Fish eDNA detection and its technical optimization: A case study of Acanthopagrus latus.

The development of a standardized eDNA detection process is the primary step in improving the accuracy and efficiency of eDNA detection. In this study, primers and probes with high specificity were selected to identify the COI gene of Acanthopagrus latus. Through experiments on the influence of different water quantities, methods of water sample preservation and water bathing times on the result of eDNA detection, the accuracy of this method for extracted water samples was improved. Specifically, a water bathing time of 6 h provided an optimal eDNA concentration from the water sample. After 6 h, the concentration began to decrease, so 6 h was determined to be the best water bathing time for A. latus. Five water extraction volumes (250 mL, 500 mL, 1 L, 2 L, and 3 L) were tested, and there was a positive correlation between water extraction volume and the DNA concentration in the water sample. Different water sample preservation methods were also compared, and it was found that at ≤7 d, the concentration obtained with the cryopreservation method for different water extraction volumes was higher than that obtained with the ethanol preservation method. In this study, we established and optimized a technical procedure for eDNA-based detection of A. latus in aquatic environments. We hope to apply this method in field investigations and provide a reference for the study of eDNA in other fishes.

Intracranial hypertension and empty Sella from adrenal adenoma and excessive and prolonged steroid usage: a case report.

BACKGROUND: There is only one documented case of intracranial hypertension (IH) and empty sella from cortisol-producing adrenal adenoma so far. And IH and empty sella caused by long-term exogenous hypercortisolism has never been reported before. The purpose of this case report is to alert clinicians to glucocorticoid-induced IH. CASE PRESENTATION: We present retrospectively a 50-year-old woman with cortisol-secreting adrenal adenoma, who progressed to intractable intracranial hypertension and a markedly expanded empty sella due to improper treatment. In 2011, the patient presented with hypertension, lack of cortisol circadian rhythm, low ACTH, a left adrenal adenoma and a partial empty sella, but did not receive low-dose dexamethasone suppression test (LDDST) and 24-h urinary cortisol. In 2014, she exhibited truncal obesity, raised cortisol, LDDST non-suppression, high urinary free cortisol and low ACTH, proving her cortisol-producing adrenal adenoma. She was simultaneously diagnosed with unexplained IH because of papilledema and elevated intracranial pressure, and her partial empty sella changed to a complete empty sella. In 2015, she underwent adrenal adenoma resection. From 2015 to 2018, she kept taking dexamethasone at least 2 mg daily without her doctors' consent. During this period, she developed transient cerebrospinal fluid rhinorrhea, and her empty sella further worsened. After switching to low dose hydrocortisone, her papilledema disappeared completely, but optic atrophy has become irreversible. CONCLUSIONS: The patient seems to be just an extreme case, but it may reveal and illustrate a general phenomenon: Both cortisol-producing adrenal adenoma and long-term exogenous hypercortisolism could cause varying degrees of elevated intracranial pressure and empty sella. Clinicians should remain vigilant for this phenomenon in patients with cortisol-producing adrenal adenoma or excessive and prolonged steroid usage and give them corresponding examinations to identify this complication.

Palmitate induces fat accumulation via repressing FoxO1-mediated ATGL-dependent lipolysis in HepG2 hepatocytes.

Obesity is closely associated with non-alcoholic fatty liver disease (NAFLD), and elevated serum palmitate is the link between obesity and excessive hepatic lipid accumulation. Forkhead box O-1 (FoxO1) is one of the FoxO family members of transcription factors and can stimulate adipose triglyceride lipase (ATGL) and suppress its inhibitor G0/G1 switch gene 2 (G0S2) expression in the liver. However, previous researches have also shown conflicting results regarding the role of FoxO1 in hepatic lipid accumulation. We therefore examined the role of FoxO1 as a downstream suppressor to palmitate-stimulated hepatic steatosis. Palmitate significantly promoted lipid accumulation but inhibited lipid decomposition in human HepG2 hepatoma cells. Palmitate also significantly reduced FoxO1, ATGL and its activator comparative gene identification-58 (CGI-58) expression but increased peroxisome proliferator-activated receptorγ (PPARγ) and its target gene G0S2 expression. FoxO1 overexpression significantly increased palmitate-inhibited ATGL and CGI-58 expression but reduced palmitate-stimulated PPARγ and its target gene G0S2 expression. FoxO1 overexpression also inhibited lipid accumulation and promoted lipolysis in palmitate-treated hepatocytes. Overall, these results indicate that FoxO1-mediated ATGL-dependent lipolysis may be an effective molecular mechanism in protecting hepatocytes from palmitate-induced fat accumulation.

The Nuclear Orphan Receptor Nur77 Alleviates Palmitate-induced Fat Accumulation by Down-regulating G0S2 in HepG2 Cells.

Excessive triglyceride accumulation in hepatocytes is the hallmark of obesity-associated nonalcoholic fatty liver disease (NAFLD). Elevated levels of the saturated free fatty acid palmitate in obesity are a major contributor to excessive hepatic lipid accumulation. The nuclear orphan receptor Nur77 is a transcriptional regulator and a lipotoxicity sensor. Using human HepG2 hepatoma cells, this study aimed to investigate the functional role of Nur77 in palmitate-induced hepatic steatosis. The results revealed that palmitate significantly induced lipid accumulation and suppressed lipolysis in hepatocytes. In addition, palmitate significantly suppressed Nur77 expression and stimulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes. Nur77 overexpression significantly reduced palmitate-induced expression of PPARγ and its target genes. Moreover, Nur77 overexpression attenuated lipid accumulation and augmented lipolysis in palmitate-treated hepatocytes. Importantly, G0S2 knockdown significantly attenuated lipid accumulation and augmented lipolysis in palmitate-treated hepatocytes, whereas G0S2 knockdown had no effect on the palmitate-induced expression of Nur77, PPARγ, or PPARγ target genes. In summary, palmitate suppresses Nur77 expression in HepG2 cells, and Nur77 overexpression alleviates palmitate-induced hepatic fat accumulation by down-regulating G0S2. These results display a novel molecular mechanism linking Nur77-regulated G0S2 expression to palmitate-induced hepatic steatosis.

Palmitate induces fat accumulation by activating C/EBPß-mediated G0S2 expression in HepG2 cells.

AIM: To determine the role of G0/G1 switch gene 2 (G0S2) and its transcriptional regulation in palmitate-induced hepatic lipid accumulation. METHODS: HepG2 cells were treated with palmitate, or palmitate in combination with CCAAT/enhancer binding protein (C/EBP)ß siRNA or G0S2 siRNA. The mRNA expression of C/EBPß, peroxisome proliferator-activated receptor (PPAR)γ and PPARγ target genes (G0S2, GPR81, GPR109A and Adipoq) was examined by qPCR. The protein expression of C/EBPß, PPARγ, and G0S2 was determined by Western blotting. Lipid accumulation was detected with Oil Red O staining and quantified by absorbance value of the extracted Oil Red O dye. Lipolysis was evaluated by measuring the amount of glycerol released into the medium. RESULTS: Palmitate caused a dose-dependent increase in lipid accumulation and a dose-dependent decrease in lipolysis in HepG2 cells. In addition, palmitate increased the mRNA expression of C/EBPß, PPARγ, and PPARγ target genes (G0S2, GPR81, GPR109A, and Adipoq) and the protein expression of C/EBPß, PPARγ, and G0S2 in a dose-dependent manner. Knockdown of C/EBPß decreased palmitate-induced PPARγ and its target genes (G0S2, GPR81, GPR109A, and Adipoq) mRNA expression and palmitate-induced PPARγ and G0S2 protein expression in HepG2 cells. Knockdown of C/EBPß also attenuated lipid accumulation and augmented lipolysis in palmitate-treated HepG2 cells. G0S2 knockdown attenuated lipid accumulation and augmented lipolysis, while G0S2 knockdown had no effects on the mRNA expression of C/EBPß, PPARγ, and PPARγ target genes (GPR81, GPR109A and Adipoq) in palmitate-treated HepG2 cells. CONCLUSION: Palmitate can induce lipid accumulation in HepG2 cells by activating C/EBPß-mediated G0S2 expression.

[Pancreatic ß-cell Dysfunction and Apoptosis Induced by Elevated Free Fatty Acids Synergize with Hyperglycemia].

OBJECTIVES: To analysis the effects of glucoxicity and lipotoxicity on the function and apoptosis of pancreatic ß-cells. METHODS: The levels of circulating glucose and free fat acids (FFAs) were elevated by infusion dextrose and fat emulsion in high-fat obese rats. The insulin resistance model obese rats were divided into four gourp: obese group with saline infusion (OB-NS group, n=7), obese group with glucose infusion (OB-GS group, n=9), obese group with Lipid emulsion infusion (OB-FFA group, n=8), obese group with glucose and lipid emulsion infusion (OB-FG group, n=9). Five rats fed with general diet were taken as normal group (NC group).Plasma FFAs and ß-hydroxybutyric acid (ß-HBA) concentrations were determined by an enzymatic colorimetric method. An intravenous glucose tolerance test (IVGTT) was performed to examine the glucose-stimulated insulin secretion in vivo and immunohistochemical staining to detect the storage volume of insulin. FFA and ß-HBA concentrations were measured at baseline and post-infusion. The apoptosis of pancreatic ß-cell was detected byin situ end labeling technique (TUNEL). RESULTS: Glucose infusion rate (GIR) of obese rats was significantly lower than that in NC group [(10.82±1.8) mg/(kg·min) vs. (25.21±1.7) mg/(kg·min), P<0.05], confirming insulin resistance rat model successfully established. The insulin secretion peak load time of OB-FG group rats delayed, and the serum insulin level was significantly lower than that of NC group and OB-NS group during IVGTT. The differences were statistically significant ( P<0.05). Compared with OB-NS and NC groups, storage volume of insulin of OB-GS group reduced, and ß cell apoptosis rate elevated significantly. CONCLUSIONS: Glucolipotoxicity could induce ketone overproduction, insulin resistance and defective insulin secretion.

α-Linolenic acid increases the G0/G1 switch gene 2 mRNA expression in peripheral blood mononuclear cells from obese patients: a pilot study.

BACKGROUND: Recent evidence has demonstrated that the G0/G1 switch gene 2 (G0S2) is an important negative regulator of the rate-limiting lipolytic enzyme adipose triglyceride lipase-mediated lipolysis. It has been revealed that α-linolenic acid (ALA), a plant-based essential omega-3 polyunsaturated fatty acids, reduces adipose tissue lipolysis. However, it is not known whether G0S2 is implicated in ALA-induced inhibition of lipolysis. The purpose of this pilot study is to investigate the effect of ALA on G0S2 gene expression in peripheral blood mononuclear cells (PBMC) of obese patients and the potential influence of G0S2 gene expression in ALA-induced inhibition of lipolysis. METHODS: A total of 26 obese patients were randomly assigned to be treated with or without ALA treatment (~4.0 g daily) for 12 weeks: the ALA-treated group (n = 14) or the untreated control group (n = 12). Plasma triglyceride (TG), free fatty acids (FFA), glycerol, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), as well as the mRNA expression levels of proliferator-activated receptor gamma (PPAR-γ), G0S2, and G protein-coupled receptor 120 (GPR120) in PBMC were repeatedly examined from fasting obese patients before and after ALA treatment. RESULTS: ALA significantly decreased plasma TG, FFA, glycerol, IL-6, and TNF-α levels and increased the mRNA expression levels of PPAR-γ, G0S2, and GPR120 in PBMC, compared with the untreated control group. In obese patients from the ALA-treated group, decreased plasma FFA (a biomarker for lipolysis) level was significantly correlated with increased PPAR-γ (a functional omega-3 fatty acids receptor) and G0S2 (a direct target gene of PPAR-γ) mRNA expression in PBMC, while decreased plasma FFA level was not correlated with increased GPR120 (another functional omega-3 fatty acids receptor) mRNA expression in PBMC. CONCLUSION: This study shows that ALA increases G0S2 gene expression in PBMC in parallel with the decrease of plasma FFA level in obese patients. Increased G0S2 gene expression might contribute to the beneficial anti-lipolytic effect of ALA in obese patients.

[Palmitate enhances toll-like receptor 4 expression and signaling in porcine vascular endothelial cells].

OBJECTIVE: To study the effect of palmitate on toll-like receptor 4 (TLR4) expression and signaling in vascular endothelial cells. METHODS: Pig iliac endothelial cells (PIECs) were incubated with palmitate. TLR4 gene expression levels were measured by quantitative real-time PCR, and TLR4 and IκBα protein expressions by Western blotting. The expression levels of TLR4 protein on the surface of PIECs were quantified using flow cytometry. ELISA was employed to detect tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) concentrations in the cell medium. RESULTS: Palmitate treatment significantly increased TLR4 mRNA and protein expression levels in PIECs compared with those in the control cells (4.73∓0.61 vs 1.25∓0.90, P<0.05; 5.79∓0.05 vs 4.07∓0.31, P<0.05). The expression levels of TLR4 on the cell surface significantly increased (38.070∓3.907 vs 29.390∓1.072, P<0.05), while IκBα protein level was significantly lowered in PIECs after palmitate treatment as compared with those in the control cells (2.04∓0.22 vs 3.98∓0.18, P<0.05). Palmitate treatment significantly elevated TNF-α (2.52∓0.30 vs 1.38∓0.26, P<0.05) and IL-6 (IL-6: 3.28∓0.32 vs 1.44∓0.28, P<0.05) concentrations in the cell culture medium. CONCLUSION: Palmitate can enhance TLR4 expression and signaling in porcine vascular endothelial cells.

[Alpha-linolenic acid improves insulin sensitivity in obese patients].

OBJECTIVE: To explore the effects of α-linolenic acid on insulin sensitivity in obese patients. METHODS: From October 2011 to April 2012, 16 patients received an oral dose of α-linolenic acid for 8 weeks.Oral glucose tolerance test (OGTT) and insulin releasing test were performed before and after treatment. Homeostasis model assessment insulin resistance (HOMA-IR) index and area under curve of insulin (AUCI) were calculated to evaluate the insulin sensitivity. The levels of serum triglyceride, free fatty acids (FFA), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured after an overnight fast. RESULTS: Obese patients had significantly elevated serum insulin, triglyceride, FFA, IL-6 and TNF-α versus the subjects in normal control group (all P < 0.05). Obese patients were also more insulin-resistant than normal subjects based on a higher HOMA-IR (P < 0.05). Decreased serum insulin, triglyceride, FFA, IL-6 and TNF-α were observed after treatment. With the administration of α-linolenic acid, HOMA-IR and AUCI significantly decreased in obese patients (HOMA-IR: 1.8 ± 0.2 vs 1.2 ± 0.3, P < 0.05; AUCI: 1151 ± 505 vs 768 ± 347, P < 0.05). CONCLUSION: α-linolenic acid increases peripheral insulin sensitivity in obese patients and it may aid the prevention and treatment of type 2 diabetes mellitus and atherosclerotic vascular diseases.

[Role of apoptosis and mitochondrial apoptotic pathway in glucolipotoxicity-induced islet beta-cell dysfunction].

OBJECTIVE: To investigate the mechanism of beta-cell dysfunction induced by glucolipotoxicity in high fat-fed obese rats. METHODS: Eighteen high-fat obese male Wistar rats were assigned into 3 groups and underwent 48-hour infusion through the jugular vein with normal saline (n=6), 20% intralipid + heparin (FFA group, n=6), or 25%glucose +20% intralipid + heparin (GS-FFA group, n=6). The plasma beta-hydroxybutyric acid (beta-HBA) was measured before and at the end of the infusion. After the infusion, the rats were sacrificed following an intravenous glucose tolerance test (IVGTT) to remove the tail of the pancreas for detection of apoptotic islet cells using TUNEL method. Immunohistochemical staining was performed to detect the expression of cytochrome c (cyt c), apoptosis-inducing factor (AIF), caspase-9 and caspase-3 in the islet cells. RESULTS: At the end of the infusion, all the rats exhibited increased plasma beta-HBA levels, which was the highest in the GS-FFA group (P<0.05). IVGTT performed after the infusion showed a significantly lower insulinogenic index in GS-FFA group than that in NS and FFA groups. Greater number of apoptotic islet cells was found in the GS-FFA group than in the FFA and NS groups (P<0.05), and the islets had significantly higher levels of cyt c, AIF, caspase-9 and caspase-3 in the former group than in the latter two groups (P<0.05). CONCLUSIONS: Hyperglycemia and high free fatty acid level synergistically impair insulin secretions to cause ketone overproduction in high fat-fed obese rats. The beta-cell dysfunction due to glucolipotoxicity is associated with increased beta-cell apoptosis and activation of mitochondrial apoptotic pathway.