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Beneficial effects of Cinnamon on hepatic lipid metabolism are impaired in hypothyroid rats
Journal of Functional Foods 50 (2018) 210–215
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Beneficial effects of Cinnamon on hepatic lipid metabolism are impaired in hypothyroid rats
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Bruna Pereira Lopesa, Thaiane Gadioli Gaiqueb, Luana Lopes Souzaa, Gabriela Silva Monteiro Paulaa, George E.G. Kluckc, Georgia C. Atellac, ⁎ Carmen Cabanelas Pazos-Mouraa,c, Karen Jesus Oliveiraa,b, a
Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro 21949-900, RJ, Brazil Biomedical Institute, Fluminense Federal University, Niterói, Rio de Janeiro 24210-130, RJ, Brazil c Medical Biochemistry Institute Leopoldo de Meis, Federal University of Rio de Janeiro, 21941-902 RJ, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cinnamon Hypothyroidism Lipid metabolism Liver Rat
Hypolipidemic effects of cinnamon (Cinnamomum zeylanicum) have been described in humans and in experimental animals. Hypothyroidism is frequently associated with hyperlipidemia. Here we investigated the effects of chronic ingestion of cinnamon on lipid metabolism of hypothyroid male Wistar rats. Rats received methimazole for 7 weeks, and treated either with cinnamon as powder added to the chow or aqueous extract of cinnamon, during the last 4 weeks of protocol, and compared to untreated hypothyroid and euthyroid rats. Cinnamon intake reduced body mass increasing fat mass and reducing body protein content of hypothyroid rats. Cinnamon ingestion did not revert the hypercholesterolemia of hypothyroid rats and promoted a further reduction in serum T3, suggesting a worsening of the hypothyroid condition. Our results indicate that the beneficial effects of cinnamon on lipid metabolism are impaired in hypothyroidism.
1. Introduction Dyslipidemia is a world-wide common disturbance and new treatment options are welcome (WHO, 2014). Cinnamon, a traditional spice composed of different polyphenols, aldehydes, sugars and flavonoids has hypoglycemic, antioxidant and anti-inflammatory properties (Anderson et al., 2004; Dorri, Hashemitabar, & Hosseinzadeh, 2018; Jarvill-Taylor, Anderson, & Graves, 2001; Sun et al., 2016). It has been also reported that cinnamon intake ameliorates, reduces fat mass and hepatic lipid accumulation in diabetic, obese and in nonalcoholic fatty liver disease patients (Askari, Rashidkhani, & Hekmatdoost, 2014; Khan et al., 2003; Van Hul et al., 2018; Ziegenfuss, Hofheins, Mendel, Landis, & Anderson, 2006). Experimental data confirmed these effects in obese and healthy rats (Couturier et al., 2010; Shalaby & Saifan, 2014; Sartorius et al., 2014; Lopes et al., 2015). Recently, we demonstrated that cinnamon intake inhibits biosynthesis and esterification of cholesterol and triacylglycerol in healthy rats (Lopes et al., 2015). Hypothyroidism has a well-known association with dyslipidemia, especially hypercholesterolemia (Duntas & Brenta, 2012). Thyroid hormones (TH) play an essential role in the regulation of lipid metabolism, modulating several steps of lipid synthesis and degradation, and
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have a major influence in cholesterol metabolism, stimulating liver cholesterol uptake, synthesis and efflux into the bile (Duntas & Brenta, 2012). These effects are dependent on direct actions of T3 (3,5,3′triiodothyronine) in the liver, mediated mainly by nuclear thyroid hormone receptor β (TRβ) (Mullur, Liu, & Brent, 2014) regulating the expression of key genes involved in lipid metabolism. Although cinnamon and thyroid hormones seem to have common targets in lipid metabolism, it has not been investigated whether cinnamon intake may ameliorate dyslipidemia present in hypothyroidism. Therefore, here we investigated the effects of chronic ingestion of cinnamon in lipid metabolism of hypothyroid rats. 2. Materials and methods 2.1. Animals and experimental design The procedures were approved by the Institutional Animal Care Committee of Fluminense Federal University and complied with the ethical guidelines of the Brazilian Society of Laboratory Animal Science. Three-month-old adult male Wistar rats were maintained under controlled conditions of temperature (24 ± 1 °C) and lighting (12 h cycle
Corresponding author at: Biomedical Institute, Fluminense Federal University, 24210-130 Rio de Janeiro, Brazil. E-mail address: [email protected]ff.br (K.J. Oliveira).
https://doi.org/10.1016/j.jff.2018.10.002 Received 13 August 2018; Received in revised form 30 September 2018; Accepted 1 October 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
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variation was 3.75%, 3.03% and 3.75%, T4, T3 and TSH, respectively.
of darkness and light, lights on at 7am). Animals were separated into four groups: EU (euthyroid)-standard chow and gavage with filtered water; HYPO (hypothyroid)-standard chow and gavage with filtered water; HYPO-P (hypothyroid + cinnamon powder)-treated with cinnamon diet and gavage with filtered water; HYPO-E (hypothyroid + aqueous extract of cinnamon)-treated with standard chow and gavage with aqueous extract of cinnamon. Hypothyroidism was induced by 0.03% methimazole (MMI - Sigma-Aldrich - Missouri, USA) administered in drinking water for 7 weeks. Cinnamon treatment started 3 weeks after MMI introduction and lasted for 4 weeks until sacrifice. Body mass was measured three times a week. At the end of the experiment, all animals were sacrificed by decapitation at fed state, serum was obtained from the trunk blood, and tissues were excised, weighed and stored at −70 °C. Adipose tissue was returned to the carcasses, which were used for body composition analysis.
2.5. Serum lipid profile Triacylglycerol (TG), total cholesterol and high-density lipoprotein cholesterol (HDL) were measured using commercial kits (Biosystems, Paraná, Brazil). Free fatty acids were measured by chromatography, as described below. 2.6. Hepatic lipid profile Lipid extraction was performed as previously described (Bligh & Dyer, 1959; Lopes et al., 2015) based on chloroform-methanol extraction. The lipids were separated by one-dimensional Thin Layer Chromatography (TLC) in silica gel plates, immersed in a charring solution (3% CuSO4 and 8% H3PO4 (v/v) (Vetec, Rio de Janeiro, Brazil). The quantification of the lipids was obtained by densitometry using Image Master Total Lab software (Amersham Pharmacia Biotec, USA).
2.2. Cinnamon aqueous extract and cinnamon diet Both extract and diet were prepared using the same batch of cinnamon bark (Cinnamomum zeylanicum), purchased from a local seller. The aqueous cinnamon extract was produced as described previously (Lopes et al., 2015). Briefly, 10 g of cinnamon was mechanically powdered and 100 mL of water was added. The suspension was maintained at 60 °C for 60 min, followed by centrifugation (1000g for 5 min). The supernatant was collected and stored at −20 °C. For the cinnamon diet, 7 g of cinnamon mechanically powdered was added to 1 kg of standard chow (Biobase, Santa Catarina, Brazil), powdered similarly. The cinnamon diet was humidified with water, modeled, stored at 4 °C and offered ad libitum. The small amount of cinnamon added to the commercial diet did not alter the nutritional composition of the diet. The protocol and concentration of cinnamon used in the diet and in the aqueous extract (400 mg cinnamon/kg body mass/day) were based those described by Kannappan, Jayaraman, Rajasekar, Ravichandran, and Anuradha (2006), Sheng, Zhang, Gong, Huang, and Zang (2008). The approximate dose of 400 mg per kg for rats is equivalent to 64 mg per kg per day in adult human, according to the body surface area normalization method for translating the drug doses used in animal studies (Reagan-Shaw, Nihal, & Ahmad, 2008). The aqueous extract and cinnamon powder were analyzed by high performance liquid chromatography (DAD-HPLC-UV) and positive and negative electrospray ionization mass spectrometry (+ESI-MS and −ESI-MS, respectively) as we described previously (Gaique et al., 2016; Lopes et al., 2015). The HPLC analyzes showed that cinnamaldehyde, the main component of cinnamon (Helal, Tagliazucchi, Verzelloni, & Conte, 2014), has a major peak in both chromatograms, although other substances such as phenylpropanoid, fatty acids and procyanidins were present (data not shown).
2.7. Analysis of hepatic mRNA expression Liver total RNA was isolated using the Trizol reagent (Invitrogen, CA, USA). The reverse transcription was performed from 1 μg of RNA and a Superscript III kit (Invitrogen, CA, USA), according to the manufacturer’s instructions. The mRNA expression was analyzed by realtime PCR using specific primers (Supplementary Material) and Rplp0, gene coding the ribosomal protein lateral stalk subunit P0 (36B4), was used as a control gene. The products were amplified on an Applied Biosystems 7500 Real-Time PCR System (Life Technologies Corp., CA, USA) using SYBR Green PCR Master Mix (Applied Biosystems, CA, USA), and the cycle parameters used were: 50 °C for 2 min and 95 °C for 10 min; 40 cycles of 95 °C for 15 s; 60 °C for 30 s and 72 °C for 45 s. Results were expressed in values relative to the values of the control groups that were set to 1, as 2−ΔΔCT method (Livak & Schmittgen, 2001). The efficiencies for each reaction were calculated from a serial dilution and only efficiencies between 0.95 and 1.05 were used. The purity of the PCR products was confirmed by the analysis of the melting curves. 2.8. Statistical analysis Data are expressed as the mean ± standard error of the mean (S.E.M.) and were analyzed by one-way ANOVA followed by a Newman Keuls post-test (GraphPad Prism Software, CA, USA). Differences were considered significant at P < 0.05. 3. Results and discussion
2.3. Protein content of the carcass
3.1. Hormonal profile, body mass gain and body composition
The protein content of the carcasses was determined as previously described (Souza et al., 2010). Briefly, eviscerated carcasses were weighed, autoclaved and homogenized in distilled water (1:1). Protein was extracted with 0.6 N KOH and quantified by Bradford method (Bradford, 1976).
The induction of hypothyroidism by MMI was efficient as shown by higher serum TSH, decreased total T4 and total T3 (Fig. 1). Treatment with cinnamon did not change serum TSH and T4 of the MMI-treated rats (Fig. 1A-B), but concentrations of T3 were further reduced to undetectable levels in both HYPO-P and HYPO-E groups. (Fig. 1C). All hypothyroid groups exhibited loss of body mass, however it was more pronounced in those treated with cinnamon (Fig. 1D). Although the white adipose tissue mass (corrected for the body mass) and protein content of the carcass did not differ between HYPO and EU groups, the adipose tissue mass was approximately 40% higher in HYPO-P and HYPO-E groups (Fig. 1E), while the protein content was lower in HYPOP group (Fig. 1F), compared to EU group. Our results indicate that cinnamon ingestion have unique consequences in lipid metabolism in the context of hypothyroidism, losing its beneficial effects in lipid homeostasis, reported in many other studies (Askari et al., 2014; Sheng et al., 2008; Ziegenfuss et al., 2006). In
2.4. Hormonal measurements Serum concentrations of total thyroxine (T4) and total T3 were measured using radioimmunoassay kits (MP Biomedicals, Santa Ana, USA). Serum thyroid-stimulating hormone (TSH) was measured using reagents acquired from National Hormone Pituitary Program (NHPP, Bethesda, USA), as described previously (Ortiga-Carvalho, Polak, McCann, & Pazos-Moura, 1996). All samples were measured within the same assay. The minimum detection levels were 1 µg/dL, 12.5 ng/dL and 0.36 ng/mL for T4, T3 and TSH, respectively. The intra-assay 211
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Fig. 1. Hormonal profile, body mass gain and body composition. Serum TSH (A), total T4 (B), total T3 (C), body mass gain (D), white adipose tissue mass (E) and total carcasses content of proteins (F). EU: euthyroid (n = 8); HYPO: hypothyroid (n = 8); HYPO-P: hypothyroid + cinnamon powder (n = 8); HYPO-E: hypothyroid + aqueous extract of cinnamon (n = 5). *P < 0.05 vs EU; #P < 0.05 vs HYPO. Values are expressed as the means ± S.E.M.
groups (Fig. 2D). Although cinnamon preparations have hypolipidemic effects in conditions of hyperlipidemia (Khan et al., 2003; Sartorius et al., 2014; Sheng et al., 2008; Van Hul et al., 2018), it did not mitigate the hypercholesterolemia present in hypothyroid rats.
addition, specific aspects of hypothyroid phenotype are modified by cinnamon chronic intake. As expected in rodents (Souza et al., 2010, 2011), hypothyroidism induced a reduction in body weight, however cinnamon exacerbated the weight loss, which was accompanied by increased fat mass and reduced lean mass, the opposite to previous findings in healthy animals (Lopes et al., 2015) and in humans with metabolic syndrome (Ziegenfuss et al., 2006).
3.3. Hepatic cholesterol metabolism Concerning the hepatic lipid profile, no changes were observed in free cholesterol (Fig. 3A), however esterified cholesterol showed a strong trend to be higher in HYPO-P and HYPO-E groups (PANOVA = 0.03; Fig. 3B). Cinnamon intake promoted higher mRNA expression of Srebf2 (Sterol regulatory element-binding protein 2) in HYPO-P and HYPO-E groups (Fig. 3C). Its target gene, Hmgcr (3-hydroxy-3-methylglutaryl-Coenzyme A reductase), was reduced in HYPO
3.2. Lipid serum profile Serum of hypothyroid groups treated or not with cinnamon exhibited higher total cholesterol (Fig. 2A), and lower HDL cholesterol (Fig. 2B), and triacylglycerol concentrations (Fig. 2C). No differences in serum concentrations of free fatty acids were observed among all
Fig. 2. Lipid serum profile. Serum concentration of total cholesterol (A), HDL cholesterol (B), triacylglycerol (C) and free fatty acids (D). EU: euthyroid (n = 8); HYPO: hypothyroid (n = 8); HYPO-P: hypothyroid + cinnamon powder (n = 8); HYPO-E: hypothyroid + aqueous extract of cinnamon (n = 5). *P < 0.05 vs EU; #P < 0.05 vs HYPO. Values are expressed as the means ± S.E.M.
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Fig. 3. Hepatic cholesterol metabolism. Hepatic content of free cholesterol (A) and esterified cholesterol (B); Hepatic mRNA expression of: Srebf2 (C), Hmgcr (D), Acat (E), Ldlr (F), Abcg5 (G) and Abcg8 (H). Results from PCR were normalized by the reference gene (Rplp0) and expressed relative to the control group. EU: euthyroid (n = 5–7); HYPO: hypothyroid (n = 5–7); HYPO-P: hypothyroid + cinnamon powder (n = 5–7); HYPO-E: hypothyroid + aqueous extract of cinnamon (n = 5). * P < 0.05 vs EU; #P < 0.05 vs HYPO. Values are expressed as the means ± S.E.M.
(Field, Albright, & Mathur, 1986; Lopez, Abisambra Socarrás, Bedi, & Ness, 2007; Shin & Osborne, 2003), which expression could be stimulated by cinnamon treatment. Cinnamon treatment did not rescue the decreased mRNA expression of Abcg5 and Abcg8 of hypothyroid animals. These transporters are responsible for direct elimination of cholesterol in the bile. Our result may suggest that total biliary excretion of cholesterol remained reduced in the groups treated with cinnamon, which may contribute to the persistent hypercholesterolemia in hypothyroid rats treated with cinnamon. Taken together, the data lead to the suggestion that in the context of hypothyroidism, cinnamon promoted increased biosynthesis and uptake of hepatic cholesterol, as well as reduced cholesterol biliary exportation, and this can explain, at least in part, the liver accumulation of esterified cholesterol observed in these animals and the persistent hypercholesterolemia.
group partially normalized in HYPO-P and increased above euthyroid levels in HYPO-E (Fig. 3D). The mRNA expression of Ldlr (low-density lipoprotein receptor), a gene involved in cholesterol uptake, was reduced in all hypothyroid groups (Fig. 3F). However, the cinnamon powder intake induced an increase in the expression of Ldlr compared to HYPO group (Fig. 3F). The mRNA expression of cholesterol transporters Abcg5 and Abcg8 (ATP-binding cassette sub-family G; Fig. 3G and H) and Acat (acetyl-CoA acetyltransferase 1; Fig. 3E) enzyme responsible for cholesterol esterification, were reduced similarly in all hypothyroid groups. Cinnamon ingestion induced greater accumulation of esterified cholesterol in the liver of hypothyroid animals that is contrary to our previous finding in euthyroid animals whose liver content of cholesterol esters were reduced when treated with cinnamon extract (Lopes et al., 2015). The higher mRNA expression of Srebf2, a key transcription factor of cholesterol biosynthesis (Landa et al., 2014), and of Hmgcr, the ratelimiting enzyme in cholesterol synthesis, suggest that cinnamon promoted a higher rate of cholesterol synthesis in the liver of hypothyroid rats. The rate of cholesterol esterification may not be altered since cinnamon treatment did not change the mRNA of Acat, an enzyme that catalyzes the esterification of cholesterol. Cinnamon powder preparation was also able to recover partially the suppressed gene expression of Ldlr caused by hypothyroidism, which may suggest that cholesterol uptake in hypothyroid animals could be ameliorated by the treatment with cinnamon powder. Importantly, as mentioned before, Srebf2, Hmgcr, and Ldlr are well known suppressed genes in hypothyroidism
3.4. Hepatic triglyceride metabolism Hepatic triacylglycerol and free fatty acids content were unaltered in HYPO group, however, cinnamon-treated groups exhibited higher triacylglycerol content (Fig. 4A) and a strong trend to increased free fatty acids hepatic levels (PANOVA = 0.05; Fig. 4B). The mRNA expression of Acaca (Acetyl-CoA carboxylase), a key enzyme in fatty acid synthesis, was lower in all MMI-treated animals. This reduction was even higher in HYPO-P and HYPO-E groups compared to HYPO group 213
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Fig. 4. Hepatic triglyceride metabolism. Hepatic content of triacylglycerol (A) and free fatty acids (B); Hepatic mRNA expression: Srebf1c (C), Acaca (D), Dgat2 (E), Ppara (F), Cpt1a (G) and Lipc (H). Results from PCR were normalized by the reference gene (Rplp0) and expressed relative to the control group. EU: euthyroid (n = 5–7); HYPO: hypothyroid (n = 5–7); HYPO-P: hypothyroid + cinnamon powder (n = 5–7); HYPO-E: hypothyroid + aqueous extract of cinnamon (n = 5). * P < 0.05 vs EU; #P < 0.05 vs HYPO. Values are expressed as the means ± S.E.M.
expression of Dgat2 was not altered, unlike that found in healthy animals treated with cinnamon, when the lower accumulation of triacylglycerol was associated with reduced expression of hepatic Dgat2 (Lopes et al., 2015). In addition, our data suggest that in hypothyroid rats, as well as in healthy rats (Lopes et al., 2015), cinnamon intake does not favor the fatty acid oxidation pathway, regulated by Ppara and its target gene Cpt1a. Furthermore, the expression of the mRNA of Lipc was reduced similarly in all hypothyroid groups, which argues against reduced lipolysis as a cause for the accumulation of hepatic triacylglycerol in cinnamon-treated groups. The liver content of triacylglycerol and free fatty acids reflects the dynamic balance of fatty acid uptake, de novo lipogenesis, oxidation and, also the liver exportation of triacylglycerol as lipoproteins. Our data suggest that in hypothyroidism context, cinnamon intake was able to increase hepatic triacylglycerol and free fatty acids by mechanisms not involving the regulation of gene expression of proteins involved in lipid metabolism in hepatic cells. We propose that the accumulation of triglycerides in the liver of cinnamon-treated hypothyroid rats may result from relocation from adipose tissues depots, which were increased in cinnamon-treated hypothyroid rats. However, a limitation of our study is that we cannot exclude the possibility that cinnamon regulates the activity of the transcription factors or of the enzymes by nongenomic pathways. Regardless of the mechanism, it is evident from our data that in hypothyroidism cinnamon intake does not promote beneficial effects on
(Fig. 4D). The expression of Dgat2 mRNA (diacylglycerol O-acyltransferase 2), an enzyme responsible for the formation of triacylglycerol, was not significantly changed after MMI or cinnamon treatment (Fig. 4E). Cpt1a (Carnitine palmitoyltransferase 1a), a key enzyme of the fatty acid oxidation pathway, was not altered by hypothyroidism or supplementation with cinnamon (Fig. 4G), but Lipc (hepatic lipase) expression was reduced in all hypothyroid groups (Fig. 4H). The mRNA expressions of master transcriptional factors involved in the regulation of fatty acid biosynthesis and oxidation, Srebf1c (Sterol regulatory element-binding protein 1c; Fig. 4C), and Ppara (Peroxisome proliferator-activated receptor α; Fig. 4F), respectively, were not affected by cinnamon treatment, although hypothyroidism decreased Ppara expression (Fig. 4F). Hypothyroidism alone did not modify the hepatic content of triacylglycerol and hepatic free fatty acids, even though they have reduced serum triacylglycerol. Interestingly, hypothyroid rats treated with cinnamon showed hepatic accumulation of triacylglycerol and free fatty acids, an opposite phenotype observed in healthy rats treated with aqueous cinnamon extract (Lopes et al., 2015), whose expression of Srebf1c, transcription factor involved in controlling lipogenic genes, was decreased (Lopes et al., 2015). Our data do not support an explanation related to increased de novo lipogenesis, since Srebf1c mRNA was unaltered, and moreover, the evaluation of the mRNA abundance of Acaca, a key enzyme in the biosynthesis of fatty acids, showed an additional decrease over that caused by hypothyroidism alone (Blennemann, Leahy, Kim, & Freake, 1994). In addition, the mRNA 214
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hepatic lipids content accumulation already described in dyslipidemic (Kanuri et al., 2009; Sartorius et al., 2014) and in healthy animal models (Lopes et al., 2015). The mechanism of action of cinnamon, its major component, cinnamaldehyde, and other less abundant compounds, are not well defined. It is well characterized that cinnamon has insulin-sensitizing effects (Askari et al., 2014; Kannappan et al., 2006; Khan et al., 2003), and our study suggests the importance of thyroid hormones for the action of cinnamon. Interestingly, the treatment with cinnamon, in both preparations, further reduced serum T3 of hypothyroid animals to undetectable levels, which is in accordance with our recent study showing lower serum T3 in healthy rats treated with cinnamon extract (Gaique et al., 2016). The mechanism and the functional consequences are unknown but raise the possibility that cinnamon may disrupt thyroid function, which remains to be fully investigated.
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4. Conclusion In conclusion, the present study shows that, contrary to what expected from studies in patients with metabolic diseases or animal models of those diseases, cinnamon chronic ingestion was unable to ameliorate the alterations in lipid metabolism of hypothyroid rats, and in some aspects, may even aggravate the disturbances. Therefore, these data suggest that normal concentration of thyroid hormones is a decisive factor for the beneficial effects of cinnamon. 5. Funding This work was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES - Código de Financiamento 001). 6. Ethics statement The procedures were approved by the Institutional Animal Care Committee of Fluminense Federal University and complied with the ethical guidelines of the Brazilian Society of Laboratory Animal Science. Conflict of interest The authors declare that there is no conflict of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2018.10.002. References Anderson, R. A., Broadhurst, C. L., Polansky, M. M., Schmidt, W. F., Khan, A., Flanagan, V. P., ... Graves, D. J. (2004). Isolation and characterization of polyphenol type-A polymers from cinnamon with insulin like biological activity. Journal of Agriculture and Food Chemistry, 52, 65–70. Askari, F., Rashidkhani, B., & Hekmatdoost, A. (2014). Cinnamon may have therapeutic benefits on lipid profile, liver enzymes, insulin resistance, and high-sensitivity C-reactive protein in nonalcoholic fatty liver disease patients. Nutrition Research, 34, 143–148. Blennemann, B., Leahy, P., Kim, T. S., & Freake, H. C. (1994). Tissue-specific regulation of lipogenic mRNAs by thyroid hormone. Molecular and Cellular Endocrinology, 28, 1–8. Bligh, E., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911–917. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. Couturier, K., Batandier, C., Awada, M., Hininger-Favier, I., Canini, F., ... Roussel, A. M. (2010). Cinnamon improves insulin sensitivity and alters the body composition in an animal model of the metabolic syndrome. Archives of Biochemistry and Biophysics, 501,
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Beneficial effects of Cinnamon on hepatic lipid metabolism are impaired in hypothyroid rats
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Bruna Pereira Lopesa, Thaiane Gadioli Gaiqueb, Luana Lopes Souzaa, Gabriela Silva Monteiro Paulaa, George E.G. Kluckc, Georgia C. Atellac, ⁎ Carmen Cabanelas Pazos-Mouraa,c, Karen Jesus Oliveiraa,b, a
Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro 21949-900, RJ, Brazil Biomedical Institute, Fluminense Federal University, Niterói, Rio de Janeiro 24210-130, RJ, Brazil c Medical Biochemistry Institute Leopoldo de Meis, Federal University of Rio de Janeiro, 21941-902 RJ, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cinnamon Hypothyroidism Lipid metabolism Liver Rat
Hypolipidemic effects of cinnamon (Cinnamomum zeylanicum) have been described in humans and in experimental animals. Hypothyroidism is frequently associated with hyperlipidemia. Here we investigated the effects of chronic ingestion of cinnamon on lipid metabolism of hypothyroid male Wistar rats. Rats received methimazole for 7 weeks, and treated either with cinnamon as powder added to the chow or aqueous extract of cinnamon, during the last 4 weeks of protocol, and compared to untreated hypothyroid and euthyroid rats. Cinnamon intake reduced body mass increasing fat mass and reducing body protein content of hypothyroid rats. Cinnamon ingestion did not revert the hypercholesterolemia of hypothyroid rats and promoted a further reduction in serum T3, suggesting a worsening of the hypothyroid condition. Our results indicate that the beneficial effects of cinnamon on lipid metabolism are impaired in hypothyroidism.
1. Introduction Dyslipidemia is a world-wide common disturbance and new treatment options are welcome (WHO, 2014). Cinnamon, a traditional spice composed of different polyphenols, aldehydes, sugars and flavonoids has hypoglycemic, antioxidant and anti-inflammatory properties (Anderson et al., 2004; Dorri, Hashemitabar, & Hosseinzadeh, 2018; Jarvill-Taylor, Anderson, & Graves, 2001; Sun et al., 2016). It has been also reported that cinnamon intake ameliorates, reduces fat mass and hepatic lipid accumulation in diabetic, obese and in nonalcoholic fatty liver disease patients (Askari, Rashidkhani, & Hekmatdoost, 2014; Khan et al., 2003; Van Hul et al., 2018; Ziegenfuss, Hofheins, Mendel, Landis, & Anderson, 2006). Experimental data confirmed these effects in obese and healthy rats (Couturier et al., 2010; Shalaby & Saifan, 2014; Sartorius et al., 2014; Lopes et al., 2015). Recently, we demonstrated that cinnamon intake inhibits biosynthesis and esterification of cholesterol and triacylglycerol in healthy rats (Lopes et al., 2015). Hypothyroidism has a well-known association with dyslipidemia, especially hypercholesterolemia (Duntas & Brenta, 2012). Thyroid hormones (TH) play an essential role in the regulation of lipid metabolism, modulating several steps of lipid synthesis and degradation, and
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have a major influence in cholesterol metabolism, stimulating liver cholesterol uptake, synthesis and efflux into the bile (Duntas & Brenta, 2012). These effects are dependent on direct actions of T3 (3,5,3′triiodothyronine) in the liver, mediated mainly by nuclear thyroid hormone receptor β (TRβ) (Mullur, Liu, & Brent, 2014) regulating the expression of key genes involved in lipid metabolism. Although cinnamon and thyroid hormones seem to have common targets in lipid metabolism, it has not been investigated whether cinnamon intake may ameliorate dyslipidemia present in hypothyroidism. Therefore, here we investigated the effects of chronic ingestion of cinnamon in lipid metabolism of hypothyroid rats. 2. Materials and methods 2.1. Animals and experimental design The procedures were approved by the Institutional Animal Care Committee of Fluminense Federal University and complied with the ethical guidelines of the Brazilian Society of Laboratory Animal Science. Three-month-old adult male Wistar rats were maintained under controlled conditions of temperature (24 ± 1 °C) and lighting (12 h cycle
Corresponding author at: Biomedical Institute, Fluminense Federal University, 24210-130 Rio de Janeiro, Brazil. E-mail address: [email protected]ff.br (K.J. Oliveira).
https://doi.org/10.1016/j.jff.2018.10.002 Received 13 August 2018; Received in revised form 30 September 2018; Accepted 1 October 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
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variation was 3.75%, 3.03% and 3.75%, T4, T3 and TSH, respectively.
of darkness and light, lights on at 7am). Animals were separated into four groups: EU (euthyroid)-standard chow and gavage with filtered water; HYPO (hypothyroid)-standard chow and gavage with filtered water; HYPO-P (hypothyroid + cinnamon powder)-treated with cinnamon diet and gavage with filtered water; HYPO-E (hypothyroid + aqueous extract of cinnamon)-treated with standard chow and gavage with aqueous extract of cinnamon. Hypothyroidism was induced by 0.03% methimazole (MMI - Sigma-Aldrich - Missouri, USA) administered in drinking water for 7 weeks. Cinnamon treatment started 3 weeks after MMI introduction and lasted for 4 weeks until sacrifice. Body mass was measured three times a week. At the end of the experiment, all animals were sacrificed by decapitation at fed state, serum was obtained from the trunk blood, and tissues were excised, weighed and stored at −70 °C. Adipose tissue was returned to the carcasses, which were used for body composition analysis.
2.5. Serum lipid profile Triacylglycerol (TG), total cholesterol and high-density lipoprotein cholesterol (HDL) were measured using commercial kits (Biosystems, Paraná, Brazil). Free fatty acids were measured by chromatography, as described below. 2.6. Hepatic lipid profile Lipid extraction was performed as previously described (Bligh & Dyer, 1959; Lopes et al., 2015) based on chloroform-methanol extraction. The lipids were separated by one-dimensional Thin Layer Chromatography (TLC) in silica gel plates, immersed in a charring solution (3% CuSO4 and 8% H3PO4 (v/v) (Vetec, Rio de Janeiro, Brazil). The quantification of the lipids was obtained by densitometry using Image Master Total Lab software (Amersham Pharmacia Biotec, USA).
2.2. Cinnamon aqueous extract and cinnamon diet Both extract and diet were prepared using the same batch of cinnamon bark (Cinnamomum zeylanicum), purchased from a local seller. The aqueous cinnamon extract was produced as described previously (Lopes et al., 2015). Briefly, 10 g of cinnamon was mechanically powdered and 100 mL of water was added. The suspension was maintained at 60 °C for 60 min, followed by centrifugation (1000g for 5 min). The supernatant was collected and stored at −20 °C. For the cinnamon diet, 7 g of cinnamon mechanically powdered was added to 1 kg of standard chow (Biobase, Santa Catarina, Brazil), powdered similarly. The cinnamon diet was humidified with water, modeled, stored at 4 °C and offered ad libitum. The small amount of cinnamon added to the commercial diet did not alter the nutritional composition of the diet. The protocol and concentration of cinnamon used in the diet and in the aqueous extract (400 mg cinnamon/kg body mass/day) were based those described by Kannappan, Jayaraman, Rajasekar, Ravichandran, and Anuradha (2006), Sheng, Zhang, Gong, Huang, and Zang (2008). The approximate dose of 400 mg per kg for rats is equivalent to 64 mg per kg per day in adult human, according to the body surface area normalization method for translating the drug doses used in animal studies (Reagan-Shaw, Nihal, & Ahmad, 2008). The aqueous extract and cinnamon powder were analyzed by high performance liquid chromatography (DAD-HPLC-UV) and positive and negative electrospray ionization mass spectrometry (+ESI-MS and −ESI-MS, respectively) as we described previously (Gaique et al., 2016; Lopes et al., 2015). The HPLC analyzes showed that cinnamaldehyde, the main component of cinnamon (Helal, Tagliazucchi, Verzelloni, & Conte, 2014), has a major peak in both chromatograms, although other substances such as phenylpropanoid, fatty acids and procyanidins were present (data not shown).
2.7. Analysis of hepatic mRNA expression Liver total RNA was isolated using the Trizol reagent (Invitrogen, CA, USA). The reverse transcription was performed from 1 μg of RNA and a Superscript III kit (Invitrogen, CA, USA), according to the manufacturer’s instructions. The mRNA expression was analyzed by realtime PCR using specific primers (Supplementary Material) and Rplp0, gene coding the ribosomal protein lateral stalk subunit P0 (36B4), was used as a control gene. The products were amplified on an Applied Biosystems 7500 Real-Time PCR System (Life Technologies Corp., CA, USA) using SYBR Green PCR Master Mix (Applied Biosystems, CA, USA), and the cycle parameters used were: 50 °C for 2 min and 95 °C for 10 min; 40 cycles of 95 °C for 15 s; 60 °C for 30 s and 72 °C for 45 s. Results were expressed in values relative to the values of the control groups that were set to 1, as 2−ΔΔCT method (Livak & Schmittgen, 2001). The efficiencies for each reaction were calculated from a serial dilution and only efficiencies between 0.95 and 1.05 were used. The purity of the PCR products was confirmed by the analysis of the melting curves. 2.8. Statistical analysis Data are expressed as the mean ± standard error of the mean (S.E.M.) and were analyzed by one-way ANOVA followed by a Newman Keuls post-test (GraphPad Prism Software, CA, USA). Differences were considered significant at P < 0.05. 3. Results and discussion
2.3. Protein content of the carcass
3.1. Hormonal profile, body mass gain and body composition
The protein content of the carcasses was determined as previously described (Souza et al., 2010). Briefly, eviscerated carcasses were weighed, autoclaved and homogenized in distilled water (1:1). Protein was extracted with 0.6 N KOH and quantified by Bradford method (Bradford, 1976).
The induction of hypothyroidism by MMI was efficient as shown by higher serum TSH, decreased total T4 and total T3 (Fig. 1). Treatment with cinnamon did not change serum TSH and T4 of the MMI-treated rats (Fig. 1A-B), but concentrations of T3 were further reduced to undetectable levels in both HYPO-P and HYPO-E groups. (Fig. 1C). All hypothyroid groups exhibited loss of body mass, however it was more pronounced in those treated with cinnamon (Fig. 1D). Although the white adipose tissue mass (corrected for the body mass) and protein content of the carcass did not differ between HYPO and EU groups, the adipose tissue mass was approximately 40% higher in HYPO-P and HYPO-E groups (Fig. 1E), while the protein content was lower in HYPOP group (Fig. 1F), compared to EU group. Our results indicate that cinnamon ingestion have unique consequences in lipid metabolism in the context of hypothyroidism, losing its beneficial effects in lipid homeostasis, reported in many other studies (Askari et al., 2014; Sheng et al., 2008; Ziegenfuss et al., 2006). In
2.4. Hormonal measurements Serum concentrations of total thyroxine (T4) and total T3 were measured using radioimmunoassay kits (MP Biomedicals, Santa Ana, USA). Serum thyroid-stimulating hormone (TSH) was measured using reagents acquired from National Hormone Pituitary Program (NHPP, Bethesda, USA), as described previously (Ortiga-Carvalho, Polak, McCann, & Pazos-Moura, 1996). All samples were measured within the same assay. The minimum detection levels were 1 µg/dL, 12.5 ng/dL and 0.36 ng/mL for T4, T3 and TSH, respectively. The intra-assay 211
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Fig. 1. Hormonal profile, body mass gain and body composition. Serum TSH (A), total T4 (B), total T3 (C), body mass gain (D), white adipose tissue mass (E) and total carcasses content of proteins (F). EU: euthyroid (n = 8); HYPO: hypothyroid (n = 8); HYPO-P: hypothyroid + cinnamon powder (n = 8); HYPO-E: hypothyroid + aqueous extract of cinnamon (n = 5). *P < 0.05 vs EU; #P < 0.05 vs HYPO. Values are expressed as the means ± S.E.M.
groups (Fig. 2D). Although cinnamon preparations have hypolipidemic effects in conditions of hyperlipidemia (Khan et al., 2003; Sartorius et al., 2014; Sheng et al., 2008; Van Hul et al., 2018), it did not mitigate the hypercholesterolemia present in hypothyroid rats.
addition, specific aspects of hypothyroid phenotype are modified by cinnamon chronic intake. As expected in rodents (Souza et al., 2010, 2011), hypothyroidism induced a reduction in body weight, however cinnamon exacerbated the weight loss, which was accompanied by increased fat mass and reduced lean mass, the opposite to previous findings in healthy animals (Lopes et al., 2015) and in humans with metabolic syndrome (Ziegenfuss et al., 2006).
3.3. Hepatic cholesterol metabolism Concerning the hepatic lipid profile, no changes were observed in free cholesterol (Fig. 3A), however esterified cholesterol showed a strong trend to be higher in HYPO-P and HYPO-E groups (PANOVA = 0.03; Fig. 3B). Cinnamon intake promoted higher mRNA expression of Srebf2 (Sterol regulatory element-binding protein 2) in HYPO-P and HYPO-E groups (Fig. 3C). Its target gene, Hmgcr (3-hydroxy-3-methylglutaryl-Coenzyme A reductase), was reduced in HYPO
3.2. Lipid serum profile Serum of hypothyroid groups treated or not with cinnamon exhibited higher total cholesterol (Fig. 2A), and lower HDL cholesterol (Fig. 2B), and triacylglycerol concentrations (Fig. 2C). No differences in serum concentrations of free fatty acids were observed among all
Fig. 2. Lipid serum profile. Serum concentration of total cholesterol (A), HDL cholesterol (B), triacylglycerol (C) and free fatty acids (D). EU: euthyroid (n = 8); HYPO: hypothyroid (n = 8); HYPO-P: hypothyroid + cinnamon powder (n = 8); HYPO-E: hypothyroid + aqueous extract of cinnamon (n = 5). *P < 0.05 vs EU; #P < 0.05 vs HYPO. Values are expressed as the means ± S.E.M.
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Fig. 3. Hepatic cholesterol metabolism. Hepatic content of free cholesterol (A) and esterified cholesterol (B); Hepatic mRNA expression of: Srebf2 (C), Hmgcr (D), Acat (E), Ldlr (F), Abcg5 (G) and Abcg8 (H). Results from PCR were normalized by the reference gene (Rplp0) and expressed relative to the control group. EU: euthyroid (n = 5–7); HYPO: hypothyroid (n = 5–7); HYPO-P: hypothyroid + cinnamon powder (n = 5–7); HYPO-E: hypothyroid + aqueous extract of cinnamon (n = 5). * P < 0.05 vs EU; #P < 0.05 vs HYPO. Values are expressed as the means ± S.E.M.
(Field, Albright, & Mathur, 1986; Lopez, Abisambra Socarrás, Bedi, & Ness, 2007; Shin & Osborne, 2003), which expression could be stimulated by cinnamon treatment. Cinnamon treatment did not rescue the decreased mRNA expression of Abcg5 and Abcg8 of hypothyroid animals. These transporters are responsible for direct elimination of cholesterol in the bile. Our result may suggest that total biliary excretion of cholesterol remained reduced in the groups treated with cinnamon, which may contribute to the persistent hypercholesterolemia in hypothyroid rats treated with cinnamon. Taken together, the data lead to the suggestion that in the context of hypothyroidism, cinnamon promoted increased biosynthesis and uptake of hepatic cholesterol, as well as reduced cholesterol biliary exportation, and this can explain, at least in part, the liver accumulation of esterified cholesterol observed in these animals and the persistent hypercholesterolemia.
group partially normalized in HYPO-P and increased above euthyroid levels in HYPO-E (Fig. 3D). The mRNA expression of Ldlr (low-density lipoprotein receptor), a gene involved in cholesterol uptake, was reduced in all hypothyroid groups (Fig. 3F). However, the cinnamon powder intake induced an increase in the expression of Ldlr compared to HYPO group (Fig. 3F). The mRNA expression of cholesterol transporters Abcg5 and Abcg8 (ATP-binding cassette sub-family G; Fig. 3G and H) and Acat (acetyl-CoA acetyltransferase 1; Fig. 3E) enzyme responsible for cholesterol esterification, were reduced similarly in all hypothyroid groups. Cinnamon ingestion induced greater accumulation of esterified cholesterol in the liver of hypothyroid animals that is contrary to our previous finding in euthyroid animals whose liver content of cholesterol esters were reduced when treated with cinnamon extract (Lopes et al., 2015). The higher mRNA expression of Srebf2, a key transcription factor of cholesterol biosynthesis (Landa et al., 2014), and of Hmgcr, the ratelimiting enzyme in cholesterol synthesis, suggest that cinnamon promoted a higher rate of cholesterol synthesis in the liver of hypothyroid rats. The rate of cholesterol esterification may not be altered since cinnamon treatment did not change the mRNA of Acat, an enzyme that catalyzes the esterification of cholesterol. Cinnamon powder preparation was also able to recover partially the suppressed gene expression of Ldlr caused by hypothyroidism, which may suggest that cholesterol uptake in hypothyroid animals could be ameliorated by the treatment with cinnamon powder. Importantly, as mentioned before, Srebf2, Hmgcr, and Ldlr are well known suppressed genes in hypothyroidism
3.4. Hepatic triglyceride metabolism Hepatic triacylglycerol and free fatty acids content were unaltered in HYPO group, however, cinnamon-treated groups exhibited higher triacylglycerol content (Fig. 4A) and a strong trend to increased free fatty acids hepatic levels (PANOVA = 0.05; Fig. 4B). The mRNA expression of Acaca (Acetyl-CoA carboxylase), a key enzyme in fatty acid synthesis, was lower in all MMI-treated animals. This reduction was even higher in HYPO-P and HYPO-E groups compared to HYPO group 213
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Fig. 4. Hepatic triglyceride metabolism. Hepatic content of triacylglycerol (A) and free fatty acids (B); Hepatic mRNA expression: Srebf1c (C), Acaca (D), Dgat2 (E), Ppara (F), Cpt1a (G) and Lipc (H). Results from PCR were normalized by the reference gene (Rplp0) and expressed relative to the control group. EU: euthyroid (n = 5–7); HYPO: hypothyroid (n = 5–7); HYPO-P: hypothyroid + cinnamon powder (n = 5–7); HYPO-E: hypothyroid + aqueous extract of cinnamon (n = 5). * P < 0.05 vs EU; #P < 0.05 vs HYPO. Values are expressed as the means ± S.E.M.
expression of Dgat2 was not altered, unlike that found in healthy animals treated with cinnamon, when the lower accumulation of triacylglycerol was associated with reduced expression of hepatic Dgat2 (Lopes et al., 2015). In addition, our data suggest that in hypothyroid rats, as well as in healthy rats (Lopes et al., 2015), cinnamon intake does not favor the fatty acid oxidation pathway, regulated by Ppara and its target gene Cpt1a. Furthermore, the expression of the mRNA of Lipc was reduced similarly in all hypothyroid groups, which argues against reduced lipolysis as a cause for the accumulation of hepatic triacylglycerol in cinnamon-treated groups. The liver content of triacylglycerol and free fatty acids reflects the dynamic balance of fatty acid uptake, de novo lipogenesis, oxidation and, also the liver exportation of triacylglycerol as lipoproteins. Our data suggest that in hypothyroidism context, cinnamon intake was able to increase hepatic triacylglycerol and free fatty acids by mechanisms not involving the regulation of gene expression of proteins involved in lipid metabolism in hepatic cells. We propose that the accumulation of triglycerides in the liver of cinnamon-treated hypothyroid rats may result from relocation from adipose tissues depots, which were increased in cinnamon-treated hypothyroid rats. However, a limitation of our study is that we cannot exclude the possibility that cinnamon regulates the activity of the transcription factors or of the enzymes by nongenomic pathways. Regardless of the mechanism, it is evident from our data that in hypothyroidism cinnamon intake does not promote beneficial effects on
(Fig. 4D). The expression of Dgat2 mRNA (diacylglycerol O-acyltransferase 2), an enzyme responsible for the formation of triacylglycerol, was not significantly changed after MMI or cinnamon treatment (Fig. 4E). Cpt1a (Carnitine palmitoyltransferase 1a), a key enzyme of the fatty acid oxidation pathway, was not altered by hypothyroidism or supplementation with cinnamon (Fig. 4G), but Lipc (hepatic lipase) expression was reduced in all hypothyroid groups (Fig. 4H). The mRNA expressions of master transcriptional factors involved in the regulation of fatty acid biosynthesis and oxidation, Srebf1c (Sterol regulatory element-binding protein 1c; Fig. 4C), and Ppara (Peroxisome proliferator-activated receptor α; Fig. 4F), respectively, were not affected by cinnamon treatment, although hypothyroidism decreased Ppara expression (Fig. 4F). Hypothyroidism alone did not modify the hepatic content of triacylglycerol and hepatic free fatty acids, even though they have reduced serum triacylglycerol. Interestingly, hypothyroid rats treated with cinnamon showed hepatic accumulation of triacylglycerol and free fatty acids, an opposite phenotype observed in healthy rats treated with aqueous cinnamon extract (Lopes et al., 2015), whose expression of Srebf1c, transcription factor involved in controlling lipogenic genes, was decreased (Lopes et al., 2015). Our data do not support an explanation related to increased de novo lipogenesis, since Srebf1c mRNA was unaltered, and moreover, the evaluation of the mRNA abundance of Acaca, a key enzyme in the biosynthesis of fatty acids, showed an additional decrease over that caused by hypothyroidism alone (Blennemann, Leahy, Kim, & Freake, 1994). In addition, the mRNA 214
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hepatic lipids content accumulation already described in dyslipidemic (Kanuri et al., 2009; Sartorius et al., 2014) and in healthy animal models (Lopes et al., 2015). The mechanism of action of cinnamon, its major component, cinnamaldehyde, and other less abundant compounds, are not well defined. It is well characterized that cinnamon has insulin-sensitizing effects (Askari et al., 2014; Kannappan et al., 2006; Khan et al., 2003), and our study suggests the importance of thyroid hormones for the action of cinnamon. Interestingly, the treatment with cinnamon, in both preparations, further reduced serum T3 of hypothyroid animals to undetectable levels, which is in accordance with our recent study showing lower serum T3 in healthy rats treated with cinnamon extract (Gaique et al., 2016). The mechanism and the functional consequences are unknown but raise the possibility that cinnamon may disrupt thyroid function, which remains to be fully investigated.
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4. Conclusion In conclusion, the present study shows that, contrary to what expected from studies in patients with metabolic diseases or animal models of those diseases, cinnamon chronic ingestion was unable to ameliorate the alterations in lipid metabolism of hypothyroid rats, and in some aspects, may even aggravate the disturbances. Therefore, these data suggest that normal concentration of thyroid hormones is a decisive factor for the beneficial effects of cinnamon. 5. Funding This work was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES - Código de Financiamento 001). 6. Ethics statement The procedures were approved by the Institutional Animal Care Committee of Fluminense Federal University and complied with the ethical guidelines of the Brazilian Society of Laboratory Animal Science. Conflict of interest The authors declare that there is no conflict of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2018.10.002. References Anderson, R. A., Broadhurst, C. L., Polansky, M. M., Schmidt, W. F., Khan, A., Flanagan, V. P., ... Graves, D. J. (2004). Isolation and characterization of polyphenol type-A polymers from cinnamon with insulin like biological activity. Journal of Agriculture and Food Chemistry, 52, 65–70. Askari, F., Rashidkhani, B., & Hekmatdoost, A. (2014). Cinnamon may have therapeutic benefits on lipid profile, liver enzymes, insulin resistance, and high-sensitivity C-reactive protein in nonalcoholic fatty liver disease patients. Nutrition Research, 34, 143–148. Blennemann, B., Leahy, P., Kim, T. S., & Freake, H. C. (1994). Tissue-specific regulation of lipogenic mRNAs by thyroid hormone. Molecular and Cellular Endocrinology, 28, 1–8. Bligh, E., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911–917. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. Couturier, K., Batandier, C., Awada, M., Hininger-Favier, I., Canini, F., ... Roussel, A. M. (2010). Cinnamon improves insulin sensitivity and alters the body composition in an animal model of the metabolic syndrome. Archives of Biochemistry and Biophysics, 501,
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