Ribonucleic acid efflux from isolated mouse liver nuclei is altered by diet and genotypically determined change in nuclear envelope composition.

Differences in immunological abnormalities like autoimmunity, abnormal T cell proliferative disorders and accelerated ageing occur between MRL/Mp-lpr/lpr(lpr/lpr) and MRL/Mp-+/+(+/+) mice as a consequence of one gene. The present study was designed to assess the effect of these differences in genotype and diet on the composition and function of the liver nuclear envelope. Mice of both strains were fed nutritionally adequate diets differing only in fatty acid composition for 4 weeks. Phospholipid fatty acid composition of the liver nuclear envelope was determined and the effect of altering the lipid composition of the nuclear membrane on nucleoside-triphosphatase (NTPase) activity, ribonucleic acid (RNA) efflux and binding of L-triiodothyronine (L-T3) was determined. Strain of mouse and level of dietary linoleic acid exhibited significant effects on the phospholipid fatty acid composition of the nuclear envelope. Levels of 18:1(n - 9) and 18:2(n - 6) were lower and 20:4(n - 6) content was higher in nuclear envelope phospholipids of lpr/lpr mice compared with mice of the +/+ strain. Mice fed the high linoleic acid diet exhibited higher levels of 18:0, 18:2(n - 6) and 20:4(n - 6) and lower levels of 16:0 and 18:1(n - 9) in liver nuclear envelope phospholipids, compared with mice fed the low linoleic acid diet. These changes in membrane composition were reflected in alteration of NTPase activity and efflux of RNA from isolated mouse liver nuclei. Nucleoside triphosphatase activity and efflux of ribonucleic acid from isolated nuclei were significantly higher in livers of the lpr/lpr strain. NTPase activity and RNA efflux from isolated nuclei were higher in the high linoleic acid fed group compared with the low linoleic acid group. A single class of binding sites for L-T3 was present in liver nuclear envelopes of these mice and Kd values were not influenced by strain or dietary linoleic acid levels. Nuclear envelopes prepared from +/+ animals exhibited a significantly higher number of binding sites for L-T3 compared with the lpr/lpr group. These observations indicate that the single gene difference characterizing lpr/lpr mice from +/+ mice results in alterations in the composition and function of the nuclear envelope. This genetic difference also alters the response of this membrane to dietary factors known to modulate characteristics and functions of the nuclear envelope.

Genotype effects on the antioxidant enzymes activity and mRNA expression in liver and kidney tissues of autoimmune-prone MRL/MpJ-lpr/lpr mice.

Congeneic pairs of MRL/lpr and MRL/++ (+/+) mice differ in incidence of autoantibodies, lymphoproliferative disease and survival, characteristics that are linked to immunological abnormalities. MRL/lpr mice have a significantly shorter life span compared to +/+ mice. Because a weak antioxidant defense and an increased generation of free radicals are generally implicated in the severity of many autoimmune disease, the present study was undertaken to compare the influence of genotype on lipid composition, lipid peroxidation and expression of mRNA, and activity of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in the livers and kidneys of these mice. The expression of SOD, GSH-Px and CAT mRNAs was significantly higher (P < 0.05) in the livers of +/+ mice, while in the kidneys only SOD expression was found significantly higher in +/+ mice when compared to MRL/lpr mice. Further, the activity of cytosolic SOD and GSH-Px was also found significantly higher (P < 0.001) in the livers of +/+ mice. Both livers and kidneys of MRL/lpr mice exhibited significantly higher levels of arachidonic acid (20:4(n-6)), significantly higher generation of thiobarbituric acid reactive substances (TBARS) and higher estimated peroxidation index than the +/+ mice. In addition, the MRL/lpr mice had higher levels of serum anti-cardiolipin antibodies. In summary, the results from the present study indicate that besides several immune-related abnormalities, the MRL/lpr mice may exhibit their inability to cope with oxidative stress due to a poor antioxidant defense system.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of genotype on the phospholipid fatty acid composition of splenic T and B lymphocytes in MRL/MpJ-lpr/lpr mice.

The MRL/MpJ-lpr/lpr mice manifest a T cell proliferative and autoimmune disorder. Similar changes occur much later in the life of MRL/MpJ-+/+ mice. MRL/MpJ-lpr/lpr (lpr/lpr) and MRL/MpJ-+/+ (+/+) mice were fed for six weeks nutritionally adequate semipurified diets containing 20% (w/w) fat, but differing in linoleic acid content. The phospholipid fatty acid composition of T and B cells was found to be dependent on genetic background of mice and level of linoleic acid in the diet. Changes in the levels of specific fatty acids like 16:0, 18:2 omega 6, 22:5 omega 3 and 22:6 omega 3 in some of the phospholipid components were observed in the MRL/MpJ-lpr/lpr strain in both the B and T cell types as compared with their normal +/+ counterpart strain. T cells of lpr/lpr mice exhibited significantly higher levels of 20:4 omega 6 than did T cells of other strain. High levels of dietary linoleic acid significantly increased incorporation of 18:2 omega 6 in T and B cells, while the effect on other fatty acids of the two types of cells varied with the phospholipid classes and fatty acids when compared with the low linoleic acid fed-group. Differences observed in the phospholipid fatty acid composition of the T and B cells of the congenic mice might contribute to differences in rate of progression of age-related changes suggesting that the autoimmune disorder might be mitigated by dietary manipulation.

Effect of blood transfusions on immune function. Part VI. Effect on immunologic response to tumor.

Transfusions are reported to increase the incidence of tumor metastasis in clinical studies and primary tumor growth in animal studies. We evaluated the effect of transfusions on immunologic response to primary and metastatic tumors in multiple rat models. One half of the animals were administered lactated Ringer's solution and one half ACI rat blood at the time of tumor challenge. In 80 rats a slow-growing colon tumor was implanted subcutaneously. At 4 months there were no significant differences in tumor size or leukocyte infiltration of the tumor. Similar results were obtained with a rapidly growing colon cancer. Analysis of T-lymphocyte subpopulations in both groups showed no differences. Rats transfused at the time of intravenous challenge with a suspension of 1 x 10(6) tumor cells had a mean survival time of 38.3 +/- 0.8 days and the control group had a mean survival time of 41.1 +/- 0.8 days (p = 0.016). One week after transfusion, natural killer cell lysis of tumor cells at a 100:1 effector/target cell ratio was 18.0% +/- 1.8% in the transfusion group and 23.0% +/- 1.3% in the control group (p = 0.034). In conclusion, transfusions in multiple rat cancer models did not affect primary tumor growth or the host's immunologic response to it but did significantly impair natural killer cell function and survival with tumor metastases.

Effects of dietary omega3 and omega6 lipids and vitamin E on proliferative response, lymphoid cell subsets, production of cytokines by spleen cells, and splenic protein levels for cytokines and oncogenes in MRL/MpJ-lpr/lpr mice.

omega3 Fatty acid rich fish oil (FO) and vitamin E may delay the progress of certain autoimmune diseases. The present study examined the mechanisms of action of omega3 lipids and vitamin E in autoimmune-prone MRL/lpr mice suffering from extensive lymphoproliferation, lupus-like symptoms, and accelerated aging. To determine whether the effects of omega3 lipids in autoimmune disease is linked to vitamin E levels, weanling female MRL/lpr and congenic control MRL/++ mice were fed diets containing 10% corn oil (CO) or 10% FO at two levels of vitamin E (75 IU or 500 IU/kg diet) for 4 months. The appearance of lymph nodes was delayed in the mice fed FO, and higher levels of FO offered further protection against the appearance of lymph nodes. Analysis of the spleen cells revealed that the cells positive for Thy.1 and Fas were significantly higher in the MRL/++ mice. The groups fed high levels of vitamin E generally exhibited higher levels of Fas. The proliferative response of splenocytes of MRL/++ mice to mitogens was significantly higher compared with MRL/lpr mice. Interleukin (IL)-10 production by spleen cells was significantly higher in FO-fed MRL/lpr mice than in CO-fed mice. In mice fed a high level of vitamin E, the production of IL-12 and tumor necrosis factor-alpha was significantly lower and IL-2 was significantly higher than in animals fed a low level of vitamin E. Proinflammatory cytokines were higher in the MRL/lpr mice and both FO and vitamin E lowered the levels of proinflammatory cytokines and lipid mediators. Western blots revealed that c-myc and c-ras were significantly lower and IL-2 and transforming growth factor (TGF)-beta1 levels were significantly higher in the spleens of MRL/++ mice. FO lowered c-myc and high levels of vitamin E in the diets normalized the levels of TGF-beta1 in MRL/lpr mice. The observations from this study suggest that both FO and vitamin E modulate the levels of specific cytokines, decrease the levels of proinflammatory cytokines, inflammatory lipid mediators, and c-myc, and increase TGF-beta1 levels in spleens of MRL/lpr mice and thus may delay the progress of autoimmune diseases.

Effects of the level of dietary fat intake and endurance exercise on plasma cytokines in runners.

PURPOSE: Chronic exercise and high fat diets have been associated with immune suppression. We have reported the effects of level of dietary fat and exercise on lymphocyte subsets, proliferative response, and in vitro production of cytokines by peripheral blood mononuclear cells of runners. The present study was planned to further investigate whether the mechanisms of action of dietary fats is through their modulation of plasma cytokines in runners. METHODS: This study compared plasma cytokines at rest and after endurance exercise at 80% of V02max in female (N = 8-10) and male (N = 8-10) runners after eating diets comprised of 17% (LF), 32% (MF), and 41% (HF) fats (4 wk each). RESULTS: The level of interleukin-1 beta (IL-1 beta) was independent of gender, exercise, and level of dietary fat. tumor necrosis factor (TNF-alpha) level was higher in the plasma of men compared with that in women runners, and the level of these two cytokines increased with increasing level of dietary fat. Plasma interleukin-2 (IL-2) level (a cytokine involved in enhancing T cell functions for host defense) was significantly higher in men compared with that in women runners and decreased in men with increase dietary fat. Plasma interleukin-6(IL-6) level was significantly lower after the endurance run, and IL-6 levels decreased with increase in dietary fat. CONCLUSIONS: Data from the present study suggest that dietary fat has differential effects on plasma cytokine levels in runners. Increasing the level of dietary fat significantly increased endurance run time and had no adverse effects on the level of plasma IL-2 and pro-inflammatory cytokines (IL-1 beta, IL-6 and TNF-alpha in runners.

Dietary fats and immune status in athletes: clinical implications.

Athletes are competitive, train at very high levels with inadequate rest, consume too few calories, avoid fats, and may be at increased risk of infections. The immune system is sensitive to both fat intake and intense exercise, suggesting that athletes may have suppressed immune function. It has been reported that many athletes consume about 25% fewer calories than the estimated expenditure, leading to low intakes of some essential micronutrients and fats. Acute exercise has been shown to increase inflammatory and decrease antiinflammatory immune factors and may increase oxidant stress. Chronic exercise appears to improve immune competence. Lipids are powerful mediators of the immune system, and they may modulate the immunosuppressive effects of strenuous exercise. Studies have shown that a low-fat high-carbohydrate diet (15% fat, 65% CHO, 20% protein of total calories), typically eaten by athletes, increases inflammatory and decreases antiinflammatory immune factors, depresses antioxidants, and negatively affects blood lipoprotein ratios. Increasing total caloric intake by 25% to match energy expenditure and the dietary fat intake to 32% in athletes appears to reverse the negative effects on immune function and lipoprotein levels reported on a low-fat diet. Increasing the dietary fat intake of athletes to 42%, while maintaining caloric intake equal to expenditure, does not negatively affect immune competency or blood lipoproteins, whereas it improves endurance exercise performance at 60-80% of VO2max in cyclists, soldiers, and runners. There is no evidence that higher fat intakes (up to 42% of total calories), in calorically balanced diets, increase the risk of cancer, but studies are needed to determine whether the beneficial effects of higher fat diets in athletes reduce their rate of infections.

Influence of the level of dietary lipid intake and maximal exercise on the immune status in runners.

Chronic exercise and high fat diets are associated with immune suppression. This study compares cellular immune responses at rest and after maximal exercise in runners after eating diets comprised of 17% low fat (LF), 32% medium fat (MF), and 41% high fat (HF) (4 wk each). VO2max increased significantly from the 17% to 41% fat diet. The leukocyte cell counts were significantly increased after exercise. In men, significantly higher proliferative response to phytohemagglutinin (PHA) (P < 0.004) was observed with MF diet, while response to pokeweed mitogen (PWM) was significantly decreased by MF and HF diets. The number of CD8+ (suppressor) T cells was significantly higher in men and exercise increased it significantly, while CD4+ (helper) T cells were not affected. Natural killer cells number was significantly increased 2.5 fold by exercise and with increase in dietary fat. The production of IL-2 by peripheral blood mononuclear cells was significantly higher in men (P < 0.0001) and increasing dietary fat significantly increased IL-2 production (P < 0.001). In men, exercise decreased the level of the proinflammatory cytokines (IL-1, IL-6 and TNF-alpha), whereas in women, with the exception of MF diet for IL-6, exercise had no effect. This study indicates that short, intense bouts of exercise in runners training 40 miles.wk-1 have mixed effects on the immune system. A high percentage of fat intake (41%) did not have any deleterious effects on the immune system of the well-trained runners.

The role of dietary fat on performance, metabolism, and health.

This paper presents a model to evaluate the nutritional status of trained athletes based on work in our laboratory as well as others. The model proposes that substrate use is set by the muscle fibers recruited, based on the exercise intensity. Second, the substrate available is primarily determined by the intramuscular stores. In trained athletes, intramuscular fat plays an important role in metabolism at exercise intensities as high as 80% of maximal aerobic power. Based on these factors, increasing the fat in the diet (while maintaining adequate intramuscular glycogen) increases VO2max and intramuscular stores of fat (presumably due to increased mitochondrial volume). These two factors result in a significant increase in the time to exhaustion at set levels of exercise (endurance). It also appears that fatigue is associated with depletion of either glycogen or fat. These conclusions hold true for athletes on diets where sufficient calories are taken in to meet demands and for exercise levels below 80% of VO2max, where primarily slow-twitch oxidative fibers are used. These data may not apply in exercise where predominantly fast-twitch fibers are used. Also, these data do not apply to runners eating a hypocaloric diet, where reducing the percentage of carbohydrates may compromise their glycogen stores. It would appear that the fat in the diet can be increased to a very high level without compromising the cardiovascular or immune systems of athletes. Moreover, it can be proposed that these data could be applied to sedentary persons, as long as they are isocaloric. This would imply that the fat consumed in the diet would be used in the muscle, as in the runners, although at a lower level. Thus, the dietary intake should be matched in both total calories and percentage of fats and carbohydrates to calories consumed by daily activity. It should be cautioned that if glycogen and fat stores are compromised, protein resynthesis is inhibited and loss of muscle mass may result. This has a negative effect on the athlete's ability to perform at high levels.

Does a threshold for the effect of dietary omega-3 fatty acids on the fatty acid composition of nuclear envelope phospholipids exist?

Existence of a dietary maximal level or threshold for incorporation of omega 3 fatty acids into membrane phospholipids is of interest as it may further define understanding of the dietary requirement for omega 3 fatty acids. To test whether feeding increasing levels of dietary omega 3 fatty acids continues to increase membrane omega 3 fatty acid content, weanling rats were fed a nutritionally adequate semipurified diet which provided increasing amounts of C20 and C22 omega 3 fatty acids, such as 20:5 omega 3 and 22:6 omega 3. Dietary 20:5 omega 3 and 22:6 omega 3 were provided by substituting a purified shark oil concentrate of high 22:6 omega 3 content for safflower oil high in 18:2 omega 6. After four weeks of feeding, nuclear envelopes from four animals in each diet group were prepared, lipid was extracted and phospholipids separated. Arachidonic acid content in membrane phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine was significantly reduced by feeding increased dietary levels of omega 3 fatty acids. Decline of 20:4 omega 6 level in phospholipid tended to stabilize when the dietary content of total omega 3 fatty acids reached 4-5% of total fatty acids. Above this level, dietary omega 3 fatty acids did not result in a further decrease in membrane content of 20:4 omega 6. Increase in membrane phospholipid content of 20:5 omega 3 occurred as the dietary intake of omega 3 fatty acids increased from 1.1% to 5% of total fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of n-3 and n-6 fatty acids on the activities and expression of hepatic antioxidant enzymes in autoimmune-prone NZBxNZW F1 mice.

Menhaden fish oil (FO) containing n-3 fatty acids dramatically extends the life span and delays the onset and progression of autoimmune disease in (NZBxNZW)F1 (B/W) female mice as compared to those fed corn oil (CO) rich in n-6 lipids. As an inefficient antioxidant defense system has been linked to autoimmune diseases, the present study was undertaken to determine whether the protective action of n-3 lipids is mediated through their antioxidant defense system. Weanling B/W mice were fed a nutritionally adequate, semipurified diet containing CO or krill oil (KO) or FO at 10% level (w/w) ad libitum until the mice were 6.5 months old. All diets contained the same level of vitamin E (21.5 mg/100 g diet). We compared the effects of feeding n-6 and n-3 lipids on survival, kidney disease, hepatic microsomal lipid composition, peroxidation, and on the activity and mRNA expression of the antioxidant enzymes catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in 6.5-month-old B/W mice. The results showed that when compared to livers from CO-fed mice, livers from KO- and FO-fed mice showed: (i) significantly higher (P < 0.001) activities and expression of CAT, GSH-Px and SOD; (ii) significantly lower (P < 0.001) arachidonic acid (20:4n-6) and linoleic acid (18:2n-6) and higher (P < 0.001) eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) levels in hepatic microsomes; and (iii) significantly lower (P < 0.001) estimated peroxidation indices and thiobarbituric acid reactive substances generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of genotype on diet-induced changes in membrane phosphatidylcholine and phosphatidylethanolamine composition of splenocytes, liver nuclear envelope and liver mitochondria.

Inbred congenic mice of strains MRL/Mp-lpr/lpr (lpr/lpr) and MRL/Mp(-)+/+ (+/+) were fed nutritionally adequate semipurified diets containing 20% (w/w) fat and differing in linoleic acid content. Levels of linoleic acid (18:2n-6) and arachidonic acid (20:4n-6) in phospholipids of splenocytes, liver mitochondria and liver nuclear envelopes were determined. Membranes of lpr/lpr mice exhibited significantly lower levels of 18:2n-6 and 20:4n-6 in phospholipids compared with the +/+ strain. The high linoleic acid diet increased incorporation of 18:2n-6 and 20:4n-6 in most phospholipid fractions of these membranes. These observations indicate that genotype as well as dietary 18:2n-6 content significantly influenced incorporation of 18:2n-6 and 20:4n-6 into membrane phospholipids. The results also suggest that membrane compositional abnormalities found in the lpr/lpr mice, which develop lymphoma and age faster than +/+ mice, are not restricted to the immune system but also extend to other organs. Differences observed in phospholipid fatty acid composition in splenocytes and liver subcellular membranes for mice fed diets differing in linoleic acid content suggest that the early expression of the lpr gene resulting in progression of autoimmunity may be delayed through dietary manipulation.

Effect of dietary fat on diabetes-induced changes in liver microsomal fatty acid composition and glucose-6-phosphatase activity in rats.

Experimental diabetes may manifest itself in a defect in liver microsomal fatty acid desaturation and increased activity of glucose-6-phosphatase (G-6-Pase). The present study was designed to determine whether these changes could be normalized by a change in the dietary fat consumed. Control and streptozotocin-induced diabetic rats were fed nutritionally adequate diets which varied in fatty acid composition. Fatty acid analysis of liver microsomal phospholipids revealed that non-diabetic control animals fed saturated fat (beef tallow) or a diet high in omega 3 fatty acids (fish oil) exhibited a significantly higher level of 18:2 omega 6 and a lower level of 20:4 omega 6 in the phosphatidylcholine and phosphatidylethanolamine fractions compared with diabetic animals. Control and diabetic animals fed the high linoleic acid diet had similar levels of 18:2 omega 6 in the microsomal phosphatidylcholine and phosphatidylserine fractions. Microsomal G-6-Pase activity was higher in diabetic than in control animals. Activity of G-6-Pase was lower in microsomes of control animals fed the soybean oil or the fish oil diet, but was not significantly reduced in diabetic animals fed high polyunsaturated fats. Blood glucose levels were similar in control groups fed the different diets, but the plasma hemoglobin Alc level was lower in diabetic animals fed the soybean oil diet. Cholesterol and triglyceride levels were lower in diabetic animals fed the fish oil-based diet. The results suggest that dietary fat manipulation has the potential to change at least some of the abnormalities in the microsomal membrane in experimental diabetes.

Multiple thyroid hormone binding sites on male rat liver nuclear matrices.

Equilibrium binding of T3 to nuclear matrices isolated from male rat liver occurred after incubation for 3h at 20 degrees C. Two binding sites, having KD's of 6 and 95 nM, were revealed by Scatchard analysis. T3 and Triac competed for the binding of [125I]T3 to the high affinity site whereas only T3 competed for binding to the lower affinity site. Reverse T3 (rT3) did not compete for the binding of T3 to either class of binding sites. The binding sites were highly DNAse-sensitive, and less sensitive to protease treatment. The effect of binding of T3 to nuclear matrices by ATP, DTT and EDTA indicated that the sites are dissimilar to previously identified cytosolic binding sites. The higher affinity site resembles the T3 receptor in affinity and thyroid hormone specificity. The second site represents a new class of thyroid hormone binding sites. Its role in the regulation of thyroid hormone action warrants further investigation.

Effects of dietary omega-3 and omega-6 lipids and vitamin E on serum cytokines, lipid mediators and anti-DNA antibodies in a mouse model for rheumatoid arthritis.

OBJECTIVE: Omega-3 (omega-3) fatty acid rich-fish oil (FO) and vitamin E (vit-E) may delay the progress of certain autoimmune diseases. The present study examined the mechanism of action of omega-3 and omega-6 lipids and vit-E on the serum cytokines and lipid mediators in autoimmune-prone MRL/lpr mice (a model for rheumatoid arthritis, RA). The lpr (lymphoproliferative) gene is overexpressed in these mice causing extensive lymphoproliferation, lupus-like symptoms and accelerated aging. METHODS: Weanling female MRL/lpr and congenic control MRL/++ mice were fed 10% corn oil (CO, omega6) or FO-based semipurified diets containing two levels of vitamin E (vit-E-75, I.U. and vit-E-500 I.U./Kg diet) for four months. At the end of the experiment, serum anti-DNA antibodies, cytokines and lipid mediators levels were determined. RESULTS: The appearance of enlarged lymph nodes was delayed in the mice fed FO, and the FO-500 IU vit-E diet offered further protection against enlargement of lymph nodes. The MRL/lpr mice exhibited significantly higher levels of serum anti-dsDNA antibodies. The FO-fed mice had significantly lower serum IL-6, IL-10, IL-12, TNF-alpha, PGE2, TXB2 and LTB4 levels compared with CO-fed mice. In mice fed 500 IU vit-E diets, the serum IL-6, IL-10, IL-12 and TNF-alpha levels were significantly lower and serum IL-1beta was significantly higher compared to 75 IU-vit-E-fed mice in CO/FO or both. The levels of anti-DNA antibodies, IL-4, IL-6, TNF-alpha, IL-10 and IL-12 were higher in the sera of MRL/lpr mice. The FO diet lowered the levels of these cytokines (except IL-4) and lipid mediators. Adding 500 IU of vit-E to the FO diet further lowered the levels of IL-6, IL-10, IL-12, and TNF-alpha. CONCLUSION: It is clear from our observations that the beneficial effects of FO can be enhanced by the addition of 500 IU of vit-E in the diet. The FO diet containing 500 IU of vit-E may specifically modulate the levels of IL-6, IL-10, IL-12 and TNF-alpha and thereby may delay the onset of autoimmunity in the MRL/lpr mouse model. The observations from this study may form a basis for selective nutrition intervention based on specific fatty acids and antioxidants in delaying the progress of RA.

Multiple thyroid hormone binding sites on rat liver nuclear envelopes.

Nuclear envelopes relatively free of plasma membrane contamination were isolated from the male rat liver. Equilibrium binding of T3 to nuclear envelopes occurred after incubation for 3 h at 20 degrees C. Scatchard analysis revealed two classes of binding sites; a high affinity site having a KD of 1.8 nM with a maximum binding capacity of 14.5 pmol/mg protein and a low affinity site having a KD of 152.1 nM with a maximum binding capacity of 346.8 pmol/mg protein. No degradation of the radioligand occurred during incubation with the nuclear envelope. T4, rT3 and Triac competed effectively for the binding of T3 to the high affinity site whereas only T4 competed well for binding to the lower affinity site. The binding site was protease sensitive but not salt extractable. Multiple T3 binding sites having similar affinities have been reported on plasma membranes. An intriguing possibility is that membrane binding sites may be involved in translocation of thyroid hormone across membrane barriers.

Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver.

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3',5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3',5'-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.

Diet fat alters the structure and function of the nuclear envelope: modulation of membrane fatty acid composition, NTPase activity and binding of triiodothyronine.

Mice were fed a diet either high or low in P/S ratio to determine the effect of altering dietary lipid on the fatty acid composition of liver nuclear envelopes and thereby on functions of the nuclear envelope. Mice fed the high P/S diet exhibited higher levels of C18:2 omega 6 and unsaturates in liver nuclear envelopes, higher specific activity of NTPase and specific binding for L-triiodothyronine at 15 degrees C and 22 degrees C compared with the low P/S diet fed group. These observations indicate that diet-induced differences in the fatty acid composition of nuclear envelope lipid affects functions of the nuclear envelope.