This is the first study to determine that the stimulatory effects of caffeine on UCP1 seen in vitro can be translated to adult humans ingesting caffeine in a commonly consumed coffee beverage. The increase in temperature of the region which co-locates with BAT observed with caffeine ingestion is indicative of an increase in BAT activity32 following a relatively low dose of caffeine33 from a single standard cup of coffee. It is supportive of our in vitro observations in human and murine stem-cell derived adipocytes, in which the abundance of UCP1 was increased, together with oxygen consumption, and markers of mitochondrial and lipid metabolism. These findings suggest that the increase in metabolic rate following caffeine ingestion could be mediated by enhanced BAT function, which precedes any change in skin temperature34.

Enhanced BAT activity in adult humans has the potential to improve lipid and carbohydrate homeostasis as well as contributing to weight loss strategies1. The extent to which individual components of the diet can activate BAT in humans is not well established, as the gold standard to assess BAT function is the uptake of radio-labelled glucose (fludeoxyglucose or 18FDG) as measured by PET-CT5,8. Normally, this has to be undertaken in fasted subjects, as feeding is accompanied by such a large increase in glucose uptake by muscle that the capacity to detect BAT is greatly reduced35. We have, therefore, developed thermal imaging as a practical method to detect in vivo changes in BAT activity and which can detect changes in response to ingestion without requiring exposure to radiation and which correlates well with the uptake of 18FDG following cold exposure24. Moreover, there is increasing evidence that dietary ingredients promote brown fat function9, responses which may be mediated by the release of secretin from the gut27.

It has been suggested that thermal imaging may not give an optimal measure of BAT36,37, however, such studies have not considered the changes in temperature of the supraclavicular region with an appropriate reference point that correlate well with BAT function as assessed by PET-CT24. In addition, single point measures of supraclavicular temperature can be misleading as they cannot reliably detect the hottest region of brown fat as identified with thermal imaging approach used here38. Although caffeine has been reported to cause changes in blood flow39,40,41, this was not expected to cause substantial changes in skin temperature, as this typically does not occur until at least 2 h after caffeine consumption34. Further studies measuring the kinetics of caffeine-induced blood flow changes can help refine its impact on thermal analysis.

Temperature, together with intact innervation42 are primary factors regulating the magnitude of change in brown and/or beige fat function, which is further modulated by the relative amount of UCP1 within a depot. Consequently, transferring mice from 22 to 5 °C results in a modest rise within interscapular BAT, but a much larger increase within inguinal beige fat43. It is thus likely that comparatively small changes in the amount of UCP1 can impact on energy balance, a proposal supported by the present study. We demonstrate that in beige adipocytes20, in which caffeine exposure increased the amount of UCP1 by ~20%, in conjunction with more mitochondria and lipid, this was accompanied by a marked rise in oxygen consumption in vitro. The magnitude of change was, however, lower than that seen when comparing mitochondria sampled from perirenal fat in humans with pheochromocytoma patients living in a warm climate44. It would thus be useful to further examine the extent to which comparable outcomes are found in vitro in brown as opposed to beige adipocytes.

Previous studies reported that caffeine inhibits differentiation, promotes lipolysis and suppresses intracellular lipid accumulation in adipocyte cultures21,45,46,47, while increasing BAT temperature, guanosine-5′-diphosphate (GDP) binding and oxygen consumption in mitochondria in mice48. The present study found that caffeine used at 1 mM upregulated not only UCP1 but also primary regulatory genes including PPARγ, PRDM16, and PGC-1α49. Caffeine, therefore, activated the main regulators of brown adipocyte thermogenesis50,51,52, but also gene expression of CD137, LHX8, P2RX5, CITED1 and COX8b, that are indicative of molecular conversion of white/beige cells into brown adipocytes. Consistent with these molecular changes induced by caffeine was the appearance of smaller lipid droplets in UCP1-expressing adipocytes, a major morphological sign of lipolysis49. The response to caffeine exposure was observed to vary in human and mouse cultures, in line with the different level of lipid accumulation noted under adipogenic induction in these models, pointing to intrinsic differences which will require further characterization. The recruitment of thermogenesis results in mitochondriogenesis, a process controlled by PGC-1α50,53,54, and was here accompanied by increased PGC-1α mRNA expression and PGC-1α protein nuclear localization indicating PGC-1α functional activity in caffeine-treated cells. In line with these observations, increased mitochondrial biogenesis and PGC-1α expression after caffeine treatment have been reported in other cell types55,56,57. PGC-1α not only triggers mitochondrial biogenesis, but also activates several components of the adaptive thermogenic program in BAT, including fatty-acid oxidation, and increases oxygen consumption through co-activation of transcription factors such as PPARγ53. These molecular adaptations with caffeine exposure were accompanied by changes in mitochondrial ultrastructure and the redistribution of adjacent small lipid droplets. The numerous lamellar cristae observed in caffeine-treated adipocytes are characteristic of activated beige/brown adipocytes that expand the surface of their inner mitochondria membrane with enhanced functional UCP14. These contact sites constitute an important way of inter-organelle communication58 in which fatty acids liberated by lipolysis could be used for β-oxidation. Future analysis of sorted lipid-laden cells could provide a more specific measure of the mitochondrial response to caffeine treatment. The TRPV receptor family can also modulate beige/brown adipocyte thermogenesis, and TRPV4 can be a negative regulator of PGC1α59, so caffeine might further induce PGC1α and UCP1 expression by antagonizing TRPV4. The capacity of caffeine-treated adipocytes to undergo mitochondrial uncoupling was confirmed by enhanced bioenergetic capacity accompanied with an increase in ATP-producing metabolic pathways, i.e. oxidative phosphorylation and glycolysis which are indicative of higher energy demands observed during the browning process20, although additional pathways besides UCP1-dependent mechanisms could also be involved. The low Ct values observed here for UCP1 may be consistent with this, and might also underline differences in between the half-life of UCP1 protein (2–20 days) and UCP1 mRNA (3 h)60,61.

Besides acting as an adenosine receptor antagonist, caffeine inhibits phosphodiesterase, increasing intracellular cAMP62, a process also activated by β-adrenergic stimulation4,63. This could be the mechanism by which caffeine enhances UCP1 function. Coupled to G proteins, β3-AR induces cAMP formation, which in turn activates protein kinase A and stimulates fat hydrolysis, with the free fatty acids released activating UCP14,64. Adenylyl cyclase (which synthesizes cAMP from ATP65) is positively coupled to β3-AR but inhibited by ARα266, which was observed here to decrease with caffeine exposure. Taken together, the morphological evidence of lipolysis, plus reduced ARα2 gene suppression induced by caffeine, supports an antilipolytic effect67. Further experiments investigating the effects of PKA inhibition could provide additional information on the role of cAMP levels in the caffeine response.

In conclusion, these results provide new complementary in vitro and in vivo evidence that caffeine (and a coffee beverage) can promote BAT function at doses compatible with human use. Similar approaches could be considered to screen other potential dietary compounds that could target UCP1 and promote BAT function. Future intervention studies can now be undertaken to assess whether caffeine-induced BAT activation in humans is dose-dependent, refine the minimal intake required for a BAT response, and explore whether comparable effects are seen in fully differentiated adipocytes and primary cells, as well as in diabetic and/or obese individuals.