Different metabolic responses of human brown adipose tissue to activation by cold and insulin

We investigated the metabolism of human brown adipose tissue (BAT) in healthy subjects by determining its cold-induced and insulin-stimulated glucose uptake and blood flow (perfusion) using positron emission tomography (PET) combined with computed tomography (CT). Second, we assessed gene expression in human BAT and white adipose tissue (WAT). Glucose uptake was induced 12-fold in BAT by cold, accompanied by doubling of perfusion. We found a positive association between whole-body energy expenditure and BAT perfusion. Insulin enhanced glucose uptake 5-fold in BAT independently of its perfusion, while the effect on WAT was weaker. The gene expression level of insulin-sensitive glucose transporter GLUT4 was also higher in BAT as compared to WAT. In conclusion, BAT appears to be differently activated by insulin and cold; in response to insulin, BAT displays high glucose uptake without increased perfusion, but when activated by cold, it dissipates energy in a perfusion-dependent manner.     

Eicosapentaenoic acid regulates brown adipose tissue metabolism in high-fat-fed mice and in clonal brown adipocytes

Brown adipose tissue (BAT) plays a key role in energy expenditure through its specialized thermogenic function. Therefore, BAT activation may help prevent and/or treat obesity. Interestingly, subcutaneous white adipose tissue (WAT) also has the ability to differentiate into brown-like adipocytes and may potentially contribute to increased thermogenesis. We have previously reported that eicosapentaenoic acid (EPA) reduces high-fat (HF)-diet-induced obesity and insulin resistance in mice. Whether BAT mediates some of these beneficial effects of EPA has not been determined. We hypothesized that EPA activates BAT thermogenic program, contributing to its antiobesity effects. BAT and WAT were harvested from B6 male mice fed HF diets supplemented with or without EPA. HIB 1B clonal brown adipocytes treated with or without EPA were also used. Gene and protein expressions were measured in adipose tissues and H1B 1B cells by quantitative polymerase chain reaction and immunoblotting, respectively. Our results show that BAT from EPA-supplemented mice expressed significantly higher levels of thermogenic genes such as PRDM16 and PGC1α and higher levels of uncoupling protein 1 compared to HF-fed mice. By contrast, both WATs (subcutaneous and visceral) had undetectable levels of these markers with no up regulation by EPA. HIB 1B cells treated with EPA showed significantly higher mRNA expression of PGC1α and SIRT2. EPA treatment significantly increased maximum oxidative and peak glycolytic metabolism in H1B 1B cells. Our results demonstrate a novel and promising role for EPA in preventing obesity via activation of BAT, adding to its known beneficial anti-inflammatory effects.     

A study of GDP binding to purified thermogenin protein from brown adipose tissue.

1. The binding of GDP to purified thermogenin protein was studied by using fluorescence spectroscopy and equilibrium dialysis. 2. GDP binding to thermogenin diminished fluorescence emission in a concentration-dependent manner that exhibited saturation. 3. Kd values for binding of nucleoside di- and tri-phosphates were lower than those for nucleoside monophosphates. 4. The GDP-induced fluorescence quenching was decreased by increasing pH, but the apparent Kd was unaltered by pH changes. 5. Equilibrium dialysis showed a Kd change from 3 to 6 microM when the pH was increased from 6.6 to 8.5. 6. The apparent pK of the fluorescence changes induced by pH (8.3) was identical with the apparent pK of the GDP-binding response. 7. The data are consistent with the existence of protonated and non-protonated forms of thermogenin protein that both bind GDP.     

Mfn2 deletion in brown adipose tissue protects from insulin resistance and impairs thermogenesis

BAT‐controlled thermogenic activity is thought to be required for its capacity to prevent the development of insulin resistance. This hypothesis predicts that mediators of thermogenesis may help prevent diet‐induced insulin resistance. We report that the mitochondrial fusion protein Mitofusin 2 (Mfn2) in BAT is essential for cold‐stimulated thermogenesis, but promotes insulin resistance in obese mice. Mfn2 deletion in mice through Ucp1‐cre (BAT‐Mfn2‐KO) causes BAT lipohypertrophy and cold intolerance. Surprisingly however, deletion of Mfn2 in mice fed a high fat diet (HFD) results in improved insulin sensitivity and resistance to obesity, while impaired cold‐stimulated thermogenesis is maintained. Improvement in insulin sensitivity is associated with a gender‐specific remodeling of BAT mitochondrial function. In females, BAT mitochondria increase their efficiency for ATP‐synthesizing fat oxidation, whereas in BAT from males, complex I‐driven respiration is decreased and glycolytic capacity is increased. Thus, BAT adaptation to obesity is regulated by Mfn2 and with BAT‐Mfn2 absent, BAT contribution to prevention of insulin resistance is independent and inversely correlated to whole‐body cold‐stimulated thermogenesis.     

A novel preadipocyte cell line established from mouse adult mature adipocytes

We have established a novel preadipocyte cell line from mouse adult mature adipocytes. The mature adipocytes were isolated from fat tissues by taking only the floating population of mature fat cells. The isolated mature adipocytes were de-differentiated into fibroblast-like cells. The in vitro studies showed that the cells could re-differentiate into mature adipocytes after over 20 passages. The in vivo transplantation study also demonstrated that the cells had the full potential to differentiate into mature adipocytes, which has not been shown for the 3T3-L1 preadipocyte cell line derived from mouse embryo. We have further analyzed the expression profile of key fat regulatory genes such as the peroxisome proliferator-activated receptorgamma or CCAAT/enhancer-binding protein gene families. We conclude that our cell line could be used as a preferred alternative to 3T3-L1, potentially reflecting the characteristics of mature adipocytes more, since the cell line is actually derived from adult mature adipocytes.     

Altered effect of insulin and catecholamines in brown adipose tissue of cold-acclimated rats.

Data are presented indicating that in brown adipose tissue (BAT) of cold-acclimated (CA), but not cold-exposed (CE) rats, there was an alteration in the relative response to catecholamines and insulin as evidenced by increased binding of alprenolol and decreased binding of insulin to plasma membrane enriched fractions. In addition, the stimulatory effect of insulin on glucose incorporation into glycogen and its inhibitory action on adenylate cyclase activity were both blunted in the CA tissues. It is proposed that shifts in the capacity of BAT to respond to catecholamines and insulin may be involved in the mechanism of cold acclimation.     

Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue

We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro. This work was supported by grants from the Japan Ministry of Education, Science, Sports, and Culture (no. 19580348) and from MEXT. HAITEKU (2007–2011).     

Differentiation of rapid and slower-acting effects of insulin on mitochondrial processes in brown adipose tissue from streptozotocin-diabetic rats.

Insulin treatment of streptozotocin-diabetic rats restores the depressed palmitoyl-group oxidation observed in brown-adipose-tissue mitochondria from diabetic rats. A relatively rapid effect of insulin (5 h) to increase carnitine-dependent oxidation of palmitoyl-CoA and to increase overt carnitine palmitoyltransferase activity is differentiated from a slower effect of the hormone (1 day) to increase palmitoylcarnitine oxidation.     

DNA synthesis in mouse brown adipose tissue is under beta-adrenergic control

The rate of DNA synthesis in mouse brown adipose tissue was followed with injections of [3H]thymidine. Cold exposure led to a large increase in the rate of [3H]thymidine incorporation, reaching a maximum after 8 days, whereafter the activity abruptly ceased. A series of norepinephrine injections was in itself able to increase [3H]thymidine incorporation. When norepinephrine was injected in combination with the alpha-adrenergic antagonist phentolamine or with the beta-adrenergic antagonist propranolol, the stimulation was fully blocked by propranolol. It is suggested that stimulation of DNA synthesis in brown adipose tissue is a beta-adrenergically mediated process and that the tissue is an interesting model for studies of physiological control of DNA synthesis.     

Lack of TRPV2 impairs thermogenesis in mouse brown adipose tissue

Brown adipose tissue (BAT), a major site for mammalian non‐shivering thermogenesis, could be a target for prevention and treatment of human obesity. Transient receptor potential vanilloid 2 (TRPV2), a Ca²⁺‐permeable non‐selective cation channel, plays vital roles in the regulation of various cellular functions. Here, we show that TRPV2 is expressed in brown adipocytes and that mRNA levels of thermogenic genes are reduced in both cultured brown adipocytes and BAT from TRPV2 knockout (TRPV2KO) mice. The induction of thermogenic genes in response to β‐adrenergic receptor stimulation is also decreased in TRPV2KO brown adipocytes and suppressed by reduced intracellular Ca²⁺ concentrations in wild‐type brown adipocytes. In addition, TRPV2KO mice have more white adipose tissue and larger brown adipocytes and show cold intolerance, and lower BAT temperature increases in response to β‐adrenergic receptor stimulation. Furthermore, TRPV2KO mice have increased body weight and fat upon high‐fat‐diet treatment. Based on these findings, we conclude that TRPV2 has a role in BAT thermogenesis and could be a target for human obesity therapy.     

Interaction between heat acclimation and exogenous insulin in brown adipose tissue of rats

Seventy-one male Wistar strain rats (7 weeks old) were kept at 5, 25, or 34° C, respectively, for 2 weeks with or without insulin administration. Insulin (Novo Lente MC) was given subcutaneously in a dose of 3.62 nmol/125 µl saline per 100 g body weight. An apparent effect of insulin treatment was noted only in heat-exposed rats, resulting in a remarkable gain in inter-scapular brown adipose tissue (BAT) mass of heat-acclimated, insulin-treated rats in terms of weight or weight per unit body weight. The BAT from heat-acclimated, insulin-treated rats had significantly higher levels of protein, DNA, RNA, and triglyceride than BAT from heat-acclimated, saline-treated rats. Therefore, it seems likely that the growth of BAT in heat-acclimated, insulin-treated rats was mostly due to the anabolic effects of insulin. The uncoupling protein mRNA was, however, present in BAT of heat-acclimated, insulin-treated rats at rather a depressed level, explaining a corresponding decrease in cold tolerance. On the other hand, the expression of insulin receptor mRNA was attenuated in BAT of rats from all the insulin-treated groups, possibly due to the down-regulation of insulin. Thus, there appeared to be some linkage among BAT, heat acclimation, and insulin.     

Protein kinases and their substrates in brown adipose tissue from newborn rats.

The 10000 X g supernatant fraction of brown fat from newborn rats catalyzed the cyclic AMP-dependent phosphorylation of both histone and a preparation of proteins from the same subcellular fraction (endogenous proteins). The apparent affinity for ATP was lower for the phosphorylation of the endogenous proteins than for the phosphorylation of histone. In order to discover whether the phosphorylation of histone and the endogenous proteins were catalyzed by different enzymes, the 100000 X g supernatant was fractionated by ion-exchange and adsorption chromatography. Three different cyclic AMP-dependent protein kinases and one cyclic AMP-independent protein kinase were separated and partially purified. Each of these enzymes catalyzed the phosphorylation of both substrates, and the difference in apparent Km for ATP remained. Neither affinity chromatography on histone-Sepharose, nor electrophoresis on polyacrylamide gels resulted in the separation of the phosphorylation of histone from that of the endogenous proteins of any of the partially purified kinases. Moreover, experiments in which the phosphorylated substrates were separated by differential precipitation with trichloroacetic acid showed that the endogenous proteins competitively inhibited the phosphorylation of lysine-rich histone. It is concluded that each of the partially purified kinase preparations contains protein kinase, which catalyzes the phosphorylation of both substrates. The difference in apparent Km for ATP was found to be due to the presence in the endogenous protein preparation of a low molecular weight compound which competes with ATP. This was not ATP nor the modulator protein. The ratio of the phosphorylation of endogenous proteins to that of histone was much higher for the cyclic AMP-independent kinase preparation than for the other enzymes. Electrophoresis of the endogenous substrates in the presence of sodium dodecyl sulphate showed that the enzyme phosphorylated a greater number of proteins than did the cyclic AMP-dependent kinases. The phosphorylation of endogenous proteins relative to that of histone was significantly lower for one of the cyclic AMP-dependent kinases than for the other two. This difference was not reflected in a different pattern of phosphorylation of the individual proteins of the endogenous mixture.