MetAP2 inhibition reduces food intake and body weight in a ciliopathy mouse model of obesity

The ciliopathies Bardet-Biedl Syndrome and Alström Syndrome are genetically inherited pleiotropic disorders with primary clinical features of hyperphagia and obesity. Methionine aminopeptidase 2 inhibitors (MetAP2i) have been shown in preclinical and clinical studies to reduce food intake, body weight, and adiposity. Here we investigated the effects of MetAP2i administration in a mouse model of ciliopathy produced by conditional deletion of the Thm1 gene in adulthood (Thm1 cko). Thm1 cko mice show decreased hypothalamic pro-opiomelanocortin expression as well as hyperphagia, obesity, metabolic disease and hepatic steatosis. In obese Thm1 cko mice, two-week administration of MetAP2i reduced daily food intake and reduced body weight 17.1% from baseline (vs. 5% reduction for vehicle). This was accompanied with decreased levels of blood glucose, insulin and leptin. Further, MetAP2i reduced gonadal adipose depots and adipocyte size and improved liver morphology. This is the first report of MetAP2i reducing hyperphagia and body weight, and ameliorating metabolic indices in a mouse model of ciliopathy. These results support further investigation of MetAP2 inhibition as a potential therapeutic strategy for ciliary-mediated forms of obesity.


Introduction
Obesity and associated insulin resistance increase risk for potentially fatal chronic diseases, including cardiovascular disease, type 2 diabetes, and non-alcoholic fatty liver disease.
As obesity is pandemic 1 , finding effective therapeutic strategies is critical to improving global health.
In preclinical and clinical studies, inhibition of methionine aminopeptidase 2 (MetAP2) has shown promising results. MetAP2 belongs to a family of metalloproteases, which cleaves the N-terminal methionine of nascent proteins. This post-translational modification induces subcellular localization changes and activation of the targeted protein 2 . Fumagillin, a natural product of Aspergillus fumigatus, irreversibly inhibits MetAP2 3 . MetAP2 inhibition causes late G1 cell cycle arrest, and inhibits cell proliferation, as well as phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK½) [4][5][6][7] . Early studies revealed anti-cancer and anti-fungal effects of fumagillin 8,9 . Subsequently, studies showed fumagillin and its analogs also have antiobesity effects, resulting in decreased body weight and adiposity and increased insulin sensitivity in high-fat diet-induced obese mice and rats 10,11 , as well as in genetic ob/ob mice 12 .
Additionally, administration of the fumagillin derivative, beloranib, to individuals with nongenetic causes of obesity and to patients with Prader-Willi Syndrome, a genetic disease that causes insatiable appetite and obesity, resulted in reduced food intake and body weight 13,14 . The effectiveness of MetAP2i in various models raises the possibility that inhibiting MetAP2 may counter other forms of obesity.
Ciliopathies are genetic disorders that arise from dysfunctional or absent cilia, and present numerous clinical features, including renal and hepatic fibrocystic disease, skeletal defects, infertility, hydrocephalus, mental disability, brain malformations, and central obesity 15 .
Primary cilia are microtubule-based, mechanosensory organelles that protrude from the apical membrane of most mammalian cells and regulate signaling pathways. Primary cilia utilize intraflagellar transport (IFT) multi-protein complexes for bi-directional movement of protein cargo along the ciliary axoneme. The IFT-B complex mediates anterograde protein transport, while the IFT-A complex is required for retrograde transport and for ciliary import of membraneassociated and signaling proteins 16,17 . Another multi-protein complex, the BBSome, transports signaling molecules to the ciliary base, and acts like an adaptor between IFT complexes and protein cargo in the ciliary export of signaling molecules. Two ciliopathies, Alström Syndrome and Bardet-Biedl Syndrome (BBS), present obesity as a central clinical feature 18,19 .
Additionally, polymorphisms in the BBS genes in the general population have been associated with obesity, and cilia length defects have been identified in adipose-derived mesenchymal stem cells from obese individuals, suggesting a more common relevance for cilia-related mechanisms [20][21][22] .
Modifying mutations in the IFT-A gene, THM1, have been reported in patients with Bardet Biedl Syndrome 23 . We have shown that global deletion of Thm1 in adult mice causes decreased hypothalamic expression of appetite-controlling pro-opiomelanocortin (Pomc), hyperphagia, obesity and metabolic syndrome 24 . Here we examine the effects of administering a fumagillin derivative to obese Thm1 conditional knock-out (cko) mice. Our results reveal reduced food intake, body weight and adipose tissue mass, as well as improved metabolic indices. These data indicate MetAP2 inhibition as a potential therapeutic strategy against obesity caused by genetic disorders of cilia.

MetAP2i treatment decreases body weight and food intake in Thm1 cko mice
To generate obese Thm1 cko mice, we induced deletion of Thm1 in male mice at five weeks of age and fed mutant mice and control littermates ab libitum throughout the 13-week duration of the study (Fig 1A). Body weight was measured weekly from 0-10 weeks post-Thm1 deletion. At 10 weeks following gene deletion, Thm1 cko mice weighed 42.2g ± 1.1g, while control littermates weighted 29.4g ± 0.7g, confirming the obese phenotype of the mutant mice ( Fig S1A). At this timepoint (week 10 of the experiment), mice were housed individually and baseline measurements of body weight and food intake were obtained daily for one week. From week 10-11 of the experiment, Thm1 cko mice showed a higher average daily food intake than control littermates, consistent with hyperphagia ( Fig 1B). Unexpectedly, we observed that individual housing induced weight loss in many of the control mice and in all of the Thm1 cko mice, and that the mutant animals showed a greater percent weight reduction (-3.8%) than control animals (-0.8%; Fig 1C; Fig S1B). Following this one-week observation period, we administered daily subcutaneous injections of MetAP2i or vehicle for two weeks (week 11-13).
MetAP2i treatment reduced food intake relative to vehicle in Thm1 cko mice ( Fig 1D).
Additionally, MetAP2i treatment caused a -17.1% body weight reduction compared to -5.0% for vehicle in Thm1 cko mice ( Fig 1D; Fig S1C). These data show that MetAP2i can counter the hyperphagia and increased body weight induced by deletion of Thm1.

MetAP2i treatment improved metabolic indices in Thm1 cko mice
Following the two-week intervention, we measured metabolic parameters, including nonfasting blood glucose and serum insulin and leptin, which we previously observed to be elevated in obese Thm1 cko mice 24 . In this study, Thm1 cko mice did not have higher non-fasting blood glucose than control littermates (Fig 2A). Yet in Thm1 cko mice, MetAP2i treatment reduced non-fasting blood glucose levels relative to vehicle treatment (Fig 2A). Serum insulin levels were higher in vehicle-treated Thm1 cko mice than in vehicle-treated control littermates, but MetAP2i treatment in Thm1 cko mice decreased insulin levels relative to vehicle treatment and to an extent that insulin levels were not significantly different from vehicle-treated control mice ( Fig 2B). Similarly, serum leptin was elevated in vehicle-treated Thm1 cko mice relative to control littermates. In Thm1 cko mice, MetAP2i treatment reduced leptin levels relative to vehicle treatment and to a degree that leptin levels were not significantly different from those of vehicle-treated control mice ( Fig 2C). These data indicate that MetAP2i can correct these metabolic parameters.

MetAP2i treatment decreases gonadal fat mass and adipocyte size in Thm1 cko mice
We next analyzed gonadal and peri-renal fat depots, which we have shown previously to be increased in obese Thm1 cko mice 24 . As expected, vehicle-treated Thm1 cko mice showed increased gonadal and renal adipose tissue mass relative to control littermates (Figs 3A-D). In Thm1 cko mice, MetAP2i treatment reduced gonadal fat mass compared to vehicle-treatment ( Fig 3A). Histology of gonadal fat pads showed an increase in gonadal adipocyte cell size in vehicle-treated Thm1 cko mice relative to control mice (Figs 3E and 3F), consistent with previous findings 24 . However, gonadal adipocyte cell size was reduced in MetAP2i-treated mutants. These data show that MetAP2i treatment partially attenuates the increased gonadal adipose tissue mass and adipocyte size caused by deletion of Thm1.

MetAP2i treatment improved liver morphology of Thm1 cko mice
Obese Thm1 cko mice can develop hepatic steatosis 24 . To determine if MetAP2i treatment affected the liver, we examined the histology of livers of vehicle-and MetAP2i-treated mice. Vehicle-treated Thm1 cko mice had vacuoles in their livers, suggesting formation of lipid droplets (Fig 4). These vacuoles were not observed in control mice. Further, the vacuoles were reduced or absent in livers of MetAP2i-treated Thm1 cko mice, suggesting that the improved metabolism resulting from MetAP2i treatment extends to the liver.

MetAP2i treatment increased renal cilia length of control and Thm1 cko mice
Perinatal deletion of Thm1 causes renal cystic disease by six weeks of age, but deletion of Thm1 in adulthood does not cause renal cysts at three months following gene deletion 25 .
Accordingly, kidney weights and morphology were similar between control and Thm1 cko mice (Figs 5A and 5C). We observed a slight decrease in KW/BW ratio of vehicle-treated Thm1 cko mice relative to vehicle-treated control mice (Fig 5B), which could be due to the increased body weight of the Thm1 cko mice. MetAP2i treatment of control or Thm1 cko mice did not affect kidney morphology (Fig. 5C). We next examined primary cilia by immunostaining for the ciliary membrane protein, ARL13B, together with incubating with the lectin, Dolichos biflorus agglutinin (DBA) to label the collecting duct. We observed previously that cilia are shortened in Thm1-deficient mice [24][25][26] . We observed that average cilia length in collecting ducts of vehicletreated Thm1 cko mice was shortened relative to vehicle-treated control mice, but we noted a wide range of cilia lengths in the Thm1 cko collecting ducts and statistical significance was not achieved (Fig 1E). Unexpectedly, we observed that cilia lengths of MetAP2i-treated mice were longer than in vehicle-treated mice, and this occurred in both control and Thm1 cko mice.

Discussion
In this study, we provide the first evidence that MetAP2 inhibition reduces food intake and body weight and improves metabolic parameters in a ciliary model of obesity. This suggests a novel potential therapeutic strategy for ciliary disorders of obesity. This also broadens the therapeutic applicability of MetAP2 inhibition. To our knowledge, this is also the first study to show an effective pharmacological intervention in a ciliopathy rodent model of obesity. Interestingly, we observed increased cilia length in renal epithelial cells of MetAP2itreated mice. A possible mechanism by which MetAP2i increased cilia length could involve its function as an ERK inhibitor, since ERK inhibition has been shown to increase cilia length. In adipose-derived mesenchymal stem cells obtained from obese individuals, cilia are shortened 21 .
However, treatment with an ERK inhibitor lengthened primary cilia and improved stemness and differentiation capacity of the cells 29 . Additionally, ERK inhibition of renal proximal tubular cells preserved cilia length during cisplatin treatment and protected the cells from cisplatin-induced apoptosis 30 . Determining in which other tissues and cells MetAP2i increased cilia length would be informative. We have shown previously that primary cilia in the Thm1 cko hypothalamic arcuate nucleus are shortened and bulbous 24 . It seems plausible that lengthening of the mutant cilia in the arcuate nucleus may potentially attenuate the misregulated appetite. We were unable to detect primary cilia in the hypothalamus using the currently available commercial antibodies, but testing such a hypothesis in the future could reveal an important potential mechanism. Further, lengthening of primary cilia in other cell types of Thm1 cko mice, such as adipose-derived mesenchymal stem cells, could also result in beneficial effects.
MetAP2i treatment also resulted in smaller adipocyte cell size, consistent with previous studies in rodent models of high fat diet-induced obesity 10,11 . Increased adipogenesis could result in smaller, metabolically healthier adipocytes. The direct effects of MetAP2i on adipogenesis are unclear. In one study, addition of fumagillin to an in vitro pre-adipocyte differentiation assay promoted adipogenesis, but had minimal effects in an in vivo adipogenesis assay 31 . In contrast in another study, addition of a fumagillin derivative to an in vitro pre-adipocyte differentiation study inhibited adipogenesis, yet fumagillin treatment of cells caused enhanced glucose uptake, suggesting metabolically healthier cells 32 . Alternatively, fumagillin has anti-angiogenic effects and a prevailing hypothesis is that changes in angiogenesis induced by fumagillin reduce adipose tissue mass. Yet, a study showed that angiogenesis changes did not drive reduction of adipose tissue mass 10 . Thus, further studies are required to determine the mechanisms by which adipocyte size and adipose tissue mass are reduced by MetAP2i.
Given safety issues, Zafgen has recently discontinued the development of MetAP2 inhibitors 33 . Our study indicates that there are plausible targets of MetAP2 inhibition that effectively reduce hyperphagia and body weight and substantially improve metabolic parameters in a ciliopathy model. This substantiates the need for greater understanding of the biology of MetAP2 34 . Currently, agonists of the melanocortin 4 receptor, which is activated by the processed protein products of Pomc, are being tested in clinical trial to target obesity in patients with BBS and Alström Syndrome 35 . Aside from this, a potential therapy for ciliopathy-induced obesity has not been demonstrated. The mechanism by which MetAP2 inhibition exerts its antiobesity effects remain elusive. Intriguingly, our data suggest MetAP2 inhibition modifies cilia length irrespective of genotype, and therefore, may alter cilia length in other models of obesity.
Whether changes in ciliary dynamics underlie the beneficial effects of MetAP2 inhibition may warrant investigation, and could reveal the therapeutic targets.

Generation of Thm1 cko mice
Thm1 cko mice were generated as described 25 . Briefly, Thm1 cko mice were generated by using a Thm1 null allele (called aln) and a floxed allele, which has LoxP sites flanking exon 4, together with a tamoxifen-inducible ROSA26-CreERT recombinase (Jackson Laboratories, Stock 004847), which is expressed globally. Cre recombinase expression was induced at 5 weeks of age by i.p. injection of 10 mg tamoxifen/40g mouse weight. Tamoxifen (Sigma T5648) was suspended in corn oil (Sigma C8267) at 30 mg/ml. Only male mice were used.

Administration of MetAP2i
Mice were fed ab libitum throughout the duration of the study. Body weight was measured weekly from 0-10 weeks post-tamoxifen injection to ascertain the obese phenotype in Thm1 cko mice. Beginning at 10 weeks post-tamoxifen injection, mice were housed individually, and food intake and body weight were measured daily until the end of the experiment. Subcutaneous injections of MetAP2i (ZGN-1258 -0.3 mg/kg/day) or saline vehicle were administered daily from 11-13 weeks post-tamoxifen injection. All animal studies were approved by KUMC IACUC.

Blood glucose and serum insulin and leptin measurements
Blood glucose was measured from tail blood using Bayer Blood Glucose Contour Strips together with the Bayer Contour Blood Glucose Meter system. For serum insulin and leptin measurements, trunk blood was collected in a Microvette CB300z blood collection tube (Kent Scientific), and serum was isolated by centrifuging blood collection tubes for 6 minutes at 800xg at 4°C using a tabletop centrifuge (PrismR, C2500-R). Insulin and leptin levels were measured using Mouse Ultrasensitive Insulin ELISA and Mouse/Rat Leptin ELISA kits (ALPCO) according to manufacturer's instructions.

Histology
Tissues were isolated and submerged in 10% formalin for 3-7 days. Fixed tissues were dehydrated through an ethanol series, paraffin-embedded and sectioned at 7µm thicknesses.
Sections were rehydrated and stained with hematoxylin and eosin using a standard protocol.
Staining was viewed and imaged using a Nikon 80i microscope equipped with a Nikon DS-Fi1 camera.
Heat antigen retrieval was performed in tri-sodium citrate solution, pH 6.0, using a steamer.
Sections were rinsed in distilled water (10X), then blocked in 2% BSA in PBS for 1 hour.
Arl13B antibody (Proteintech) was diluted in blocking buffer (2% BSA in PBS) and tissue sections were incubated with primary antibody at 4°C overnight. Sections were washed 3X in PBS, then incubated with Dolichos biflorus agglutinin (DBA; Vector Laboratories) for 1 hour at room temperature. Sections were washed in PBS 3X, then incubated with Alexa Fluor 594, goat anti-rabbit secondary antibody (ThermoFisher) for 1 hour at room temperature. Following 3 washes in PBS, tissue sections were then mounted with Fluoromount G containing DAPI mounting media (Electron Microscopy Services). Immuno-labeled tissues were viewed and imaged using a Leica TCS SPE confocal microscope configured on a DM550 Q upright microscope.
Statistics: Graphpad Prism 8 was used to perform statistical analyses, which included two-tailed unpaired t-tests, and ANOVA followed by Tukey's test.  Thm1 cko