Abstract.-A radioimmunoassay has been developed for determining the serum levels of carcinoembryonic antigen of the human digestive system in patients with cancer of the colon and rectum. The assay is simple to perform and has a high degree of reproducibility and specificity. The test detects a concentration of 2.5 ng of carcinoembryonic antigen per ml of serum and this has provided the first demonstration of a circulating tumor-specific antigen in the sera of cancer patients.The carcinoembryonic antigen (CEA), first described in this laboratory, has been found exclusively in adenocarcinomata arising from the entodermallyderived digestive system epithelium, and in embryonic and fetal digestive tissues in the first two trimesters of gestation." 2 The CEA has been characterized as a protein-polysaccharide complex" I which is soluble in 1.0 M perchloric acid and 50 per cent ammonium sulfate. Purified CEA isolated from different metastatic tumors was found to be of consistent amino acid and carbohydrate composition.3Serologic studies revealed that 70 per cent of patients suffering from primary, nonmetastatic cancers of the digestive system, and a comparable proportion of women during pregnancy and the immediate liost-partum period contained circulating anti-CEA antibodies in their sera.4 However, in patients where the tumor had undergone spread from its primary site in the digestive system to other organs, no anti-CEA antibodies could be demonstrated in any case. There are at least two possible explanations for this observation.First, it may be that a large mass of tumor serves as an antibody "sink," adsorbing the anti-CEA antibodies as the blood circulates through the tumor tissue.Experiments have, in fact, shown that the CEA is intimately associated with the tumor cell surface and would, therefore, be readily available to interact in this fashion with its corresponding antibodies.5The second possibility is that the tumor tissue may release CEA directly into the circulation. With a large tumor mass, the corresponding serum concentration of the antigenic material would be relatively high and might lead to CEAanti-CEA complexes in antigen excess. The anti-CEA antibody constituents would, under these circumstances, no longer be available for participation in subsequent serologic reactions. The purpose of the present study was to develop a radioimmunoassay for the detection of circulating CEA in an attempt to confirm the second hypothesis.Materials and Methods.-Purified CEA: Purification of the CEA was carried out by a modification of a technique which has been previously described.' Homogenates of metastatic cancer tissue which had originated within the digestive system were extracted in 1.0 M perchloric acid at room temperature for 20 min. After removal of the resultant 161 MEDICAL SCIENCES: THOMSON ET AL. precipitate by centrifugation, the supernatant was exhaustively dialyzed against water, lyophylized, and redissolved in a small volume of distilled water. The final, purified CEA fraction was obtained by sequent...

AMP-activated protein kinase (AMPK) has been identified as a regulator of gene transcription, increasing mitochondrial proteins of oxidative metabolism as well as hexokinase expression in skeletal muscle. In mice, muscle-specific knockout of LKB1, a component of the upstream kinase of AMPK, prevents contraction- and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced activation of AMPK in skeletal muscle, and the increase in hexokinase II protein that is normally observed with chronic AICAR activation of AMPK. Since previous reports show a cAMP response element in the promoter region of the hexokinase II gene, we hypothesized that the cAMP-response element (CRE) binding protein (CREB) family of transcription factors could be targets of AMPK. Using radioisotopic kinase assays, we found that recombinant and rat liver and muscle AMPK phosphorylated CREB1 at the same site as cAMP-dependent protein kinase (PKA). AMPK was also found to phosphorylate activating transcription factor 1 (ATF1), CRE modulator (CREM), and CREB-like 2 (CREBL2), but not ATF2. Treatment of HEK-293 cells stably transfected with a CREB-driven luciferase reporter with AICAR increased luciferase activity approximately threefold over a 24-h time course. This increase was blocked with compound C, an AMPK inhibitor. In addition, AICAR-induced activation of AMPK in incubated rat epitrochlearis muscles resulted in an increase in both phospho-acetyl-CoA carboxylase and phospho-CREB. We conclude that CREB and related proteins are direct downstream targets for AMPK and are therefore likely involved in mediating some effects of AMPK on expression of genes having a CRE in their promoters.

Thomson DM, Porter BB, Tall JH, Kim H-J, Barrow JR, Winder WW. Skeletal muscle and heart LKB1 deficiency causes decreased voluntary running and reduced muscle mitochondrial marker enzyme expression in mice. Am J Physiol Endocrinol Metab 292: E196 -E202, 2007. First published August 22, 2006; doi:10.1152/ajpendo.00366.2006.-LKB1 has been identified as a component of the major upstream kinase of AMP-activated protein kinase (AMPK) in skeletal muscle. To investigate the roles of LKB1 in skeletal muscle, we used muscle-specific LKB1 knockout (MLKB1KO) mice that exhibit low expression of LKB1 in heart and skeletal muscle, but not in other tissues. The importance of LKB1 in muscle physiology was demonstrated by the observation that electrical stimulation of the muscle in situ increased AMPK phosphorylation and activity in the wild-type (WT) but not in the muscle-specific LKB1KO mice. Likewise, phosphorylation of acetyl-CoA carboxylase (ACC) was markedly attenuated in the KO mice. The LKB1KO mice had difficulty running on the treadmill and exhibited marked reduction in distance run in voluntary running wheels over a 3-wk period (5.9 Ϯ 0.9 km/day for WT vs. 1.7 Ϯ 0.7 km/day for MLKB1KO mice). The MLKB1KO mice anesthetized at rest exhibited significantly decreased phospho-AMPK and phospho-ACC compared with WT mice. KO mice exhibited lower levels of mitochondrial protein expression in the red and white regions of the quadriceps. These observations, along with previous observations from other laboratories, clearly demonstrate that LKB1 is the major upstream kinase in skeletal muscle and that it is essential for maintaining mitochondrial marker proteins in skeletal muscle. These data provide evidence for a critical role of LKB1 in muscle physiology, one of which is maintaining basal levels of mitochondrial oxidative enzymes. Capacity for voluntary running is compromised with muscle and heart LKB1 deficiency. adenosine 3Ј-cyclic monophosphate-activated protein kinase; muscle specific LKB1 knockout mouse; muscle mitochondria; citrate synthase AMP-ACTIVATED PROTEIN KINASE (AMPK) is a major regulator of skeletal muscle energy metabolism (3,5,27,29). It is activated in response to exercise and muscle contraction and other conditions of metabolic stress when AMP concentration increases (12,19,26,30). When active, AMPK works to restore cellular energy balance by promoting ATP-generating processes such as fatty acid oxidation and glucose uptake, while inhibiting anabolic processes, such as protein synthesis, that consume ATP (3-5, 14, 27-29). In addition to its role in maintaining energy homeostasis during exercise, AMPK is also thought to play an important role in many adaptations to chronic exercise such as elevations in protein levels of GLUT4, hexokinase II, and mitochondrial proteins (2,7,9,25,31,34). AMPK is a heterotrimer composed of a catalytic ␣-subunit and regulatory ␤-and ␥-subunits. Binding of AMP to the ␥-subunit of AMPK promotes phosphorylation at Thr 172 on its ␣-subunit, which is requisite for its activity. Several...

AMP-activated protein kinase (AMPK) is an energy sensing/signaling protein that, when activated, increases ATP production by stimulating glucose uptake and fatty acid oxidation while at the same time inhibiting ATP = consuming processes such as protein synthesis. Chronic activation of AMPK inhibits expression of lipogenic enzymes in the liver and enhances expression of mitochondrial oxidative enzymes in skeletal muscle. Deficiency of muscle LKB1, the upstream kinase of AMPK, results in greater fluctuation in energy charge during muscle contraction and decreased capacity for exercise at higher work rates. Because AMPK enhances both glucose uptake and fatty acid oxidation in skeletal muscle, it has become a target for prevention and treatment of type 2 diabetes and obesity.

Skeletal muscle mass declines with age, as does the potential for overload-induced fast-twitch skeletal muscle hypertrophy. Because 5'-AMP-activated protein kinase (AMPK) activity is thought to inhibit skeletal muscle protein synthesis and may therefore modulate muscle mass and hypertrophy, the purpose of this investigation was to examine AMPK phosphorylation status (a marker of AMPK activity) and its potential association with the attenuated overload-induced hypertrophy observed in aged skeletal muscle. One-week overload of fast-twitch plantaris and slow-twitch soleus muscles was achieved in young adult (8 mo; n = 7) and old (30 mo; n = 7) Fischer344 x Brown Norway male rats via unilateral gastrocnemius ablation. Significant (P < or = 0.05) age-related atrophy (as measured by total protein content) was noted in plantaris and soleus control (sham-operated) muscles. In fast-twitch plantaris muscles, percent hypertrophy with overload was significantly attenuated with age, whereas AMPK phosphorylation status as determined by Western blotting [phospho-AMPK (Thr172)/total AMPK] was significantly elevated with age (regardless of loading status). There was also a main effect of loading on AMPK phosphorylation status in plantaris muscles (overload > control). Moreover, a strong and significant negative correlation (r = -0.82) was observed between AMPK phosphorylation status and percent hypertrophy in the overloaded plantaris muscles of all animals. In contrast to the plantaris, overload-induced hypertrophy of the slow-twitch soleus muscle was similar between ages, and AMPK phosphorylation in this muscle was also unaffected by age or overload. These data support the possibility that an age-related elevation in AMPK phosphorylation may partly contribute to the attenuated hypertrophic response observed with age in overloaded fast-twitch plantaris muscle.

Regulation of protein translation through Akt and the downstream mammalian target of rapamycin (mTOR) pathway is an important component of the cellular response to hypertrophic stimuli. It has been proposed that 5'-AMP-activated protein kinase (AMPK) activation during muscle contraction may limit the hypertrophic response to resistance-type exercise by inhibiting translational signaling. However, experimental manipulation of AMPK activity during such a stimulus has not been attempted. Therefore, we investigated whether AMPK activation can attenuate the downstream signaling response of the Akt/mTOR pathway to electrically stimulated lengthening muscle contractions. Extensor digitorum longus muscles (n = 8/group) were subjected to a 22-min bout of lengthening contractions by high-frequency sciatic nerve electrical stimulation (STIM) in young adult (8 mo) Fischer 344 x Brown Norway male rats. Forty minutes before electrical stimulation, rats were subcutaneously injected with saline or 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR; 1 mg/g body wt), an AMPK activator. Stimulated and contralateral resting muscles were removed at 0, 20, and 40 min post-STIM, and AMPK, acetyl CoA carboxylase (ACC), Akt, eukaryotic initiation factor 4E-binding protein (4E-BP1), 70-kDa ribosomal protein S6 kinase (S6K1), and eukaryotic elongation factor 2 (eEF2) phosphorylations were assessed by Western blot. AICAR treatment increased (P < or = 0.05) post-STIM AMPK (Thr172) and ACC phosphorylation (Ser79/221), inhibited post-STIM S6K1 (Thr389) and 4E-BP1 (gel shift) phosphorylation, and elevated post-STIM eEF2 phosphorylation (Thr56). These findings suggest that translational signaling downstream of Akt/mTOR can be inhibited after lengthening contractions when preceded by AMPK activation and that energetic stress may be antagonistic to the hypertrophic translational signaling response to loaded muscle contractions.

AMPK (5’-adenosine monophosphate-activated protein kinase) is heavily involved in skeletal muscle metabolic control through its regulation of many downstream targets. Because of their effects on anabolic and catabolic cellular processes, AMPK plays an important role in the control of skeletal muscle development and growth. In this review, the effects of AMPK signaling, and those of its upstream activator, liver kinase B1 (LKB1), on skeletal muscle growth and atrophy are reviewed. The effect of AMPK activity on satellite cell-mediated muscle growth and regeneration after injury is also reviewed. Together, the current data indicate that AMPK does play an important role in regulating muscle mass and regeneration, with AMPKα1 playing a prominent role in stimulating anabolism and in regulating satellite cell dynamics during regeneration, and AMPKα2 playing a potentially more important role in regulating muscle degradation during atrophy.

Lipid metabolism is important for health and insulin action, yet the fundamental process of regulating lipid metabolism during muscle contraction is incompletely understood. Here, we show that liver kinase B1 (LKB1) muscle-specific knockout (LKB1 MKO) mice display decreased fatty acid (FA) oxidation during treadmill exercise. LKB1 MKO mice also show decreased muscle SIK3 activity, increased histone deacetylase 4 expression, decreased NAD+ concentration and SIRT1 activity, and decreased expression of genes involved in FA oxidation. In AMP-activated protein kinase (AMPK)α2 KO mice, substrate use was similar to that in WT mice, which excluded that decreased FA oxidation in LKB1 MKO mice was due to decreased AMPKα2 activity. Additionally, LKB1 MKO muscle demonstrated decreased FA oxidation in vitro. A markedly decreased phosphorylation of TBC1D1, a proposed regulator of FA transport, and a low CoA content could contribute to the low FA oxidation in LKB1 MKO. LKB1 deficiency did not reduce muscle glucose uptake or oxidation during exercise in vivo, excluding a general impairment of substrate use during exercise in LKB1 MKO mice. Our findings demonstrate that LKB1 is a novel molecular regulator of major importance for FA oxidation but not glucose uptake in muscle during exercise.