Marc E Tischler

Interests

Teaching

Medical Biochemistry, Nutrition and Endocrinology

Courses

Scholarly Contributions

Chapters

• Tischler, M. E. (1992). CHAPTER 9 – Estimation of Protein Synthesis and Proteolysis in Vitro. In Modern Methods in Protein Nutrition and Metabolism(pp 225-248). doi:10.1016/B978-0-12-519570-6.50013-9

Journals/Publications

• Tischler, M. E., & Slentz, M. J. (2007). Impact of Weighlessness on Muscle Function. Gravitational and Space Research, 8(2).
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  The most studied skeletal muscles which depend on gravity, "antigravity" muscles, are located in the posterior portion of the legs. Antigravity muscles are characterized generally by a different fiber type composition than those which are considered nonpostural. The gravity-dependent function of the antigravity muscles makes them particularly sensitive to weightlessness (unweighting) resulting in a substantial loss of muscle protein, with a relatively greater loss of myofibrillar (structural) proteins. Accordingly a-actin mRNA decreases in muscle of rats exposed to microgravity. In the legs, the coleus seems particularly responsive to the lack of weight-bearing associated with space flight. The loss of muscle protein leads to a decreased cross-sectional area of muscle fibers, particularly of the slow-twitch, oxidative (SO) ones compared to fast-twitch glycolytic (FG) or oxidative-glycolytic (FOG) fibers. In some muscles, a shift in fiber composition from SO to FOG has been reported in the adaptation to spaceflight. Changes in muscle composition with spaceflight have been associated with decreased maximal isometric tension (P0) and increased maximal shortening velocity. In terms of fuel metabolism, results varied depending on the pathway considered. Glucose uptake, in the presence of insulin, and activities of glycolytic enzymes are increased by space flight. In contrast, oxidation of fatty acids may be diminished. Oxidation of pyruvate, activity of the citric acid cycle, and ketone metabolism in muscle seem to be unaffected by microgravity.
• O'Keefe, M. P., Perez, F. R., Sloniger, J. A., Tischler, M. E., & Henriksen, E. J. (2004). Enhanced insulin action on glucose transport and insulin signaling in 7-day unweighted rat soleus muscle. Journal of applied physiology (Bethesda, Md. : 1985), 97(1), 63-71.
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  Hindlimb suspension (HS), a model of simulated weightlessness, enhances insulin action on glucose transport in unweighted rat soleus muscle. In the present study, we tested the hypothesis that these changes in glucose transport in 3- and 7-day HS soleus of juvenile, female Sprague-Dawley rats were due to increased functionality of insulin signaling factors, including insulin receptor (IR), IR substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3-kinase), and Akt. Insulin-stimulated (2 mU/ml) glucose transport was significantly (P < 0.05) enhanced in 3- and 7-day HS soleus by 59 and 113%, respectively, compared with weight-bearing controls. Insulin-stimulated tyrosine phosphorylation of IR and Ser(473) phosphorylation of Akt was not altered by unweighting. Despite decreased (34 and 64%) IRS-1 protein in 3- and 7-day HS soleus, absolute insulin-stimulated tyrosine phosphorylation of IRS-1 was not diminished, indicating relative increases in IRS-1 phosphorylation of 62 and 184%, respectively. In the 7-day HS soleus, this was accompanied by increased (47%) insulin-stimulated IRS-1 associated with the p85 subunit of PI3-kinase. Interestingly, the enhanced insulin-stimulated glucose transport in the unweighted soleus was not completely inhibited (89-92%) by wortmannin, a PI3-kinase inhibitor. Finally, protein expression and activation of p38 MAPK, a stress-activated serine/threonine kinase associated with insulin resistance, was decreased by 32 and 18% in 7-day HS soleus. These results indicate that the increased insulin action on glucose transport in the 7-day unweighted soleus is associated with increased insulin signaling through IRS-1 and PI3-kinase and decreased p38 MAPK protein expression. However, PI3-kinase-independent mechanisms must also play a small role in this adaptive response to HS.
• O'keefe, M. P., Perez, F. R., Kinnick, T. R., Tischler, M. E., & Henriksen, E. J. (2004). Development of whole-body and skeletal muscle insulin resistance after one day of hindlimb suspension. Metabolism: clinical and experimental, 53(9), 1215-22.
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  Hindlimb suspension (HS) of rats is a model of simulated weightlessness and induces dynamic alterations in insulin action. In the present study, the effect of acute (1-day) HS on whole-body glucose tolerance and insulin action on skeletal muscle glucose transport was assessed in juvenile, female Sprague-Dawley rats. Compared to weight-bearing control rats, 1-day HS animals displayed significantly decreased glucose tolerance and diminished whole-body insulin sensitivity. Glucose transport activity in the 1-day unweighted soleus muscle was significantly decreased (P
• Tischler, M., Weinstein, R. B., Eleid, N., LeCesne, C., Durando, B., Crawford, J. T., Heffner, M., Layton, C., O'Keefe, M., Robinson, J., Rudinsky, S., Henriksen, E. J., & Tischler, M. E. (2002). Differential half-maximal effects of human insulin and its analogs for in situ glucose transport and protein synthesis in rat soleus muscle. Metabolism: clinical and experimental, 51(8).
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  Analogs of human insulin have been used to discriminate between responses of metabolic and mitogenic (growth-related) pathways. This study compared the stimulatory effects of human insulin (HI) and 2 analogs (X2, B-Asp(9), B-Glu(27) and H2, A-His(8),B-His(4),B-Glu(10), B-His(27)) on glucose uptake and protein synthesis in rat soleus muscle in situ. Glucose uptake, estimated by intramuscular (IM) injection of 2-deoxy[1,2-3H]glucose with or without insulin, was maximally increased at 10(-6) mol/L for HI and X2 and 10(-7) mol/L for H2. HI had a larger effect (318%) than either X2 (156%) or H2 (124%). The half-maximal effect (ED(50)) values for HI, X2, and H2 were 3.3 x10(-8) mol/L, 1.7 x 10(-7) mol/L, and 1.6 x 10(-9) mol/L, respectively. Protein synthesis, estimated by protein incorporation of [(3)H]phenylalanine injected into muscles with or without insulin, was maximally increased at 10(-5) mol/L for HI and 10(-6) for X2 and H2. HI had a larger effect in stimulating protein synthesis (34%) than either X2 (25%) or H2 (19.8%). The ED(50) values for HI, X2, and H2 were 3.0 x 10(-7) mol/L, 3.2 x 10(-7) mol/L, and 1.0 x 10(-9) mol/L, respectively. The biological potency of each analog (ED(50)insulin/ED(50)analog) showed X2 to be less potent than HI for both glucose uptake (0.2) and protein synthesis (0.9), whereas H2 is more potent than HI with ratios of 20 and 300, respectively. These data suggest that this approach for studying insulin responsiveness in a single muscle in situ may be a useful tool for investigating insulin signaling in muscle in vivo.
• Tischler, M. E., Satarug, S., Munoz, K. A., Henriksen, E. J., & Aannestad, A. (1997). Insulin attenuates atrophy of unweighted soleus muscle by amplified inhibition of protein degradation.. Metabolism: clinical and experimental, 46(6), 673-9. doi:10.1016/s0026-0495(97)90012-3
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  Unweighting atrophy of immature soleus muscle occurs rapidly over the first several days, followed by slower atrophy coinciding with increased sensitivity to insulin of in vitro protein metabolism. This study determined whether this increased sensitivity might account for the diminution of atrophy after 3 days of tall-cast hindlimb suspension. The physiological significance of the increased response to insulin in unweighted muscle was evaluated by analyzing in vivo protein metabolism for day 3 (48 to 72 hours) and day 4 (72 to 96 hours) of unweighting in diabetic animals either injected with insulin or not treated. Soleus from nontreated diabetic animals showed a similar loss of protein during day 3 (-16.2%) and day 4 (-14.5%) of unweighting, whereas muscle from insulin-treated animals showed rapid atrophy (-14.5%) during day 3 only, declining to just -3.1% the next day. Since fractional protein synthesis was similar for both day 3 (8.6%/d) and day 4 (7.0%/d) of unweighting in insulin-treated animals, the reduction in protein loss must be accounted for by a slowing of protein degradation due to circulating insulin. Intramuscular (IM) injection of insulin (600 nmol/L) stimulated in situ protein synthesis similarly in 4-day unweighted (+56%) and weight-bearing (+90%) soleus, even though unweighted muscle showed a greater in situ response of 2-deoxy-[3H]glucose uptake to IM injection of either insulin (133 nmol/L) or insulin-like growth factor-I (IGF-I) (200 nmol/L) than control muscle. These findings suggest that unweighted muscle is selectively more responsive in vivo to insulin, and that the slower atrophy after 3 days of unweighting was due to an increased effect of insulin on inhibiting protein degradation.
• Tischler, M., Weinstein, R. B., Slentz, M. J., Webster, K., Takeuchi, J. A., & Tischler, M. E. (1997). Lysosomal proteolysis in distally or proximally denervated rat soleus muscle. The American journal of physiology, 273(4 Pt 2).
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  We examined the mechanism of accelerated proteolysis in denervated rat soleus muscles. The soleus was denervated by severing either the tibial nerve (proximal, short stump) or sciatic nerve (distal, long stump) at 24, 48, 72, or 96 h before excision. Twenty-four hours after denervation, the extent of atrophy was similar for proximal and distal denervation, although lysosomal latency declined in both groups. After 48 and 72 h, denervation resulted in a decline in protein content, an increase in in vitro protein degradation, and a decline in lysosomal latency, all of which were greater in proximally denervated than in contralateral distally denervated muscles. These differences between acute responses of proximally and distally denervated muscles suggest the retention of some factor in the longer nerve stump that attenuates atrophy. After 96 h, total protein loss, protein degradation, and lysosomal latency were similar for proximal and distal denervation, suggesting the loss of axoplasmic flow from the long nerve stump.
• Toth, A., Tischler, M. E., Pal, M., & Johnson, P. C. (1996). Are there oxygen-deficient regions in resting skeletal muscle?. The American journal of physiology, 270(6 Pt 2), H1933-9. doi:10.1152/ajpheart.1996.270.6.h1933
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  The purpose of this study was to determine whether there are local regions in resting skeletal muscle in which the oxygen delivery is insufficient to support oxidative metabolism. This hypothesis was tested by stopping the blood supply to the exteriorized cat sartorius muscle for 5 min while monitoring NADH fluorescence in localized tissue areas 15-25 microns in diameter. A rise in fluorescence was taken to indicate a shift to anaerobic metabolism. Tissue sites in the arteriolar and venular regions of the capillary network were selected for study. After flow stoppage, fluorescence did not change for an average of 48 +/- 22 (SD) s and then rose over a period of 61 +/- 27 s to an average value 55 +/- 19% above control for arteriolar and venular sites combined. Fluorescence began to rise within 5 s of flow stasis in only 1 of 61 sites and within 10 s in 2 sites. There was no difference in the time course or magnitude of fluorescence changes at arteriolar and venular sites. The data indicate that in resting skeletal muscle, oxygen supply appears sufficient to support oxidative metabolism in over 95% of the tissue.
• Tischler, M., Wu, M., & Tischler, M. E. (1995). Utilization of [14C]phenylalanine derived from arylphorin or free amino acid in Manduca sexta pharate adults. Archives of insect biochemistry and physiology, 28(3).
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  The role of arylphorin as a storage protein was studied using 14C-arylphorin. 14C-arylphorin was produced optimally by incubating one-half fat body from Manduca sexta fifth instar larvae at 22 degrees C for 24 h, in 1 ml of medium containing amino acids at 25% of their physiological concentration with [U-14C]-phenylalanine (phe) provided initially without nonlabeled phenylalanine. Nonlabeled phe was provided after 1 h at 16% of its physiological concentration. The specific activity of 14C-arylphorin produced in vitro was 30 times greater than that generated in vivo. Injection of 14C-arylphorin into pharate adults was used to study the distribution of 14C-phe derived from this protein into 14CO2 and tissues for comparison with injection of free 14C-phe during the middle (days 6 to 12 pharate adult) and late (days 12 to 17 pharate adult) stages of adult development. Appearance of 14CO2 from 14C-arylphorin as compared to 14C-phenylalanine showed a slower time course during both the middle and late stages of development, in keeping with the time needed for degradation of the protein. In accord with faster phe turnover near the end of adult development, total 14CO2 production was greater and the retention of 14C in hemolymph and fat body was less compared to the middle stage of development regardless of whether 14C-arylphorin or 14C-phe was injected. In the middle stage of development, the appearance of 14C in the cuticle and head parts was greater, whereas incorporation into abdomen and thorax was less than during the late stage of development. Since the pattern of 14C distribution from 14C-arylphorin and 14C-phe was similar, one major function of arylphorin must be as a storage protein replenishing the supply of free amino acids used for synthesis of adult tissues. These results also suggest a limited contribution of M. sexta arylphorin to formation of the cuticle subsequent to day-6 pharate adult.
• Tischler, M., & Tischler, M. E. (1994). Effect of the antiglucocorticoid RU38486 on protein metabolism in unweighted soleus muscle. Metabolism: clinical and experimental, 43(11).
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  Unweighting the hindlimbs by tail suspension of juvenile rats leads to atrophy of the soleus muscle, especially during the third day of unweighting. Although a previous study using adrenalectomized animals suggested a minimal role of glucocorticoids in this atrophy, the inability of adrenalectomized animals to release other adrenal hormones could have been important. Therefore, the influence of oral administration of RU38486 [11-beta-(4-dimethylaminophenyl) 17-beta-hydroxy 17-alpha(prop-1-ynyl) estra 4,9-dien 3-one], a selective glucocorticoid antagonist, on protein metabolism in the unweighted soleus was studied. The effectiveness of RU38486 treatment was demonstrated in hindlimb-suspended rats as the drug abolished the increase in soleus glutamine synthetase activity shown previously to be caused by elevated circulating glucocorticoids. The slower weight gain of suspended rats was unaffected by the drug. After 3 days of unweighting, the difference in protein content from weighted soleus muscle was not diminished significantly by RU38486 (-25%, vehicle only; -18%, RU38486-treated). However, in both weight-bearing and suspended animals, RU38486 seemed to promote protein accretion between days 2 and 3 of the experiment; ie, unweighted muscle seemed to lose less protein. All suspended animals showed slower (-58% to -64%) fractional in vivo rates of synthesis. RU38486 did not affect these percent differences in fractional protein synthesis after either 2 or 3 days of unweighting. The apparent improvement in protein balance likely resulted from a decline in protein degradation in both the weight-bearing (-26%) and unweighted (-35%) soleus.(ABSTRACT TRUNCATED AT 250 WORDS)
• Tischler, M., Henriksen, E. J., Munoz, K. A., Aannestad, A. T., & Tischler, M. E. (1994). Cardiac protein content and synthesis in vivo after voluntary running or head-down suspension. Journal of applied physiology (Bethesda, Md. : 1985), 76(6).
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  The adaptive responses of myocardial protein metabolism to chronic increases in work load were evaluated in juvenile female Sprague-Dawley rats. Rats were studied under four conditions: normal weight bearing (N), voluntary wheel running (WR) for < or = 4 wk, head-down-tilt suspension for 7 days (HS), or wheel running (2 or 3 wk) followed by 7 days of suspension (WR-HS). WR activity plateaued after 2 wk at 16 km/day and was maintained through week 4. WR did not affect normal whole body growth. Protein metabolism was studied by measuring heart protein content and in vivo fractional rate of protein synthesis with the [3H]phenylalanine "flooding dose" method. Two weeks of WR increased (P < 0.05) absolute heart protein content (22%) and protein synthesis (21%) relative to age-matched N group values. These differences in protein content and synthesis were maintained for > or = 4 wk. Rats failed to gain significant body weight during suspension. Heart protein content increased (P < 0.05) by 12% to 26% as did protein synthesis (14% to 22%) in HS compared with N group. In WR-HS group, cardiac protein content and protein synthesis were maintained at significantly elevated levels. These findings indicate that 1) high-volume WR by young rats provides a convenient noninvasive method for producing rapid and substantial cardiac hypertrophy, which results, at least in part, from enhanced cardiac protein synthesis; and 2) head-down suspension of sedentary juvenile rats leads to increased cardiac protein synthesis, which helps to increase cardiac protein content despite a lack of whole body growth.
• Wyckoff, D., Tischler, M. E., Payne, C. M., Glasser, L., Fiederlein, R. L., Cromey, D. W., & Bohnert, O. (1994). Programmed cell death of the normal human neutrophil: an in vitro model of senescence.. Microscopy research and technique, 28(4), 327-44. doi:10.1002/jemt.1070280408
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  The present study provides experimental data which indicate that the neutrophil is ideal for studying programmed cell death or apoptosis in vitro. Neutrophils can be obtained from human peripheral blood in large numbers with minimal experimental manipulation and are easily separated from other leukocytes, providing nearly pure cell suspensions. The neutrophil life span in vitro is sufficiently short to allow observations to be made within eight hours after experimental manipulation. Neutrophils can also be easily maintained in serum-free, chemically defined media which can be systematically altered, thereby defining specific variables that influence the apoptotic process. Since the neutrophils do not need an exogenous trigger to undergo programmed cell death, it is also an excellent model to study senescence. It was determined from this study that neutrophils undergo apoptosis most efficiently at 37 degrees C, a temperature requirement for physiologic cell death. Neutrophils undergo apoptosis at a slightly faster rate and maintain membrane integrity better when incubated in a tissue culture medium (e.g., RPMI 1640) compared with a balanced salt solution (e.g., HBBB). Cycloheximide, an inhibitor of protein synthesis, was shown to accelerate apoptosis in a dose-dependent manner. The presence of Zn++ significantly decreased the rate of apoptosis, whereas the presence of Ca++ and Mg++ had no apparent effect. These studies indicate that the process of senescence, culminating in cell death, is subject to modulation by a variety of agents and experimental conditions. In addition, the ultrastructural features of neutrophils undergoing programmed cell death in vitro were compared in detail to those occurring in vivo and were found to be comparable.
• Tischler, M., Munoz, K. A., Satarug, S., & Tischler, M. E. (1993). Time course of the response of myofibrillar and sarcoplasmic protein metabolism to unweighting of the soleus muscle. Metabolism: clinical and experimental, 42(8).
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  Contributions of altered in vivo protein synthesis and degradation to unweighting atrophy of the soleus muscle in tail-suspended young female rats were analyzed daily for up to 6 days. Specific changes in myofibrillar and sarcoplasmic proteins were also evaluated to assess their contributions to the loss of total protein. Synthesis of myofibrillar and sarcoplasmic proteins was estimated by intramuscular (IM) injection and total protein by intraperitoneal (IP) injection of flooding doses of 3H-phenylalanine. Total protein loss was greatest during the first 3 days following suspension and was a consequence of the loss of myofibrillar rather than sarcoplasmic proteins. However, synthesis of total myofibrillar and sarcoplasmic proteins diminished in parallel beginning in the first 24 hours. Therefore sarcoplasmic proteins must be spared due to a decrease in their degradation. In contrast, myofibrillar protein degradation increased, thus explaining the elevated degradation of the total pool. Following 72 hours of suspension, protein synthesis remained low, but the rate of myofibrillar protein loss diminished, suggesting a slowing of degradation. These various results show (1) acute loss of protein during unweighting atrophy is a consequence of decreased synthesis and increased degradation of myofibrillar proteins, and (2) sarcoplasmic proteins are spared due to slower degradation, likely explaining the sparing of plasma membrane receptors. Based on other published data, we propose that the slowing of atrophy after the initial response may be attributed to an increased effect of insulin.
• Woodman, C. R., Tischler, M. E., Stump, C. S., Munoz, K. A., Kirby, C. R., & Henriksen, E. J. (1993). Elevated interstitial fluid volume in soleus muscles unweighted by spaceflight or suspension.. Journal of applied physiology (Bethesda, Md. : 1985), 75(4), 1650-3. doi:10.1152/jappl.1993.75.4.1650
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  Recent evidence by Kandarian et al. (J. Appl. Physiol. 71: 910-914, 1991) indicates that prolonged (28 days) unweighting of the rat soleus by hindlimb suspension results in a substantial increase in interstitial fluid volume (IFV), as defined by the inulin space. The lack of any significant difference in absolute IFV values between unweighted and control groups suggested that this elevated IFV was a consequence of muscle atrophy. Using young female rats, we directly tested this hypothesis by comparing the early responses of soleus muscle weight and IFV with unweighting by tail-cast suspension or actual exposure to microgravity during spaceflight. Significant differences from control were first observed after 3 days of suspension unweighting for soleus wet weight (-14%; P < 0.01) and IFV (+35%; P < 0.01) and increased further after 6 days (-32% and +53%, respectively; both P < 0.001). After 5.4 days of spaceflight, soleus wet weight was 38% less and IFV was 52% greater than control (both P < 0.001). A highly significant negative correlation between soleus wet weight and IFV for all groups was observed (r = -0.70, P < 0.001). These data indicate that elevated soleus IFV develops at an early time point during unweighting and that there is a direct relationship between the magnitude of this increase in IFV and the extent of muscle atrophy. This relationship also exists in soleus muscles unweighted by exposure to a microgravity environment.
• Woodman, C. R., Tischler, M. E., Stump, C. S., Munoz, K. A., Kirby, C. R., & Henriksen, E. J. (1993). Spaceflight on STS-48 and earth-based unweighting produce similar effects on skeletal muscle of young rats.. Journal of applied physiology (Bethesda, Md. : 1985), 74(5), 2161-5. doi:10.1152/jappl.1993.74.5.2161
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  Our knowledge of the effects of unweighting on skeletal muscle of juvenile rapidly growing rats has been obtained entirely by using hindlimb-suspension models. No spaceflight data on juvenile animals are available to validate these models of simulated weightlessness. Therefore, eight 26-day-old female Sprague-Dawley albino rats were exposed to 5.4 days of weightlessness aboard the space shuttle Discovery (mission STS-48, September 1991). An asynchronous ground control experiment mimicked the flight cage condition, ambient shuttle temperatures, and mission duration for a second group of rats. A third group of animals underwent hindlimb suspension for 5.4 days at ambient temperatures. Although all groups consumed food at a similar rate, flight animals gained a greater percentage of body mass per day (P < 0.05). Mass and protein data showed weight-bearing hindlimb muscles were most affected, with atrophy of the soleus and reduced growth of the plantaris and gastrocnemius in both the flight and suspended animals. In contrast, the non-weight-bearing extensor digitorum longus and tibialis anterior muscles grew normally. Earlier suspension studies showed that the soleus develops an increased sensitivity to insulin during unweighting atrophy, particularly for the uptake of 2-[1,2-3H]deoxyglucose. Therefore, this characteristic was studied in isolated muscles within 2 h after cessation of spaceflight or suspension. Insulin increased uptake 2.5- and 2.7-fold in soleus of flight and suspended animals, respectively, whereas it increased only 1.6-fold in control animals. In contrast, the effect of insulin was similar among the three groups for the extensor digitorum longus, which provides a control for potential systemic differences in the animals.(ABSTRACT TRUNCATED AT 250 WORDS)
• Tischler, M., Kirby, C. R., Woodman, C. R., Woolridge, D., & Tischler, M. E. (1992). Cyclic adenosine monophosphate accumulation and beta-adrenergic binding in unweighted and denervated rat soleus muscle. Metabolism: clinical and experimental, 41(7).
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  Unweighting, but not denervation, of muscle reportedly "spares" insulin receptors, increasing insulin sensitivity. Unweighting also increases beta-adrenergic responses of carbohydrate metabolism. These differential characteristics were studied further by comparing cyclic adenosine monophosphate (cAMP) accumulation and beta-adrenergic binding in normal and 3-day unweighted or denervated soleus muscle. Submaximal amounts of isoproterenol, a beta-agonist, increased cAMP accumulation in vitro and in vivo (by intramuscular [IM] injection) to a greater degree (P less than .05) in unweighted muscles. Forskolin or maximal isoproterenol had similar in vitro effects in all muscles, suggesting increased beta-adrenergic sensitivity following unweighting. Increased sensitivity was confirmed by a greater receptor density (Bmax) for [125I]iodo-(-)-pindolol in particulate preparations of unweighted (420.10(-18) mol/mg muscle) than of control or denervated muscles (285.10(-18) mol/mg muscle). The three dissociation constant (Kd) values were similar (20.3 to 25.8 pmol/L). Total binding capacity (11.4 fmol/muscle) did not change during 3 days of unweighting, but diminished by 30% with denervation. This result illustrates the "sparing" and loss of receptors, respectively, in these two atrophy models. In diabetic animals, IM injection of insulin diminished cAMP accumulation in the presence of theophylline in unweighted muscle (-66% +/- 2%) more than in controls (-42% +/- 6%, P less than .001). These results show that insulin affects cAMP formation in muscle, and support a greater in vivo insulin response following unweighting atrophy. These various data support a role for lysosomal proteolysis in denervation, but not in unweighting, atrophy.
• Toth, A., Tischler, M. E., Pal, M., Koller, A., & Johnson, P. C. (1992). A multipurpose instrument for quantitative intravital microscopy.. Journal of applied physiology (Bethesda, Md. : 1985), 73(1), 296-306. doi:10.1152/jappl.1992.73.1.296
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  An in vivo microscope system has been developed that can measure fluorescence emission and/or light absorption at up to five wavelengths in a tissue area of 18-30 microns diam while imaging adjacent microcirculatory vessels with a video system. The system also incorporates a computer-controlled stage and data acquisition system for rapid and repeated measurements from a number of tissue sites. The tissue area monitored for fluorescence or absorption can be defined further by a confocal arrangement of the microscope optics. Tests of the system for NADH fluorescence measurements show good agreement between the fluorescence at 450 nm and NADH concentration in vitro and in skeletal muscle. The instrument can also be used simultaneously for spectrophotometric determination of O2 saturation and hematocrit in microcirculatory vessels. In vitro tests indicate suitable accuracy for such measurements. The open architecture and modular arrangement of the instrument facilitates its use for a variety of simultaneous measurements of parenchymal cell and microcirculatory function.
• Tischler, M. E., Tome, M. E., Tischler, M. E., Rosenberg, S., Kirby, C. R., & Chase, P. B. (1991). Mechanisms of accelerated proteolysis in rat soleus muscle atrophy induced by unweighting or denervation.. The Physiologist, 34(1 Suppl), S177-8.
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  A hypothesis proposed by Tischler and coworkers (Henriksen et al., 1986; Tischler et al., 1990) concerning the mechanisms of atrophy induced by unweighting or denervation was tested using rat soleus muscle from animals subjected to hindlimb suspension and denervation of muscles. The procedure included (1) measuring protein degradation in isolated muscles and testing the effects of lysosome inhibitors, (2) analyzing the lysosome permeability and autophagocytosis, (3) testing the effects of altering calcium-dependent proteolysis, and (4) evaluating in vivo the effects of various agents to determine the physiological significance of the hypothesis. The results obtained suggest that there are major differences between the mechanisms of atrophies caused by unweighting and denervation, though slower protein synthesis is an important feature common for both.
• Tischler, M., Munoz, K. A., & Tischler, M. E. (1991). The effect of a space food bar diet on body and muscle mass in normal and hind-limb suspended rats. Aviation, space, and environmental medicine, 62(9 Pt 1).
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  A food bar diet is used for rats in space flight. Since ground based studies have only been performed with the typical rat chow in dry pellet form, we tested whether the food bar diet allows normal growth and normal response of muscle protein content to unloading. These parameters were measured in normal and tail-cast hind limb suspended rats fed standard pellets or food bars. Body mass following 5 d of hind limb unloading was similar in bar-fed (97.9 +/- 4.8 g) and pellet-fed (92.6 +/- 3.4 g) animals (p greater than 0.05). In addition, gains in body mass were comparable between bar-fed (5.3 g/d) and pellet-fed (5.1 g/d) animals. Food bar consumption over 6 d increased from 10.5 to 12.0 g/d animal. During 5 d of hind limb suspension, food bar consumption increased from 13.2 +/- 1.4 to 19.1 +/- 1.4 g/d per animal. In agreement with previous studies, hind limb unloading reduced soleus muscle mass and protein content per 100 g body mass in both diet groups (p less than 0.05). Protein content per 100 g body mass was unchanged for the plantaris, extensor digitorum longus and tibialis anterior muscles during suspension in both diet groups. Rodent consumption of a food bar diet results in normal gains in body mass and muscle protein when compared to a standard pellet diet, and does not alter the atrophic response of skeletal muscle to unloading.
• Tischler, M. E., Henriksen, E. J., Tome, M. E., Tischler, M. E., Satarug, S., Rosenberg, S., Kirby, C. R., Henriksen, E. J., & Chase, P. B. (1990). Different mechanisms of increased proteolysis in atrophy induced by denervation or unweighting of rat soleus muscle.. Metabolism: clinical and experimental, 39(7), 756-63. doi:10.1016/0026-0495(90)90113-q
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  Mechanisms of accelerated proteolysis were compared in denervated and unweighted (by tail-cast suspension) soleus muscles. In vitro and in vivo proteolysis were more rapid and lysosomal latency was lower in denervated than in unweighted muscle. In vitro, lysosomotropic agents (eg, chloroquine, methylamine) did not lessen the increase in proteolysis caused by unweighting, but abolished the difference in proteolysis between denervated and unweighted muscle. Leucine methylester, an indicator of lysosome fragility, lowered latency more in denervated than in unweighted muscle. 3-Methyladenine, which inhibits phagosome formation, increased latency similarly in all muscles tested. Mersalyl, a thiol protease inhibitor, and 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which antagonizes sarcoplasmic reticulum release of Ca2+, reduced accelerated proteolysis caused by unweighting without diminishing the faster proteolysis due to denervation. Calcium ionophore (A23187) increased proteolysis more so in unweighted than control muscles whether or not Ca2+ was present. Different mechanisms of accelerated proteolysis were studied further by treating muscles in vivo for 24 hours with chloroquine or mersalyl. Chloroquine diminished atrophy of the denervated but not the unweighted muscle, whereas mersalyl prevented atrophy of the unweighted but not of the denervated muscle, both by inhibiting in vivo proteolysis. These results suggest that (1) atrophy of denervated, but not of unweighted, soleus muscle involves increased lysosomal proteolysis, possibly caused by greater permeability of the lysosome, and (2) cytosolic proteolysis is important in unweighting atrophy, involving some role of Ca2(+)-dependent proteolysis and/or thiol proteases.
• Tischler, M. E., Satarug, S., Rosenberg, S. B., Henriksen, E. J., & Eisenfeld, S. H. (1990). Insulin effects in denervated and non-weight-bearing rat soleus muscle.. Muscle & nerve, 13(7), 593-600. doi:10.1002/mus.880130706
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  Previous reports indicated that glucose uptake in denervated muscle is resistant to insulin, while in non-weight-bearing (unweighted) muscle this effect of insulin is enhanced. To extend the comparison of these differences, insulin effects on amino acid uptake and protein metabolism were studied in soleus muscles subjected to denervation or unweighting. Denervated muscle showed insulin resistance of both 2-deoxy[1,2-3H]glucose and alpha-[methyl-3H]aminoisobutyric acid uptake whereas unweighted muscle showed an increased or normal response, respectively. Atrophy was greater in denervated than in unweighted muscle, apparently due to faster protein degradation. The stimulation of protein synthesis and the inhibition of protein degradation by insulin was generally less in denervated than in unweighted muscle. Since metabolic measurements in denervated-unweighted muscles did not differ from those in denervated-weight-bearing muscles, effects of denervation must be independent of leg posture.
• Tischler, M., Kirby, C. R., & Tischler, M. E. (1990). Beta-adrenergic effects on carbohydrate metabolism in the unweighted rat soleus muscle. Journal of applied physiology (Bethesda, Md. : 1985), 69(6).
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  The effects of insulin on carbohydrate metabolism in atrophied rat soleus muscle are increased after unweighting by tail-cast suspension. This work has been extended by testing the effect of unweighting on the response of carbohydrate metabolism to isoproterenol, a beta-adrenergic agonist. Isoproterenol promoted glycogen degradation more in the unweighted than in the weight-bearing soleus but showed no differences in the extensor digitorum longus, which is unresponsive to hindlimb unweighting. In soleus muscles depleted of glycogen, to avoid varied inhibitory effects of glycogen on glycogen synthesis, isoproterenol inhibited this process more in the unweighted muscle. Isoproterenol did not have a greater inhibitory effect on net uptake of 2-deoxy-D[1,2-3H]glucose by the unweighted muscle. Measurements of intracellular 2-deoxy-[3H]glucose 6-phosphate and 3-O-methyl-D-[1-3H]glucose, which cannot be phosphorylated, showed that isoproterenol inhibited glucose phosphorylation but not transport. This effect could be explained by an increase of glucose 6-phosphate, an inhibitor of hexokinase. At 100 microU insulin/ml but not at a lower amount (10 microU/ml), isoproterenol inhibited hexose phosphorylation more in the control than in the unweighted muscle. This result may be explained by greater insulin antagonism in the unweighted muscle owing to increased insulin sensitivity. However, insulin antagonism of isoproterenol stimulation of glycogenolysis or inhibition of glycogenesis was not altered by unweighting. Therefore, for some aspects of carbohydrate metabolism, the unweighted muscle has an increased response to beta-adrenergic activation, just as this muscle shows increased responses to insulin.
• Wu, M., Tischler, M. E., Hodsden, S., & Cook, P. (1990). Ecdysteroids affect in vivo protein metabolism of the flight muscle of the tobacco hornworm (Manduca sexta).. Journal of insect physiology, 36(10), 699-708. doi:10.1016/0022-1910(90)90043-f
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  Ecdysteroid growth promotion of the dorsolongitudinal flight muscle of Manduca sexta was studied by measuring in vivo protein metabolism using both "flooding-dose" and "non-carrier" techniques. These procedures differ in that the former method includes injection of non-labelled phenylalanine (30 micromoles/insect) together with the [3H]amino acid. Injected radioactivity plateaued in the haemolymph within 7 min. With the flooding-dose method, haemolymph and intramuscular specific radioactivities were similar between 15 min and 2 h. Incorporation of [3H]phenylalanine into muscle protein was linear with either method between 30 and 120 min. Fractional rates (%/12 h) of synthesis with the flooding-dose technique were best measured after 1 h because of the initial delay in radioactivity equilibration. Estimation of body phenylalanine turnover with the non-carrier method showed 24-53%/h which was negligible with the flooding-dose method. Since the two methods yielded similar rates of protein synthesis, the large injection of non-labelled amino acid did not alter the rate of synthesis. Because the flooding-dose technique requires only a single time point measurement, it is the preferred method. The decline and eventual cessation of flight-muscle growth was mostly a consequence of declining protein synthesis though degradation increased between 76-86 h before eclosion and was relatively rapid. This decline in muscle growth could be prevented by treating pupae with 20-hydroxyecdysone (10 micrograms/insect). Protein accretion was promoted by a decline of up to 80% in protein breakdown, which was offset in part by a concurrent though much smaller decrease in protein synthesis. Therefore, ecdysteroids may increase flight-muscle growth by inhibiting proteolysis.
• Tischler, M., Fagan, J. M., & Tischler, M. E. (1989). Effects of oxygen deprivation on incubated rat soleus muscle. Life sciences, 44(10).
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  Isolated soleus muscle deprived of oxygen produces more lactate and alanine than oxygen-supplied muscle. Oxygenated muscle synthesized glutamine, while anoxic muscle used this amino acid. Oxygen deprivation decreased adenine nucleotides leading to the efflux of nucleosides. Protein synthesis and degradation responded differently to anoxia. Synthesis almost completely ceased, while proteolysis increased. Therefore, protein degradation in soleus muscle is enhanced when energy supplies and oxygen tension are low.
• Tischler, M., Henriksen, E. J., Kirby, C. R., & Tischler, M. E. (1989). Glycogen supercompensation in rat soleus muscle during recovery from nonweight bearing. Journal of applied physiology (Bethesda, Md. : 1985), 66(6).
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  The time course of glycogen changes in soleus muscle recovering from 3 days of nonweight bearing by hindlimb suspension was investigated. Within 15 min and up to 2 h, muscle glycogen decreased. Coincidentally, muscle glucose 6-phosphate and the fractional activity of glycogen phosphorylase, measured at the fresh muscle concentrations of AMP, increased. Increased fractional activity of glycogen synthase during this time was likely the result of greater glucose 6-phosphate and decreased glycogen. From 2 to 4 h, when the synthase activity remained elevated and the phosphorylase activity declined, glycogen levels increased (glycogen supercompensation). A further increase of glycogen up to 24 h did not correlate with the enzyme activities. Between 24 and 72 h, glycogen decreased to control values, possibly initiated by high phosphorylase activity at 24 h. At 12 and 24 h, the inverse relationship between glycogen concentration and the synthase activity ratio was lost, indicating that reloading transiently uncoupled glycogen control of this enzyme. These data suggest that the activities of glycogen synthase and phosphorylase, when measured at physiological effector levels, likely provide the closest approximation to the actual enzyme activities in vivo. Measurements made in this way effectively explained the majority of the changes in the soleus glycogen content during recovery from nonweight bearing.
• Tischler, M., Jaspers, S. R., Henriksen, E. J., Satarug, S., & Tischler, M. E. (1989). Effects of stretching and disuse on amino acids in muscles of rat hind limbs. Metabolism: clinical and experimental, 38(4).
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  Effects of stretching on muscle amino acids were tested in unloaded soleus by casting the foot in dorsiflexion on one limb of tail-casted, hindquarter-suspended rats. For comparison with unloading, amino acids also were measured in shortened extensor digitorum longus (EDL) in the same casted limb and in denervated leg muscles. Concentrations of tyrosine and glutamate were lower, while aspartate, ammonia, and the ratio of glutamine to glutamate were greater in the stretched than in the freely moving, unloaded soleus, but stretched did not differ from weight-bearing, control muscle. Therefore, stretching the soleus muscle prevented changes in certain amino acids due to unloading. Aspartate, ammonia, glutamine, and the ratio of glutamine to glutamate were lower in the shortened EDL than in the freely moving muscle of the contralateral limb, or in the control muscle. When denervated, these leg muscles also showed lower aspartate, ammonia, and ratio of glutamine to glutamate relative to innervated muscles. Since muscle shortening or denervation produced amino acid changes that mimicked the effects of unloading on the soleus, these responses must reflect the effect of muscle disuse. These data suggested that lower ammonia might cause the lower ratio of glutamine to glutamate with disuse. Because the fresh muscle energy charge, one factor which controls AMP deaminase, generally was not affected by disuse, altered deamination of glutamate via glutamate dehydrogenase may explain the variations in muscle ammonia.(ABSTRACT TRUNCATED AT 250 WORDS)
• Tischler, M., Jaspers, S. R., Henriksen, E., Jacob, S., & Tischler, M. E. (1989). Metabolism of branched-chain amino acids in leg muscles from tail-cast suspended intact and adrenalectomized rats. Metabolism: clinical and experimental, 38(2).
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  Degradation of branched-chain amino acids was studied in muscles of unloaded hind limbs from rats subjected to six days of tail-cast suspension. The total production of 14CO2 from uniformly labeled 14C-leucine, isoleucine, or valine, and the fluxes through leucine aminotransferase and alpha-ketoisocaproate dehydrogenase, which were measured using L-1-14C-leucine, were generally greater in the soleus and extensor digitorum longus muscles of unloaded than of weight-bearing hind limbs. Adrenalectomy abolished any difference in flux through the aminotransferase, whereas the administration of cortisol to adrenalectomized animals restored the greater flux in the unloaded soleus muscle. Adrenalectomy partially diminished the greater flux through alpha-ketoisocaproate dehydrogenase in the unloaded soleus, whereas cortisol (2 mg/100 g body weight) treatment increased this difference. In the extensor digitorum longus, adrenalectomy abolished the differences in both enzyme fluxes due to hind limb suspension. In this muscle, cortisol treatment increased these fluxes to a similar extent in both weight-bearing and suspended, adrenalectomized animals so that the normal difference was not restored. These results suggest that leucine catabolism in hind limb muscles of suspended rats was influenced primarily by increased circulating glucocorticoid hormones, which are elevated twofold to fourfold in these animals.
• Wu, M., Tischler, M. E., Mccready, S., Hodsden, S., & Cook, P. (1989). Ecdysteroids influence growth of the dorsolongitudinal flight muscle in the tobacco hornworm (Manduca sexta). Journal of Insect Physiology, 35(12), 1017-1022. doi:10.1016/0022-1910(89)90025-5
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  Abstract We have considered whether the decline of the ecdysteroid titre in the latter stages of adult Manduca development may induce cessation of growth of the flight muscle. Specific stages [Stages 6–13; Schwartz and Truman, Dev. Biol. 99 , 103–114 (1983)] during the last 100 h of development were timed to allow construction of a growth curve for the dorsolongitudinal flight muscle. Pupa weight and muscle mass were unchanged in successive stages. The muscle ratio of wet weight to dry weight and the extracellular volume declined during these stages, reflecting loss of fluids. A growth curve plotted as total muscle protein content against the time before eclosion showed a gradual decline in the growth rate from 150 μg protein/h during Stage 6 to 50 μg protein/h during Stage 11. Growth ceased during Stage 12. Injection of 20-hydroxyecdysone (10 μg/insect) at Stage 10 or Stage 11 significantly increased the total muscle protein accretion by 6- and 3-fold, respectively, during these stages. However, 20-hydroxyecdysone failed to promote growth during Stage 12. A dose curve showed that 5 μg ecdysteroid/g insect produced a near maximal response. These results suggest that the fall of ecdysteroid during adult development leads to reduced growth of the flight muscle and that shortly before eclosion this muscle becomes unresponsive to these hormones.
• Tischler, M. E., & Henriksen, E. J. (1988). Glucose uptake in rat soleus: effect of acute unloading and subsequent reloading.. Journal of applied physiology (Bethesda, Md. : 1985), 64(4), 1428-32. doi:10.1152/jappl.1988.64.4.1428
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  The effect of acutely reduced weight bearing (unloading) on the in vitro uptake of 2-[1,2-3H]deoxy-D-glucose was studied in the soleus muscle by tail casting and suspending rats. After just 4 h, the uptake of 2-deoxy-D-glucose fell (-19%, P less than 0.01) and declined further after an additional 20 h of unloading. This diminution at 24 h was associated with slower oxidation of [14C]glucose and incorporation of [14C]glucose into glycogen. Unlike after 1 day, at 3 days of unloading basal uptake of 2-deoxy-D-glucose did not differ from control. Reloading of the soleus after 1 or 3 days of unloading increased uptake of 2-deoxy-D-glucose above control and returned it to normal within 6 h and 4 days, respectively. These effects of unloading and recovery were caused by local changes in the soleus, because the extensor digitorum longus from the same hindlimbs did not display any alterations in uptake of 2-deoxy-D-glucose or metabolism of glucose. This study demonstrates that alterations in contractile activity, brought about by unloading or recovery from unloading, can influence the regulation of glucose transport in the soleus.
• Tischler, M. E., Satarug, S., Henriksen, E. J., & Furst, P. (1988). Responses of lysosomal and non-lysosomal proteases to unloading of the soleus.. Advances in experimental medicine and biology, 240, 235-42. doi:10.1007/978-1-4613-1057-0_28
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  Atrophy of skeletal muscle can be induced by a number of interventions, including denervation of the hindlimb,1 hindlimb immobilization,2 and total mechanical unloading by either tail-cast suspension,3harness suspension,4 or exposure to microgravity. 5One probable causal factor for this loss of muscle mass is an increased rate of degradation of myofibrillar proteins. When coupled with an unchanged or decreased rate of protein synthesis, this leads to a net protein breakdown and thus muscle atrophy. While the exact mechanism for this intramuscular protein breakdown is still unclear,6 it is most likely due to either an increase in the proteolytic capacity of the muscle cell, an increased susceptibility of substrate proteins to proteolytic enzymes, or a combination of the two.6
• Tischler, M. E., Satarug, S., Jacob, S., Henriksen, E. J., & Cook, P. (1988). Problems in analysis of data from muscles of rats flown in space.. The Physiologist, 31(1 Suppl), S10-3.
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  Comparison of hindlimb muscles of rats flown on Spacelab-3 or tail-traction-suspended showed that 11-17 h reloading post-flight might have altered the results. Soleus atrophied, plantaris, gastrocnemius and extensor digitorum longus grew slower, and tibialis anterior grew normally. In both flight and simulated soleus and plantaris, higher tyrosine and greater glutamine/glutamate ratio indicated negative protein balance and increased glutamine production, respectively, relative to controls. Aspartate was lower in these muscles. Reloading generally decreased tyrosine, but increased aspartate and glutamine/glutamate. These data showed that at 12 h of reloading after flight is characterized by reversal to varying extents of effects of unloading.
• Tischler, M., Henriksen, E. J., & Tischler, M. E. (1988). Time course of the response of carbohydrate metabolism to unloading of the soleus. Metabolism: clinical and experimental, 37(3).
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  The time course of the response of carbohydrate metabolism to unloading was studied in the soleus muscle of rats subjected to tail-cast suspension. In the fresh soleus, just 12 hours of unloading led to higher concentrations of glycogen and lower activity ratios of both glycogen synthase and glycogen phosphorylase. These changes were still evident on day 3. This initial accumulation of glycogen was likely due to its decreased degradation in response to muscle disuse. Thereafter, the increased glycogen concentration apparently diminished the activity ratio of glycogen synthase, leading to a subsequent fall in the total glycogen content after day 1. After 24 hours of unloading, when no significant atrophy was detectable, there was no differential response to insulin for in vitro glucose metabolism. As reported for day 6 (reference 6), on day 3 the soleus atrophied significantly and displayed a greater sensitivity to insulin for most of these parameters compared to the weight-bearing control muscle. However, insulin sensitivity for glycogen synthesis was unchanged. These results showed that the increased sensitivity to insulin of the unloaded soleus is associated with the degree of muscle atrophy, likely due to an increased insulin binding capacity relative to muscle mass. This study also showed that insulin regulation of glucose uptake and of glycogen synthesis is affected differentially in the unloaded soleus muscle.
• Tischler, M., Jaspers, S. R., & Tischler, M. E. (1988). Insulin effect on amino acid uptake by unloaded rat hindlimb muscles. Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et métabolisme, 20(2).
• Tischler, M., Jaspers, S. R., Fagan, J. M., Satarug, S., Cook, P. H., & Tischler, M. E. (1988). Effects of immobilization on rat hind limb muscles under non-weight-bearing conditions. Muscle & nerve, 11(5).
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  Dorsiflexion of one unloaded hind limb caused hypertrophy of the soleus relative to weight-bearing controls and faster growth of the plantaris and gastrocnemius muscles relative to the contralateral freely moving muscles. Unloading of the soleus muscle diminished primarily myofibrillar proteins whereas stretching increased all proteins. Stretching the soleus increased RNA, accelerated, especially, in vitro synthesis of sarcoplasmic proteins, and diminished in vitro proteolysis. Both in vivo and in vitro results showed slower synthesis and faster degradation in the freely moving than in the weight-bearing soleus muscle, faster synthesis and slower degradation in the stretched than in the freely moving soleus muscle, and faster degradation in the stretched than in the weight-bearing soleus muscle. Hence, stretching of the soleus muscle prevented changes in mass and protein metabolism produced by unloading. Shortening of the extensor digitorum longus muscle produced less muscle growth, slowed in vitro protein synthesis, and lowered RNA relative to the contralateral, freely moving muscle.
• Tischler, M. E., Satarug, S., Fagan, J. M., & Cook, P. (1987). Rat muscle protein turnover and redox state in progressive diabetes.. Life sciences, 40(8), 783-90. doi:10.1016/0024-3205(87)90306-7
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  Protein synthesis and degradation, and redox state were measured in soleus and extensor digitorum longus muscles of rats up to 12 days after injection of streptozotocin. Muscle growth was slower in these animals apparently due to slower protein synthesis throughout the duration of diabetes. Up to day 4 after injection of streptozotocin or withdrawal of insulin from treated, diabetic animals, the muscle ratio of lactate/pyruvate, an indicator of the cytoplasmic NAD+ redox couple, was lower and protein degradation was faster than in control muscles. Thereafter, the ratio of lactate/pyruvate was greater and protein degradation was slower than in size- or age-matched control muscles. Insulin treatment in vitro or in vivo increased lactate/pyruvate and decreased proteolysis. Therefore, in muscles of streptozotocin-diabetic rats, the initial increase and later fall in proteolysis, and the inhibition of proteolysis by insulin, may correlate with opposite changes in NADH/NAD+.
• Tischler, M. E., & Fagan, J. M. (1986). Reduction-oxidation state and protein degradation in skeletal muscle of fasted and refed rats.. The Journal of nutrition, 116(10), 2028-33. doi:10.1093/jn/116.10.2028
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  Redox state and protein degradation were measured in isolated muscles of fasted (up to 10 d) and refed (up to 4 d) 7- to 14-wk-old rats. Protein degradation in the extensor digitorum longus muscle, but not in the soleus muscle, was greater in the fasted rats than in weight-matched muscle from fed rats. The NAD couple was more oxidized in incubated and fresh extensor digitorum longus muscles and in some incubated soleus muscles of fasted rats than in weight-matched muscle from fed rats. In the extensor digitorum longus muscle of refed or prolonged fasted rats, protein degradation was slower and the NAD couple was more reduced than in the fed state. Therefore, oxidation of the NAD couple was associated with increased muscle breakdown during fasting, whereas reduction of the NAD couple was associated with muscle conservation and deposition.
• Tischler, M. E., & Jaspers, S. R. (1986). Role of glucocorticoids in the response of rat leg muscles to reduced activity.. Muscle & nerve, 9(6), 554-61. doi:10.1002/mus.880090613
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  Adrenalectomy did not prevent atrophy of rat soleus muscle during 6 days of tail cast suspension. Cortisol treatment enhanced this atrophy and caused atrophy of the weight-bearing soleus and both extensor digitorum longus (EDL) muscles. Unloading led to increased sarcoplasmic protein concentration in the soleus, but cortisol administration increased the myofibrillar (+stromal) protein concentration in both muscles. Suspension of hindlimbs of adrenalectomized animals led to faster protein degradation, slower sarcoplasmic protein synthesis, and faster myofibrillar protein synthesis in the isolated soleus, whereas with cortisol-treated animals, the difference in synthesis of myofibrillar proteins was enhanced and that of sarcoplasmic proteins was abolished. Both soleus and EDL of suspended, cortisol-treated animals showed faster protein degradation. It is unlikely that any elevation in circulating glucocorticoids was solely responsible for atrophy of the soleus in this model, but catabolic amounts of glucocorticoids could alter the response of muscle to unloading.
• Tischler, M. E., Jaspers, S. R., & Jacob, S. (1986). Metabolism of amino acids by the atrophied soleus of tail-casted, suspended rats.. Metabolism: clinical and experimental, 35(3), 216-23. doi:10.1016/0026-0495(86)90204-0
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  Amino acid metabolism was investigated in atrophied soleus muscle from rats subjected to six days of tail-cast, hindlimb suspension. The fresh-frozen unloaded muscle showed higher concentrations of tyrosine and glutamate but lower amounts of aspartate, glutamine, ammonia, and a lower ratio of glutamine to glutamate than normal muscle. The atrophied muscle also showed faster in vitro production of alanine and tyrosine, and slower utilization of glutamate and aspartate. Despite a greater activity of glutamine synthetase, synthesis of glutamine was slower in the soleus muscle of suspended rats than in control muscle. Provision of ammonium chloride and/or glutamate showed that this slower synthesis of glutamine in the atrophied soleus probably was due to limiting amounts of free ammonia and not of glutamate. Flux through AMP deaminase was probably slower as demonstrated by the maintenance of a greater pool of total adenine nucleotides and by the slower release of nucleosides by the incubated soleus muscle of suspended v control rats. The extensor digitorum longus muscles of suspended animals showed greater glutamine production, glutamine synthetase activity, and aspartate utilization than control muscles. Data from muscles of intact, adrenalectomized and adrenalectomized, cortisol-treated rats suggested that the greater glutamine synthetase activity was mediated possibly by higher circulating glucocorticoid hormones and a greater response of the soleus muscle to these hormones. Glutamine synthesis in skeletal muscle may be regulated primarily by the availability of ammonia, which is associated with the degradation of adenine nucleotides, and secondarily by the amount of glutamine synthetase and glutamate in the tissue.
• Tischler, M. E., Johnson, D., & Henriksen, E. J. (1986). Increased response to insulin of glucose metabolism in the 6-day unloaded rat soleus muscle. Journal of Biological Chemistry, 261(23), 10707-10712. doi:10.1016/s0021-9258(18)67443-5
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  Hind leg muscles of female rats (85-99 g) were unloaded by tail cast suspension for 6 days. In the fresh-frozen unloaded soleus, the significantly greater concentration of glycogen correlated with a lower activity ratio of glycogen phosphorylase (p less than 0.02). The activity ratio of glycogen synthase also was lower (p less than 0.001), possibly due to the higher concentration of glycogen. In isolated unloaded soleus, insulin (0.1 milliunit/ml) increased the oxidation of D-[U-14C]glucose, release of lactate and pyruvate, incorporation of D-[U-14C]glucose into glycogen, and the concentration of glucose 6-phosphate more (p less than 0.05) than in the weight-bearing soleus. At physiological doses of insulin, the percent of maximal uptake of 2-deoxy-D-[1,2-3H]glucose/muscle also was greater in the unloaded soleus. Unloading of the soleus increased by 50% the concentration of insulin receptors, due to no decrease in total receptor number during muscle atrophy. This increase may account for the greater response of glucose metabolism to insulin in this muscle. The extensor digitorum longus, which generally shows little response to unloading, displayed no differential response of glucose metabolism to insulin.
• Tischler, M. E., Ost, A. H., & Coffman, J. (1986). Protein turnover in adipose tissue from fasted or diabetic rats.. Life sciences, 39(16), 1447-52. doi:10.1016/0024-3205(86)90549-7
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  Protein synthesis and degradation in vitro were compared in epididymal fat pads from animals deprived of food for 48 h or treated 6 or 12 days prior with streptozotocin to induce diabetes. Although both fasting and diabetes led to depressed (-24% to -57%) protein synthesis, the diminution in protein degradation (-63% to -72%) was even greater, so that net in vitro protein balance improved dramatically. Insulin failed to inhibit protein degradation in fat pads of these rats as it does for fed animals. Although insulin stimulated protein synthesis in fat pads of fasted and 12 day diabetic rats, the absolute change was much smaller than that seen in the fed state. The inhibition of protein degradation by leucine also seems to be less in fasted animals, probably because leucine catabolism is slower in fasting. These results show that fasting and diabetes may improve protein balance in adipose tissue but diminish the regulatory effects of insulin.
• Tischler, M. E., & Allen, D. K. (1985). Comparison of thioltransferase (glutathione:disulfide oxidoreductase) from various rat tissues.. Enzyme, 34(4), 220-3. doi:10.1159/000469389
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  Thioltransferase activity was detected in kidney, heart, epididymal fat pads and skeletal muscles, and was inhibited at high concentrations (greater than 1.6 mmol/l) of reduced glutathione. With several disulfide substrates the Vmax and Km for thioltransferase in kidney, heart, soleus and extensor digitorum longus were considerably smaller than in liver. The enzyme from tibialis anterior showed similar kinetic constants as from liver with certain substrates but not with L-cystine.
• Tischler, M. E., & Henriksen, E. J. (1985). Possible mechanism for changes in glycogen metabolism in unloaded soleus muscle.. The Physiologist, 28(6 Suppl), S131-2.
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  Carbohydrate metabolism has been shown to be affected in a number of ways by different models of hypokinesia. In vivo glycogen levels in the soleus muscle are known to be increased by short-term denervation and harness suspension. In addition, exposure to 7 days of hypogravity also caused a dramatic increase in glycogen concentration in this muscle. The biochemical alterations caused by unloading that may bring about these increases in glycogen storage in the soleus were sought.
• Tischler, M. E., Fagan, J. M., D, A., & Allen, D. (1985). Relationship of the redox state to muscle protein degradation.. Progress in clinical and biological research, 180, 363-72.
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  With increasing muscle size, incubated or fresh-frozen leg muscles showed a more reduced redox state and slower proteolysis. The ln of these data gave a linear correlation showing faster proteolysis under more oxidized conditions. In incubated diaphragms, the inhibitory effects of insulin and catecholamines on proteolysis were associated with a more reduced state. Fasting, trauma or cortisol treatment led to accelerated proteolysis and a more oxidized state. Long term fasting and refeeding supported this relationship, as did streptozotocin diabetes. Like the NAD+ and NADP+ redox couples, the glutathione couple seemed to fit this relationship. Use of proteinase inhibitors showed that the redox state probably mediated the effects of the various factors on proteolysis rather than vice versa. Muscle contains thioltransferase which catalyzes the formation of glutathione-protein mixed disulfides. We propose that increased formation of oxidized glutathione and its interaction with muscle proteins may act as a signal for the initiation of proteolysis.
• Tischler, M. E., Jacob, S., Henriksen, E. J., & Cook, P. H. (1985). Muscle protein and glycogen responses to recovery from hypogravity and unloading by tail-cast suspension.. The Physiologist, 28(6 Suppl), S193-4.
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  Previous studies in this laboratory using the tail-bast hindlimb suspension model have shown that there are specific changes in protein and carbohydrate metabolism in the soleus muscle due to unloading. For example, 6 days of unloading caused a 27 percent decrease in mass and a 60 percent increse in glycogen content in the soleus muscle, while the extensor digitorum longus muscle was unaffected. Also, fresh tissue tyrosine and its in vitro release from the muscle are increased in the unloaded soleus, indicating that this condition causes a more negative protein balance. With these results in mind, studies to investigate the effect of hypogravity on protein and carbohydrate metabolism in a number of rat hindlimb muscles were carried out.
• Tischler, M. E., Jacob, S., Henriksen, E. J., & Cook, P. H. (1985). Responses of amino acids in hindlimb muscles to recovery from hypogravity and unloading by tail-cast suspension.. The Physiologist, 28(6 Suppl), S191-2.
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  Amino acids were assayed in muscles from rats exposed to 7 days of hypogravity and 12 h of gravity (F) or 6 days of suspension with (R) or without (H) 12 h of loading. In these groups, lower aspartate was common only to the soleus (SOL) relative to control muscles, the smallest difference being in group R. This difference in aspartate for F and H, but not for R, correlated with lower malate suggesting diminution of citric acid cycle intermediates. The R SOL value was increased over the H SOL. Therefore desite 12 h of loading, the F SOL was more comparable to the H SOL. The role of stress in preventing recovery of the F SOL was apparent from the ratios of glutamine/glutamate. Synthesis of glutamine is enhanced by glucocorticoids and is reflected by an increased ratio. In 5 of the 6 F muscles studied, this ratio was greater than in controls. In contrast, the ratio in all R muscles was similar to controls and showed recovery from the values in H muscles. Hence the post-flight treatment of F rats may have produced additional stress. Despite this stress, in some respects the SOL responses to hypogravity were similar to its responses to unloding by suspension.
• Tischler, M. E., Jaspers, S. R., & Fagan, J. M. (1985). Biochemical response to chronic shortening in unloaded soleus muscles.. Journal of applied physiology (Bethesda, Md. : 1985), 59(4), 1159-63. doi:10.1152/jappl.1985.59.4.1159
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  One leg of tail-casted suspended rats was immobilized in a plantar-flexed position to test whether chronic shortening of posterior leg muscles affected the metabolic response to unloading. The immobilized plantaris and gastrocnemius muscles of these animals showed approximately 20% loss of muscle mass in contrast to simply a slower growth rate with unloading. Loss of mass of the soleus muscle during suspension was not accentuated by chronic shortening. Although protein degradation in the isolated soleus muscle of the plantar-flexed limb was slightly faster than in the contralateral free limb, this difference was offset by faster synthesis of the myofibrillar protein fraction of the chronically shortened muscle. Total adenine nucleotides were 17% lower (P less than 0.005) in the chronically shortened soleus muscle following incubation. Glutamate, glutamine, and alanine metabolism showed little response to chronic shortening. These results suggest that, in the soleus muscle, chronic shortening did not alter significantly the metabolic responses to unloading and reduced activity.
• Tischler, M. E., Jaspers, S. R., Jacob, S., & Henriksen, E. J. (1985). Responses of skeletal muscle to unloading--a review.. The Physiologist, 28(6 Suppl), S13-6.
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  Suspension models were used to study muscle response to reduced activity. During 6 days of tail casting, the soleus (SOL) atrophies while the extensor digitorum longus grows relatively normally. After discounting those changes in both muscles due primarily to increased secretion of adrenal hormones, the following conclusions regarding the specific responses of the SOL could be drawn: (1) Atrophy is probably due primarily to increased protein degradation; (2) Decreased synthesis of glutamine may result from reduced availability of ammonia due to diminished use of ATP; (3) Greater muscle glycogen seems to reflect an increased response to insulin of glucose uptake which leads to greater glucose metabolism; and (4) Faster catabolism of branched-chain amino acids can be attributed to enhanced flux through ketoacid dehydrogenase. Studies by others using tail casted suspended rats showed in the SOL: (1) a gradual switch from type 1 to type 2 fibers; (2) increased acid protease activity; and (3) altered muscle function and contractile duration. Using harness suspended rats, others showed in the SOL: (1) significant atrophy; (2) increased numbers of glucocorticoid receptors; and (3) no change in muscle fatigability.
• Tischler, M. E., Jaspers, S. R., Jacob, S., Henriksen, E. J., & Cook, P. (1985). Response of rat hindlimb muscles to 12 hours recovery from tail-cast suspension.. The Physiologist, 28(6 Suppl), S129-30.
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  Previous work has shown a number of biochemical changes which accompany atrophy or reduced muscle growth in hindlimb of tail-casted, suspended rats. These results clearly show that altered muscle growth was due to changes in protein turnover. Accordingly, the rise in soleus tyrosine following unloading reflects the more negative protein balance. Other major changes we found included slower synthesis of glutamine as indicated by lower ratios of glutamine/glutamate and reduced levels of aspartate which coincide with slower aspartate and ammonia metabolism in vitro. In conjunction with the study of SL-3 rats, which were subjected to 12 h of post-flight gravity, a study of the effects of 12 h eight bearing on metabolism of 6-day unloaded hindlimb muscles was carried out.
• Tischler, M. E., & Cammisa, H. M. (1984). Metabolism of protein, amino acids, and glucose and their response to insulin in atria and cardiac myocytes of traumatized rats.. Metabolism: clinical and experimental, 33(6), 515-20. doi:10.1016/0026-0495(84)90005-2
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  Soft tissue injury to one hindlimb of rats was used to test the metabolic response of atrial and ventricular muscle to trauma. Effects of insulin on muscle metabolism were also studied. In myocytes and atria from normal animals, insulin increased protein synthesis and decreased protein degradation. For myocytes of rats at one and two days after trauma, this effect of insulin on proteolysis could not be detected. Over the next two days, the inhibitory effect returned to normal. Insulin also did not increase protein synthesis on day 1, but did thereafter. In atria, in contrast to heart cells, the inhibitory effect of insulin on proteolysis was enhanced at two and three days after trauma, and its stimulation of protein synthesis was unaltered. Insulin increased carbohydrate metabolism in both myocytes and atria of normal rats and traumatized rats after 2 days, and trauma did not alter this response. In myocytes, but not atria, trauma led to a faster oxidation of leucine and a significant rise in the production of alanine. Production of glutamine and glutamate was not affected in either tissue. These results show that the metabolic responses to trauma of atrial and ventricular muscle differ considerably.
• Tischler, M. E., & Jaspers, S. R. (1984). Atrophy and growth failure of rat hindlimb muscles in tail-cast suspension.. Journal of applied physiology: respiratory, environmental and exercise physiology, 57(5), 1472-9. doi:10.1152/jappl.1984.57.5.1472
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  Atrophy and growth failure of muscle in a tail-cast suspension model were evaluated in hindlimbs of female Sprague-Dawley rats. Based on measurements of food consumption, animal growth rate, urinary excretion of urea and ammonia, and muscle size, 6 days seemed to be the optimum duration of suspension for studying muscle unloading. After 6 days, the soleus, plantaris, and gastrocnemius muscles from suspended animals were 27, 10, and 11% smaller (P less than 0.05), respectively, than those from tail-casted weight-bearing animals. The extensor digitorum longus and tibialis anterior muscles were unaffected by suspension (less than or equal to 6 days) while the triceps brachii hypertrophied (8%, P less than 0.05). Wet weight-to-dry weight ratios were smaller in the plantaris (-0.19, P less than 0.05) and gastrocnemius (-0.19, P less than 0.05) muscles from suspended rats. In the plantaris, this difference coincided with a higher protein concentration (+12 mg/g, P less than 0.001). In vitro measurements of protein metabolism in the soleus muscles of suspended rats showed both slower protein synthesis (P less than 0.05) and faster protein degradation (P less than 0.05), whereas these processes were unaltered in the extensor digitorum longus muscles.
• Tischler, M. E., Fagan, J. M., & Allen, D. K. (1984). Reduction-oxidation state as a mediator of hormonal control of muscle proteolysis*. Journal of Molecular and Cellular Cardiology, 16, 36-36. doi:10.1016/s0022-2828(84)80139-x
• Tischler, M. E., Ost, A. H., Coffman, J., & Cammisa, H. M. (1984). Metabolism of amino acids, protein and glucose in fat pads of traumatized rats.. Life sciences, 35(4), 449-54. doi:10.1016/0024-3205(84)90656-8
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  Soft tissue injury to one hindlimb of rats was used to test the response to trauma of metabolism in epididymal fat pads. Degradation of [1-14)C]leucine was lower on day 2 after injury, but not on days 1 or 3, whether or not glucose or insulin were provided. Although trauma did not affect the basal rate of release of 14CO2, lactate or pyruvate from fat pads incubated with [U-14C] glucose, the stimulation by insulin of these processes was smaller in fat pads of 2 day traumatized than of normal animals. These results suggest that trauma due to injury may decrease the capacity for utilization of leucine and glucose by adipose tissue. Release of alanine, glutamine and glutamate by fat pads incubated with leucine was also lower on day 2. This decreased efflux could not be accounted for by changes in net protein breakdown or in pyruvate availability and probably reflected their reduced de novo synthesis due to the diminished release of nitrogen from leucine.
• Tischler, M. E., & Fagan, J. M. (1983). Response to trauma of protein, amino acid, and carbohydrate metabolism in injured and uninjured rat skeletal muscles.. Metabolism: clinical and experimental, 32(9), 853-68. doi:10.1016/0026-0495(83)90198-1
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  Soft tissue injury to one hindlimb produced trauma in rats without affecting their food intake or weight gain. Histologic examination showed damage to the soleus and gastrocnemius muscles but not to the extensor digitorum longus muscle. The protein content of the injured soleus muscle was lower than that of the contralateral soleus at one day after injury, and was reflected in vitro by a faster rate of protein degradation. The injured soleus also showed greater rates of protein synthesis, glucose uptake, glycolysis, oxidation of glucose, pyruvate, and leucine, and de novo synthesis of alanine. During three days after the injury, urinary nitrogen excretion increased progressively and was paralleled by a faster rate of protein degradation in uninjured muscles incubated with glucose, insulin, and amino acids. In these muscles, the inhibition of protein degradation by insulin diminished, while its stimulation of protein synthesis was unaffected. This insensitivity of proteolysis to insulin in trauma can explain the increased rate of this process. The oxidation of glucose and pyruvate were lower in the diaphragms of traumatized than of normal rats incubated with leucine, while glycolysis and uptake of 2-deoxyglucose did not differ. The degradation of leucine and isoleucine was greater in the diaphragms of traumatized animals and was associated with a faster de novo synthesis of alanine. For the uninjured soleus muscles of the traumatized rats, the slower rates of oxidation of glucose, glycolysis, and uptake of 2-deoxyglucose in the presence of insulin showed an insensitivity of glucose metabolism to this hormone. In contrast, no differences were seen in these various metabolic processes between the extensor digitorum longus muscles of traumatized and normal rats. These data suggest that the response of skeletal muscles to trauma may depend on their physiologic and biochemical characteristics.
• Tischler, M. E., & Fagan, J. M. (1982). Relationship of the reduction-oxidation state to protein degradation in skeletal and atrial muscle.. Archives of biochemistry and biophysics, 217(1), 191-201. doi:10.1016/0003-9861(82)90493-3
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  Abstract Changes in proteolysis were correlated with the cell reduction-oxidation state in rat diaphragm and atrium. Protein degradation was measured in the presence of cycloheximide as the linear release of tyrosine into the medium. Intracellular ratios of lactate/pyruvate, total NADH NAD , and malate/pyruvate were used as indicators of the muscle reduction-oxidation state. Incubation of diaphragms with leucine (0.5–2.0 m m ) or its transamination product, sodium α-ketoisocaproate (0.5 m m ), resulted in a lower rate of proteolysis and a higher ratio of lactate/pyruvate and NADH NAD . These effects of leucine could be abolished by inhibiting its transamination with l -cycloserine. Unlike leucine, neither isoleucine nor valine alone produced any change in these parameters. Incubation of diaphragms with glucose (20 m m ) or atria with sodium lactate (2 m m ) produced a diminution of tyrosine release from the muscles and a rise in the ratio of total NADH NAD . Similarly, in incubated diaphragms of fasted rats, the anabolic effects of insulin, epinephrine and isoproterenol on protein degradation were associated with a higher malate/pyruvate ratio. In catabolic states, such as fasting, cortisol treatment of fasted, adrenalectomized rats or traumatization, enhanced muscle proteolysis was observed. Fresh-frozen diaphragms from these rats had both lower lactate/pyruvate and malate/pyruvate ratios than did muscles from control animals. These data show that diminution of proteolysis in diaphragm is accompanied by an increase of the NAD(P)H NAD(P) ratios. In contrast to these findings, chymostatin and leupeptin, which inhibit directly muscle proteinases, caused a decrease of the lactate/pyruvate and malate/pyruvate ratios. These results suggest that protein degradation in diaphragm and atrium is linked to the cellular redox state.
• Tischler, M. E., Goldberg, A. L., & Desautels, M. (1982). Does leucine, leucyl-tRNA, or some metabolite of leucine regulate protein synthesis and degradation in skeletal and cardiac muscle?. Journal of Biological Chemistry, 257(4), 1613-1621. doi:10.1016/s0021-9258(19)68081-6
• Tischler, M. E. (1981). Hormonal regulation of protein degradation in skeletal and cardiac muscle.. Life sciences, 28(23), 2569-76. doi:10.1016/0024-3205(81)90713-x
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  Abstract A number of hormones produce either anabolic or catabolic effects on protein degradation in muscle. These effects can account for the changes in muscle proteolysis associated with a variety of physiological and pathological states. Thus the balance of hormones within the organism seems to play an important role in the overall regulation of this process. In the fed state, insulin may be the single most important factor maintaining low rates of proteolysis, whereas the catabolic effects of the glucocorticoid hormones in fasting seem to predominate. The proportions of these hormones may be important not only during starvation, but also in trauma and in diseases associated with their altered production and secretion (e.g., diabetes, Cushing's syndrome). Hyperthyroidism too causes catabolic effects on muscle proteolysis.
• Tischler, M. E., & Goldberg, A. L. (1980). Amino acid degradation and effect of leucine on pyruvate oxidation in rat atrial muscle.. The American journal of physiology, 238(5), E480-6. doi:10.1152/ajpendo.1980.238.5.e480
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  Both left and right atria from fasted rats produced significant amounts of 14CO2 during incubation with U-14C-labeled leucine, isoleucine, valine, alanine, glutamate, glutamine, aspartate, asparagine, proline, threonine, or lysine. This pattern of amino acid metabolism resembles that of skeletal muscle. Production of 14CO2 from [1-14C]leucine was 2.5-fold greater in atria from fasted than from fed rats and was due to greater alpha-ketoisocaproic dehydrogenase activity in the tissue from fasted animals. At normal plasma concentrations, leucine reduced the oxidation of glucose and lactate in atria from fasted but not from fed rats by inhibiting pyruvate oxidation and without altering the rate of glycolysis. Leucine also reduced glucose oxidation when added in the presence of ketone bodies or other amino acids and stimulated the release of lactate into the medium. Although the leucine skeleton can be completely oxidized to CO2 and thus can serve as an alternative fuel in fasting in place of glucose, oxidation of leucine (like glucose or lactate oxidation) accounts only for a very small fraction of the total oxygen consumption of the resting atria.
• Tischler, M. E., & Goldberg, A. L. (1980). Leucine degradation and release of glutamine and alanine by adipose tissue. Journal of Biological Chemistry, 255(17), 8074-8081. doi:10.1016/s0021-9258(19)70609-7
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  Adipose tissue from fed rats rapidly degrades [l14C]leucine to 14C02. This process is stimulated by glucose (but not by pyruvate) and by insulin, which also enhances the degradation of isoleucine and valine. Fasting and protein deficiency markedly reduced the catabolism of [l-‘4C]leucine in epididymal fat pads by decreasing the decarboxylation of a-ketoisocaproic acid and the transamination of leucine. In tissue homogenates, the rates of these reactions decreased by 63 and 47%, respectively, after 2 days of food deprivation. Within only 1 day of food deprivation, total decarboxylation of leucine in tissue homogenates was 73% slower. In adipose tissue from the fasted animals, leutine oxidation was also stimulated by glucose but not by insulin. In fat pads from fed (but not fasted) rats, leucine decreased the oxidation of pyruvate and thus may spare use of glucose-derived acetyl coenzyme A for lipogenesis. When fed and fasted rats were injected with [1-14CJleucine, their rates of expiration of 14C02 did not differ, but when they were injected with p-‘4C]leucine, 14C02 expiration was greater in the fasted rats. Therefore, food deprivation did not affect the total amount of leucine degradation by the organism but seemed to increase the oxidation of leucine by muscle, while decreasing its conversion to triglycerides. The incubated fat pads appear to synthesize and release large amounts of glutamine and smaller quantities of alanine and glutamate. Addition of leucine increased release into the medium of glutamine and, to a lesser extent, that of alanine and glutamate (without affecting tissue protein balance or the release of other amino acids). In fasting, when degradation of leucine fell, the accumulation of glutamine and alanine in the medium decreased. Isoleucine and valine were degraded more slowly than leucine and stimulated glutamine and alanine release to a lesser extent. In adipose tissue, as in muscle, production of glutamine, as well as alanine and glutamate, seems to be the route for disposal of amino groups released in the transamination of branched-chain amino acids.
• Tischler, M. E., & Goldberg, A. L. (1980). Production of alanine and glutamine by atrial muscle from fed and fasted rats.. The American journal of physiology, 238(5), E487-93. doi:10.1152/ajpendo.1980.238.5.e487
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  During incubation in vitro, rat atrial and rabbit papillary muscles synthesized and released large amounts of alanine and even higher amounts of glutamine. The amino groups used in the synthesis of alanine and glutamine appeared to be derived from the catabolism of other amino acids, specifically, leucine, isoleucine, valine, aspartate, glutamate, asparagine, arginine, and proline. The total amount of glutamine and alanine synthesized increased by 45% in atrial muscle from fasted rats, in which the oxidation of branched-chain amino acids is enhanced. In atria from fed rats, addition of leucine stimulated by about 20% the production of both alanine and glutamine, probably by providing additional amino groups for transamination with alpha-ketoglutarate. However, in cardiac muscle from fasted rats and rabbits, leucine increased the production of glutamine, but not of alanine. Isoleucine and valine, by contrast, enhanced production of both alanine and glutamine in these tissues. In the presence of leucine, low levels of pyruvate seem to limit the production of alanine, apparently because leucine, unlike isoleucine or valine, promotes the conversion of pyruvate to lactate.
• Williamson, J. R., Viale, R. O., Tischler, M. E., Murphy, E., & Coll, K. E. (1979). Kinetics and regulation of the glutamate-aspartate translocator in rat liver mitochondria.. Journal of Biological Chemistry, 254(17), 8369-8376. doi:10.1016/s0021-9258(19)86900-4
• Williamson, J. R., Tischler, M. E., & Hecht, P. (1977). Determination of mitochondrial/cytosolic metabolite gradients in isolated rat liver cells by cell disruption.. Archives of biochemistry and biophysics, 181(1), 278-93. doi:10.1016/0003-9861(77)90506-9
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  Abstract A new technique is described for determining the distribution of metabolites between the cytosol and mitochondria. Rapid lysis of the cell plasma membrane is obtained by forcing isolated liver cells under high pressure through a small diameter needle. The cells, after disruption by the shearing forces generated during the turbulent flow through the needle, are exposed to mitochondrial anion transport inhibitors to prevent efflux of mitochondrial metabolites. Maximal release of cytosolic metabolites was obtained when release of lactate dehydrogenase was greater than 70%, which corresponded with minimal release of mitochondrial enzymes (5–9%). A measured Reynolds number between 7600 and 8000 was indicative of optimal disruption. Mitochondria in the disrupted cells were still functional, as shown by the ability of ADP to stimulate respiration when glutamate plus malate were provided as substrates. Measurement of the subcellular volumes yielded values of 2.0 and 0.2 ml/g dry wt, respectively, for the cytosol and mitochondria. Calculation of the mitochondrial ΔpH (pHin-pHout) in the isolated liver cell based on 22 individual measurements of mitochondria/cytosol gradients for citrate, isocitrate, α-ketoglutarate, malate, glutamate, and pyruvate yielded a value of 0.41 ±0.03. The excellent relationship of these gradients to a common ΔpH lends credence to the technique. Cytosolic and mitochondrial ATP ADP ratios were similar in liver cells isolated from starved and fed rats. Fed rat liver cells, however, had a higher cytosolic adenine nucleotide content (16.8 μmol/g dry wt) than those from starved rats (14.5 μmol/ g dry wt) whereas the mitochondrial content was the same (16 nmol/mg of mitochondrial protein). Data obtained by the disruption technique are compared with other previously published data obtained using either digitonin treatment of isolated hepatocytes or nonaqueous solvent extraction of lyophilized freeze-clamped perfused livers.
• Williamson, J. R., Tischler, M. E., & Hecht, P. (1977). Effect of ammonia on mitochondrial and cytosolic NADH and NADPH systems in isolated rat liver cells.. FEBS letters, 76(1), 99-104. doi:10.1016/0014-5793(77)80129-4
• Williamson, J. R., Tischler, M. E., Friedrichs, D., & Coll, K. E. (1977). Pyridine nucleotide distributions and enzyme mass action ratios in hepatocytes from fed and starved rats.. Archives of biochemistry and biophysics, 184(1), 222-36. doi:10.1016/0003-9861(77)90346-0
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  Abstract Hepatocytes isolated from fed or starved rats were rapidly lysed using the recently described technique of turbulent flow (M. E. Tischler, P. Hecht, and J. R. Williamson, 1977, Arch. Biochem. Biophys. , 181 , 278–292) . Pyridine nucleotide and metabolite contents were measured in the particulate fraction of both whole and disrupted cells after centrifugation through silicone oil. Lactate/pyruvate, β-hydroxybutyrate/acetoacetate, isocitrate/α-ketoglutarate, and malate/pyruvate ratios were determined for calculation of the free NADH NAD + and NADPH NADP + ratios in the cytosol and mitochondria. Lactate/pyruvate ratios measured in the extracellular and cytosolic compartments were in good agreement. Ratios of β-hydroxybutyrate/acetoacetate measured in the extracellular, cytosolic, and mitochondrial compartments also agreed well. Addition of ammonia to fed or starved rat liver cells incubated with lactate, pyruvate, β-hydroxybutyrate, and acetoacetate caused an oxidation of both the NAD and NADP redox states in the mitochondria and cytosol, although the NADP system was oxidized to a greater extent. Calculation of the free NADH and NAD concentrations in the cytosol provided values of about 1 and 400 to 500 μ m , respectively, under control conditions. The concentrations of free NADH and NAD in the mitochondria were considerably higher, being 300 to 400 μ m and 4 to 6 m m , respectively. The free andm bound NAD systems in both the cytosol and mitochondria were more oxidized in the presence of ammonia. NAD and NADP redox potential differences across the mitochondrial membrane (Δ E h ) were not significantly affected by ammonia addition and were generally similar in cells from both fed and starved rats: −52 and −56 mV for the NAD system and −19 to −29 mV for the NADP system. For the NAD system the cytosolic potential was −260 mV in the absence of ammonia and −250 mV in its presence, the mitochondrial values being −315 and −303 mV, respectively. The average cytosolic NADP potential, on the other hand, was −400 mV in the absence and −384 mV in the presence of ammonia. The mitochondrial fractions yielded NADP potentials of −420 mV in the absence of ammonia with both fed and starved rats. Ammonia decreased the mitochondrial NADP potential to −404 mV in fed rats and to −415 mV in starved rats. The calculated free NADH NAD + and NADPH NADP + ratios as well as metabolite concentrations were used to evaluate the mass action ratios of both cytosolic and mitochondrial enzymes. Cytosolic alanine aminotransferase remained near equilibrium in the absence and presence of ammonia, while cytosolic and mitochondrial aspartate aminotransferase reactions deviated up to fivefold. The glutamate dehydrogenase reaction was in near equilibrium with the NAD system, but deviated by three to four orders of magnitude from equilibrium with the NADP system in the direction favoring glutamate synthesis rather than deamination. Cytosolic malate dehydrogenase deviated from equilibrium by about one order of magnitude, while mitochondrial malate dehydrogenase and citrate synthase deviated by two to six orders of magnitude. These data emphasize the importance of regulation of the citric acid cycle at the citrate synthase step.
• Williamson, J. R., Tischler, M. E., Pachence, J. M., & Noue, K. F. (1976). Mechanism of glutamate-aspartate translocation across the mitochondrial inner membrane.. Archives of biochemistry and biophysics, 173(2), 448-61. doi:10.1016/0003-9861(76)90282-4
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  Abstract In order to study the mechanism of the glutamate-aspartate translocator, rat liver mitochondria were loaded with either glutamate or aspartate. In the presence of ascorbate plus tetramethyl- p -phenylenediamine as an electron donor at the third energy conservation site, exchange of external glutamate for matrix aspartate is highly favored over the reverse exchange. In the absence of an energy source, although the asymmetry of the exchange rates is much smaller, it is still observable. Further studies have shown that the proton uptake accompanying influx of glutamate in exchange for aspartate efflux occurs by protonation of a group on the carrier (p K = 7.9) at the external side of the inner mitochondrial membrane, followed by deprotonation at the matrix surface. It is postulated that glutamate binds to the protonated form of the carrier and aspartate to the deprotonated form. Because of the alkaline p K , aspartate efflux is inhibited with increased matrix [H + ] due to limitation of the availability of deprotonated carrier for aspartate binding. For the reverse exchange, aspartate uptake is inhibited by increasing external [H + ]. Thus the rate of aspartate uptake by mitochondria is apparently impeded both by a proton motive force (Δ p ) unfavorable to entry of ions with net negative charge as well as by the small proportion of deprotonated carrier at the outer surface of the membrane. This conclusion is further illustrated by inhibition of the aspartate-aspartate exchange with increased [H + ] and by addition of an energy source. The glutamate-glutamate exchange, however, showed a slight stimulation by increased [H + ] and was unaffected by the energy state. The model initially proposed for the carrier, in which a neutral glutamate-carrier complex exchanges for a negatively charged aspartate-carrier complex, is tested further. Glutamate uptake was noncompetitively inhibited by external aspartate, which indicates that aspartate and glutamate bind to separate forms of the carrier. Intramitochrondrial glutamate at a concentration of 18 m m , however, had no effect on aspartate efflux. Arrhenius plots for the glutamate-aspartate and aspartate-glutamate exchanges were linear over the range of temperatures tested (1–35 °C and 5–25 °C, respectively) and provided an average value of 14.3 kcal/mol for the energy of activation. In addition, there appear to be two pools, exchangeable and nonexchangeable, of matrix aspartate available to the translocator, since extramitochondrial radiolabeled aspartate can equilibrate only with unlabeled matrix aspartate at low aspartate loading (1–2 nmol of aspartate/mg of protein). The physiological significance of the data is discussed.
• Tischler, M. E., & Lanoue, K. F. (1974). Electrogenic characteristics of the mitochondrial glutamate-aspartate antiporter.. Journal of Biological Chemistry, 249(23), 7522-7528. doi:10.1016/s0021-9258(19)81269-3
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  Abstract Rat liver mitochondria were loaded with aspartate by treating glutamate-loaded mitochondria with oxalacetate. Aspartate efflux was initiated by addition of extramitochondrial glutamate and was accompanied by the uptake of an equivalent amount of glutamate and protons. Proton uptake, however, did not occur in the presence of an uncoupling agent, and net transport of aspartate was facilitated by the presence of a source of energy from the oxidation of ascorbate and tetramethylphenylenediamine. In the absence of uncoupling agents or a source of energy, exchange of aspartate for glutamate resulted in acidification of the mitochondrial matrix and efflux of an equivalent amount of potassium. The data indicate that exchange of aspartate and glutamate across the mitochondrial membrane is an electrogenic process. The most plausible model is one in which a neutral glutamate-carrier complex transports glutamate from outside the mitochondria to inside. The aspartate-carrier complex formed on the inner surface of the mitochondrial membrane should then be negatively charged and transport of the anionic carrier-aspartate complex would be facilitated by an electrical potential across the mitochondrial membrane.
• Tischler, M. E., & Fisher, R. R. (1973). Oxidation of reduced nicotinamide hypoxanthine dinucleotide phosphate by intact rat liver mitochondria.. Biochimica et biophysica acta, 305(2), 199-205. doi:10.1016/0005-2728(73)90169-2