The enzymic isolation of adult rat hepatocytes in a functional and viable state

Fry, Jeffrey R.; Jones, Carol; Wiebkin, Philip; Bellemann, Peter; Bridges, James W.

doi:10.1016/s0003-2697(76)80003-6

Cited by 135 publications

(27 citation statements)

References 16 publications

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“…Mouse hepatocytes were obtained as described by others (26). Ca-Green 5N and Fluo-4 were purchased from Molecular Probes (Eugene, OR); cyclosporin H was purchased from Eton Bioscience (San Diego); 4-hydroxynonenal (HNE) was obtained from Cayman Chemical (Ann Arbor, MI); and all other chemicals were obtained from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Limited Mitochondrial Permeabilization Is an Early Manifestation of Palmitate-induced Lipotoxicity in Pancreatic β-Cells

Koshkin

Dai

Doucette

et al. 2008

Journal of Biological Chemistry

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Involvement of the mitochondrial permeability transition (MPT) pore in early stages of lipotoxic stress in the pancreatic ␤-cell lines MIN6 and INS-1 was the focus of this study. Both long term (indirect) and acute (direct) effects of fatty acid (FA) application on ␤-cell susceptibility to Ca 2؉ -induced MPT induction were examined using both permeabilized and intact ␤-cells. Long term exposure to moderate (i.e. below cytotoxic) levels of the saturated FA palmitate sensitized ␤-cell mitochondria to MPT induced by Ca 2؉ . Long term exposure to palmitate was significantly a more efficient inducer of MPT than the unsaturated FA oleate, although upon acute application both caused similar MPT activation. Application of antioxidants, inhibitors of the ceramide pathway, or modifiers of membrane fluidity did not protect ␤-cell mitochondria from FA exposure. However, significant protection was provided by co-application of the unsaturated FA oleate in a phosphatidylinositol 3-kinase-dependent manner. Characterization of MPT pore opening in response to moderate palmitate treatment revealed the opening of a unique form of MPT in ␤-cells as it encompassed features of both low and high conductance MPT states. Specifically, this MPT showed solute selectivity, characteristic of a low conductance MPT; however, it affected mitochondrial respiration and membrane potential in a way typical of a high conductance MPT. Activation of the full-size/high conductance form of MPT required application of high levels of FA that reduced growth and initiated apoptosis. These findings suggest that in the ␤-cell, MPTs can act as both initiators of cell death and as versatile modulators of cell metabolism, depending on the mode of the MPT pore induced. Mitochondrial permeability transition (MPT)2 is the calcium (Ca 2ϩ )-dependent opening of a nonspecific pore in the mitochondrial inner membrane in response to a variety of inducers and co-inducers, including inorganic phosphate (P i ), thiol oxidants, fatty acids (FA), and others. Opening of the "full-size" or "high conductance" MPT causes equilibration across the inner membrane of ions and all solutes up to 1.5 kDa, which induces mitochondrial swelling and the release of mitochondrial proteins capable of apoptosis activation (1-5). MPT can also operate in a more "limited size" or "low conductance" mode, which allows passage of only small ions across the inner mitochondrial membrane (6, 7). Because opening of the low conductance MPT does not allow passage of large molecules, but does allow the passage of small ions, mitochondrial ion gradients and energy production are affected. As such, it is thought that the low conductance MPT functions not to induce cell death but to aid in the fine regulation of cell metabolism (6). Extensive evidence documents the high conductance MPT as a cause of generalized mitochondrial dysfunction and cell death, and an important mechanism in disease pathologies such as hepatotoxicity (8), cardiac ischemia/reperfusion (9, 10), and neuronal injury (10, 11). In contrast,...

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Section: Methodsmentioning

confidence: 99%

Limited Mitochondrial Permeabilization Is an Early Manifestation of Palmitate-induced Lipotoxicity in Pancreatic β-Cells

Koshkin

Dai

Doucette

et al. 2008

Journal of Biological Chemistry

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“…Hepatocytes were isolated by a modified method of collagenase/hyaluronidase (50 and 100 rag, respectively, per 100 ml Hank's solution) digestion of liver slices, which is especially suitable for harvesting a large number of viable cells from small liver pieces, e.g. biopsy samples [8]. The total cell yield and the viability index (percentage of viable cells) were calculated in duplicate with a haemocytometer; viability was based on the ability of cells to exclude the dye, trypan blue [9].…”

Section: Isolation and Cultivation Of Hepatocytesmentioning

confidence: 99%

Effect of insulin on glycogen and protein synthesis in monolayer cultures of hepatocytes from normal and alloxan diabetic rats

et al. 1977

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Summary. The effects of insulin on net glycogen synthesis and amino acid incorporation into protein were studied in cultured hepatocytes from adult normal and alloxan diabetic rats. Insulin stimulated glycogen synthesis in monolayer cells throughout a four day culture period and enhanced leucine incorporation into protein more effectively in normal cells with high glycogen levels than in cultured diabetic cells. These differences correlate well with the observed cellular ultrastructures which were maintained much better in the presence of insulin. Restoration of the morphological changes of alloxan diabetic hepatocytes to normal liver cell structures can be observed at any time during the culture period by giving insulin continuously.Key words: Insulin, glycogen, amino acid, alloxan diabetes, cultured hepatocytes.It has been generally accepted that insulin plays an essential role in the regulation of carbohydrate and protein metabolism in liver tissue. The specific nature of its action at cellular level has been previously investigated in perfused liver [1,2] and in hepatocyte suspensions [3,4], but various attempts to obtain hepatocytes capable of efficient glycogen synthesis during a several day culture period have not been successful. Cultured hepatocytes have, however, become increasingly popular as a valuable system for investigating many liver biochemical runePresent address. Department of Biochemistry, University of Surrey, Gufldford, Surrey GU2 5XH, England 2 Pathologisches Instltut der Universitat Tfibmgen, D-7400 Ttibingen 9 tions in vitro [5,6]. We set out to develop a hepatocyte preparation capable of glycogen synthesis from normal gluconeogenic substrates. It was, therefore, of considerable interest to see if these cultured hepatocytes, especially from diabetic animals could synthesize cellular glycogen in the presence of insulin, and if amino acid incorporation into protein could be stimulated by the hormone even after several days in culture. In the present study we report the stimulatory effect of insulin on hepatic glycogen and protein synthesis and interrelate it with ultrastructural alterations in monolayer cultures of normal and diabetic rat hepatocytes. Materials and Methods Isolation and Cultivation of HepatocytesMale Sprague-Dawley rats weighing 100-150g were used throughout. All rats were kept on a standard laboratory diet (Altromin | and tap water ad libitum. The animals had easy access to food from a dish placed on the floor. This procedure was recently reported to enhance significantly liver glycogen content [7]. Hepatocytes were isolated by a modified method of collagenase/hyaluronidase (50 and 100 rag, respectively, per 100 ml Hank's solution) digestion of liver slices, which is especially suitable for harvesting a large number of viable cells from small liver pieces, e.g. biopsy samples [8]. The total cell yield and the viability index (percentage of viable cells) were calculated in duplicate with a haemocytometer; viability was based on the ability of cells to exclude the dye, tryp...

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“…Transgenic mice and nontransgenic controls were anesthetized with metofane, and hepatocytes were obtained as described (23). Briefly, the inferior vena cava was cannulated, and the portal vein was severed.…”

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confidence: 99%

Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma.

Hagen

Huang

Curnutte

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

283

190

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A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in the liver was used to determine the extent of oxidative DNA damage that occurs during chronic HBV infection. This mouse strain develops a chronic necroinflammatory liver disease that mimics the inflammation, cellular hyperplasia, and increased risk for cancer that is evident in human chronic active hepatitis. When perfused in situ with nitroblue tetrazolium, an indicator for superoxide formation, the liver of transgenic mice displayed intense formazan deposition in Kupffer cells, indicating oxygen radical production, and S-phase hepatocytes were commonly seen adjacent to the stained Kupffer cells. Similar changes were not observed in nontransgenic control livers. To determine whether these events were associated with oxidative DNA damage, genomic DNA from the livers of transgenic mice and nontransgenic controls was isolated and examined for 8-oxo-2'-deoxyguanosine, an oxidatively modified adduct of deoxyguanosine. Results showed a significant, sustained accumulation in steady-state 8-oxo-2'-deoxyguanosine that started early in life exdusively in the transgenic mice and increased progressively with advancing disease. The most pronounced increase occurred in livers exhibiting microscopic nodular hyperplasia, adenomas, and hepatocellular carcinoma. Thus, HBV transgenic mice with chronic active hepatitis display greatly increased hepatic oxidative DNA damage. Moreover, the DNA damage occurs in the presence of heightened hepatocellular proliferation, increasing the probability of fixation of the attendant genetic and chromosomal abnormalities and the development of hepatocellular carcinoma.Chronic inflammatory diseases affect millions of people world-wide and may be a primary factor in the development of up to one-third of all cancers (1). A major association between a persistent infection and cancer is evident in chronic active hepatitis B virus (HBV) infection (2). Epidemiological studies show that incidence of hepatocellular carcinoma (HCC) correlates strongly to geographical areas of endemic HBV infection (3). Furthermore, patients with chronic hepatitis have a much higher incidence of liver cirrhosis and HCC than individuals not infected with HBV (4). Also, HBV antigens in sera of patients are associated with HCC (5). Because the virus infects >300 million people throughout the world, HBV has been described as second only to tobacco as a known human carcinogen (6).Despite the apparent epidemiological association of chronic active hepatitis infection and cancer, the mechanisms involved are still not entirely understood. Research has been hampered due to the long latency period between the onset of infection and cancer development, and the lack of suitable animal models that mimic chronic active human hepatitis.HBV does not contain any known acutely transforming oncogenes, and only rarely do integrated HBV sequences activate cellular protooncogenes (7-10). In contrast, persistent inflammation associated with chro...

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The enzymic isolation of adult rat hepatocytes in a functional and viable state

Cited by 135 publications

References 16 publications

Limited Mitochondrial Permeabilization Is an Early Manifestation of Palmitate-induced Lipotoxicity in Pancreatic β-Cells

Limited Mitochondrial Permeabilization Is an Early Manifestation of Palmitate-induced Lipotoxicity in Pancreatic β-Cells

Effect of insulin on glycogen and protein synthesis in monolayer cultures of hepatocytes from normal and alloxan diabetic rats

Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma.

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