Measuring Optokinetic Reflex and Vestibulo-Ocular Reflex in Unilateral Vestibular Organ Damage Model of Zebrafish.

One-sided vestibular disorders are common in clinical practice; however, their models have not been fully established. We investigated the effect of unilateral or bilateral deficits in the vestibular organs on the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) of zebrafish using in-house equipment. For physical dislodgement of the otoliths in the utricles of zebrafish larvae, one or both utricles were separated from the surrounding tissue using glass capillaries. The video data from VOR and OKR tests with the larvae was collected and processed using digital signal processing techniques such as fast Fourier transform and low-pass filters. The results showed that unilateral and bilateral damage to the vestibular system significantly reduced VOR and OKR. In contrast, no significant difference was observed between unilateral and bilateral damage. This study confirmed that VOR and OKR were significantly reduced in zebrafish with unilateral and bilateral vestibular damage. Follow-up studies on unilateral vestibular disorders can be conducted using this tool.

Laminaria Japonica Polysaccharide Improved the Productivities and Systemic Health of Ducks by Mediating the Gut Microbiota and Metabolome.

This study investigated the beneficial effects of a Laminaria japonica polysaccharide (LJPS) on the systemic health of ducks by modulating the gut microbiome and metabolome. Our findings demonstrated that the LJPS supplementation enhanced the overall growth performance and physiological immune and antioxidant index of ducks. In addition, the LJPS-fed group significantly increased abundances of intestinal Bacteroides and Prevotellaceae with decreased α-diversity than that in the control group. Among the total of 1840 intestinal metabolites, 186 metabolites were identified to be differentially regulated by LJPS feeding (upregulated 143 metabolites and downregulated 43 metabolites), which is closely associated with some of the growth-related metabolic pathways. Lastly, the correlation analysis recapitulates that the beneficial effects of LJPS underlie the alterations in intestinal microbiota and metabolites. Taken together, LJPS supplementation improved the physiological parameters and richness of some beneficial microbes and upregulated certain metabolic pathways, which facilitated better productivities and systemic health of ducks.

Protective effects of esomeprazole against cisplatin-induced ototoxicity: an in vitro and in vivo study.

In this study, we aimed to identify novel compounds that could afford protection against cisplatin-induced ototoxicity by employing both cell- and zebrafish (Danio rerio)-based screening platforms. We screened 923 US Food and Drug Administration-approved drugs to identify potential compounds exhibiting protective effects against cisplatin-induced ototoxicity in HEI-OC1 cells (auditory hair cell line). The screening strategy identified esomeprazole and dexlansoprazole as the primary hit compounds. Subsequently, we examined the effects of these compounds on cell viability and apoptosis. Our results revealed that esomeprazole and dexlansoprazole inhibited organic cation transporter 2 (OCT2), thus providing in vitro evidence that these compounds could ameliorate cisplatin-induced ototoxicity by directly inhibiting OCT2-mediated cisplatin transport. In vivo, the protective effects were validated using zebrafish; esomeprazole was found to decrease cisplatin-induced hair cell damage in neuromasts. Furthermore, the esomeprazole-treated group showed a significantly lower number of TUNEL-positive cells than the cisplatin-treated group. Collectively, our findings revealed that esomeprazole exerts a protective effect against cisplatin-induced hair cell damage in both HEI-OC1 cells and a zebrafish model.

Molecular profiling of the stroke-induced alterations in the cerebral microvasculature reveals promising therapeutic candidates.

Stroke-induced cerebral microvascular dysfunction contributes to aggravation of neuronal injury and compromises the efficacy of current reperfusion therapies. Understanding the molecular alterations in cerebral microvessels in stroke will provide original opportunities for scientific investigation of novel therapeutic strategies. Toward this goal, using a recently optimized method which minimizes cell activation and preserves endothelial cell interactions and RNA integrity, we conducted a genome-wide transcriptomic analysis of cerebral microvessels in a mouse model of stroke and compared these transcriptomic alterations with the ones observed in human, nonfatal, brain stroke lesions. Results from these unbiased comparative analyses have revealed the common alterations in mouse stroke microvessels and human stroke lesions and identified shared molecular features associated with vascular disease (e.g., Serpine1/Plasminogen Activator Inhibitor-1, Hemoxygenase-1), endothelial activation (e.g., Angiopoietin-2), and alterations in sphingolipid metabolism and signaling (e.g., Sphigosine-1-Phosphate Receptor 2). Sphingolipid profiling of mouse cerebral microvessels validated the transcript data and revealed the enrichment of sphingomyelin and sphingoid species in the cerebral microvasculature compared to brain and the stroke-induced increase in ceramide species. In summary, our study has identified novel molecular alterations in several microvessel-enriched, translationally relevant, and druggable targets, which are potent modulators of endothelial function. Our comparative analyses have revealed the presence of molecular features associated with cerebral microvascular dysfunction in human chronic stroke lesions. The results shared here provide a detailed resource for therapeutic discovery of candidates for neurovascular protection in stroke and potentially, other pathologies exhibiting cerebral microvascular dysfunction.

Chemical induction of gut ß-like-cells by combined FoxO1/Notch inhibition as a glucose-lowering treatment for diabetes.

OBJECTIVE: Lifelong insulin replacement remains the mainstay of type 1 diabetes treatment. Genetic FoxO1 ablation promotes enteroendocrine cell (EECs) conversion into glucose-responsive ß-like cells. Here, we tested whether chemical FoxO1 inhibitors can generate ß-like gut cells. METHODS: We used Ngn3-or Villin-driven FoxO1 ablation to capture the distinctive developmental effects of FoxO1 on EEC pool. We combined FoxO1 ablation with Notch inhibition to enhance the expansion of EEC pool. We tested the ability of an orally available small molecule of FoxO1 inhibitor, Cpd10, to phenocopy genetic ablation of FoxO1. We evaluated the therapeutic impact of genetic ablation or chemical inhibition of FoxO1 on insulin-deficient diabetes in Ins2Akita/+ mice. RESULTS: Pan-intestinal epithelial FoxO1 ablation expanded the EEC pool, induced ß-like cells, and improved glucose tolerance in Ins2Akita/+ mice. This genetic effect was phenocopied by Cpd10. Cpd10 induced ß-like cells that released insulin in response to glucose in gut organoids, and this effect was enhanced by the Notch inhibitor, DBZ. In Ins2Akita/+ mice, a five-day course of either Cpd10 or DBZ induced intestinal insulin-immunoreactive ß-like cells, lowered glycemia, and increased plasma insulin levels without apparent adverse effects. CONCLUSION: These results provide proof of principle of gut cell conversion into ß-like cells by a small molecule FoxO1 inhibitor, paving the way for clinical applications.

A FOXO1-dependent transcription network is a targetable vulnerability of mantle cell lymphomas.

Targeting lineage-defined transcriptional dependencies has emerged as an effective therapeutic strategy in cancer treatment. Through screening for molecular vulnerabilities of mantle cell lymphoma (MCL), we identified a set of transcription factors (TFs) including FOXO1, EBF1, PAX5, and IRF4 that are essential for MCL propagation. Integrated chromatin immunoprecipitation and sequencing (ChIP-Seq) with transcriptional network reconstruction analysis revealed FOXO1 as a master regulator that acts upstream in the regulatory TF hierarchy. FOXO1 is both necessary and sufficient to drive MCL lineage commitment through supporting the lineage-specific transcription programs. We further show that FOXO1, but not its close paralog FOXO3, can reprogram myeloid leukemia cells and induce B-lineage gene expression. Finally, we demonstrate that cpd10, a small molecule identified from an enriched FOXO1 inhibitor library, induces a robust cytotoxic response in MCL cells in vitro and suppresses MCL progression in vivo. Our findings establish FOXO1 inhibition as a therapeutic strategy targeting lineage-driven transcriptional addiction in MCL.

Pharmacological conversion of gut epithelial cells into insulin-producing cells lowers glycemia in diabetic animals.

As a highly regenerative organ, the intestine is a promising source for cellular reprogramming for replacing lost pancreatic ß cells in diabetes. Gut enterochromaffin cells can be converted to insulin-producing cells by forkhead box O1 (FoxO1) ablation, but their numbers are limited. In this study, we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine. Accordingly, lineage-tracing experiments show that, upon genetic or pharmacologic FoxO1 ablation, the Paneth/goblet lineage can also undergo conversion to the insulin lineage. We designed a screening platform in gut organoids to accurately quantitate ß-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process in mice and human adult intestinal organoids. We identified a triple blockade of FOXO1, Notch, and TGF-ß that, when tested in insulin-deficient streptozotocin (STZ) or NOD diabetic animals, resulted in near normalization of glucose levels, associated with the generation of intestinal insulin-producing cells. The findings illustrate a therapeutic approach for replacing insulin treatment in diabetes.

Single-agent FOXO1 inhibition normalizes glycemia and induces gut ß-like cells in streptozotocin-diabetic mice.

OBJECTIVES: Insulin treatment remains the sole effective intervention for Type 1 Diabetes. Here, we investigated the therapeutic potential of converting intestinal epithelial cells to insulin-producing, glucose-responsive ß-like cells by targeted inhibition of FOXO1. We have previously shown that this can be achieved by genetic ablation in gut Neurogenin3 progenitors, adenoviral or shRNA-mediated inhibition in human gut organoids, and chemical inhibition in Akita mice, a model of insulin-deficient diabetes. METHODS: We profiled two novel FOXO1 inhibitors in reporter gene assays, and hepatocyte gene expression studies, and in vivo pyruvate tolerance test (PTT) for their activity and specificity. We evaluated their glucose-lowering effect in mice rendered insulin-deficient by administration of streptozotocin. RESULTS: We provide evidence that two novel FOXO1 inhibitors, FBT432 and FBT374 have glucose-lowering and gut ß-like cell-inducing properties in mice. FBT432 is also highly effective in combination with a Notch inhibitor in this model. CONCLUSION: The data add to a growing body of evidence suggesting that FOXO1 inhibition be pursued as an alternative treatment to insulin administration in diabetes.

Influence of the ecological environment on the structural characteristics and bioactivities of polysaccharides from alfalfa (Medicago sativa L.).

Polysaccharides from alfalfa (Medicago sativa L.) (APS) exhibit a variety of bioactivities; however, little information is available on the effects of the ecological environment on the structural characteristics and bioactivities of APS. This study aimed to investigate the structural characteristics and bioactivities of two APS types isolated from alfalfa; these APSs were obtained from alfalfa cultured in normal soil (APS1) or saline-alkali soil (APS2). Results indicated that the two kinds of APS had the same monomer compositions in different molar proportions, where APS2 had greater content of arabinose and galacturonic acid than APS1. Furthermore, APS1 exhibited a greater molar mass of 1.77 × 105 g mol-1 as compared to 1.01 × 105 g mol-1 for APS2. Likewise, APS1 and APS2 had highly branched molecules with crosslinking nets composed of similar monomer residues but with different glycosidic linkages. Additionally, both APS significantly inhibited both adipogenesis and lipid accumulation in 3T3-L1 cells by downregulating mRNA expression of Ppar-γ, C/ebp-α, and Fas; APS2 had superior antiadipogenic effects as compared to APS1. Altogether, the ecological environment impacts the structural characteristics and biofunctions of APS, making them potential candidates for antiadipogenic use through functional food. These findings provide a novel perspective for the selection of phytogenic polysaccharides with specific bioactivities by considering growth environmental conditions.

Correction: Influence of the ecological environment on the structural characteristics and bioactivities of polysaccharides from alfalfa (Medicago sativa L.).

Correction for 'Influence of the ecological environment on the structural characteristics and bioactivities of polysaccharides from alfalfa (Medicago sativa L.)' by Chongyu Zhang et al., Food Funct., 2022, https://doi.org/10.1039/d2fo00371f.

Diet Type Impacts Production Performance of Fattening Lambs by Manipulating the Ruminal Microbiota and Metabolome.

The pelleted total mixed ration (PTMR) has a positive effect on the productivity of fattening lambs. However, whether the beneficial effects are underpinned by altering the rumen microbiota and metabolome that remain unclear. This study aimed to investigate correlations among growth performance, ruminal microbiota, and ruminal metabolome of lambs fed PTMR diet. A total of 100 crossbred (Dorper sheep × Fine-wool sheep) ram lambs at 55 days of age with similar body weight (BW) (13.2 ± 0.5 kg) were randomly allocated to 10 pens that were fed either PTMR (PTMR group) or unpelleted total mixed ration (UPTMR group) with the same dietary ingredients and nutritional contents. The average daily gain (ADG) and average daily feed intake (ADFI) were determined during the 62-day experimental period and ruminal pH, volatile fatty acid (VFA) concentrations, microbiota, and metabolome in the rumen of the lambs were examined at the end of the experiment. Compared to those of the UPTMR group, the PTMR group had greater ADFI (P = 0.002), ADG (P = 0.003), and feed efficiency (G/F) (P < 0.05). Similarly, feeding PTMR increased the concentration of total VFA (TVFA) and the molar proportion of propionate, but decreased the proportion of butyrate and acetate to propionate ratio in the rumen of lambs compared to that in lambs from the UPTMR group (P < 0.05). In addition, the PTMR group demonstrated lowered alpha-diversity of the ruminal microbiota and enhanced the relative abundance of Fibrobacter (P < 0.05), Veillonellaceae (P < 0.05), and the abundance of Rikenellaceae (P = 0.064) in the rumen compared with those in the UPTMR group. Feeding lambs with PTMR significantly upregulated the metabolic pathways involving tryptophan, histidine, cysteine and methionine, ß-alanine, tyrosine metabolisms, and steroid biosynthesis. Moreover, the abundance of the microbiota strongly correlated with the altered performance, ruminal VFA, metabolites, and metabolic pathways of lambs. Taken together, feeding PTMR shaped the ruminal microbiota of lambs with decreased diversity, while improving relative abundance of some specific microbes and upregulating certain growth-related metabolic pathways, which contributed to the augmented growth performance and G/F of fattening lambs. Thus, feeding PTMR to fattening lambs for superior production performance and G/F is recommended.

Structural Characteristics and Immunomodulatory Effects of a Long-Chain Polysaccharide From Laminaria japonica.

Polysaccharides derived from Laminaria japonica (LJPS) have shown a variety of beneficial effects on improving human health; however, the structural features and bioactivities of long-chain LJPS remain unclear. This study aimed to investigate the structural characteristics and bioactivities of a novel long-chain LJPS. Results showed that the LJPS was composed of Fuc, Rha, Ara, Gal, Glc, Xyl, Man, Fru, Rib, GalA, GluA, GlcA, and ManA, with a molar ratio of 35.71:1.48:0.28:13.16:0.55:2.97:6.92:0.58:0.41:0.14:3.16:15.84:18.79. Of these, Fuc, Gal, Man, GlcA, and ManA were the predominant components with an accumulated proportion of 93.6%. The LJPS was found to consist of seven types of the monomer residues, and the main interchain glycosidic linkages were ß -D-(1 → 2), α -D-(1 → 3), (1 → 4), and (1 → 6), and the molecular mass was 5.79 × 104 g/mol. Regarding the molecular conformation, LJPS was a multi-branched, long-chain macromolecule, and appeared in a denser crosslinking network with highly branched and helix domains in the terms of morphology. Additionally, the LJPS had no toxicity to mouse macrophage cells and exhibited biphasic immuno-modulating capacity. The present findings suggested that the long-chain LJPS might be an attractive candidate as an immunopotentiating and anti-inflammatory functional food, and this study also provides a feasible approach to decipher the structural characteristics and spatial conformations of plant-derived polysaccharides.

Dietary Energy Level Impacts the Performance of Donkeys by Manipulating the Gut Microbiome and Metabolome.

Considerable evidence suggests that dietary energy levels and gut microbiota are pivotal for animal health and productivity. However, little information exists about the correlations among dietary energy level, performance, and the gut microbiota and metabolome of donkeys. The objective of this study was to investigate the mechanisms by which dietary energy content dictates the growth performance by modulating the intestinal microbiome and metabolome of donkeys. Thirty-six nine-month-old male Dezhou donkeys with similar body weights were randomly assigned to two groups fed low- or high-energy diets (LE or HE). The results showed that donkeys fed HE had increased (p < 0.05) the average daily gain (ADG) and feed efficiency (G/F) compared with those that received LE diet. The gut microbiota in both groups was dominated by the phyla Firmicutes and Bacteroidetes regardless of the dietary energy level. However, feeding HE to donkeys significantly decreased (p < 0.05) the ratio of Firmicutes to Bacteroidetes (F/B). Compared to the LE group, feeding HE specifically increased the abundances of unidentified_Prevotellaceae (p = 0.02) while decreasing the richness of unidentified_Ruminococcaceae (p = 0.05). Compared to the LE group, feeding the HE diet significantly (p < 0.05) upregulated certain metabolic pathways involving the aspartate metabolism and the urea cycle. In addition, the increased bacteria and metabolites in the HE-fed group exhibited a positive correlation with improved growth performance of donkeys. Taken together, feeding the HE diet increased the richness of Prevotellaceae and upregulated growth-related metabolic pathways, which may have contributed to the ameliorated growth performance of donkeys. Thus, it is a recommendable dietary strategy to feed HE diets to fattening donkeys for superior product performance and feed efficiency.

The modulatory effects of alfalfa polysaccharide on intestinal microbiota and systemic health of Salmonella serotype (ser.) Enteritidis-challenged broilers.

Salmonella serotype (ser.) Enteritidis infection in broilers is a main foodborne illness that substantially threatens food security. This study aimed to examine the effects of a novel polysaccharide isolated from alfalfa (APS) on the intestinal microbiome and systemic health of S. ser. Enteritidis-infected broilers. The results indicated that broilers receiving the APS-supplemented diet had the improved (P < 0.05) growth performance and gut health than those fed no APS-supplemented diet. Supplementation with APS enhanced (P < 0.05) the richness of gut beneficial microbes such as Bacteroidetes, Barnesiella, Parabacteroides, Butyricimonas, and Prevotellaceae, while decreased (P < 0.05) the abundance of facultative anaerobic bacteria including Proteobacteria, Actinobacteria, Ruminococcaceae, Lachnospiraceae, and Burkholderiaceae in the S. ser. Enteritidis-infected broilers. The Bacteroides and Odoribacter were identified as the two core microbes across all treatments and combined with their syntrophic microbes formed the hub in co-occurrence networks linking microbiome structure to performance of broilers. Taken together, dietary APS supplementation improved the systemic health of broilers by reshaping the intestinal microbiome regardless of whether S. ser. Enteritidis infection was present. Therefore, APS can be employed as a potential functional additives to inhibit the S. ser. Enteritidis and enhance the food safety in poultry farming.

Peanut sprout rich in p-coumaric acid ameliorates obesity and lipopolysaccharide-induced inflammation and the inhibition of browning in adipocytes via mitochondrial activation.

Obesity is accompanied by adipose tissue inflammation that subsequently reduces thermogenic potential in brown and beige (brown-like) adipocytes. We previously reported that peanut sprout (PS) inhibited triglyceride accumulation via fatty acid oxidation in adipocytes. However, it is unknown whether PS reverses diet-induced obesity/inflammation and protects against the inflammation-induced inhibition of browning. To investigate this, C57BL/6 male mice, as an in vivo model, were randomly assigned to three different diets and fed for 8 weeks: (i) low-fat diet (LF, 11% kcal from fat), (ii) high-fat diet (HF, 61% kcal from fat), or (iii) HF diet with PS (4% PS in diet, HF + PS). As an in vitro model, lipopolysaccharides (LPS)-induced macrophages and 3T3-L1 adipocytes in the absence (white adipocytes) or presence of dibutyryl-cAMP (Bt-cAMP, beige adipocytes) were used. The supplementation of PS improved HF-diet-mediated body weight gain, dyslipidemia, and hyperglycemia as compared to the HF group. Although there was a marginal impact on visceral hypertrophy, PS reversed the adipocyte inflammation. In parallel, LPS-mediated induction of inflammation was impeded by PS extract (PSE) in macrophages and adipocytes. PSE also protected against LPS-induced suppression of adipocyte browning in Bt-cAMP-treated adipocytes with mitochondrial activation. The phenolic acid analysis showed that among the constituent of PSE, p-coumaric acid (PCA) was identified as a polyphenol that showed a similar effect to PSE. PCA treatment was also able to maintain a higher temperature than the control group upon cold exposure. Taken together, PCA-enriched PS attenuated HF-diet-induced obesity and protected against LPS-induced inflammation and the inhibition of browning via mitochondrial activation.

RMR-Related MAP2K6 Gene Variation on the Risk of Overweight/Obesity in Children: A 3-Year Panel Study.

From a pilot GWAS, seven MAP2K6 (MEK6) SNPs were significantly associated with resting metabolic rate (RMR) in obese children aged 8-9 years. The aim of this study was to investigate how RMR-linked MEK6 variation affected obesity in Korean children. With the follow-up students (77.9%) in the 3-year panel study, the changes of the variables associated with obesity (such as anthropometrics, blood biochemistry, and dietary intake) were collected. After the MEK6 SNPs were screened by Affymetrix Genome-Wide Human SNP array 6.0, the genotyping of the seven MEK6 SNPs was performed via SNaPshot assay. As the prevalence of obesity (≥85th percentile) increased from 19.4% to 25.5%, the rates of change of the variables RMR, body mass index (BMI), waist circumference (WC), systolic blood pressure (SBP), and dietary intake (energy and carbohydrate intakes) increased. The rate of overweight/obesity was higher in all mutant alleles of the seven MEK6 SNPs than it was in the matched children without mutant alleles. However, over the 3-year study period, RMRs were only significantly increased by the mutants of two single nucleotide polymorphisms (SNPs), rs996229 and rs756942, mainly related to male overweight/obesity as both WC and SBP levels increased. In the mutants of two of the SNPs, the odds ratio of overweight/obesity risk was six times higher in the highest tercile of fat intake and SBP than those of the lowest tercile. For personalized medicine to prevent pediatric obesity, SBP, WC, and dietary fat intake should be observed, particularly if boys have mutants of MEK6 SNPs, rs9916229, or rs756942.

Potential Antidiabetic Effects of Seaweed Extracts by Upregulating Glucose Utilization and Alleviating Inflammation in C2C12 Myotubes.

Seaweed is known to have various health-promoting effects. However, the mechanisms underlying seaweed's antidiabetic effects remain unclear. We investigated the potential antidiabetic effects of seaweed water extracts and further examined their mechanism(s) using C2C12 mouse skeletal muscle cells. Briefly, we screened the physiochemical properties of seven seaweed extracts by comparing the antioxidant and α-glucosidase inhibitory effects. Among them, three seaweed extracts, Undaria pinnatifida sporophyll (UPS), Codium fragile (CF), and Gracilaria verrucosa (GV), were selected for further testing of their possible antidiabetic effects with underlying mechanisms using C2C12 myotubes. Consistent with the superior α-glucosidase inhibition of the three seaweed extracts, the extracts also enhanced glucose utilization in myotubes compared to the control. The upregulated glucose uptake by the seaweed extracts was reversed by an AMP-activated protein kinase (AMPK) inhibitor, compound C, in the UPS- and CF-treated groups. Furthermore, all three seaweed extracts significantly promoted the phosphorylation of AMPK which was completely blocked by pretreating with compound C. In addition, all three extracts reduced lipopolysaccharide-simulated TNF-α production in C2C12 cells. Our results demonstrated that all three seaweed extracts exhibited antidiabetic properties through not only the inhibition of glucose absorption but also the promotion of glucose utilization. Moreover, the regulation of inflammatory cytokine production by the extracts suggested their potential anti-inflammatory property which might play a critical role in protecting insulin sensitivity in a chronic inflammatory state. Taken together, UPS, CF, and GV are a promising source to modulate the glucose absorption and utilization in muscle cells partially via the AMPK pathway.

FOXO1 inhibition synergizes with FGF21 to normalize glucose control in diabetic mice.

OBJECTIVE: Forkhead box protein O1 (FOXO1) plays a key role in regulating hepatic glucose production, but investigations of FOXO1 inhibition as a potential therapeutic approach have been hampered by a lack of selective chemical inhibitors. By profiling structurally diverse FOXO1 inhibitors, the current study validates FOXO1 as a viable target for the treatment of diabetes. METHODS: Using reporter gene assays, hepatocyte gene expression studies, and in vivo studies in mice, we profiled our leading tool compound 10 and a previously characterized FOXO1 inhibitor, AS1842856 (AS). RESULTS: We show that AS has significant FOXO1-independent effects, as demonstrated by testing in FOXO1-deficient cell lines and animals, while compound 10 is highly selective for FOXO1 both in vitro and in vivo and fails to elicit any effect in genetic models of FOXO1 ablation. Chronic administration of compound 10 improved insulin sensitivity and glucose control in db/db mice without causing weight gain. Furthermore, chronic compound 10 treatment combined with FGF21 led to synergistic glucose lowering in lean, streptozotocin-induced diabetic mice. CONCLUSIONS: We show that the widely used AS compound has substantial off-target activities and that compound 10 is a superior tool molecule for the investigation of FOXO1 function. In addition, we provide preclinical evidence that selective FOXO1 inhibition has potential therapeutic benefits for diabetes as a monotherapy or in combination with FGF21.