Class Note for BME 510 at UA
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The Journal of Biological Chemistry 12a Supplemental Material can be ound at http lwww jbc orgcgicontent ullM706127200DC1 THE JOURNAL OE BlOLOGlCAL CHEMlSTRV VOL 282 NO 45 pp 32844 732855 November 9 2007 2007 by The Americah Socrety for Biochemistry ahd Molecular Biology lhc Primed ih the USA Overexpression of the Cytosolic Form of Phosphoenolpyruvate Carboxykinase GTP in Skeletal Muscle Repatterns Energy Metabolism in the Mouse Received for publicationJuly 252007 and in revised form August 21 2007 Published JBC Papers in Press August 23 2007 DOI 101074jbcll706127200 Parvin Hakimi Jianqi Yang Gemma Casadesus Duna Massillonl Fatima TolentinoSilvaH Colleen K Nye Marco E CabreraH David R Hageni Christopher B Utter Yacoub Baghdyi David H Johnson David L Wilson John P Kirwan Satish C Kalhan and Richard W Hansoni1 From the Departments of Biochemistry Nutrition Pediatrics Neuroscience and HBiomedical Engineering Case Western Reserve University School ofMedicine Cleveland Ohio 441064935 and the Department of GastroenterologyHepatology and Pathobiology Cleveland Clinic Foundation Cleveland Ohio 44195 Transgenic mice containing a chimeric gene in which the CDNA for phosphoenolpyruvate carboxykinase GTP PEPCK C EC 41132 was linked to the Liz skeletal actin gene promoter express PEPCK C in skeletal muscle 1 3 unitsg Breeding two founder lines together produced mice with an activity of PEPCK C of 9 units g of muscle PEPCK Cm s mice These mice were seven times more active in their cages than controls On a mouse tread mill PEPCK Cm s mice ran up to 6 km at a speed of 20 mmin whereas controls stopped at 02 km PEPCK Cm s mice had an enhanced exercise capacity with aVOZImx of 156 i 80 mlkg min a maximal respiratory exchange ratio of 091 i 003 and a blood lactate concentration of 37 i 10 mM after running for 32 min at a 25 grade the values for control animals were 112 i 21 mlkg min 099 i 008 and 81 i 50 mM respectively The PEPCK Cm s mice ate 60 more than controls but had half the body weight and 10 the body fat as determined by magnetic resonance imaging In addition the number of mitochondria and the content of triglycer ide in the skeletal muscle of PEPCK Cm s mice were greatly increased as compared with controls PEPCK Cm s mice had an extended life span relative to control animals mice up to an age of 25 years ran twice as fast as 6 12 month old control animals We conclude that overexpression of PEPCK C repatterns energy metabolism and leads to greater longevity PEPCK C2 is involved in gluconeogenesis in the liver and kidney cortex and in glyceroneogenesis in liver and white and This work was supported by National Institutes of Health Grants DK058620 and DK025541 to R W H GM66309 to M EC AG12834 to J P K EB004070 to D L W and CA43703 to the Case Western Reserve Univer sity Comprehensive Cancer Center by a Provost Vision Fund grant to sup port the Case Western Reserve University School of Medicine Behavioral Core Laboratory to G C by Grant NNJ06HD81G to MEC from the NASA and by an Ohio BRTT The Biomedical Structure Functional and Molecular Imaging Enterprise grant The costs of publication ofthis article were defrayed in part by the payment of page charges This article must therefore be hereby marked advertisement in accordance with 18 USC Section 1734 solely to indicate this fact This article was selected as a Paper of the Week El The on Iine version ofthis article available at httpwwwjbcorg contains a video with sound showing mice performing a treadmill test 1 To whom correspondence should be addressed Tel 216 368 3880 Fax 216 368 4544 E mail rwhcaseedu 2 The abbreviations used are PEPCK C the cytosolic form of phosphoenol pyruvate carboxykinase GTP bGH bovine growth hormone PPAR per 32844 JOURNAL OF BIOLOGICAL CHEMISTRY brown adipose tissue see Ref 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 adre nal gland lung and muscle its metabolic role in these tissues remains obscure To study the physiological function of PEPCK C the gene has been overexpressed or ablated in spe cific tissues of the mouse When PEPCK C was overexpressed in white adipose tissue the mice had increased rates of glycero neogenesis in their adipose tissue and became obese 3 In con trast 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 overexpressed the gene for PEPCK C in the skeletal muscle of transgenic mice to test the metabolic and physiolog ical consequences Skeletal muscle was selected as a target organ because there is no clear indication of the metabolic out come of having a high activity of PEPCK C in this tissue Skel etal 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 ongoing in our laboratory3 that glyceroneogenesis occurs in skeletal muscle This pathway is an abbreviated version of gluconeogenesis which involves the synthesis of glycerol S phosphate used for triglyceride syn thesis from precursors other than glucose and glycerol How ever glyceroneogenesis has not previously been reported to occur in skeletal muscle so the extent of the metabolic effect of overexpressing 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 oxisome proliferator activated receptor RER respiratory exchange ratio WT wild type CRE CAMP response element CREB cAMP response ele ment binding protein P enolpyruvate phosphoenolpyruvate 3 C K Nye R W Hanson and S C Kalhan unpublished results TE as 1quot VOLUME 28239 NUMBER 45 39 NOVEMBER 9 2007 9003 L JeuneAON uo Me an euozuv JO Allelenun 112 61039oql39MMM won pepeoiumoa 4 39 5 The Journal of Biological Chemistry 1 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 ux that is required to generate the energy to support muscle con traction An increase in citric acid cycle anions occurs largely due to anaplerosis However any four or five carbon interme diate 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 the 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 crit ical role of PEPCK C in energy metabolism in mammalian skel etal muscle has not been tested Our results indicate that transgenic mice that overexpress the gene for PEPCK C about 9 units g of 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 a high concentration of triglycer ide in their skeletal muscles The mice overexpressing 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 EXPERIMENTAL PROCEDURES Generation and Analysis of Transgenic Mice A chimeric gene was constructed that included 2 kb of the oz skeletal actin gene promoter 9 that was linked to a 1976 bp segment of the cDNA for PEPCK C from the mouse followed by a 710 bp frag ment of the 3 untranslanted region of the bovine growth hor mone bGH mRNA see Fig 1A The oz skeletal actin gene promoter was a kind gift from Dr Laurence H Kedes University of Southern California This gene promoter was selected since it has been shown to limitthe transcription oflinked structural genes to skeletal muscle with a low level of expression in brown adi pose tissue 10 The mice were produced at the Case Western Reserve University School of Medicine Transgenic Mouse Core Facility by a procedure described in detail previously 11 12 Of the 34 mice that were produced six positive founders des ignated as lines A B C D E and F were identified by Southern blotting DNA was isolated from the tails of the mice by lysis overnight at 55 C in a buffer containing 50 mM KCl 10 mM Tris HCl pH 83 25 mM MgClg 01 gelatin 045 Nonidet P 40 045 Tween 20 and 24 mgml proteinase K The DNA was digested with Pstl and the resulting fragments were sepa rated by electrophoresis using 1 agarose gel and transferred to Gene Screen Plus PerkinElmer Life Sciences A 690 bp frag ment of the human oz skeletal actin gene promoter Fig 1A which showed no significant homology to sequences in the mouse genome as judged by National Center for Biotechnology Information NCBI BLAST prediction was used as a hybrid ization probe To increase the activity of PEPCK C in the skel etal muscle founder lines C and D were bred together to create a new line designated as line CD see Fig 1C Homozygous mice in the line CD C D were used for most of the experiments Animal Care The mice were maintained in the Case West ern Reserve University Animal Resource facility under the NOVEMBER 9 2007VOLUIVIE 28239 NUMBER 45 PEPCKC in Skeletal Muscle supervision of full time veterinarians The research described in this study was approved by the Case Western Reserve Uni versity School of Medicine Institutional Animal Care and Use Committee RNA Analysis Total RNA was extracted from mouse tissues using the QuickPrep total RNA kit Amersham Biosciences 13 and Northern blotting was performed as described in detail previously 13 Brie y 20 Lg of total RNA was separated by electrophoresis on an agarose gel transferred to Gene Screen Plus membrane and hybridized with a 10 kb Smal frag ment of the PEPCK C cDNA The DNA probe was labeled using oz 32PdATP Determination of PEPCK C Various mouse tissues were homogenized in 025 M sucrose containing 5 mM Tris HCl at pH 74 and 1 mM dithiothreitol A cytosol fraction prepared by centrifuging the homogenate at 30 000 X gfor 30 min at 4 0C was used to determine the activity of PEPCK C by the method of Ballard and Hanson 14 The levels of PEPCK C from liver heart kidney and skeletal muscle were determined using Western blotting The antibody to PEPCK C was kindly pro vided by Dr F I Ballard Adelaide Australia Assay of Metabolites in the Blood and Tissaes Mice were anesthetized by an intraperitoneal injection of avertin 05 ml of 20 mgml solution25 g of body weight The concentration of glucose in the blood was determined using an Encore glucom eter Bayer Corp Plasma was generated from whole blood using MlCROTAlNER plasma separator tubes BD Bio sciences The concentration of triglyceride fractionated bili rubin B hydroxybutyrate albumin total protein blood urea nitrogen cholesterol creatinine creatine kinase triglyceride and free fatty acid was determined by Veterinary Diagnostic Services Marshfield Laboratories Muscle triglycerides were extracted using a modification of the method of Folch et al 15 Brie y fresh muscle samples were freed from any visible non muscle material and weighed out in duplicate Each sample was homogenized via lKA ULTRA TURRAX T 25 basic tissue blender and total lipids were extracted by incubating the tissue homogenate in a 21 chloroformzmethanol solution for 48 h at 4 0C The two phases were further defined by the addition of MgCl2 and centrifuga tion The supernatant was extracted and discarded and the remaining layer was taken to dryness under air The triglycer ides were saponified using methanolic KOH 05 N at 70 C for 75 min acidified with 6 N HCl The resulting fatty acids were extracted three times with hexane The aqueous phase contain ing glycerol was dried using a Labconco CentriVap concentra tor at 25 0C The concentration of glycerol was determined using a fluorometric assay essentially as described by Wieland 16 that value was used for the calculation of the concentra tion of triglyceride in the tissue Home Cage Activity Testing All mice were individually caged 6 weeks prior to the onset of behavioral testing Prior to home cage activity measurements the mice were observed in their cages for 15 min three times a day to rule out unusual behaviors that could confound the home cage activity measure ments such as locomotor or grooming stereotypy tremors unusual nest building and overall locomotor function unusual gait events Two observations were carried out during the light JOURNAL OF BIOLOGICAL CHEMISTRY 32845 9003 L JeuneAON uo Me an euozuv jo Aimenun 112 61039oqI39MMM won pepeowmoa 4 39 5 The Journal of Biological Chemistry 1 PEPCKC in Skeletal Muscle phase and one was carried out during the dark phase Five mice were observed at each 15 min time point with each animal being observed for 30 s intervals All of the mice were brought into the testing room 24 h prior to the onset of testing for acclimatization to the room and test ing conditions which included the replacement of the standard waterfood holding top with a clear top and placing feed and uid source Hydrogel inside the cage All mice were tested during the same trial Animal cages were placed on a table directly below a charged coupled device CCD camera Pana sonic and observations were carried out for 22 h consecu tively starting at 3 pm and concluding at 1 pm using a track ing system Noldus Ethovision 31 Pro Leesburg VA Distance traveled velocity and number of rearings as well as turn angle and angular velocity to further rule out stereotypy related running were automatically calculated by the tracking system Data were nested in 1 h intervals to simplify statistical analysis Upon termination of testing wire tops with standard food and water were placed back on the cages and the mice were returned to their housing room Maximal Exercise Capacity Exercise performance and exercise capacity were determined in fed gender matched and age matched PEPCK Cmus mice and controls The mice were subjected to strenuous exercise on a rodent treadmill Columbus Instruments Oxymax System Columbus OH using standard exercise running tests to evaluate aerobic capacity V02m max imal running endurance and maximal running speed To encourage the mice to run the treadmill was equipped with an electrical shock grid at the rear of the treadmill The shock grid was set to deliver 02 mA which caused an uncomfortable shock but did not physically harm or injure the animals When the mice reached exhaustion as defined by their inability to run for 10 s the electric shock was discontinued Types of Treadmill T esting We included three types of strenuous exercise measurements treadmill testing in this study First we assessed the ability of untrained PEPCK Cmus and control mice to run for distance The animals were initially acclimated to the treadmill environment for 30 min For warm up and for further familiarization with treadmill run ning the mice were required to run at a relatively easy pace of 10 mmin for 30 min Then the speed of the treadmill was increased to 20 mmin and we recorded the exercise duration and distance the mice could run until exhaustion Exhaustion was defined operationally as the time at which the mouse was unable or refused to maintain its running speed despite encouragement by mild electrical stimulation Second we determined the V02mx of both types of mice Prior to running the mice were acclimated to the enclosed treadmill and its surroundings for 60 min During this period measurements of whole body oxygen consumption V02 car bon dioxide production VCO2 and the respiratory exchange ratio RER were obtained while the animals rested quietly The mice then began walking on the treadmill at a speed of 5 mmin and a grade of 0 for a 10 min warm up The grade ofthe tread mill was then set to 25 and the speed was increased 2 mmin every 2 min until the mouse reached exhaustion Both VO2 and VCO2 were monitored continuously throughout the exercise test Substrate utilization during exercise was assessed from the 32846 JOURNAL OF BIOLOGICAL CHEMISTRY RER data A blood sample was taken before and after exercise for the determination of lactate Third PEPCK Cmus and control mice of various ages were acclimated to the treadmill for 30 min followed by a 30 min warm up period at a treadmill speed of 10 mmin set at a grade of 0 We then increased the speed ofthe treadmill by 1 mmin every min and determined the maximum speed that the mice could run until exhaustion Magnetic Resonance Imaging Analysis of Total Body Fat A fed PFPCK Cmus mouse and two control animals were imaged using a 7T30 cm Bruker BioSpin small animal magnetic reso nance scanner A multislice multiecho spin echo acquisition was used to obtain high resolution coronal images of a whole mouse using a 120 mm rat volume coil to transmit and receive with T1 weighting and respiratory gating time to repetition echo time 9009 ms 512 X 256 matrix 110 X 41 mm field of view 33 slices To separate visceral and subcutaneous adipose tissue compartments the internal cavity within the abdominal wall was manually segmented using a common software pack age Analyze 60 Mayo Clinic Minneapolis MN Fat was hyper intense in these images allowing us to segment adipose tissue by interactively setting a threshold value Visceral and subcutaneous adipose tissue volumes were computed from the number of voxels times the volume of a single voxel Similar single slice images were acquired for publication rapid acqui sition with retocussed echoes TRTE 100012 ms 512 X 512 matrix 100 X 60 mm FOV lmage acquisitions were repeated over 3 weeks to examine measurement repeatability Histological and Electron Microscopy Analysis of Tissaes Skeletal muscle was isolated from overnight fasted PEPCK Cmus and control mice and fixed immediately in formalin solu tion Sigma in liquid N2 or in Tissue Tek OTC compound Sakura Finetek Inc for staining by hematoxylin and eosin for succinate dehydrogenase and NADH dehydrogenase or for electron microscopy respectively The histochemical and elec tron microscopy analysis was performed at the Case Western Reserve University School of Medicine by the Cytology Labo ratory and the Electron Microscopy Facility E ect of Aging on the Capacity of PEPCK C m s Mice to Ran PEPCK Cmus mice and controls were The Journal of Biological Chemistry fie PS I I I sl l I l quotI uskeletal actin mouse PEI C39KC cDNA bGH promoter poly A quotariuus Icnglli liq kb I 7 I I d I quot AI l i m Founders A WT B C D Jfl M w 3quot I e l I a l lb I L quota L E 1 5 u L S L PEPC KC mRNA 39 a V 39 Ifquot Skeletal Muscle zssiRm i i v s IssiRXA i IU RNA gel stained with ethidium bromide PEPCKC in Skeletal Muscle C cl Df x Cl l cl 01 x Cw DH Cu Di cl Du 11 6 216 216 415 1l15 1516 Discarded va39 Dl CT 0quot 45 c 0quotquot cu Dl Cm 0on cu DH co Du C39 DI C4 Ba 9 IS I 103 l 12 34 H 108 i 002 H NI the activity of PEPCKC in skeletal muscle unitsgram tissue E PEI CK C quotmquot I E PCKC m39 1 l 3 ll 3 ll 2 a in 3 399 a pa E 5 3 t 3 1 5 3 E i E 2 e 72 2 s c 2 1 a x J vi 3 I PEPCK C FIGURE 1 Generation of PEPCKCmus miceA a chimeric gene containing 2 kb ofthe human or skeletal actin gene promoter linked to the cDNA for PEPCK C from the mouse followed by the 3 end of bGH see quotExperimental Procedures for details The hatched bar represents the DNA probe that was used for Southern blotting genomic DNA from the mice 3 a schematic illustration of two copies of the transgene integrated into the host cell genome Pstl digestion ofgenomic DNA results in a 19 kb fragment and fragments ofvarious lengths depending upon the location ofother Pstl sites in the genome The hatched bar represents the DNA probe that was used for Southern blotting genomic DNA from the mice The Southern blot shown in B is for DNA from founder lines A B C D and wild type WT mice The pattern of DNA for the cross between founder lines C and D CD is also shown in the last two lanes the homozygous CD mice are termed PEPCK Cm mice C a schematic representation of the CD cross to generate the PEPCK Cmus mice used in these studiesThis panel illustrates the level of activity of PEPCK C in the skeletal muscle of mice at various stages of interbreeding Mice heterozygous for either the C or the D genotypes had an activity ofPEPCK C between 12 and 34 unitsg of skeletal muscle it was not possible to distinguish between the speci c genotypes ofthese mice by Southern blotting The values for the activity of PEPCK C for the PEPCK Cmus mice and wild type animals are presented as the mean i the SE ofthe mean for the number of mice shown in parentheses D a Northern blot ofthe PEPCK C mRNA in the skeletal muscle of CD mice and control animals WT The PEPCK C mRNA in the liver is also presented as a positive control E a Western blot of the PEPCK C from the liver heart kidney and skeletal muscle 5 Muscle of PEPCK Cmus mice and controls WT PEPCK C activity in their muscles Fig 1C the animals were subsequently used to determine the consequences of overex pressing PEPCK C on the metabolism of skeletal muscle After nine generations of crossbreeding of founder lines C and D we created a homozygous line of mice which we termed PEPCK Cmus mice Expression of PEPCK C in Muscle The expression of the transgene was assessed by both Northern and Western blotting of several tissues isolated from PEPCK Cmus mice and controls Fig 1 D and E PEPCK C mRNA transcribed from the trans gene was detected in skeletal muscle of PEPCK Cmus mice no PEPCK C mRNA from the transgene was noted in muscle from the control animals The same pattern was observed by West ern blotting using an antibody specific for PEPCK C The level of mRNA for PEPCK C noted in the skeletal muscle of mice differed in our lines of PEPCK Cmus mice during the process of NOVEMBER 9 2007 VOLUME 28239 NUMBER 45 v a breeding founder lines C and D together Fig 1D This was re ected in a variation in the activity of PEPCK C in the muscle of these mice Fig 1C Homozygous PEPCK Cmus mice had 9 unitsg of PEPCK C activity in gastrocnemius soleus and diaphragm in contrast the same muscles from con trol mice have about 008 unitsg of PEPCK C activity Table 1 Since the gene for PEPCK C was expressed from the oz skel etal actin gene promoter all the skeletal muscle types deter mined had the same activity In addition the heart from the PEPCK Cmus mice had 074 unitsg of enzyme activity the activity of PEPCK C is normally undetectable in the hearts of mice We routinely noted that the activity of PEPCK C was higher in the livers of the PEPCK Cmus mice than controls Table 1 This increased activity was not due to transcription from the transgene since a specific cDNA probe composed of the 805 bp Smal EcoRl fragment of the bGH gene located at JOURNAL OF BIOLOGICAL CHEMISTRY 32847 9003 L JeuneAON uo AJBJqH euozuv i0 AllSJeAlUn 112 DJO39Oql39MMM Luou pepeowmoa The Journal of Biological Chemistry 1 PEPCKC in Skeletal Muscle TABLE 1 The activity of PEPCKC in selected tissues Selected tissues were collected and the activity of PEPCKC determined as described under Experimental Procedures The phrase Skeletal musclequot refers to mixed thigh muscle The activity ofPEPCKC is expressed as the mean t SE for the number of animals indicated in parentheses The unit of activity is defined as one umole of substrate converted to productmin at 37 C PE PCK Cmus unitsg n Skeletal muscle 915 i 103 4 Soleus 928 i 126 4 Gastrocnemius 886 i 204 4 Diaphragm 800 i 193 4 Heart 074 i 033 2 Liver 500 033 11 Skeletal muscle WT 008 i 002 8 Liver WT 309 013 6 the 3 end of the transgene did not hybridize to hepatic mRNA data not shown The elevated hepatic PEPCK C activity was most likely due to induced transcription of the endogenous PEPCK C gene in the liver of the transgenic mice caused by the high level of energy utilization noted in these mice Subsequent studies of the physiological effect of overexpression of the gene for PEPCK C in the muscle concentrated on the use of PEPCK Cmus mice with about 9 units g of activity of the enzyme in their skeletal muscles Activity in the Home Cage During the process of generating a homozygous line of PEPCK Cmus mice from the C and D founder lines we noted a very marked increase in the physical activity of the animals as compared with controls the mice ran continuously in their cages A systematic analysis of the PEPCK Cmus mice was thus undertaken Our preliminary observations did not detect any obvious signs of stereotypy like locomotion excessive grooming or abnormal gait all of which are potential confounders during home cages activity measure ments of the PEPCK Cmus mice or controls Turn angle and angular velocities were similar in both groups thus ruling out spinning a common example of stereotypy like locomotion in the experimental group Home cage activity measurements indicated that PEPCK Cmus mice were markedly more active in their home cages as compared with control animals Fig 2 This was indicated by a greatly increased distance traveled and an increased rearing frequency Likewise PEPCK Cmus mice had significantly faster movement in the cage as compared with controls Fig 2 The mobility parameter tracks the location of the pixels which are identified as belonging to the tracked animal in the current sample frame and compares them with the pixels in the previous frame A large difference more than a user defined threshold is identified as strongly mobile a small difference less than the threshold is immobile and values in between are mobile This allows for a refined and quantifiable categoriza tion of movement or lack thereof in experimental subjects In this regard our data indicate that although control and PEPCK Cmus mice spent a similar percentage of time in the mobile range PEPCK Cmus mice spent significantly less time immobile and significantly more time as strongly mobile as compared with control mice Fig 2 The fact that PEPCK Cmus mice were faster than controls is in accordance with our strong mobility findings However the fact that these animals spent similar 32848 JOURNAL OF BIOLOGICAL CHEMISTRY son 75 400 5 an 3 100 E 5 z e 200 74 i 2 39 9 100quot r nu u d 39 A E 750 393quot E 2 1 h 5 5 c c i f E 0 500 39le i E 33 L 1 a 3 quot 1 c 150 ill 7 b a i 1 WT PEPCK cm39 WT PICI CKC39m FIGURE 2 The home cage activity of PEPCKCmus mice The activity of PEPCK Cmus mice and controls WT maintained with a 12 h lightdark cycle was determined as outlined under quotExperimental Procedures The measurements made over 22 h included the distance covered the velocity of the movement the rearing frequency and the percentage of time the mice were strongly mobile portions of time in the mobile range and showed significantly more rearings rules out the possibility that the increased levels of activity distance traveled were simply due to a constant higher velocity of the mice Treadmill Testing We next tested the ability of PEPCK Cmus mice to run for distance on a treadmill Fig 3 Untrained PEPCK Cmus mice ran for up to 6 km at a speed of 20 mmin whereas controls ran for only 02 km at the same speed before exhaustion The PEPCK Cmus mice also retained an extraordi nary ability to perform strenuous exercise at an age greater than 24 months see Fig 9 A sound Video of these mice running is included as supplemental data Determining theRER Oxygen consumption carbon dioxide generation both are used to calculate the RER and alterations in the concentration oflactate in the blood of PEPCK Cmus and control mice during strenuous exercise were next measured with animals running on a mouse treadmill using the second exercise protocol that is described in detail under Experimen tal Procedures After a 60 min period of acclimation the mice began walking on the treadmill at a speed of5 The Journal of Biological Chemistry 1 4 3 Distance run km 2 1 0 WT P EPCKC In mice FIGURE 3 PEPCKCmus mice run for a long distance on a treadmill Untrained 3 month old PEPCK Cmus mice and controls WT were tested for their ability to run long distances The mice were placed on a treadmill at a grade of 0 and run at 20 mmin until exhaustion as described in the first protocol under quotExperimental Procedures In the supplemental data please note that a video is available that documents the remarkable ability of the PEPCK Cmus mice to run for long distances TABLE 2 Metabolic parameters of PEPCKCmus mice and controls after strenuous exercise Values are the means i SD for the number ofmice shown in the parenthesis The VO2 oxygen consumption VOZmax maximal V02 VCO2 carbon dioxide pro duction VCO2mm maximal CO2 production and RER were determined as described under Experimental Procedures p lt 005 Variables Wild type PE PCK Cmus n 10 n 9 VO2 at rest mlkg 1min 1 469 i 96 477 i 109 VCO2 at rest mlkg 1min 1 373 i 934 409 i 141 RER VCOZVOZ at rest 079 i 005 077 i 005 Blood lactate at rest mM 490 i 034 370 i 017 vomx mlkg 1min 1 1123 209 1564 806 vco2 at volm mlkg 1min 1 1108 210 1420 848 RER at volm 099 008 091 003 Blood lactate mM after exercise 812 i 50 370 i 100 Maximum speed mmin at 25 slope 234 i 479 366 i 760 Maximum running time min 192 i 476 319 i 763 to 091 at exhaustion 319 min of running In contrast the control animals n 10 began the experiment with an RER of 079 which rapidly increased to 099 at exhaustion 19 min of strenuous exercise Fig 4 is a graphical representation of the difference between a selected PEPCK Cmus mouse and a control animal that illus trates the extraordinary metabolic characteristics of the PEPCK Cmus mice This animal ran for 43 min until exhaus tion as compared with 13 min by its control littermate and it had an RER of 082 at exhaustion the control animal had an RER of 107 What is especially dramatic is the difference in the concentration of lactate in the blood Although both the PEPCK Cmus mouse and the control began the period of exer cise with nearly equal values of blood lactate concentration at exhaustion the concentration of lactate in the blood of the con trol animal rose to 17 mM whereas 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 con trol mice rapidly move from fatty acid metabolism to the utili NOVEMBER 9 2007 VOLUIVIE 28239 NUMBER 45 v PEPCKC in Skeletal Muscle Lactate in blood Rest Exercise PEPCKC quotWquot 38 m 39I 28 mM 1 1 Wild type 44 m39I 70 mM 10 39 E 2 Wild type 09 39 RER I PEPCK C quotm 0839 07 05 5 I 5 0 5 1D 15 20 25 30 35 40 7 7quot quotv Rest Exercise time min FIGURE 4 A graphical plot of fuel utilization by a PEPCKCmus and control mouse during strenuous exercise This gure is a graphical plot of the alter ations in the RERofan untrained PEPCK Cmus mouseand a control animaIThe data are drawn from a larger group of PEPCK Cmus mice n 9 and control Iittermates n 10 that is presented in Table 3The RER was assessed using a speed ramped treadmill entirely enclosed in an environmental chamber and equipped to measure changes in oxygen consumption and carbon dioxide outputThe mice were acclimated to the chamber for 60 min after which the speed of the treadmill set at a 25 slope was set at 10 mmin and for 30 min and then increased 2 mmin every 2 min until the mice reached exhaustion see the second protocol described under quotExperimental Procedures A blood sample was taken before and after exercise for the measurement of lactate Both the rate of oxygen consumption and the rate of carbon dioxide generation by the mice were monitored continuously throughout the exer cise period zation 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 C m s M ice 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 mag netic resonance imaging Fig 5B The adipose tissue volumes were 04 i 02 ml for visceral depots and 13 i 08 ml for subcutaneous This compares with 07 i 03 for visceral depots and 12 i 04 for subcutaneous adipose tissue for a 6 month old control mouse A control mouse of similar age and genetic background has 2 3 times as much visceral and subcutaneous fat as the PEPCK Cmus mouse 27 i 11 and 27 i 09 respec tively Standard error indicates the variability obtained from imaging these same mice weekly over a 3 week period The Relationship between Triglyceride Con tent of the M MSCle and PEPCK C Activity in PEPCK Cm M ice Mice with vary ing 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 008 unitsg in a control and 120 252 and 390 units g of muscle in the various PEPCK Cmus mice Fig 6 The concentration of triglyceride in the muscle of the animals cor relates 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 JOURNAL OF BIOLOGICAL CHEMISTRY 32849 9003 L JeuneAON uo Aieiqn BUOZIJV JO MISJGAIUn 112 61039oqI39MMM won pepeowmoa The Journal of Biological Chemistry fie PEPCKC in Skeletal Muscle A 4quot t4 l5 20 3 lt A r K E 3quot m quotl Z F L it 4 5 E 3 5 T 5 3 20 10 a m l a E V E m g G v i III I 1 I WT PEPCK C 395 W39T PEPCKCquotquot B PEPCK quotquot WT WT 18 months old 18 months old 6 months old 27 1 11 07 1 03 Subcutan m 13 t 03 27 t 09 12 i 04 fat ml FIGURE 5 Food intake and relative body fat of PEPCK Cmus mice and con trols A The average daily food consumption rightpanel as related to the body weight of the mice was calculated for ve control WT and 17 PEPCK Cmus mice daily over a 3 week period The body weight left ofthe same mice was determined three times a week over a 3 week period The values are presented as the mean i SE of the mean 3 magnetic resonance imaging showing hyper intense adipose tissue due to T1 weighting Animals are an 18 month old PEPCK Cmus mouse CD18 an 18 month old control WT and a 6 month old control WT Volumes of visceral and subcutaneous adipose tissues were calculated as outlined under quotExperimental Procedures tissue although this remains to be determined experimentally In agreement with the biochemical measurements skeletal muscle from PEPCK Cmus mice analyzed by hematoxylin and eosin staining H 9E staining had high concentrations of lipid as compared with 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 0fPEPCK C m s Mice The concen tration 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 3 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 four 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 32850 JOURNAL OF BIOLOGICAL CHEMISTRY Triglyceride Limol lg muscle 008 120 252 390 PEPCK C Activity unitslg muscle FIGURE 6The relationship between the concentration of triglyceride and the activity of PEPCK C in the skeletal muscle of PEPCKCmus mice and control WT animals The activity of PEPCK C and the concentration of trig lyceride in skeletal muscle were determined as described under quotExperimen taI Procedures The unit of activity is 1 umol of substrate converted to prod uct per min at 37 C A H 8 E w ining WT PEPCK C 39quotm mice Succinmc Dchydrugcnusc w NADH Dehydrogenase r A I WT PEPCKC quotquot mice FIGURE 7 Histological analysis of skeletal muscle from PEPCKCmus mice and controls A hematoxylin and eosin staining H amp Estaning of skeletal muscle showing Iipid inclusions in the muscle of the PEPCK Cmus mice 3 succinate dehydrogenase staining C NADH dehydrogenase staining AII slides are at gtlt200 magnification The Mascle 0f PEPCK C m s Mice Contains More Mito chondria The dramatic difference in fuel utilization during strenuous exercise suggests a profound shift in energy metabo lism 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 Fig 7 B and C These results are con VOLUIVIE 28239 NUMBER 45 39 NOVEMBER 9 2007 9003 L JeuneAON uo Anaan euozuv 0 AJISJGAIUn 112 61039oql39MMM won pepeowmoa The Journal of Biological Chemistry 4 TABLE 3 The concentration of metabolites in blood PEPCKC in Skeletal Muscle PEPCIQC LE mice and control animals were fed ad libitum or fasted for 18 h and their blood was collected The analysis of metabolites was performed by Marshfield Laboratories as outlined under Experimental Procedures The results are expressed as the means i SE for the number of animals indicated in parentheses The p value is calculated for PEPCIQC us mice and wild type animals Fed Fasted Wild type PE PCK Cmus p value Wild type PEPCK Cmus p value Cholesterol mgd1 1056 127 5 658 102 9 003 1 833 82 6 602 87 6 008 Free fatty acid mEqliter 056 009 7 039 006 10 010 062 023 6 050 010 6 066 Triglycerides mgd1 957 177 7 587 75 10 005 1 646 223 7 483 80 6 053 Ketone bodies mgd1 191 023 7 282 077 10 035 62 12 6 63 20 6 096 Glucose mgd1 1507 81 3 2177 66 3 lt001 1190 73 5 1364 50 9 009 Blood urea nitrogen mgd1 293 i 29 7 291 i 18 10 095 218 i 46 5 270 i 28 5 036 Creatinine mgd1 011 002 7 016 003 10 022 012 002 5 013 002 6 066 Creatine kinase unitliter 2762 10856 3248 690 10 070 1215 131 6 4460 13025 002 Total bilirubin mgd1 027 012 7 011 001 10 013 076 038 5 010 000 2 035 Total protein gdl 486 020 7 460 007 10 018 476 014 5 433 044 3 025 Albumin gdl 273 0117 293 014 10 032 306 018 5 253 026 3 014 a Statistical significance sistent with a greater number of mitochondria in the skeletal A muscle This is supported by the increased mitochondrial DNA 6 in skeletal muscle from three PEPCK Cmus mice as compared 33 with control animals Fig 8A The electron microscopy image 2 2 shown in Fig 8B demonstrates a marked increase in mitochon dria in the soleus muscle of the PEPCK Cmus mice relative to controls 23 Aging ofthe PEPCK C m s Mice We noted that the PEPCK 3 I 7 i Cm mice survived far longer and looked healthier than con 2 trols and that both male and female PEPCK Cmus mice were reproductively active at 21 months of age one PEPCK Cmus mouse gave birth at 30 months of age data not shown We 0 J J therefore tested the running ability of PEPCK Cmus and control mice at various ages using the third protocol described in detail under Experimental Procedures The mice were given a period of 30 min acclimation to the treadmill followed by a 30 min warm up period in which they were run at a speed of 10 mmin with no elevation of the treadmill The speed of the treadmill was increased by 1 mmin every min until the mice were exhausted Fig 9 At all ages tested the PEPCK Cmus mice performed significantly better than control animals Twelve to 18 month old PEPCK Cmus mice ran at an average maximum speed of 45 mmin as compared with 22 mmin for 6 month old control mice PEPCK Cmus mice from 18 months of age to older than 24 months also ran at a higher rate of speed than control animals that were 2 6 months of age DISCUSSION The oz skeletal actin gene promoter has been widely used for the expression of genes specifically in skeletal muscle of trans genic mice 9 10 17 The gene promoter from 2000 to 239 drives a high level of expression in all types of striated muscle and is regulated in a tissue specific manner during development 9 Transcription from the oz skeletal actin gene promoter in the mouse hind limb is initiated at about embry onic day 14 when it replaces oz cardiac actin as the major sar comeric actin Clapham et al 10 used the mouse oz skeletal actin gene promoter to drive the expression of the structural gene for uncoupling protein 3 UCP B in the skeletal muscle of transgenic mice They reported expression of the UCP 3 trans gene in skeletal muscle with a small 5 fraction of the activity also noted in brown adipose tissue There was no UCP 3 NOVEMBER 9 2007 VOLUME 28239 NUMBER 45 v WT PEPC K C quot1quot mice 5 5 7 l I ii 11 quot5 x 7 39l 39f 7 i 391 WT PE PC KCquotquotquot mouse FIGURE 8 Increased mitochondrial content in skeletal muscle of PEPCK Cmquots mice A the relative concentration of mitochondrial DNA in skeletal muscle of PEPCK Cmus mice and controls WT The inset is a Southern blot of DNA isolated from three PEPCK Cmus mice and two control animals 3 elec tron micrographs of soleus muscle from PEPCK Cmus mouse and a control animal WT showing an increased number of mitochondria arrow mRNA as determined by reverse transcription PCR in 12 other tissues from their mice We also noted expression of the gene for PEPCK C mainly in skeletal muscle of our transgenic mice PEPCK C mRNA was not detected by Northern blotting in the hearts of any of the four founder lines measured but was noted in mice generated after crossing founder lines C and D PEPCK Cmus mice We assume that transcription from the oz skeletal actin gene promoter occurred in the hearts of the PEPCK Cmus mice due to cardiac hypertrophy induced in the PEPCK Cmus mice by their marked hyperac tivity The effect that the expression of the gene for PEPCK C JOURNAL OF BIOLOGICAL CHEMISTRY 32851 9003 L JeuneAON uo Aieiqn euozuv 0 Allelenun 112 61039oqi39MMM won pepeowmoa 4 39 5 The Journal of Biological Chemistry e PEPCKC in Skeletal Muscle WT 5 PEPCKC quot mice 5 a 51 40 2 3 7i 4 4 3 0 Age month l2 26 6l2 12l8 l824 gt24 FIGURE 9 Running ability of PEPCK Cmus mice with age Trained PEPCK Cmus mice and controls WT ofvarying ages were tested for their ability to run on a treadmill using the third protocol as described in detail under quotExperi mental Procedures The mice were acclimated to the treadmill at a grade of 0 for 30 min at a speed of 10 mmin after which time the speed of the treadmill was increased 1 mmin every min until the mice reached exhaus tion The number ofanimals tested is indicated in parentheses Maximum speed in mm 3 la c l has on cardiac function remains to be determined However it is likely that the majority of the metabolic and behavioral changes noted in the PEPCK Cmus mice are due to metabolic alterations resulting from the overexpression of PEPCK C in skeletal muscle and that the presence of PEPCK C in the heart is an adaptation to the greatly increased physical activity that characterize these mice It is remarkable that the overexpression of a single enzyme involved in a metabolic pathway should result in such a pro found alteration in the phenotype of the mouse There are sev eral recent examples of marked alterations in energy metabo lism in transgenic mice these involve PPAR5 a transcription factor 17 or PGC loz 18 or PGC 1B 19 transcriptional co regulators The overexpression of the genes for these proteins would be expected to alter expression of a number of genes in the muscle The gene for PPAR5 was driven by the oz skeletal actin gene promoter and the transgene was expressed in skel etal muscle 17 These mice had increased type 1 muscle fibers and demonstrated an enhanced exercise performance The genes for PGC loz and PGC 1B were transcribed in transgenic mice from the muscle creatine kinase gene promoter and resulted in an increase in type 1 muscle fibers PGC loz 18 and an increase in type II fibers PGC lB 19 in the skeletal muscle ofthe animals Mice that overexpress PPAR5 ran for 15 km before exhaustion 17 as compared with the 5 6 km noted with the PEPCK Cmus mice Fig 3 One of the most notable physiological differences caused by the overexpression of PEPCK C was the 40 increase in VO2max in these animals as compared with controls This level of oxidative capacity is comparable with data reported for trained mice that were selectively bred through 10 generations to produce mice with outstanding running ability 20 The ele vated 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 manipu lation 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 32852 JOURNAL OF BIOLOGICAL CHEMISTRY the oxidative capacity of the muscle during exercise and pro viding 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 091 and a V02mx of 156 ml kg min at exhaustion ie after 37 min of running at an increasing speed on a treadmill set at a 250 incline Over this entire period of strenuous exercise the PEPCK Cmus mice generated no net lactate control animals had a blood lactate concentration of 812 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 13 i 01 versus 17 i 03 mgg oftissuep 02 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 of28 mmin did not accu mulate 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 dehydro genase complex to acetyl CoA which is subsequently oxidized in the citric acid cycle They reported that rats running at a speed of 28 mmin for 30 min had a 2 fold increase in both PEPCK C and pyruvate dehydrogenase complex in their skele tal 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 fac tors 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 concen tration of P enolpyruvate in the skeletal muscle is reduced by 50 after 30 min of exercise 21 How Does OverexpressingPEPCK CAlterEnergy Metabolism in Skeletal MuscleP 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 spon taneous it was evident as early as 2 weeks after birth It is not clear however how an overexpression of a single enzyme can so drastically repattern 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 ux 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 cit ric acid cycle activity Some years ago Hahn and Novak 22 VOLUME 28239 NUMBER 45 39 NOVEMBER 9 2007 9003 L JeuneAON uo Aieiqn euoziiv 0 MISJeAlun ie 61039oql39MMM won pepeowmoa 4 39 5 The Journal of Biological Chemistry Jig 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 P enol pyruvate 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 nucleo tides across the inner mitochondrial membrane or the conver sion of GTP to ATP by nucleoside diphosphokinase in the mitochondria and its subsequent transport and conversion back to GTP The intracellular location of the different iso forms of this enzyme in muscle is not clear although studies suggest that hepatic nucleoside diphosphokinase 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 ATPADP translocase 23 However the requirement for gua nine 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 overexpression 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 ux with subse quent 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 037 and 026 unitsg of PEPCK C activity in quadriceps and dia phragm of the rat this activity was induced 8 fold in the quad riceps 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 P enolpyruvate 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 with control mice data not shown NOVEMBER 9 2007 VOLUME 28239 NUMBER 45 PEPCKC in Skeletal Muscle Cataplerosis may also be important during or after strenuous exercise when the concentration of citric acid cycle intermedi ates in the mitochondria of skeletal muscle greatly increases There is a rapid 10 fold increase in the concentration of inter mediates 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 ux and thus ATP generation via the respi ratory chain might be limited by the concentration of interme diates 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 abla tion 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 overexpression results in anaplerosis PEPCK C is a reversible enzyme in vitro it has an Keq of 037 M at 30 C so that one could speculate that the reaction generates oxalacetate from P enolpyruvate to replen ish 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 glyc eroneogenesis Skeletal muscle synthesizes and deposits con siderable 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 rat3 Surprisingly even when rats are fed a diet high in carbohydrate the glycer ide 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 overexpression of PEPCK C in skel etal 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 C m s M ice 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 2 weeks after birth It has been well established that contractile activity such as endurance exercise results in mitochondrial biogenesis in skeletal muscle and the develop ment of type 1 muscle fibers 33 In addition feeding mice a high fat diet and giving heparin to increase the concentration of free fatty acid in the blood induced mitochondrial biogenesis in skeletal muscle 34 This is due in part to an up regulation of PPARy 35 PPARoz 36 and PGC loz 37 which interact to promote the oxidative capacity of skeletal muscle by stimulat ing the transcription of genes that lead to mitochondrial bio JOURNAL OF BIOLOGICAL CHEMISTRY 32853 9003 L JeuneAON uo Me an euozuv JO Aimenun 112 61039oql39MMM won pepeowmoa The Journal of Biological Chemistry Jo PEPCKC in Skeletal Muscle genesis see Ref 33 for a review PPARoz PPARy and PGC 1oz act upstream of several genes that code for the transcription factors NRF 1 NRF 2a and Tfam which themselves induce mitochondrial biogenesis Wu et al 38 reported that trans ducer of regulated CREB binding proteins a co activator of CREB can induce PGC 1oz gene transcription and induce mitochondrial biogenesis in muscle cells Transducer of reg ulated CREB binding proteins has also been shown to function as a calcium and cAMP sensitive mediator that coordinates the effects of the two pathways on gene transcription 39 This links the calcium which is released from the sarcoplasmic retic ulum in response to contractile activity with the activation of the transcriptional process that induces mitochondrial biogen esis in skeletal muscle Exercise stimulates PGC 1oz by activa tion of the p38 mitogen activated protein kinase MAPK path way via calcium signaling through the calcineurinmyocyte enhancer factor 2 MEF2 signaling cascade 40 Calcium may also exert a positive regulatory effect on PGC 1oz gene tran scription via the CRE site on the gene promoter by activating the calciumcalmodulin dependent protein kinase 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 A number of recent studies have shown that AMP acti vated protein kinase is necessary for mitochondrial biogenesis via an induction of the PGC 1oz NRF pathway 43 45 Taken together the current information on the mechanisms respon sible 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 Cm mice as the initiating factor lncreased 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 C m s 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 ongoing Alternatively the elevated creatine kinase may be re ective of apoptosis and accelerated muscle remodeling that are a normal part of exer cise induced adaptations in skeletal muscle 49 Aging of the PEPCK C m s 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 SlRT1 which activates transcription of the gene for PGC 1oz Although 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 32854 JOURNAL OF BIOLOGICAL CHEMISTRY control animals Fig 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 have also 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 Cm mice that cause the behavior described in detail in this report Acknowledgments We thank Dr Brian Hoit for generously allowing us to use the mouse treadmill Nancy Edgehouse for the histological analysis and Dr Hisashi Fujioka for the electron microscopy of the muscle We also appreciate the help oinaoyingKong and Tertius Tuy in this study REFERENCES 1 Hanson R W and Patel Y M eds 1994 P 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