Tore Bengtsson

Three years with adult human brown adipose tissue

The presence of active brown adipose tissue in adult humans has been recognized in general physiology only since 2007. The intervening three years established that the depots originally observed by 18F‐fluoro‐deoxy‐glucose positron emission tomography (FDG PET) scanning techniques really are brown adipose tissue depots because they are enriched for uncoupling protein 1 (UCP1). Reports of low apparent prevalence of brown adipose tissue based on retrospective studies of hospital records of FDG PET scans markedly underestimate true prevalence because such studies only reflect acute activity state; consequently, such retrospective studies cannot be conclusively analysed for factors influencing activity and amount of brown adipose tissue. Dedicated studies show that the true prevalence is 30–100%, depending on cohort. Warm temperature during the investigation—as well as adrenergic antagonists—inhibit tissue activity. There is probably no sexual dimorphism in the prevalence of brown adipose tissue. Outdoor temperature may affect the amount of brown adipose tissue, and the amount is negatively correlated with age and obesity. The presence of brown adipose tissue is associated with cold‐induced nonshivering thermogenesis, and the tissue may be a major organ for glucose disposal. The decline in brown adipose tissue amount with increasing age may account for or aggravate middle‐age obesity. Maintained activation of brown adipose tissue throughout life may thus protect against obesity and diabetes.

New Powers of Brown Fat: Fighting the Metabolic Syndrome

An understanding of the full powers of brown adipose tissue (BAT) is only successively being accumulated. In a paper in Nature Medicine, Bartelt et al. (2011) add further impressive aspects to the potential powers of BAT in the combat against the metabolic syndrome by demonstrating its vast capacity for triglyceride clearance and glucose disposal.

beta1 to beta3 switch in control of cyclic adenosine monophosphate during brown adipocyte development explains distinct beta-adrenoceptor subtype mediation of proliferation and differentiation.

To explain the distinctive pharmacological profiles observed for adrenergic stimulation of cell proliferation (β1) and cell differentiation (β3), the adrenergic control of cAMP accumulation was investigated during brown adipocyte development. In preadipocytes, norepinephrine (NE) increased cAMP levels but theβ 3-agonists BRL-37344 and CGP-12177 did not; in contrast, when the cells had differentiated into mature brown adipocytes, a large cAMP response to the β3-agonists had emerged and was now double that to NE (although the affinity of NE had increased 10-fold).β 1-messenger RNA (mRNA) levels were high in both pre- and mature brown adipocytes; β3-mRNA did not appear until maturation but then abruptly. Although β1-receptors remained detectable by [3H]CGP-12177 binding in the mature brown adipocytes, the cAMP response to NE (based on propranolol inhibitory potency) switched from β1 to β3. Even the established β1-agonist dobutamine acted throughβ 3-receptors in the mature brown adipocytes. The increases in c...

β-Adrenoceptors, but not α-adrenoceptors, stimulate AMP-activated protein kinase in brown adipocytes independently of uncoupling protein-1

Aims/hypothesisBrown adipocytes provide a potentially important model system for understanding AMP-activated protein kinase (AMPK) regulation, where adrenergic stimulation leads to mitochondrial uncoupling through uncoupling protein-1 (UCP1) activity. AMPK is a sensor of energy homeostasis and has been implicated in glucose and lipid metabolism in several insulin-sensitive tissues. The aim of this study was to characterise the potential role of AMPK in adrenergically mediated glucose uptake and to find out whether UCP1 is involved in the adrenergic activation of AMPK.MethodsWe used primary brown adipocytes differentiated in culture and measured AMPK phosphorylation and glucose uptake following adrenergic activation.ResultsTreatment of adipocytes with noradrenaline (norepinephrine) caused phosphorylation of AMPK via β-adrenoceptors and not α1- or α2-adrenoceptors. This effect was not β3-adrenoceptor specific, since responses remained intact in adipocytes from β3-adrenoceptor knock-out mice. These effects were also mimicked by forskolin and cAMP analogues. Treatment of cells with adenine 8-β-d-arabinofuranoside, an AMPK inhibitor, partially blocked β-adrenoceptor-mediated increases in glucose uptake. Brown adipocytes are characterised by the production of UCP1, which can uncouple the mitochondria. Using adipocytes from Ucp1+/+ and Ucp1−/− mice, we showed that noradrenaline-mediated phosphorylation of AMPK does not require the presence or activity of UCP1.Conclusions/interpretationThese results suggest a pathway where increases in cAMP mediated by β-adrenoceptors leads to activation of AMPK in brown adipocytes, which contributes in part to β-adrenoceptor-mediated increases in glucose uptake, an effect independent of the presence or function of UCP1.

Regulation of AMP-activated protein kinase activity by G-protein coupled receptors : Potential utility in treatment of diabetes and heart disease

G-protein coupled receptors (GPCRs) comprise the largest and most diverse family of membrane receptors in the human genome, relaying information from a vast array of external stimuli. GPCRs are targets for approximately 30% of all current therapeutic agents. Recently some GPCRs have been shown to mediate part of their effects through activation of AMP-activated protein kinase (AMPK), a sensor of whole body energy status that plays a pivotal role in whole body energy balance by integrating signals in the periphery and central nervous system. It regulates glucose and lipid metabolism, food intake and body weight, making it an attractive target for the treatment of diseases such as type 2 diabetes and obesity. It mediates the effects of several important adipokines such as leptin and adiponectin and is thought to be responsible for the antidiabetic effects of metformin and thiazolidinediones. A diverse number of GPCRs (including adrenoceptors, cannabinoid receptors, ghrelin receptors, melanocortin receptors) modulate AMPK activity. This review focuses on the regulation of AMPK by GPCRs and signaling intermediates of GPCR signaling such as cyclic AMP and calcium, and how GPCR signaling can modulate AMPK activity by several different mechanisms, and the therapeutic implications of AMPK activation by GPCRs.

Glucose uptake in brown fat cells is dependent on mTOR complex 2–promoted GLUT1 translocation

β3-Adrenoceptors promote glucose uptake in brown adipose tissue via both cAMP-mediated increases in GLUT1 transcription and mTORC2-stimulated translocation of newly synthesized GLUT1 to the plasma membrane.

Multiple signalling pathways involved in β2-adrenoceptor-mediated glucose uptake in rat skeletal muscle cells

β‐adrenoceptor (AR) agonists increase 2‐deoxy‐[3H]‐D‐glucose uptake (GU) via β2‐AR in rat L6 cells. The β‐AR agonists, zinterol (β2‐AR) and (−)‐isoprenaline, increased cAMP accumulation in a concentration‐dependent manner (pEC50=9.1±0.02 and 7.8±0.02). Cholera toxin (% max increase 141.8±2.5) and the cAMP analogues, 8‐bromo‐cAMP (8Br‐cAMP) and dibutyryl cAMP (dbcAMP), also increased GU (196.8±13.5 and 196.4±17.3%). The adenylate cyclase inhibitor, 2′,5′‐dideoxyadenosine (50 μM), significantly reduced cAMP accumulation to zinterol (100 nM) (109.7+35.0 to 21.6+4.5 pmol well−1), or forskolin (10 μM) (230.1±58.0 to 107.2±26.3 pmol well−1), and partially inhibited zinterol‐stimulated GU (217±26.3 to 176.1±20.4%). The protein kinase A (PKA) inhibitor, 4‐cyano‐3‐methylisoquinoline (100 nM), did not inhibit zinterol‐stimulated GU. The PDE4 inhibitor, rolipram (10 μM), increased cAMP accumulation to zinterol or forskolin, and sensitised the GU response to zinterol, indicating a stimulatory role of cAMP in GU. cAMP accumulation studies indicated that the β2‐AR was desensitised by prolonged stimulation with zinterol, but not forskolin, whereas GU responses to zinterol increased with time, suggesting that receptor desensitisation may be involved in GU. Receptor desensitisation was not reversed by inhibition of PKA or Gi. PTX pretreatment (100 ng ml−1) inhibited insulin or zinterol‐stimulated but not 8Br‐cAMP or dbcAMP‐stimulated GU. The PI3K inhibitor, LY294002 (1 μM), inhibited insulin‐ (174.9±5.9 to 142.7±2.7%) and zinterol‐ (166.9±7.6 to 141.1±8.1%) but not 8 Br‐cAMP‐stimulated GU. In contrast to insulin, zinterol did not cause phosphorylation of Akt. The results suggest that GU in L6 cells involves three mechanisms: (1) an insulin‐dependent pathway involving PI3K, (2) a β2‐AR‐mediated pathway involving both cAMP and PI3K, and (3) a receptor‐independent pathway suggested by cAMP analogues that increase GU independently of PI3K. PKA appears to negatively regulate β2‐AR‐mediated GU.

Mouse β3a‐ and β3b‐adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways

This study characterizes the mouse β3a‐adrenoceptor (AR) and the splice variant of the β3‐AR (β3b‐AR) expressed in Chinese hamster ovary cells (CHO‐K1). Stable clones with high (∼1200), medium (∼500) or low receptor expression (∼100 fmol mg protein−1) were determined by saturation binding with [125I]‐(−)‐cyanopindolol. Competition binding studies showed no significant differences in affinity of β‐AR ligands for either receptor. Several functional responses of each receptor were measured, namely extracellular acidification rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The β3‐AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non‐conventional partial β‐AR agonist CGP12177 and the β‐AR agonist (−)‐isoprenaline caused concentration‐dependent increases in EAR in cells expressing either splice variant. CL316243 caused concentration‐dependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor. PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the β3b‐AR but not in cells expressing the β3a‐AR at all levels of receptor expression. CL316243 increased Erk1/2 phosphorylation with pEC50 values and maximum responses that were not significantly different in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3Kγ inhibitor), PP2 (c‐Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both β3a‐ or β3b‐AR transfected cells. These results suggest that in CHO‐K1 cells, the β3b‐AR, can couple to both Gs and Gi to stimulate and inhibit cyclic AMP production respectively, while the β3a‐AR, couples solely to Gs to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to Gi or the generation of cyclic AMP.

Characterization of the β‐adrenoceptor subtype involved in mediation of glucose transport in L6 cells

The receptor that mediates the increase in glucose transport (GT) in response to β‐adrenoceptor (β‐AR) agonists was characterized in the rat skeletal muscle cell line L6, using the 2‐deoxy‐[3H]‐D‐glucose assay. The β3‐AR agonist BRL37344 (pEC50=6.89±0.21), the β‐AR agonist isoprenaline (pEC50=8.99±0.24) and the β2‐AR agonist zinterol (pEC50=9.74±0.15) increased GT as did insulin (pEC50=6.93±0.15). The highly selective β3‐AR agonist CL316243 only weakly stimulated GT. The pKB values calculated from the shift of the pEC50 values of the agonists in the presence of the β1‐AR selective antagonist CGP 20712A or the β3‐AR selective antagonist SR 59230A were not indicative of activation of β1‐ or β3‐ARs. Only (−)‐propranolol and the β2‐AR selective antagonist ICI 118551 caused marked rightward shifts of CR curves to isoprenaline (pKB=10.2±0.2 and 9.6±0.3), zinterol (pKB=9.0±0.1 and 9.4±0.3) and BRL 37344 (pKB=9.4±0.3 and 8.4±.2), indicating participation of β2‐ARs. The pharmacological analysis was supported by reverse transcription and polymerase chain reaction analysis of L6 mRNA, which showed high levels of expression of β2‐AR but not β1‐ or β3‐AR in these cells. Forskolin and dibutyryl cyclic AMP produced negligible increases in GT while the phosphatidylinositol‐3 kinase inhibitor, wortmannin, significantly decreased both insulin‐ and zinterol‐stimulated GT, suggesting a possible interaction between the insulin and β2‐AR pathways. This study demonstrates that β2‐ARs mediate the increase in GT in L6 cells to β‐AR agonists, including the β3‐AR selective agonist BRL 37344. This effect does not appear to be directly related to increases in cyclic AMP but requires P13K.

β1-adrenergic receptors increase UCP1 in human MADS brown adipocytes and rescue cold-acclimated β3-adrenergic receptor knockout mice via nonshivering thermogenesis

With the finding that brown adipose tissue is present and negatively correlated to obesity in adult man, finding the mechanism(s) of how to activate brown adipose tissue in humans could be important in combating obesity, type 2 diabetes, and their complications. In mice, the main regulator of nonshivering thermogenesis in brown adipose tissue is norepinephrine acting predominantly via β(3)-adrenergic receptors. However, vast majorities of β(3)-adrenergic agonists have so far not been able to stimulate human β(3)-adrenergic receptors or brown adipose tissue activity, and it was postulated that human brown adipose tissue could be regulated instead by β(1)-adrenergic receptors. Therefore, we have investigated the signaling pathways, specifically pathways to nonshivering thermogenesis, in mice lacking β(3)-adrenergic receptors. Wild-type and β(3)-knockout mice were either exposed to acute cold (up to 12 h) or acclimated for 7 wk to cold, and parameters related to metabolism and brown adipose tissue function were investigated. β(3)-knockout mice were able to survive both acute and prolonged cold exposure due to activation of β(1)-adrenergic receptors. Thus, in the absence of β(3)-adrenergic receptors, β(1)-adrenergic receptors are effectively able to signal via cAMP to elicit cAMP-mediated responses and to recruit and activate brown adipose tissue. In addition, we found that in human multipotent adipose-derived stem cells differentiated into functional brown adipocytes, activation of either β(1)-adrenergic receptors or β(3)-adrenergic receptors was able to increase UCP1 mRNA and protein levels. Thus, in humans, β(1)-adrenergic receptors could play an important role in regulating nonshivering thermogenesis.