An updated method for the isolation and culture of primary calf hepatocytes

An elevated plasma non-esterified fatty acids (NEFA) concentration is an important indicator of negative energy balance, which is often associated with metabolic diseases such as fatty liver and ketosis. Angiopoietin-like protein 4 (ANGPTL4) is a hepatokine that plays important roles in the regulation of lipid metabolism. However, the direct effects of high concentrations of NEFA on ANGPTL4 expression and secretion in hepatocytes from cows are not entirely clear. The objective of this study was to investigate the effects of different NEFA concentrations on ANGPTL4 expression and secretion in calf hepatocytes cultured in vitro. NEFA was added to the culture solution at final concentrations of 0.6, 1.2, 1.8, and 2.4 mmol/L. After 24 h of continuous culture, ANGPTL4 mRNA and protein expression levels in the hepatocytes were determined by real-time polymerase chain reaction and western blot, respectively. ANGPTL4 secretion in the supernatant was determined by enzyme-linked immunosorbent assay. The results showed similar levels of ANGPTL4 expression and secretion following treatment with 0.6 and 1.2 mmol/L NEFA, which were higher than those observed for the controls (P < 0.05). ANGPTL4 expression and secretion were also similar at 1.8 and 2.4 mmol/L NEFA and significantly higher than those observed for controls (P < 0.01). These data suggest that high concentrations of NEFA significantly promote ANGPTL4 expression and secretion in bovine hepatocytes. In particular, this promotion occurs in a dose-dependent manner and may be involved in the pathological processes of energy metabolism disorders of dairy cows in the peripartum period.

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“…Hepatocytes were cultured using the collagenase perfusion method. Cell culture methods were performed as described by Zhang et al [15].…”

Section: Methodsmentioning

confidence: 99%

Non-esterified fatty acids promote expression and secretion of angiopoietin-like protein 4 in calf hepatocytes cultured in vitro

et al. 2014

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“…Hepatocytes were cultured using the collagenase perfusion method described previously [8,9]. Briefly, the liver (caudate lobe) was obtained under sterile conditions from a Holstein newborn calf anesthetised with thiamylal sodium.…”

Section: Methodsmentioning

confidence: 99%

Effect of Non-Esterified Fatty Acids on Fatty Acid Metabolism-Related Genes in Calf Hepatocytes Culturedin Vitro

Liu

Zhang

Long

et al. 2013

Cell Physiol Biochem

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Background: NEFA plays numerous roles in the metabolism of glucose, lipids, and proteins. A number of experimental studies have shown that NEFA may have an important role in fatty acid metabolism in the liver, especially in dairy cows that experience negative energy balance (NEB) during early lactation. Methods: In this study, using fluorescent quantitative RT-PCR, ELISA, and primary hepatocytes cultured in vitro, we examined the effect of NEFA (0, 0.2, 0.4, 0.8, 1.6, and 3.2 mmol/L) on fatty acid metabolism by monitoring the mRNA and protein expression of the following key enzymes: long chain acyl-CoA synthetase (ACSL), carnitine palmitoyltransferase IA (CPT IA), long chain acyl-CoA dehydrogenase (ACADL), and acetyl-CoA carboxylase (ACC). Results: The mRNA and protein expression levels of ACSL and ACADL markedly increased as the concentration of NEFA in the media was increased. The mRNA and protein expression levels of CPT IA were enhanced significantly when the NEFA concentrations increased from 0 to 1.6 mmol/L and decreased significantly when the NEFA concentrations increased from 1.6 to 3.2 mmol/L. The mRNA and protein expression of ACC decreased gradually with increasing concentrations of NEFA. Conclusion: These findings indicate that increased NEFA significantly promote the activation and β-oxidation of fatty acids, but very high NEFA concentrations may inhibit the translocation of fatty acids into mitochondria of hepatocytes. This may explain the development of ketosis or liver lipidosis in dairy cows. CPT IA might be the key control enzyme of the fatty acid oxidation process in hepatocytes.

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“…Calf (one-day-old, female, 40 kg, health, fasting) primary hepatocytes were isolated and cultured as previously described [22,23]. Briefly, a scalpel was used to obtain the caudate lobe of the liver from a Holstein calf that was anesthetized with thiamylal sodium under sterile conditions.…”

Section: Isolation Of Cow Primary Hepatocytesmentioning

confidence: 99%

“…Briefly, a scalpel was used to obtain the caudate lobe of the liver from a Holstein calf that was anesthetized with thiamylal sodium under sterile conditions. The preparation methods of perfusion solutions A, B and C were according to the study of Zhang et al [22]. The liver was then quickly placed on a sterile bench, and bloodstains on the surface were removed with perfusion solution A, revealing the blood vessel on the cross section of the caudate process.…”

Section: Isolation Of Cow Primary Hepatocytesmentioning

confidence: 99%

NEFAs activate the oxidative stress-mediated NF-κB signaling pathway to induce inflammatory response in calf hepatocytes

Shi

Deng

et al. 2015

The Journal of Steroid Biochemistry and Molecular Biology

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An updated method for the isolation and culture of primary calf hepatocytes

Cited by 29 publications

References 26 publications

Non-esterified fatty acids promote expression and secretion of angiopoietin-like protein 4 in calf hepatocytes cultured in vitro

Non-esterified fatty acids promote expression and secretion of angiopoietin-like protein 4 in calf hepatocytes cultured in vitro

Effect of Non-Esterified Fatty Acids on Fatty Acid Metabolism-Related Genes in Calf Hepatocytes Culturedin Vitro

NEFAs activate the oxidative stress-mediated NF-κB signaling pathway to induce inflammatory response in calf hepatocytes

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