Artículo Analogo (BCTP)

Pharmacological Evaluation of a b-HydroxyphosphonateAnalogue of L-Carnitine in Obese Zucker fa/fa Rats

Jorge Reyes-Esparza1, Brissa Mendoza-Rivera1, Ricardo De la Cruz-Cordero2, Jorge L. Rosado3, Miguel . Duarte-Vzquez2,Mario G. Solis4, Odn Vite-Vallejo1 and Lourdes Rodrguez-Fragoso1

1Facultad de Farmacia, Universidad Autnoma del Estado de Morelos, Cuernavaca, Morelos, Mxico, 2Nucitec, S. A. de C. V., Peuelas,Queretaro, Mexico, 3CINDETEC A.C., Parque Industrial Quertaro, Quertaro, Mexico and 4Department of Pathology, General Hospital of

Tlahuac, Villa Centroamericana y del Caribe, Tlahuac, Mexico

(Received 10 May 2012; Accepted 17 September 2012)

Abstract: In this study, we evaluated the effect of an analogue of L-carnitine on parameters involved with Metabolic Syndromein obese Zucker rats. Twenty-four rats were treated for 5 weeks with L-carnitine (300 mg/kg) and its analogue at two concentra-tions (100 and 250 mg/kg) to assess their impact on glucose, triglycerides and cholesterol in liver and blood samples, as well asthe amount of liver glycogen. Liver slices were also analysed. The analogue reduced the levels of glucose, triglycerides and cho-lesterol in liver and the level of triglycerides in serum. At 100 mg/kg, the analogue proved more effective than L-carnitine inimproving the biochemical alterations present in liver. The amount of liver glycogen content was higher in obese animals treatedwith both L-carnitine and the analogue. No changes on insulin and leptin were observed in animals treated. L-carnitine and itsanalogue reduced the microvesicular fatty infiltration in liver. This study demonstrated that the analogue tested is more potentand efficient than L-carnitine and improves the pharmacological profile of L-carnitine.

Metabolic syndrome, syndrome X or insulin resistance syn-drome has been the focus of attention during the past 80 years[1]. Its importance lies in its multiple, interrelated risk factorsfor metabolic origin, which can lead to the development ofdiseases such as atherosclerosis, type 2 diabetes mellitus,dyslipidaemia, high blood pressure, elevated plasma glucose,prothrombotic state and pro-inflammatory state [2]. Other fac-tors associated with metabolic syndrome are abdominal obes-ity, insulin resistance and cardiovascular diseases (CVD) [3].The main causes for the increase in the disease are obesityand diabetes mellitus among populations with a sedentary lifepattern [4,5].

L-carnitine is synthesized by mammals as a result of thesynthesis of amino acids such as lysine or methionine and isalso obtained from the diet [6,7]. Carnitine is an essential fac-tor in transporting the long-chain fatty acids (acyl CoA) fromthe cytoplasm into the mitochondria where the beta-oxidationtakes place [8]. A large body of evidence suggests that carni-tine and its derivatives acetyl-L-carnitine and propionyl-L-carnitineenhance glucose utilization by stimulating the activity of pyru-vate dehydrogenase complex [9], which is a key enzymaticcomplex in glucose oxidation, because intramitochondrialacetyl-CoA can be converted with carnitine into acetyl-L-carnitine (ALC) via the carnitine acetyltransferase that isthen transported out of the mitochondria [10]. L-Carnitine isan amine key substrate in a family of carnitine acyltransfer-ases, transferred reversibly between active units of acyl carni-tine and co-enzyme A to preserve homoeostasis for a wide

range of traffic that are crucial for acyl intermediary metabo-lism and cellular regulation [11].Various analogues of L-carnitine have been synthesized to

study their mode of action and the structural features of theirbinding sites [12,13]. The phosphorus analogues of naturallyoccurring amino acids, which are produced by certain organ-isms, are of great interest in bioorganic and medicinal chem-istry. The replacement of the carboxylic acid functionalgroup in biologically important molecules by phosphonicacids continues to attract much interest in these fields [1416]. Much of the progress in this area has been associatedwith the phosphorus analogues of amino acids. These com-pounds have a tetrahedral configuration due to the presenceof the phosphorus atom, so they serve as stable analogues ofthe unstable tetrahedral intermediate formed in enzymaticprocesses. In a previous work [17], we evaluated the efficacyand safety of two analogues of L-carnitine in insulin-resistantWistar rats with a high fructose diet. That study demon-strated that both analogues maintained the pharmacologicalproperties of L-carnitine and could improve the alterationspresent in insulin-resistant treats; however and according tothe results, they varied in potency, effectiveness and toxicity.The purpose of this study was to evaluate the effect of b-hydroxy phosphonate analogue of L-carnitine under differentparameters involved with metabolic syndrome in obese Zuc-ker rats.

Materials and Methods

Chemical synthesis of L-carnitine analogue. The starting material wasb-hydroxyphosphonate 4, obtained by modifying the method reportedby Ordez et al. [18]. The b-hydroxyphosphonate 4 was used in thenext reactions directly without isolation as a diastereomeric mixture.

Author for correspondence: Lourdes Rodrguez-Fragoso, Flavio GarcaNo. 32, Presidentes Ejidales CP 04470, Mexico, D.F. (fax + 0052 7773297089, e-mail [email protected]).

2012 The AuthorsBasic & Clinical Pharmacology & Toxicology 2012 Nordic Pharmacological Society

Basic & Clinical Pharmacology & Toxicology, 2013, 112, 222228 Doi: 10.1111/bcpt.12019

A diastereomeric mixture of compound 4 was hydrogenolized andmethylated, producing a racemic mixture of compound 6, 2-R and 2-S,respectively, in proportion 85:15. The b-hiydroxyphosphonate 3 wasobtained diastereomerically pure (determined by 1H NMR and 31PNMR) by fractional crystallization. Figure 1 shows the processinvolved in the synthesis of L-carnitine analogue. L-carnitine and L-carnitine analogue were obtained from Nucitec S.A de C.V.(Queretaro, Mexico) and dissolved in sterile deionized water prior toexperimental use.

Animals and type of diets. Male Zucker rats (obese and lean) wereobtained at 5 weeks of age from Harlan Laboratories Inc., MexicoCity, Mexico. The lean animals were not genotyped and could havebeen either +/+ or +/ for the leptin receptor deletion. The animalswere housed in a temperature- and humidity-controlled environmentand were allowed food (Standard Purina Chow Diet, Mexico) andwater ad libitum. Body-weight, glucose, triglycerides and cholesterolwere monitored throughout the study. The experiments wereconducted in accordance with the Guide for the Care and Use forLaboratory Animals [19]. The Institutional Animal Care and UseCommittee from the Biotechnology Institute of the NationalAutonomous University of Mexico approved all procedures.

Pharmacological treatments. After acclimatization, the animals weredivided into the following groups, each one consisting of six rats. Thelean Zucker rats received a control diet and water ad libitum andcomprised the control group. The obese Zucker rats were divided intofour groups. One group received a control diet and water ad libitum, asecond group received L-carnitine (250 mg/kg body-weight/day p.o. in500 ll of water), a third group received the analogue at 100 mg/kgbody-weight/day p.o. in 500 ll of water, and the fourth groupreceived the analogue at 250 mg/kg body-weight/day p.o. in 500 ll ofwater. All treatments were administered 5 days a week for theduration of 5 weeks. L-carnitine and its analogue were obtained fromNUCITEC S.A. de C.V. and dissolved in sterile deionized water priorto experimental use. The cholesterol, glucose and triglyceride serumlevels were monitored every 15 days. Food consumption and body-weight were quantified every week. An oral glucose tolerance test(3 g/kg) was performed after 5 weeks of treatment. After treatments,animals were starved overnight and killed under light chloroformanaesthesia. Liver tissue and blood samples were collected from eachanimal and kept at 4C until further studies.

Biochemical analysis. Plasma from blood samples was collected bycentrifugation, and triglycerides, cholesterol and glucose levels werequantified by colorimetric methods (Triglycerides SL, CholesterolPAP SL and Glucose PAP SL; ELITech, Morelos, Mexico). Glycogenwas assessed using the antrone method described by Fong et al. [20].

Hepatic triglycerides were also quantified through a colorimetricmethod (Triglycerides SL; ELITech). Insulin and leptin werequantified through ELISA kits (Rat/ Insulin and Rat/Leptin Kits;Millipore, MO, USA).

Histopathological analysis. Liver tissue fragments were fixed in 10%formaldehyde solution, dissolved in phosphate-saline buffer (pH 7.4),dehydrated in alcohol and embedded in paraffin. Four-micrometreparaffin sections were stained with haematoxylin and eosin (HE) andsubjected to histopathological examination.

Statistical analysis. Results are expressed as means standard errorof the mean (S.E.M.). Baseline and post-treatment variables [peakand area under the curve (AUC) glucose] were compared using apaired t-test. AUC was calculated using the trapezoidal method.Significant differences were detected by one-way analysis ofvariance using the Graph Pad Instat program (Graph Pad SoftwareV2.03; Graph Pad Software Inc., San Diego, CA, USA). The TukeyKramer multiple comparison test was applied when significantvariations were found. Differences were considered significant atp < 0.05.

Results

Body-weight gain and food consumption of obese Zucker rats.The obese Zucker fa/fa rats were characterized by weight gainand an increase in liver and serum triglyceride and cholesterollevels throughout the period of the experiment (data notshown). Body-weight gain and food consumption for both thecontrol and the obese groups are shown in fig. 2. After5 weeks, obese Zucker fa/fa rats had gained 239 9.8 g(fig. 2A), an increase in 84% compared with lean rats(p < 0.05). At the end of treatment, animals treated with L-carnitineand the analogue (100 and 250 mg/kg) had gained 245 18,245 20 and 230 17 g in each case, 7788% when com-pared with lean rats (p < 0.05); no significant differences werefound between the experimental groups. All animals had agradual increase in body-weight, as can be seen in fig. 2A,even though food amounts remained constant throughoutentire study and regardless of treatment.

Biochemical findings in obese Zucker rats.Figure 3A shows the serum and liver glucose levels in obeseZucker rats treated with carnitine and the analogue. No changesin glucose levels were observed in serum from animals treatedwith carnitine and analogue at 100 mg/kg. However, a signifi-cant increase (14%) was observed in animals receiving the ana-logue in 250 mg/kg doses. As we can see, obese animalstended to increase their hepatic glucose levels, but this was notstatistically significant. Obese animals treated with L-carnitinefor 5 weeks did not show significant changes in liver glucoselevels when compared with obese rats. A reduction in liver glu-cose was nevertheless observed in obese animals treated with100 mg/kg (30%) of the analogue (p < 0.05). Figure 3B showsthe results of the analysis of liver glycogen content. As shown,the amount of liver glycogen changed in obese rats, resultingin a 3.4 times increase when compared with the control group(p < 0.05). Animals treated with L-carnitine showed anincrease in 48.3% in liver glycogen (p < 0.05), while animals

P(OMe)2

NBn2

OH O

rac-4

-hydroxyphosphonate 3 (Analog of L-carnitine)

P(OMe)2

NH2

OH O

H2, Pd/C 10%MeOH, 37-40 C

P(OMe)2

+NMe3

OH O

I-

P(OMe)2

+NMe3

OH O

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Crystallization

rac-5CH3I, K2CO3MeOH, 37-39 C

rac-6

Fig. 1. Representative scheme for the synthesis of the b-hiydroxyphosph-onate analogue.


PHARMACOLOGICAL EVALUATION OF AN ANALOGUE OF L-CARNITINE 223

treated with 100 and 250 mg/kg of the analogue also showed a49.3% and 48% increase in glycogen liver content when com-pared with obese rats (p < 0.05).To know the effect of treatment on oral glucose tolerance,

we administered a bolus of 3 g/kg of glucose to animals andquantified the plasma glucose concentrations. As can be seenin fig. 4, obese animals had high concentrations of glucose,remaining almost constant between 15 and 120 min., whencompared with the control group (AUC 54926 3547 versus8751 987) (p < 0.05). On the other hand, animals treatedwith L-carnitine showed a similar pattern to that observed inthe control group (AUC 6711 1890 versus 8751 987),and there was a reduction in 88% in AUC when comparedwith obese rats (p < 0.05). The patterns shown by analogue-treated animals, however, depended on the dose. Those treatedwith 100 mg/kg had a pattern similar to that of the controlgroup (AUC 6892 2100 versus 8751 987) and a reductionin 87.5% in AUC was observed in comparison with obese rats(p < 0.05). The pattern of those treated with 250 mg/kg, onthe other hand, was very similar to that of the obese rats(AUC 48791 4598 versus 54926 3547).Figure 5 shows the insulin and leptin levels found in all

animals. As we can see, the insulin levels in obese animalsshowed an important increase (20 times) (p < 0.05). Animalstreated with L-carnitine did not show any changes when com-pared with obese rats. Animals treated with 100 or 250 mg/kgof the analogue tended to reduce slightly the insulin levels;however, they were not significant. On the other hand, the lep-tin levels in obese animals showed also an important increase(13 times) (p < 0.05); however, neither L-carnitine nor ana-logue modified the leptin levels.Figure 6A shows that cholesterol serum levels increased by

62% in obese rats when compared with the control group.However, obese animals treated with L-carnitine and 100 or250 mg/kg of the analogue did not show significant changes

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Hepatic

A

B

Fig. 3. Pharmacological effects of the analogue on glucose and glyco-gen from obese Zucker rats. (A) Hepatic and serum levels of glucose.(B) Hepatic glycogen content. Each bar represents the mean S.E.M.in experiments performed in duplicate. All groups consisted of six ani-mals. For abbreviations, see fig. 2. *p < 0.05: statistically significantlydifferent from the control group; #p < 0.05: statistically significantlydifferent from obese rats. &p < 0.05: statistically significantly differentfrom L-carnitine group.

Fig. 4. Effects of the analogue on glucose plasma concentrations inobese Zucker rats after administering 3 g/kg glucose. Each bar repre-sents the mean S.E.M. in experiments performed in duplicate. Allgroups consisted of six animals. For abbreviations, see fig. 2.*p < 0.05: statistically significantly different from the control group;#p < 0.05: statistically significantly different from obese rats.&p < 0.05: statistically significantly different from L-carnitine group.

A

B

Fig. 2. Effect of the analogue on weight gain and food intake. (A) Cor-poral weight was quantified every week during 5 weeks. (B) Food con-sumption was monitored every day during 5 weeks. All groupsconsisted of six animals. A100 = analogue at 100 mg/kg; A250 = ana-logue at 250 mg/kg. *p < 0.05: statistically significantly different fromthe control group; #p < 0.05: statistically significantly different fromobese rats.


224 JORGE REYES-ESPARZA ET AL.

in cholesterol levels when compared to obese rats. Figure 6Bshows cholesterol levels increased by 66% in livers fromobese rats when compared with the control group (p < 0.05).Animals treated with L-carnitine showed a reduction in 15% inhepatic cholesterol; however, animals receiving 100 and250 mg/kg of the analogue showed, respectively, a 60% and50% reduction in hepatic cholesterol levels when comparedwith obese rats (p < 0.05).Hepatic and serum triglyceride levels are shown in fig. 7. A

five times increase in triglyceride serum levels was observedfor obese rats when compared to the control group (p < 0.05).Obese animals treated for 5 weeks with L-carnitine showed a

16% reduction in triglyceride levels when compared tountreated obese rats (p < 0.05) (fig. 7A). On the other hand,obese animals treated with 100 or 250 mg/kg of the analogueshowed a reduction in triglyceride levels of 36.6% and 30%,respectively, when compared to obese rats (p < 0.05).Figure 7B shows triglyceride levels in liver from the controland obese groups. Hepatic triglycerides showed a 2.5 timesincrease in obese rats (p < 0.05). No reductions were observedin animals treated with L-carnitine. However, obese animalstreated with 100 and 250 mg/kg of the analogue showed,respectively, a 25% and 23% reduction in hepatic triglycerideswhen compared with obese animals (p < 0.05).

Histopathological findings.Figure 8 shows representative histological changes in the liv-ers of the different groups. By week 5, obese Zucker ratsshowed hepatocellular injury characterized by centrilobular,microvesicular fatty infiltration, ballooning degeneration andpleomorphic nuclei (fig. 8B). Obese rats treated with L-carnitineshowed a reduction in centrilobular, microvesicular fattyinfiltration and nuclear changes, along with cellular degenera-tion and the presence of inflammatory cells (fig. 8C). How-ever, obese rats treated with 100 mg/kg of the analogueshowed significant improvement in the degree of fat accumu-lation in the liver and liver specimens displayed normal liverhistology and scattered inflammatory cells (fig. 8D). Obeserats treated with 250 mg/kg of the analogue also showed areduction in fat accumulation in liver, but in less extension(fig. 8E).

Fig. 6. Effects of the analogue on hepatic and serum cholesterol levelsin obese Zucker rats. Each bar represents the mean S.E.M. in exper-iments performed in duplicate. All groups consisted of six animals.For abbreviations, see fig. 2. *p < 0.05: statistically significantly dif-ferent from the control group; #p < 0.05: statistically significantly dif-ferent from obese rats. &p < 0.05: statistically significantly differentfrom L-carnitine group.

Fig. 7. Effects of the analogue on hepatic and serum triglycerides lev-els in obese Zucker rats. Each bar represents the mean S.E.M. inexperiments performed in duplicate. All groups consisted of six ani-mals. For abbreviations, see fig. 2. *p < 0.05: statistically significantlydifferent from the control group; #p < 0.05: statistically significantlydifferent from obese rats. &p < 0.05: statistically significantly differentfrom L-carnitine group.

Fig. 5. Effects of the analogue on insulin and leptin serum levels inobese Zucker rats. Each bar represents the mean S.E.M. in experi-ments performed in duplicate. All groups consisted of six animals. Forabbreviations, see fig. 2. *p < 0.05: statistically significantly differentfrom the control group; #p < 0.05: statistically significantly differentfrom obese rats. &p < 0.05: statistically significantly different fromL-carnitine group.



Discussion

Metabolic syndrome has been recognized in medical literaturefor over 80 years. It does not constitute a single illness.Instead, it can be defined as a group of health problemscaused by genetic and environmental factors and the common,fundamental pathogenic component of which is insulin resis-tance. These problems may occur simultaneously or one byone, but their joint appearance in a single individual is signifi-cant given that these patients are more prone to CVD in gen-eral and to coronary disease in particular [21,22].

L-carnitine, a constituent of plasma and tissues, is biosynthe-sized from lysine and methionine in the liver [6]. It plays anessential role in the transportation of free fatty acids into themitochondrial matrix for subsequent b-oxidation [6]. L-carnitineoccurs naturally in food items, especially red meat and fish.The amounts ingested in a normal diet are, however, insuffi-cient to meet the requirements of the entire body [8]. A biosyn-thetic pathway for L-carnitine has already been identified inmammals, including human beings. Our study evaluated ab-hydroxyphosphonate analogue of L-carnitine to identify itspharmacological effects on certain parameters of metabolicsyndrome.Genetically, obese (fa/fa) Zucker rats have proven being a

useful model for the study of metabolic syndrome becausethey display several metabolic alterations, including obesity,impaired glucose tolerance and liver steatosis [2325]. Thepresent study showed that obese rats suffered an increase intriglycerides and cholesterol (in both serum and liver) as wellas liver glycogen and fatty liver and that L-carnitine treatmentimproved liver cholesterol, serum TG, glucogen and oral glu-cose tolerance in this model. The present results demonstratedthat the b-hydroxyphosphonate analogue improved the phar-

macological effects compared with the original molecule(L-carnitine) but had differential effects depending on the dose.Doses of 250 mg/kg of the analogue only modified triglycer-ide levels in serum as well as triglycerides and cholesterol inliver; the 100 mg/kg dose had a more powerful effect on tri-glyceride levels in serum while also reducing glucose, trigly-cerides and cholesterol in liver. Our results suggest that lowdoses of the analogue had a better in vivo pharmacologicaleffect than those of L-carnitine. According to these results,100 mg/kg of the analogue was more effective and potent thanL-carnitine when tested on obese Zucker rats. Analogue dosesof 250 mg/kg were less effective.The role of carnitine in the regulation of fatty acid and carbo-

hydrate metabolism is very complex. It is an essential cofactorin the transfer of fatty acyl groups into the mitochondrial matrix,where they undergo beta-oxidation [6,12]. Carnitine also plays arole in the transfer of acetyl and other short acyl groupsfrom peroxisomes to mitochondria for further oxidation [13]. Ithas been shown that dietary L-carnitine supplementationdecreased liver triglycerides as a result of an enhanced capacityfor b-oxidation [26]. As a result, it may help stimulate hepaticglucose output and, therefore, may improve glucose status.Recent studies suggest that L-carnitine has a beneficial impacton the liver in cases of insulin resistance; it has modulatoryeffects on the post-receptor level because it is able to enhancetyrosine phosphorylation status [27]. We cannot discard the pos-sibility that the b-hydroxyphosphonate analogue of L-carnitinemay act through the same mechanism. Results show that bothdoses of the analogue reduced triglyceride levels in blood andliver. Therefore, we can assume that this analogue of L-carnitinemight interfere with processes involved in b-oxidation and accu-mulation of lipotoxic metabolites that might contribute to mito-chondrial dysfunction and insulin resistance.

A B

C D E

Fig. 8. Effect of the analogue on liver slices histopathology. (A) Lean rat, (B) Obese rat, (C) Obese + carnitine, (D) Obese + A100 and (E)Obese + A250. Liver slices were taken after 5 weeks of treatment. Arrows means microvesicular fatty infiltration; asterisk means the presence ofinflammatory cells. Haematoxilin and Eosin stain, magnification 9250.


226 JORGE REYES-ESPARZA ET AL.

Obesity is the most significant single risk factor for thedevelopment of fatty liver, both in children and in adults;obesity is also predictive of the presence of fibrosis, poten-tially progressing into advanced liver disease [28]. From apathogenic point of view, insulin resistance plays a centralrole in the accumulation of triglycerides inside the hepatocytesand the initiation of the inflammatory cascade. Here, we foundthat 100 mg/kg of the analogue reduces liver damage andmicrovesicular fatty infiltration. Carnitine has also proveneffective in reducing plasma lipids and liver triglycerides inthe livers of rats chronically treated with ethanol [29]. How-ever, the exact mechanism through which carnitine reducessteatosis is still unknown. Recent findings suggest that thehepatoprotective mechanism of carnitine involves dampeningof Kupffer cell TNF-a production [30]. Therefore, we cannotdiscard the possibility that the analogue can ameliorate liverinjury through that mechanism.Obese Zucker rats are considered representative of obesity-

associated type diabetes in human beings. They are hyperlipi-daemic, grossly obese, hyperinsulinaemic, insulin-resistant andnot prone to ketosis [31]. These animals are not always hyper-glycaemic but exhibit an abnormal glucose tolerance similar tothat observed in human beings [32]. These rat glucose levelsare actually normal, or only slightly higher than normal.Therefore, these animals are not the best models to studyeffective treatments to control alterations of glucose homoeo-stasis. Our study did not find important changes in the glucoseserum, insulin serum and liver levels of obese rats. But theanimals displayed an abnormal plasma glucose concentrationduring test tolerance, which was associated with impaired glu-cose tolerance; in that sense, our results agreed with previousreports. Although L-carnitine did not modify the glucose andinsulin levels in serum and liver after 5 weeks of treatment,the animals showed a normal rate of glucose tolerance. Bycontrast, 100 mg/kg of the analogue reduced not only glucoseconcentrations in serum and liver but also resulted in a nor-mal glucose tolerance. This means that, in low doses, the b-hydroxyphosphonate analogue is able to modify carbohydratemetabolism better than L-carnitine.Most mammalian cells store glycogen as a reserve for the

production of glucose 6-phosphate as a metabolic fuel for gly-colysis. In liver, glycogen is mainly stored as a glucose reser-voir for other tissues [33]. There is ample evidence humanbeings with type 2 diabetes suffer from excess hepatic glucoseproduction and an abnormal hepatic glycogen metabolism.Studies with Zucker rats have also shown dramatic findingsrelate to regarding abnormal glycogen synthesis and storage[31]. Our results agree with this. It has been found that ATPproduction and glycogen synthesis increase with the additionof L-carnitine to cultured hepatocytes from new born rats [34].L-carnitine improves insulin-mediated glucose disposal eitherin healthy individuals or in type 2 diabetic patients. There aretwo possible mechanisms in the metabolic effect of carnitine:the first is a regulation of acetyl and acyl cellular traffickingfor correctly meeting the energy demand; the second is a con-trol action not only in the synthesis of key glycolytic and glu-coneogenic enzymes, but also all glycogen-metabolizing

enzymes. Our results showed that the amount of glycogen inliver was higher in L-carnitine-treated Zucker rats than in thosetreated with the analogue. These results suggest that the ana-logue may produce the same effect on glycogen metabolism.Zucker rats have displayed markedly elevated circulating

leptin levels and develop severe obesity with hyperphagia,defective nonshivering thermogenesis and preferential deposi-tion of energy in adipose tissue [3538]. It has been observedthat L-carnitine reduces obesity caused by high fat in C57BL/6J mice [39]. Clinically, it has also been found that patientstreated with sibutramine/carnitine achieve significant weightloss with a good safety profile [40]. L-carnitine has an antiobe-sity action, which could modulate lipid metabolism by stimu-lating lipolysis and oxidation, accompanied by correspondingchanges in gene expression and the expression and suppres-sion of the expression of adipogenic genes [41]. Our studyfound that Zucker rats reached twice the body-weight of theirlean counterparts; their weight gain was associated with anaccumulation in central fat and an increase in liver weight(data not shown). Neither L-carnitine nor the analogue modi-fied weight gain in Zucker rats. The discrepancy between ourresults and previous work may be due to the animal modelused in the present study. Zucker rats present a mutation inthe leptin receptor, which is the molecular base of their char-acteristic phenotype (loss of satiety) [42]. L-carnitine and itsanalogue may have an influence on fat and carbohydratemetabolism but not on the central nervous system mechanismof obesity and hyperphagia in Zucker rats. Our result showedthat although food consumption was constant during 5 weeks,there was a progressive body-weight gain. Therefore, weightgain was not dependent on food intake, but on the metabolismand genetic alterations of the obese rats, as was corroborate bythe leptin levels.This study demonstrates that the b-hydroxyphosphonate

analogue employed is more potent and efficient than L-carnitineand improves the pharmacological properties of L-carnitine, aswell as it can improve the alterations present in fa/fa Zuckerrats. According to the present data, the analogues mecha-nisms of action could be similar to those reported forL-carnitine.

AcknowledgementsWe wish to thank to CONACYT Mexico for its financial

support (Project 153324 and 179848).

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