Drug-induced and postnatal hypothyroidism impairs the accumulation of diacylglycerol in liver and liver cell plasma membranes
© Krasilnikova et al; licensee BioMed Central Ltd. 2002
Received: 10 June 2002
Accepted: 16 August 2002
Published: 16 August 2002
Thyroid hormones are well known modulators of signal transduction. The effect of hyper- and hypo-thyroidism on diacylglycerol/protein kinase C (DAG/PKC) signaling in cardiomiocytes has been determined. Triiodothyronine (T3) has been shown to prevent the α1-adrenoreceptor-mediated activation of PKC but does not alter the stimulation of enzyme and hepatic metabolism by phorbol ethers. It has been suggested that the elevation of endogenous DAG in senescent or hypothyroid cells changes the PKC-dependent response of cells to phorbol esters and hormones. In the present study, was examined the formation of DAG and activation of PKC in liver cells from rats of different thyroid status.
The results obtained provide the first demonstration of DAG accumulation in liver and cell plasma membranes at age- and drug-dependent thyroid gland malfunction. The experiments were performed in either the [14C]CH3COOH-labeled rat liver, liver slices or hepatocytes labeled by [14C] oleic acid and [3H]arachidonic acid or [14C]palmitic acid as well as in the isolated liver cell plasma membranes of 90- and 720-day-old rats of different thyroid status. The decrease of T4 and T3 levels in blood serum of 720-day-old rats and mercazolil-treated animals was associated with increases of both the DAG mass in liver and liver cell plasma membranes and newly synthesized [14C]DAG level in liver and isolated hepatocytes. Hypothyroidism decreased PKC activity in both membrane and cytosol as well as phospholipid and triacylglycerol synthesis in liver. These hypothyroidism effects were restored in liver by injection of T4. T4 administration to the intact animals of different ages decreased the DAG level in liver and isolated plasma membranes and the content of newly synthesized DAG in liver. The reduction of DAG level in liver was not associated with increasing free fatty acid level. DAG labeling ratio 14C/3H in liver slices of rats of different thyroid state sharply differed from PL. DAG was relatively enriched in [14C]oleic acid whereas PL were enriched in [3H]arachidonic acid.
The above data have indicated that thyroid hormones are important physiological modulators of DAG level in rat liver and cell plasma membranes. Age- and drug-induced malfunction of thyroid gland resulted in a prominent decrease of glycerolipid synthesis which may promote DAG accumulation in liver.
sn-1,2-Diacylglycerol plays a key role in lipid biosynthesis and signal transduction in a variety of mammalian cells. Thus, to maintain cellular homeostasis, intracellular DAG levels must be tightly regulated. This is illustrated by evidence that inappropriate accumulation of DAG contributes to cellular transformation. For example, cell lines that overexpress PLCγ have a malignant phenotype . Also, cells transformed with one of several oncogenes have elevated DAG levels . Most of the evidences for this pathological effect centers on excessive and/or prolonged activation of PKC, which is a common feature of the transformed state, both in tumors and in cell cultures. Accumulation of DAG with increased membrane-associated PKC is the mechanism of spontaneous hepatocarcinogenesis in choline-deficient rats [3, 4]. Aortic endothelial cells have been shown to contain particularly high basal levels of polyunsaturated DAG together with a very high degree of membrane-associated PKC, which is largely insensitive to further activation . Glucose-induced de novo synthesis of DAG and sustained isozyme-selective PKC activation (especially PKC-β) contribute to the pathogenesis of diabetic micro- and macroangiopathy . The fed state was associated with increased DAG level and PKC activity in muscle tissue of insulin-resistant obese Zucker rats . These changes in PKC are likely to exacerbate the hyperglycaemia and hypertriglyceridaemia at obesity-induced diabetes. Hypothyroidism is frequently known to coexist with diabetes . Decreased thyroid hormone levels are often observed in the experimentally diabetic animals . However, little has been known about the effect of hypothyroidism on DAG content in cells. It has been demonstrated that activity of PLC and PLD was depressed , and DAG level decreased  in thyroxine-induced cardiac hypertrophy. In contrast, experimental hyperthyroidism caused a significant increase in inositol trisphosphate formation and PLC activation in the perfused hearts while hypothyroidism was associated with a decrease in this activity . Hypothyroidism increased the basal PLC-linked inositol phospholipid hydrolysis in rat hypothalamus, whereas L-T4 supplementation to hypothyroid rats resulted in a complete restoration of hypothalamic inositol phosphate formation to the value of euthyroid control .
Numerous investigations demonstrated a novel role of thyroid hormones as modulators of signal transduction. PKC is critical to the mechanism by which thyroid hormones rapidly induce phosphorylation and nuclear translocation of mitogen-activated protein kinase and subsequently potentiate both the antiviral and immunomodulatory actions of IFNγ in cultured cells  and regulate the exchange of signaling phospholipids (PL) in hepatocytes [15, 16]. It was found that L-T4 rapidly induced the biphasic DAG accumulation and PKC activation in liver slices and isolated hepatocytes . The effect of L-T4 on PLC, -D, PKC, and DAG accumulation was too rapid: from seconds to a few minutes. Perfusion of the liver of hypothyroid rats with L-T4-containing solution during 10 min increased the content of DAG in the liver . However, the effect of hypothyroidism on basal level of DAG in liver has not been determined.
In the present study, we have examined DAG level and PKC activity in liver and isolated membranes during sustained thyroid hormone stimulation and at different thyroid state of organism. Degradation of thyroid gland function with aging or under the mercazolil action decreased PL and TAG synthesis in liver and increased the DAG level in liver slices and isolated liver cell plasma membranes. Hypothyroidism markedly decreased PKC activity. L-T4 administration to intact or mercazolil-treated rats decreased DAG level and increased PL and TAG synthesis in liver. These results provide the evidence that thyroxine is crucial in lowering DAG by converting them to PL and TAG during sustained hormone action.
Results and Discussion
The present work is aimed at the elucidation of the influence of thyroid functional status on the DAG level in liver and plasma membranes. To determine the role of thyroid hormones in the regulation of DAG level in liver, a comparison of euthyroid, mercazolil- and mercazolil+L-T4-treated rats or intact animals after injection of L-T4 has been made.
Blood serum thyroxine and triiodothyronine content in rats of different age and thyroid status
88.0 ± 6.9
1.5 ± 0.07
58.2 ± 3.2*
0.82 ± 0.0*
48.6 ± 0.9**
0.3 ± 0.08**
20.6 ± 4.26**
410 ± 51.0***
5.86 ± 0.48***
270 ± 10.0***
3.9 ± 0.09***
14C/3H ratio of the 1,2-diacylglycerol and phospholipid classes in liver slices of rats of different thyroid status
2.33 ± 0.19
4.33 ± 0.56
3.17 ± 0.38
1.24 ± 0.16
0.94 ± 0.16
0.93 ± 0.18
0.69 ± 0.20
1.18 ± 0.15
0.44 ± 0.09
1.32 ± 0.17
0.90 ± 0.09
0.44 ± 0.09
0.52 ± 0.15
0.39 ± 0.03
0.50 ± 0.12
0.32 ± 0.04
0.44 ± 0.04
0.33 ± 0.07
Overnight 12-O-tetradecanoylphorbol 13-acetate (TPA) pretreatment appeared to down-regulate predominantly the alfa-isoform of PKC in rat hepatocytes and to suppress cell signalling in response to vasopressin, TPA and Ca2+ ionophore A23187 . Previous data indicated that just the phorbol 12-myristate 13-acetate but not its inactive analog, phorbol 12-myristate 13-acetate 4-O-methyl ether, activated linoleic acid incorporation into PL  and phosphoinositide synthesis de novo in liver slices and isolated hepatocytes . PKC-dependent signalling has been indicated in plasma membranes of young intact 90-day-old rats. No effect has been found in plasma membranes isolated from liver of 720-day-old mercazolil-treated rats. These findings suggest that the desensitization of the response to agonist in cell of hypothyroid rats is dependent on PKC down regulation at such pathological state of organism.
In conclusion, results of the present study indicate thyroid hormone as a potentially important physiological modulator of DAG level in rat liver. Unlike our previous work  were thyroxine (in physiological concentration) was shown to induce PLC/PLD activation and short-lived DAG accumulation in liver cells, we detected the prominent DAG level decrease under unphysiological doses of hormone administration to rats. Age- and mercazolil-dependent decline of thyroid hormone level in blood serum coincides with elevation of DAG content in liver and liver cell plasma membranes. In contrast, under hypothyroid status of organism synthesis of PL and TAG decreases in liver. The reduction of glycerolipid synthesis and DAG level elevation can be corrected by thyroxine treatment of hypothyroid rats. Thus, thyroid hormone-dependent changes in DAG content are likely associated with glycerolipid synthesis. Extremely high basal DAG level in hypothyroid liver coincides with a declined PKC activity in both cytosol and membrane of liver cells. Stable alterations in DAG metabolism in hypothyroid liver are suggested to lead to disturbances in agonist responsiveness of liver cells.
Materials and Methods
[14C]oleic acid (58 mCi/mmol) and [3H]arachidonic acid (60 Ci/mmol) – Amersham Corp., [14C]palmitic acid (58 mCi/mmol), [γ-32P]ATP (1000 Ci/mmol) and [14C]CH3COOH (25 mCi/mmol) – BPO Isotop (Russia); DEAE-52-cellulose from Whatman (England); silica gel from Woelm (Germany). Phosphatidylserine was isolated from ox brain; other lipid standards and histone H1 were obtained from Sigma (USA). T4 and mercazolil (1-methyl,2-mercaptoimidozol) were from Zdorov'e Trudyaschikhsya (Kharkov, Ukraine). T4 and T3 radioimmunoassay kits were from Minsk (Belarussia). Other chemicals used were of chemically pure grade.
Adult 90- and 720-day-old male Wistar rats which had a free access to food and water and were kept at 24°C on a cycle of 12 h light/12 h darkness were used for experiments. Mercazolil was injected intraperitoneally (1 mg/100 g weight) in 0.9% NaCl to the experimental animals every day during 16 days-experiment. In some cases, the mercazolil-treated rats were injected intraperitoneally by T4 (10 μg/100 g weight) 48 h prior to killing. Besides, T4 (200 μg/100 g weight) was injected to the normal rats 15, 30 and 60 min prior to killing or three times a week in which case the last hormone injection was made 48 h prior to killing. Control rats received 0.9% NaCl of the same volume. The animals were starved overnight prior to experiment.. The thyroid state of rats was monitored by radioimmunological determination of the T4 and T3 in blood serum.
Experiments with liver slices
The 1 mCi of [14C]CH3COOH was intraperitoneally injected to rats four times every 30 minutes for 2 hours . The liver was perfused with 0.9% NaCl, then removed and washed in Krebs-Henseleit buffer, pH 7.4, containing 2 mM CaCl2 and 0.2% BSA. Pre-labeled slices of liver were used for [14C]-DAG and [14C]-FFA analysis. Besides, the liver slices were labeled by incubation in Eagle medium containing 10% fetal calf serum, 100 units/litre streptomycin, 100 units/litre penicillin, 13 mg/ml gentamycin and 2.5 μCi/ml of [14C]oleic acid and 2.5 μCi/ml [3H]arachidonic acid or 2.5 μCi/ml [14C]palmitic acid alone for 1 h in 95% O2/5% CO2 atmosphere at 37°C. The lipids were extracted and analyzed as described below.
Cell suspension experiments
Hepatocytes were isolated from the 90- and 720-day-old male Wistar rats which had a free access to food and water and were kept at 24°C on a cycle of 12 h light/12 h darkness by the method described in . Preparation of hepatocytes was started between 9:00 and 10:00 a.m. Cells (107/ml) were labeled by incubation in Eagle medium containing 10% fetal calf serum, 100 units/liter streptomycin, 100 units/liter penicillin, 13 mg/ml gentamycin and 2.5 μCi/ml of [14C]oleic acid for 3 h in 95% O2/5% CO2 atmosphere at 37°C. Before lipid extraction, cells were washed twice with a Krebs-Henseleit buffer pH 7.4, containing 2 mM CaCl2, 25 mM HEPES, 0.1% BSA. The lipids were extracted and analyzed as described below.
Isolation of liver cell plasma membranes
Plasma membranes were prepared using differential centrifugation through various concentration of sucrose and characterized by their specific marker enzymes as described in . The lipids were extracted and analyzed as described below.
Extraction and separation of lipids
Lipids were extracted according to Folch et al.  and phosphoinositides as described in . The chloroform phase was collected and dried under N2 at 37°C. The lipids were redissolved in chloroform/methanol (1:2, v/v) and applied on TLC plates. For DAG and FFA isolation the solvent system: hexane/diethyl ether/acetic acid (80:20:2, v/v) was used; for PC and PE separation – chloroform/methanol/acetic acid/water (25:15:4:2, v/v) and for PI, PIP and PIP2 – chloroform/methanol/NH4OH (50:40:10, v/v). Appropriate standards were applied on each plate for quantification. The gel spots containing [14C/3H]lipids were scraped and transferred to scintillation vials. Radioactivity was measured by scintillation counter.
Protein kinase C enzyme assay
Activities of protein kinase C in the cytosol and in the crude membrane fraction of liver cells were determined after partial enzyme purification by chromatography on DEAE-cellulose . The activity of protein kinase C was determined by the transfer of 32P from [γ-32P]ATP into H1 histone in the presence of phosphatidylserine and Ca2+ (0.2 mM). Since histone is a poor substrate for calcium-insensitive isoforms of PKC, the predominant isoforms of PKC detected were cPKC and aPKC. The enzyme activity was expressed as pmoles of phosphate transferred from [γ-32P]ATP into H1 histone per minute. The protein content was determined by Bradford method .
– protein kinase C
– phosphatidylinositol 4-phosphate
– phosphatidylinositol 4,5-bisphosphate
– free fatty acids
– phosphatidic acid phosphatase
– 12-O-tetradecanoylphorbol 13-acetate.
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