Predicting exon criticality from protein sequence.

Alternative splicing is frequently involved in the diversification of protein function and can also be modulated for therapeutic purposes. Here we develop a predictive model, called Exon ByPASS (predicting Exon skipping Based on Protein amino acid SequenceS), to assess the criticality of exon inclusion based solely on information contained in the amino acid sequence upstream and downstream of the exon junctions. By focusing on protein sequence, Exon ByPASS predicts exon skipping independent of tissue and species in the absence of any intronic information. We validate model predictions using transcriptomic and proteomic data and show that the model can capture exon skipping in different tissues and species. Additionally, we reveal potential therapeutic opportunities by predicting synthetically skippable exons and neo-junctions arising in cancer cells.

Impact of guanidine-containing backbone linkages on stereopure antisense oligonucleotides in the CNS.

Attaining sufficient tissue exposure at the site of action to achieve the desired pharmacodynamic effect on a target is an important determinant for any drug discovery program, and this can be particularly challenging for oligonucleotides in deep tissues of the CNS. Herein, we report the synthesis and impact of stereopure phosphoryl guanidine-containing backbone linkages (PN linkages) to oligonucleotides acting through an RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks. We found that the incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO)-based molecules. One of these backbone types, called PN-1, also yielded profound silencing benefits throughout the mouse brain and spinal cord at low doses, improving both the potency and durability of response, especially in difficult to reach brain tissues. Given these benefits in preclinical models, the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics.

In this study, the authors explore the impact of nitrogen-containing (PN) backbones on oligonucleotides that promote RNase H-mediated degradation of a transcript in the central nervous system (CNS). Using Malat1, a ubiquitously expressed non-coding RNA that is predominately localized in the nucleus, and C9orf72, a challenging RNA target requiring a more nuanced targeting strategy, as benchmarks, they show that chimeric oligonucleotides containing stereopure PS and one of the more promising PN backbones (PN-1) have more potent and durable activity throughout the CNS compared with more traditional PS-modified molecules in mouse models. They demonstrate that potency and durability benefits in vivo derive at least in part from increased tissue exposure, especially in more difficult to reach regions of the brain. Ultimately, these benefits enabled the authors to demonstrate pharmacodynamic effects on Malat1 and C9orf72 RNAs in multiple brain regions with relatively low doses.

Control of backbone chemistry and chirality boost oligonucleotide splice switching activity.

Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype. These data demonstrate that chemical optimization alone can profoundly impact oligonucleotide pharmacology and highlight the potential for continued innovation around the oligonucleotide backbone. More specifically, we conclude that chimeric stereopure oligonucleotides are a promising splice-switching modality with potential for the treatment of neuromuscular and other genetic diseases impacting difficult to reach tissues such as the skeletal muscle and heart.

Variant-selective stereopure oligonucleotides protect against pathologies associated with C9orf72-repeat expansion in preclinical models.

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.

Stereochemistry Enhances Potency, Efficacy, and Durability of Malat1 Antisense Oligonucleotides In Vitro and In Vivo in Multiple Species.

Purpose: Antisense oligonucleotides have been under investigation as potential therapeutics for many diseases, including inherited retinal diseases. Chemical modifications, such as chiral phosphorothioate (PS) backbone modification, are often used to improve stability and pharmacokinetic properties of these molecules. We aimed to generate a stereopure MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) antisense oligonucleotide as a tool to assess the impact stereochemistry has on potency, efficacy, and durability of oligonucleotide activity when delivered by intravitreal injection to eye. Methods: We generated a stereopure oligonucleotide (MALAT1-200) and assessed the potency, efficacy, and durability of its MALAT1 RNA-depleting activity compared with a stereorandom mixture, MALAT1-181, and other controls in in vitro assays, in vivo mouse and nonhuman primate (NHP) eyes, and ex vivo human retina cultures. Results: The activity of the stereopure oligonucleotide is superior to its stereorandom mixture counterpart with the same sequence and chemical modification pattern in in vitro assays, in vivo mouse and NHP eyes, and ex vivo human retina cultures. Findings in NHPs showed durable activity of the stereopure oligonucleotide in the retina, with nearly 95% reduction of MALAT1 RNA maintained for 4 months postinjection. Conclusions: An optimized, stereopure antisense oligonucleotide shows enhanced potency, efficacy, and durability of MALAT1 RNA depletion in the eye compared with its stereorandom counterpart in multiple preclinical models. Translational Relevance: As novel therapeutics, stereopure oligonucleotides have the potential to enable infrequent administration and low-dose regimens for patients with genetic diseases of the eye.

Investigational Assay for Haplotype Phasing of the Huntingtin Gene.

Novel treatments for Huntington's disease (HD), a progressive neurodegenerative disorder, include selective targeting of the mutant allele of the huntingtin gene (mHTT) carrying the abnormally expanded disease-causing cytosine-adenine-guanine (CAG) repeat. WVE-120101 and WVE-120102 are investigational stereopure antisense oligonucleotides that enable selective suppression of mHTT by targeting single-nucleotide polymorphisms (SNPs) that are in haplotype phase with the CAG repeat expansion. Recently developed long-read sequencing technologies can capture CAG expansions and distant SNPs of interest and potentially facilitate haplotype-based identification of patients for clinical trials of oligonucleotide therapies. However, improved methods are needed to phase SNPs with CAG repeat expansions directly and reliably without need for familial genotype/haplotype data. Our haplotype phasing method uses single-molecule real-time sequencing and a custom algorithm to determine with confidence bases at SNPs on mutant alleles, even without familial data. Herein, we summarize this methodology and validate the approach using patient-derived samples with known phasing results. Comparison of experimentally measured CAG repeat lengths, heterozygosity, and phasing with previously determined results showed improved performance. Our methodology enables the haplotype phasing of SNPs of interest and the disease-causing, expanded CAG repeat of the huntingtin gene, enabling accurate identification of patients with HD eligible for allele-selective clinical studies.

Insulin-like growth factor 2 (IGF2) protects against Huntington's disease through the extracellular disposal of protein aggregates.

Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here, we studied the impact of IGF2 signaling on protein aggregation in models of Huntington's disease (HD) as proof of concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients and spinocerebellar ataxia cases. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 was independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human postmortem brain tissue and blood samples from HD patients showed a reduction in IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein species.

Genotyping single nucleotide polymorphisms for allele-selective therapy in Huntington disease.

BACKGROUND: The huntingtin gene (HTT) pathogenic cytosine-adenine-guanine (CAG) repeat expansion responsible for Huntington disease (HD) is phased with single nucleotide polymorphisms (SNPs), providing targets for allele-selective treatments. OBJECTIVE: This prospective observational study defined the frequency at which rs362307 (SNP1) or rs362331 (SNP2) was found on the same allele with pathogenic CAG expansions. METHODS: Across 7 US sites, 202 individuals with HD provided blood samples that were processed centrally to determine the number and size of CAG repeats, presence and heterozygosity of SNPs, and whether SNPs were present on the mutant HTT allele using long-read sequencing and phasing. RESULTS: Heterozygosity of SNP1 and/or SNP2 was identified in 146 (72%) individuals. The 2 polymorphisms were associated only with the mHTT allele in 61% (95% high density interval: 55%, 67%) of individuals. CONCLUSIONS: These results are consistent with previous reports and demonstrate the feasibility of genotyping, phasing, and targeting of HTT SNPs for personalized treatment of HD.

Integrative analyses of proteomics and RNA transcriptomics implicate mitochondrial processes, protein folding pathways and GWAS loci in Parkinson disease.

BACKGROUND: Parkinson disease (PD) is a neurodegenerative disease characterized by the accumulation of alpha-synuclein (SNCA) and other proteins in aggregates termed "Lewy Bodies" within neurons. PD has both genetic and environmental risk factors, and while processes leading to aberrant protein aggregation are unknown, past work points to abnormal levels of SNCA and other proteins. Although several genome-wide studies have been performed for PD, these have focused on DNA sequence variants by genome-wide association studies (GWAS) and on RNA levels (microarray transcriptomics), while genome-wide proteomics analysis has been lacking. METHODS: This study employed two state-of-the-art technologies, three-stage Mass Spectrometry Tandem Mass Tag Proteomics (12 PD, 12 controls) and RNA-sequencing transcriptomics (29 PD, 44 controls), evaluated in the context of PD GWAS implicated loci and microarray transcriptomics (19 PD, 24 controls). The technologies applied for this study were performed in a set of overlapping prefrontal cortex (Brodmann area 9) samples obtained from PD patients and sex and age similar neurologically healthy controls. RESULTS: After appropriate filters, proteomics robustly identified 3558 unique proteins, with 283 of these (7.9 %) significantly different between PD and controls (q-value < 0.05). RNA-sequencing identified 17,580 protein-coding genes, with 1095 of these (6.2 %) significantly different (FDR p-value < 0.05); only 166 of the FDR significant protein-coding genes (0.94 %) were present among the 3558 proteins characterized. Of these 166, eight genes (4.8 %) were significant in both studies, with the same direction of effect. Functional enrichment analysis of the proteomics results strongly supports mitochondrial-related pathways, while comparable analysis of the RNA-sequencing results implicates protein folding pathways and metallothioneins. Ten of the implicated genes or proteins co-localized to GWAS loci. Evidence implicating SNCA was stronger in proteomics than in RNA-sequencing analyses. CONCLUSIONS: We report the largest analysis of proteomics in PD to date, and the first to combine this technology with RNA-sequencing to investigate GWAS implicated loci. Notably, differentially expressed protein-coding genes were more likely to not be characterized in the proteomics analysis, which lessens the ability to compare across platforms. Combining multiple genome-wide platforms offers novel insights into the pathological processes responsible for this disease by identifying pathways implicated across methodologies.

Increased metabolic flexibility and complexity in a long-lived growth hormone insensitive mouse model.

The goal of this study was to test whether the "loss of the complexity" hypothesis can be applied to compare the metabolic patterns of mouse models with known differences in metabolic and endocrine function as well as life span. Here, we compare the complexity of locomotor activity and metabolic patterns (energy expenditure, VO2, and respiratory quotient) of the long-lived growth hormone receptor gene deleted mice (GHR(-/-)) and their wild-type littermates. Using approximate entropy as a measure of complexity, we observed greater metabolic complexity, as indicated by greater irregularity in the physiological fluctuations of the GHR(-/-) mice. Further analysis of the data also revealed lower energy costs of locomotor activity and a stronger relationship between locomotor activity and respiratory quotient in the GHR(-/-) mice relative to controls. These findings suggest underlying differences in metabolic modulation in the GHR(-/-) mice revealed especially through measures of complexity of their time-dependent fluctuations.

Secreted frizzled-related protein 5 suppresses adipocyte mitochondrial metabolism through WNT inhibition.

Preadipocytes secrete several WNT family proteins that act through autocrine/paracrine mechanisms to inhibit adipogenesis. The activity of WNT ligands is often decreased by secreted frizzled-related proteins (SFRPs). Sfrp5 is strongly induced during adipocyte differentiation and increases in adipocytes during obesity, presumably to counteract WNT signaling. We tested the hypothesis that obesity-induced Sfrp5 expression promotes the development of new adipocytes by inhibiting endogenous suppressors of adipogenesis. As predicted, mice that lack functional SFRP5 were resistant to diet-induced obesity. However, counter to our hypothesis, we found that adipose tissue of SFRP5-deficient mice had similar numbers of adipocytes, but a reduction in large adipocytes. Transplantation of adipose tissue from SFRP5-deficient mice into leptin receptor-deficient mice indicated that the effects of SFRP5 deficiency are tissue-autonomous. Mitochondrial gene expression was increased in adipose tissue and cultured adipocytes from SFRP5-deficient mice. In adipocytes, lack of SFRP5 stimulated oxidative capacity through increased mitochondrial activity, which was mediated in part by PGC1α and mitochondrial transcription factor A. WNT3a also increased oxygen consumption and the expression of mitochondrial genes. Thus, our findings support a model of adipogenesis in which SFRP5 inhibits WNT signaling to suppress oxidative metabolism and stimulate adipocyte growth during obesity.

Pharmacologic inhibition of ghrelin receptor signaling is insulin sparing and promotes insulin sensitivity.

Ghrelin influences a variety of metabolic functions through a direct action at its receptor, the GhrR (GhrR-1a). Ghrelin knockout (KO) and GhrR KO mice are resistant to the negative effects of high-fat diet (HFD) feeding. We have generated several classes of small-molecule GhrR antagonists and evaluated whether pharmacologic blockade of ghrelin signaling can recapitulate the phenotype of ghrelin/GhrR KO mice. Antagonist treatment blocked ghrelin-induced and spontaneous food intake; however, the effects on spontaneous feeding were absent in GhrR KO mice, suggesting target-specific effects of the antagonists. Oral administration of antagonists to HFD-fed mice improved insulin sensitivity in both glucose tolerance and glycemic clamp tests. The insulin sensitivity observed was characterized by improved glucose disposal with dramatically decreased insulin secretion. It is noteworthy that these results mimic those obtained in similar tests of HFD-fed GhrR KO mice. HFD-fed mice treated for 56 days with antagonist experienced a transient decrease in food intake but a sustained body weight decrease resulting from decreased white adipose, but not lean tissue. They also had improved glucose disposal and a striking reduction in the amount of insulin needed to achieve this. These mice had reduced hepatic steatosis, improved liver function, and no evidence of systemic toxicity relative to controls. Furthermore, GhrR KO mice placed on low- or high-fat diets had lifespans similar to the wild type, emphasizing the long-term safety of ghrelin receptor blockade. We have therefore demonstrated that chronic pharmacologic blockade of the GhrR is an effective and safe strategy for treating metabolic syndrome.

Characterization of the insulin sensitivity of ghrelin receptor KO mice using glycemic clamps.

BACKGROUND: We and others have demonstrated previously that ghrelin receptor (GhrR) knock out (KO) mice fed a high fat diet (HFD) have increased insulin sensitivity and metabolic flexibility relative to WT littermates. A striking feature of the HFD-fed GhrR KO mouse is the dramatic decrease in hepatic steatosis. To characterize further the underlying mechanisms of glucose homeostasis in GhrR KO mice, we conducted both hyperglycemic (HG) and hyperinsulinemic-euglycemic (HI-E) clamps. Additionally, we investigated tissue glucose uptake and specifically examined liver insulin sensitivity. RESULTS: Consistent with glucose tolerance-test data, in HG clamp experiments, GhrR KO mice showed a reduction in glucose-stimulated insulin release relative to WT littermates. Nevertheless, a robust 1st phase insulin secretion was still achieved, indicating that a healthy ß-cell response is maintained. Additionally, GhrR KO mice demonstrated both a significantly increased glucose infusion rate and significantly reduced insulin requirement for maintenance of the HG clamp, consistent with their relative insulin sensitivity. In HI-E clamps, both LFD-fed and HFD-fed GhrR KO mice showed higher peripheral insulin sensitivity relative to WT littermates as indicated by a significant increase in insulin-stimulated glucose disposal (Rd), and decreased hepatic glucose production (HGP). HFD-fed GhrR KO mice showed a marked increase in peripheral tissue glucose uptake in a variety of tissues, including skeletal muscle, brown adipose tissue and white adipose tissue. GhrR KO mice fed a HFD also showed a modest, but significant decrease in conversion of pyruvate to glucose, as would be anticipated if these mice displayed increased liver insulin sensitivity. Additionally, the levels of UCP2 and UCP1 were reduced in the liver and BAT, respectively, in GhrR KO mice relative to WT mice. CONCLUSIONS: These results indicate that improved glucose homeostasis of GhrR KO mice is characterized by robust improvements of glucose disposal in both normal and metabolically challenged states, relative to WT controls. GhrR KO mice have an intact 1st phase insulin response but require significantly less insulin for glucose disposal. Our experiments reveal that the insulin sensitivity of GhrR KO mice is due to both BW independent and dependent factors. We also provide several lines of evidence that a key feature of the GhrR KO mouse is maintenance of hepatic insulin sensitivity during metabolic challenge.

Daily energy balance in growth hormone receptor/binding protein (GHR -/-) gene-disrupted mice is achieved through an increase in dark-phase energy efficiency.

The goal of this study was to examine factors that contribute to energy balance in female GHR -/- mice. We measured energy intake, energy expenditure (EE), fuel utilization, body mass (M(b)) changes and physical activity in 17month-old female GHR -/- mice and their age-matched wild type littermates. The GHR -/- mice were smaller, consumed more food per unit M(b), had greater EE per unit M(b) and had an increase in 24-h EE/M(b) that was similar to the increase in their surface-area-to-volume ratio. Locomotor activity (LMA) was reduced in the GHR -/- mice, but the energetic cost associated with their LMA was greater than in wild type controls. Furthermore, M(b) and LMA were independent explanatory covariates of most of the variance in EE, and when adjusted for M(b) and LMA, the GHR -/- mice had higher EE during both the light and dark phases of the daily cycle. Respiratory quotient was lower in GHR -/- mice during the light phase, which indicated a greater utilization of lipid relative to carbohydrate in these mice. Additionally, GHR -/- mice had higher ratios of caloric intake to EE at several intervals during the dark phase, and this effect was greater and more sustained in the final 3h of the dark phase. Therefore, we conclude that GHR -/- mice are able to overcome the substantial energetic challenges of dwarfism through several mechanisms that promote stable M(b). Relative to wild type mice, the GHR -/- mice consumed more calories per unit M(b), which offset the disproportionate increase in their daily energy expenditure. While GHR -/- mice oxidized a greater proportion of lipid during the light phase in order to meet their energy requirements, they achieved greater energy efficiency and storage during the dark phase through a combination of higher energy consumption and lower LMA.

The 24-hour respiratory quotient predicts energy intake and changes in body mass.

To define the relationship between the respiratory quotient (RQ) and energy intake (EI) and to determine the impact of spontaneous locomotor activity (LMA) in the development of diet-induced obesity (DIO), we fed C57BL/6 mice a high-fat diet (HFD) for either 4 days or 17 wk and analyzed them using indirect calorimetry. Importantly, changes in body mass during calorimetry (DeltaM(b)) significantly covaried with RQ and EI; adjusting the data for DeltaM(b) permitted an analysis of the energy-balanced state. The 24-h RQ strongly predicted 24-h EI, and the slope of this relationship was diet dependent (HFD or chow) but independent of the HFD feeding period. Early-stage DIO was characterized by dark-period hyperphagia and fat storage, offset by greater light-period lipid oxidation; later stage DIO mice had a milder hyperphagia and lower substrate flexibility. Consequently, whereas 24-h RQ equaled the food quotient of the HFD in both early- and late-stage DIO, the range of RQ values was negatively correlated with, and mostly explained by, 24-h EI only in late-stage DIO. Lean and early-stage DIO mice had similar LMA values that were reduced in late-stage DIO. However, LMA significantly explained variance in total energy expenditure (EE) in only early-stage DIO mice. This indicated that the link between LMA and EE was a transient adaptive response to early DIO, whereas the later loss of LMA did not explain body weight gain in C57BL/6 DIO mice.

Enhanced gastrointestinal motility with orally active ghrelin receptor agonists.

The orexigenic peptide ghrelin has been shown to have prokinetic activity in the gastrointestinal (GI) system of several species, including humans. In this series of experiments, we have evaluated the prokinetic activity of novel, small-molecule ghrelin receptor (GhrR) agonists after parenteral and peroral dosing in mice and rats. Gastric emptying, small intestinal transport, and fecal output were determined after intraperitoneal and intracerebroventricular dosing of GhrR agonists, using ghrelin as a positive control. These same parameters were evaluated after oral gavage dosing of the synthetic agonists. Regardless of dose route, GhrR agonist treatment increased gastric emptying, small intestinal transit, and fecal output. However, fecal output was only increased by GhrR agonist treatment if mice were able to feed during the stimulatory period. Thus, GhrR agonists can stimulate upper GI motility, and the orexigenic action of the compounds can indirectly contribute to prokinetic activity along the entire GI tract. The orexigenic and prokinetic effects of either ghrelin or small-molecule GhrR agonists were selective for the GhrR because they were absent when evaluated in GhrR knockout mice. We next evaluated the efficacy of the synthetic GhrR agonists dosed in a model of opiate-induced bowel dysfunction induced by a single injection of morphine. Oral dosing of a GhrR agonist normalized GI motility in opiate-induced dysmotility. These data demonstrate the potential utility of GhrR agonists for treating gastrointestinal hypomotility disorders.

Improved insulin sensitivity and metabolic flexibility in ghrelin receptor knockout mice.

Stimulation of the ghrelin receptor (GhrR) by ghrelin results in a variety of metabolic changes including increased food intake, fat storage and insulin resistance. Loss of ghrelin signaling is protective against diet-induced obesity, suggesting that ghrelin plays a significant homeostatic role in conditions of metabolic stress. We examined glycemic control in GhrR -/- mice fed a high-fat diet, and used indirect calorimetry to assess fuel substrate usage and energy expenditure. GhrR -/- mice fed a high-fat diet had several measures of greater insulin sensitivity, including: lower fasted blood glucose and plasma insulin, lower %Hb(A1c), lower insulin levels during glucose tolerance tests, and improved performance in hyperinsulinemic-euglycemic and hyperglycemic clamp studies. GhrR -/- mice fed a high-fat diet did not develop hepatic steatosis and had lower total cholesterol, relative to controls. Furthermore, GhrR -/- mice demonstrated a lower intestinal triglyceride secretion rate of dietary lipid. GhrR -/- mice have higher respiratory quotients (RQ), indicating a preference for carbohydrate as fuel. The range of RQ values was wider in GhrR -/- mice, indicating greater metabolic flexibility and insulin sensitivity in these animals. We therefore propose that loss of ghrelin signaling promotes insulin sensitivity and metabolic flexibility, and protects against several fatty diet-induced features of metabolic syndrome due to convergent changes in the intake, absorption and utilization of energy.

Wnt10b inhibits obesity in ob/ob and agouti mice.

The Wnt family of secreted signaling molecules has profound effects on diverse developmental processes, including the fate of mesenchymal progenitors. While activation of Wnt signaling blocks adipogenesis, inhibition of endogenous Wnt/beta-catenin signaling by Wnt10b promotes spontaneous preadipocyte differentiation. Transgenic mice with expression of Wnt10b from the FABP4 promoter (FABP4-Wnt10b) have less adipose tissue when maintained on a normal chow diet and are resistant to diet-induced obesity. Here we demonstrate that FABP4-Wnt10b mice largely avert weight gain and metabolic abnormalities associated with genetic obesity. FABP4-Wnt10b mice do not gain significant body weight on the ob/ob background, and at 8 weeks of age, they have an approximately 70% reduction in visceral and subcutaneous adipose tissues compared with ob/ob mice. Similarly, on the lethal yellow agouti (A(y)) background, FABP4-Wnt10b mice have 50-70% less adipose tissue weight and circulating leptin at 5 months of age. Wnt10b-Ay mice are more glucose tolerant and insulin sensitive than A(y) controls, perhaps due to reduced expression and circulation of resistin. Reduced expression of inflammatory cytokines may also contribute to improved glucose homeostasis.

Effects of Wnt signaling on brown adipocyte differentiation and metabolism mediated by PGC-1alpha.

Activation of canonical Wnt signaling inhibits brown adipogenesis of cultured cells by impeding induction of PPARgamma and C/EBPalpha. Although enforced expression of these adipogenic transcription factors restores lipid accumulation and expression of FABP4 in Wnt-expressing cells, additional expression of PGC-1alpha is required for activation of uncoupling protein 1 (UCP1). Wnt10b blocks brown adipose tissue development and expression of UCP1 when expressed from the fatty acid binding protein 4 promoter, even when mice are administered a beta3-agonist. In differentiated brown adipocytes, activation of Wnt signaling suppresses expression of UCP1 through repression of PGC-1alpha. Consistent with these in vitro observations, UCP1-Wnt10b transgenic mice, which express Wnt10b in interscapular tissue, lack functional brown adipose tissue. While interscapular tissue of UCP1-Wnt10b mice lacks expression of PGC-1alpha and UCP1, the presence of unilocular lipid droplets and expression of white adipocyte genes suggest conversion of brown adipose tissue to white. Reciprocal expression of Wnt10b with UCP1 and PGC-1alpha in interscapular tissue from cold-challenged or genetically obese mice provides further evidence for regulation of brown adipocyte metabolism by Wnt signaling. Taken together, these data suggest that activation of canonical Wnt signaling early in differentiation blocks brown adipogenesis, whereas activating Wnt signaling in mature brown adipocytes stimulates their conversion to white adipocytes.

Regulation of osteoblastogenesis and bone mass by Wnt10b.

Wnts comprise a family of secreted signaling proteins that regulate diverse developmental processes. Activation of Wnt signaling by Wnt10b inhibits differentiation of preadipocytes and blocks adipose tissue development; however, the effect of Wnt10b on other mesenchymal lineages has not been defined. To explore the physiological role of Wnt signaling in bone development, we analyzed FABP4-Wnt10b mice, which express the Wnt10b transgene in marrow. Femurs from FABP4-Wnt10b mice have almost four times as much bone in the distal metaphyses and are mechanically stronger. These mice maintain elevated bone mass at least through 23 months of age. In addition, FABP4-Wnt10b mice are protected from the bone loss characteristic of estrogen deficiency. We used pharmacological and genetic approaches to demonstrate that canonical Wnt signaling stimulates osteoblastogenesis and inhibits adipogenesis of bipotential mesenchymal precursors. Wnt10b shifts cell fate toward the osteoblast lineage by induction of the osteoblastogenic transcription factors Runx2, Dlx5, and osterix and suppression of the adipogenic transcription factors C/EBPalpha and PPARgamma. One mechanism whereby Wnt10b promotes osteoblastogenesis is suppression of PPARgamma expression. Finally, Wnt10b-/- mice have decreased trabecular bone and serum osteocalcin, confirming that Wnt10b is an endogenous regulator of bone formation.