A Study Of The Impact Of Low Temperature-Induced Visceral Fat Browning On Murine Metabolism And Potential Mechanisms

BackgroundThe prevalence of obesity has reached pandemic proportions, and the population who suffered from obesity is still rising. Metabolic disorders, including insulin resistance, diabetes, dyslipidemia, and cardiovascular diseases caused by the excess storage and dysfunction of fat are putting public health under threat. Adipose tissue is closely associated with the pathology of obesity and its various complications.Two types of adipose tissue exist, white and brown. White adipose tissue serves as an energy reservoir in the traditional point of view, now it is also recognized as an endocrine organ since it secretes various adipokines to regulate whole body metabolism. The main function of brown adipose tissue is dissipating heat to maintain the core body temperature. The mitochondria within the brown adipose tissue are rich in uncoupling protein 1, which can dissipate chemical energy as heat through uncoupling oxidative respiration and ATP synthesis. The main location of white adipose tissue is intrabdominal and subcutaneous. White adipocytes are characterized by an unilocular large lipid droplet with little cytoplasm. Brown adipose tissue locates in the interscapular region of rodents and newborns. The brown adipocyte contains multilocular lipid droplets and abundant mitochondria. Environmental cues such as cold and PPARy agonists lead to the browning of white adipose tissue, meaning beige adipocytes appear within the depot. Beige adipocytes resemble brown adipocytes in appearance for the multilocular lipid droplets. However, UCP1 expression in beige adipocytes depends on stimulation, which makes them differ from classic brown adipocytes. UCP1 expression levels in fully activated beige adipocytes are similar to that of brown adipocytes. As a result, beige adipocytes are also able to generate heat via mitochondria uncoupling. That’s why brown and beige adipocytes are called thermogenic adipocytes. Brown adipocytes were believed to no longer exist in adult human until 2009. Functional brown adipose were identified in adult human with 18F-PET/CT scanning. Now this field is replenished with recognizable beige adipocytes in human brown adipose tissue, confirming beige fat exists in adults.Activated sympathetic nervous system by cold can release catacholamine, which binds to β3 adrenoceptor then leading to the activation of Gs-cAMP-PKA pathway and downstream Perilipin, hormone-sensitive lipase and adipose triglyceride lipase. The result is increased lipolysis and great amount of released free fatty acids. Both brown and beige adipocytes can take up free fatty acids either from hydrolysis of their stored lipid or the circulation to oxidation and UCP1-mediated heat production. Besides, activated beige and brown adipocytes are also able to take up glucose from the circulation, thus may increase insulin sensitivity. Based on these, we speculate modulation of white adipose tissue browning and brown adipose tissue can bring benefits to metabolism.Although visceral fat and subcutaneous fat all belong to white adipose tissue, the accumulation of visceral fat is more closely associated with metabolic diseases. The sensitivity of browning is depot specific. Under cold acclimation, subcutaneous adipose tissue is more likely turn brown than epididymal adipose tissue. The browning resistance of epididymal adipose tissue has earned the name as pure white adipose tissue. However, previous cold experiments were mainly conducted around 4℃, whether lower temperature can enhance eWAT browning is unknown. This thesis aims to determine the browning of eWAT along with subcWAT and BAT under different cold conditions in normal and high fat diet-induced obese mice.Objectives1. To elucidate the impact of cold acclimation on various adipose depots in normal mice, especially visceral adipose tissue;2. To examine the impact of cold acclimation on various adipose depots in high fat diet-induced obese mice, especially visceral adipose tissueMethodsAnimalAll animal studies were approved by the Local Ethical Committee of Shandong University Qilu Hospitaland complied with the Management Rules of the Chinese Ministry of Health. Male 7-to 8-week-old C57BL/6 mice were purchased from the Beijing HFK Bioscience Co.Part Ⅰ Cold acclimation for normal miceThe cold facilities were designed for a 12 h-light and 12 h-dark rhythm. Before cold exposure, all mice were adapted at 18℃ for 1 week, then transferred to 4℃ for another week. One group remained at 4℃ for the entire experimental duration. Other groups of mice were kept at -5℃ during daytime (12 h) and at 4℃ during nighttime (12 h) for 3 consecutive days. Mice were then exposed to -10℃ for 12 h during daytime and to 4℃ for 12 h during nighttime for the rest of the week. One group was further maintained at -10℃ for 24 h for 3 weeks. The -10℃/-20℃ group was transferred to -20℃ for 12 h daytime and -10℃ for 12 h nighttime during the next 3 weeks. For -10℃- and -10℃/-20℃ -exposed animals, fresh drinking water was frequently changed. Extra snow and frost was put in the cage floor to make sure mice had free access to moisture. For thermoneutrality, mice were directly transferred to 30℃ and maintained at this temperature for the duration equivalent to cold exposure.Part Ⅱ High fat diet-induced obese mice experimentMale,7-8-week-old C57BL/6 mice were fed a high fat diet containing 60% fat, after 4 months they were randomly divided into 30℃,4℃,-10℃ and -10℃/-20℃. The cold acclimation was the same as part Ⅰ.Measurement of core body temperature and blood pressureCore body temperature was measured via a rectal temperature probe thermometer between 10:00 and 12:00 during the daytime. Tail artery blood pressure was measured via small animal blood pressure meter through a cuff method.Micro-PET imagingMice fasted for about 3 h and were injected with 200～300 μCi 18F-FDG through the tail vein. After 1 h, adipose depots were quickly dissected and scanned by the Inveon Dedicated PET System.Tissue sample collectionMice were starved for 6 to 8 h. Blood samples were collected through the cardiac apex of pentobarbital-anesthetized animals. After sacrifice, various adipose depots and organs were quickly dissected. Some tissues were kept in liquid nitrogen and stored at -80℃ until further extraction of RNA and protein. A fraction of tissues was fixed in 4% paraformaldehyde (PFA) for future histological analysis.Measurement of lipolysis and serum leptinTo assess lipolysis, eWAT was surgically dissected and were incubated with or without 10 mM isoproterenol. The released glycerol was measured. Serum leptin levels were measured using commercial ELISA kits.Histology and immunohistochemistryHematoxylin and eosin staining was performed according to standard protocol. Adipose tissue slides were stained with UCP1, Prohibitin, Perilipin A and Endomucin.Gene microarray analysis, qRT-PCR and Western BlotTotal RNA was extracted from eWAT in triplicates. Affymetrix Gene Chip Mouse Exon 1.0 ST Array was used. Total RNA was extracted from various adipose tissues according to the manufacturer’s instruction. qPCR was used to assess relative mRNA expression levels of Ucpl, Dio2, Cidea, Cox7al, Leptin, Adiponectin, Irisin, Resistin, Pgcla, Prdm16, and Ebf2. Total proteins were extracted from adipose tissues and UCP1 expression levels were examined.Statistical AnalysisAll data were presented as the mean ± SEM. Data analysis involved unpaired Student’s t test, one-way ANOVA and two-way ANOVA. A p<0.05 was considered statistically significant.ResultsThe impact of cold acclimation on food intake, body weight, BMI, adipose tissue weight, core body temperature, and blood pressure in normal miceThe body weight and BMI were decreased with reducing temperature relative to the thermoneutral condition although food intake was markedly increased. eWAT, subcutaneous WAT and iBAT mass were all reduced in cold conditions. eWAT and subcutaneous WAT mass was reduced with droping temperature. The core body temperature and blood pressure were similar between all groups.The impact of cold acclimation on browning of different adipose depots in normal miceHE staining and lipid droplet membrane protein Perilipin A staining of different visceral adipose depots including eWAT, rWAT, and mWAT revealed that adipocytes sizes were reduced and multilocular lipid droplets began to appear. UCPl staining was confirmed positive. Along with reducing temperatures, mitochondria marker prohibitin and microvessel marker endomucin staining was increased. Cold acclimation also increased UCP1, prohibitin and endomucin staining in subcWAT and iBAT. These markers either rose in subcWAT or remain undistinguishable in iBAT with reducing temperature. Furthermore, qPCR and WB analysis confirmed that cold promotes UCPl expression.Gene microarray analysis and qPCRMicroarray analysis showed that cold caused changes of many metabolic pathways and promoted lipolysis-related gene expression. qPCR of eWAT and subcWAT showed that cold increased expression of Dio2, Cidea and Cox7al, decreased expression of adipokine Leptin, Adiponectin, and Irisim, while Resistin remain unaltered. Transcription factors Pgcla were mildly altered, while Prdml6 and Ebf2 either decreased or unchanged. Except Dio2, cold significantly decreased expression levels of Cidea, Cox7al, Leptin, Adiponectin, Irisin, Resistin, Pgcla, Prdml6 and Ebf2. These results indicate that cold may exerts different effects on different adipose depots.The impact of cold acclimation on food intake, body weight, BM I, adipose tissue weight in high fat diet-induced obese miceThe body weight and BMI were decreased with reducing temperature relative to the thermoneutral condition although food intake was markedly increased. eWAT, subcutaneous WAT and iBAT mass were all reduced in cold conditions. eWAT and subcutaneous WAT mass was reduced with droping temperature while iBAT remain similar between all cold groups.The impact of cold acclimation on browning of different adipose depots in high fat diet-induced obese miceHE staining and lipid droplet membrane protein Perilipin A immunohistochemical staining of different visceral adipose depots including eWAT, rWAT, and mWAT revealed that adipocytes sizes were reduced and multilocular lipid droplets began to appear. Positive UCP1 staining was confirmed. Along with reducing temperatures, mitochondria marker prohibitin and microvessel marker endomucin staining was increased. Cold acclimation also increased UCP1, prohibitin and endomucin staining in subcWAT and iBAT. These markers either increased in subcWAT or remain undistinguishable in iBAT with reducing temperature. Furthermore, qPCR and WB analysis confirmed that cold promotes UCP1 expression.The impact of cold acclimation on adipose tissue mRNA levels of thermogenic genes, adipokines and transcriptional factors in high fat diet-induced obese miceqPCR of eWAT and subcWAT showed that cold increased expression of Dio2, Cidea and Cox7al, decreased expression of adipokine Leptin, Adiponectin, and Irisin. Transcription factors Pgcla were mildly upregulated, while Prdml6 and Ebf2 decreased. Cold significantly decreased expression levels of Cidea, Cox7al, Leptin, Adiponectin, Irisin, Resistin, Pgc1α, Prdm16 and Ebf2 in iBAT. These results indicated that cold may exerts different effects on different adipose depots.The impact of cold acclimation on lipolysisIn vitro analysis of glycerol released from eWAT with or without isoproterenol stimulation showed elevated lipolysis during cold acclimation in both normal and obese mice.Conclusions1. Cold can predispose normal and diet-induced obese mice to smaller adipose depots despite of increased food intake;2. Cold promotes browning of visceral adipose tissue, subcutaneous adipose tissue and activates brown adipose tissue in normal mice. Visceral fat is less sensitive to cold-induced browning than subcutaneous fat;3. Cold promotes browning of visceral adipose tissue, subcutaneous adipose tissue and activates brown adipose tissue in high fat diet-induced mice. The obese adipose tissue is less sensitive to cold-induced browning than normal lean fat;4. Cold can change expression profile in visceral fat, resulting in enhanced lipolysis-associated genes expression and accelerated lipolysis;5. Cold can modulate mRNA levels of thermogenic genes, adipokines and browning-related transcription factors in different adipose depots, and the regulatory effect is depot-specific.BackgroundThe world is facing exploding obesity crisis, with a population larger than 1.9 billion being overweight or obese. The health problems caused by obesity and insulin resistance, type 2 diabetes, dyslipidemia, cardiovascular disease and fatty liver have put a clear threat to the society. On the contrary, little progress has been made on interventions effective to defend weight gain. The occurrence of obesity and its complications is closely associated with adipose tissue. When energy intake exceeds expenditure, adipose tissue stores the extra energy as fat. Excess expansion of adipose tissue leads to metabolic disorders.Adipose tissue is able to store and release lipid in quick response to metabolic changes, thus its physiologic functions is closely related with whole body metabolism. Adipocytes dysfunction is the pathophysiologic basis of various metabolic disorders, including obesity. Three kinds of adipocytes exist within the body, white, brown and beige. They differ in origin, morphology, function, and location. White adipocytes are composed of a unilocular lipid droplet in the center and a thin layer of cytoplasm at the periphery. Their main function is storage of triglycerides and secretion of adipokines. Brown adipocytes contain multilocular lipid droplets and lots of mitochondria, and their main function is producing heat through substrate oxidation and UCP1 mediated uncoupling. Beige adipocytes resemble the brown morphology and are also called inducible brown adipocytes for their ability to express UCP1 and dissipate heat in response to external stimulation. Factors like catecholamines, FGF-21 and irisin are able to induce browning of white adipose tissue, meaning beige adipocytes appear in white adipose tissue.Beige adipocytes have been proven to play an important role in mediating energy homeostasis. Research showed that obese patients have less brown and beige fat, and impaired heat production capacity upon cold stimulation. Obese resistant Sv129 mice disclosed elevated beige adipocyte contents compared with data from C57BL/6J mice, which predisposes the latter strain to diet-induce obesity. Knockout type 1A BMP receptor in all Myf5+-derived cells causes brown adipose tissue paucity, and white adipose tissue undergoes browning to compensate the heat production deficiency. Adipocyte-specific PRDM16 knockout suppresses white adipose tissue browning with little influence on brown adipose tissue, leading to aggravated obesity, insulin resistance and fatty liver disease by high fat diet. Based on these, we infer that white fat browning can improve whole body metabolism.Insulin resistance (IR) caused by obesity is a main contributor to the epidemic of type 2 diabetes mellitus. Dysmetabolism occurred in the excessive fat results in lipid overspill, and ectopic fat accumulates in liver or muscle, this is often referred as lipotoxicity. Adipocytes can secrete a variety of adipokines, which trigger inflammation thus promotes insulin resistance and dyslipidemia. Adipose tissue, especially visceral fat whose blood drained directly into the liver, can regulate hepatocytes metabolism and inflammation through secreting adipokines such as adiponectin, leptin, IL-6, IL-1β, and saturated or unsaturated free fatty acids. Expansion of fat eventually leads to metabolic disorders in liver, promotes the pathogenesis of nonalcoholic fatty liver (NAFLD). People living at high altitude have lower fasting blood glucose and better glucose tolerance compared to those live near the sea level. Besides, the prevalence of obesity and diabetes in people at high altitude is lower. But the reason remained unclear. We speculate that the differences may be related to activation of adipose tissue under lower environmental temperature. Shah RV et al. found that regardless of BMI, accumulation of visceral adipose tissue is associated with cardiovascular risk factors and coronary artery calcification. Compared with subcutaneous fat, visceral fat has a stronger correlation with the incidence of metabolic syndrome. In 2012 JAMA published results from a large scale Dallas Heart Study, claiming that visceral fat mass in obese patients is closely associated with the degree of insulin resistance and future risk in developing type 2 diabetes. Another study found that NAFLD incidence is lower in BAT positive population than in those whose BAT was undetectable. Based on these we conclude that adipose tissue, especially visceral adipose tissue plays an important role in the pathogenesis of IR and NAFLD. Interventions targeted visceral adipose tissue may attenuate these complications.This thesis aims to study the impact of low temperature induced visceral fat browning on whole body metabolism, including metabolic thermogenesis, lipiS metabolism, insulin sensitivity, and liver lipid deposition in normal fed and high fat diet-induced obese mice and to explore potential mechanisms.Objectives1. To study the impact of cold acclimation on whole body metabolism in normal and high fat diet-induced obese mice;2. To explore the potential mechanisms underlying visceral adipose tissue browning induced metabolic improvements.MethodsAnimalMale 7-to 8-week-old C57BL/6 mice were purchased from the Beijing HFK Bioscience Co. The UCPl knockout mice were from Jackson Laboratory. All animal studies complied with the Management Rules of the Chinese Ministry of Health and were approved by the Local Ethical Committee of Qilu Hospital, Shandong University.Part Ⅰ Cold acclimation of normal fed mice, reference paper I.Part Ⅱ Cold acclimation of high fat diet-induced obese mice, reference paper I.Part Ⅲ eWAT removal and transplantation experimentBefore 18℃ adaptation, eWAT was surgically removed as described. For the sham operation, the abdominal cavity was incised and eWAT was mobilized, but not excised. Both groups underwent the cold acclimation as described above. For the transplantation part, mice living at thermoneutrality were given equal amount of beige eWAT or thermoneutral eWAT, non-shivering thermogenesis were recorded afterwards.Part Ⅳ UCP1-/- mice experimentUCP1-/- mice were challenged with cold.Part Ⅴ P3 adrenoceptor agonist experimentC57BL/6 mice maintained at room temperature were randomly divided into two groups, one group was given β3 adrenoceptor agonist CL316243, another group received equal amount of normal saline.Measurement of blood glucose, glucose tolerant test and insulin tolerant testBlood glucose strips and the Accu-Chek glucose meter were used to measure fasting glucose and to monitor blood glucose levels during insulin and glucose tolerance tests. Prior to insulin tolerance test, all mice fasted for 6 h, then were intraperitoneally (i.p.) injected with 0.5 U human insulin/kg body weight for healthy lean mice and 0.75 U human insulin/kg body weight for obese mice. Blood glucose levels were determined before injection and at 15,30,60,90 and 120 min after the injection. For the glucose tolerance test, all mice fasted for 12 h and were i.p. injected with 2 g glucose/kg body weight. Blood glucose levels were determined before and at 15,30,60,90 and 120 min after the injection.Indirect calorimetryAnimals were anaesthetized and oxygen consumption as well as carbon dioxide release were measured for 30 min at 33℃. After measuring the basal metabolic rate, mice were injected with norepinephrine (1 mg/kg, s.c). O2 consumption and CO2 release were recorded for the following 90 min.eWAT removal and transplantationMice were fixed after anesthesia, surgical operation was conducted through a median abdominal incision, after that epididymal fat was exposed and removed through blunt separation. The abdominal cavity was opened and epididymal adipose tissue was mobilized, but not excised in the sham group. For the transplantation experiment, eWAT from the donor mice was quickly dissected, weighed and washed by normal saline for several times. Then the minced fat pieces were bedded in the folds of recipient mice eWAT.Mice genotyping and core body temperature measurementDNA was extracted from mice tail, agarose gel electrophoresis was performed after PCR amplification. The knockout genotype was determined according to fragment sizes. Core body temperature was measured via a rectal temperature probe thermometer between 10:00 and 12:00 during the daytime.Tissue sample collectionmice were starved for 6 to 8 h. Blood samples were collected through the cardiac apex of pentobarbital-anesthetized animals. After sacrifice, various adipose depot11 and organs were quickly dissected. Some tissues were kept in liquid nitrogen and stored at -80℃ until further extraction of RNA and protein. A fraction of tissues was fixed in 4% paraformaldehyde (PFA) for future histological analysis.Measurement of blood lipid and serum insulinBlood levels of total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C) and non-esterified fatty acid (NEFA) were measured. Fasting serum insulin was measured using commercial ELIS A kits.Measurement of creatine kinase, creatine and creatine phosphate in eWATeWAT exposed from various temperatures were homogenized on ice to examine the creatine kinase activity, creatine and creatine phosphate contents.Histology and immunohistochemistryHematoxylin and eosin staining was performed according to standard protocol. Adipose tissue slides were stained with UCP1, Prohibitin, Perilipin A and Endomucin. Liver cryosections were stained with oil red O.qRT-PCRTotal RNA was extracted from eWAT according to the manufacturer’s instruction. qPCR was used to assess relative mRNA expression levels of Ucpl, Dio2, Cidea, Cox7al, Leptin, Adiponectin, Irisin, Resistin, Pgcla, Prdm16, and Ebf2.Statistical AnalysisAll data were presented as the mean ± SEM. Data analysis involved unpaired Student’s t test, one-way ANOVA and two-way ANOVA as appropriate. A P<0.05 was considered statistically significant.ResultsThe impact of cold acclimation on normal mice metabolismNST showed markedly elevated oxygen consumption and carbon dioxide during cold acclimation, with -10℃ and -10℃/-20℃ group higher than 4℃ group, which indicates low temperature accelerates metabolism and adaptive thermogenesis. Blood TG, NEFA and LDL-C were markedly decreased during cold acclimation. Blood TG was significantly decreased with reducing temperatures. TC and HDL-C showed tendency to decrease with reducing temperature, while -10℃/-20℃ group is significantly lower than all other groups. Fasting blood glucose levels showed no differences between all groups, while fasting insulin level in -10℃/-20℃ group is lower than other groups. Glucose tolerance test and insulin tolerance test showed blood glucose levels of -10℃ and -10℃/-20℃ group were lower than 30℃ and 4℃ group, and the area under the curve was also smaller. These indicate that cold could improve glucose tolerance and insulin sensitivity in mice.The impact of cold acclimation on metabolism in high fat diet-induced obese miceCold could accelerate the metabolic rate of obese mice, with -10℃ and -10℃/-20℃ group consumpting more oxygen and releasing more carbon dioxide than 4℃ group. Blood TG, TC and LDL-C were markedly decreased during cold acclimation. TG levels were significantly lower in -10℃ and -10℃/-20℃ than 4℃ group. Fasting glucose and insulin levels were decreased during cold conditions. Cold improved glucose tolerance and insulin tolerance of obese mice. Liver weight was reduced during cold acclimation. Lipid droplets content was markedly reduced as measured by oil red o staining with reducing temperature.Cold enhanced creatine-driven substrate cycle in visceral fat, but UCPl-mediated thermogenesis was still the dominant resource of heatCold increased creatine kinase activity in eWAT, the lower the temperature, the higher the activity of creatine kinase. The creatine content was increased and creatine phosphate content decreased in eWAT during cold, with the highest creatine and lowest creatine phosphate content in -10℃/-20℃ group, indicating that the CK/Cr/PCr pathway was activated. However, UCP1-/- mice showed reduced core body temperatures during cold adaptation, and they cannot survive freezing environment due to insufficient thermogenesis. These proved the importance of UCPl-mediated thermogenesis in defending extreme cold.The impact of activated eWAT on whole body metabolismThe mortality rate of mice lacking eWAT (eWL) during cold acclimation is higher compared with Sham-operated group. The NST capacity and insulin sensitivity were also impaired in eWL compared with Sham. Compared with inactive eWAT, transplantation of active eWAT enhanced mice NST capacity.The impact of P3 adrenoceptor agonist CL316243 on adipose tissue and metabolismCL treatment successfully mimicked the effect of cold on adipose tissue. Compared with control, CL treatment reduced body weight, BMI and adipose tissue mass. Histology analysis of eWAT, subcWAT and iBAT showed decreased adipocyte sizes, increased UCP1, mitochondria prohibitin and vessel endomucin staining. CL treatment promoted thermogenic genes expression in eWAT while reduced adipokines and several browning related transcription factors mRNA levels. Moreover, CL treatment significantly reduced blood TG, TC and LDL-C levels as well as increased insulin sensitivity.Conclusions1. Low temperature induced visceral fat browning can enhance metabolism and NST capacity in normal and high fat diet induced mice;2. Cold decreases blood TG levels, improves dyslipidemia, glucose tolerance and insulin tolerance in normal and high fat diet-induced obese mice. Cold also reduces liver lipid deposition in obese mice;3. Beige visceral fat can use both UCP1-mediated and creatine-driven substrate cycle for thermogenesis;4. Cold may act through β3 adrenoceptor to induce browning of visceral fat and regulate metabolism.