In previous fMRI studies, visual food-related stimuli, as compared to non-food stimuli, reliably elicited activation within occipital, insular, and frontal cortices, the striatum, and the amygdala [24
]. Here, images of desirable foods, as compared to brick walls, elicited bilateral activation in all of these regions and in the following subcortical regions activated in some, but not all prior studies: thalamus, midbrain, hippocampus and posterior cerebellum. Although a few previous studies reported cerebellar activation [25
], activation in occipital cortex was consistently reported. Some occipital clusters may have extended into neighboring posterior cerebellum, as seen here, and also in all four patient groups of a recent report which made no mention of the cerebellum (compare our to Figure 1 of [29
]). These observations suggest that cerebellar activation by food-related visual stimuli may be underreported.
Visual food-related stimuli activate more of the brain when participants, especially women [31
], are hungry than when then are satiated [32
]. As images of high-calorie foods elicited more hunger after leptin replacement was withheld than when replacement was ongoing in year 5 of this study [7
], we expected the images of food to produce a greater distribution of brain activation when replacement was withheld. When assessing the effects of leptin on brain activation by food pictures that same year, however, about six times as many voxels exhibited greater activation during leptin replacement, when hunger was low, as compared to when replacement was withheld and hunger was high (1288 voxels in Table 4 vs. 219 voxels in Table 3 of [7
]). Moreover, 51% of the voxels that were more activated during leptin replacement, including those with the largest effects, were in the cerebellum, where regional cerebral blood flow, a measure of neural activation, is attenuated during satiety in normal-weight men [34
], and in both lean and obese women [35
Our recent finding that withholding leptin reversibly decreased GM concentration in several brain regions of these patients, particularly in the cerebellum [8
], offered a possible explanation. BOLD signal may be lower after withholding leptin in an area with less GM signal, possibly indicating reduced neuronal density or size. In addition, the weight and the body fat of the patients increased when leptin was withheld [7
], and both obesity and body fat content have been associated with GM deficits in cross-sectional studies [36
]. Because the contribution of leptin to these associations remains unclear, we previously evaluated effects of withholding leptin on brain structure [8
] by employing the two-covariate design (duration of leptin replacement and BMI) used in the present study. Withholding leptin replacement reversed the increases in GM concentration observed within the cerebellum and anterior cingulate gyrus during the first 18 months of leptin replacement. GM changes in the anterior cingulate gyrus were best explained by the changes in BMI, whereas changes in the cerebellum were best explained by the duration of leptin replacement.
In the study reported here, duration of leptin replacement showed direct covariation with brain activation elicited by food-related images in the posterior inferior cerebellar hemispheres, most prominently at GM/white-matter boundaries in lobules IX and VIIIB (, top; , middle panel). Post hoc analyses revealed a significant effect (p < 0.001) in 5.0 % of the voxels in lobule IX bilaterally, and 4.0 % of the voxels of lobule VIIIB. This effect overlapped with the area where we have shown covariation between duration of leptin replacement and GM structure [8
]. At the voxel where the structural covariation was strongest (−29, −57, −42), duration of leptin replacement was also correlated with the BOLD response to food-related cues in the present investigation (t =3.52, p < 0.002). This observation supports the idea that reduction in GM structural signal when leptin is withheld may contribute to reduced local activation by food-related stimuli.
In contrast, brain activity elicited by food-related images was associated with higher BMI in a superior part of the posterior cerebellar hemispheres, in lobules VI and Crus1, with clear L > R asymmetry, and in the precuneus of the posterior parietal lobe. Structural changes were not associated with either BMI or duration of leptin replacement in these areas [8
]. Post hoc analyses indicated a significant effect (p < 0.001) in 10.1% of lobule VI bilaterally (17.6 % in the left cerebellar hemisphere) and 6.8% of Crus I bilaterally (11.0 % in the left cerebellar hemisphere). The corresponding association with duration of leptin replacement involved only 3.7 % of lobule VI bilaterally and 2.4 % of Crus I. Because cerebro-cerebellar connections are primarily contralateral, most language and motor tasks produce R>L activation. However, L>R activation in lobules VI and Crus1 associated with emotional processing and executive tasks has been reported [11
]. The precuneus has been linked with visual processing, anticipation, and imagery. We interpret the greater activity associated with BMI as representing an increased emotional response to food-related stimuli when leptin-deficiency increases hunger and body fat.
Cerebellar function has been implicated in the fine-tuning of sensorimotor processing, but an emerging reconceptualization implicates the posterior lobe of the cerebellum, where the current findings are localized, in higher-level cognitive and emotional tasks [11
]. The cerebellum has also been shown to detect blood-borne nutritional signals directly, and to be activated during anticipation of food [40
]. In six diet-regulated obese inpatients assessed before and after stabilization at a 10% reduced body weight, dieting was associated with increased fMRI-measured activation by visual food stimuli in the dorsal posterior lobe of the cerebellum, and leptin administration reversed the increase [41
]. Since leptin also reversed the increased hunger associated with the weight-reduced state [41
], the effect was consistent with the cerebellar activation associated with BMI that we observe and attribute to increased hunger when leptin was withheld [7
]. While administration of leptin for five weeks to the weight-reduced inpatients, as compared to administration of placebo, decreased activation in dorsal posterior cerebellum, it also increased activation in nearby voxels [41
]. The increases may have been mediated by leptin-associated increases in GM structure like those we have reported [8
], since leptin was administered in doses designed to reverse the relative hypoleptinemia induced by weight reduction [41
Although leptin also increased activation in extracerebellar structures of the inpatients, the participants were not leptin-deficient [41
] although they may have been leptin-resistant. In addition, very different statistical thresholds were employed as compared to our study. In the final across-subjects analysis, a voxel threshold of p < 0.05 and a spatial extent threshold of 5 or more contiguous voxels were used [41
]. We employed more conservative thresholds; voxel p < 0.001 with spatial extent p < 0.05 after multiple-comparisons correction for whole-brain search volume (>115 voxels). The z-scores for effects of leptin replacement corresponding to the T-scores in our range from 3.83 to 4.57, as compared to scores ranging from 1.69 to 3.30 in Table 3 of the earlier study [41
]. In our study, BOLD response was also directly related to the number of days of leptin replacement with peak Z-scores > 3 in most of the extracerebellar structures where leptin increased activation in the earlier study [41
], but these effects did not survive multiple-comparison correction for whole-brain volume, and therefore cannot be interpreted as evidence without an a priori hypothesized effect location.
Although it has been argued that the high levels of OB-Rb leptin receptors in the human cerebellum may indicate a function unrelated to body weight homeostasis [42
], our findings, together with the other studies discussed above, suggest that both structural and functional effects of leptin on cerebellar plasticity may in fact be part of the mechanism by which leptin alters food consumption. They also underscore the importance of considering structural effects when interpreting functional brain response.
Extrahypothalamic effects of leptin are consistent with neurochemical actions, including activation of synaptic NR2A-containing N
-methyl-D-aspartate (NMDA) receptors and the mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) in multiple brain areas where leptin regulates synaptic morphology [43
].The MAPK/ERK [46
], phosphatidylinositol 3-kinase (PI3K) [47
] and nuclear factor kappa-B transcription factor (NF-kB) pathways [48
] have been shown to contribute to pro-survival responses that make leptin a potent neurogenic factor in hippocampal [49
] and cortical neurons [51
]. Through activation of a Janus kinase signal transducer and activator of transcription-3 (JAK/STAT3) / PI3K / Protein Kinase B (PKB) -signaling cascade, leptin protects hippocampal neurons from apoptosis induced by removal of trophic support and excitotoxic and oxidative insults [52
]. In the cerebellum specifically, leptin promotes survival of Purkinje cells [10
], and facilitates NMDA receptor-mediated calcium influx in granule cells [53
], to activate the JAK2/STAT3 pathway and to reduce PKB activation [54
]. At a systems level, effects of leptin on NMDA receptors could have extensive functional influence because of the wide distribution of these receptors in brain and their role in excitatory synaptic transmission.
Limitations of the current study include the rarity of congenital leptin deficiency, and the fact that common obesity is often accompanied by leptin resistance, as it was for our patient C. This patient required 13–17 times as much leptin daily as the others to achieve comparable weight loss, and has been less able than the others to maintain a stable BMI (see ). The fact that this individual showed changes in brain response to food cues, despite relative hypothalamic leptin resistance, can be explained by the higher dosing and tissue-specificity of leptin resistance. In diet-induced, obese hyperleptinemic mice, the arcuate nucleus is the major site of leptin resistance, whereas other hypothalamic and extrahypothalamic sites remain leptin-responsive [55
]. Since the first-level individual results for effects of leptin on brain function were similar in all three patients, patient C may be relatively more leptin-resistant at the level of the arcuate nucleus, but more leptin-responsive in extrahypothalamic regions such as the cerebellum.
Although the interaction of functional and structural effects of withholding leptin would be best quantified via a combined multimodal analysis, the small sample size precluded this type of analysis with currently available statistical tools [56
]. A strength of this study is that it evaluated the consequences of the complete absence of leptin, and of its replacement, both in the short- and long-term, in a human model.
In conclusion, this study demonstrates two opposite and reversible effects of leptin replacement on cerebellar response to food cues in leptin-deficient adults. Leptin supplementation decreases hunger and BMI [7
]. In this study, the BMI decreases were associated with decreases in activity within lobules VI and Crus1 of the posterior cerebellar hemispheres after presentation of food-related stimuli (). We tentatively interpret this effect as due to decreased motivational salience of the stimuli when participants are not as hungry. At the same time, a longer as compared to a shorter duration of ongoing leptin supplementation was associated with more activation ventrally in cerebellar lobules IX, VIIIB and VI. This may be a consequence of the reversible increase in GM structure at these locations which we have shown to be produced by leptin in these patients [8
More generally, these findings contribute to emerging evidence for plasticity of brain structure-function relationships over a few weeks [58
] or even hours [59
], and suggest that leptin may have therapeutic value in modulating plasticity-dependent brain functions. The results reported here also highlight the possibility of a hitherto underexplored but critical role for the cerebellum in the regulation of leptin-mediated cognitive processes related to food intake.