article: Bardet-Biedl syndrome (BBS) is a highly pleiotropic autosomal recessive disorder associated with a wide range of phenotypes including obesity. However, the underlying mechanism remains unclear. Here, we show that neuronal BBSome is a critical determinant of energy balance through its role in the regulation of the trafficking of the long signaling form of the leptin receptor (LRb). Targeted disruption of the BBSome by deleting the Bbs1 gene from the nervous system causes obesity in mice, and this phenotype is reproduced by ablation of the Bbs1 gene selectively in the LRb-expressing cells, but not from adipocytes. Obesity developed as a consequence of both increased food intake and decreased energy expenditure in mice lacking the Bbs1 gene in LRb-expressing cells. Strikingly, the well-known role of BBS proteins in the regulation of ciliary formation and function is unlikely to account for the obesogenic effect of BBS1 loss as disruption of the intraflagellar transport (IFT) machinery required for ciliogenesis by deleting the Ift88 gene in LRb-expressing cells caused a marginal increase in body weight and adiposity. Instead, we demonstrate that silencing BBS proteins, but not IFT88, impair the trafficking of the LRb to the plasma membrane leading to central leptin resistance in a manner independent of obesity. Our data also demonstrate that postnatal deletion of the Bbs1 gene in the mediobasal hypothalamus can cause obesity in mice, arguing against an early neurodevelopmental origin of obesity in BBS. Our results depict a novel mechanism underlying energy imbalance and obesity in BBS with potential implications in common forms of human obesity.
  Like many other medical conditions, monogenic obesity syndromes have been invaluable in understanding the biological bases of body weight regulation and dissection of the processes underlying excess fat stores [1,2]. Bardet-Biedl syndrome (BBS) is a highly pleiotropic autosomal recessive disorder in which obesity is a predominant presenting feature [3,4]. Other primary clinical features displayed by BBS patients are retinopathy, polydactyly, learning disabilities and hypogenitalism. BBS is genetically heterogenous with 20 genes (BBS1-BBS20) identified to date [5], with multiple protein–protein interactions occurring between the encoded proteins [6]. Indeed, 8 BBS proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 and BBS18 [also known as BBIP10]) form a stable complex, the BBSome, which mediates protein trafficking to the ciliary membrane and perhaps to other membrane compartments [7,8]. BBS3 is a small Ras GTPase and controls BBSome recruitment to the membrane and BBSome ciliary entry [9]. Three other BBS proteins (BBS6, BBS10 and BBS12) form another complex with CCT/TRiC family of group II chaperonins (termed chaperonin-like proteins, CLP) and mediate BBSome assembly [10]. The discovery that various BBS proteins interact to form complexes is consistent with the overlapping phenotypes arising from mutations in different BBS genes. BBS proteins have been known to play a prominent role in the regulation of cilia formation and function by coordinating movement rates of different particles of intraflagellar transport (IFT) machinery which moves cargo proteins within the cilium [11,12]. The importance of BBS proteins for ciliary function is further supported by the fact that BBS genes are conserved in ciliated organisms, but not in non-ciliated organisms [13,14]; the action of BBSome as a membrane coat complex that recruits resident proteins to cilia [9]; and the ciliary defects associated with loss of BBS genes [15]. Cilia are hair-like cellular projections present in virtually all cell types of the mammalian body [4,16]. These evolutionarily conserved organelles play a fundamental role in the regulation of several biological processes. A wide spectrum of human genetic disorders including BBS, termed ciliopathies, has been attributed to ciliary defects [16]. It should be noted, however, that obesity is not universally associated with ciliopathies [4,17]. BBS mouse models, which present many of the features found in BBS patients including obesity, have been useful in gaining insights into the etiology of this syndrome [15,18–21]. We previously determined that obesity in BBS mutant mice (Bbs1M390R, Bbs2-/-, Bbs4-/- and Bbs6-/-) was associated with hyperphagia and leptin resistance [15,21]. We studied the mechanism of leptin resistance in BBS mutant mice by normalizing their body weight and circulating leptin levels by calorie-restriction [22]. Despite attaining normal serum leptin levels, leptin failed to reduce food intake or activate hypothalamic leptin receptor (LRb) signaling such as Stat3 (signal transducer and activator of transcription-3) [22]. This impairment was accompanied by decreased hypothalamic expression of anorexigenic proopiomelanocortin (Pomc) gene without alterations in the expression of orexigenic agouti protein-related protein (Agrp) or neuropeptide y (Npy) genes [21,22]. Here, we show that disruption of BBS proteins selectively in the nervous system, hypothalamus or LRb-expressing cells, but not in adipocytes, is sufficient to cause obesity. Remarkably, the obesogenic effect of disrupting BBS proteins is unrelated to cilia as interference with ciliogenesis, by disruption of IFT, in LRb cells caused a minimal weight gain. Mechanistically, we demonstrate that silencing the expression of BBS proteins, but not IFT, reduce the surface expression of the LRb leading to leptin resistance independently from obesity.
  To gain insight into the role of BBS proteins in the nervous system in the regulation of energy homeostasis we used a conditional knockout mouse model where exon 3 of the Bbs1 gene is floxed [23]. Breeding Bbs1fl/fl and NestinCre mice created mice deficient in Bbs1 gene only in the nervous system as indicated by the loss of Bbs1 gene expression in the brain, but not in peripheral tissues (Fig 1A and 1B). Moreover, using co-immunoprecipitation assays we found that NestinCre/Bbs1fl/fl mice have disrupted BBSome in the nervous system as indicated by the inability of BBS2 to pull down BBS9 in the brain whereas in testes the interaction between BBS2 and BBS9 was not altered (Fig 1C) indicating that in NestinCre/Bbs1fl/fl mice disruption of the BBSome was specific to the nervous system. From the time of weaning (4 weeks) up to 8 weeks of age, NestinCre/Bbs1fl/fl mice weighed the same as the littermate controls. However, by about 9 weeks of age, NestinCre/Bbs1fl/fl mice began gaining significantly more weight than the littermate controls (Fig 1D). Notably, the obesity phenotype was more pronounced in the females relative to males (S1A and S1B Fig). At 25 weeks of age, the female NestinCre/Bbs1fl/fl mice weighed about 53% more than the controls while the male NestinCre/Bbs1fl/fl mice weighed about 18% more than their littermate controls. The increased body weight in NestinCre/Bbs1fl/fl mice was associated with substantial increase in fat mass as measured by MRI in 12- and 25-week old mice (Fig 1E and 1F). This was further confirmed by weighing individual fat pads at sacrifice (S1C Fig). However, there was no difference in lean mass as determined by MRI (Fig 1F). Consistent with the sex difference in body weight, the increased weight of fat pads in NestinCre/Bbs1fl/fl mice was more pronounced in females relative to males (S1D and S1E Fig). These findings are consistent with what is observed in the mice bearing global deletion of BBS genes. Feeding studies indicated that NestinCre/Bbs1fl/fl mice are hyperphagic. Measurement of food intake during 5 days showed that obese NestinCre/Bbs1fl/fl mice eat about 35% more than the controls (Fig 1G). We also examined the gene expression profile of the key hypothalamic orexigenic and anorexigenic neuropeptides involved in the regulation of energy homeostasis. We found that mRNA levels of AgRP and NPY were normal, whereas the mRNA level of POMC was significantly reduced in NestinCre/Bbs1fl/fl mice (Fig 1H). Together, these findings demonstrate the importance of Bbs1 gene in the nervous system for energy homeostasis and the obesity phenotype of BBS. Next, we tested whether the obesity phenotype can be recapitulated by deleting the Bbs1 gene in tissues other than the nervous system. In cultured adipocytes, inactivation of BBS genes accelerated cell division, caused aberrant differentiation and promoted adipogenesis and fat accumulation [24,25] pointing to a possible contribution of adipose tissue to BBS-associated obesity. Thus, we crossed the Bbs1fl/fl mice with Adiponectin (Adip) Cre mice in which Cre expression is restricted to adipocytes [26]. However, there was no difference in body weight, fat pad weights or adipocytes size between AdipCre/Bbs1fl/fl mice and littermate controls (S2A–S2D Fig). These findings indicate that the changes that occur in adipocytes lacking BBS genes in vitro may not translate in vivo. Alternatively, it is possible that early loss of the Bbs1 gene in adipocytes may have caused compensatory adaptations that protect the mice from metabolic alterations and obesity. This is unlikely given that lifelong Bbs1 gene deficiency in the nervous system caused obesity. The influential role of leptin in the regulation of energy homeostasis [27] led us to ask whether deletion of the Bbs1 gene specifically in the LRb-expressing cells will recapitulate the obesity phenotype of BBS. For this, we used LRbCre mice which provide specificity of Cre expression to LRb expressing cells (see Fig 2A and Refs [28,29]). First, we assessed whether LRb cells express Bbs genes. We used Fluorescence Activated Cell Sorting (FACS) procedure [30,31] to isolate LRb-positive cells (S3 Fig). We found that FACS-purified LRb containing cells (td-Tomato+) of the hypothalamus express all the Bbs genes tested including those encoding proteins of the BBSome (Bbs1,2, 4,5, 7,8 and 9) and the CLP complex (Bbs6,10 and 12) (Fig 2B). At 4 and 5 weeks of age, there was no difference in body weight between LRbCre/Bbs1fl/fl mice and littermate controls (Fig 2C). At around 6 weeks of age, LRbCre/Bbs1fl/fl mice start gaining more weight than the controls. The divergence in body weight continues as the mice grow (Fig 2C). At 25 weeks of age, the LRbCre/Bbs1fl/fl mice weighed about 31% more than the controls (Fig 2C) and become morbidly obese as they age (Fig 2D). In contrast to the NestinCre/Bbs1fl/fl mice, the weight gain trend was similar in male and female LRbCre/Bbs1fl/fl mice relative to their littermate controls (S4A and S4B Fig). Fat mass and plasma leptin levels were not different in young (<6-week old) LRbCre/Bbs1fl/fl mice relative to controls (Fig 2E–2G), but older LRbCre/Bbs1fl/fl mice develop adiposity as indicated by the increased fat mass (Figs 2E, 2F and S4C and S4D), plasma leptin (Fig 2G) and size of adipocytes (Fig 2H and 2I). Liver mass was also increased in LRbCre/Bbs1fl/fl mice (Fig 2J) which was associated with accumulation of fat as revealed by Oil-Red-O staining (Fig 2K). Similar to the NestinCre/Bbs1fl/fl mice, the LRbCre/Bbs1fl/fl mice exhibited normal expression of hypothalamic orexigenic Agrp and Npy genes while the expression of anorexigenic Pomc gene was significantly reduced (Fig 2L). Interestingly, hypothalamic Pomc gene expression was also decreased in young LRbCre/Bbs1fl/fl mice (0. 56±0. 07 AU vs 1. 00±0. 14 AU in controls, P = 0. 007). Of note, Bbs1 gene deletion did not appear to affect cilia in LRb expressing neurons (S4E Fig). Collectively, these findings highlight the importance of BBS genes in LRb-containing cells for body weight regulation in a manner independent of cilia. Food intake was no different between LRbCre/Bbs1fl/fl and control mice at 4 (P = 0. 4), 5 (P = 0. 35) and 6 weeks (P = 0. 22) of age. However, after 6 weeks of age, the LRbCre/Bbs1fl/fl mice eat consistently more than the controls (Fig 3A). Cumulatively, the LRbCre/Bbs1fl/fl mice eat about 20% more than the controls. Bomb calorimetric analyses of fecal caloric content showed that LRbCre/Bbs1fl/fl mice have normal digestive efficiency (absorbing 79±1% vs. 78±1% in control mice, P = 0. 20), indicating that the elevated food intake of the LRbCre/Bbs1fl/fl mice translate into an increase in total caloric absorption and availability. To examine whether hyperphagia can explain the increased adiposity in LRbCre/Bbs1fl/fl mice, we performed a pair-feeding study by providing each LRbCre/Bbs1fl/fl mouse the same amount of food eaten daily by the littermate control beginning at 5 weeks of age. At 25 weeks, body weight and fat pad masses of pair-fed LRbCre/Bbs1fl/fl mice were substantially lower relative to the LRbCre/Bbs1fl/fl mice fed ad libitum (Fig 3B and 3C) indicating that hyperphagia contribute to the obesity phenotype of LRbCre/Bbs1fl/fl mice. However, body weight and fat pad masses of the 25 weeks old pair-fed LRbCre/Bbs1fl/fl mice remained significantly elevated compared to age-matched littermate controls (Fig 3B and 3C). Similar trend was observed in a subset of LRbCre/Bbs1fl/fl mice that were pair-fed until 15 weeks of age (S5A and S5B Fig). These findings suggest that LRbCre/Bbs1fl/fl mice have reduced energy expenditure. In favor of such possibility, 24-h body temperature, measured by radiotelemetry during 7 days, was consistently lower in the LRbCre/Bbs1fl/fl mice as compared to the controls (Fig 3D). Next, we used indirect calorimetry to assess energy expenditure in young weight-matched LRbCre/Bbs1fl/fl mice (23. 7±1. 2 g) and littermate controls (23. 2±0. 3 g). Both O2 consumption (Fig 3E) and heat production (Fig 3F) were significantly lower in the obese LRbCre/Bbs1fl/fl mice relative to controls. Young LRbCre/Bbs1fl/fl mice tend to have lower O2 consumption (2. 68±0. 17 ml/100g/min) and heat production (8. 04±0. 47 kcal/kg/h) relative to age-matched controls (2. 92±0. 07 ml/100g/min and 8. 53±0. 20 kcal/kg/h, respectively), but this was not statistically different (P = 0. 08 and 0. 16, respectively). Together, these findings demonstrate that both hyperphagia and decreased energy expenditure contribute to the obesity phenotype of the LRbCre/Bbs1fl/fl mice. Of note, NestinCre/Bbs1fl/fl mice (weight-matched to littermate controls) also exhibit a significantly reduced O2 consumption and heat production (S5C and S5D Fig). The presence of obesity and hyperleptinemia in the LRbCre/Bbs1fl/fl mice prompted us to test whether these mice are leptin resistant. We found that intraperitoneal (i. p.) administration of leptin (1 μg/g body weight, twice daily) was less effective in reducing food intake and body weight in obese LRbCre/Bbs1fl/fl mice relative to controls (S6A and S6B Fig). To test the contribution of obesity to leptin resistance in LRbCre/Bbs1fl/fl mice, we performed several experiments. First, we tested leptin sensitivity in LRbCre/Bbs1fl/fl mice that remained lean throughout their lifespan. For this, beginning at weaning we restricted the amount of food LRbCre/Bbs1fl/fl mice could eat (by feeding them 70–80% of daily amount of food eaten by the littermate controls) so that they could not become obese. This strategy prevented the development of obesity in the LRbCre/Bbs1fl/fl mice as indicated by the normalization of plasma leptin levels (3. 0±0. 3 ng/mL in calorie restricted LRbCre/Bbs1fl/fl mice vs. 3. 1±0. 5 ng/mL in control mice). Calorie-restricted LRbCre/Bbs1fl/fl mice were resistant to the anorectic and weight-reducing effects induced by intracerebroventricular (i. c. v.) injection of leptin (Fig 4A–4C). Moreover, leptin-evoked activation of hypothalamic Stat3 (p-Stat3) was significantly reduced in the calorie-restricted LRbCre/Bbs1fl/fl mice as assessed by Western blotting (Fig 4D). We also tested leptin sensitivity in young LRbCre/Bbs1fl/fl mice before the development of obesity and hyperleptinemia (Fig 2E–2G). Treatment with i. p. leptin decreased food intake and body weight in the young LRbCre/Bbs1fl/fl mice, but these responses were substantially attenuated compared to the control mice (Fig 4E and 4F). We also used immunohistochemistry to assess Stat3 activation in the arcuate nucleus of young LRbCre/Bbs1fl/fl mice which were bred with the td-Tomato reporter mice to fluorescently label the LRb-expressing cells. Relative to controls, young LRbCre/Bbs1fl/fl mice displayed a substantially reduced leptin-induced hypothalamic Stat3 activation by immunohistochemistry (Fig 4G and 4H). Collectively, these data demonstrate that leptin resistance in mice lacking the Bbs1 gene in the LRb-expressing cells is not secondary to obesity. We considered the possibility that inactivation of the Bbs1 gene may alter the expression of the LRb or number of LRb-expressing cells leading to leptin resistance. However, the mRNA level of the LRb in the hypothalamic explants was not significantly different in the LRbCre/Bbs1fl/fl mice (1. 38±0. 7 AU) relative to the controls (1. 00±0. 2 AU, p = 0. 3) indicating that leptin resistance in the LRbCre/Bbs1fl/fl mice is not due to decreased LRb gene expression. The number of LRb-expressing cells in the hypothalamic arcuate nucleus was also comparable between the LRbCre/Bbs1fl/fl mice and controls (S6C Fig). Alternatively, leptin resistance can be caused by overabundance of the negative feedback inhibitors of the LRb signaling such as the suppressor of cytokine signaling 3 (SOCS3) [32] or the protein-tyrosine phosphatase 1B (PTP1B) [33]. We found that levels of hypothalamic SOCS3 mRNA were not altered while the mRNA level of PTP1B was reduced in the LRbCre/Bbs1fl/fl animals (S6D Fig) excluding these negative inhibitors of LRb signaling as a mechanism of leptin resistance in the LRbCre/Bbs1fl/fl mice. We also investigated the potential role of the LR gene-related protein (LRGRP) a negative regulator of LRb transport to the cell surface and previously implicated in the development of leptin resistance in diet-induced obesity [34]. However, the mRNA levels of LRGRP tended to be lower in the hypothalamic explants of the LRbCre/Bbs1fl/fl mice relative to littermate controls (S6D Fig). We asked whether postnatal deletion of the Bbs1 gene alters body weight and adiposity. The evidences including the decreased Pomc gene expression points to the mediobasal hypothalamus as a potential site underlying energy imbalance in BBS. Thus, we tested the effect on body weight of deleting the Bbs1 gene in the mediobasal hypothalamus which includes the arcuate and ventromedial nuclei. We stereotaxically targeted the mediobasal hypothalamus bilaterally with an adeno-associated virus expressing Cre (AAV-Cre) using a previously validated strategy [35]. Because td-Tomato/Bbs1fl/fl mice were used in this study, induction of td-Tomato (assessed at the conclusion of the experiments in every animal) was used as an indication of Cre expression (Fig 5A). Bbs1fl/fl mice (7–10 weeks old) received bilateral microinjections into the mediobasal hypothalamus of either AAV-Cre or an AAV expressing enhanced green fluorescence protein (AAV-GFP), used as controls. Body weight was not different between the 2 groups at baseline or during the first 4–5 weeks after stereotaxic injections (Fig 5B). However, after 5 weeks, Bbs1fl/fl mice treated with AAV-Cre began to gain more weight than the AAV-GFP infused group. After 9 weeks, body weight was consistently elevated in the AAV-Cre injected mice (Fig 5B). Fat mass measured at 18 weeks after stereotaxic injections was significantly elevated in AAV-Cre treated mice relative to AAV-GFP controls (Figs 5C, 5D and S7) whereas lean mass was comparable between the 2 groups (Fig 5D). Measurement of food intake showed that AAV-Cre treated mice eat significantly more than the controls (Fig 5E). Thus, postnatal ablation of the Bbs1 gene specifically in the mediobasal hypothalamus is sufficient to cause hyperphagia and obesity. To test whether loss of other cilia-related genes in LRb-containing neurons recapitulate the obesity phenotype induced by ablation of the Bbs1 gene, we crossed the mice bearing the conditional allele of the Ift88 gene [36] with the LRbCre mice. As expected, LRbCre/Ift88fl/fl mice displayed loss of cilia (ACIII staining) specifically in the LRb expressing neurons. Indeed, in the LRbCre/Ift88fl/fl mice cilia were unaffected in non-LRb-containing cells whereas virtually no cilia or severely truncated cilia were detected in the LRb expressing cells (Fig 6A), which is consistent with the requirement of IFT for ciliogenesis [11,12]. The LRbCre/Ift88fl/fl mice exhibited a slight, but significantly elevated body weight (Fig 6B) and fat mass (Fig 6C and 6D) than their littermate controls. Of note, the increase in body weight and adiposity was more noticeable in the males than the females LRbCre/Ift88fl/fl mice (S8A–S8D Fig). Food intake was not different between LRbCre/Ift88fl/fl mice and littermate controls (Fig 6E) suggesting that the increased adiposity in LRbCre/Ift88fl/fl mice is not caused by hyperphagia. Rather, a decrease in energy expenditure seems to account for the increased adiposity in LRbCre/Ift88fl/fl mice as indicated by the slight, but significantly lower body temperature (Fig 6F), O2 consumption (Fig 6G) and heat production (Fig 6H) in these mice. Hypothalamic expression levels of Agrp, Npy and Pomc genes were not altered in LRbCre/Ift88fl/fl mice (Fig 6I). Leptin-induced decrease in food intake was indistinguishable between LRbCre/Ift88fl/fl mice and littermate controls during the 4-day treatment (Fig 6J). The decrease in body weight evoked by leptin was significantly attenuated in the LRbCre/Ift88fl/fl mice during the first day, but not during the remaining 3 days of treatment (Fig 6K). These data demonstrate minimal leptin resistance in adult LRbCre/Ift88fl/fl mice which is likely due to the increased adiposity as leptin sensitivity was not altered in lean young LRbCre/Ift88fl/fl mice as indicated by the comparable decrease in food intake and body weight in young (6–8 weeks old) LRbCre/Ift88fl/fl mice relative to controls (S8E and S8F Fig). These findings indicate that leptin resistance does not account for the increased adiposity associated with the disruption of the Ift88 gene. This implies that LRb-independent mechanisms contribute to the obesity associated with ciliopathies other than BBS. We considered the possibility that BBS proteins may be involved in transporting the LRb to the cell membrane and that loss of the Bbs1 gene may cause abnormal trafficking and localization of the LRb. We previously demonstrated that BBS1 protein specifically interacts with the LRb, but not with LRa, in transiently transfected cells [22]. To substantiate this biochemical interaction further in vivo, we generated a new transgenic mouse model that expresses a Flag-tagged BBS1 protein. Co-immunoprecipitation assays using an antibody recognizing both the LRb and LRa (S9A Fig) showed that Flag-BBS1 interacts with the endogenous LRb, but not LRa, in protein lysates prepared from transgenic mouse brains (Fig 7A). Of note, Flag-BBS1 and LRb can also be reciprocally immunoprecipitated using hypothalamic protein lysates, but not in protein lysates from the cortex (S9B Fig). Using HEK-293 cells, we examined whether knockdown of BBS1 expression impacts LRb cell surface expression. Cells were transiently co-transfected with Flag-tagged LRb together with increased dose of BBS1 shRNA (S10A Fig). Flag-LRb cell surface expression was quantified using a surface protein biotinylation assay. We observed a dose-dependent decrease in Flag-LRb cell surface levels in response to BBS1 knockdown (Fig 7B and 7C). In contrast, the total amount of Flag-LRb was not affected. Consistent with the reduced Flag-LRb in the plasma membrane, leptin-activation of Stat3 and S6K was attenuated in the BBS1 knockdown cells compared to control cells (S10B and S10C Fig). These results demonstrate that BBS1 knockdown alters leptin action via reduction in the number of LRb in the plasma membrane. This effect is specific as knockdown of the BBS1 gene did not alter the trafficking of the endogenous transferrin receptor (in same experiment as Flag-LRb) or the HA-tagged LRa (in separate experiments) (Fig 7B and 7C). Next, we determined the requirement of other BBSome proteins for LRb trafficking. For this, we tested whether knockdown of the BBS2 gene, another component of the BBSome, interferes with LRb trafficking to the cell membrane. ShRNA-mediated knockdown of the BBS2 gene (S10D Fig) reduced surface expression of Flag-LRb, but not HA-LRa or endogenous transferrin receptor (Fig 7D and 7E). Again, this was associated with an attenuated leptin-evoked Stat3 stimulation (S10E Fig). These results demonstrate the importance of the BBSome in mediating LRb trafficking to the cell membrane. It has been shown that the BBSome mediates cilia membrane protein trafficking through Rab8a, a member of Rab superfamily proteins. We reasoned that Rab8 may also be required for the trafficking of the LRb to the plasma membrane. Cells transfected with a dominant negative form of Rab8a [7] exhibited a significantly reduced Flag-LRb plasma membrane level (Fig 7F and 7G). Interestingly, dominant negative form of Rab8a also reduced the surface levels of HA-LRa and transferrin receptor indicating that Rab8a plays a general role in membrane protein trafficking. Finally, we tested whether siRNA-mediated knockdown of IFT88 expression alters LRb trafficking to the cell surface levels. However, we found that disruption of IFT88 expression (S10F Fig) had no impact on the membrane levels of Flag-LRb or transferrin receptor (Fig 7H and 7I). These findings demonstrate that IFT88 has no effect on LRb trafficking to the cell membrane. Thus, the role of BBS proteins in mediating LRb trafficking to the cell membrane is specific.
  The current study identifies neuronal BBS proteins and the BBSome as key regulators of energy homeostasis through their role in mediating the trafficking of the LRb. We found that mice lacking the Bbs1 gene in the nervous system develop obesity. Interestingly, Bbs1 gene ablation from the LRb-expressing cells or in the mediobasal hypothalamus is sufficient to cause to obesity in mice. Moreover, we show that mice lacking the Bbs1 gene in LRb cells develop obesity as a consequence of both hyperphagia and decreased energy expenditure. On the other hand, ablation of the Ift88 gene, which is required for ciliogenesis, in LRb-expressing cells caused only marginal weight gain and adiposity. We further show that loss of BBSome subunits, but not IFT88, impairs the trafficking of the LRb to the plasma membrane leading to leptin resistance independently of cilia and obesity. Collectively, these findings demonstrate that obesity in BBS arises from leptin resistance in association with disrupted trafficking of the LRb to the plasma membrane. Recently, we reported the requirement of BBS proteins for insulin receptor trafficking to the cell membrane and demonstrated that disruption of BBS proteins interfere with insulin receptor surface expression leading to insulin resistance and dysregulated glucose metabolism [37]. Taken together, our findings represent a paradigm shift in the way the role and function of cilia proteins such as BBS are viewed. Indeed, these proteins are commonly thought to be solely associated with ciliary function. However, our studies indicate that BBS proteins are directly involved in the trafficking of receptors to the plasma membrane. Several mouse models that lack functional BBS genes globally display several features seen in BBS patients including obesity [15,18,21]. Our demonstration that the obesity phenotype can largely be reproduced by deleting the Bbs1 gene either throughout the nervous system, in the mediobasal hypothalamus or selectively in the LRb-expressing cells, but not in adipocytes, highlight the importance of the neurogenic mechanisms for BBS-associated obesity. Interestingly, the sex difference in the development of obesity observed previously in global knockout and knockin mice [15,18–21] was recapitulated in mice lacking the Bbs1 gene throughout the nervous system, but not when the gene deletion was restricted to the LRb cells suggesting the contribution of cell types other than those expressing the LRb to the sexual dimorphism of obesity in BBS. The expression of BBS genes begin in early development and continue throughout adult life in numerous types of tissue including the nervous system [38–44]. Of note, pre- and post-natal interference with neuronal pathways regulating metabolism can lead to disparate phenotypes [45]. In addition, it has been suggested that obesity in BBS may be caused by neurodevelopmental defects that arises as a consequence of prenatal loss of the BBS genes [46]. This is mainly based on the high prevalence of behavioral, neurocognitive and neuroanatomical abnormalities in individuals with BBS, which can manifest in early years [47]. However, our data demonstrate that postnatal deletion of the Bbs1 gene in the mediobasal hypothalamus can cause obesity in mice, arguing against an early neurodevelopmental origin of obesity in BBS. Analysis of mice lacking the Bbs1 gene in the LRb-expressing cells showed that obesity develops as a consequence of both increased food intake and lower energy expenditure. Data also revealed that these mice have reduced leptin sensitivity and LRb signal transduction in a manner independent of obesity. These findings along with the reduced hypothalamic expression of direct targets of the LRb such as Pomc gene [4] in BBS mice points to leptin resistance as an underlying mechanism of energy imbalance in BBS. This is further supported by our finding that BBS proteins and the BBSome are required for the trafficking of the LRb to the plasma membrane. In contrast to our findings, however, a recent report has argued that leptin resistance in BBS mice is secondary to obesity [46]. This was based on normal induction of hypothalamic Stat3 activation and anorexia by exogenous leptin in young Bbs4-/- mice. However, the changes that occur when leptin resistance develops as a secondary consequence of obesity such as in diet-induced obesity are not compatible with what is occurring in BBS. For instance, while increased expression of hypothalamic Socs3 [32] and Lrgrp [34] are hallmark features of diet-induced obesity and key factors in the development of common leptin resistance, the expression of these genes is not altered or tended to be lower in BBS mice. The demonstration that BBS patients exhibit greater circulating leptin levels than weight- and age-matched non-BBS controls [48,49], indicative of pronounced leptin resistance, bolster the notion that dysfunctional leptin signaling is a key pathophysiological mechanism of obesity in BBS. Cilia-related proteins other than BBS have been implicated in the regulation of energy homeostasis and trafficking of the LRb. Induced ablation of the kinesin-2 subunit KIF3A or IFT88 throughout the nervous system or in the hypothalamus cause obesity in mice [50,51]. Furthermore, the LRb was found to localize in the cilia-basal body in cells and appears required for ciliogenesis [51]. The Retinitis Pigmentosa GTPase Regulator-Interacting Protein-1 Like (RPGRIP1L) gene, which encodes a protein localized at the transition zone of the primary cilium was shown to interact physically with the LRb in the vicinity to the cilium upon leptin stimulation [52]. Moreover, mice hypomorphic for Rpgrip1l display altered LRb localization and reduced leptin sensitivity and LRb signaling associated with increased body weight and adiposity [52]. These evidences combined with the importance of BBSome for ciliary function, led us to postulate that deleting cilia, through ablation of the Ift88 gene, from LRb-expressing cells would have the same outcome as loss of Bbs1 gene. However, loss of cilia in LRb-containing cells caused only a mild increase in body weight and adiposity, which was driven mainly by a reduction in energy expenditure as food intake was not altered. Leptin sensitivity and LRb trafficking were also unaffected by Ift88 gene ablation. Similarly, mice with induced ablation of the Ift88 gene in the nervous system exhibited normal responses to leptin when rendered lean by calorie restriction [46]. These findings indicate that processes independent of leptin signaling contribute to the obesity that arises from defects in cilia-related proteins other than BBS [17]. In summary, the present study demonstrates that BBS proteins expressed in the nervous system play a central role in the regulation of energy homeostasis by mediating the trafficking of the LRb. The findings also point to impairment in leptin signaling due to mishandling of the LRb as a major mechanism for energy imbalance leading to obesity in BBS. Another important observation from the current investigation relates to the differential role of the BBS and IFT proteins in the regulation of food intake, energy expenditure and LRb trafficking.
  Care of the mice used in the experiments met the standards set forth by the National Institutes of Health in their guidelines for the care and use of experimental animals. The study was approved by Institutional Animal Care and Use Committee (IACUC) of the University of Iowa (protocol number 1301003). All animal testing was approved by The University of Iowa Animal Care and Use Committee. Mice expressing the Bbs1fl/fl [23], Ift88fl/fl [36], NestinCre [53] and LRbCre [29] mice were obtained from our colonies. AdipCre and ROSA (Stopfl/fl-tdTomato) reporter transgenic mice were obtained from the Jackson Laboratory. To study the interaction between the LRb and BBS1 protein in the brain, we generated a new transgenic mouse line expressing Flag- and S-tagged human BBS1 gene under the control of human β–actin promoter. Briefly, human BBS1 gene was tagged with 3X Flag tag and 2X S tag in its N-terminus and inserted into pSETC-1 vector with human β–actin promoter. The transgene expression was ubiquitous with varying levels in different tissues including the brain. All mice used in this study were on a mixed 129SvEv and C57B/6J backgrounds. Adult mice (12–16 weeks of age) were used unless indicated otherwise. To specifically delete the Bbs1 gene from the nervous system or LRb cells we crossed transgenic NestinCre or LRbCre male mice with Bbs1fl/fl female mice. NestinCre/Bbs1fl/wt and LRbCre/Bbs1fl/wt offspring were subsequently crossed with Bbs1fl/wt mice to generate NestinCre/Bbs1fl/fl and LRbCre/Bbs1fl/fl mice, respectively. Similar strategy was used to delete the Bbs1 gene from adipocytes (using the AdipCre mice) and to selectively delete the Ift88 gene in the LRb-expressing cells using Ift88fl/fl mice. Presence of the Cre transgene, Bbs1 and Ift88 alleles, and td-Tomato of conditional knockout mice were detected by PCR analysis of tail DNA using the primer sequences provided in S1 Table. Mice were housed in groups of 3–5 per cage and maintained on 12-h light-dark cycle with lights on at 6 am. Room temperature was maintained at 22°C. Food and water were available ad libitum except when the mice were fasted, pair-fed or had their food restricted as described below. Stereotaxic surgery procedures were performed as previously described [35]. Briefly, 7- to 10-week-old female Bbs1fl/fl mice were anesthetized with i. p. administration of ketamine/xylazine cocktail, and restrained in a Kopf stereotaxic apparatus (Tujunga, CA, USA). A small hole was drilled into the skull under aseptic conditions and a glass micropipette connected to an iontophoresis machine was positioned via the stereotaxic manipulator. Approximately 50 nl of AAV expressing GFP or Cre recombinase was slowly administered into each side of the mediobasal hypothalamic area (-1. 50 mm from bregma, ±1. 10 mm lateral and -5. 90 mm from the surface of the cortex with 8° angle). After bilateral microinjections, the skin incision was closed with surgical staples. Mice were weighed once a week until 25 weeks of age. At the end of study, mice were transcardially perfused with 4% paraformaldehyde and cryopreserved in 20% sucrose. Brains were then sectioned into 40-μm coronal sections and injection sites were examined in every brain (by Zeiss LSM710) using td-Tomato as a read out of Cre-recombinase (“hit” microinjections). Mice were weighed once a week, beginning at weaning (4 weeks of age) until 25 weeks of age. Total fat mass and lean mass was determined by MRI under anesthesia (91 mg/kg ketamine and 9. 1 mg/kg xylazine, i. p.) using a Varian Unity/Inova 4. 7 T small-bore MRI system (Varian Inc.). The acquisition consisted of a T1-weighted fast spin-echo sequence (repetition time/echo time = 625/12 ms) with an in-plane resolution of 0. 13 x 0. 25 mm2 and a slice thickness of 1 mm acquired in the axial and coronal planes. In addition, different fat depots and organs were dissected at death and weighed. To measure food intake, mice were housed individually in regular cages and allowed to acclimate for at least 3 days prior to measurements of 24-hour food intake. In pair-feeding studies, each LRbCre/Bbs1fl/fl animal was given the same amount of food consumed by sex-matched wild-type littermate the day before, beginning at weaning (4 weeks of age) until 15 or 25 weeks of age. Parallel groups of LRbCre/Bbs1fl/fl mice were fed ad libitum. In the calorie restriction experiment, each LRbCre/Bbs1fl/fl mouse was given 75–80% of the daily food intake of its control littermate, starting at 4 weeks until 14–16 weeks of age. Normal plasma leptin level of LRbcre/Bbs1fl/fl mice relative control littermates was used as an indication of successful normalization of body weight by calorie restriction. Food intake and body weight response to leptin were compared between wild-type and LRbCre/Bbs1fl/fl mice. Baseline body weight and food intake of individually housed mice were measured daily for 5 consecutive days before beginning treatment with vehicle or leptin (1 μg/μl/g body weight i. p. , twice per day (8 am and 5 pm) for 4 days). To test the effect of i. c. v. injection of leptin, a cannula was implanted into the lateral cerebral ventricle as described previously [35]. Mice were allowed at least 1 week to recover from surgery before i. c. v. injection of vehicle (saline, 2 μl) or leptin (2 μg/mouse). Body weight and food intake were recorded 4 and 24 hours after i. c. v. injection. VO2 was measured in mice housed individually in their home cages with free access to food and water as we reported previously [53]. Regular filter lids were replaced with well-sealed filter lids. Room air drawn through each cage was dried by passage through two successive columns of CaSO4 (Drierite, Arcos, Newark, NJ) desiccant, then analyzed for O2 (model S-3A/II, AEI Technologies, Pittsburgh, PA) content. Radiotelemetric measurement was used to record body temperature and locomotor activity in conscious mice using TA10TA-F20 transmitter (Data Sciences, St. Paul, MN) which was implanted in the peritoneal cavity under anesthesia. After 7 days recovery, body temperature and locomotor activity were collected continuously for 6 days and analyzed using Data Science Dataquest software. For histological assessment of fat and liver, mice were perfused with fresh saline, followed by 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), and peri-gonadal fat pad and liver were dissected, post fixed in 4% paraformaldehyde at 4°C overnight. Tissue blocks were sectioned (10 μm) on a sliding microtome, mounted onto gelatin-coated slides, and stained with standard hematoxylin (peri-gonadal fat) or Oil-Red-O method (liver). Slides were visualized and pictures were obtained using Olympus BX-51 light microscope (ESICO, Davenport, IA). Size of adipocytes was analyzed using National Institutes of Health Image J software (http: //rsbweb. nih. gov) as reported previously [53]. At least three slides from each animal were analyzed. To measure tissue expression of the Bbs1 gene, total RNA was isolated from 4 different brain regions (hypothalamus, cortex, hippocampus and brainstem), as well as white adipose tissue, liver, kidney and skeletal muscle using RNeasy Plus Mini Kit from Qiagen. Total RNA (1 μg in final volume of 20 μl) was used to synthesize first-strand cDNAs with iScript cDNA Synthesis kit (Bio-Rad). cDNA (5 μl) and primers (0. 4 mmol/l) were then added in a final volume of 25 μl PCR mixture (Platinum Supermix, Invitrogen), and amplified in a iQ5 Multicolor Real Time PCR Detection System (Bio-Rad). The PCR conditions for all genes were as follows: denaturation for 5 min at 95°C, 30 cycles for 30 seconds at 95°C, and 30 seconds at 60°C. β–actin mRNA expression was used as an internal control to normalize mRNA expression of these genes. Primer sequences are provided in S1 Table. The sense primer for the Bbs1 locates to the 3rd exon, which is flunked by two loxP sites, and produces a 536-bp band in Cre-negative mice and no band in Cre-positive mice. To analyze expression of various hypothalamic genes, mice were sacrificed by CO2 asphyxiation, and total RNA was isolated using RNeasy Plus Mini Kit from Qiagen. Total RNA (5 μg of in final volume of 100 μl) was used to synthesize first-strand cDNAs with the Super-Script pre-amplification system. Then, 10 μl of cDNA and 0. 4 mmol/L of primers were added in a final volume of 25 μl PCR mixture (iQ SYBR Green supermix, Bio-Rad), and amplified in an iQ5 Multicolor Real Time PCR Detection System (Bio-Rad). The PCR conditions for all genes were as follow: denaturation for 5 min at 95°C, then 40 cycles for 30 seconds at 95°C and 30 seconds at 60°C. S18 ribosomal RNA expression was used as internal control to normalize mRNA expression of these genes. Primer sequences are provided in S1 Table. In order to study the expression of the genes encoding the BBSome and CLP subunits in LRb positive cells, LRb neurons were sorted with flow cytometry. We first established the gating intensities of the tdTomato-positive cells by comparing the fluorescence characteristics of dissociated hypothalamic cells from wild-type mice versus NestinCre/tdTomato mice (S3A Fig). Briefly, hypothalami from 15 mice were dissected, combined, and frozen in liquid nitrogen. Sliced hypothalami were incubated with 1 ml Acutase (Gibco, A11105-01) at 4°C overnight without shaking, and then passed 10 times through a 0. 75 mm diameter glass pipette. Single cells were separated with discontinuous Percoll (Sigma, P1644) density gradient (bottom layer, 3. 426 ml Hibernate A, 824. 5 μl Percoll and 97. 8 μl of 1 M NaCl, middle layer, 3. 6 ml Hibernate A, 650. 5 μl Percoll and 76. 5 μl of 1M NaCl and Top layer, 3. 77 ml Hibernate A, 480. 3 μl Percoll and 59. 5 μl of 1M NaCl) with centrifuge at 2115 rpm for 20 minutes. The pellet containing single cells was resuspended in PBS for flow cytometry. Cell sorting was performed with a Becton Dickinson FACS Aria II (San Jose, CA). Fifty mW of 561 nm light was used to excite td-Tomato while using a 582/15 bandpass filter to collect emissions. Dual parameter plots of forward scatter pulse area (FSC-A) versus side scatter pulse area (SSC-A) and forward scatter pulse area (FSC-A) versus forward scatter pulse width (FSC-W) were used to eliminate dead cells, debris, and aggregates. Twenty thousand gated events were collected first to determine where to set the td-Tomato sort gate hypothalamic cells of LRbcre/td-Tomato mice (S3B–S3D Fig). Two hundred fifty thousand LRb (td-Tomato positive) cells were obtained from 18 million cells, and 150 ng of total RNA was isolated using RNeasy Mini Kit from Qiagen. Total RNA in final volume of 100 μl was used to synthesize first-strand cDNAs with the Super-Script pre-amplification system. To assess the expression of the genes encoding the BBSome and CLP proteins, a two-step amplification technique was used. cDNA (10 μl) and primers (0. 4 mmol/l) were added in a final volume of 50 μl PCR mixture (Platinum supermix, Invitrogen), and amplified in a T100 Thermal Cycler PCR Detection System (Bio-Rad). The PCR conditions for all genes were as follows: denaturation for 5 min at 95°C, then 20 cycles for the first round PCR amplification, 30 seconds at 95°C 45 seconds at 65°C and 30 seconds at 72°C. From second round PCR amplification, 10 μl of PCR product from first round PCR application was added in a final volume of 50 μl PCR mixture, denaturation for 5 min at 95°C, 30 cycles for 30 seconds at 95°C, 45 seconds at 65°C and 30 seconds at 72°C. Primer sequences are provided in S1 Table. To examine the effect of ablating the Bbs1 gene on the BBSome formation, whole brain and testes were collected from NestinCre/Bbs1fl/fl mice and control littermates. Brain and testes were homogenized with protein lysate buffer (50 mM HEPES, pH 7. 5,150 mM NaCl, 1 mM MgCl2,1 mM CaCl2,10 mM NaF, 5 mM EDTA, 1% Triton, 2 mM sodium orthovanadate and Roche cocktail protease inhibitor tablet). Interaction between BBS2 and BBS9 was used to detect the BBSome complex by immunoprecipitation. Briefly, BBS2 protein was immunoprecipitated with an anti-BBS2 antibody (Santa Cruz, goat-anti-BBS2, sc-49381 (C-16) ), the immunocomplex was then separated in a 9% SDS PAGE gel, electrotransferred to a polyvinylidene fluoride membrane, and BBS9 and BBS2 were detected by Western blotting using rabbit antibodies recognizing BBS9 (Sigma, HPA02189,1: 1,000 dilution) or BBS2 (Proteintech Group, 11188-2-AP, 1: 1,000 dilution). To test LRb interaction with BBS1 in the brain of transgenic mice expressing Flag-tagged BBS1 gene, whole brain was used. Protein samples (800 μg) were subjected to immunoprecipitation assay with 5 μg anti-LR receptor antibody (Santa Cruz, sc-8391), which recognizes both the LRb and LRa. Immunocomplex was separated in 6% SDS PAGE gel, electrotransferred to a polyvinylidene fluoride membrane, then probed with primary antibodies, M2 anti-Flag antibody (Sigma, F1804,1: 1,000), anti-LR antibody (Santa Cruz, sc-8391,1: 1,000) followed by a secondary goat anti-rabbit or goat anti-mouse antibody (1: 10,000). Protein expression was visualized with ECL detection kit (GE healthcare). To detect cell surface receptor expression, cell surface biotinylation assays were performed in HEK 293 cells transiently co-transfected with the pcDNA3-Flag-LRb (Flag-LRb) or pcDNA3-HA-LRa (with a control plasmid on an LKO1) and a plasmid expressing the BBS1-shRNA, BBS2-shRNA or Rab8-DN with X-tremeGENE 9 transfection reagent (Roche). Ift88 siRNA On-Target smartpool (Thermo Scientific) was transfected by Lipofectamine RNAiMAX transfection reagent (Invitrogen). To determine silencing efficacy of BBS1-shRNA and BBS2-shRNA, HA-tagged BBS1 or HA-tagged BBS2 were co-transfected with BBS1-shRNA or BBS2-shRNA plasmid DNA, then their protein expression was examined by Western blotting analysis with HA antibody. For Ift88 siRNA silencing efficacy, real-time RT-PCR was used to measure Ift88 mRNA levels (see primer sequences in S1 Table). Forty eight hours post transfection, the cells were incubated with cleavable EZ-Link Sulfo-NHS-SS-Biotin (Thermo Scientific) for 30 min on ice, and biotinylated proteins were recovered using streptavidin-agarose beads (GE Healthcare). Beads were resuspended in Laemmli buffer, and bound proteins were separated by SDS-PAGE and detected by immunoblotting using mouse M2 anti-Flag (Sigma) to detect Flag-LRb or 3F10 anti-HA (Roche) to detect HA-LRa or mouse anti-transferrin receptor (Invitrogen Inc.) monoclonal antibody. Total Flag-LRb, HA-LRa or transferrin receptor expression in cell lysates was used to normalize cell surface receptors expression. To measure circulating leptin, plasma was obtained from blood collected from the mice by centrifuging at 2,040g for 10 min. Plasma leptin concentrations were measured by ELISA using a commercially available kit (Bio-Plex Pro Assays, Bio-Rad). Activation of hypothalamic Stat3 by i. p. or i. c. v. leptin was determined by Western blot analysis. Mice were sacrificed with CO2 asphyxiation and the mediobasal hypothalamus was quickly removed. Western blot was also used to examine activation of Stat3 by leptin (10 μM for 15 minutes) in HEK 293 cells transiently transfected by shRNA against BBS1 or BBS2 genes. Proteins were extracted by homogenizing the aorta in tissue lysate buffer (50 mM HEPES, pH 7. 5,150 mM NaCl, 1 mM MgCl2,1 mM CaCl2,10 mM NaF, 5 mM EDTA, 1% Triton, 2 mM sodium orthovanadate and Roche cocktail protease inhibitor tablet). Protein samples (20 μg) were subjected to 9% SDS PAGE gel, electrotransferred on a polyvinylidene fluoride membrane, then probed with primary antibodies against phospho-Stat3 (Cell Signaling, #9131S, 1: 1,000) or Stat3 (Cell Signaling, #9132S 1: 1,000) followed by a secondary anti-rabbit antibody (1: 10,000). Visualization was performed with enhanced chemiluminescence (ECL) detection kit (GE healthcare) followed by autoradiography. Protein expression intensity was analyzed with Image J software. Mice anesthetized with ketamine and xylazine cocktail were perfused with PBS (5 ml/min; 50 ml) followed by 4% paraformaldehyde/HistoChoice Tissue Fixative (Amresco) in PBS (2. 5 ml/min; 50 ml) using Harvard PHD 22/2000 Syringe Pump. Entire brain was excised and incubated in the same fixative overnight at 4°C. Fixed brains were washed 3 times with PBS and incubated in 30% sucrose/PBS overnight with one change of solution after 4–6 hours of initial incubation. Brains were vibratome-sectioned with 40 μm thickness. Immunochemistry was performed on brain sections to detect phosphorylated Stat3 as described previously [35] using a 1: 250 dilution of a rabbit polyclonal anti-p-Stat3 antibody (Cell Signaling, #9131S). A cyclase III antibody (Santa Cruz, sc-32113 (N-14), 1: 250 dilution) was used to label cilia in the brain. Processed brain sections were mounted using VectaShield mounting medium with DAPI. Images were visualized using confocal microscopy (Zeiss LSM710). Staining of p-Stat3 in the arcuate nucleus was quantified using Image J software. Data are expressed as means ± SEM and analyzed using Student’s t-test or 1- or 2-way analysis of variance (ANOVA). When ANOVA reached significance, a post-hoc comparison was performed. A P<0. 05 was considered statistically significant.
summary: The brain plays an important role in maintaining energy homeostasis. The hormone leptin is a critical afferent signal in metabolic homeostasis through its action in the brain. Here we show that neuronal Bardet-Biedl syndrome (BBS) proteins, encoded by genes that cause obesity when mutated, govern energy homeostasis through the control of cell surface expression of the leptin receptor. Selective disruption of BBS proteins causes obesity in mice and impairs the transport of the leptin receptor to the plasma membrane leading to leptin resistance in a manner independent of obesity. These results establish BBS proteins as a fundamental mechanism underlying transport of the leptin receptor and explain why BBS patients develop obesity.
keywords: body weight
  medicine and health sciences
  brain
  hormones
  animal models
  membrane proteins
  model organisms
  physiological parameters
  obesity
  cellular structures and organelles
  research and analysis methods
  lipids
  leptin
  fats
  mouse models
  hypothalamus
  cell membranes
  peptide hormones
  biochemistry
  cell biology
  anatomy
  cilia
  physiology
  biology and life sciences
title: The BBSome Controls Energy Homeostasis by Mediating the Transport of the Leptin Receptor to the Plasma Membrane