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A ketone ester diet increased brain malonyl CoA and uncoupling protein 4 and 5 while decreasing food intake in the normal Wistar rat.

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A ketone ester diet increased brain malonyl CoA and uncoupling protein 4 and 5 while decreasing food intake in the normal Wistar rat.
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  Ketone ester diet increases brain malonyl CoA A Ketone Ester Diet Increased Brain Malonyl CoA and UncouplingProtein 4 and 5 While Decreasing Food Intake in the Normal Wistar Rat* Yoshihiro Kashiwaya ‡ , Robert Pawlosky ‡ , William Markis ‡ , M. Todd King ‡ , ChristianBergman ‡ , Shireesh Srivastava ‡ , Andrew Murray £ , Kieran Clarke ¶ , Richard L. Veech ‡1   From ‡  Laboratory of Metabolic Control, NIAAA, National Institutes of Health, DHHS, Rockville, Maryland 20852-9047, £  Department of Physiology, Development & Neuroscience, University of Cambridge, Downing St., Cambridge CB2 3EG, UK and the ¶   Department of Physiology, Anatomy &Genetics, University of Oxford, Oxford, OX1 3PT, U.K. Running title: Ketone ester diet increases brain malonyl CoA 1  Address correspondence to: Richard L. Veech MD DPhil. Laboratory of Metabolic Control, NIAAA, NIH, DHHS 5625 Fishers Lane, Rm #2S28 Rockville, MD 20852-9047 U.S.A, phone 301-443-4620 Fax: 301-443-0930 email:rveech@mail.nih.gov   2 The abbreviations used are: DHAP, dihydroxy-acetone-phosphate; GABA, gamma-aminobutyric acid;UCP, uncoupling protein; P i , inorganic phosphate; Cit, citrate; Isocit, isocitrate; GOT, glutamicoxaloacetic transaminase;*This work was supported in part by the Defense Advanced Research Projects Administration, USDepartment of Defense. Three groups of male Wistar rats were pairfed NIH-31 diets for 14 days to which wereadded 30% of calories as corn starch, palm oil,or  R -3-hydroxybutyrate-  R -1,3-butanediolmonoester (3HB-BD ester). On the 14 th day, theanimals brains were removed by freeze blowingand brain metabolites measured. Animals fedthe ketone ester diet had elevated mean bloodketone bodies of 3.5 mM and lowered plasmaglucose, insulin and leptin. In spite of thedecreased plasma leptin, feeding the ketoneester diet  ad lib decreased voluntary food intaketwo fold for 6 days while brain malonyl CoAwas increased by about 25% in ketone-fedgroup but not in the palm oil fed group. Unlikethe acute effects of ketone body metabolism inthe perfused working heart, there was noincreased reduction in brain free mitochondrial[NAD + ]/[NADH] ratio nor in the free energy of ATP hydrolysis which was compatible with theobserved 1.5 fold increase in brain uncouplingprotein 4 and 5. Feeding ketone ester or palmoil supplemented diets decreased brain L -glutamate by 15-20% and GABA by about 34%supporting the view that fatty acids as well asketone bodies can be metabolized by the brain. The metabolism of ketone bodies in theworking perfused heart increased the supply of mitochondrial NADH and the Δ G of ATPhydrolysis (1). Other than the observation thatketone bodies can replace glucose as the major energy substrate in brain (2), little is known aboutthe precise effects of ketone metabolism in brain in vivo . The elevation of ketone bodies (3-hydroxybutyrate and acetoacetate) and free fattyacids through ketogenic diets have been used for almost a century to treat drug refractory epilepsy(3;4). In addition, it has been suggested that mildketosis might be an effective treatment for anumber of neurodegenerative and other diseases(5-7). We therefore undertook a broad survey of the effects of a ketone ester and a fatsupplemented diet on several of the pathways of intermediary and energy metabolism in rat brain.Prevention of post mortal changes, necessaryfor the accurate determination of  in vivo redox and phosphorylation states in brain, require rapidinactivation of tissue which was accomplished byfreeze blowing (8;9). In addition to the[lactate]/[pyruvate] ratio, we report differences inthe [succinate]/[fumarate] ratio as an additionalindicator of hypoxic changes induced by different brain collection methods used in brain metabolismanalysis.Elevation of blood ketone bodies, by either fasting or high fat diets, results in elevation of both blood ketone bodies and free fatty acids.Therefore, prior to this report, it has not been possible to investigate ketone body metabolism in brain independent of the effects induced byelevation of plasma free fatty acids. 1   http://www.jbc.org/cgi/doi/10.1074/jbc.M110.138198The latest version is at JBC Papers in Press. Published on June 7, 2010 as Manuscript M110.138198   Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.   a t   C  am b r i   d  g e U ni  v  er  s i   t   y L i   b r  ar  y  , on J  ul   y 1 4  ,2  0 1  0 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   Ketone ester diet increases brain malonyl CoA More recently, ketogenic diets have been usedin the treatment of obesity where it has beenshown that high protein, low carbohydrate dietsdecreased appetite, sensation of hunger and foodintake in hospitalized patients(10). Theintraventricular infusion of 3-hydroxybutyrate(11;12) or intravenous administration of its precursor 1,3-butanediol (13), have previously been shown to decrease food intake in the rat ashas intraperitoneal injections of 3-hydroxybutyrateor 1,3-butanediol in the pigmy goat (14).Subcutaneous administration of 3-hydroxybutyrate, but not acetoacetate, has also been shown todecrease food intake in the rat (15). Increasedhypothalamic malonyl CoA associated withadministration of the fatty acid synthase inhibitor,C75, decreased food intake (16-19) for 1 day innormal lean mice but for up to 6 days in the obeseob/ob leptin deficient mouse (20).Classic ketogenic diets containing minimalamounts of carbohydrate and large amounts of saturated fats are unpalatable, leading to poor  patient compliance. More importantly, these dietslead to elevated blood cholesterol (21) and freefatty acids (22;23), both of which have welldocumented adverse effects. Therefore,alternative methods of elevating blood ketone body levels were needed. Accordingly, wesynthesized a monoester comprised of  D - β -hydroxybutyrate and  R -1,3-butanediol.  R -1,3- butanediol is converted by liver to ketone bodies(24). We report here, for the first time, the use andanalysis of a ketone ester supplemented diet onvarious pathways of brain intermediarymetabolism and contrast these effects with thoseresulting from a starch or palm oil supplementeddiet. EXPERIMENTAL PROCEDURES  Animals and Diets – Male Wistar ratsweighing 280 to 310 g (n = 6-12) were obtainedfrom Charles River Laboratories, Wilmington MA.All experiments were reviewed and approved bythe Animal Care and Use Committee of NIAAA, NIH. Diets were prepared by grinding NIH-31fortified rodent diet to powder in a Waring blender and mixing the diet with “sugar free” maltodextrincontaining gelatin (Jell-O™), water and thevarious components listed in Table 1. Vitaminsand minerals (AIN-93 GMX, Bio-serv) wereadded according to guidelines set by the AmericanInstitute of Nutrition in 1993 (25). Feeding Protocol and Sample Collection – One group of animals was meal fed the 3 diets(Starch, Fat, and Ketone Ester) ad lib for 3 hoursand food intake monitored to determine the effectsof diets on voluntary food intake. Because thegroup receiving the ketone ester ate less food in a3 hour  ad lib meal, a second group of animals was pair fed for 3 hours each morning for 14 days,matching the caloric intake of the starch and fatsupplemented diets to that of the ketone ester supplemented diet. Initially, the food intake in the pair fed animals was 10 g per rat per day butincreased gradually to 15-20 g per day. Inaddition, the bodyweight of the animals decreased by about 10 % but stabilized in all three dietgroups after 4 days. Following the meal on the14 th day, brains were freeze blown and bloodcollected in heparinized syringes. The arterial plasma pH and pCO 2 was measured using an i-STAT blood gas analyzer (Abbott Laboratories).The whole blood was centrifuged at 5,000 x g for 10 minutes at 4°C. Plasma was collected fromsupernatant and stored at - 80°C until analysis. Comparison of Brain Extraction Techniques – Measurements of lactate, pyruvate, succinate, andfumarate from brain samples harvested with thefreeze blowing technique (n=4) or by rapidremoval of the brain followed by immersion inliquid N 2 (n=4) and the calculation of the freecytosolic [NAD + ]/[NADH] ratio were performedas previously described (8;9).    Enzymatic analysis – Brain samples were prepared by perchloric acid extraction as previously described (8). Enzymatic analyseswere performed using methods described byPassonneau and Lowry (26) Glycolyticintermediates, ATP, creatine, phospho-creatine, plasma ketone bodies and plasma glucose weremeasured enzymatically (27). All other metabolites were measured enzymatically asdescribed previously (1). GC-MS Measurements – Perchloric acidextracts of frozen brain were used to measurecitrate, isocitrate, α -ketoglutarate, succinate,fumarate, malate, glutamate, glutamine, aspartate, N acetyl aspartate and gamma amino butyric acid,GABA. They were analyzed as the silyl ether derivatives, quantified using 13 C-labeled standardsfor each analyte using gas chromatography-mass 2    a t   C  am b r i   d  g e U ni  v  er  s i   t   y L i   b r  ar  y  , on J  ul   y 1 4  ,2  0 1  0 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   Ketone ester diet increases brain malonyl CoA spectrometry (GC-MS) as previously described(28). Briefly, the mass spectrometer was operatedin the electron impact mode (70eV) and thequadrupole mass analyzer scanned for ions whichcorresponded to a loss of 15 mass units (-CH 3 )from the molecular ion and the base peak of eachanalyte and its corresponding 13 C-labelled internalstandard using selected ion monitoring. The ratioof peak area count of the 13C-labelled internalstandard to that of the analyte was used to quantifyits concentration according to our previous report(28). A sample chromatogram is given in Figure 1. Sample extraction procedure and capillaryelectrophoresis mass spectral (CE-MS)determination of acyl-coA compounds and acetylcholine – Frozen brain tissue samples wereextracted using a modified chloroform-methanolextraction procedure and analyzed by capillaryelectrophoresis mass spectrometry (CE-MS)according to the method of Soga (29) with theaddition of  13 C-labeled CoA internal standards for quantification as described previously (28). Plasma peptide measurements – Plasmainsulin and leptin were measured using a rat plasma EIA kit (from Alpco diagnostics andLingo/Millipore respectively) according to themanufacturer’s instructions. Calculations – The free [Mg 2+ ] (30), phosphorylation potential, free cytosolic [ADP],[AMP], [Pi], [oxaloacetate], the Δ G of ATPhydrolysis, cytosolic and mitochondrial redoxstates were calculated as previously described(1;31). The adjustment of the equilibriumconstants for tissue pH and free [Mg 2+ ] were doneas previously described (32).  Brain mitochondrial UCP4 and UCP5immunoblotting – In each case, 30 µg total brain protein was loaded onto a 12% polyacrylamide gel,and proteins were separated by running the gel at100 V before transferring the protein on to anitrocellulose membrane. Polyclonal rabbit anti-UCP4 and anti-UCP5 primary antibodies (SantaCruz Biotechnology, Santa Cruz, CA, USA) wereused at concentrations of 1:1000 in 5% milk TBS-Tween. The secondary antibody used in bothcases was anti-rabbit IgG peroxidase conjugate polyclonal antibody (Autogen Bioclear, Wiltshire,UK) at a concentration of 1:3500 in 5% milk TBS-Tween. For all blots, the films were scanned anddensitometry measurements quantified using Un-Scan-It gel digitizing software (Silk Scientific,Orem, UT, USA). UCP4 and UCP5 levels werenormalized to total protein using Ponceau S stain(Sigma, St Louis, MO). Statistical analysis- The number of samplesanalyzed for each metabolite, n, varied from 6 to 8.The results are presented as means ± SEM. Anon-parametric statistical procedure, Mann-Whitney U test, was used to determine thesignificance of the difference between means. RESULTS  Effect of ketone ester on plasma measurements of ketones  – Rats fed a starch or fat supplementeddiet (Table 1) had mean total blood ketone bodiesof 0.05 to 0.08 mM. Rats fed a ketone ester supplemented diet had a mean total ketone bodylevel of 3.5 mM (n = 6) and  R -1,3-butanediolconcentration of 0.02 mM three hours after the beginning of the meal.  Effect of ketone ester on arterial blood pH  – The pH of the arterial blood plasma was measured andremained unchanged at 7.35 in all groups withCO 2 lowered from 47 mmHg in the starch and fatfed groups to 35 mmHg in the ketone ester fedgroup.  Effect of ketone ester on plasma glucose, insulin,and leptin – As compared to the fat supplementeddiet group, the ketone ester fed group decreased blood glucose from 4.8 mM to 2.8 mM or about 44%even though carbohydrate content of the dietswere matched (Table 1). Plasma insulin was alsodecreased by a factor of 2 (Table 2). Theanorexigenic adipose tissue peptide leptin wasdecreased from 3.1 to 1.8 ng/ml plasma.  Effect of diets on daily food intake – Rats fed theketone ester supplemented diet ad lib consumedsignificantly less food in a 3 hour meal comparedto rats fed either starch or fat supplemented dietson days 2 through 6 as judged by Mann WhitneyU test (P> 0.04) n = 6. Furthermore, on day 1, both ketone ester and fat supplemented diet groupsconsumed significantly less than the starchsupplemented diet groups (Figure 2). Comparison of metabolite levels in freeze blownand “rapidly frozen” brains – When comparingthe reduced substrates from different brainharvesting procedures (freeze blown vs. quick frozen), there was a 1.5 fold increase in lactate anda 4 fold increase in succinate in brains that weredropped into liquid N 2 compared to freeze-blown 3    a t   C  am b r i   d  g e U ni  v  er  s i   t   y L i   b r  ar  y  , on J  ul   y 1 4  ,2  0 1  0 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   Ketone ester diet increases brain malonyl CoA  brains (Table 3). There were no differences in thelevels of the oxidized substrates, pyruvate andfumarate .   Effect of ketone ester diet on brain glycolyticintermediates – Brain glycolytic intermediateswere measured in the freeze-blown brains. Theconcentrations of brain glucose and the glycolyticintermediates 3-phosphoglycerate and L -lactatewere significantly decreased in rats fed ketoneester diet (Table 4). The [lactate]/[pyruvate] ratiowas significantly decreased in the ketone ester fedgroup as compared to starch group. Feeding a dietsupplemented with 30% of calories as fatsignificantly increased brain [lactate]concentration (Table 4).  Brain Krebs cycle and CoA intermediates – Theconcentration of Krebs cycle intermediates in brain was unchanged by any of the three dietstested (Table 5). Malonyl CoA concentrationalone was increased by feeding a ketone ester supplemented diet, but not by a fat supplementeddiet.    Brain amino acids and neurotransmitter content  – Feeding a ketone ester diet and a fat supplementeddiet both resulted in a decrease of about 40% in brain L -glutamate and GABA when compared to astarch supplemented diet. There were nosignificant differences in brain aspartate or acetylcholine between the three diets.   The equilibrium constants, K  eq  , and measured brain tissue metabolite ratios, Γ, for enzymereactions with glutamate or glutamine assubstrates –    From the values in Tables 5 and 6, an in vivo ratio of metabolites ( Γ ) was calculated for glutamic oxaloacetic transaminase, glutaminesynthase, glutaminase, and glutamatedecarboxylate and compared to the K  eq   measuredin vitro for each reaction. The metabolite ratiomeasured in tissue, Γ , for the reactants of theglutamic oxaloacetic transaminase reaction (EC2.6.1.1) was 10 to 12 and agreed well with the K  eq  of this reaction, 6.61 (33;34). In contrast, theother reactions, in which L -glutamine or GABAare substrates, had tissue ratios that were differentfrom K  eq by two or more orders of magnitude.    Brain high energy intermediates – There weresmall, but non-significant decreases in total brainATP and P-creatine in rats fed the fatsupplemented diet (Table 8) in comparison to thegroup fed the starch supplemented diet. Theketone ester fed group showed a significantdecrease in both P Creatine and N-acetyl-aspartatecontent when compared with the starchsupplemented diet group (Table 8).  Brain uncoupling protein content  – In the brains of ketone diet fed rats, levels of mitochondrial UCP4(Figure 3) were 45% greater than in the brains of fat fed rats (p < 0.01) and 50% greater than in the brains of rats fed the starch supplemented diet (p <0.001). Similarly, levels of mitochondrial UCP5(Figure 3) were elevated in ketone diet fed rat brains by 66% compared with fat fed rats (p <0.001) and 59% compared with starch-fed rats (p <0.01).  Free nucleotide ratios and concentrations – Feeding ketone ester supplemented diets led to asignificant oxidation in the free cytoplasmic[NAD + ]/[NADH] (Table 9) compatible with anincrease in UCP 4 and 5 (Figure 3). There wereno changes in either the mitochondrial NAD- or coenzyme Q- couple. The Δ G of ATP hydrolysiswas -58.4 to -59.2 in all diet groups estimatedfrom the component of the GAP dehydrogenase 3 phosphoglycerate kinase reactions or the creatinekinase reaction and measured P i . There was noconsistent change in the Δ G of ATP hydrolysis in brain in the 3 diet groups studied. The freecytosolic [AMP] was not changed in any dietarygroup (Table 9).   DISCUSSION Feeding a ketone ester supplemented dietelevated blood ketone bodies to 3.5 mM (Table 2)while decreasing both blood glucose and insulin toabout half the value in rats fed a fat or starchsupplemented diet. This suggests that ketonesincrease insulin sensitivity as was observed previously in the perfused heart (1). A previousstudy has demonstrated increased insulinsensitivity in insulin responsive tissues duringketone infusions in man (35).The ketone ester supplemented diet groupshowed a decrease in food intake (Figure 2) notresulting from an increase in plasma leptin (Table2), implying a different mechanism for decreasedfood intake than that produced by increased leptin.Decreased plasma leptin has been observed previously during short term fasting (36), whichalso is associated with mild ketosis.The ketone ester supplemented diet led to anincrease in brain malonyl CoA compared to the 4    a t   C  am b r i   d  g e U ni  v  er  s i   t   y L i   b r  ar  y  , on J  ul   y 1 4  ,2  0 1  0 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   Ketone ester diet increases brain malonyl CoA starch supplemented group (Table 5). In addition,feeding a palm oil supplemented diet did notincrease malonyl CoA even though themetabolism of fat should increase the availabilityof acetyl CoA in the brain. An increase in brainmalonyl CoA would increase the rate of fatty acidsynthesis (37), which could satisfactorily explainwhy feeding small amounts of  β -hydroxybutyrateinduced myelination and reversed quadriplegia inmultiple acyl CoA dehydrogenase deficiency patients (38).Malonyl CoA is also known to be ananorexigenic metabolite and to be associated withdecreased food intake (16;17). Our data suggeststhat feeding ketone body esters, which decreasedfood intake for 6 days (Figure 2), has a longer lasting effect in normal lean animals than the fattyacid synthase inhibitor C75, which decreased foodintake for only 1 day (20).Since the brain has a high demand for acontinuous supply of oxygen to preserve the invivo energetics, methods of tissue inactivation,extraction, and preservation may alter thecytosolic and mitochondrial redox potentials aswell as the phosphorylation potential by alteringconcentrations of oxygen sensitive metabolites.Any delay in inactivating brain metabolism leadsto an increase in the [lactate]/[pyruvate] and the[succinate]/[fumarate] ratio (Table 3) reflecting areduction in an NAD and co-enzyme Q linked near equilibrium redox couples. Accuratedetermination of succinate is of importance sincesuccinate increases hypoxia-inducible factor 1alpha subunit (HIF1- α) secondary to productinhibition of prolyl hydroxylase (EC 1.14.11.2)(39).The changes in brain glutamate and GABAafter feeding a diet supplemented with palm oil(fat diet, Table 1) are in agreement with earlier reports that palmitate can be taken up andmetabolized by rat brain (40). Animals fed a fatsupplemented diet, as ketone fed animals, haddecreased brain GABA, but did not have increaseduncoupling protein (Figure 3) nor increased brainmalonyl CoA (Table 5). This difference is likelydue to the duel effects of malonyl CoA as theimmediate precursor of fatty acid synthesis (37)and also malonyl CoA’s ability to inhibit carnitine palmitoyl CoA transferase (41) decreasing fattyacid transport into mitochondria to undergo β  oxidation. The resultant buildup of fatty acid inthe cytoplasm would activate the peroxisome proliferator-activated receptor (PPAR) nuclear transcription factors (42;43).Feeding a ketone ester diet compared withstarch fed animals significantly decreased both L -glutamate and GABA in the ketone ester and fatfed rats but brain L -aspartate was not changed byeither diet (Table 6). Our data, therefore, does notsupport the hypothesis that the anti-epileptic actionof the ketogenic diet results from increases in theinhibitory brain neurotransmitter, GABA (44-46);rather it suggests that a decrease in the stimulatorymetabolite L -glutamate as a potential mechanismfor the antiepileptic effects of the ketogenic diet.Labeling studies suggest that there are twodistinct L -glutamate pools in brain: a rapidlyturning over  L -glutamate pool in glial cellcomprising about 10 % of brain L -glutamate and alarger neuronal pool comprising about 90 % of  brain L -glutamate (47;48). It has also been postulated that the large pool of brain L -glutamatecould reflect the mitochondrial redox state of free[NAD + ]/[NADH] in brain (49;50). We show(Table 6 and 7) that measurements of total brain L -glutamate, when combined with total tissuemeasurements of the reactants of the aspartateamino transferase reaction (EC 2.6.1.1), Γ , yieldeda value of 10 to 12, very close to the actualequilibrium constant of the aspartate aminotransferase reaction of 6.6 . This suggests thatmeasured brain L -glutamate, L -aspartate, α -ketoglutarate and oxaloacetate are all localized ina neuronal compartment containing that enzyme.In contrast, glutamine is synthesized from NH 4+ and L -glutamate by glutamine synthase (EC6.3.1.2) which is exclusively localized in thecytoplasm of astrocytes (51). Our measurementsof total tissue contents show that the brainreactions involving L -glutamine, glutaminase (EC3.5.1.2) and glutamine synthase (EC 6.3.1.2), wereall 4 to 5 orders of magnitude from equilibrium(Table 7). This is compatible with the hypothesisthat glutamine synthase reaction is simply out of equilibrium or, more likely, that L -glutamine islargely sequestered in the glial space and absentfrom neuronal space. The inference that glutaminemay be low in neuronal space is compatible withthe observation that glutaminase, the enzymeconverting glutamine to glutamate and NH 4+ , is of high activity in neurons (52). This inference isfurther supported by the observation that the 5    a t   C  am b r i   d  g e U ni  v  er  s i   t   y L i   b r  ar  y  , on J  ul   y 1 4  ,2  0 1  0 www. j   b  c . or  gD  ownl   o a d  e d f  r  om 
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