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Supplemental material, Supplementary_material_JBR for The Influence of Melatonin on the Daily 24-h Rhythm of Putative Reference Gene Expression in White Adipose Tissues by Tatienne Neder Figueira da Costa, Sandra Andreotti, Talita da Silva Mendes de Farias, Fábio Bessa Lima and Paula Bargi-Souza in Journal of Biological Rhythms

Obesity results from critical periods of positive energy balance characterized by caloric intake greater than energy expenditure. This disbalance promotes adipose tissue dysfunction which is related to other comorbidities. Melatonin is a low-cost therapeutic agent and studies indicate that its use may improve obesity-related disorders. To evaluate if the melatonin is efficient in delaying or even blocking the damages caused by excessive ingestion of a high-fat diet (HFD) in mice, as well as improving the inflammatory profile triggered by obesity herein, male C57BL/6 mice of 8 weeks were induced to obesity by a HFD and treated for 10 weeks with melatonin. The results demonstrate that melatonin supplementation attenuated serum triglyceride levels and total and LDL cholesterol and prevented body mass gain through a decreased lipogenesis rate and increased lipolytic capacity in white adipocytes, with a concomitant increment in oxygen consumption and Pgc1a and Prdm16 expression. Altogether, these effects prevented adipocyte hypertrophy caused by HFD and reflected in decreased adiposity. Finally, melatonin supplementation reduced the crown-like-structure (CLS) formation, characteristic of the inflammatory process by macrophage infiltration into white adipose tissue of obese subjects, as well as decreased the gene expression of inflammation-related factors, such as leptin and MCP1. Thus, the melatonin can be considered a potential therapeutic agent to attenuate the metabolic and inflammatory disorders triggered by obesity.

Macrophages play a pivotal role in the development of emphysema and depending on the microenvironment stimuli can be polarized into M1- or M2-like macrophage phenotypes. We compared macrophage polarizations in cigarette smoke (CS)- and porcine pancreatic elastase (PPE)-induced emphysema models. C57BL/6 mice were subdivided into four experimental groups. In the PPE group, animals received an intranasal instillation of PPE (0.677 IU); in the saline group, animals received an intranasal instillation of saline (0.9%). Animals from both groups were euthanized on day 28. In the CS group, animals were exposed to CS for 30 min, twice a day, 5 days per week for 12 weeks. In the control group, animals received filtered air. We observed an increase in total macrophages for both experimental models. For M1-like macrophage markers, we observed an increase in TNF-α+ and IFN-γ+ cells, Cxcl-9 and Cxcl-10 expressions in PPE and CS groups. Only in the CS group, we detected an increased expression of IL-12b. For M2-like macrophages markers we observed a down regulation in IL-10, IL-4, IL-13, Arg1 and Fizz1 and an increase of TGF-β+ cells in the PPE group, while for the CS group there was an increase in TGF-β+ cells and IL-10 expression. All exposure groups were compared to their respective controls. In summary, we demonstrated that CS- and PPE-induced models resulted in different microenvironmental stimuli. CS exposure induced an environmental stimulus related to M1- and M2-like macrophage phenotypes similar to previous results described in COPD patients, whereas the elastase-induced model provided an environmental stimulus related only to the M1 phenotype., Summary: The CS-induced model showed M1 and M2-related genes and chemokines, whereas the PPE-induced model showed a downregulation of M2- and an increase in M1-related genes and chemokines.

Scope We tested herein the hypothesis that peroxisome proliferator activated receptor γ (PPARγ) is a major mediator of omega-3 (n-3) protective actions against high-fat diet (HFD) induced obesity, glucose intolerance, and adipose tissue inflammation. Methods and results C57BL6 wild-type and fat-1 transgenic (fat-1) mice were fed a low-fat diet (LFD) or HFD, treated or not with PPARγ antagonist, and evaluated for energy balance, adiposity, glucose tolerance, and adipose tissue inflammation. Fat-1 mice were protected from obesity, fasting hyperglycemia, glucose intolerance, and adipose tissue inflammation. PPARγ inhibition completely abolished fat-1 protection against HFD-induced glucose intolerance, but not obesity or adipose tissue inflammation. To investigate the role of myeloid cell as mediator of n-3 beneficial metabolic actions, mice with deletion (LyzM-PPARγ(KO)) or nondeletion (LyzM-PPARγ(WT)) of PPARγ in myeloid cells were fed either LFD or HFD (lard) or an HFD rich in n-3 (fish oil). Our findings indicate that myeloid cell associated PPARγ is not involved in the attenuation of HFD-induced glucose intolerance and adipose tissue inflammation induced by n-3. Conclusion High endogenous n-3 fatty acid levels protect from HFD obesity, glucose intolerance, and adipose tissue inflammation. Among these, only protection against glucose intolerance is mediated by non-myeloid cell PPARγ.

Melatonin, the main hormone produced by the pineal gland, is secreted in a circadian manner (24-hr period), and its oscillation influences several circadian biological rhythms, such as the regulation of clock genes expression (chronobiotic effect) and the modulation of several endocrine functions in peripheral tissues. Assuming that the circadian synchronization of clock genes can play a role in the regulation of energy metabolism and it is influenced by melatonin, our study was designed to assess possible alterations as a consequence of melatonin absence on the circadian expression of clock genes in the epididymal adipose tissue of male Wistar rats and the possible metabolic repercussions to this tissue. Our data show that pinealectomy indeed has impacts on molecular events: it abolishes the daily pattern of the expression of Clock, Per2, and Cry1 clock genes and Pparγ expression, significantly increases the amplitude of daily expression of Rev-erbα, and affects the pattern of and impairs adipokine production, leading to a decrease in leptin levels. However, regarding some metabolic aspects of adipocyte functions, such as its ability to synthesize triacylglycerols from glucose along 24 hr, was not compromised by pinealectomy, although the daily profile of the lipogenic enzymes expression (ATP-citrate lyase, malic enzyme, fatty acid synthase, and glucose-6-phosphate dehydrogenase) was abolished in pinealectomized animals.

Diabetes mellitus is a product of low insulin sensibility and pancreatic β-cell insufficiency. Rats with streptozotocin-induced diabetes during the neonatal period by the fifth day of age develop the classic diabetic picture of hyperglycemia, hypoinsulinemia, polyuria, and polydipsia aggravated by insulin resistance in adulthood. In this study, we investigated whether the effect of long-term treatment with melatonin can improve insulin resistance and other metabolic disorders in these animals. At the fourth week of age, diabetic animals started an 8-wk treatment with melatonin (1 mg/kg body weight) in the drinking water at night. Animals were then killing, and the sc, epididymal (EP), and retroperitoneal (RP) fat pads were excised, weighed, and processed for adipocyte isolation for morphometric analysis as well as for measuring glucose uptake, oxidation, and incorporation of glucose into lipids. Blood samples were collected for biochemical assays. Melatonin treatment reduced hyperglycemia, polydipsia, and polyphagia as well as improved insulin resistance as demonstrated by constant glucose disappearance rate and homeostasis model of assessment-insulin resistance. However, melatonin treatment was unable to recover body weight deficiency, fat mass, and adipocyte size of diabetic animals. Adiponectin and fructosamine levels were completely recovered by melatonin, whereas neither plasma insulin level nor insulin secretion capacity was improved in diabetic animals. Furthermore, melatonin caused a marked delay in the sexual development, leaving genital structures smaller than those of nontreated diabetic animals. Melatonin treatment improved the responsiveness of adipocytes to insulin in diabetic animals measured by tests of glucose uptake (sc, EP, and RP), glucose oxidation, and incorporation of glucose into lipids (EP and RP), an effect that seems partially related to an increased expression of insulin receptor substrate 1, acetyl-coenzyme A carboxylase and fatty acid synthase. In conclusion, melatonin treatment was capable of ameliorating the metabolic abnormalities in this particular diabetes model, including insulin resistance and promoting a better long-term glycemic control.

A melatonina, produzida pela pineal e secretada de maneira cíclica num período de 24 horas, possui ação na sincronização dos ritmos biológicos. Admitindo-se que a expressão circadiana dos clock genes está envolvida na regulação do metabolismo energético e que este padrão oscilatório está sob influência da melatonina, avaliamos o processo de incorporação de glicose em lipídeos no tecido adiposo de ratos controles e pinealectomizados e verificamos a interferência da pinealectomia na expressão dos genes relógio. Os resultados mostram que a pinealectomia alterou a expressão circadiana dos genes Clock, Per2 e Cry1, aumentou a expressão de Rev-erb (Zt 8) e alterou a expressão de Ppar. Ainda, provocou aumento nas concentrações séricas de corticosterona e diminuição nas de leptina. No entanto, o processo de incorporação de glicose em lipídeos não foi alterado. Sendo assim, concluímos que apesar da pinealectomia ter afetado eventos moleculares e metabólicos, quatro semanas de cirurgia ainda é curto para evidenciar repercussões metabólicas mais significativas no animal. Melatonin, which is produced by pineal gland and secreted in a cyclic fashion over 24 hours, acts in biological rhythms synchronization. Thus, assuming that circadian expression of clock genes is involved in the regulation of energy metabolism and that oscillatory pattern is under influence of melatonin, we analyzed the process of glucose incorporation into lipids in adipose tissue of pinealectomized and controls rats and we determined whether pinealectomy interferes on the expression of clock genes in this tissue. Our results show that pinealectomy alters the circadian pattern of Clock, Per2, Cry1 and Ppar expression, while increases the Rev-erb gene expression (Zt 8). Moreover, the pinealectomy promoted increased corticosterone and decreased leptin plasma levels. However, the incorporation of glucose into lipids over 24 hours was not altered by pinealectomy. Thus, we conclude that despite the pinealectomy have affected molecular and metabolic events, four weeks of surgery is still too short to induce more significant metabolic effects in animals.

O objetivo do trabalho foi investigar se há ritmicidade dos eventos lipolíticos e lipogênicos no TA peri-epididimal e caracterizar se a pinealectomia interfere nesse processo. O sacrifício dos animais, controles e pinealectomizados, foi realizado de quatro em quatro horas. Em relação a lipogênese observaram-se diferenças significativas entre os horários analisados, no entanto, a comparação entre grupos, não mostrou diferenças significativas, indicando que a pinealectomia não interfere nesse processo metabólico do TAB. O estudo da lipólise mostrou que existem algumas diferenças significativas entre os horários, contudo, a pinealectomia também não interferiu no ritmo desse processo. A utilização do programa COSINOR, para análise da existência de ritmos, evidenciou que existe um ritmo, tanto na lipogênese quanto na lipólise, mas que não ocorre de maneira circadiana, ou seja, não ocorre em intervalos de 24h ±4. Também não foram encontradas diferenças significativas, entre os grupos, quando analisadas: consumo alimentar, ingestão hídrica e evolução do peso corporal. The objective was to investigate whether there rhythmicity of events in lipolytic and lipogenic TA peri-epididymal and characterize whether pinealectomy interferes with this process. The sacrifice of animals, controls and pinealectomized was performed every four hours. In relation to lipogenesis observed significant differences between the times tested, however, the comparison between groups showed no significant differences, indicating that pinealectomy does not intervene in the metabolic process of WAT. The study of lipolysis showed that there are some significant differences between the times, however, pinealectomy did not affect the rhythms of this process. The use of COSINOR program for analysis of the existence of rhythms, suggest that there is a rhythm in both lipogenesis and lipolysis, but that does not occur in a circadian manner, i.e., does not occur at intervals of 24 ± 4. We also found no significant differences between groups when analyzed food intake, water intake and body weight evolution.

Aims Maternal hyperglycemia during pregnancy can lead to fetal changes, like macrosomia or obesity in adult life. Experimental models of diabetes have been studied to evaluate the consequences of offspring lipid metabolism. This study aimed to investigate the metabolic changes in adipose tissue of offspring of streptozotocin-induced diabetic mothers during neonatal period. Main methods Diabetes was induced in female rats by streptozotocin administration on 5th day of life. In adulthood, female rats were bred with control male rats. Male puppies were sacrificed on 12th week of life and epididymal (EP) and subcutaneous (SC) adipose fat pads were excised and weighted. Adipocytes were isolated and evaluated for basal and insulin-stimulated 2-deoxyglucose uptake, oxidation of glucose into CO2, and incorporation of glucose into lipids and lipolytic capacity. Key findings Body weight, EP fat pad weight and diameter of adipocytes from offspring of diabetic mothers were increased in comparison to offspring of control mothers. EP adipocytes from offspring of diabetic mothers presented increased basal and insulin stimulated glucose uptake in comparison to control ones. Similar pattern was observed for glucose oxidation into CO2 and incorporation into lipids. However, significant difference in lipolytic capacity in vitro was not observed. Protein content of GLUT4, insulin receptor and acetyl-CoA carboxylase was significantly increased in EP fat pad of offspring of diabetic mothers in relation to control group. Significance Metabolic programming occurred in the adipose tissue of offspring of diabetic mothers, increasing its capacity to store lipids with no changes in lipolytic capacity.

Biological rhythms, besides being under melatonin control, are also controlled by genetic mechanisms, which inform to neuronal and peripheral structures about circadian rhythms. Circadian system ar...

We investigated whether palmitoleic acid, a fatty acid that enhances whole body glucose disposal and suppresses hepatic steatosis, modulates triacylglycerol (TAG) metabolism in adipocytes. For this, both differentiated 3T3-L1 cells treated with either palmitoleic acid (16:1n7, 200 μM) or palmitic acid (16:0, 200 μM) for 24 h and primary adipocytes from wild-type or PPARα-deficient mice treated with 16:1n7 (300 mg·kg−1·day−1) or oleic acid (18:1n9, 300 mg·kg−1·day−1) by gavage for 10 days were evaluated for lipolysis, TAG, and glycerol 3-phosphate synthesis and gene and protein expression profile. Treatment of differentiated 3T3-L1 cells with 16:1n7, but not 16:0, increased basal and isoproterenol-stimulated lipolysis, mRNA levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) and protein content of ATGL and pSer660-HSL. Such increase in lipolysis induced by 16:1n7, which can be prevented by pharmacological inhibition of PPARα, was associated with higher rates of PPARα binding to DNA. In contrast to lipolysis, both 16:1n7 and 16:0 increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose without affecting glyceroneogenesis and glycerokinase expression. Corroborating in vitro findings, treatment of wild-type but not PPARα-deficient mice with 16:1n7 increased primary adipocyte basal and stimulated lipolysis and ATGL and HSL mRNA levels. In contrast to lipolysis, however, 16:1n7 treatment increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose in both wild-type and PPARα-deficient mice. In conclusion, palmitoleic acid increases adipocyte lipolysis and lipases by a mechanism that requires a functional PPARα.