RESUMEN

Triiodothyronine (T3) stimulates a robust increase (>40-fold) in transcription of the malic enzyme gene in chick embryo hepatocytes. Previous work has shown that optimal T3 regulation of malic enzyme transcription is dependent on the presence of an accessory element (designated as region E) that immediately flanks a cluster of five T3 response elements in the malic enzyme gene. Here, we have analyzed the binding of nuclear proteins to region E and investigated the mechanism by which region E enhances T3 responsiveness. In nuclear extracts from hepatocytes, region E binds heterodimeric complexes consisting of the homeodomain proteins PBX and MEIS1. Region E contains four consecutive PBX/MEIS1 half-sites. PBX-MEIS1 heterodimers bind the first and second half-sites, the third and fourth half-sites, and the first and fourth half-sites. The configuration conferring the greatest increase in T3 responsiveness consists of the first and fourth half-sites that are separated by 7 nucleotides. Stimulation of T3 response element functions by region E does not require the presence of additional malic enzyme sequences. In pull-down experiments, PBX1a and PBX1b specifically bind the nuclear T3 receptor-alpha, and this interaction is enhanced by the presence of T3. A T3 receptor-alpha region containing the DNA binding domain plus flanking sequences (amino acids 21-157) is necessary and sufficient for binding to PBX1a and PBX1b. These results indicate that PBX-MEIS1 complexes interact with nuclear T3 receptors to enhance T3 regulation of malic enzyme transcription in hepatocytes.

Asunto(s)

RESUMEN

Triiodothyronine (T3) stimulates a 7-fold increase in transcription of the acetyl-CoA carboxylase-alpha (ACCalpha) gene in chick embryo hepatocytes. Here, we characterized an ACCalpha T3 response element (ACCalpha-T3RE) with unique functional and protein binding properties. ACCalpha-T3RE activated transcription both in the absence and presence of T3, with a greater activation observed in the presence of T3. In nuclear extracts from hepatocytes incubated in the absence of T3, ACCalpha-T3RE bound protein complexes (complexes 1 and 2) containing the liver X receptor (LXR) and the retinoid X receptor (RXR). In nuclear extracts from hepatocytes incubated in the presence of T3 for 24 h, ACCalpha-T3RE bound a different set of complexes. One complex contained LXR and RXR (complex 3) and another contained the nuclear T3 receptor (TR) and RXR (complex 4). Mutations of ACCalpha-T3RE that inhibited the binding of complexes 1 and 2 decreased transcriptional activation in the absence of T3, and mutations of ACCalpha-T3RE that inhibited the binding of complexes 3 and 4 decreased transcriptional activation in the presence of T3. The stimulation of ACCalpha transcription caused by T3 was closely associated with changes in the binding of complexes 1-4 to ACCalpha-T3RE. These data suggest that T3 regulates ACCalpha transcription by a novel mechanism involving changes in the composition of nuclear receptor complexes bound to ACCalpha-T3RE. We propose that complexes containing LXR/RXR ensure a basal level of ACCalpha expression for the synthesis of structural lipids in cell membranes and that complexes containing LXR/RXR and TR/RXR mediate the stimulation of ACCalpha expression caused by T3.

Asunto(s)

RESUMEN

High-carbohydrate feeding and triiodothyronine (T3) increase the abundance of acetyl-CoA carboxylase-alpha (ACC alpha) mRNA in avian hepatocytes, whereas starvation, glucagon, and medium-chain fatty acids decrease the abundance of ACC alpha mRNA. These changes in ACC alpha mRNA levels are mediated by alterations in the rate of transcription of the ACC alpha gene. In liver, ACC alpha transcription is initiated from two promoters, promoter 1 and promoter 2, resulting in transcripts that contain heterogeneity in their 5'-untranslated regions. Here, we investigated the role of promoter 1 and promoter 2 in mediating nutrient- and hormone-induced changes in ACC alpha mRNA abundance by measuring the level of transcripts expressed from promoter 1 and promoter 2 using a ribonuclease protection assay. The results indicated that both promoter 1 and promoter 2 were regulated by starvation/refeeding in livers of intact chicks and by T3, glucagon, and medium-chain fatty acids in chick embryo hepatocyte cultures and that alterations in the activity of promoter 2 accounted for a greater proportion of the changes in total ACC alpha mRNA abundance caused by nutrient and hormone treatment. Five DNase-hypersensitive sites were also identified between -500 and +1 bp relative to the transcription start site of promoter 2 in livers of intact chicks and in chick embryo hepatocyte cultures. In transient transfection analyses, this region of DNase hypersensitivity conferred regulation of transcription by T3, glucagon, and medium-chain fatty acids in chick embryo hepatocytes. Data from this study demonstrate that diet-induced changes in the activities of promoter 1 and promoter 2 in livers of intact chicks are mimicked in chick embryo hepatocyte cultures by manipulating the concentrations of T3, glucagon and medium-chain fatty acids in the culture medium and that cis-acting sequences mediating the effects of nutrients and hormones on promoter 2 activity are located immediately upstream of the transcription start site of this promoter.

Asunto(s)

RESUMEN

Triiodothyronine (T3) stimulates a marked increase (> 40-fold) in transcription of the malic enzyme gene in chick embryo hepatocytes (CEH), but has no effect on malic enzyme transcription in chick embryo fibroblasts (CEF) that express nuclear T3 receptors (TR) at levels which are similar to those of CEH. Heterodimerization of the TR with other nuclear proteins is a potential mechanism for the regulation of T3 action. For example, heterodimers of retinoid X receptors (RXR) and TR bind to T3 response elements (T3RE) with higher affinity and modulate transcription more effectively than TR homodimers. In the present report, we investigated the role of RXR in mediating differences in T3 responsiveness of the malic enzyme gene between CEH and CEF. Data from gel mobility shift analyses demonstrated that endogenous TRs from CEH and CEF bind to the major T3RE of the malic enzyme gene primarily as heterodimers with RXR alpha or a protein highly related to RXR alpha. The total binding activity of RXR alpha/TR complexes in CEF was decreased relative to that observed in CEH. Cell-type dependent differences in RXR alpha/TR complex formation were greater in cells incubated in the presence of T3 because T3 treatment increased RXR alpha/TR binding activity in CEH but had no effect on RXR alpha/TR binding activity in CEF. Decreased RXR alpha/TR complex formation in CEF relative to CEH was associated with a reduction in the abundance of RXR alpha protein and RXR alpha mRNA in the former cell-type. Expression of exogenous RXR alpha in CEF increased the T3 responsiveness of the malic enzyme promoter by about 4-fold. In contrast, expression of exogenous RXR alpha in CEH had no effect on the regulation of malic enzyme transcription by T3. These observations support the hypothesis that alterations in RXR alpha expression contribute to cell-type dependent differences in T3 responsiveness of the malic enzyme gene.

Asunto(s)

RESUMEN

Transcription of fatty acid synthase (FAS) and malic enzyme (ME) in avian liver is low during starvation or feeding a low-carbohydrate, high-fat diet and high during feeding a high-carbohydrate, low-fat diet. The role of glucose in the nutritional control of FAS and ME was investigated by determining the effects of this metabolic fuel on expression of FAS and ME in primary cultures of chick embryo hepatocytes. In the presence of triiodothyronine, glucose (25 mM) stimulated an increase in the activity and mRNA abundance of FAS and ME. These effects required the phosphorylation of glucose to glucose 6-phosphate but not further metabolism downstream of the aldolase step of the glycolytic pathway. Xylitol mimicked the effects of glucose on FAS and ME expression, suggesting that an intermediate of the pentose phosphate pathway may be involved in mediating this response. The effects of glucose on the mRNA abundance of FAS and ME were accompanied by similar changes in transcription of FAS and ME. These data support the hypothesis that glucose plays a role in mediating the effects of nutritional manipulation on transcription of FAS and ME in liver.

Asunto(s)

RESUMEN

Stimulation of malic enzyme transcription by triiodothyronine (T3) is robust (> 60-fold) in chick embryo hepatocytes, weak (5-fold) in chick embryo fibroblasts that stably overexpress the nuclear T3 receptor-alpha, and still weaker (1-fold) in chick embryo fibroblasts which contain nuclear T3 receptor levels that are similar to those of chick embryo hepatocytes. Using DNase I hypersensitivity, functional transfection, and in vitro DNA-binding analyses, four cis-acting elements were identified in the malic enzyme 5'-flanking DNA that conferred differences in nuclear T3 receptor activity between chick embryo hepatocytes and chick embryo fibroblasts. These cell-specific regulatory elements are located at -3895/-3890, -3761/-3744, -3703/-3686, and -3474/-2715 bp and overlap with DNase I hypersensitive sites that are observed in chromatin of chick embryo hepatocytes. Each element enhances T3 responsiveness of the malic enzyme promoter in chick embryo hepatocytes but has no effect on T3 responsiveness in chick embryo fibroblasts. Three of the cell-specific regulatory elements flank a previously identified DNA fragment (-3889 to -3769 bp; Hodnett et al., Arch. Biochem. Biophys. 334, 309-324, 1996) that contains one major and four minor T3 response elements. The cell-specific regulatory element at -3703/-3686 bp binds to the liver-enriched factor, CCAAT/enhancer-binding protein-alpha, whereas cell-specific regulatory elements at -3895/-3890 and -3761/-3744 bp bind proteins of unknown identity. While the cell-specific regulatory element at -3761/-3744 bp contains sequences that resemble binding sites for CCAAT/enhancer-binding protein, activator protein-1, cyclic AMP response element binding protein, and NF-1, none of these proteins appear to bind to this DNA fragment. These data suggest that cell-specific differences in T3 responsiveness of the malic enzyme gene are mediated in large part by nonreceptor proteins that augment the transcriptional activity of the nuclear T3 receptor in hepatocytes.

Asunto(s)

RESUMEN

The mechanisms by which triiodothyronine (T3), glucose, insulin, and glucagon regulate acetyl-CoA carboxylase expression in primary cultures of chick embryo hepatocytes have been investigated. Incubating hepatocytes with T3 in the absence of glucose caused a fourfold increase in acetyl-CoA carboxylase activity. Addition of glucose (20 mM) enhanced the T3-induced increase in acetyl-CoA carboxylase activity by threefold but had no effect on enzyme activity in the absence of T3. The effects of T3 and glucose on acetyl-CoA carboxylase activity were accompanied by similar changes in acetyl-CoA carboxylase mRNA levels, indicating that regulation occurred at a pretranslational step. Xylitol mimicked the effect of glucose on acetyl-CoA carboxylase mRNA abundance, suggesting that an intermediate(s) of the nonoxidative branch of the pentose phosphate pathway may be involved in mediating this response. Insulin accelerated the accumulation of acetyl-CoA carboxylase mRNA abundance caused by T3 and glucose but had no effect on steady-state levels of acetyl-CoA carboxylase mRNA in the absence or presence of T3. Glucagon caused a 65% decrease in the accumulation of acetyl-CoA carboxylase mRNA in hepatocytes incubated with T3 and glucose. The effects of T3, glucose, insulin, and glucagon on the abundance of acetyl-CoA carboxylase mRNA were accounted for by changes in the transcription rate of the acetyl-CoA carboxylase gene. These data support the hypothesis that T3, glucose, insulin, and glucagon play a role in mediating the effects of nutritional manipulation on transcription of acetyl-CoA carboxylase in liver.

Asunto(s)

RESUMEN

Transcription of acetyl-CoA carboxylase in avian liver is low during starvation or after consumption of a low-carbohydrate, high-fat diet and high during consumption of a high-carbohydrate, low-fat diet. The role of fatty acids or metabolites derived from fatty acids in the nutritional control of acetyl-CoA carboxylase transcription was investigated by determining the effects of long- and medium-chain fatty acids on acetyl-CoA carboxylase expression in primary cultures of chick embryo hepatocytes. Palmitate, oleate, and arachidonate caused a decrease in acetyl-CoA carboxylase activity in hepatocytes incubated with triiodothyronine (T3). The inhibition of acetyl-CoA carboxylase activity caused by arachidonate was accompanied by a similar decrease in transcription of the acetyl-CoA carboxylase gene. In contrast, neither palmitate nor oleate were effective in modulating acetyl-CoA carboxylase transcription. These results are consistent with arachidonate or a metabolite derived therefrom mediating the effects of diets containing high levels of n-6 polyunsaturated fatty acids on acetyl-CoA carboxylase transcription in liver. Hexanoate and octanoate also inhibited acetyl-CoA carboxylase activity in the presence of T3. The magnitude of the hexanoate- or octanoate-induced decrease in acetyl-CoA carboxylase activity was greater than that observed for long-chain fatty acids. Hexanoate and octanoate inhibited acetyl-CoA carboxylase activity at a transcriptional step, and did so within 2 h of addition of fatty acid. Addition of carnitine partially reversed the inhibitory effects of octanoate on acetyl-CoA carboxylase expression, suggesting that a metabolite of octanoate is involved in mediating this response. 2-Bromooctanoate was a more potent inhibitor of acetyl-CoA carboxylase expression than octanoate or hexanoate. We postulate that a metabolite of hexanoate and octanoate, possibly a six or eight carbon acyl-CoA, plays a role in the nutritional regulation of acetyl-CoA carboxylase transcription.

Asunto(s)

RESUMEN

Feeding previously starved chicks with a high-carbohydrate, low-fat diet stimulates a 9-fold increase in both the rate of synthesis of acetyl-CoA carboxylase (ACC) and the abundance of its mRNA in liver. To define the steps involved in mediating diet-induced changes in the abundance of ACC mRNA, transcriptional activity was measured with the nuclear run-on assay and multiple DNA probes specific to the ACC gene. ACC transcription was low in livers of starved chicks; feeding them with a high-carbohydrate, low-fat diet induced ACC transcription, increasing it 11-fold. An increase in transcription was detectable at 1 h, was maximal at 5 h and remained high for 26 h. Feeding previously starved chicks with a low-carbohydrate, high-fat diet stimulated a smaller increase (4-fold) in the abundance of ACC mRNA and the transcription of ACC than feeding with a high-carbohydrate, low-fat diet. The half-life of ACC mRNA in liver, as estimated from the kinetics of accumulation and decay of ACC mRNA during high-carbohydrate feeding and starvation, was not changed significantly by dietary manipulation. ACC mRNA was expressed at low levels in heart, pectoral muscle, kidney and brain. The abundance of ACC mRNA in these tissues was not affected by nutritional manipulation. These results demonstrate that nutritional control of the abundance of ACC mRNA in the chicken is liver-specific and is mediated primarily by changes in the rate of transcription of the ACC gene.

Asunto(s)

Asunto(s)

RESUMEN

The role of the alpha-thyroid hormone receptor (TR alpha) in regulation of transcription of the gene for chicken malic enzyme was analyzed in fibroblast cell lines normally unresponsive to triiodothyronine (T3). The gene for this transcription factor was introduced stably and overexpressed using a replication-competent retroviral vector. In chick embryo fibroblasts (CEF), overexpression of TR alpha decreased malic enzyme activity by 90% in the absence of T3. Addition of T3 almost completely restored malic enzyme activity to the level of similarly treated control CEF infected with virus lacking TR alpha. These TR alpha-induced changes in malic enzyme activity were mediated by alterations in transcription of the malic enzyme gene. Similar results were obtained when transcriptional activity of TR alpha was analyzed using a transient co-transfection system. Thus, the unliganded TR alpha is a transcriptional repressor of the malic enzyme gene; binding of T3 to the receptor abolishes this repression. In contrast, stable overexpression of TR alpha in QT6 cells had no effect on malic enzyme expression in the absence or presence of T3. Nuclear T3 binding was equally high in CEF and QT6 cells overexpressing TR alpha. These findings suggest that cell-specific factors control the ability of TR alpha to regulate the malic enzyme gene. Overexpression of TR alpha in CEF had no effect on the expression of fatty acid synthase and acetyl-CoA carboxylase, lipogenic enzymes that are stimulated by T3 in hepatocytes in culture. Thus, gene-specific factors also may control the transcriptional activity of TR alpha.

Asunto(s)

RESUMEN

This paper reviews work from our laboratory on the molecular mechanisms involved in the nutritional and hormonal regulation of avian malic enzyme. The activity of hepatic malic enzyme, one of the set of "lipogenic" enzymes, is high in well-fed chickens and low in starved chickens. In chick embryo hepatocytes in culture, insulin and triiodothyronine (T3) are positive effectors and glucagon, acting via cyclic AMP, is a negative effector. Hormone concentrations in blood are consistent with insulin and T3 playing the major positive roles, and glucagon a major negative role, in regulating hepatic malic enzyme activity during the transitions between the fed and the starved states. New results indicate that insulin-like growth factor 1 also stimulates accumulation of malic enzyme. Our strategy has been to trace the intracellular signalling pathway from its distal end, altered enzyme activity, towards its proximal end, interaction of humoral factors with their appropriate cellular receptors. Nutrition- and hormone-induced changes in malic enzyme activity are due to altered concentrations of malic enzyme protein which, in turn, are due to altered rates of synthesis of malic enzyme. Synthesis of malic enzyme is controlled by regulating the level of malic enzyme mRNA which, in turn is regulated at initiation of transcription. The next step in this analysis will be to identify cis-acting sequence elements in the malic enzyme gene which bestow upon it a selective response to nutritional state and hormones. We are using transient expression systems and avian retroviral vectors to test the function of cis-acting elements involved in the regulation of transcription.

Asunto(s)

RESUMEN

Obese (ob/ob) mice exhibit impaired hepatic thyroid hormone action that is mediated, at least in part, by a reduced nuclear 3,5,3'-triiodothyronine (T3) receptor occupancy. The possibility that lowered occupancy in obese mice may be caused by decreased transport of T3 across the hepatic plasma membrane was examined by measuring the unidirectional influx of [125I]T3 into livers of 8- to 10-wk-old obese and lean mice using a tissue-sampling portal vein-injection technique. Influx of [125I]thyroxine (T4), a substrate for T4 5'-deiodinase, was also measured. Unidirectional clearance of T3 and T4 was 64 and 80% lower, respectively, in obese mice than in lean mice. Hepatic T3 and T4 uptake was nonsaturable in both lean and obese mice, suggesting that transport occurs by lipid-mediated free diffusion. Clearance of another lipid-soluble hormone, hydrocortisone, was also lower in obese mice than in lean mice. Decreased membrane permeability to the above hormones in obese mice may result from reported changes in membrane lipid composition. In conclusion, decreased hepatic thyroid hormone uptake may contribute to impaired thyroid hormone action and T3 production in livers of obese mice.

Asunto(s)

RESUMEN

Obese (ob/ob) mice exhibit reduced adaptive thermogenesis associated with an impairment of thyroid hormone action. The mechanism underlying the latter defect was investigated by comparing the binding characteristics and occupancy of solubilized nuclear 3,5,3'-triiodothyronine (T3) receptors from livers of lean and obese mice. Scatchard analysis showed minimal differences in Bmax and Kd between phenotypes at both 4 and 8-10 wk of age, indicating that reduced hepatic thyroid hormone expression in obese mice is not caused by alterations in nuclear receptor concentration or affinity. In contrast, nuclear T3 receptor occupancy (endogenous T3 associated with the specific receptor divided by Bmax) was 14 and 23% lower in 4- and 8- to 10-wk-old obese mice, respectively. Together with reported changes in hepatic thyroid hormone-sensitive enzymes, these data are consistent with a diminished nuclear T3 signal initiating thyroid hormone action in obese mice. Decreased nuclear T3 receptor occupancy may be secondary to a low transport of plasma T3 to the nuclear pool. In conclusion, impaired hepatic thyroid hormone action in obese mice is mediated in part at least by a reduction in nuclear T3 receptor occupancy.

Asunto(s)

RESUMEN

Rats fed low protein diets often exhibit an elevation in adaptive thermogenesis that may result in part from activation of thyroid hormone action at the level of peripheral deiodination of thyroxine (T4) to 3,5,3'-triiodothyronine (T3). This possibility was examined by comparing the kinetic parameters of iodothyronine 5'-deiodination in hepatic and renal microsomes and the efficiency of energy retention in weanling rats fed diets containing 5% (LP-5), 8% (LP-8) or 22% (NP) casein. Energy balance measurements revealed that efficiency of energy retention was reduced in rats fed LP-5, whereas no significant change in adaptive thermogenesis was observed in rats fed LP-8. In the LP-5 group the maximal activity of hepatic 5'-deiodinase per milligram of microsomal protein was not different from that of the NP group; however, the Vmax of the renal enzyme was 21% higher. In both tissues, the Km of the reaction was not affected by dietary treatment. Hepatic and renal 5'-deiodination in rats fed LP-8 was similar to that in rats fed LP-5. When compared to corresponding NP controls, each of the low protein dietary groups (LP-5, LP-8) had a higher serum total T3 concentration, whereas serum free T3 and total T4 concentrations were unaffected by the dietary treatments. The findings demonstrate that iodothyronine 5'-deiodination and circulating thyroid hormone concentrations vary in a similar manner in LP-5 and LP-8 animals even though these two groups exhibit different responses in energy efficiency relative to NP controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

RESUMEN

Obese (ob/ob) mice exhibit impaired cold- and diet-induced thermogenesis, which may result in part from a defect in thyroid hormone action at the level of peripheral deiodination of thyroxine (T4) to 3,5,3'-triiodothyronine (T3). The latter possibility was examined by comparing kinetic parameters of 5'-deiodination (5'-D) in hepatic and renal microsomes of lean and obese mice during various physiological conditions. 5'-D Was adaptive to changes in age (1 to 8-10 wk), environmental temperature (14, 25, and 33 degrees C), and thyroid hormone status in both lean and obese mice. The magnitude of enzyme response, however, varied between phenotypes. 5'-D Response to age and environmental temperature was also dependent on tissue type, indicating that different isozymes for 5'-D may exist in liver and kidney. Under basal conditions at 25 degrees C, maximal hepatic and renal 5'-D was lower in obese mice than in lean mice. Differences in Vmax were observed as early as 1 wk of age. Km values for 5'-D were similar in lean and obese mice. These findings suggest that T3 availability to thermogenic target tissues may be impaired in obese mice, which may contribute to diminished thyroid hormone expression and heat production in these animals.

Asunto(s)

RESUMEN

3-Methylhistidine excretion in vivo and in vitro was monitored in hypervitaminotic and pair-fed control rats. Feeding with excess of retinyl palmitate (40 000 i.u./day per 100 g body wt.) significantly increased urinary 3-methylhistidine and creatinine output during a 4-day treatment interval. 3-Methylhistidine release from perfused rat hindquarters was also elevated after 5 days of vitamin treatment. To determine whether the adrenals were involved in mediating the above response, a study was conducted on adrenalectomized and sham-operated rats. Excessive vitamin A intake stimulated 3-methylhistidine excretion in vivo and in vitro in both adrenalectomized and sham-operated animals, thus suggesting that the vitamin A-induced acceleration in myofibrillar protein breakdown was not mediated by the adrenals. In both groups of rats, vitamin A treatment had no effect on the rate of protein synthesis, on the basis of incorporation in vitro of [3H]phenylalanine into muscle protein. Additional studies revealed that the addition of excess retinol to the perfusion medium (10 i.u./ml) had no significant effect on the rates of 3-methylhistidine release or [3H]phenylalanine incorporation in vitro. Finally, high doses of cortisol (7 mg/day per 100g body wt.) administered to intact rats for 5 days significantly increased rates of 3-methylhistidine excretion, both in vivo and in vitro.

Asunto(s)

RESUMEN

Myofibrillar protein degradation was measured in 4-week-old normal (line 412) and genetically muscular-dystrophic (line 413) New Hampshire chickens by monitoring the rates of 3-methylhistidine excretion in vivo and in vitro. A method of perfusing breast and wing muscles was developed and the rate of 3-methylhistidine release in vitro was measured between 30 and 90min of perfusion. During this perfusion period, 3-methylhistidine release from the muscle preparation was linear, indicating that changes in 3-methylhistidine concentration of the perfusate were the result of myofibrillar protein degradation. Furthermore, the viability of the perfused muscle was maintained during this interval. After 60min of perfusion, ATP, ADP and creatine phosphate concentrations in pectoral muscle were similar to muscle freeze-clamped in vivo. Rates of glucose uptake and lactate production were constant during the perfusion. In dystrophic-muscle preparations, the rate of 3-methylhistidine release in vitro (nmol/h per g of dried muscle) was elevated 2-fold when compared with that in normal muscle. From these data the fractional degradation rates of myofibrillar protein in normal and dystrophic pectoral muscle were calculated to be 12 and 24% respectively. Daily 3-methylhistidine excretion (nmol/day per g body wt.) in vivo was elevated 1.35-fold in dystrophic chickens. Additional studies revealed that the anti-dystrophic drugs diphenylhydantoin and methylsergide, which improve righting ability of dystrophic chickens, did not alter 3-methylhistidine release in vitro. This result implies that changes in myofibrillar protein turnover are not the primary lesion in avian muscular dystrophy. From tissue amino acid analysis, the myofibrillar 3-methylhistidine content per g dry weight of muscle was similar in normal and dystrophic pectoral muscle. More than 96% of the 3-methylhistidine present in pectoral muscle was associated with the myofibrillar fraction. Dystrophic myofibrillar protein contained significantly less 3-methylhistidine (nmol/g of myofibrillar protein) than protein from normal muscle. This observation supports the hypothesis that there may be a block in the biochemical maturation and development of dystrophic muscle after hatching. Free 3-methylhistidine (nmol/g wet wt.) was elevated in dystrophic muscle, whereas blood 3-methylhistidine concentrations were similar in both lines. In summary, the increased myofibrillar protein catabolism demonstrated in dystrophic pectoral muscle correlates with the increased lysosomal cathepsin activity in this tissue as reported by others.

Asunto(s)