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J Biol Chem. 2007 Aug 23. Transgenic mice, containing a chimeric gene in which the cDNA for phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (EC 4.1.1.32) was linked to the alpha-skeletal actin gene promoter, express PEPCK-C in skeletal muscle (1~3 units/g). Breeding two founder lines together produced mice with an activity of PEPCK-C of 9 units/g muscle (PEPCK-C(mus) mice). These mice were seven times more active in their cages than controls. On a mouse treadmill, PEPCK-C(mus) mice ran up to 6 km at a speed of 20 m/min while controls stopped at 0.2 km. PEPCK-C( mus) mice had an enhanced running ability, with a VO(2) max of 156 +/- 8.0 ml/kg/min, a maximal Respiratory Exchange Ratio (RER) of 0.91 +/- 0.03 and a blood lactate concentration of 3.7 +/- 1.0 mM after running for 32 min at a 25 masculine grade; the values for control animals were 112 +/- 21 ml/kg/min, 0.99 +/- 0.08, and 8.1 +/- 5.0 mM respectively. The PEPCK-C(mus) mice eat 60% more than controls, but had half the body weight and 10% the body fat as determined by MRI. In addition, the number of mitochondria and the content of triglyceride in the skeletal muscle of PEPCK-C(mus) mice was greatly increased as compared to controls. PEPCK-C(mus) mice had an extended life span relative to control animals; mice up to an age of 2.5 years ran twice as fast as 6-12 month old control animals. We conclude that over-expression of PEPCK-C repatterns energy metabolism and leads to greater longevity. From the full text article: PEPCK-C is involved in gluconeogenesis in the liver and kidney cortex and in glyceroneogenesis in liver, white and brown adipose tissue (see (1) for a review). However, this enzyme is also present in a broad variety of mammalian tissues (2), including the small intestine, colon, mammary gland, adrenal gland, lung and muscle; its metabolic role in these tissues remains obscure. To study the physiological function of PEPCK-C, the gene has been over-expressed or ablated in specific tissues of the mouse. When PEPCK-C was over-expressed in white adipose tissue, the mice had increased rates of glyceroneogenesis in their adipose tissue and became obese (3). In contrast, ablating the expression of PEPCK-C in adipose tissue resulted in mice with lipodystrophy (4). However, a systematic study involving other mammalian tissues where the enzyme has been detected has not been undertaken. We have over-expressed the gene for PEPCK-C in the skeletal muscle of transgenic mice to test the metabolic and physiological consequences. Skeletal muscle was selected as a target organ because there is no clear indication of the metabolic outcome of having a high activity of PEPCK-C in this tissue. Skeletal muscle does not synthesize and release glucose, although there have been reports over the years that the tissue can make glycogen de novo, since both PEPCK-C and fructose-1-6-bisphosphatase activities have been found in skeletal muscle (5-6). We have evidence from research on-going in our laboratory (Nye, Hanson and Kalhan, unpublished results) that glyceroneogenesis occurs in skeletal muscle. This pathway is an abbreviated version of gluconeogenesis, which involves the synthesis of glycerol-3-phosphate (used for triglyceride synthesis) from precursors other than glucose and glycerol. However, glyceroneogenesis has not previously been reported to occur in skeletal muscle, so the extent of the metabolic effect of over-expressing PEPCK-C in this tissue was not immediately apparent. PEPCK-C is a major cataplerotic enzyme (7). However, it is also capable of synthesizing oxalacetate, thus replenishing the citric acid cycle, so it has the potential of being an anaplerotic enzyme. In either case, it would be predicted that increasing the activity of PEPCK-C in skeletal muscle would increase citric acid cycle flux in the animal; ablation of hepatic PEPCK-C greatly reduces citric acid cycle flux (8). This is especially important in tissues such as skeletal muscle, in which the levels of citric acid cycle intermediates vary widely during strenuous exercise to accommodate the major increase in total cycle flux that is required to generate the energy to support muscle contraction. An increase in citric acid cycle anions occurs largely due to anaplerosis. However, any four or five carbon intermediate that enters the citric acid cycle must be removed since it cannot be completely oxidized to carbon dioxide in the cycle. It is thus likely that PEPCK-C contributes to both the generation and subsequent removal of citric acid cycle anions (7). If this is the case, the enzyme is an important component of citric acid cycle function in muscle. Until this study, the concept of a critical role of PEPCK-C in energy metabolism in mammalian skeletal muscle has not been tested. Our results indicate that transgenic mice that over-express the gene for PEPCK-C (about 9 units/g muscle) have a greatly enhanced level of physical activity, which extends well into old age (24 months or older). This is due, in part, to an increased number of mitochondria and high concentration of triglyceride in their skeletal muscles. The mice over-expressing the gene for PEPCK-C also have very little body fat, despite eating 60% more than controls. The biochemical basis for this effect was investigated. [...] Figure 4 is a graphical representation of the difference between a selected PEPCK-Cmus mouse and a control animal that illustrates the extraordinary metabolic characteristics of the PEPCK-Cmus mice. This animal ran for 43 min until exhaustion, as compared to 13 min by its control littermate, and it had an RER of 0.82 at exhaustion (the control animal had an RER of 1.07). What is especially dramatic is the difference in the concentration of lactate in the blood. While both the PEPCK-Cmus mouse and the control began the period of exercise with nearly equal values of blood lactate concentration, at exhaustion the concentration of lactate in the blood of the control animal rose to 17 mM, while the lactate in the blood of the PEPCK-Cmus mouse remained at the same low level noted before exercise. We conclude that the PEPCK-Cmus mice rely heavily on fatty acids as a source of energy for their muscles during exercise and thus do not generate lactate during this period, despite the strenuous nature of the exercise. The control mice rapidly move from fatty acid metabolism to the utilization of muscle glycogen as a fuel; this results in a marked rise in the concentration of lactate in the blood of these animals. Food intake and body composition of the PEPCK-Cmus mice. The body weight of both PEPCK-Cmus mice and controls was determined and related to the average daily food intake of the animals (Fig. 5A). The three PEPCK-Cmus mice tested ate, on a body weight basis, an average of 60% more food than controls. Despite eating more, 18-month old PEPCK-Cmus mice weighed less and had dramatically less body fat, as determined by MRI (Fig. 5B). The adipose tissue volumes were 0.4 +/- 0.2 ml for visceral depots and 1.3 +/- 0.8 ml for subcutaneous. This compares with 0.7 +/- 0.3 for visceral depots and 1.2 +/- 0.4 for subcutaneous adipose tissue for a 6-month-old control mouse. A control mouse of similar age and genetic background has 2 to 3 times as much visceral and subcutaneous fat as the PEPCK-Cmus mouse (2.7 +/- 1.1 and 2.7 +/- 0.9 respectively). Standard error indicates the variability obtained from imaging these same mice weekly over a 3-week period. The relationship between triglyceride content of the muscle and PEPCK-C activity in PEPCK-Cmus mice. Mice with varying levels of PEPCK-C activity were selected for analysis. These animals were generated from individual mice from the C and D founder lines. The activity of PEPCK-C in the muscle of these animals was 0.08 units/g in a control, 1.20, 2.52 and 3.90 units/g muscle in the various PEPCK-Cmus mice (Fig. 6). The concentration of triglyceride in the muscle of the animals correlates well with the activity of PEPCK-C determined in the skeletal muscle. The observed relationship between the level of PEPCK-C in the muscle and the concentration of triglyceride is likely due to an increase in the rate of glyceroneogenesis in the tissue, although this remains to be determined experimentally. In agreement with the biochemical measurements, skeletal muscle from PEPCK-Cmus mice, analyzed by H & E staining, had high concentrations of lipid as compared to controls (Fig. 7A). It seems likely that this high concentration of triglyceride in the skeletal muscle of the PEPCK-Cmus mice provides the fuel needed to sustain their extraordinary level of activity. Metabolites in the blood of PEPCK-Cmus mice. The concentration of a number of metabolites as well as the activity of creatine kinase was determined in the blood of fed and fasted PEPCK-Cmus and control mice (Table III). The most striking difference was the greatly increased activity of creatine kinase in the blood of fasted PEPCK-Cmus mice; the activity of this enzyme in the blood of these animals was almost 4 times that of controls. There were also lower levels of cholesterol, free fatty acids and triglyceride in the blood of fed PEPCK-Cmus mice. The concentration of glucose in the blood of fed PEPCK-Cmus mice was increased over the values noted in control mice. The muscle of PEPCK-Cmus mice contains more mitochondria. The dramatic difference in fuel utilization during strenuous exercise suggests a profound shift in energy metabolism in the muscle. Histochemical analysis of skeletal muscle from PEPCK-Cmus mice and a control animal indicated a marked increase in the activity of succinate dehydrogenase and NADH dehydrogenase (Figs. 7B and 7C). These results are consistent with a greater number of mitochondria in the skeletal muscle. This is supported by the increased mitochondrial DNA in skeletal muscle from three PEPCK-Cmus mice, as compared to control animals (Fig. 8A). The electron microscopy image shown in Figure 8B, demonstrates a marked increase in mitochondria in the soleus muscle of the PEPCK-Cmus mice relative to controls. [...] It is remarkable that the over-expression of a single enzyme involved in a metabolic pathway should result in such a profound alteration in the phenotype of the mouse. There are several recent examples of marked alterations in energy metabolism in transgenic mice; these involve PPARδ, a transcription factor (17), or PGC-1α (18) or PGC-1β (19), transcriptional co-regulators. The over-expression of the genes for these proteins would be expected to alter expression of a number of genes in the muscle. The gene for PPARδ was driven by the α-skeletal actin gene promoter, and the transgene was expressed in skeletal muscle (17). These mice had increased type 1 muscle fibers and demonstrated an enhanced exercise performance. The genes for PGC-1α and PGC-1β were transcribed in transgenic mice from the muscle creatine kinase gene promoter and resulted in an increase in type 1 muscle fibers (PGC-1α) (18) and an increase in type II fibers (PGC-1β) (19) in the skeletal muscle of the animals. Mice that over-express PPARδ, ran for 1.5 km before exhaustion (17), as compared with the 5 to 6 km noted with the PEPCK-Cmus mice (See Fig. 3). One of the most notable physiological differences caused by the over-expression of PEPCK-C was the 40% increase in VO2 max in these animals compared to controls. This level of oxidative capacity is comparable to data reported for trained mice that were selectively bred through 10 generations to produce mice with outstanding running ability (20). The elevated oxidative capacity in the PEPCK-Cmus mice cannot be attributed to exercise training per se, but was likely due to the high daily-activity levels of these mice and the genetic manipulation, which when combined, led to the dramatic increase in mitochondrial biogenesis noted in skeletal muscle and the greatly increased concentration of triglyceride in the muscle. These cellular changes most likely had the effect of enhancing the oxidative capacity of the muscle during exercise and providing additional fuel to support energy metabolism. In this regard, the PEPCK-Cmus mice did not accumulate lactate in their blood during maximal exercise and were able to use fatty acids as an energy source during intensive exercise. They had an RER of 0.91 and a VO2 max of 156 ml/kg/min at exhaustion (i.e. after 37 min of running at an increasing speed on a treadmill set at a 25° incline). Over this entire period of strenuous exercise, the PEPCK-Cmus mice generated no net lactate; control animals had a blood lactate concentration of 8.12 mM. The increase in oxidative capacity of the muscle of the PEPCK-Cmus mice is most likely supported by more complete oxidation of glucose/glycogen and the high concentration of triglyceride noted in the muscles (up to 10 times that of control animals). It is well known that endurance training results in an increased glycogen store and an elevated utilization of fatty acids relative to carbohydrate by skeletal muscle. However, fed PEPCK-Cmus mice have slightly lower glycogen stores (1.3 +/- 0.1 vs. 1.7 +/- 0.3 mg/g tissue, P= 0.2) but greater levels of triglyceride in their skeletal muscle than controls and use fatty acids extensively during prolonged exercise. Dohm et al. (21) reported that rats running at a speed of 28 m/min did not accumulate either lactate or pyruvate in their blood, presumably because low-intensity exercise can be accomplished via aerobic metabolism. The aerobic metabolism of lactate and pyruvate requires pyruvate decarboxylation via the pyruvate dehydrogenase complex to acetyl CoA, which is subsequently oxidized in the citric acid cycle. They reported that rats running at a speed of 28m/min for 30 min had a 2-fold increase in both PEPCK-C and pyruvate dehydrogenase complex in their skeletal muscles; the activity of both enzymes decreased markedly within 5 min after the cessation of exercise. Since there are no known allosteric regulators of PEPCK-C in any tissue, the factors that are responsible for the rapid alterations in its activity in skeletal muscle are not clear. However, the effect was dramatic enough to suggest that alterations in the activity of PEPCK-C could be an important factor in the response of the animal to exercise. This is supported by the observation that the concentration of PEP in the skeletal muscle is reduced by 50% after 30 min of exercise (21). How does over-expressing PEPCK-C alter energy metabolism in skeletal muscle? PEPCK-Cmus mice have a high level of physical activity, supported in part by larger triglyceride reserves in their skeletal muscle, more complete oxidation of carbohydrate as evidenced by attenuated lactate production in response to exercise, more mitochondria and enhanced food intake. The increased activity of the PEPCK-Cmus mice is spontaneous; it was evident as early as two weeks after birth. It is not clear, however, how an over-expression of a single enzyme can so drastically re-pattern energy metabolism in the mice. There are several previously suggested mechanisms, which could partly account for the profound changes in energy metabolism. First, the increase in physical activity requires ATP to drive muscle contraction. This ATP is produced by an increased flux of intermediates through the citric acid cycle. Since PEPCK-C uses GTP and generates GDP and succinyl CoA synthase requires GDP, a possible link exists between PEPCK-C and citric acid cycle activity. Some years ago, Hahn and Novak (22) noted that brown adipose tissue had four times the PEPCK-C activity found in white adipose tissue (based on cellular protein content) and suggested that the "extra" PEPCK-C activity is involved in a cycle in which the enzyme uses the GTP generated in the citric acid cycle by succinyl CoA synthase to form PEP from oxalacetate, which is converted to pyruvate by pyruvate kinase. Pyruvate can then be decarboxylated to acetyl CoA by pyruvate dehydrogenase complex and used to generate energy in the citric acid cycle. Since PEPCK-C is in the cytosol, this scheme would require the movement of guanine nucleotides across the inner mitochondrial membrane, or the conversion of GTP to ATP by nucleoside diphosphokinase (NDK) in the mitochondria and its subsequent transport and conversion back to GTP. The intracellular location of the different isoforms of this enzyme in muscle is not clear, although studies suggest that hepatic NDK is present outside the mitochondrial matrix. There is also evidence that GTP may be transported directly from the mitochondrial matrix on an atractyloside-insensitive carrier, but the rate of transport via this carrier is slower than the well-characterized ATP/ADP translocase (23). However, the requirement for guanine nucleotides in non-dividing tissues is low, so that the transport process may be sufficient to meet the physiological requirements for guanine nucleotides in tissues such as skeletal muscle. If such a cycle exists in mammalian skeletal muscle, the over-expression of PEPCK-C could greatly enhance the rate of citric acid cycle flux and contribute to the generation of ATP required to support the increased physical activity noted with the PEPCK-Cmus mice. Second, PEPCK-C could be involved in either cataplerosis or anaplerosis. Cataplerosis is the removal of citric acid cycle anions that accumulate when the carbon skeletons of amino acids enter the cycle for ultimate degradation (7). This process is especially important in muscle, where exercise and protein turnover generates considerable amino acid flux, with subsequent oxidation (24-25). In addition, metabolic processes such as hepatic and renal gluconeogenesis and glyceroneogenesis are fundamentally cataplerotic since they involve the removal of citric acid cycle anions for biosynthesis. Viewed in this way, it is not surprising that normal skeletal muscle would have some PEPCK-C activity. Newsholme and Williams (26) reported 0.37 and 0.26 units/g of PEPCK-C activity in quadriceps and diaphragm of the rat; this activity was induced 8-fold in the quadriceps after 72 h of starvation. The synthesis of alanine from pyruvate in skeletal muscle is another example of a cataplerotic process. Snell and Duff (27) demonstrated that glutamate and valine stimulated the release of alanine by rat diaphragm and that this stimulation could be blocked by the addition of 3-mercaptopicolinate, an inhibitor of PEPCK-C (28). They proposed that PEPCK-C converted the oxalacetate, which was generated in the citric acid cycle from the metabolism of glutamate and valine, to PEP, which was subsequently converted to pyruvate via M-type pyruvate kinase and then transaminated to alanine by alanine aminotransferase. However, we could not detect an increase in the concentration of alanine in the blood or muscle of the PEPCK-Cmus mice as compared to control mice (data not shown). Cataplerosis may also be important during or after strenuous exercise when the concentration of citric acid cycle intermediates in the mitochondria of skeletal muscle greatly increases. There is a rapid, 10-fold increase in the concentration of intermediates of the citric acid cycle (anaplerosis) in muscle at the onset of moderate to intense exercise, which declines with strenuous exercise (24). It has been hypothesized that the rate of citric acid cycle flux, and thus ATP generation via the respiratory chain, might be limited by the concentration of intermediates in the cycle (24-25, 29-30) ; the presence of PEPCK-C in muscle may provide a mechanism for the removal of citric acid cycle intermediates during or after exercise. In this regard, ablation of PEPCK-C activity in the liver greatly decreased citric acid cycle flux (8, 31), so that it is likely that an increase in the activity of the enzyme would have the opposite effect. Of course, it is possible that PEPCK-C over-expression results in anaplerosis. PEPCK-C is a reversible enzyme in vitro (it has an Keq of 0.37 M at 30°C) so that one could speculate that the reaction generates oxalacetate from PEP to replenish the citric acid cycle in vivo. This would also produce GDP, which could stimulate the activity of succinyl CoA synthase and enhance citric acid cycle flux. However, many tissues contain substantial activity of pyruvate carboxylase, the major anaplerotic enzyme, which generates oxalacetate directly in the mitochondria. A third possible role of PEPCK-C in skeletal muscle is glyceroneogenesis. Skeletal muscle synthesizes and deposits considerable triglyceride to support energy metabolism. We have demonstrated that glyceroneogenesis, not glycolysis, is the major source of the glyceride-glycerol found in triglyceride in the soleus and gastrocnemius muscle of the rat (Nye, Hanson and Kalhan, unpublished results). Surprisingly, even when rats are fed a diet high in carbohydrate, the glyceride-glycerol isolated from the triglyceride in these muscles is derived from glyceroneogenesis and not from glycolysis. Thus, PEPCK-C, the key step in glyceroneogenesis, is most likely involved in triglyceride-fatty acid cycling in skeletal muscle (32). We also assume that over-expression of PEPCK-C in skeletal muscle of the PEPCK-Cmus mice causes the deposition of the observed triglyceride by providing the 3-phosphoglycerol required for its synthesis. We plan to determine the rate of glyceroneogenesis in the skeletal muscle of these mice to directly assess this possibility. Mitochondrial biogenesis in PEPCK-Cmus mice. Our studies clearly demonstrate that skeletal muscle of adult, PEPCK-Cmus mice have more mitochondria than control animals of the same age. We have not determined the developmental pattern of mitochondrial biogenesis but assume that this occurs early in life, since we noted that PEPCK-Cmus mice are highly active within the first two weeks after birth. It has been well-established that contractile activity, such as endurance exercise results in mitochondrial biogenesis in skeletal muscle and the development of type I muscle fibers (33). In addition, feeding mice a high fat diet and giving heparin to increase the concentration of FFA in the blood induced mitochondrial biogenesis in skeletal muscle (34). This is due in part to an up-regulation of PPARγ (35), PPARα (36) and PGC-1α (37), which interact to promote the oxidative capacity of skeletal muscle by stimulating the transcription of genes that lead to mitochondrial biogenesis (see reference (33) for a review). PPARα, PPARγ and PGC-1α act upstream of several genes that code for the transcription factors NRF-1 and NRF-2a and Tfam, which themselves induce mitochondrial biogenesis. Wu et al (38) reported that transducer of regulated CREB-binding proteins (TORCs), a co-activator of CREB, can induce PGC-1α gene transcription and induce mitochondrial biogenesis in muscle cells. TORCs has also been shown to function as a calcium- and cAMP-sensitive mediator that co-ordinate the effects of the two pathways on gene transcription (39). This links the calcium, which is released from the sarcoplasmic reticulum in response to contractile activity, with the activation of the transcriptional process that induces mitochondrial biogenesis in skeletal muscle. Exercise stimulates PGC-1γ by activation of the p38 mitogen activated protein kinase (MAPK) pathway via calcium signaling through the calcineurin/myocyte enhancer factor 2 (MEF2) signalling cascade (40). Calcium may also exert a positive regulatory effect on PGC-1γ gene transcription via the CRE site on the gene promoter, by activating the calcium/calmodulin-dependent protein kinase (CaMK) pathway (41- 42). Finally, strenuous exercise depletes ATP in skeletal muscle and increases the concentration of AMP via adenylate kinase; the result is an activation of AMP-activated protein kinase (AMPK). A number of recent studies have shown that AMPK is necessary for mitochondrial biogenesis via an induction of the PGC-1γ-NRF- pathway (43-45). Taken together, the current information on the mechanisms responsible for mitochondrial biogenesis supports an energy-driven stimulus (perhaps related to increased fatty acid availability), such as that which occurs in the skeletal muscle of the PEPCK-Cmus mice as the initiating factor. Increased physical activity caused by an enhanced rate of citric acid cycle flux could be a critical point in the repatterning of energy metabolism noted in these mice. Blood Metabolites in the PEPCK-Cmus mice. An elevated activity of creatine kinase in the blood is a widely used marker of skeletal muscle damage. It is commonly observed after intense or extreme exercise (46-47). The elevated resting creatine kinase levels in the PEPCK-Cmus mice may be indicative of muscle damage due to the continuous, repeated stress caused by high levels of activity. One of us (48) previously reported that exercise-induced muscle damage is associated with increased circulating creatine kinase and insulin resistance. The elevated glucose response to feeding in the PEPCK-Cmus mice supports the possibility that, despite being lean and highly active, these mice may be insulin resistant. A more detailed evaluation to follow-up on these observations is on-going. Alternatively, the elevated creatine kinase may be reflective of apoptosis and accelerated muscle remodeling that are a normal part of exercise-induced adaptations in skeletal muscle (49). Aging of the PEPCK-Cmus mice. It is well established that caloric restriction leads to increased life span in species ranging from yeast (50) to rodents (51) and increases mitochondrial biogenesis in human skeletal muscle (52); this may be due to an up-regulation of SIRT1, which activates transcription of the gene for PGC-1α. While we have not carried out a detailed aging study on our mice, we have noted that PEPCK-Cmus mice live longer and are more energetic at an older age than are control animals (see Figure 9), despite having a greater daily food intake. Holloszy (53) reported that female rats given access to voluntary running wheels had improved survival. These rats had an increase in food intake that accompanied the increase in exercise. We also have noted that 30 month-old female PEPCK-Cmus mice gave birth to normal sized litters. A more detailed analysis of this phenomenon, with more mice, will be required to conclusively support this preliminary observation. Finally, a major question, which is unanswered by the present study, is what alterations occur in the brains of the PEPCK-Cmus mice that cause the behavior described in detail in this report. Categories: 2007, PWS genes, Energy metabolism, Fatty acid metabolism, Gluconeogenesis, Carbohydrate metabolism, Glyceroneogenesis, Glycogenolysis, Diabetes, Krebs cycle, Mitochondria, NRF-1, PPARalpha, PPARbeta, PEPCK, PGC-1alpha, Cataplerosis, Anaplerosis |