Stilbenes

Pterostilbene (PTS) is the dimethylated derivative of resveratrol (RSV) and its potential weight-lowering capacity has only recently gained attention. Interest in PTS rose when Rimando et al.^{(Reference Rimando, Nagmani and Feller27)} demonstrated that PTS strongly induced PPARα activity in a rat hepatocyte cell line overexpressing a PPRE-luciferase reporter gene^{(Reference Rimando, Nagmani and Feller27)}. PTS at 100 μm displayed 2-fold higher PPARα induction than the pharmacologic PPARα agonist ciprofibrate. The anti-hyperlipidaemic activity of PTS was verified in hamsters where PTS-fortification of high fat diet (HFD; 25 mg PTS/kg food) led to lower total and LDL-cholesterol levels^{(Reference Rimando, Nagmani and Feller27)}. PPARα is a nuclear transcription factor, which is highly enriched in the liver as well as BAT v. WAT where it controls a myriad of genes involved in fatty acid uptake as well as β- and peroxisomal lipid oxidation^{(Reference Kersten28,Reference Rakhshandehroo, Knoch and Müller29)} . The group of Maria Portillo examined the effect of PTS on adipose tissue in rat models of genetic and dietary-induced obesity^{(Reference Etxeberria, Hijona and Aguirre30,Reference Gómez-Zorita, Fernández-Quintela and Lasa31)} . In Zucker fatty rats, the daily oral application of 15 mg PTS/kg for 6 weeks resulted in significantly less total fat (10 % v. 13⋅2 %) compared to untreated animals^{(Reference Etxeberria, Hijona and Aguirre30)}. During the dietary intervention, Wistar rats were fed an adipogenic high fat–high sucrose (20 % each) diet enriched with PTS to ensure an estimated daily intake of either 15 mg/kg or 30 mg/kg BW. Although no effect on weight gain or BW was observed, total adipose tissue mass was dose-dependently reduced in comparison with control animals (40⋅3 g, 36⋅6 g, 47⋅5 g)^{(Reference Gómez-Zorita, Fernández-Quintela and Lasa31)}. Moreover, enzymatic carnitine palmitoyltransferase 1α activity was higher in the liver while fatty acid synthase activity was blunted in WAT^{(Reference Gómez-Zorita, Fernández-Quintela and Lasa31)}. The described phenotype might result from an interplay between increased fatty acid oxidation due to higher fatty acid import into mitochondria via carnitine palmitoyltransferase 1α and reduced adipocyte lipogenesis due to low fatty acid synthase activity. Unfortunately, the only published human clinical trial with PTS as investigatory compound could not reproduce these findings from rats. Instead, 8 weeks of daily PTS ingestion (0, 100 or 250 mg daily) increased LDL-cholesterol and consequently total cholesterol levels in a cohort of male and female hypercholesterolemic Caucasian and African Americans^{(Reference Riche, Riche and Blackshear32)}. Overall, a mild but significant effect of PTS on BMI was observed when participants were stratified for cholesterol medication. BAT possesses a tremendous oxidative capacity and relies heavily on substrate e.g. fatty acid oxidation to fulfil its thermogenic function. PPARα agonists are known to induce UCP1 expression in murine BAT^{(Reference Barbera, Schluter and Pedraza33)} and β-AR signalling increases PPARα expression in brown adipocytes^{(Reference Mottillo, Bloch and Leff34)}. To date, three publications have addressed the impact of PTS-fortified diet on WAT browning or BAT functionality in rodent models. Aguirre et al.^{(Reference Aguirre, Milton-Laskibar and Hijona35)} found that 15 or 30 mg PTS/kg BW upregulated various brown and mitochondrial marker genes in male Zucker fa/fa rats when administered daily for 6 weeks^{(Reference Aguirre, Milton-Laskibar and Hijona35)}. These changes were accompanied by increased PPARα and UCP1 protein abundance in the BAT. Additionally, higher carnitine palmitoyltransferase 1α activity was measured in the intrascapular BAT lysate of PTS-rats compared to controls^{(Reference Aguirre, Milton-Laskibar and Hijona35)}. These molecular alterations imply that PTS enhances the oxidative capacity of BAT in genetically obese rats that translated to a beneficial phenotype as total adipose tissue weight and BW were reduced in PTS-rats^{(Reference Aguirre, Milton-Laskibar and Hijona35)}. This conclusion is substantiated by data from Nagao and co-workers^{(Reference Nagao, Jinnouchi and Kai36)} who applied a 10× higher PTS dose (300 mg/kg BW daily) as a food supplement to male, genetically hyperphagic rats, which again caused lower body fat accumulation^{(Reference Nagao, Jinnouchi and Kai36)}. Compared to control-fed animals, PTS stimulated oxygen consumption (3⋅91 v. 3⋅69 litre/100 g BW daily) and enhanced EE (80⋅4 v. 77⋅1 litre/100 g BW daily) after 4 weeks of feeding^{(Reference Nagao, Jinnouchi and Kai36)}. The observed reduction in the respiratory quotient (0⋅82 v. 0⋅85) of PTS-treated rats was in agreement with higher lipid oxidation during the metabolically active dark phase^{(Reference Nagao, Jinnouchi and Kai36)}. Interestingly, Nagao et al.^{(Reference Nagao, Jinnouchi and Kai36)} not only confirmed that PTS is a PPARα agonist, but also discovered that PTS at 50–250 μm increased sirtuin 1 (SIRT1) activity^{(Reference Nagao, Jinnouchi and Kai36)}. SIRT1 is a NAD+-dependent protein deacetylase and modulates the functional activity of various enzymes and transcription factors. Deacetylation of PPARγ by SIRT1 is crucial for browning of WAT as it enables the recruitment of the PPARγ coactivator PRDM16, which functions as a master regulator of the thermogenic gene programme in WAT^{(Reference Seale, Conroe and Estall37,Reference Qiang, Wang and Kon38)} . In turn, food deprivation or pharmacological SIRT1 activation have been shown to blunt the PPARγ-dependent expression of lipogenic genes in WAT through SIRT1-mediated blockade of the PPARγ-coactivator NCoR^{(Reference Picard, Kurtev and Chung39)}. This repressive effect on PPARγ promotes lipolysis and reduces fat storage in WAT^{(Reference Picard, Kurtev and Chung39)}. SIRT1 additionally affects mitochondrial biogenesis by two converging mechanisms. First, SIRT1 deacetylates and activates liver kinase B1, an upstream kinase and activator of AMPK, which in turn phosphorylates PPARγ co-activator 1α (PGC1α)^{(Reference Lan, Cacicedo and Ruderman40)}. Secondly, SIRT1 directly deacetylates PGC1α^{(Reference Rodgers, Lerin and Haas41)}. Consistent with the relevance of SIRT1 in WAT browning, chronic PTS administration to HFD-fed mice (90 mg/kg daily) caused lower weight gain and a trend towards browning of the inguinal WAT as measured by upregulation of browning-specific marker genes (CIDEA, PGC1α, EBF2, PPARγ and TBX1)^{(Reference La Spina, Galletta and Azzolini42)}. The transcriptional and morphological changes during WAT remodelling in response to cold exposure are independent of PPARα as PPARα knockout mice display equal browning when compared with wild-type animals^{(Reference Defour, Dijk and Ruppert43)}. Collectively, it seems that SIRT1 agonism by PTS is the dominant molecular trigger, which confers the physiological effects of long-term PTS consumption such as browning of WAT, enhanced oxidative capacity, lipid mobilisation and increased EE. The pharmacokinetic analysis of orally applied PTS (168 mg/kg BW) in rats revealed a high bioavailability of about 80 % with a peak plasma PTS concentration of approximately 30 μm^{(Reference Kapetanovic, Muzzio and Huang44)}. This dosage corresponds to an approximated human equivalent dose of 27 mg/kg^{(Reference Nair and Jacob45)} and up to 250 mg/kg BW of PTS is evidentially safe for human use^{(Reference Riche, McEwen and Riche46)}.

RSV attracted the interest of scientific community after Howitz et al.^{(Reference Howitz, Bitterman and Cohen47)} identified RSV as a powerful small molecule activator of SIRT1^{(Reference Howitz, Bitterman and Cohen47)}. SIRT1 operates as one of the molecular regulators essential for the beneficial physiological effects of energetic restriction^{(Reference Bordone and Guarente48)}. This discovery introduced the idea that RSV might be a natural substance able to ameliorate obesity by mimicking a low energy state. Lagouge et al.^{(Reference Lagouge, Argmann and Gerhart-Hines49)} first demonstrated that a RSV-enriched (400 mg/kg BW daily) diet impairs weight gain in male HFD-fed mice^{(Reference Lagouge, Argmann and Gerhart-Hines49)}. RSV mice displayed enhanced oxygen consumption, improved cold tolerance during an acute cold challenge as well as increased mitochondrial content in BAT. A gene-enrichment analysis confirmed the induction of genes related to mitochondrial biogenesis and function after RSV therapy in muscle. The measured increase in EE was not attributable to spontaneous locomotor activity, suggesting that RSV modifies BAT functionality as a driver of adaptive thermogenesis. Another study explored the effects of lifelong RSV-containing HFD (0⋅04 % w/w) on metabolic health in middle-aged, male mice^{(Reference Baur, Pearson and Price50)}. Here, RSV intake did not affect BW, possibly due to the lower concentrations used, but significantly attenuated the signs of ageing such as elevated fasting blood glucose levels, insulin resistance and pathological organ changes^{(Reference Baur, Pearson and Price50)}. In a before–after study performed in six male, non-human primates, 4 weeks daily RSV ingestion (200 mg/kg BW) suppressed BW gain and enhanced RMR by 29 % compared to baseline^{(Reference Dal-Pan, Blanc and Aujard51)}. In addition, the body temperature difference between the active phase and the hypothermic light phase was reduced, suggesting a thermogenic activity^{(Reference Dal-Pan, Blanc and Aujard51)}. To date, direct, robust evidence for a contribution of BAT to the systemic effects of RSV is scarce. Two months of daily RSV feeding of male mice (400 mg/kg BW) on a standard diet led to lower epididymal and retroperitoneal adipose tissue weight, higher oxygen consumption and about 2-fold upregulation of UCP1 and SIRT1 mRNA expression in BAT of RSV mice^{(Reference Andrade, Frade and Guimarães52)}. The authors hypothesise that fat stores are mobilised to accommodate the increased fatty acid demand of more active BAT, which in turn lowers adiposity. However, no histological sections with UCP1-immunohistochemistry, UPC1 protein levels or mitochondrial parameters were presented to strengthen such a statement. The laboratory of Du^{(Reference Wang, Liang and Yang53)} demonstrated the formation of brown-like adipocytes in the presence of 10 μm RSV, when stromal vascular cells isolated from WAT were differentiated with a brown adipogenic cocktail^{(Reference Wang, Liang and Yang53)}. These brown-like adipocytes upregulated an array of essential brown marker genes. This finding was affirmed in female CD-1 mice, where RSV (0⋅1 % in HFD) slowed weight gain and induced the same thermogenic gene set in the inguinal WAT when compared to controls^{(Reference Wang, Liang and Yang54)}. Consistent with the known morphological features of browning, the inguinal WAT of RSV-fed mice exhibited adipocytes with multilocular lipid droplets and a shift towards smaller cells. RSV-treated mice and cells further displayed increased metabolic rates and enhanced lipid oxidation. More intriguingly, all the specified molecular alterations and the consequent physiological outcomes were not observed when experiments were repeated in the absence of AMPK^{(Reference Wang, Liang and Yang54)}. Using the same experimental set-up, Wang et al.^{(Reference Wang, Liang and Yang54)} counted more brown adipocytes in histological BAT sections, which implies that RSV might provoke the formation of brown adipocytes, in vivo. In parallel, RSV feeding resulted in higher UCP1 and PRDM16 protein content as well as the augmented levels of phosphoAMPK^{(Reference Wang, Liang and Yang54)}. Similar findings including reduced amounts of acetylated PGC1α were reported for the brown fat in male rats treated with 30 mg/kg BW^{(Reference Alberdi, Rodríguez and Miranda55)}.

Contrary to the overwhelming amount of literature addressing the benefits of RSV in animal models, no human clinical trials exist that specifically use EE, BAT activation or weight management as readouts. A plethora of studies (supplementary material) conducted in different target groups with daily RSV doses from 75 up to 2000 mg did not detect any effects of RSV on key metabolic parameters, anthropometric measures or molecular downstream markers of SIRT1 activation^{(Reference Poulsen, Vestergaard and Clasen56–Reference Crandall, Oram and Trandafirescu58)}. In strong contrast stands a cross-over trial completed in obese, healthy men, where daily RSV (150 mg) intake for 30 d modestly mimicked the physiological effects resembling energetic restriction^{(Reference Timmers, Konings and Bilet59)}. The participants had lower resting and sleeping metabolic rates (2–4 % lower EE), higher daily respiratory quotient as indicator for improved metabolic flexibility, higher SIRT1 and phosphoAMPK protein abundance in muscle biopsies plus elevated carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone-uncoupled respiration. This striking discrepancy in study outcome might be due to such factors as the extent of obesity, degree of insulin resistance, age, sex, sample size, parallel v. crossover design (seasonal impact) and blood RSV concentrations^{(Reference Poulsen, Vestergaard and Clasen56–Reference Timmers, Konings and Bilet59)}.

The structural similarity between PTS and RSV implies that they might share the same molecular mode of action, SIRT1 agonism. Nonetheless, the methoxy groups in PTS protect it from hepatic metabolisation and possibly cause the high oral bioavailability of PTS (80 %)^{(Reference Kapetanovic, Muzzio and Huang44)}. In contrast, the low oral bioavailability of RSV is one of its major drawbacks and might account for the poor reproducibility as well as comparability of human RSV trials. Although RSV is well absorbed in the intestine through passive diffusion (70 %), the phenolic groups are rapidly glucuronidated or sulphonated in the liver and leave only 1⋅5 % of RSV unmodified^{(Reference Springer and Moco60)}. Notably, inter-species differences exist in the formation of glucuronidated RSV (g-RSV) metabolites. In rats and mice, 90 % of an applied RSV dose is glucuronidated compared to 65 % in human subjects and dogs. Lastly, enzymatic stereoselectivity leads to distinct ratios of g-RSV types: the dominant type (RSV-4’G) in human subjects and dogs is undetectable in mice and rats^{(Reference Maier-Salamon, Böhmdorfer and Thalhammer61)}. Consequently, findings from rodent studies are difficult to translate to human subjects and other models might be required to assess the physiological bioactivity/efficacy of RSV metabolites. The accepted difficulty in producing reproducible and meaningful effects with RSV in human subjects makes it currently unlikely to be upgraded from an over the counter nutritional supplement to a pharmacological remedy.

Flavonoids

Quercetin is particularly abundant (16⋅5mg/g) in the onion peel extract (OPE; 16⋅5 mg/g), which is frequently used as a quercetin matrix in animal experiments^{(Reference Kim and Kim62)}. OPE reduced diet-induced obesity by 6 % and visceral fat in male rats when added to HFD (3⋅6 g/kg food) for 8 weeks^{(Reference Moon, Do and Kim63)}. Ting et al.^{(Reference Ting, Chang and Shiau64)} described an even more pronounced weight phenotype (20 % difference in weight gain) for a quercetin-rich supplement in male HFD-fed rats (185, 270 or 925 mg/kg BW daily), demonstrating that quercetin likely is the active ingredient in the OPE^{(Reference Ting, Chang and Shiau64)}. In an extensive study with Zucker fatty rats, quercetin was provided daily via the food (10 mg/kg BW) and profoundly reduced weight gain when combined with either standard diet (12 v. 26 %) or HFD (26 v. 33 %) over a 10 week period^{(Reference Rivera, Morón and Sánchez65)}. Quercetin-treated rats showed improved insulin sensitivity, reduced endothelial nitric oxide synthase protein and inflammatory TNFα release in the visceral WAT. Unfortunately, the aforementioned studies did not evaluate EE or physical activity as potential contributors.

In male mice, HFD enriched with 5 g OPE/kg for 8 weeks did not attenuate weight or fat mass compared to plain HFD but upregulated markers of WAT browning^{(Reference Lee, Parks and Kang66)}. One possibility for the lack of a weight phenotype could be due to a stronger obese phenotype or different quercetin contents in the OPE (68 v. 276 mg/g dry weight). Browning of murine, male WAT following 12 weeks HFD feeding with pure quercetin (1 g/kg food) was confirmed by Kuipers et al.^{(Reference Kuipers, van Dam and Held67)}. Classical BAT was not affected by the treatment and browning was insufficient to modulate BW or EE, in accordance with the results from Stewart et al.^{(Reference Stewart, Soileau and Ribnicky68)} at a relatively high quercetin dose of 8 g/kg HFD^{(Reference Stewart, Soileau and Ribnicky68)}. A recent report^{(Reference Choi, Kim and Yu69)} describes browning of WAT alongside enhanced BAT-UCP1 content in male mice fed with HFD + 0⋅05 % quercetin^{(Reference Choi, Kim and Yu69)}. Plasma norepinephrine concentrations were about three times higher in quercetin-exposed animals, which suggests an intensified sympathetic nervous activity as the underlying effector. Furthermore, protein kinase A (PKA) protein levels were elevated in subcutaneous WAT of quercetin mice. However, cAMP levels or phosphoPKA substrates, which are surrogate markers of adrenergic stimulation, were not determined. It remains to be shown whether these molecular adaptations translate into a distinct phenotype as body weight or EE were not measured^{(Reference Choi, Kim and Yu69)}. Higher postprandial norepinephrine levels were also found in male calves fed quercetin (50 mg/kg BW) supplemented milk replacement for 1 week^{(Reference Gruse, Görs and Tuchscherer70)}. In human liver lysates, quercetin and its glycoside rutin showed catechol-O-methyltransferase (COMT) inhibitory activity at IC₅₀ of 5⋅3 and 10⋅8 μm, respectively and could potentiate norepinephrine action^{(Reference Gugler and Dengler71)}. Furthermore, quercetin dose dependently (1–250 μm) reinforced norepinephrine and isoproterenol-induced lipolysis in isolated male rat adipocytes by increasing cAMP concentrations^{(Reference Kuppusamy and Das72)}. Even though the effects of quercetin on BW are inconclusive, quercetin might assist in improving metabolic homoeostasis. Notably, inter-species differences in quercetin efficacy are obvious as most studies in rats showed a weight-lowering effect after 8–12 weeks of intervention, which was mostly absent in mice. Surprisingly, an extended quercetin feeding diet regimen over 12 weeks (1 g/kg HFD) in male mice added up to reduced terminal BW because of a lower adipose tissue mass^{(Reference Dong, Zhang and Zhang73)}. In these mice, adipose tissue-resident macrophages and mast cells were significantly lowered, which caused attenuated visceral adipose tissue as well as systemic inflammation. Furthermore, UCP1 mRNA was upregulated in the BAT after quercetin treatment, suggesting a potential thermogenic effect^{(Reference Dong, Zhang and Zhang73)}. At a molecular level, quercetin led to higher SIRT1 protein amounts and phosphoAMPK levels in the visceral WAT^{(Reference Dong, Zhang and Zhang73)}. The anti-inflammatory property of activated SIRT1 is well established^{(Reference Cho and Lumeng74,Reference Yoshizaki, Milne and Imamura75)} . Induction of the SIRT1–liver kinase B1–AMPK pathway as a mediator of quercetin's anti-inflammatory activity was confirmed in bone-derived macrophages stimulated with lipopolysaccharides to mimic obesity-associated inflammation^{(Reference Comalada, Ballester and Bailón76)}. Under this condition, quercetin (20 μm) increased SIRT1, phosphoAMPK and phospho liver kinase B1 protein levels in parallel to a heightened intracellular AMP:ATP ratio^{(Reference Comalada, Ballester and Bailón76)}.

Although promising effects regarding BW and inflammatory states are evident in rodent studies, the current available human clinical trials do not reproduce this picture. An up to date meta-analysis covering doses from 100 up to 1000 mg daily and intervention durations from 2 to 12 weeks did not find any beneficial clinical effects of OPE or quercetin intake on human BW^{(Reference Huang, Liao and Dong77)}. The absence of any human physiologically relevant effect is underlined by the studies summarised in the supplementary material, mainly performed in Korean subjects^{(Reference Kim, Cha and Lee78–Reference Lee, Cha and Lee80)}.

It is striking that circulating quercetin concentrations reported in rodent studies appear much higher than in human subjects. In male mice, 5⋅3 g/kg BW daily quercetin related to 90 μm of fasting quercetin levels^{(Reference Zhang, Angst and Park81)} and a lower dose (100 mg/kg BW) to 10 μm after 12 weeks feeding^{(Reference Dong, Zhang and Zhang73)}. In male and female human subjects, 150 mg daily over 5 weeks resulted in a mean fasting plasma quercetin concentration of 0⋅270 μm^{(Reference Egert, Bosy-Westphal and Seiberl82)}, 500 mg daily over 12 weeks in about 1⋅3 μm^{(Reference Jin, Nieman and Shanely83)}. Surprisingly, the mouse dose of 100 mg/kg BW translates to a human equivalent dose of 8 mg/kg BW and in human subjects, 500 mg daily reflects 6⋅5 mg/kg BW as calculated by the mean BW in this cohort. Consequently, although the doses lie in a comparable range, the circulating quercetin concentration in human subjects is roughly ten times lower. This comparison highlights potential inter-species differences in quercetin metabolism, absorption and excretion that affects final plasma concentrations and potentially the therapeutic outcome. Considering that human quercetin intake is safe up to 5 g daily^{(Reference Lu, Crespi and Liu84)}, clinical trials of extended duration (17 weeks) and at higher doses are required to conclude on the efficacy of quercetin in human subjects.

Luteolin has been extensively studied due to its anti-inflammatory activity and research focusing on weight loss following luteolin intake is in its early stages and limited to rodent studies. Distinct long-term dietary interventions with luteolin-enriched HFD (12 weeks, 0⋅01 % w/w or 16 weeks, 0⋅005 % w/w) significantly reduced BW gain in male mice^{(Reference Xu, Zhang and Dong85–Reference Zhang, Han and Zhang87)}. This phenotype was due to reduced visceral and subcutaneous WAT accumulation with a lower proinflammatory state of the WAT (e.g. macrophage infiltration, lower M1:M2 ratio)^{(Reference Xu, Zhang and Dong85–Reference Zhang, Han and Zhang87)}. Kwon et al.^{(Reference Kwon, Jung and Park86)} measured enhanced faecal lipid output in the luteolin group and higher rates of lipolysis in the WAT, which likely influenced adiposity^{(Reference Kwon, Jung and Park86)}. Luteolin was shown to elevate PPARγ protein levels and transcriptional activity in 3T3-L1 adipocytes^{(Reference Ding, Jin and Chen88)}. Modulation of PPARγ might contribute to the healthier adipose tissue expansion but cannot explain the reduction in adipose tissue mass as established pharmacological PPARγ agonists (thiazolidinediones) tend to increase fat mass^{(Reference de Souza, Eckhardt and Gagen89)}. It is reasonable to assume that the effect of luteolin on BW is polymodal and due to many physiological changes such as attenuated inflammation, reduced intestinal lipid absorption and more lipolytic WAT that fuels activated BAT. The latter assumption comes from a study by Zhang et al.^{(Reference Zhang, Zhang and Wang90)} who demonstrated higher EE in luteolin-HFD (0⋅01 % w/w) fed male mice compared to untreated mice^{(Reference Zhang, Zhang and Wang90)}. The histological examination of the BAT revealed an increased density of UCP1+ cells in the luteolin group combined with an induction of the thermogenic programme at the mRNA and protein levels. Moreover, browning of subcutaneous WAT was detected. Interestingly, this phenotype was preserved when HFD was replaced by standard diet and proves the inherent thermogenic activity of luteolin^{(Reference Zhang, Zhang and Wang90)}. At the molecular level, luteolin stimulated AMPK phosphorylation in vivo and the beneficial effects of luteolin on thermogenic genes was abolished in primary brown and white adipocytes when cells were pre-exposed to the AMPK inhibitor compound C. It appears that luteolin promotes an activated brown phenotype through the AMPK–SIRT1–PGC1α axis and human clinical studies are necessary for proof of concept/efficacy. Newest findings by Zhang et al.^{(Reference Zhang, Wang and Yin91)} illustrate a significant role of mast cells in energy homoeostasis^{(Reference Zhang, Wang and Yin91)}. Genetic ablation or pharmacologic inactivation of mast cells in male mice amplified acute norepinephrine-triggered oxygen consumption and increased body temperature. In both conditions, massive browning occurred in the subcutaneous WAT together with higher UCP1 protein levels and the upregulation of thermogenic genes^{(Reference Zhang, Wang and Yin91)}. The authors claim that serotonin secretion from mast cells inhibits platelet derived growth factor receptor α + progenitor cell proliferation that can give rise to beige/brite adipocytes. In agreement with the anti-inflammatory features common to quercetin and luteolin as well as the attenuation of mast cells’ infiltration into WAT following the chronic consumption of these compounds, one can speculate that quercetin and/or luteolin enhance thermogenesis and systemic EE by WAT browning, due to lower mast cell-derived serotonin release.

Catechins

Catechins are named after the catechu, an aqueous extract obtained from the wood of the acacia tree (Senegalia catechu) and rich in these polyphenolic substances. (−)-Epigallocatechin gallate (EGCG) and (−)-epigallocatechin are the most prevalent catechins in green tea infusions^{(Reference Graham92–Reference Arts, van de Putte and Hollman94)}. The first notion that green tea/catechin consumption might affect BW traces back to a study investigating the effect of a standardised green tea extract on 24-h EE in healthy men^{(Reference Dulloo, Duret and Rohrer95)}. Surprisingly, three times daily intake of a combined caffeine (50 mg) plus EGCG (90 mg) preparation increased total 24-h EE by 3⋅5 % whereas the caffeine (50 mg) only group did not show any effects^{(Reference Dulloo, Duret and Rohrer95)}. These findings were confirmed by Rudelle et al.^{(Reference Rudelle, Ferruzzi and Cristiani96)}, who measured a comparable 4⋅6 % elevation (444 kJ daily) in the 24-h EE in lean men and women after a 3 d treatment diet regimen consisting of 282 mg EGCG and 300 mg caffeine daily^{(Reference Rudelle, Ferruzzi and Cristiani96)}. Bérubé-Parent et al.^{(Reference Bérubé-Parent, Pelletier and Doré97)} next aimed to identify the necessary EGCG content in green tea capsules at a fixed caffeine dose (200 mg) to maximise the mixture's impact on 24-h EE in men^{(Reference Bérubé-Parent, Pelletier and Doré97)}. Consistent with previous reports, three times daily ingestion of the mixture enhanced 24-h EE at any EGCG concentration (minimum 270 mg daily up to maximum 1200 mg daily); however, no significant dose–response was observed. It seems plausible that the thermogenic activity of the ingested green tea mimetic is mediated either solely by EGCG and/or by a synergistic action of caffeine and EGCG, whereas the effective amount of EGCG reaches a threshold beyond which no additional benefits on EE are to be expected. A 3 d trial focusing on the differential effects of EGCG or caffeine alone on EE in men (mean BMI 31) did not find any relevant impact at different EGCG concentrations (300 v. 600 mg) nor for caffeine (200 mg) or in combination^{(Reference Thielecke, Rahn and Böhnke98)}. Nevertheless, EGCG enhanced fat oxidation as determined by the respiratory quotient 2 h after an overnight fast by 7 % in the 300 mg EGCG group as well as in the postprandial phase, 2 h post-meal by 33 %. The contradictory results concerning EE may be due to the mode of administration e.g. capsules v. beverage, the ethnicity of the participants^{(Reference Hursel, Viechtbauer and Westerterp-Plantenga99)}, the degree of overweight/obese subjects and/or the utilised caffeine dose. Accordingly, EGCG-caffeine supplements seem to be more effective on weight loss or weight maintenance in subjects with low habitual caffeine intake^{(Reference Hursel, Viechtbauer and Westerterp-Plantenga99,Reference Westerterp-Plantenga, Lejeune and Kovacs100)} . A meta-analysis evaluating the influence of short-term EGCG consumption on EE and fat oxidation concluded that EGCG moderately boosts metabolic rate as indicated by significantly lower respiratory quotient and higher EE^{(Reference Kapoor, Sugita and Fukuzawa101)}.

A large body of literature^{(Reference Yoneshiro, Matsushita and Hibi102–Reference Chantre and Lairon107)} (supplementary material) has examined whether the possible thermogenic activity of catechins translates to a weight loss phenotype during extended supplement interventions. Overall, the effect of catechins on weight maintenance or loss as measured by BW, BMI and waist circumference is significant when combined with caffeine whereas only a mild clinical impact can be implied based on the described effect sizes^{(Reference Hursel, Viechtbauer and Westerterp-Plantenga99,Reference Phung, Baker and Matthews108)} .

The exact molecular events as well as the physiological targets, which trigger fat loss and elevated EE in response to catechin ingestion are still under discussion. However, the general consent supported by solid experimental evidence points at the activation and/or recruitment of BAT as likely effectors. In male rats, the supplementation of HFD with green tea extract (20 mg/g) over 2 weeks lowered body fat accumulation and boosted EE^{(Reference Choo109)}. This phenotype was accompanied by higher BAT weight with increased total protein content, which is indicative for enhanced thermogenic BAT capacity. In turn, the positive impact of the green tea extract was abolished when the food was spiked with the β-AR inhibitor, propranolol^{(Reference Choo109)}, suggesting that noradrenergic stimulation is required for the observed phenotype. In line with this, EGCG or tea catechins do not directly affect UCP1 mRNA expression levels in BAT. Imaizumi and coworkers^{(Reference Nomura, Ichinose and Jinde110)} reported that in an 8 week dietary intervention a minor (70 %) increase in UCP1 mRNA only when the tea catechins (40 % EGCG, 0⋅1 % caffeine) were administered in low fat diet to male rats^{(Reference Nomura, Ichinose and Jinde110)}. Functionally relevant UCP1 protein abundance was not examined. In comparison, an EGCG (94 %) enriched green tea extract did not alter BAT-UCP1 mRNA levels in male mice after 4 weeks feeding with HFD although total body fat content was reduced^{(Reference Klaus, Pültz and Thöne-Reineke111)}. An initial hypothesis proposed that the thermogenic action of EGCG originates from its ability to inhibit the COMT, resulting in higher norepinephrine levels in the postsynaptic cleft and therefore higher adrenergic input to BAT. COMT methylates, inactivates and initiates the degradation of catecholamines and thereby controls the availability as well as activity of these neurotransmitters. Catechins were shown to exert COMT-inhibitory activity in human subjects, mouse and rat liver extracts, although the determined IC₅₀ values range from 70 nm up to 1, 15 or 55 μm depending on the substrate, catechin type or enzyme preparation used^{(Reference Nagai, Conney and Zhu112–Reference Zhu, Shim and Nagai114)}. Nevertheless, the oral bioavailability of catechins is very low in men and women and plasma concentrations account for approximately 0⋅18 % of ingested catechins (400 mg) from black tea^{(Reference Warden, Smith and Beecher115)}. A pharmacokinetic analysis of daily green tea consumption (615 mg daily) detected peak plasma catechin levels 2 h post-prandially reaching roughly 85 ng/ml whereas EGCG levels were about 50 nm^{(Reference Takahashi, Miyashita and Suzuki116)}. Additionally, catechins are estimated to be cleared from the body within 10–12 h after intake^{(Reference Yang, Chen and Lee117)}. Considering these pharmacokinetic parameters, circulating catechin concentrations are unlikely to reach the required IC₅₀ to exert a meaningful inactivation of COMT. Lorenz et al.^{(Reference Lorenz, Paul and Moobed118)} proved that even a high dose of EGCG (750 mg) does not inhibit COMT activity in vivo when measured in human erythrocytes^{(Reference Lorenz, Paul and Moobed118)}.

An elegant, placebo-controlled crossover study performed in men^{(Reference Yoneshiro, Matsushita and Hibi102)} revealed the direct involvement of BAT in the acute thermogenic effect of a catechin-rich beverage (615 mg). The participants were allocated to either low- or high-BAT groups based on their BAT activity measured with fluorodeoxyglucose positron emission tomography–computed tomography after 2 h of cold acclimatisation. Only the high BAT group displayed a significant increase in EE after the consumption of the catechin drink while there was no difference between the other groups^{(Reference Yoneshiro, Matsushita and Hibi102)}. Using the same technique, the authors found that prolonged daily catechin supply can increase cold-induced thermogenesis in the low-BAT group, which is paralleled by enhanced fat oxidation^{(Reference Yoneshiro, Matsushita and Hibi102)}. Also, in healthy Japanese women a daily 540 mg dose of catechins increased BAT density in the supraclavicular region^{(Reference Nirengi, Amagasa and Homma103)}. These data indicate that catechins can recruit BAT to a physiologically relevant extent. Similar to capsinoids or capsaicinoids (described later), catechins might act through the transient receptor potential (TRP) vanilloid 1 (TRPV1) channels as first anticipated due to their astringent taste. TRPV1 are located on sensory neurons that signal to the central nervous system causing increased catecholamine release to stimulate BAT functionality. In vitro, EGCG has been shown to activate TRP channels in primary cultures of murine dorsal root ganglion neurons as well as in intestinal STC-1 cells^{(Reference Kurogi, Miyashita and Emoto119,Reference Kurogi, Kawai and Nagatomo120)} . The possible excitation of sensory neurons by catechins provides a plausible explanation for the observed threshold-dose of effective EGCG. Chronic exposure of TRP to high levels of capsinoids can induce receptor desensitisation and a comparable phenomenon might happen with high catechin use^{(Reference Sanz-Salvador, Andrés-Borderia and Ferrer-Montiel121)}. In addition, intestinal catechin levels are not affected by the low systemic bioavailability and are likely sufficient to elicit TRP. Interestingly, there seems to be a significant interaction between EGCG (100 μm) and a priming dose of the β3-receptor agonist ephedrine (0⋅1 μm) on oxygen consumption in male rats' BAT explants. The effect of EGCG on BAT respiratory rates was higher in the presence of ephedrine and was more profound with increasing ephedrine levels^{(Reference Dulloo, Seydoux and Girardier122)}. TRP channels are multimodal sensors that integrate distinct signals to fine tune their activity and modulate cellular events. PKA is the downstream effector kinase of β-AR signalling and known to positively affect TRPV1 efficiency^{(Reference Ramsey, Delling and Clapham123)}. Thus, EGCG signalling via TRPV1 might be effectively enforced by the β3-AR–PKA axis. However, up to date corresponding human data are lacking to support the hypothesis concerning catechins and TRP.

Isoflavones: phytoestrogens

Soya represents the most dietary relevant isoflavone source with daidzein and genistein being the most prevalent forms and thus focus of this review. The positive nutritional profile of soya, specified by high protein, high isoflavone and low SFA contents, has long raised interest in weight management strategies, in particular due to favourable satiety and appetite control^{(Reference Leidy, Todd and Zino124)}. Genistein/daidzein share structural similarity to the female sex hormone 17β-oestradiol (E2), which not only masters female reproduction but also profoundly integrates into energy homoeostasis by controlling appetite as well as BAT and WAT functionality through central and peripheral action. Centrally, E2 acts on the ventromedial hypothalamus where it inhibits AMPK-signalling via estrogen receptor (ER) α and enhances sympathetic tone towards BAT and upregulates BAT UCP1 and PGC1α^{(Reference Martínez de Morentin, González-García and Martins125)}. Similar to postmenopausal women, loss of E2 signalling in ovariectomised rats stimulates hyperphagia with resultant weight gain. E2 administration reverses this phenotype by increasing EE through the ERα–sympathetic nervous system–BAT axis and decreasing food intake. E2 is a membrane permeable, hydrophobic molecule and able to tune brown adipocyte function directly by signalling through ERα, ERβ or g-protein coupled receptor 30^{(Reference Prossnitz and Barton126,Reference Suetsugi, Su and Karlsberg127)} . In cultured brown adipocytes isolated from male mice, E2 increases β3-AR mRNA expression and exerts the opposite effect on inhibitory α2-levels, which could potentiate sympathetic inputs^{(Reference Monjo, Rodríguez and Palou128)}. E2 further promotes mitochondrial biogenesis by inhibiting phosphatase and tensin homolog, causing more active AKT-signalling and nuclear translocation of nuclear respiratory factor 1, the head of mitochondrial gene transcription^{(Reference Mattingly, Ivanova and Riggs129,Reference Rodríguez-Cuenca, Monjo and Gianotti130)} . Therefore, the question arises whether the oestrogenic potency of dietary isoflavones is effective at shaping energy homoeostasis to a physiologically relevant extent such as weight loss or stabilisation.

In male mice, HFD supplementation with the isoflavone-rich fraction (9 g/kg) of the Kudzu flower lowered weight gain as well as WAT and BAT weight after 7 weeks. Histological analysis of BAT from treated mice revealed more UPC1+ cells, suggesting that isoflavones activate BAT to burn more fatty acids^{(Reference Kamiya, Nagamine and Sameshima-Kamiya131)}. Comparable findings were reported for male CD-1 mice fed with a soya-enriched (25 % w/w), high phytoestrogenic diet (HP; 150 ppm daidzein, 190 ppm genistein) when compared to a soya-free, low phytoestrogenic diet^{(Reference Cederroth, Vinciguerra and Kühne132)}. This dietary intervention resulted in a 7⋅6 % lower weight gain in HP mice compared to low phytoestrogenic controls with significantly reduced adipose tissue mass. The BAT of HP mice was denser, concomitant with higher EE and apparent cold resistance during an acute cold challenge^{(Reference Cederroth, Vinciguerra and Kühne132)}. As anticipated from the efficacy of exogenous oestradiol administration to ovariectomised rats, an isoflavone-rich diet (200 μg/g) prevented the severity of weight gain in these animals post-surgery^{(Reference Russell, Grimes and Cruthirds133)}. Similar findings, including the upregulation of UCP1 protein levels and higher plasma T3 concentrations, were reported by Lephart et al.^{(Reference Lephart, Porter and Lund134)} using a diet with an isoflavone content of 600 ppm in male rats^{(Reference Lephart, Porter and Lund134)}. They showed that dietary isoflavone ingestion is effective in a model of low E2 and enhances metabolic rates at least partially due to brown fat activation. Several groups investigated the potency of the individual isoflavone types on weight development and EE in various models. In lean or obese rats, enrichment of high fat or standard diet with daidzein (50 mg/kg BW, 14 d) stabilised BW or induced weight loss compared to controls^{(Reference Crespillo, Alonso and Vida135)}. Dose-dependent weight loss after daidzein intake (30 d) was also observed in obese male mice at 50 mg/kg BW (42⋅9 to 33⋅7 g) and at 100 mg/kg BW (42⋅5 to 32⋅4 g) but not at 25 mg/kg BW when compared to vehicle controls^{(Reference Guo, Wu and Su136)}. Although EE was not measured, UCP1 immunofluorescence imaging showed higher UCP1 content in the BAT of the daidzein-exposed group^{(Reference Crespillo, Alonso and Vida135)}. In vitro, daidzein stimulated hormone sensitive lipase mediated glycerol release from primary adipocytes from male rats indicating that elevated adipocyte lipolysis supports daidzein-induced weight loss^{(Reference Guo, Wu and Su136,Reference Szkudelska, Szkudelski and Nogowski137)} . Elevated fatty acid generation in brown adipocyte could enhance EE by directly activating UCP1. Additionally, daidzein was shown to inhibit phosphodiesterase activity at an IC₅₀ of 50 μm^{(Reference Ko, Shih and Lai138)}, a critical enzyme which arrests β-AR signalling by inactivating cAMP.

The literature is more extensive for the efficacy of genistein, probably due to the higher oestrogenicity^{(Reference Chrzan and Bradford139,Reference An, Tzagarakis-Foster and Scharschmidt140)} . In female mice, 8 weeks genistein treatment with HFD (0⋅25 % w/w) resulted in reduced weight gain and lower WAT expansion than in control HFD animals. Within the WAT, brite adipocyte marker genes were upregulated, which was probably centrally controlled^{(Reference Zhou, Xiao and Zhang141)}. Urocortin-3 (UCN3) was among the differentially regulated hypothalamic genes when HFD v. HFD + genistein and HFD v. control animals were compared. UCN3 is an anorexigenic peptide and a known player in energy homoeostasis. Male UCN3 transgenic mice are protected from HFD-induced weight gain^{(Reference Jamieson, Cleasby and Kuperman142)} and intracerebroventricular UCN3 administration to male rats provoked thermogenesis^{(Reference Telegdy, Adamik and Tóth143)} as well as higher BAT UCP1 expression levels^{(Reference Kotz, Wang and Levine144)}. In another recent study, genistein-exposed (0⋅2 % w/w) male mice displayed higher EE and the appearance of UCP1+ cells within the subcutaneous WAT compared to chow-fed controls, confirming the role of genistein in adipose browning^{(Reference Palacios-González, Vargas-Castillo and Velázquez-Villegas145)}. We confirmed a cell autonomous effect of both genistein and daidzein in mature immortalised brown adipocytes, which enhanced UPC1 promoter activity in a UPC1-driven luciferase assay and led to higher UCP1 activity as measured by UCP1-immunofluorescence intensity^{(Reference Buhlmann, Horváth and Houriet146)}. In 4-week old male and female mice, daily administration of genistein (200 mg/kg BW) for 15 d lowered body weight by 4 and 4⋅3 %, respectively due to reduced food intake as well as reduced adipogenic gene expression mediated by ERβ^{(Reference Penza, Montani and Romani147)}. In human fetal brown fat, ERα is the dominant oestrogen receptor type^{(Reference Velickovic, Cvoro and Srdic148)} but the ratio is unknown in adulthood. Ligand assays unveiled higher affinity of genistein and daidzein for ERβ than ERα, demonstrating that isoflavones preferentially activate the oestrogen-response element via ERβ. The EC₅₀s of ERα for genistein (15 μm) and daidzein (>300 μm) are supraphysiological and beyond circulating levels achievable^{(Reference Kostelac, Rechkemmer and Briviba149)} with dietary isoflavone supplementation (100 mg isoflavone capsules daily; thereof 14 mg daidzein and 3–4 mg genistein)^{(Reference van der Velpen, Hollman and van Nielen150)}. Given these concentrations, it is unlikely that isoflavones exert their physiological impact through ERα, fitting to the overall impression that E2-treatment via sympathetic nervous system-ERα signalling elicits more pronounced alterations in energy homoeostasis. Nevertheless, an ERβ-agonist (β-LGND2) conferred anti-obesity effects in male HFD-fed mice^{(Reference Ponnusamy, Tran and Harvey151)}. Pharmacological ERβ activation (30 mg/kg BW daily, s.c.) elevated EE and acute cold tolerance by 14 %. The thermogenic β-LGND2-mediated response culminated in a fat mass loss of 50–60 % compared to control animals. These beneficial effects were dramatically blunted, although not absent, in ERβ-knockout mice. At the molecular level, β-LGND2 stimulated WAT browning and enhanced the mitochondrial respiratory capacity of white adipocytes.

No human clinical trial so far included EE or BAT activation as outcome measures. Most reports^{(Reference Liu, Ho and Chen152–Reference Sites, Cooper and Toth158)} (supplementary material) focus on anthropometric measures and body composition as surrogates for alterations in energy homoeostasis. The interpretation of human trials investigating the efficacy of isoflavone mixtures or individual compounds is complex as interactive factors such as sex, pre-/postmenopausal state, habitual isoflavone exposure/ethnicity and interindividual variation in metabolising enzymes (hepatic or intestinal, gut microflora) need to be taken into account. Within their food matrix, isoflavones exist usually as glycosides (genistin and daidzin)^{(Reference Barnes, Prasain and D'Alessandro159)}. However, they are rapidly hydrolysed to their aglycones after ingestion and undergo intestinal and hepatic metabolisation generating distinct metabolites, among others equol^{(Reference Barnes, Prasain and D'Alessandro159)}. Equol is built from daidzein by intestinal bacteria and is more bioactive than its precursor^{(Reference Atkinson, Frankenfeld and Lampe160)}. Surprisingly, all experimental animals are able to generate equol but not every human subject is an equol producer^{(Reference Muthyala, Ju and Sheng161)}. Subject to differences in the gut microbiome, only 20–35 % of Western individuals are equol-producers, whereas in the Asian region the rate is 50–60 %^{(Reference Arai, Uehara and Sato162–Reference Liu, Qin and Liu164)}. There is also a sex and age bias towards postmenopausal women in the published trials. In this target group, dietary isoflavones might simply aid in minimising or compensating the effects of declining endogenous oestrogen levels and mitigate postmenopausal weight gain. Presently, two meta-analyses dissect in depth the efficacy of isoflavones on body composition and weight development. These studies provide a clearer view on the effects in different subpopulations or influencing factors such as dosing, weight status or trial duration. The meta-analysis from Zhang et al.^{(Reference Zhang, Chen and Guo165)} concentrates specifically on postmenopausal non-Asian women and included nine trials for BW^{(Reference Zhang, Chen and Guo165)}. Soya isoflavone supplementation was associated with a significant lower BW and subgroup analyses revealed that the difference is more pronounced in normal weight than obese women as well as with lower doses (<100 mg daily)^{(Reference Zhang, Chen and Guo165)}. A second analysis performed by Akhlaghi et al.^{(Reference Akhlaghi, Zare and Nouripour166)} includes both sexes, all ethnicities independent of age and comprised forty-seven trials with soya and seventeen trials with isoflavones as investigational subjects^{(Reference Akhlaghi, Zare and Nouripour166)}. BMI in response to isoflavone intake tended to be lower (P = 0⋅085), especially with lower doses and shorter trial length. The subgroup analysis suggests that postmenopausal and Caucasian women likely benefit more from isoflavone intake. Contrarily, soya intake does not affect BW, fat mass or waist circumference. The latter two are also not modified by isoflavone supplementation^{(Reference Akhlaghi, Zare and Nouripour166)}. Taken together, it seems that isoflavones have a mild beneficial effect on BW development and seem to be particularly beneficial in postmenopausal women to account for the reduced endogenous oestrogen levels. Although lower doses below 100 mg daily are more effective, they still exceed the amounts achievable through normal dietary sources, even in populations with high daily isoflavone intake (25–50 mg)^{(Reference Nagata167)}. Thus, isoflavone fortification or supplements would be required to fulfil a physiological relevant intake.

Alkaloids

Alkaloids exert a plethora of pharmacological activities crucial for human pharmacotherapy (e.g. morphine, quinine and vinblastine)^{(Reference Matsuura, Fett-Neto, Gopalakrishnakone, Carlini and Ligabue-Braun168)} and the alkaloids capsaicin (CAP) and berberine (BBR) have been shown to affect BAT activity or induce browning of WAT.

Capsaicinoids, especially CAP, confer the pungent sensation of red chilli from the Capsicum genus^{(Reference Orellana-Escobedo, Garcia-Amezquita and Olivas169,170)} . The thermogenic properties of CAP-rich food are reflected in a variety of physiological responses such as elevated body temperature, flushing, vasodilation and the concomitant onset of cooling mechanisms such as sweating. CAP stimulates afferent sensory neurons involved in thermo- and nociception by binding to TRPV1, which increases intracellular Ca²⁺ concentrations that trigger membrane depolarisation^{(Reference Bevan and Szolcsányi171)}. Many groups have examined the effect of acute or chronic capsaicinoids/capsinoids on EE, fat oxidation, weight management and the contribution of thermogenically active BAT, with diverging outcomes. Early studies investigating the influence of spicy foods using chilli sauce (3 g) or red pepper (30 mg CAP) on metabolic rates in men reported significant increases in postprandial EE by 25–32 % compared to control meals^{(Reference Henry and Emery172,Reference Yoshioka, Lim and Kikuzato173)} . This metabolic effect was abolished when the participants were pre-treated with the β3-AR antagonist propranolol^{(Reference Yoshioka, Lim and Kikuzato173)}, indicating the involvement of sympathetic nerve activity. Higher sympathetic:parasympathetic nervous system activity was confirmed in Caucasian men after the ingestion of a red pepper-spiked (6 g) appetiser compared to a control snack^{(Reference Yoshioka, St-Pierre and Drapeau174)}. In contrast, Smeets et al.^{(Reference Smeets and Westerterp-Plantenga175)} did not find elevated diet-induced thermogenesis after a CAP-containing lunch (approximately 5⋅15 mg) nor altered substrate oxidation in men and women^{(Reference Smeets and Westerterp-Plantenga175)}. However, the effect size might be too small, given a 6-fold lower CAP dose. Another placebo-controlled trial in men failed to identify a significant difference in the RMR of healthy subjects treated acutely with increasing amounts of dihydrocapsiate (0, 3 or 9 mg)^{(Reference Galgani and Ravussin176)}. In vitro, CAP agonises TRPV1 at an effective dose (EC₅₀) of 50–100 nm. Contrarily, the EC₅₀ of capsiate and dihydrocapsiate are 580 and 670 nm, respectively^{(Reference Iida, Moriyama and Kobata177,Reference Sasahara, Furuhata and Iwasaki178)} . This discrepancy in potency between CAP and capsinoids combined with varying doses might resolve the absence of an effect in the dihydrocapsiate trial. A meta-analysis^{(Reference Ludy, Moore and Mattes179)} covering the efficacy of capsiate and CAP intake on EE in human subjects reveals that capsiate enhances lipid utilisation and EE while CAP is only effective on fat oxidation. The effects of CAP on EE was only relevant in high dose trials (135–150 mg CAP), when CAP intake was stratified for dosage^{(Reference Ludy, Moore and Mattes179)}. For both substances, sympathetic nervous activity was stimulated where this outcome was recorded^{(Reference Ludy, Moore and Mattes179)}. Orally administrated CAP and capsiate (10 mg/kg BW) equally boosted oxygen consumption and serum catecholamine levels in mice (unknown sex) when compared to untreated animals^{(Reference Ohnuki, Haramizu and Oki180)}. Kawabata et al.^{(Reference Kawabata, Inoue and Masamoto181)} identified that the acute intragastric administration of capsinoids or CAP (10 mg/kg BW, each) to male mice increased metabolic rate and stimulated fat oxidation in wild type, but not TRPV1 knockout mice, demonstrating the necessity of the vanilloid receptor as thermogenic mediator^{(Reference Kawabata, Inoue and Masamoto181)}. Adrenergic input is a potent activator of the thermogenic BAT programme as well as subcutaneous WAT browning and TRPV1 is expressed in both adipose types. Additionally, murine TRPV1 levels are upregulated in both BAT and WAT following a CAP-enriched diet^{(Reference Baskaran, Krishnan and Fettel182–Reference Kang, Goto and Han184)}. Therefore, it was soon hypothesised that enforced BAT functionality or a bigger brown/brite adipocyte pool contributes to the physiologic effects of CAP. A direct effect of CAP on BAT activity was already implied in 1988, when Yoshida et al.^{(Reference Yoshida, Yoshioka and Wakabayashi185)} demonstrated that the intramuscular injection of 3 mg/kg CAP to rats elevated intrascapular BAT temperature locally and increased mitochondrial oxygen consumption^{(Reference Yoshida, Yoshioka and Wakabayashi185)}. Similarly, 3 h intragastric CAP or capsinoid administration by Kawabata et al.^{(Reference Kawabata, Inoue and Masamoto181)} (see earlier) heated up core and local BAT temperatures only in wild-type but not TRPV1 null mice^{(Reference Kawabata, Inoue and Masamoto181)}. A role of BAT in capsiate action is further supported by an acute capsiate exposure for 30 min that upregulated UCP1 mRNA levels in the BAT and evolved to higher UCP1 protein abundance in the BAT from male mice after 2 weeks of capsiate supplementation^{(Reference Masuda, Haramizu and Oki186)}. A detailed study by Baskaran et al.^{(Reference Baskaran, Krishnan and Fettel182)} substantiates the interplay between CAP, active BAT and TRPV1 in male mice^{(Reference Baskaran, Krishnan and Fettel182)}. The addition of CAP to HFD (0⋅01 % w/w) over a 32 week intervention period prevented weight gain by almost 35 % compared to HFD controls and led to a final BW comparable to mice on standard diet. This impressive BW phenotype was not observed in TRPV1-knockout animals with the same diet regimen. Other rodent studies with males addressing obesity prevention by the supplementation of HFD (0⋅014 %, rat)^{(Reference Kawada, Hagihara and Iwai187)} or standard diet (10 mg/kg, mouse)^{(Reference Ohnuki, Haramizu and Oki180)} with CAP or capsiate up to 14 d equally reported lowered weight gain and stimulated fat loss from WAT compared to controls^{(Reference Ohnuki, Haramizu and Oki180,Reference Kawada, Hagihara and Iwai187)} . Regarding obesity treatment, CAP feeding attenuated additional weight gain in already obese male mice during a 10 week follow-up period (36⋅5 v. 41 g), resulting in reduced adiposity^{(Reference Kang, Goto and Han184)}. Furthermore, CAP feeding lowered circulating TAG levels, which put forward the idea that CAP mobilises lipids from adipose depots and enhances lipid oxidation, likely in the BAT^{(Reference Ohnuki, Haramizu and Oki180,Reference Kawada, Hagihara and Iwai187)} . Apart from elevated EE^{(Reference Ohnuki, Haramizu and Oki180,Reference Baskaran, Krishnan and Fettel182,Reference Baskaran, Krishnan and Ren183)} , the RER of CAP-HFD male mice was significantly higher^{(Reference Baskaran, Krishnan and Fettel182,Reference Baskaran, Krishnan and Ren183)} and points towards increased carbohydrate oxidation. Others found enhanced fat^{(Reference Kawabata, Inoue and Masamoto181,Reference Masuda, Haramizu and Oki186)} and reduced carbohydrate^{(Reference Kawabata, Inoue and Masamoto181)} oxidation after CAP-administration, which underlines a higher metabolic flexibility in these animals. Indications for metabolically more activated CAP-BAT were further evident in the heightened glycerol release from this tissue as surrogate for lipolysis in comparison with HFD-BAT^{(Reference Baskaran, Krishnan and Fettel182)}. The lipolytic principle of CAP is very likely mediated by augmented catecholamine secretion and signalling through the β-AR–PKA–hormone-sensitive lipase pathway. Acute stimulation of brown adipocytes from male CAP-HFD mice with 1 μm CAP dramatically increased intracellular calcium influx, which was blunted in TRPV1-lacking adipocytes^{(Reference Baskaran, Krishnan and Fettel182)}. A downstream analysis of the molecular events revealed that CAP activates TRPV1 leading to higher calcium levels and the activation of CaMKII, which in turn phosphorylates and activates AMPK. AMPK stimulates SIRT1 activity triggering the deacetylation of PGC1α and PPARγ as ultimate enhancers of BAT functionality. Baskaran and coworkers^{(Reference Baskaran, Krishnan and Ren183)} demonstrated the browning of the inguinal WAT in response to CAP diet, based on the same molecular mechanism^{(Reference Baskaran, Krishnan and Ren183)}.

Human clinical trials evaluating the long-term effect of CAP/capsiate on weight development are rare (supplementary material). A study by Janssens et al.^{(Reference Janssens, Hursel and Martens188)} implies a role of CAP supplementation (2⋅56 mg/meal) in weight maintenance after weight loss in male and female subjects. Accordingly, the acute thermogenic effect of CAP could be sufficient to compensate for the reduction in EE that goes along with weight loss and energetic restriction^{(Reference Janssens, Hursel and Martens188)}. In a US placebo controlled-trial with obese male and female participants (mean BMI 30 kg/m²) the daily ingestion of 6 mg capsinoids over a 12 weeks trended in a BW change (0⋅9 v. 0⋅5 kg) and significantly reduced visceral adiposity^{(Reference Snitker, Fujishima and Shen189)}. However, more of these long-term studies with higher doses or in relation to weight stabilisation after weight loss are required to delineate the pharmacological potential of capsinoids. A recent crossover study directly captured BAT-activity during an acute capsinoid (12 mg) trigger in men and women by luorodeoxyglucose positron emission tomography–magnetic resonance in comparison with cold-exposure using a cold vest (14⋅5°C). The mean standardised uptake value (SUV) for the capsinoid group did not reach the accepted threshold (SUV = 2) to classify a subject as BAT-positive, contrary to the cold-exposed group (mean SUV = 2⋅9). Only after an adjustment of the grey scale values, a mild uptake of glucose was notable in the capsinoid group. Nevertheless, the mean EE post-capsinoids administration was higher compared to baseline levels and this increase was significantly greater in subjects allocated as BAT-positive based on cold-exposed BAT detectability^{(Reference Sun, Camps and Goh190)}. Unfortunately, this study lacked a placebo control although the capsinoids were ingested as capsule containing additionally a mixture of rapeseed oil and medium chain TAG. A placebo-controlled, crossover trial with eighteen Japanese men verified an acute increase in EE after capsinoids intake (9 mg) in BAT-positive subjects when compared to placebo controls and BAT-negative individuals^{(Reference Yoneshiro, Aita and Kawai191)}.

In conclusion, it appears that the physiological outcomes associated with spicy food/CAP ingestion are the sum of four primary events: (1) enhanced sympathetic activity due to the stimulation of vagal afferent neurons and catecholamine levels, which (2) promote lipolysis in WAT and BAT and (3) activate non-thermogenesis in BAT; (4) the direct, cell autonomous trigger on adipocytes via TRPV1–AMPK–SIRT1 to enhance the thermogenic phenotype. The documented bioavailability (50–90 % absorption) of capsaicinoids is high^{(Reference Kawabata, Inoue and Masamoto181)}. In mice, circulating plasma CAP concentration 1 h after an oral dose of 10 mg/kg BW reaches approximately 3⋅8 μm^{(Reference Rollyson, Stover and Brown192)}. This value falls into a concentration range that induced browning in vitro in 3T3-L1 and indicates that circulating CAP levels are sufficient to elicit direct cellular effects^{(Reference Baboota, Singh and Sarma193)}.

BBR is arguably the most prominent known phytochemical capable of modulating BAT function and several preclinical studies have delivered interesting outcomes regarding the therapeutic impact of BBR on obesity-associated metabolic diseases. BBR has a long history of use in traditional medicines and interest in the metabolic side of BBR awoke only in 2004 when the blood cholesterol-lowering activity of BBR in hypercholesterolemic patients was published^{(Reference Kong, Wei and Abidi194)}. Another study underpinned the therapeutic potential of BBR in genetically and fat-induced obese rodents^{(Reference Lee, Kim and Kim195)}. In male db/db mice a daily dose of BBR (5 mg/kg BW, i.p.) caused a 13 % weight loss with a 10 % reduction in fat mass within 26 d of treatment alongside with improved whole body metabolic homoeostasis^{(Reference Lee, Kim and Kim195)}. The same physiological effects were observed in male HFD-fed rats treated with either vehicle or BBR (380 mg/kg BW, orally)^{(Reference Lee, Kim and Kim195)}. In WAT, BBR downregulated lipogenic genes while oxidative and genes regulating mitochondria formation/function were enhanced in muscle and BAT (PPARα and PGC1α)^{(Reference Lee, Kim and Kim195)}. In 3T3-L1 cells, they subsequently identify that BBR acts as an AMPK activator, stimulating the phosphorylation of AMPK and acetyl-CoA carboxylase^{(Reference Lee, Kim and Kim195)}, possibly by inhibiting complex I causing a rise in the cellular AMP:ATP ratio^{(Reference Turner, Li and Gosby196)}. These findings motivated further research exploiting the effects of BBR on BAT activity as AMPK plays an accepted role in BAT-mediated thermogenesis and EE^{(Reference Perdikari, Kulenkampff and Rudigier197–Reference Wu, Zhang and Li199)}. Zhang et al.^{(Reference Zhang, Zhang and Li200)} first reported a thermogenic effect by BBR^{(Reference Zhang, Zhang and Li200)}. Apart from the established metabolic and weight improvements, BBR-therapy to male db/db mice (5 mg/kg BW, i.p.) increased rectal temperature (about 1⋅5°C), consistently enhanced metabolic rate and defended body heat loss during cold exposure compared to vehicle treatment. Most strikingly, BBR dramatically enforced BAT thermogenesis directly as reflected by higher 18-FDG uptake monitored using micro-positron emission tomography–magnetic resonance. This activated BAT state was supported by reduced BAT mass, higher mitochondrial content and molecular evidence such as enhancement of the BAT-specific mRNA and protein signatures (PGC1α, UCP1, nuclear respiratory factor 1 and carnitine palmitoyltransferase) through activation of AMPK^{(Reference Zhang, Zhang and Li200)}. BBR likewise stimulated browning of the inguinal WAT. Interestingly, immunohistological analysis revealed higher tyrosine hydroxylase levels in the WAT and BAT, suggesting the contribution of augmented sympathetic output to the observed phenotype. When the animals were housed under thermoneutral conditions (30°C) to minimise BAT function, the beneficial effects of BBR administration were blunted, which strongly supports the necessity of active sympathetic activity as physiologic mediator of BBR activity^{(Reference Zhang, Zhang and Li200)}. WAT-browning and elevated EE were also noted in male mice on HFD treated for 5 weeks with BBR (5 mg/kg BW i.p.) or vehicle^{(Reference Sun, Xia and Yan201)}. Additionally, BBR upregulated hepatic FGF21 gene expression resulting in a concomitant elevation in plasma FGF21 levels, which was dependent on activated SIRT1, a known regulator of FGF21 transcription^{(Reference Li, Wong and Giles202)}. FGF21 was shown to promote WAT browning as well as the thermogenic BAT programme elsewhere and might depict an alternative mechanism of action of BBR^{(Reference Hondares, Iglesias and Giralt203,Reference Fisher, Kleiner and Douris204)} . Wu et al.^{(Reference Wu, Xia and Duan205)} clearly show the requirement of intact AMPK signalling in adipose tissues for an effective BBR treatment as the physiological responses to BBR administration were absent in adiponectin-Cre driven AMPK-floxed male mice^{(Reference Wu, Xia and Duan205)}.

In the same work^{(Reference Wu, Xia and Duan205)}, the effect of 1 month dietary BBR supplementation (3× 500 mg daily) on human BAT activity was examined in overweight individuals. Using cold-stimulated fluorodeoxyglucose positron emission tomography–computed tomography imaging, they prove that human BAT is activated by BBR as mean SUV (2⋅6–3⋅3), BAT mass (14⋅1–25⋅5 cm³) and activity (103⋅1–228⋅2 ml × SUV_ave × g/ml) were all significantly increased in a paired before–after analysis. Patients (n 2) with no detectable BAT before treatment were not BAT-positive after 1 month of BBR intake. Thus, BBR is not sufficient to stimulate BAT formation or induce dormant brown adipocytes. The percentage change of BAT activity before–after BBR negatively correlated with the measured changes in BW and highlight the potency of BBR to combat overweight by means of BAT activation. A future pharmacological application of BBR to tackle obesity and associated metabolic complications is supported by additional human clinical trials. An open-label study in China investigated the effect of a lifestyle intervention (LSI) with or without daily BBR (3× 500 mg) or pioglitazone (15 mg) treatment on BW and metabolic health in individuals suffering from non-alcoholic fatty liver disease with diabetes or impaired glucose tolerance^{(Reference Yan, Xia and Wang206)}. At the end of the intervention, the BBR group lost significantly more weight (−4⋅29 v. –1⋅44 kg) and waist circumference (−4⋅84 v. –2⋅14 cm) than LSI only, but also when compared to LSI plus pioglitazone. As there was no significant difference between LSI + pioglitazone and LSI + BBR on insulin sensitivity, glucose tolerance or glycated Hb levels, BBR improved glucose homoeostasis comparably to a known antidiabetic remedy with the additive effect of weight loss^{(Reference Yan, Xia and Wang206)}. BBR was equally effective in placebo-controlled trials with Chinese type 2 diabetic and dyslipidaemic patients (1 g daily)^{(Reference Zhang, Li and Zou207)} or poor glycaemic control (1⋅5 g daily)^{(Reference Yin, Xing and Ye208)}. The BBR group displayed significant improvements in BW (66⋅4 v. 70⋅5 kg), BMI (24⋅3 v. 25⋅4 kg/m²) and glycaemic control (fasting blood glucose 5⋅6 v. 6⋅4 mm/, HbA1c 6⋅6 v. 7⋅3 %) compared to controls at the end^{(Reference Zhang, Li and Zou207)}. In a small pilot study (n 7) with obese Caucasian individuals over 13 weeks (1500 mg BBR daily) moderate changes in BW (2⋅3 %), BMI (2⋅9) and fat content (3⋅6 %) were determined compared to baselines values. Although these changes seem promising, they did not reach statistical significance likely due to the small sample size^{(Reference Hu, Ehli and Kittelsrud209)}. The available studies centring on BW control or weight loss are sparse and dominated by Asian subjects (supplementary material). Nevertheless, the reported outcomes are overly encouraging and underline the need for further efforts aiming at different ethnicities and larger sample sizes. Furthermore, it would be interesting to study whether BBR enhances EE in human subjects. One of the current drawbacks is the extremely poor bioavailability of BBR in human and animal studies. In human subjects, oral BBR intake (300 mg daily) resulted in a mean plasma BBR concentration of 0⋅3 ng/ml and an acute BBR (400 mg) challenge led to a maximal concentration of 0⋅4 ng/ml^{(Reference Hua, Ding and Chen210)}. BBR combined with polyethylene glycol enhanced intestinal BBR absorption in rats by a factor of 3, indicating that the pharmacokinetic properties of BBR are adjustable with appropriate encapsulation strategies^{(Reference Chen, Miao and Fan211)}. Improved BBR bioavailability is desirable as it affects the effective treatment dose and might reduce some of the mild side effects (e.g. constipation and intestinal discomfort) observed with the currently employed BBR dose of 1500 mg daily. Alternatively, the efficacy of the more absorbable analogue dihydroberberine with five times higher bioavailability than BBR in rats could be investigated for human use^{(Reference Feng, Shou and Zhao212)}.