glucagonlike peptide1 ( glp1 ) was first identified in 1983 , cloned by graham bell as part of the preproglucagon sequence , with the incretin effect being published in 19871 , 2 , 3 . as the other incretin , glucosedependent insulinotropic polypeptide ( gip ) , has little effect on insulin secretion in patients with type  2 diabetes , drugs giving pharmacological levels of glp1 receptor ( glp1r ) agonists have become successful therapies in the treatment of type  2 diabetes , providing glucose control as well as weight loss4 . glp1 was first shown to lower food intake in rodent animal models in 1996 , followed by a study in humans in 19985 , 6 , 7 . the study in humans showed a reduction in energy intake , and also reported that the mechanism involved a reduction in appetite along with an increase in satiety and a reduction in feelings of hunger , as shown by use of a visual analog scale questionnaire7 . the most recently approved glp1r agonists for management of type  2 diabetes are dulaglutide and albiglutide , which have been developed for onceweekly administration . however , as more compounds are approved and results from an increasing number of large , randomized , controlled , doubleblinded trials have been published , treatmentassociated weight loss in patients with type  2 diabetes differs between compounds . liraglutide is a oncedaily compound acylated with a fatty acid to facilitate noncovalent binding to albumin in  vivo as the protraction mechanism8 . although there were some differences in glycemic control , a consistent finding was that liraglutide resulted in greater weight loss than dulaglutide and albiglutide , which are much larger molecules modified with covalent addition of either a crystallizable fragment or an albumin molecule8 , 9 , 10 . furthermore , of the five glp1r agonists now approved in treatment of type  2 diabetes in various regions of the world , liraglutide is the only one that has also been investigated and approved for weight management , as adjunct to diet and exercise11 . the present minireview focuses on a novel pathway in the brain that might mediate the weightlowering effect of glp1r agonists , and reviews these data in relation to the rather large existing literature for glp1r action in the brain as well as in the periphery12 . using the novel technique of singleplane illumination microscopy ( spim ) , whereby the entire perfused brain is scanned after peripheral administration of fluorescently labeled liraglutide ( liraglutide ) in live mice , twodimensional digital images of the entire brain were obtained and assembled into a threedimensional image for optimal analysis of spatial distribution . figure  1 shows a dorsoventral ( figure  1a ) , or sagittal ( figure  1b ) , threedimensional plane view of liraglutide distribution in the mouse brain . as can be seen , regions with clear uptake were determined to be the paraventricular nucleus of the hypothalamus ( pvn ; figure  1c , d ) ; the arcuate nucleus ( arc ) and the median eminence ( me ) of the hypothalamus ( figure  1f , g ) ; and the area postrema ( ap ) in the hindbrain , whereas there was no uptake in the nucleus tractus solitarus in the hindbrain ( figure  1i , j ) . the signal in all sites of uptake were found to depend entirely on expression of the glp1r , as no uptake was detectable at these sites in glp1r mice ( figure  1e , h , k ) . uptake was also found in the organum vasculosum of the lamina terminalis , the subfornical organ , the supraoptic nucleus and the supraoptic decussation , as well as the choriod plexus . except for the choriod plexus , the uptake patterns for liraglutide in the pancreas and the hypothalamus were compared with the patterns of the specific glp1r antagonist exendin(939 ; figure  2 ) . a different flourophore was used for these experiments , enabling costaining for insulin or cocaine and amphetaminestimulated transcript ( cart ) , respectively . in the pancreas , liraglutide was found to colocalize entirely with pancreatic cells , as evidenced by an overlap with insulin staining . also , the liraglutide signal was present within the cells as evidence of internalization with the glp1r ( figure  2i , j ) . in contrast , exendin(939 ) was retained at the plasma membrane of the cells ( figure  2k , l ) . in the brain , liraglutide also appeared to be present within the neurons ( figure  2m , n ) , whereas exendin(939 ) appeared to be retained at the membrane . however , in the brain , internalization is more difficult to assess because of the high density of neuronal fibers . to further examine the precise localization of liraglutide in the arc , costaining with the cart neuropeptide that labels cart / proopiomelanocortin ( pomc ) neurons was carried out ( figure  3 ) . liraglutide was detected specifically in the arc in the cytoplasm of neurons positive for cart ( figure  3 ) . however , a few cells were only positive for liraglutide , showing that another cell type might be targeted by liraglutide in the hypothalamus . representative whole brain images viewed in the ( a ) dorsoventral or ( b ) sagittal plane from c57bl/6j mice given liraglutide ( unspecific staining has been removed from the left side of the brain ) . the brain tissue was scanned at 620  nm and 710  nm , representing both autofluorescence from the tissue ( gray ) and specific signal ( green ) . the ( c , f , i ) red regions are shown at ( d , g , j ) higher magnification , respectively . ( d , e , g , h , j , k ) highmagnification views of a single section from ( d , g , j ) c57bl/6j or ( e , h , k ) glp1r 
  mice given liraglutide . liraglutide was detectable in ( c , d ) paraventricular nucleus of the hypothalamus ( pvn ) , ( f , g ) the median eminence ( me ) , the arcuate nucleus ( arc ) , and ( i , j ) area postrema ( ap ) . ( e , h , k ) in mice lacking a functional glucagonlike peptide1 receptor ( glp1r ) , no liraglutide signal could be detected in any of these regions . scale bars , 200  m ( a , b , c , f , i ) ; 50  m ( d , e ) ; 100  m ( g , h , j , k ) . ( a  l ) representative images of mouse islets stained with hoechst nuclear stain ( blue ) , insulin ( green ) and liraglutide / exendin(939 ) ( red ) . ( a , c , e , d ) insulin and liraglutide / exendin(939)positive cells . ( b , d , f , h ) the same images as in ( a , c , e , d ) with only hoechst and liraglutide / exendin(939 ) signal . ( a  d ) in c57bl/6j mice , both liraglutide and exendin(939 ) were detected in cells expressing insulin ; ( e  h ) however , in mice lacking a functional glucagonlike peptide1 receptor , no liraglutide or exendin(939 ) signal could be detected in insulin expressing cells . ( i , j , n ) highmagnification images showed that liraglutide was internalized and the fluorescent signal was located in the cytoplasm , ( k , l , p ) while exendin(939 ) remained at the plasma membrane . in the brain , ( m , n ) liraglutide had access to arcuate nucleus ( arc ) , in which it bound the glucagonlike peptide1 receptor and internalized , ( o , p ) whereas exendin(939 ) labeled the same population of cells , but without internalization . scale bars , 100  m ( m , o ) , 50  m ( a  h ) , 10  m ( i  l , n , p ) . ( a  c ) hypothalamic sections from rats injected with liraglutide ( red ) and stained with hoechst nuclear stain ( blue ) and cocaine and amphetaminestimulated transcript ( green ) . ( b , c ) highmagnification confocal images showed accumulation of fluoro liraglutide in the cytoplasm of cocaine and amphetaminestimulated transcriptpositive cells ( arrows ) . ( c ) the same image as in ( b ) with only liraglutide signal . scale bars , 25  m ( b , c ) ; 100  m ( a ) . it has been suggested that physiologically the inhibitory effect of glp1 on gastric emptying might be more important than the incretin effect13 . delayed gastric emptying leading to prolonged gastric distention might induce shortterm satiety , and could therefore be the relevant mechanism for the postprandial changes in appetite induced by physiological doses of glp1 . however , such mechanisms are unlikely to cause the lasting effects on fasting and postprandial appetite seen with treatment with longacting glp1r agonists in patients with and without type  2 diabetes14 , 15 , 16 . with continued exposure beyond the duration of a normal postprandial period , similarly , liraglutide and other longacting glp1r agonists have little effect on gastric emptying , making it unlikely that this is the main weight loss mechanism18 , 19 . the brain is the integrating site for appetite regulation , and numerous studies have documented the expression and importance of glp1rs in the brain , and its importance in the physiological regulation of appetite20 . glp1 is also produced in the hindbrain , and has been proposed as a physiological appetite reduction signal21 . peripherallyacting glp1r agonists might communicate with the brain through the nodose ganglion where glp1rs are expressed , either in the form of glp1 released postprandially from the l  cells in the gastrointestinal tract or in the form of an exogenously dosed glp1r agonist . sisley et  al.22 described two novel animal models where mice were genetically engineered to be knockdown models for brain or nodose ganglion glp1r expression , respectively . they found that the majority of liraglutideinduced weight loss required brain glp1r expression , whereas similar weight loss was obtained in normal and nodose ganglion glp1r expression knockdown mice . hindbrain glp1rs expressed in the ap are accessible to peripherallydosed liraglutide as shown in figure  1j . while the exact role of ap glp1rs have not been determined , numerous studies support the role of hindbrain glp1 and glp1rs in appetite regulation where they communicate through projections to other sites of the brain , and take part in a complex system integrating homeostatic and likely also hedonic parts of appetite regulation23 , 24 , 25 . secher et  al.12 showed that the ap was not required for the weight loss effect of liraglutide . however , as documented in numerous studies , the hindbrain might still be an important site for integration of physiological effects of glp1 ; it is just not required for mediating the effects of pharmacological doses of a glp1r agonist on bodyweight loss . as also aforementioned , some studies have examined the importance of the vagus nerve in the appetite effect of glp1 . acute dosing studies have shown that the vagus nerve is involved in mediating a reduction in food intake in both rodent models and in humans14 , 26 . however , studies in animals with chronic exposure have shown that the vagus nerve is not required12 , 16 . no human studies have used a chronic dosing regime and addressed the importance of the vagus nerve . the study by secher et  al.12 is the first to show that a peripherallydosed glp1r agonist ( liraglutide ) can directly access and affect the hypothalamus in the rodent brain . data in that study showed that liraglutide might have important effects on the cart / pomc neurons that it is shown to access directly . using the native ligand , glp1 , glp1rs in cart / pomc neurons using electrophysiological methods , glp1 was shown to cause dosedependent membrane depolarization and an increased firing rate of spontaneous action potentials . furthermore , the effect was shown to be postsynaptic , in agreement with a potential direct effect of peripherallydosed liraglutide . importantly , liraglutide was shown to increase the messenger ribonucleic acid levels of cart in the arc , and to keep levels of neuropeptide  y ( npy ) and agoutirelated peptide ( agrp ) at the level of normal control animals , whereas reducedfeed control animals weightmatched to those on liraglutide had increased levels of both npy and agrp . the effect on agrp / npy is suggested to be indirect inhibition of the agrp / npy neurons through a local inhibitory gammaaminobutyric acid neuron , as agrp / npy neurons do not have glp1rs . the main primary neurons in the arc are the cart / pomc neurons and the agrp / npy neurons . appetiteinhibiting neurons contain pomc peptides , such as melanocytestimulating hormone and cart . melanocytestimulating hormone acts on melanocortin anoxigenic mc3 and mc4 receptors . appetitestimulating neurons in the arc contain npy , which acts on the orexigenic  y1 and y5 receptors ; and agrp , which is an antagonist on mc3/4 receptors . npy and agrp are the most important regulatory peptides made in the same neurons in the arc that both act to increase food intake ; similarly important are neurons in arc that coexpress pomc and cart signals , which reduce food intake27 , 28 , 29 , 30 , 31 . the direct access of liraglutide to this part of the brain , along with the uptake in cart / pomc neurons and the effects of increasing levels of cart , and maintenance of the low levels of npy and agrp , highlight a likely important new pathway of previously , mainly insulin and leptin have been shown to be transported into the brain , and to have direct effects in the hypothalamus ; recent evidence proposes that specialized tanycytes are mediating the uptake through the me28 , 32 , 33 . schaeffer et  al.34 recently showed circulating acylated ghrelin to be transported to the arc and to have directs effects there , much like what is now proposed for glp1r agonists , such as liraglutide . it is likely that the vast distribution of glp1rs in the brain , along with integration of peripheral signals to the hindbrain , makes up a complicated interplay between the hypothalamus and hindbrain structures , including the nucleus tractus solitarus where glp1 is produced and which receives vagal afferent inputs . inputs from higherorder areas of the brain involved in reward and cognition are integrated as well , with resultant effects on meal size and frequency , gut handling of ingested food , and energy expenditure . however , the way native glp1 affects postprandial appetite under physiological conditions is different from the way pharmacological exogenous doses of longacting glp1r agonists affect appetite . as such , it is important to discriminate between those studies where glp1 or glp1r agonists are dosed directly into localized places in the brain , and those where pharmacological doses are injected peripherally . with so many different glp1r sites of expression in the brain , it will be very complicated to evaluate the exact importance of each individual site in the total resulting appetite regulation . use of geneticallyengineered mice might be able to show some further details , but then the precise relationship to humans will need to be investigated . in fact , the only striking difference in glp1r expression between rodents and primates in the brain is that there seems to be much more glp1r expression in the primate brain in areas involved in hedonic aspects of food intake , such as the amygdala and the bed nucleus of the stria terminalis35 . although it is unclear how all these signals integrate , glp1 has been shown to reduce both basal and induced reward , and to change food preference in rodent animal models36 , 37 , 38 . raun et  al.36 showed that when rats dosed with liraglutide for 3  months were given a choice between different kinds of candy and chocolate vs normal chow , they selectively chose to eat less chocolate and candy , and more chow . glp1r agonists have been shown to lead to taste aversion in rodents , and to nausea in humans ; however , in rodents these effects are very shortlived ( typically only 12  days ) , so the effect shown by raun et  al.36 is unlikely to be a taste aversion effect . hansen et  al.37 compared liraglutide with sibutramine in a model where rats were offered a choice between a chocolate and hazelnut spread ( nutella ; ferrero spa , pino torinese , italy)/peanut butter and chow paste / regular chow ; both compounds led to a reduction in nutella / peanut butter paste , but only liraglutide led to an increase in chow intake , as in raun et  al . nausea is a relatively common sideeffect with glp1r agonist treatment in humans with and without type  2 diabetes , but also transient and dosedependent  although it does last longer than in rodents , it is not believed to be the mediator of chronic weight loss39 . no human studies have yet investigated whether longterm glp1r agonist treatment in humans leads to changes in food preferences and/or eating behavior , but studies using functional magnetic resonance imaging scanning have shown an involvement of parts of the brain known for involvement in hedonic aspects of food intake40 , 41 . most of the aforementioned areas in the brain , to which liraglutide has direct access , and further areas described in secher et  al.12 
 , are socalled circumventricular organs ; that is , areas of the brain that have no classical blood the ap , me , the subfornical organ and the organum vasculosum of the lamina terminalis are circumventricular organs . although it is logical that a peptide , such as glp1 , and other druglike glp1r agonists can bind to glp1rs in circumventricular organs , it was perhaps less expected that pharmacological doses of a glp1r agonist , such as liraglutide , could reach areas in the brain that are protected by the blood  brain barrier . the pvn and the arc are welldescribed areas for glp1r action in the brain , but direct access from the periphery has not been described before . brain barrier , but in light of the very specific binding in select regions as shown here , perhaps those older kinds of studies need to be rethought as a methodology where the entire brain parenchyma is extracted seems less well suited for a very specific pattern of uptake42 , 43 . one is that certain parts of the hypothalamus , like the ventromedial arc and the pvn , might be supplied directly by fenestrated capillaries : this could be consistent with the pattern of uptake seen with liraglutide44 , 45 . the other hypothesis is based on leptin and ghrelin studies , where specialized ependymal / glia cells called tanycytes , expressed in the me , form part of an access gate for specific signals whose receptors are also expressed in the me32 , 46 . after having passed the me , the compounds end up in the cerebrospinal fluid ( csf ) where another specialized glia cell type , ependymocytes , has cilia that facilitate fluid movement in the bottom of the third ventricle ; thereafter , compounds such as liraglutide could access the arc from the csf . interestingly , a human study has investigated the presence of liraglutide in the csf and found measurable amounts of approximately 30  pmol / l47 . although the plasma concentration of liraglutide is much higher than that , because > 99% of liraglutide is bound to albumin as part of the protraction mechanism , a csf average concentration of 30  pmol / l might be relevant for a pharmacological effect . in conclusion , new evidence from animal models has highlighted that pharmacological levels of peripherallydosed glp1r agonists can access the hypothalamus directly and have local effects on key primary neurons in arc , leading to an increase in satiety signals and a decrease in hunger signals . these data highlight a potential new important pathway that likely integrates with several other brain pathways where glp1rs are expressed . human mechanistic data showed that liraglutide increased feelings of satiety and decreased hunger , leading to an overall reduction of appetite and energy intake . more studies are required to further understand the differences in weight loss efficacy between structurally different glp1r agonists . all authors are fulltime employees of , and minor employeebased shareholders in , novo nordisk , the company that developed the liraglutide compound .
abstractliraglutide is a glucagonlike peptide1 receptor ( glp1r ) agonist marketed for the treatment of type  2 diabetes . besides lowering blood glucose 
 , liraglutide reduces bodyweight , and has recently also been approved for the obesity indication . 
 acutely , glp1 markedly reduces gastric emptying , and this effect was previously believed to at least partly explain the effect on bodyweight loss . 
 however , recent studies in both humans and animals have shown that glp1r agonists , such as liraglutide , that lead to pharmacological concentrations for 24  h / day only have a minor effect on gastric emptying ; such an effect is unlikely to have lasting effects on appetite reduction . 
 liraglutide has been shown to have direct effects in the arcuate nucleus of the rodent brain , activating proopiomelanocortin neurons and increasing levels of the cocaine and amphetaminestimulated transcript neuropeptide messenger ribonucleic acid , which correlate nicely to clinical studies where liraglutide was shown to increase feelings of satiety . however , despite the lack of a glp1r on agoutirelated peptide / neuropeptide  y neurons , liraglutide also was able to prevent a hunger associated increase in agoutirelated peptide and neuropeptide  y neuropeptide messenger ribonucleic acid , again with a strong correlation to clinical studies that document reduced hunger feelings in patients while taking liraglutide . 
 studies using fluorescent labeled liraglutide , as well as other glp1r agonists , and analysis using singleplane illumination microscopy show that such mediumsized peptidebased compounds can directly access not only circumventricular organs of the brain , but also directly access discrete regions in the hypothalamus . 
 the direct effects of longacting glp1r agonists in the hypothalamus are likely to be an important new pathway in understanding glp1r agonist mediated weight loss .