Temperature Changes in Brown Adipocytes Detected with a Bimaterial Microcantilever

Mammalian cells must produce heat to maintain body temperature and support other biological activities. Methods to measure a cell’s thermogenic ability by inserting a thermometer into the cell or measuring the rate of oxygen consumption in a closed vessel can disturb its natural state. Here, we developed a noninvasive system for measuring a cell’s heat production with a bimaterial microcantilever. This method is suitable for investigating the heat-generating properties of cells in their native state, because changes in cell temperature can be measured from the bending of the microcantilever, without damaging the cell and restricting its supply of dissolved oxygen. Thus, we were able to measure increases in cell temperature of <1 K in a small number of murine brown adipocytes (n = 4–7 cells) stimulated with norepinephrine, and observed a slow increase in temperature over several hours. This long-term heat production suggests that, in addition to converting fatty acids into heat energy, brown adipocytes may also adjust protein expression to raise their own temperature, to generate more heat. We expect this bimaterial microcantilever system to prove useful for determining a cell’s state by measuring thermal characteristics.     

Direct Evidence of Brown Adipocytes in Different Fat Depots in Children

Recent studies suggested the persistence of brown adipocytes in adult humans, as opposed to being exclusively present in infancy. In this study, we investigated the presence of brown-like adipocytes in adipose tissue (AT) samples of children and adolescents aged 0 to 18 years and evaluated the association with age, location, and obesity. For this, we analysed AT samples from 131 children and 23 adults by histological, immunohistochemical and expression analyses. We detected brown-like and UCP1 positive adipocytes in 10.3% of 87 lean children (aged 0.3 to 10.7 years) and in one overweight infant, whereas we did not find brown adipocytes in obese children or adults. In our samples, the brown-like adipocytes were interspersed within white AT of perirenal, visceral and also subcutaneous depots. Samples with brown-like adipocytes showed an increased expression of UCP1 (>200fold), PRDM16 (2.8fold), PGC1α and CIDEA while other brown/beige selective markers, such as PAT2, P2RX5, ZIC1, LHX8, TMEM26, HOXC9 and TBX1 were not significantly different between UCP1 positive and negative samples. We identified a positive correlation between UCP1 and PRDM16 within UCP1 positive samples, but not with any other brown/beige marker. In addition, we observed significantly increased PRDM16 and PAT2 expression in subcutaneous and visceral AT samples with high UCP1 expression in adults. Our data indicate that brown-like adipocytes are present well beyond infancy in subcutaneous depots of non-obese children. The presence was not restricted to typical perirenal locations, but they were also interspersed within WAT of visceral and subcutaneous depots.     

Calcium-Induced Alteration of Mitochondrial Morphology and Mitochondrial-Endoplasmic Reticulum Contacts in Rat Brown Adipocytes

Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of [Ca²⁺]_i homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ^® treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ^® drinking or tap water (control) drinking for three days. Alizarin Red S staining showed increased Ca²⁺ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ^® decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ^® treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca²⁺ diet affected mitochondrial fusion as mitofusin 1 (MFN1) and mitofusin 2 (MFN2) were increased, and mitochondrial fission as dynamin related protein 1 (DRP1) was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER). The level of uncoupling protein-1 (UCP1) was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ^® stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes.Key words: Brown adipocyte, mitochondrial dynamics, calcium, endoplasmic reticulum     

Proteome Imbalance of Mitochondrial Electron Transport Chain in Brown Adipocytes Leads to Metabolic Benefits

1. Download high-res image (171KB)
2. Download full-size image

     

H-ras Induces Glucose Uptake in Brown Adipocytes in an Insulin- and Phosphatidylinositol 3-Kinase-Independent Manner

Fetal brown adipocytes (parental cells) expressed mainly Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being much lower. At physiological doses, insulin stimulation for 15 min increased 3-fold glucose uptake and doubled the amount of Glut4 protein located at the plasma membrane. Moreover, phosphatidylinositol (PI) 3-kinase activity was induced by the presence of insulin in those cells, glucose uptake being precluded by PI 3-kinase inhibitors such as wortmannin or LY294002. H-ras^lys12-transformed brown adipocytes showed a 10-fold higher expression of Glut1 mRNA and protein than parental cells, Glut4 gene expression being completely down-regulated. Glucose uptake increased by 10-fold in transformed cells compared to parental cells; this uptake was unaltered in the presence of insulin and/or wortmannin. Transient transfection of parental cells with a dominant form of active Ras increased basal glucose uptake by 5-fold, no further effects being observed in the presence of insulin. However, PI 3-kinase activity (immunoprecipitated with anti-αp85 subunit of PI 3-kinase) remained unaltered in H-ras permanent and transient transfectants. Our results indicate that activated Ras induces brown adipocyte glucose transport in an insulin-independent manner, this induction not involving PI 3-kinase activation.     

Lecithin:Cholesterol Acyltransferase (LCAT) Deficiency Promotes Differentiation of Satellite Cells to Brown Adipocytes in a Cholesterol-dependent Manner

Our laboratory previously reported that lecithin:cholesterol acyltransferase (LCAT) and LDL receptor double knock-out mice (Ldlr^−/−xLcat^−/− or DKO) spontaneously develop functioning ectopic brown adipose tissue (BAT) in skeletal muscle, putatively contributing to protection from the diet-induced obesity phenotype. Here we further investigated their developmental origin and the mechanistic role of LCAT deficiency. Gene profiling of skeletal muscle in DKO newborns and adults revealed a classical lineage. Primary quiescent satellite cells (SC) from chow-fed DKO mice, not in Ldlr^−/−xLcat^+/+ single-knock-out (SKO) or C57BL/6 wild type, were found to (i) express exclusively classical BAT-selective genes, (ii) be primed to express key functional BAT genes, and (iii) exhibit markedly increased ex vivo adipogenic differentiation into brown adipocytes. This gene priming effect was abrogated upon feeding the mice a 2% high cholesterol diet in association with accumulation of excess intracellular cholesterol. Ex vivo cholesterol loading of chow-fed DKO SC recapitulated the effect, indicating that cellular cholesterol is a key regulator of SC-to-BAT differentiation. Comparing adipogenicity of Ldlr^+/+xLcat^−/− (LCAT-KO) SC with DKO SC identified a role for LCAT deficiency in priming SC to express BAT genes. Additionally, we found that reduced cellular cholesterol is important for adipogenic differentiation, evidenced by increased induction of adipogenesis in cholesterol-depleted SC from both LCAT-KO and SKO mice. Taken together, we conclude that ectopic BAT in DKO mice is classical in origin, and its development begins in utero. We further showed complementary roles of LCAT deficiency and cellular cholesterol reduction in the SC-to-BAT adipogenesis.     

Comparison of the Lipolytic Effects of Norepinephrine and BRL 37344 in Rat Brown and White Adipocytes

The lipolytic effects of norepinephrine (a non-selective β-agonist) and BRL 37344 (a selective β₃-agonist) were compared in isolated rat brown and white adipocytes. Norepinephrine and BRL 37344 maximally stimulated lipolysis in brown and white adipocytes, approximately 10 times above basal values. However, adipocyte sensitivity for BRL 37344 was greater than that for norepinephrine, particularly in brown adipocytes [the EC₅₀ values (nM) for BRL 37344 and norepinephrine were 5 ± 1 and 103 ± 31 in brown adipocytes (P <0.01) versus 56 ± 9 and 124 ± 17 in white adipocytes (P <0.05), respectively]. On the other hand, the lipolytic effects of norepinephrine were totally blocked by 20–40 times superior concentrations of propranolol or bupranolol in brown as well as in white adipocytes. In contrast, the lipolytic effects of BRL 37344 were fully inhibited by concentrations of propranolol or bupranolol that were 200–1000 superior to the β₃ agonist concentration. The results demonstrate that: (1) the (β₃-agonist BRL 37344 is as effective as norepinephrine for maximally stimulating lipolysis in rat brown and white adipocytes, (2) both adipocyte types are more sensitive to the lipolytic effects of BRL 37344 than to those of norepinephrine, (3) although bupranolol is a better antagonist than propranolol on BRL 37344-stimulated lipolysis, it cannot be considered as a specific β₃-antagonist, (4) brown adipocytes are 10 times more sensitive than white adipocytes to the lipolytic effects of BRL 37344, suggesting an important role of β₃-receptors in brown adipose tissue.     

UCP1 Induction during Recruitment of Brown Adipocytes in White Adipose Tissue Is Dependent on Cyclooxygenase Activity

Background

The uncoupling protein 1 (UCP1) is a hallmark of brown adipocytes and pivotal for cold- and diet-induced thermogenesis.

Methodology/Principal Findings

Here we report that cyclooxygenase (COX) activity and prostaglandin E₂ (PGE₂) are crucially involved in induction of UCP1 expression in inguinal white adipocytes, but not in classic interscapular brown adipocytes. Cold-induced expression of UCP1 in inguinal white adipocytes was repressed in COX2 knockout (KO) mice and by administration of the COX inhibitor indomethacin in wild-type mice. Indomethacin repressed β-adrenergic induction of UCP1 expression in primary inguinal adipocytes. The use of PGE₂ receptor antagonists implicated EP₄ as a main PGE₂ receptor, and injection of the stable PGE₂ analog (EP_3/4 agonist) 16,16 dm PGE₂ induced UCP1 expression in inguinal white adipose tissue. Inhibition of COX activity attenuated diet-induced UCP1 expression and increased energy efficiency and adipose tissue mass in obesity-resistant mice kept at thermoneutrality.

Conclusions/Significance

Our findings provide evidence that induction of UCP1 expression in white adipose tissue, but not in classic interscapular brown adipose tissue is dependent on cyclooxygenase activity. Our results indicate that cyclooxygenase-dependent induction of UCP1 expression in white adipose tissues is important for diet-induced thermogenesis providing support for a surprising role of COX activity in the control of energy balance and obesity development.     

Continuous Changes in Triacylglycerol Molecular Species Composition by Fatty Acids in Porcine Adipocytes

It has been demonstrated that the composition of molecular species of adipose tissue triacylglycerols (TGs) from farm animals are not equally synthesized and that some molecular species are preferentially synthesized. The objective of the present study was to determine whether exogenous fatty acids (FAs) would affect the TG composition. To this end, the composition of TG molecular species stored in porcine adipocytes differentiated with several long-chain FAs was analyzed by gas chromatography. The addition of each FA for 6 d increased TG molecular species having two or three added FAs. However, the molecular species compositions at 15 d after the addition of each FA resembled those of cells with no added FAs. Moreover, some common molecular species in all experimental cells increased, as well as cells with no added FAs. It was concluded that the addition of FAs increases the contents of specific molecular species, but does not affect the synthetic processes of individual TG molecular species.     

NAT8L (N-Acetyltransferase 8-Like) Accelerates Lipid Turnover and Increases Energy Expenditure in Brown Adipocytes

NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes.     

White Matter Changes in Patients with Amnestic Mild Cognitive Impairment Detected by Diffusion Tensor Imaging

Compared to normal aging adults, individuals with amnestic mild cognitive impairment (aMCI) have significantly increased risk for progressing into Alzheimer’s disease (AD). Autopsy studies found that most of the brains of aMCI cases showed anatomical features associated with AD pathology. The recent development of non-invasive neuroimaging technique, such as diffusion tensor imaging (DTI), makes it possible to investigate the microstructures of the cerebral white matter in vivo. We hypothesized that disrupted white matter (WM) integrity existed in aMCI. So we used DTI technique, by measuring fractional anisotropy (FA) and mean diffusivity (MD), to test the brain structures involved in patients with aMCI. DTI scans were collected from 40 patients with aMCI, and 28 normal controls (NC). Tract-based spatial statistics (TBSS) analyses of whole-brain FA and MD images in each individual and group comparisons were carried out. Compared to NC, aMCI patients showed significant FA reduction bilaterally, in the association and projection fibers of frontal, parietal, and temporal lobes, corpus callosum, bilateral corona radiation, right posterior thalamic radiation and right sagittal stratum. aMCI patients also showed significantly increased MD widespreadly in the association and projection fibers of frontal, parietal and temporal lobes, and corpus callosum. Assessment of the WM integrity of the frontal, parietal, temporal lobes, and corpus callosum by using DTI measures may aid early diagnosis of aMCI.     

Methionine Changes in Bacteriorhodopsin Detected by FTIR and Cell-Free Selenomethionine Substitution

Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more methionine residues undergo a structural change during the BR→M portion of the BR photocycle. Selenomethionine was incorporated into BR using a cell-free protein translation system containing an amino acid mixture with selenomethionine substituted for methionine. BR→M FTIR difference spectra recorded for unlabeled and selenomethionine-labeled cell-free expressed BR closely resemble the spectra of in vivo expressed BR. However, reproducible changes occur in two regions near 1284 and 900 cm⁻¹ due to selenomethionine incorporation. Isotope labeled tyrosine was also co-incorporated with selenomethionine in order to confirm these assignments. Based on recent x-ray crystallographic studies, likely methionines which give rise to the FTIR difference bands are Met-118 and Met-145, which are located inside the retinal binding pocket and in a position to constrain the motion of retinal during photoisomerization. The assignment of methionine bands in the FTIR difference spectrum of BR provides a means to study methionine-chromophore interaction under physiological conditions. More generally, combining cell-free incorporations of selenomethionine into proteins with FTIR difference spectroscopy provides a useful method for investigating the role of methionines in protein structure and function.     

Leptin Production by Encapsulated Adipocytes Increases Brown Fat,Decreases Resistin,and Improves Glucose Intolerance in Obese Mice

The neuroendocrine effects of leptin on metabolism hold promise to be translated into a complementary therapy to traditional insulin therapy for diabetes and obesity. However, injections of leptin can provoke inflammation. We tested the effects of leptin, produced in the physiological adipocyte location, on metabolism in mouse models of genetic and dietary obesity. We generated 3T3-L1 adipocytes constitutively secreting leptin and encapsulated them in a poly-L-lysine membrane, which protects the cells from immune rejection. Ob/ob mice (OB) were injected with capsules containing no cells (empty, OB^[Emp]), adipocytes (OB^[3T3]), or adipocytes overexpressing leptin (OB^[Lep]) into both visceral fat depots. Leptin was found in the plasma of OB^[Lep], but not OB^[Emp] and OB^[3T3] mice at the end of treatment (72 days). The OB^[Lep] and OB^[3T3] mice have transiently suppressed appetite and weight loss compared to OB^[Emp]. Only OB^[Lep] mice have greater brown fat mass, metabolic rate, and reduced resistin plasma levels compared to OB^[Emp]. Glucose tolerance was markedly better in OB^[Lep] vs. OB^[Emp] and OB^[3T3] mice as well as in wild type mice with high-fat diet-induced obesity and insulin resistance treated with encapsulated leptin-producing adipocytes. Our proof-of-principle study provides evidence of long-term improvement of glucose tolerance with encapsulated adipocytes producing leptin.     

Glycerol-3-phosphate Acyltransferase Isoform-4 (GPAT4) Limits Oxidation of Exogenous Fatty Acids in Brown Adipocytes

Glycerol-3-phosphate acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4^−/− mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4^−/− mice fed a 45% fat diet was 12% higher. Core body temperature was 1 ºC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4^−/− mice did not result from increased heat loss, because both cold tolerance and response to a β3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4^−/− BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4^−/− brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of β-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state.     

Rosiglitazone Modulates Insulin-Induced Plasma Membrane Area Changes in Single 3T3-L1 Adipocytes

The antioxidant activity of mitochondria-targeted small molecules, SkQ1 and MitoQ (conjugates of a lipophilic decyltriphenylphosphonium cation with an antioxidant moiety of a plastoquinone and ubiquinone, respectively), was studied in aqueous solution and in a lipid environment, i.e., micelles, liposomes and planar bilayer lipid membranes. Reactive oxygen species (ROS) were generated by azo initiators or ferrous ions with or without tert-butyl-hydroperoxide (t-BOOH). Chemiluminescence, fluorescence, oxygen consumption and inactivation of gramicidin peptide channels were measured to detect antioxidant activity. In all of the systems studied, SkQ1 was shown to effectively scavenge ROS. The scavenging was inherent to the reduced form of the quinone (SkQ1H(2)). In the majority of the above model systems, SkQ1 exhibited higher antioxidant activity than MitoQ. It is concluded that SkQ1H(2) operates as a ROS scavenger in both aqueous and lipid environments, being effective at preventing ROS-induced damage to membrane lipids as well as membrane-embedded peptides.