EffEct of L-carnitinE administration on sErum insuLin and adiponEctin LEvELs , and AMPK , APPL 1 and PPAR γ gEnE ExprEssion in stZ-inducEd diabEtic rat LivEr

1Cardiovascular research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of medical Sciences, Kerman, Iran; e-mail: bshahouzehi@yahoo.com; 2department of Virology, afzalipour School of medicine, Kerman University of medical Sciences, Kerman, Iran; e-mail: khabatzanbil@gmail.com; 3Physiology research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of medical Sciences, Kerman, Iran; e-mail: soheilaminizadeh@gmail.com; 4Physiology research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of medical Sciences, Kerman, Iran; *e-mail: ymab125@yahoo.com


D
iabetes is a worldwide chronic disorder which considered as one of the most prevalent metabolic complication.It is characterized by remarkable elevation of blood glucose levels and disturbed lipid homeostasis [1,2].In 2013, about 382 million individual burden diabetes and it has been estimated that this number will increase and reach about 592 million by 2035 [2].
L-carnitine (β-hydroxy-γ-N-trimethyl ammonium butyrate) is a non-nutrient supplement that is synthesized from essential amino acids, lysine and methionine, in liver, brain and kidney of most mammals [3,4].L-carnitine facilitates long-chain fatty acids transportation into the mitochondria as acylcarnitine and helps fatty acids to entered betaoxidation pathway.In mitochondria, these fatty acids were oxidized and produced coenzyme A (CoA).These obtained CoA entered to tricarboxylic acid (TCA) cycle and generate energy in the mitochondrial respiratory chain.Therefore, L-carnitine has an important role in fatty acids energy release and is very important for lipid metabolism [5].
doi: https://doi.org/10.15407/ubj89.06.048Following L-carnitine supplementation, fat utilization was promoted and TG levels were decreased [6].It was reported that diabetic rats have reduced serum L-carnitine levels [7].Cha Y. S. reviewed L-carnitine metabolic actions and has reported that L-carnitine affects obesity and blood glucose, but results obtained from other studies were controversial and L-carnitine some effects remained uncertain [8].Hadadinezhad S. and colleagues showed that L-carnitine attenuated fasting blood glucose but there was no effect on hemoglobin A 1C and C-peptide in patients with diabetes mellitus [9].Also, it has been reported that high dose of L-carnitine reduced diabetes severity and also improved cardiac function [10].Xia Y. et al. showed that oral administration of L-carnitine to mice was increased β-oxidation of fatty acids and also in liver maintained mitochondrial function [11].
According to previous studies, L-carnitine showed valuable effects in diabetes and it has been reported that reduced glucose and lipid levels.But data about hypoglycemic effects of L-carnitine in dia betes are inconsistent [1,10,15,16] and the mecha nism of L-carnitine beneficial effects are not fully understood, therefore, we designed this study to evaluate oral L-carnitine supplementation effects on adiponectin and insulin circulating levels and also, expression of amPK, PPARγ and aPPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding (PTB) domain and leucine zipper motif) genes in liver of STZ-induced diabetic rats.materials and methods materials.Streptozotocin (STZ, S0130) and Lcarnitine (C0283) were purchased from Sigma.Adiponectin (KRP0041) and insulin (ERINS) measured by specific ELISA kits.
Preparation of reagents.STZ prepared in citrate buffer (0.1 N, pH 4.5) and introduced to animals by a single i.p injection and L-carnitine was prepared in phosphate buffer (pH 7.4) and administrated orally to diabetic rats.
methods.All animal procedures were conducted in accordance with the requirements of European Convention for the protection of vertebrate Animals Used for Experimental and other Scientific Purposes (Strasbourg, 1986).Twenty four male Wistar rats (190-210 g weight and 75-95 days of age) were randomly selected and after a week acclimatization were divided into three groups (n = 8).Group 1 control did not receive any treatment, group 2 received a single dose of STZ (50 mg/kg) by i.p injection to induce diabetes, group 3 received a single dose of STZ (50 mg/kg) by i.p injection and also 600 mg/kg/day L-carnitine orally for 5 weeks.Before beginning the L-carnitine treatment we make sure that the animals in groups 2 and 3 were diabetic, for this reason, 72 h after STZ injection the blood glucose was measured and animals that showed blood glucose more than 250 mg/dl after 12 h fasting were entered to the study.At the end of the study (after 35 days) and after 12 h fasting overnight, animals were anesthetized with ether and sacrificed, the blood samples were collected and maintained 1 h at room temperature and centrifuged and then serum was collected to measure the glucose, adiponectin, and insulin levels.Also, the liver tissues were dissected and washed with cold saline and immediately frozen by liquid nitrogen and kept in -80 °C [8,15].determination of serum adiponectin and insulin levels.Specific ELISA kits were used to assay adiponectin and insulin in serum.All measurements were according to the manufacturer's protocol.
Semi-quantitative RT-PCR.About 75 mg of liver tissue was removed from storage for total RNA extraction.Liver tissue homogenization was performed by Ultrasonic Processor (Hielscher, UP200H).Total RNA was extracted by guanidine isothiocyanate-phenol-chloroform method (RNX plus reagent).For cDNA synthesis, 500 nanograms of total RNA was used by M-MuLV reverse transcriptase and oligo-dT primers.PCR was performed in Bio-Rad MJ Mini Personal thermal cycler.PCR reaction contained 200 nanograms of cDNA, 50 pM of target gene forward and reverse primers , 10 ul PCR master mix, the final volume was 20 μl.Each gene's expression performed duplicate.The PCR cycles were as follow; 95 °C for 10 min, then 30 cycles of 94 °C for 45 s, 60 °C for 1 min, and 45 s at 72 °C, and 5 min final extension at 72 °C.For analysis of the PCR products, we performed agarose gel elec-trophoresis on 2% agarose gel (100 v, 45 min), then by using gel documentation instrument.Primers (Table 1) were purchased from Sinagen (Sinagen Inc., Tehran, Iran).Bands were visuali zed and were quantified by using Image J software [17].
Statistical analysis.Statistical analyses were performed by SPSS 19 software (SPSS Inc., Chicago, USA).The data are expressed as mean ± SEM.For comparison between groups, we used one-way analy sis of variance (ANOVA) followed by the Tukey's post hoc test.

results and discussion
our results showed that long-term L-carnitine supplementation reduces blood glucose significantly.Also, L-carnitine leads to significant elevation of adi ponectin levels in diabetic rats, there was a mild but significant elevation of insulin after Lcarnitine administration.Body weight was significantly reduced in the diabetic control group and the diabetic group which received L-carnitine compared to control group (Table 2).Also, L-carnitine showed remarkab le effects on amPK, aPPL1 and PPARγ gene expression, and up regulated these genes compared to diabetic group (Figure , a-C).
Diabetes is one of the most prevalent metabolic diseases which affect many people worldwide [1].L-carnitine is an endogenous vitamin-like compound which synthesized by most mammals and is very important for glucose and lipid homeostasis.It was reported that L-carnitine levels were remarkably reduced in diabetes models [1,10,15,18].It was also demonstrated that L-carnitine in serum and cardiac tissue of diabetic animal were reduced [19].But data about L-carnitine hypoglycemic effects are controversial.Abdel-Razek H. reported that s.c.administration of L-carnitine improved glucose utilization in diabetic rats [1].It demonstrated that long term L-carnitine supplementation reduced TG and cholesterol but hyperglycemia was still present in diabetic rats [15].on the other hand, Bazotte R. B. and Lopes-Bertolini G. showed that L-carnitine administration showed no significant effects on blood glucose and total cholesterol but normalized blood TG levels in diabetic rats [16].Rodrigues B. and colleagues were suggested that high dose of L-carnitine reduced diabetes severity and also improved cardiac function [10].We showed that long-term supplementation of L-carnitine (600 mg/ kg/day, orally) after five weeks, significantly improved some parameters in STZ-induced diabetic rats.We found that L-carnitine attenuated blood glucose levels compared to the diabetic non-treated group which are in contrast with Patel J. et al. and Bazotte R. B. and Lopes-Bertolini G. studies.The previous study demonstrated that L-carnitine has remarkable anti-diabetic effects and it probab ly correlated to L-carnitine antioxidant activity [18].In the diabetic animal model, 500 mg/kg Lcarnitine which was administrated by i.p injection for a month showed anti-oxidant activity [20].Therefore, anti-diabetic and hypoglycemic effects of Lcarnitine could be partly related to its anti-oxidative effects.

T a b l e 1. Sequences of RT-PCR primers
oral administration of L-carnitine increased beta-oxidation of fatty acids and also in liver maintained mitochondrial function.Long-chain acylcarnitine has detergent effects on mitochondria membrane.L-carnitine supplementation reduces the chance of long-chain acylcarnitine accumulation in mitochondria space and therefore, protects membrane against destruction.Diabetes is along with oxidative stress which affects antioxidative defense against RoS [11,21].Therefore, mitochondria protective effects of L-carnitine can be considered as Lcarnitine beneficial effects observed in STZ-induced diabetic rats.
Adiponectin levels were extensively studied in diabetes and other metabolic diseases.Adiponectin was decreased in diabetes, insulin resistance and atherosclerosis and also reduced adiponectin levels related to diabetes and other metabolic complications development.Also, it was reported that PPAR activators improved diabetes and this effect was adiponectin-dependent [22][23][24].We found that L-carnitine long-term administration to diabetic rats improved diabetes and this was related to adiponectin levels in serum which significantly increased after L-carnitine supplementation.As beneficial effects of adiponectin over diabetes are well proved, therefore, elevated adiponectin levels compared to diabetes control group in our study could be considered as responsible for beneficial effects of long-term L-carnitine supplementation in diabetes.
PPARγ activation in liver attenuates intracellular lipid contents, promotes insulin sensitivity, improve insulin receptor signaling in diabetes, increase insulin secretion and inhibit gluconeogenesis.Also, TZDs which are strong insulin sensitizers' drugs, act via PPARγ agonism [13,14].Besides TZDs ben ficial effects in diabetes there are some deteriorating effects following TZDs administration which include hepatotoxicity and hepatic failure [25].Our results showed that PPARγ gene expression eleva ted by L-carnitine supplementation in diabetic rats, therefore L-carnitine exerts insulin-sensitizing effects by upregulation of PPARγ.
AMPK is an energy sensor which plays a pivotal role in energy homeostasis.AMPK responds to AMP:ATP ratio in cells.AMPK activates catabolic pathways such as fatty acids beta-oxidation, and inhibits synthetic pathways such as lipogenesis and gluconeogenesis in the liver [14].Adiponectin exerts its function by binding to membrane receptors and following adiponectin binding to its receptor, the intracellular domain of receptors recruit APPL1 [26].Deepa S. S. and Dong L. Q. showed that APPL1 is implicated in adiponectin signaling in the liver [27] and also, Mao X. and colleagues showed that adiponectin stimulates interaction of APPL1 with adiponectin receptors [28].We showed that L-carnitine administration to diabetic rats increased aPPL1 gene expression.It has been reported that L-carnitine supplementation in non-diabetic subjects increased adiponectin levels [29], here we found that L-carnitine oral administration was able to increase adiponectin levels in diabetic rats.Therefore, ele vated adiponectin interacts with its receptor and recruits APPL1 which involved in downstream effects of adiponectin such as AMPK activation and insulin sensitizing effects.
expression of amPK (A), aPPL1 (B) and PPARγ (C) genes as evaluated by RT-PCR.Data are expressed as mean±SEM.* Statistically significant compared to the control group, # statistically significant compared to the diabetes group.P < 0.05 was conside red as significant(n = 8)