard deviation for BMI was 35.05 ±â€¯4.26 in this obese population. There was a positive significant association between ANGPTL6 and vitamin D intake (p < 0.001). Also, there was a marginal association between RBP4 and vitamin A (p = 0.063) intake, but after adjustment age, and fat mass, we found a significant association (p = 0.008). However, the associations between dietary antioxidants, progranulin, and ANGPTL6 were not statistically significant. CONCLUSIONS: ANGPTL6 and RBP4 levels directly associated with dietary vitamins D and A intake, respectively. But, according to the results, the association between ANGPTL6 and vitamin D was bidirectional. The suggested associations probably can be useful in the development of interventional studies for management of chronic diseases.

ANGPTL6-mediated angiogenesis promotes alpha fetoprotein-producing gastric cancer progression.

Alpha-fetoprotein (AFP)-producing gastric cancer (AFPGC) is regarded as a rare but highly malignant gastric adenocarcinoma subtype and its clinic pathological presentation mimics hepatocellular carcinoma. However, the underlying mechanism of this disease remains elusive. The level of ANGPTL6 in AFPGC cell lines is much higher than that of common types of gastric cancer cells. A high level of ANGPTL6 confers a poor prognosis and is correlated with the expression of CD34 (an endothelial cell marker). ANGPTL6 promotes endothelial cell migration and tube formation, Moreover, ANGPTL6 knockdown inhibits cancer cell apoptosis and invasiveness. Mechanistically, ANGPTL6 activates the ERK1/2 and AKT pathways. Treatment of ERK1/2 or AKT inhibitor can attenuated cell migration and tube formation. ANGPTL6 loss results in tumor growth in vivo. Our study revealed that ANGPTL6 is an important driver gene of angiogenesis in AFPGC development. These findings provide not only an effective biomarker for diagnosis but also an attractive therapeutic target for use in AFPGC patients.

Increased Serum Angiopoietin-Like 6 Ahead of Metabolic Syndrome in a Prospective Cohort Study.

BACKGROUND: Despite being an anti-obesity hepatokine, the levels of serum angiopoietin-like 6 (ANGPTL6) are elevated in various metabolic diseases. Thus, ANGPTL6 expression may reflect metabolic burden and may have compensatory roles. This study investigated the association between serum ANGPTL6 levels and new-onset metabolic syndrome. METHODS: In total, 221 participants without metabolic syndrome were randomly selected from a rural cohort in Korea. Baseline serum ANGPTL6 levels were measured using an enzyme-linked immunosorbent assay. Anthropometric and biochemical markers were analyzed before and after follow-up examinations. RESULTS: During an average follow-up period of 2.75 (interquartile range, 0.76) years, 82 participants (37.1%) presented new-onset metabolic syndrome and had higher ANGPTL6 levels before onset than those without metabolic syndrome (48.03±18.84 ng/mL vs. 64.75±43.35 ng/mL, P=0.001). In the multivariable adjusted models, the odds ratio for the development of metabolic syndrome in the highest quartile of ANGPTL6 levels was 3.61 (95% confidence interval, 1.27 to 10.26). The use of ANGPTL6 levels in addition to the conventional components improved the prediction of new-onset metabolic syndrome (area under the receiver operating characteristic curve: 0.775 vs. 0.807, P=0.036). CONCLUSION: Increased serum ANGPTL6 levels precede the development of metabolic syndrome and its components, including low high density lipoprotein, high triglyceride, and high glucose levels, which have an independent predictive value for metabolic syndrome.

Leptin regulates the expression of angiopoietin-like 6.

Angiopoietin-like 6 (ANGPTL6) is a hepatokine that antagonizes obesity and insulin resistance by increasing energy expenditure. Despite its beneficial effects on metabolism, human studies have shown a paradoxical increase in ANGPTL6 level in the serum of patients with metabolic diseases, which has been interpreted as a compensatory upregulation. However, the regulatory mechanism of ANGPTL6 remains unclear. Since upregulation of ANGPTL6 is induced on metabolic stress, we investigated the hepatic expression of ANGPTL6 by leptin, a representative adipokine of obesity. Mice on a high-fat diet showed increased serum leptin levels and hepatic Angptl6 expression, which were attenuated by exercise training. A single leptin injection also induced hepatic ANGPTL6 expression and increased serum ANGPTL6 levels. In an in vitro model using primary hepatocytes, leptin treatment significantly upregulated ANGPTL6 expression at the mRNA and protein levels, as well as the amount of secreted ANGPTL6 protein in conditioned media. Similarly, exercise training on human participants also showed diminished serum levels of leptin and ANGPTL6. Altogether, these results strongly indicated that hepatic ANGPTL6 expression was determined by leptin.

Rare Coding Variants in ANGPTL6 Are Associated with Familial Forms of Intracranial Aneurysm.

Intracranial aneurysms (IAs) are acquired cerebrovascular abnormalities characterized by localized dilation and wall thinning in intracranial arteries, possibly leading to subarachnoid hemorrhage and severe outcome in case of rupture. Here, we identified one rare nonsense variant (c.1378A>T) in the last exon of ANGPTL6 (Angiopoietin-Like 6)-which encodes a circulating pro-angiogenic factor mainly secreted from the liver-shared by the four tested affected members of a large pedigree with multiple IA-affected case subjects. We showed a 50% reduction of ANGPTL6 serum concentration in individuals heterozygous for the c.1378A>T allele (p.Lys460Ter) compared to relatives homozygous for the normal allele, probably due to the non-secretion of the truncated protein produced by the c.1378A>T transcripts. Sequencing ANGPTL6 in a series of 94 additional index case subjects with familial IA identified three other rare coding variants in five case subjects. Overall, we detected a significant enrichment (p = 0.023) in rare coding variants within this gene among the 95 index case subjects with familial IA, compared to a reference population of 404 individuals with French ancestry. Among the 6 recruited families, 12 out of 13 (92%) individuals carrying IA also carry such variants in ANGPTL6, versus 15 out of 41 (37%) unaffected ones. We observed a higher rate of individuals with a history of high blood pressure among affected versus healthy individuals carrying ANGPTL6 variants, suggesting that ANGPTL6 could trigger cerebrovascular lesions when combined with other risk factors such as hypertension. Altogether, our results indicate that rare coding variants in ANGPTL6 are causally related to familial forms of IA.

Up-regulation of selenoprotein P and HIP/PAP mRNAs in hepatocytes by intermittent hypoxia via down-regulation of miR-203.

Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human hepatocytes (JHH5, JHH7, and HepG2) to experimental IH or normoxia for 24 h, measured mRNA levels by real-time reverse transcription polymerase chain reaction (RT-PCR), and found that IH significantly increased the mRNA levels of selenoprotein P (SELENOP) - a hepatokine - and hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) - one of REG (Regenerating gene) family. We next investigated promoter activities of both genes and discovered that they were not increased by IH. On the other hand, a target mRNA search of micro RNA (miRNA) revealed that both mRNAs have a potential target sequence for miR-203. The miR-203 level of IH-treated cells was significantly lower than that of normoxia-treated cells. Thus, we introduced miR-203 inhibitor and a non-specific control RNA (miR-203 inhibitor NC) into HepG2 cells and measured the mRNA levels of SELENOP and HIP/PAP. The IH-induced expression of SELENOP and HIP/PAP was abolished by the introduction of miR-203 inhibitor but not by miR-203 inhibitor NC. These results demonstrate that IH stress up-regulates the levels of SELENOP in human hepatocytes to accelerate insulin resistance and up-regulates the levels of HIP/PAP mRNAs to proliferate such hepatocytes, via the miR-203 mediated mechanism.

ANGPTL6 expression is coupled with mitochondrial OXPHOS function to regulate adipose FGF21.

Recent studies revealed that the inhibition of mitochondrial oxidative phosphorylation (OXPHOS) is coupled with the mitochondrial unfolded protein response, thereby stimulating the secretion of non-cell autonomous factors, which may control systemic energy metabolism and longevity. However, the nature and roles of non-cell autonomous factors induced in adipose tissue in response to reduced OXPHOS function remain to be clarified in mammals. CR6-interacting factor 1 (CRIF1) is an essential mitoribosomal protein for the intramitochondrial production of mtDNA-encoded OXPHOS subunits. Deficiency of CRIF1 impairs the proper formation of the OXPHOS complex, resulting in reduced function. To determine which secretory factors are induced in response to reduced mitochondrial OXPHOS function, we analyzed gene expression datasets in Crif1-depleted mouse embryonic fibroblasts. Crif1 deficiency preferentially increased the expression of angiopoietin-like 6 (Angptl6) and did not affect other members of the ANGPTL family. Moreover, treatment with mitochondrial OXPHOS inhibitors increased the expression of Angptl6 in cultured adipocytes. To confirm Angptl6 induction in vivo, we generated a murine model of reduced mitochondrial OXPHOS function using adipose tissue-specific Crif1-deficient mice and verified the upregulation of Angptl6 and fibroblast growth factor 21 (Fgf21) in white adipose tissue. Treatment with recombinant ANGPTL6 protein increased oxygen consumption and Pparα expression through the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in cultured adipocytes. Furthermore, the ANGPTL6-mediated increase in Pparα expression resulted in increased FGF21 expression, thereby promoting ß-oxidation. In conclusion, mitochondrial OXPHOS function governs the expression of ANGPTL6, which is an essential factor for FGF21 production in adipose tissue and cultured adipocytes.

Angiopoietin-like protein 6 in patients with obesity, type 2 diabetes mellitus, and anorexia nervosa: The influence of very low-calorie diet, bariatric surgery, and partial realimentation.

AIM OF THE STUDY: Angiopoietin-like protein 6 (ANGPTL6) is a circulating protein with a potential role in energy homeostasis. The aim of the study was to explore the changes in ANGPTL6 levels in patients with obesity (Body mass index, BMI > 40 kg/m2) with and without type 2 diabetes mellitus (T2DM) undergoing dietary intervention (very low calorie diet - VLCD) and in a subgroup of T2DM patients after bariatric surgery. Additionally, we examined changes in ANGPTL6 in anorexia nervosa (AN) patients at baseline and after partial realimentation. We also explored the changes in ANGPTL6 mRNA expression in subcutaneous adipose tissue (SAT) of obese subjects. MATERIALS AND METHODS: The study included 23 non-diabetic obese patients, 40 obese patients with T2DM (27 underwent VLCD and 13 underwent bariatric surgery), 22 patients with AN, and 37 healthy control subjects. RESULTS: ANGPTL6 levels of AN patients were increased relative to the control group (68.6 ± 9.9 ng/ml) and decreased from 110.2 ± 13.3 to 73.6 ± 7.1 ng/ml (p = 0.004) after partial realimentation. Baseline ANGPTL6 levels in patients with obesity and T2DM did not differ from the control group. VLCD decreased ANGPTL6 levels only in obese patients with T2DM. Bariatric surgery induced a transient elevation of ANGPTL6 levels with a subsequent decrease to baseline levels. ANGPTL6 mRNA expression transiently increased after bariatric surgery and returned to baseline levels after 12 months. CONCLUSIONS: Collectively, our data suggest that serum ANGPTL6 levels and ANGPTL6 mRNA expression in SAT are affected by metabolic disorders and their treatment but do not appear to directly reflect nutritional status.