NL2019390B1 - Screening Method - Google Patents

Abstract

The present invention provides a method of screening a test compound for insulin resistance modulating activity. Corresponding methods for identifying a compound for treating diabetes and/or metabolic syndrome, and methods for identifying a target of a drug for the treatment of the same are also provided. Methods for optimising the formulation of a drug for the treatment of diabetes and/or metabolic syndrome are also provided herein.

Description

Screening Method

The present invention provides a method of screening a test compound for insulin resistance modulating activity. Corresponding methods for identifying a compound for treating diabetes and/or metabolic syndrome, and methods for identifying a target of a drug for the treatment of the same are also provided. Methods for optimising the formulation of a drug for the treatment of diabetes and/or metabolic syndrome are also provided herein.

Background

Metabolic disorders including metabolic syndrome, pre-diabetes, diabetes (particularly type 2 diabetes) and cardiovascular disease are becoming a significant health issue worldwide.

Metabolic syndrome (also known as insulin resistance syndrome, or syndrome X) refers to a set of risk factors that arise from insulin resistance accompanying abnormal adipose deposition and function. It is a risk factor for coronary heart disease as well as for diabetes (e.g. type II diabetes (TIID)), dyslipidemia, hypertension and several cancers.

The prevalence of TIID is rapidly reaching pandemic levels (Hu 2011, Ng et al. 2013). Current treatment methods of TIID are still relatively limited, with insulin sensitizers such as metformin being most widely used (Chen et al., 2017, Powers, 2012). Although metformin is currently the most prescribed treatment, it has multiple targets in vivo and therefore its function (particularly in relation to insulin resistance) is still poorly understood.

Traditional research into insulin resistance and TIID is based on human cell culture and various animal models (mainly rodents), including mutant mouse strains of the leptin and leptin receptor genes (ob/ob and db/db strains respectively) and mice that are diabetic after being subjected to high fat diets. Zucker fatty rats and ZDF rats have also been widely used to study TIID, obesity and the function of leptin signalling in metabolic syndrome (Reed & Scribner 1990).

Zebrafish models have been proposed as alternative test systems for studying insulin resistance in metabolic disorders such as metabolic syndrome and/or TIID, with the generation of numerous transgenic and knockout lines (Gut et al. 2017). Although diabetic adult zebrafish models that are based on a high fat feeding systems have already been established (Michel et al., 2016; Zang et al. 2017), there is a lack of alternative early stage larval models. Early stage larval models can be useful in fast and large scale screening assays. As shown by Marin-Juez et al. (2014) zebrafish larva are highly suited to study insulin resistance, and thus are a promising model system to study TIID in a non-feeding situation. Marin-Juez etal., also identified SHP-1 in zebrafish larvae as a key factor in insulin resistance (Marin-Juez et al. 2014).

Although some progress has been made in identifying the mechanisms underlying metabolic disorders such as metabolic syndrome and TIID, further investigation of the biological pathways involved is warranted. In addition, there is a real need for the identification of new treatment options for such metabolic disorders.

Brief summary of the disclosure

The identification of new medications suitable for the treatment of metabolic disorders such as metabolic syndrome and/or diabetes has been slow due to the complex nature, and high costs associated with, testing anti-diabetic drugs in rodent models. The inventors have now developed a method to test new compounds for their ability to modulate insulin resistance in zebrafish larvae, based on several methods for glucose analysis. The novel method is based on the fact that leptin B deficient zebrafish larvae appear to be totally insulin resistant, resulting in a diabetic phenotype. They show that Metformin is highly effective for treating this diabetic phenotype. Using their novel method, the inventors have also identified that the phosphatase inhibitor NSC87877 is a potent antidiabetic drug. In contrast to metformin, NSC87877 was also active at very early larval stages and even at embryonic stages of leptin B deficient zebrafish development. Gene knockdown studies in the leptin B mutant background indicate that SHP-1 is the most likely target responsible for the antidiabetic effect of NSC87877. Using the novel early larval stage test system described herein, the inventors have shown that a high throughput method automated method can be used to screen compounds (e.g. small molecule libraries of up to hundred compounds per day) that may be useful for modulating insulin resistance (and thus may be useful in treating metabolic disorders such as metabolic syndrome and/or diabetes).

The invention has been demonstrated using leptin B deficient zebrafish larva and has shown that a leptin B zebrafish model may be used at very early stages e.g. at 4 hours post fertilisation (hpf) in e.g., an automated screening method for identifying test compounds which modulate insulin resistance. However, the invention equally applies to leptin receptor deficient zebrafish larva, due to the interaction between leptin B and its receptor. Similarly, the invention is not limited to the larval stages of the specified mutant zebrafish, as non-larval stages of development (including adult mutant fish) may also be used.

As described below in more detail, the invention is also applicable to other appropriate (i.e. leptin B deficient and/or leptin receptor deficient) teleosts.

Accordingly, the invention provides a method of screening a test compound for insulin resistance modulating activity, said method comprising: a. contacting the test compound with a leptin B deficient or a leptin receptor deficient teleost; and b. determining the effect of the test compound on basal glucose levels in the teleost or on glucose uptake in the teleost.

Suitably, the teleost is an embryo or a larva.

Suitably, the teleost is a zebrafish.

Suitably, the contacting step occurs when the teleost is less than 3 days post fertilisation (dpf).

Suitably, the contacting step comprises injecting the teleost with, or immersing the teleost in, a solution comprising the test compound.

Suitably, the contacting step comprises injecting the test compound through the chorion of the teleost.

Suitably, the determining step comprises contacting the teleost with a glucose solution and measuring glucose uptake in the teleost, wherein an increase in glucose uptake compared to a control, or compared to a predetermined level, identifies the test compound as having the ability to reduce insulin resistance.

Suitably, the teleost is dechorionated and immersed in the glucose solution.

Suitably, the determining step comprises contacting the teleost with an insulin solution and subsequently measuring the effect on basal glucose levels in the teleost, wherein a decrease in basal glucose level over time or compared to a control or a predetermined level identifies the test compound as having the ability to reduce insulin resistance.

Suitably, the glucose is labelled.

Suitably, the glucose is fluorescently labelled.

Suitably, the method is an automated method.

In a further aspect, the invention provides a method for identifying a compound for treating diabetes and/or metabolic syndrome, said method comprising determining the effect of a compound on insulin resistance in accordance with a method of the invention, and selecting a compound which has the ability to reduce insulin resistance.

Suitably, the method is an automated method.

Suitably, the compound is robotically injected through the chorion of the teleost.

Suitably, the contacting step is conducted when the teleost is less than 22 hours post fertilisation (hpf).

Suitably, the glucose is labelled and the teleost is analysed using vertebrate automated screening technology.

Suitably, the method is high throughput.

In a further aspect, the invention provides a method of identifying a target of a drug for the treatment of diabetes and/or metabolic syndrome, said method comprising: a. modifying the gene or protein expression of a potential target in a leptin B deficient or a leptin receptor deficient teleost; b. contacting the teleost with the drug; and c. determining the effect of the drug on a basal glucose level in the teleost, or on glucose uptake in the teleost.

Suitably, step a. comprises using a morpholino for the potential target.

Suitably, the determining step comprises contacting the teleost with a glucose solution and measuring glucose uptake in the teleost, wherein a decrease in glucose uptake compared to a control or compared to a predetermined level identifies the potential target as a target for the drug.

Suitably, the determining step comprises contacting the teleost with insulin solution and subsequently measuring the effect on the basal glucose level in the teleost, wherein an increase in glucose level over time or compared to a control or a predetermined level, identifies the potential target as a target for the drug.

In a further aspect, the invention provides a method for optimising the formulation of a drug for the treatment of diabetes and/or metabolic syndrome, said method comprising: a. contacting a first formulation of the drug with a first leptin B deficient or a leptin receptor deficient teleost; b. determining the effect of the first formulation on a basal glucose level in the teleost, or on glucose uptake in the teleost; c. contacting a second formulation of the drug with a second leptin B deficient or a leptin receptor deficient teleost; d. determining the effect of the second formulation on a basal glucose level in the teleost, or on glucose uptake in the teleost; and e. comparing the basal glucose levels, or glucose uptake determined in steps b) and d), and selecting the formulation which results in a higher glucose uptake or a lower glucose basal level.

Suitably, one or more of the following parameters are altered in the second formulation when compared to the first formulation: drug dose, release rate, buffers.

Suitably, the teleost is an embryo or a larva.

Suitably, the teleost is a zebrafish.

Suitably, the contacting step(s) occurs when the teleost is less than 3 dpf.

Suitably, the contacting step(s) comprises injecting the teleost with, or immersing the teleost in, a solution comprising the drug.

Suitably, the contacting step(s) comprises injecting the formulation through the chorion of the teleost.

Suitably, the determining steps comprise contacting the teleost with a glucose solution and measuring glucose uptake in the teleost.

Suitably, the teleost is dechorionated and immersed in a glucose solution.

Suitably, the determining steps comprise contacting the teleost with an insulin solution and subsequently measuring the effect on the basal glucose level in the teleost.

Suitably, the glucose is labelled.

Suitably, the glucose is fluorescently labelled.

Suitably, the method is an automated method.

Suitably, the drug is robotically injected through the chorion of the teleost.

Suitably, the contacting step is conducted when the teleost is less than 22 hours post fertilisation (hpf).

Suitably, the glucose is labelled and the teleost is analysed using vertebrate automated screening technology.

Suitably, the method is high throughput.

In a further aspect, the invention provides for the use of a leptin B deficient or a leptin receptor deficient teleost embryo or larvae as a model of diabetes or metabolic syndrome.

In a further aspect, the invention provides for the use of a leptin B deficient or a leptin receptor deficient teleost to: a. screen test compounds for their ability to modulate insulin resistance; b. identify a compound for treating diabetes and/or metabolic syndrome; c. identify a target of a drug for treatment of diabetes and/or metabolic syndrome; d. optimise a drug formulation or regimen for the treatment of diabetes and/or metabolic syndrome; and/or e. identify the location(s) of drug activity in the teleost.

Suitably, the teleost is an embryo or larva.

Suitably, the teleost is a zebrafish.

Suitably, the teleost is less than 3 dpf.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

Various aspects of the invention are described in further detail below.

Brief description of the drawings

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1A shows the sgRNA and target in exon 2 which generated a leptin B knock out zebrafish mutant line.

Figure 1B shows an overview of the outcrossing and incrossing process of generating the lepB mutant.

Figure 2A shows an insulin injection method: Zebrafish larvae at 4 dpf received 1 nl of human recombinant insulin or PBS, into the caudal vein. The samples are collected at 0, 30 and 240 min post injection, to measure the basal glucose level in the body, using an ELISA Glucose Assay Kit.

Figure 2B shows a glucose immersion method: Zebrafish larvae at 4dpf are immersed in a medium containing 250mM of glucose or mannitol. The samples are collected after 0 min, 120 min of glucose immersion and 120 min and 240 min of washing in clean egg water, to measure free glucose level in the body, using an ELISA Glucose Assay Kit. This provides an alternative and less invasive method of testing insulin resistance in zebrafish larvae compared to the injection method described above.

Figure 2C shows a fluorescent glucose injection method: Zebrafish embryos at 24 hpf are injected into the yolk with fluorescently labelled glucose. After one hour post injection, accumulation of glucose in the brain of wildtype (WT) and mutant zebrafish larvae can be observed via VAST bioimager.

Figure 2D shows a drug treatment method: A drug (e.g. NSC87877) can be administered into a zebrafish embryo via transchorion injections at very early stages (1 hpf). Drugs can also be administered during later stages (3/4dpf) via immersion.

Figure 3A shows the insulin injection method of Fig. 2A: Zebrafish larvae are sensitive to human insulin, showing inhibition of gluconeogenesis and hypoglycaemia, when exposed to a high insulin dose. Zebrafish larvae reach their physiological glucose level after 4 hours post insulin injection. In the lepB mutants the basal glucose level is already significantly higher from the beginning and increases constantly after 4 hours post injection (hpi).

Figure 3B shows results of the glucose immersion method of Fig. 2B: Zebrafish larvae treated with a high dose of glucose develop hyperglycaemia after 2 hours post glucose immersion, and reach their physiological level after 4 hours washing in clean egg water. In lepB deficient fish glucose levels remain higher, indicating their diabetic phenotype.

Figure 3C shows results of a repeated glucose immersion: WT zebrafish larvae develop hyperglycaemia when immersed in a high glucose concentration. After a first glucose immersion and washing (in accordance with Fig. 2B), zebrafish larvae were again exposed for 60 min to glucose and then washed for 120 min in a clean egg medium. After a second immersion, WT larvae developed progressive hyperglycaemia with an increased free glucose level.

Figure 3D shows results of the fluorescent glucose injection method of Fig. 2C: In WT larvae fluorescent glucose diffuses via the yolk barrier and accumulates in the brain, whereas in the lepB larvae glucose remains in the yolk, which indicates their diabetic phenotype even at the early developmental stages.

Figure 3E shows that VAST microscopy can be used for high throughput screening using the fluorescent glucose injection method.

Figure 4A shows the effect of Metformin treatment on glucose concentration. Using glucose immersion, the influence of Metformin on WT and lepB zebrafish larvae was tested. In both groups Metformin leads to a decrease in glucose level, rescuing the mutant phenotype.

Figure 4B shows the effect of Metformin treatment following fluorescent glucose injection (Quantification). After quantification of the fluorescent signal in the brain, there is no significant difference between lepB and Metformin treated embryos.

Figure 4C shows the results of insulin injection on glucose levels. Zebrafish larvae are sensitive to human insulin and develop insulin resistance when treated with a high dose of human insulin. In the WT larvae treated with non-specific protein tyrosine phosphatase inhibitor NSC87877, free glucose level decreases after a second glucose injection, preventing the zebrafish larvae from developing insulin resistance.

Figure 4D shows, using the glucose immersion method, the effect of NSC87877 treatment on glucose concentration. Using glucose immersion, the influence of non-specific protein tyrosine phosphatase inhibitor NSC87877 on WT and lepB zebrafish larvae was tested. In both cases treatment with NSC87877 leads to a decrease in glucose level and rescue of the mutant phenotype.

Figure 4E shows, using the fluorescent glucose injection method, the results of NSC87877 treatment (representative pictures): treatment with NSC87877 increases glucose uptake in the brain and reverts diabetic phenotype in lepB mutants.

Figure 4F shows, using the fluorescent glucose injection method, the results of NSC87877 treatment on glucose levels (quantification). After quantification of the fluorescent glucose signal in the brain, there is a significant difference between lepB and NSC87877 treated embryos, indicating that NSC87877 may be a more effective drug against the diabetic phenotype at the early developmental stages, than metformin.

Figure 5A shows, using the fluorescent glucose injection method, the results of Metformin treatment (representative pictures): the inventors did not observe the same Metformin effect at the earlier stages of the zebrafish development. The LepB mutant remains diabetic after Metformin treatment at early stages of development.

Fig. 5B shows, using the fluorescent glucose injection method, the results of Metformin (representative pictures): the inventors did not observe the same effect at the earlier stages of zebrafish development. LepB mutant remains diabetic after Metformin treatment.

Figure 5C shows early stage glucose injections (8 hpf). Glucose uptake from the yolk into the cells is observed at the very early stages of development (8 hours post fertilization). Fluorescent glucose was injected into the yolk and imaged 1 hour after the injection. Glucose diffuses and accumulates in the cells of developing zebrafish embryos.

Figure 5D shows early stages glucose injections (8 hpf). Glucose uptake from the yolk into the cells at the very early stages (8 hpf) is disrupted in the lepB mutants. Fluorescent glucose was injected into the yolk and imaged 1 hour after the injection. Glucose remains in the yolk of the developing zebrafish embryos. After NSC87877 injections the phenotype was rescued even at an early developmental stage.

Figure 6A shows, using fluorescent glucose injection, that the ptpn6 morpholino knockdown increases glucose uptake by the brain and reverts the diabetic phenotype in lepB mutants.

Figure 6B shows quantification of the ptpn6 morpholino knockdown: After quantification of the fluorescent signal in the brain, a significant difference between lepB mutants and ptpn6 rescued mutants can be noticed.

Figure 7 shows automatisation of the procedure of fluorescent glucose uptake. The antidiabetic compound was robotically injected transchorionally into 4 hpf embryos using an automatic robot injector. At 24 hpf, the embryos were dechorionated enzymatically and injected with fluorescent glucose, again using automatic robot injector. Injected embryos were analysed using vertebrate automated screening technology (VAST). The fluorescence of the head regions of a large number of larvae was quantified using automated image analysis.

Detailed description

Leptin is a cytokine produced in humans mainly by mature adipocytes in white adipose tissue and to a lesser extent by the skeletal muscles, placenta, ovaries, bone marrow and stomach.

In the brain, it regulates food intake, appetite behaviours and energy expenditure. Due to the wide-ranging role of leptin, mutations in its signalling pathway lead to abnormalities, which are known to be main factors influencing development of diabetic phenotype symptoms in rodents (Wang et al. 2014). Alterations in leptin signalling pathways may be caused by up regulation of SOCS3 expression, although it remains unclear what causes the increase in SOCS expression. One theory is that high fat diet induced leptin resistance is inhibited in SOCS3 heterozygous knockout mice or neuron-specific SOCS knockout mice (Mori et al. 2004). Leptin activity is also correlated with protein tyrosine phosphatases; key regulatory mechanisms in many signal transduction pathways leading to proliferation, differentiation and eventually cell death (Ullrich & Schlessinger, 1990). SHP-1 (also called PTPN6) is expressed mainly in hematopoietic cells, but also expressed at low levels in epithelial cells (Matthews et al. 1992), whereas the structurally closely related SHP-2 (also called PTPN11) is ubiquitously expressed, and is also expressed in the cells that express SHP-1 (Feng et al. 1993). It has been demonstrated that the insulin signalling pathway is strictly correlated with protein tyrosine phosphatases (PTPs) that dephosphorylate and inactivate the insulin receptor, acting as a link between the leptin and insulin signalling pathways. Protein tyrosine phosphatase 1B (PTP1B) has emerged as a promising novel therapeutic target for the treatment of TIID, as it plays an important role in the negative regulation of insulin signal transduction pathways (Tamrakar et al. 2014). Furthermore, expression of hypothalamic PTP1B is up regulated in leptin resistant animals (White et al. 2009). Knockout of low-molecular-weight tyrosine phosphatase in rodents results in attenuation of high-fat diet-induced diabetes (Stanford et al. 2017).

The inventors have analysed the function of leptin and SHP-1 in insulin resistance in zebrafish larvae and have surprisingly found that leptin B deficient zebrafish are totally insulin resistant, resulting in a diabetic phenotype from the very early stages of development. The inventors have also shown that the insulin resistant phenotype is reversible, and that the well-known insulin sensitizer, metformin, is highly effective for reducing insulin resistance in the leptin B deficient zebrafish.

The inventors have therefore developed a novel method of screening a test compound for insulin resistance modulating activity using a leptin B deficient or leptin receptor deficient zebrafish. They have used their novel method to demonstrate that the phosphatase inhibitor NSC87877 is capable of reducing insulin resistance, and that this capability occurs at an early time point of development. Furthermore, gene knockdown studies in the leptin B mutant background indicate that SHP-1 is the most likely target responsible for the anti-diabetic effect of NSC87877.

Generating a Leptin B or leptin receptor deficient teleost

The present invention provides various methods and uses for a leptin B deficient or leptin receptor deficient teleost model. A teleost model of the invention has a number of advantages over rodent models. For example, a teleost model of the invention (e.g. a leptin B deficient or leptin receptor deficient teleost) will follow a normal circadian rhythm. Without wishing to be bound by theory, it is believed that glucose metabolism is strongly regulated by circadian rhythm. Hence, a teleost model of the invention (e.g. a leptin B deficient or leptin receptor deficient teleost) will provide a more reliable system for analysing glucose metabolism which is more representative of human physiology.

Furthermore, a teleost model of the invention may be more cost-effective compared to high fat rodent models where the rodent is fed a high fat diet to generate a diabetic phenotype. Advantageously, the teleost model may be easily be used to generate a high throughput screening method as described elsewhere herein.

Furthermore, due to the unexpected utility from an early stage (e.g. from 4hpf in zebrafish), a leptin B deficient or leptin receptor deficient teleost model may be used in automated methods.

As used herein, the term "teleost" means a vertebrate of or belonging to the Teleostei or Teleostomi, a group consisting of numerous fishes having bony skeletons and rayed fins. Teleosts include, for example, zebrafish (Danio rerio), Medaka, Giant rerio, and puffer fish. Suitably, the teleost may be a zebrafish. For fish species which have a tetraploid genome such as common carp more than one leptin B gene or LepR receptor gene might have to be inactivated. Using methods such as CRISPR/CAS technology this is now possible.

The sequences of leptin proteins and leptin genes from teleosts have been well characterised (see for example Prokop et al, Leptin and leptin receptor: Analysis of a structure to function relationship in interaction and evolution from humans to fish, Peptides. 2012 Dec; 38(2): 326-336). The gene and protein sequences for a number of teleosts are publicly available.

Two distinct leptin genes have been found in a number of teleosts including: medaka (Oryzias latipes) and zebrafish (Danio rerio). The two leptin proteins expressed by these genes (leptin A and leptin B) share low interspecies amino acid sequence identity. The duplicity of genes has been described for several fish such as atlantic salmon, Japanese medaka, common carp and zebrafish and the gene sequences for each are known. For example, the gene sequence for leptin B in zebrafish (Danio rerio) may be found in the NCBI database under Gene ID: 564348 and the protein sequence may be found under UniProtKB: Q108T6.

Likewise, the leptin receptor in teleosts has been well characterised. For example, the gene sequence for the leptin receptor in zebrafish (Danio rerio) may be found in the NCBI database under Gene ID: 567241. The genomic sequence can also be found under GenBank: BX649263.06 and the protein sequence may be found under UniProtKB: C4WYH6.

Accordingly, it is a matter of routine for a person of ordinary skill in the art to generate a leptin B deficient and/or leptin receptor deficient teleost by reducing the leptin B and/or leptin receptor mRNA or protein level in the teleost (e.g. by mutation of the leptin B and/or leptin receptor gene(s)).

As used herein “leptin B deficient teleost” refers to a teleost wherein leptin B mRNA levels or leptin B protein levels are reduced by at least 40% compared to a control (wild-type) teleost. Suitably, leptin B mRNA levels or leptin B protein levels may be reduced by at least 50% or at least 60% or at least 70% or at least 80% or at least 90% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or 100% compared to the control. Suitably, a leptin B deficient teleost may be insulin resistant.

As used herein “leptin receptor deficient teleost” or “leptin R deficient teleost” refers to a teleost wherein leptin receptor mRNA levels or leptin receptor protein levels are reduced by at least 40% compared to a control (wild-type) teleost. Suitably, leptin receptor mRNA levels or leptin receptor protein levels may be reduced by at least 50% or at least 60% or at least 70% or at least 80% or at least 90% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100%. Suitably, a leptin receptor deficient teleost may be insulin resistant.

In the context of these definitions, a control (wildtype) teleost is a naturally occurring teleost of the same species as the leptin B deficient or leptin receptor deficient teleost, preferably from the same strain as the leptin B deficient or leptin receptor deficient teleost.

In the context of these definitions, “leptin B mRNA” and “leptin B protein” refer to the wildtype leptin B mRNA/protein. By way of example, the wildtype leptin B protein sequence for zebrafish may be found at uniprotKB: Q108T6. Wildtype mRNA and protein sequences for other teleosts are readily identifiable by a person of ordinary skill in the art.

In the context of these definitions, “leptin receptor mRNA” and “leptin receptor protein” refer to the wildtype leptin receptor mRNA/protein. By way of example, the wildtype leptin receptor protein sequence for zebrafish may be found at uniprotKB: C4WYH6. Wildtype mRNA and protein sequences for other teleosts are readily identifiable by a person of ordinary skill in the art.

As used herein, “insulin resistant teleost” refers to a teleost having a significantly higher basal level of glucose (in pmol/larva) compared to a control wildtype teleost following hyperinsulineamia. Suitably, the hyperinsulineamia may be induced by injection of 1 nL human recombinant insulin (Sigma-Aldrich, the Netherlands) into the caudal aorta of a 4 dpf teleost using a glass capillary as described in Juez et al., 2014. Suitably, basal levels of glucose may be quantitatively analysed using whole body lysates and a standard glucose assay kit (Cayman chemical USA), with fluorescence being measured at 540 nm using a BioTek plate reader equipped with GEN5 software (v.2.04, BioTek, Winooski, VT, USA). The teleost may be considered insulin resistant if the basal glucose level at 0, 30 and/or 240 minutes post insulin injection is significantly higher than the corresponding basal glucose level of a wildtype teleost of the same species at the same time points. Suitably, the basal glucose level may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% at least 70%, at least 80%, at least 90% or at least about 100% higher in an insulin resistant teleost compared to the corresponding wildtype.

In addition, or in the alternative, an “insulin resistant teleost” may have a significantly increased basal level of glucose at 270 minutes post injection of insulin (compared to at 0 minutes post insulin injection) under the conditions recited in the paragraph above. Suitably, the basal glucose level may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30% at 270 minutes post injection of insulin (compared to at 0 minutes post insulin injection) under the conditions recited in the paragraph above.

Suitably, the leptin B deficient and/or leptin receptor deficient teleost may have a modified leptin B and/or leptin receptor gene such that the leptin B and/or leptin receptor mRNA levels or protein levels are reduced compared to a control wildtype teleost. For example, the specified gene(s) may be modified via site directed or random mutagenesis, or modified via any other routine method known for gene modification in the art.

Suitably, the leptin B gene and/or the leptin receptor gene in the teleost may silenced. Alternatively, the expression product (such as mRNA and/or protein) of the teleost gene may be decreased.

Any suitable method may be employed to generate a leptin B and/or leptin receptor deficient teleost, for example use of: zinc finger nucleases, TALEN, CRISPR silencing, morpholinos, or RNAi.

Suitably, a leptin B deficient or leptin receptor deficient teleost may be generated by introducing a loss of function mutation into the relevant gene (e.g. by introducing a mutation into the reading frame of the gene so that a functional protein cannot be produced). Methods of generating a loss of function mutation in a teleost gene are well known in the art, and include, for example, zinc finger nuclease cleavage, TALEN, and random mutation. Zinc finger nuclease cleavage has been described, for example, in W02010076939. The mutated teleost gene, when produced in vitro, can be introduced into the teleost by methods such as microinjection.

Zinc finger nucleases (ZFNs) involves modular assembly of DNA-binding domains that typically contain three individual zinc finger repeats that can each recognize a 3 base pair DNA sequence, which are then linked to the restriction endonuclease Fokl. Since Fokl must dimerize in order to cleave DNA, a pair of ZFNs can be used to target non-palindromic DNA sites.

Suitably, mutations to generate a leptin B deficient and/or leptin receptor deficient teleost may be introduced into the teleost genome randomly, for example by following methods known in the art. A teleost comprising a desired mutation may subsequently be identified using a screening method, for example based on insulin sensitivity, or using any other suitable method. A person of ordinary skill in the art is readily aware of other methods for analysing DNA mutations in a teleost gene, such as PCR-based amplification or pyrosequencing (see W02007002204).

Suitably, the expression products (such as mRNA and/or protein) of the leptin B gene and /or leptin receptor gene may be reduced in the teleost in accordance with any known method. For example, the expression product of the teleost gene can be reduced by the use of regulatory proteins, such as repressors, to inhibit transcription of the teleost gene. In some embodiments, the expression product of the teleost gene can be reduced by destabilizing the mRNA transcribed from the teleost gene.

Suitably, the expression products of the leptin B and/or leptin receptor teleost gene(s) may be reduced through inhibition of translation of the mRNA derived from the target teleost gene by means of regulatory proteins, antisense molecules, morpholinos, or/and RNAi molecules.

Antisense molecules include, for example, short DNA, RNA or nucleic acid analog fragments (such as, for example, PNAs, LNAs, phosphorothioate oligonucleotides, morpholino oligonucleotides, 2-fluoro-RNAs or mixed compounds thereof) with a nucleic acid sequence of about 10 nucleotides or more which is complementary to a partial area of the mRNA derived from the target teleost gene. Suitable RNAi molecules include, for example, double-stranded RNA molecules with a length of about 10 base pairs or more, such as about 18 base pairs or more, or about 20 base pairs or more. The RNAi molecules can either be manufactured synthetically or in a vector-based manner in the target cells (Elbashir et al., Nature 411: 494-498, 2001; Sui et al., Proc. Natl. Acad. Sci. USA 99: 5515-5520,2002). The sequence of the RNAi molecules is selected in such a manner that it corresponds to specific sequence areas of the mRNA derived from the teleost gene.

Suitably, the teleost leptin B and/or leptin receptor gene(s) in the teleost may be silenced, for example through CRISPR silencing, morpholino, or RNAi. CRISPR silencing has been described, for example, at EP2336362 and W02012164565. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are short, multiple repeats of base pair sequences across a single DNA loci. For example, each CRISPR sequence contains a series of base pairs followed by the same or similar base pairs in reverse order and then a space region of approximately 30 base pairs. CRISPRs rely on crRNA and tracrRNA for sequence-specific silencing such that Cas9 (for example) can serve as an RNA guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA target recognition. Methods for generating suitable single guide RNAs against a desired target are a matter of routine to a person of ordinary skill in the art. The first circa. 20 nucleotide are programmed to hybridise with a desired target just prior to a PAM motif for the CRISPR/cas system that is used. Figure 1 shows a target sequence of the zebrafish genome and an example of the programmable portion of sgRNA that can be used to generate a leptin B deficient zebrafish. Various databases exist to identify other sgRNAs that can be used to knock out a known gene. Hence, CRISPR methodology can be routinely used to knock out the leptin B gene and/or the leptin receptor gene of any teleost.

Suitably, a leptin B deficient teleost and/or leptin receptor deficient teleost may be generated by inhibiting the expression product of the respective gene. Inhibitors can include, for example, protein, peptides, small molecules, antagonist antibodies, etc. The inhibitor may lead to destabilization of the protein product of the teleost gene and/or inhibition of the activity of the protein product of the teleost gene.

Suitably, a morpholino can be used to modify the expression of a teleost gene as compared to that of a teleost that has not been subjected to the morpholino (control/“wildtype” teleost). A "morpholino" or "morpholino oligonucleotide," as used herein, is an oligonucleotide composed of a 6-member morpholine ring that replaces ribose or deoxyribose rings, where (i) the structures are linked together by phosphorus- containing linkages, one to three atoms long, joining the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) purine or pyrimidine base-pairing moieties effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide. The purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil, thymine or inosine. The synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337, all of which are incorporated herein by reference.

Screening methods

In one aspect, the invention provides a method of screening a test compound for insulin resistance modulating activity, said method comprising: a. contacting the test compound with a leptin B deficient or a leptin receptor deficient teleost; and b. determining the effect of the test compound on basal glucose levels in the teleost or on glucose uptake in the teleost.

Suitably, the teleost is contained in an aqueous medium in a container such as a microtiter well, e.g. in a multi-well plate, e.g., a 96- well plate.

The teleost may be pretreated prior to exposure to the test compound, for example to facilitate the penetration and/or contacting of the compound.

Contacting

As used herein, "contacting" includes physically contacting the teleost with a test compound (for example by immersion in a solution containing the test compound) and/or introducing (for example by injecting or via ingestion) the test compound into the teleost.

Suitably, the teleost may be immersed in a solution comprising the test compound. The test compound may be administered to the teleost by dissolving the compound in a solution (e.g. media) containing the teleost. Alternatively, the compound may first be dissolved in the solution and the live teleost submerged in the solution subsequently.

Suitably, the test compound may be administered to the teleost by injection, electroporation, lipofection, or ingestion or by using holistic cell loading technology in which particles coated with the biological molecule are introduced into the cell or tissue of interest as a bolus using a high-pressure gun. Suitably, the test compound may be administered to the teleost by microinjecting the compound into the live teleost. Suitably, the contacting step may comprise injecting the test compound through the chorion of the teleost. Suitable vehicles for injection include, but are not limited to, E3 buffer and/or DMSO.

The test compound may be one or a number (e.g. a mixture) of different compounds.

The test compound may be brought into contact with the teleost alone, in conjunction with a solvent (e.g., dimethylsulfoxide (DMSO) or the like) ora carrier (including, e.g., a peptide, lipid or solvent carrier), or water, or in conjunction with another compound. Suitably, the test compound may be dissolved in any suitable solvent. Suitably, the solvent may be DMSO.

Suitably, any appropriate amount of the test compound may be contacted with a teleost. For example, a concentration of at least 1μΜ or at least 2μΜ or at least 5μΜ or at least 10 μΜ of the test compound may be used. Suitably, about 10μΜ of the test compound may be used.

Suitably, the effect of the test compound may be measured with respect to a control where the method steps and reagents are identical except for the absence of a test compound.

Suitably, the screening method may utilise a teleost at any stage of its life-cycle, including an embryo, larva or adult. Advantageously, the contacting step may occur when the leptin B deficient or a leptin receptor deficient teleost is an embryo or a larva. It has been surprisingly discovered that a leptin B deficient or a leptin receptor deficient teleost may be insulin resistant from a very early stage of its development. The Examples provided herein illustrate a method by which a test compound may be injected into the chorion of a zebrafish as early as 4 hour post fertilisation (hpf) in order to screen for the compound’s ability to modulate insulin resistance.

As used herein, “embryo” refers to a teleost up to 2 days post fertilisation and “larva” refers to a teleost from 2 to about 20 dpf.

Suitably, the contacting step may occur when the teleost is less than 4dpf, or less than 3dpf, or less than 2dpf, or less than 1 dpf, or less than 22 hpf, or less than 12hpf, or less than 8hpf, or less than 6hpf, or less than 4 hpf. Suitably, the contacting step may occur from about 4hpf to about 4dpf, or from about 4hpf to about 2dpf, or from about 4hpf to about 22hpf.

In some embodiments, the teleosts are incubated at a temperature that is the range of 24 to 32 degrees °C. In one embodiment, the temperature is slightly higher than room temperature, e.g., about 26 to 30 °C, or about 28-29 °C).

Determining

The methods of the present invention comprise determining the effect of the test compound on basal glucose levels in the teleost or on glucose uptake in the teleost.

Effect on glucose uptake

Suitably, the determining step may comprise contacting the teleost with a glucose solution and measuring glucose uptake in the teleost, wherein an increase in glucose uptake compared to a control, or compared to a predetermined level, identifies the test compound as having the ability to reduce insulin resistance.

Advantageously, the leptin B deficient or leptin receptor deficient teleost used in the screening methods is insulin resistant and, therefore, will not take up or significantly uptake glucose. Accordingly, an increase in glucose uptake compared to a control, or compared to a predetermined level, identifies the test compound as having the ability to reduce insulin resistance.

Suitably, if the teleost is chorionated during the contacting step, it may be dechorionated by any known method before contact with the glucose solution. For example, the teleost may be enzymatically dechorionated with e.g., pronase. Dechorionizing the teleost may be advantageous where immersion methods are used to measure glucose uptake during the determining step.

Suitably, the teleost may be contacted with the glucose solution in a similar manner as described above for contacting with the test compound.

For example, the teleost may be injected (for example into the yolk) with a glucose solution or a (dechorionated) teleost may be immersed in a glucose solution.

Suitably, if the teleost is immersed in a glucose solution, the immersion step may be for at least 10 minutes, or at least 20 minutes, or at least 30 minutes, or at least 1 hour, or at least 1.5 hours, or at least 2 hours. Suitably, the immersion step may be for about 2 hours.

Suitably, subsequent to the immersion step the teleost may be washed one or more times in a suitable solution such as egg water. Suitably, the teleost may be washed 2 or 3 times. The teleost may then be incubated in an appropriate solution (e.g. egg water) prior to determining glucose uptake.

Suitably, glucose uptake may be measured at any time period after the washing step(s). Suitably, glucose uptake may be measured after about at least 30 minutes or about at least 1 hour or about at least 2 hours or about at least 3 hours or about at least 4 hours of incubation in a suitable solution (e.g. egg water) following the washing step(s).

Whatever the contact method, any suitable concentration of glucose may be employed. For example, for immersion, a concentration of at least 20mM, or at least 50mM, or at least 100mM or at least 200mM glucose solution may be used. Suitably, a concentration of about 50mM to about 500Mm, or from about 100mM to about 300mM or about 200Mm may be used.

Suitably, the glucose solution may be injected into the chorion of the teleost. Any suitable concentration and volume of glucose may be employed. For example, a concentration of at least 1 mg/ml or least 2 mg/ml may be used. Suitably, about 2.5 mg/ml may be used. A suitable volume for injection can readily be identified by a person of ordinary skill in the art (see for example Spaink et al., Methods 62 (2013) 246-254, and Carvalho et al., PLoS One. 2011 Feb 16;6(2):e16779). Non-limiting examples of appropriate volumes include a range of from about 0.5nL to 2nL, and thus includes about 0.5 nL, or about 0.75 nL, or about 1 nL, or about 1.25 nL, or about 1.5 nL, or about 1.75 nL, or about 2 nL.

Glucose uptake may subsequently be determined. Suitably, glucose uptake may be measured using any known method in the art. The identification of suitable methods is well within the routine capabilities of a person of ordinary skill in the art and includes the methods exemplified herein.

In methods of determining glucose uptake, glucose or any appropriate glucose analog may be used. Suitably, the glucose or glucose analog may be labelled.

As used herein the term "labeled", refers to direct labeling of glucose or a glucose analog by coupling (i.e., physically linking) a detectable substance to the glucose or the glucose analog as well as indirect labeling of the glucose or the glucose analog by reactivity with a detectable substance.

Suitably, the glucose or glucose analog may be labelled with a fluorescent marker. Various labels and fluorescent markers are known. Suitably, the glucose analog may be the fluorescently labelled glucose analog 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (available from Life Technologies).

The level of glucose or glucose analog in the teleost may be determined by techniques known in the art, such as enzyme linked immunosorbent assays (ELISAs), immunoprecipitation, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, nuclear magnetic resonance, NMR and MRI, Mass spectrometry, in vivo glucose sensor proteins based on fluorescence (for instance FRET, fluorescence resonance energy transfer, probes). Such methods are routine in the art, see for example Veetil etal., 2012 and Yu etal., 2017).

Effect on basal glucose levels

Suitably, the determining step may comprise contacting the teleost with an insulin solution and subsequently measuring the effect on basal glucose levels in the teleost, wherein a decrease in basal glucose level over time or compared to a control or a predetermined level in the presence of a test compound identifies the test compound as having the ability to reduce insulin resistance.

Advantageously, the leptin B deficient or leptin receptor deficient teleost used in the screening methods is insulin resistant and, therefore, will not significantly decrease basal glucose levels following contact with insulin. Accordingly, a decrease in basal glucose level over time or compared to a control or a predetermined level identifies the test compound as having the ability to reduce insulin resistance.

Suitably, the teleost may be contacted with insulin in a similar manner as described above for contacting with the test compound.

For example, the teleost may be injected (for example into the yolk or caudal aorta) with an insulin solution.

Suitably, basal glucose levels may be measured at any time period after contacting with insulin. Suitably, basal glucose levels are measured at about 0 minutes, or after about at least 30 minutes, or about at least 1 hour or about at least 2 hours or about at least 3 hours or about at least 4 hours of contact with the insulin. Basal glucose levels may be measured at more than one time point.

Whatever the contact method, any suitable concentration of insulin may be employed. For example, the teleost may be injected with an insulin solution in any suitable amount. For example, abut 1 nl insulin may be used. Any suitable insulin may be used, including human recombinant insulin. For example the methodology of Juez etal. (2014) may be used.

The basal level of glucose in the teleost may be determined by techniques known in the art, such as enzyme linked immunosorbent assays (ELISAs), immunoprecipitation, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, nuclear magnetic resonance, NMR and MRI, Mass spectrometry, in vivo glucose sensor proteins based on fluorescence (for instance FRET, fluorescence resonance energy transfer, probes). Such methods are routine in the art, see for example Veetil etal., 2012 and Yu etal., 2017).

It is noted that there are also various commercial blood glucose measurement devices available fortesting diabetes, several of which have been used successfully by the inventors with zebrafish larval extracts. The level of glucose in total larval extracts is a measure for the level of uptake of glucose since glucose in tissues is rapidly converted into derivatives such as glucose 6 phosphate. Advantageously, methods such as NMR and mass spectrometry have the capacity to detect glucose per se as well as the appropriate derivatives.

Suitably, quantitative analysis of basal glucose levels may be analyzed from whole body lysates. For example any suitable glucose assay kit may be used (e.g. one obtained from Cayman, Chemical, USA), with assay kits which measure glucose levels by fluorescence being preferred.

In all methods of the invention, a test compound may be identified as reducing insulin resistance if it results in: • an increase in glucose uptake (for example an increase of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, or more) compared to a control or predetermined level; or • a decrease in basal glucose levels following incubation with insulin (for example a decrease of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, or more) compared to a control or predetermined level.

In the screening methods of the invention, a negative “control” may be included. Suitably, the negative control may be an identical teleost used in the methods and subject to the same reaction conditions with the exception that the test compound is not administered to the control.

Furthermore, a reference sample may be used to ensure that the method is working effectively. For example, mannitol may be used to confirm that the reaction conditions are sufficient for uptake of a control compound (i.e. mannitol) into the teleost. Advantageously, use of this reference sample may reduce false negative results. Alternatively, or in addition, an effective amount of metformin or NSC87877 may be used under identical reaction conditions as the test compound (and instead of the test compound), as a positive reference sample.

In the screening methods of the invention the “predetermined level” refers to a previously calculated threshold of glucose uptake or basal glucose level in a leptin B deficient and/or leptin receptor deficient teleost in the absence of the test compound, under otherwise identical reaction conditions.

High throughput

The methods described herein can be useful for screening compounds that may modulate insulin resistance, for example in a high throughput screening context. In such methods, each of the plurality of leptin B deficient or leptin receptor deficient teleosts are contacted with a different compound and the effect of each of the test compounds on basal glucose levels in each teleost or on glucose uptake in the teleost is measured. These may be compared to a control teleost or a predetermined level to identify test compounds which reduce insulin resistance.

Suitably, the plurality of compounds may comprise at least about 50 compounds, including for example at least about any of 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more different compounds.

Advantageously, screening for a test compound which modulates insulin resistance using a teleost model which can be utilised at an early stage (preferably from before 22hpf) allows the screening method to easily scaled up to a high throughput method. Furthermore, advantageously such a system allows for automation of all steps in the method as e.g. robotic injection of the test compound into an embryo can be reliably conducted.

Automation

The methods of the invention can readily be automated. One suitable method of automating the screening methods of the invention is shown in Figure 6.

The inventors have shown in recent years that zebrafish larvae are highly amenable to high throughput screening approaches including robotic injection and automated fluorescence screening technologies such as COPAS and VAST (Veneman etal., 2014; Guo etal., 2017).

The invention is particularly suitable for assays that use Vertebrate Automated Screening Technology (VAST). VAST is a microscope mounted system that enables the application of zebrafish high-throughput screening. The VAST Biolmager contains a capillary that holds a zebrafish for imaging. Through the rotation of the capillary, multiple axial-views of a specimen can be acquired. For the VAST Biolmager, fluorescence and/or confocal microscopes are used. Quantitation of a specific signal as derived from a label in one fluorescent channel requires insight in the zebrafish volume to be able to normalize quantitation to volume units. Full details of the method can be found in Veneman etal 2014 and Guo etal 2017 incorporated herein for reference.

Suitably, teleost of the invention may be collected and aligned onto any suitable surface e.g. a multiwell plate. Each well may contain a teleost.

Suitably, a test compound may be robotically injected through the chorion of the teleost. Suitably, a plurality of test compounds may be tested in a single plate, with each different test compound being robotically injected through the chorion of a teleost of the invention in its own well.

Suitably, the automated method may allow for duplicate testing of a specific test compound to increase the reliability of the results.

Suitably, a positive control and/or negative control may be added to each plate.

Suitably, the robotic injection of the test compound(s) may be conducted on leptin B deficient and/or leptin receptor deficient teleost(s) which are less than 22hpf, preferably less than 12hpf, preferably less than 5hpf, preferably at about 4hpf.

Suitably, the injected teleost(s) are then incubated under suitable conditions for at least two hours, or at least 4 hours, or at least 6 hours, or at least 12 hours, or for about 20 hours in a suitable solution (e.g. egg water).

Suitably, following incubation the teleost(s) are dechorionated by any suitable automated means. For example, they may be enzymatically dechorionated.

Suitably, the teleost(s) may be robotically injected with labelled glucose or a glucose analog (or immersed in labelled glucose or a glucose analog). Suitably, the glucose or glucose analog may be fluorescently labelled.

Suitably, the teleost(s) may be subjected to one or more washes, and optional subsequent incubations (e.g. in egg water).

Automated screening following addition of labelled glucose or a glucose analog may be carried out by any known method, such as by VAST Biolmager.

Diabetes and metabolic syndrome

In one aspect, the present invention provides a method for identifying a compound for treating diabetes and/or metabolic syndrome, said method comprising determining the effect of a compound on insulin resistance in accordance with the invention (e.g. by a screening method as detailed in the section above), and selecting a compound which has the ability to reduce insulin resistance.

Thus, the present invention provides a leptin B deficient and/or leptin receptor deficient teleost as a model of metabolic syndrome and/or diabetes. By determining and selecting test compounds which have the ability to reduce insulin resistance in a leptin B deficient and/or leptin receptor deficient teleost, compounds for treating diabetes and/or metabolic syndrome in a subject can be identified. Advantageously, a model in accordance with the invention may be more predictive of potential compounds for treating diabetes and/or metabolic syndrome in human compared to mouse models, because the circadian rhythm in teleosts is aligned with that in humans.

As used herein, “subject” refers to an individual, e.g. a human. The terms “subject”, “patient” and “individual” are used interchangeably herein. The subject may have or be at risk of having a metabolic disorder.

As used herein, “metabolic disorder” refers to a known group of diseases that adversely affect metabolism. Metabolic disorders include, for example, pre-diabetes, diabetes (particularly Type 2 diabetes), cardiovascular disease and metabolic syndrome.

As used herein, “metabolic syndrome” is a multiplex risk factor that arises from insulin resistance accompanying abnormal adipose deposition and function. It is a risk factor for coronary heart disease as well as for diabetes, fatty liver and several cancers. According to guidelines from the National Heart, Lung, and Blood Institute (NHLBI) and the American Heart Association (AHA), metabolic syndrome is diagnosed when a patient has at least three of the following five conditions: • Fasting glucose >100 mg/dL (or receiving drug therapy for hyperglycemia) • Blood pressure >130/85 mm Hg (or receiving drug therapy for hypertension) • Triglycerides >150 mg/dL (or receiving drug therapy for hypertriglyceridemia) • HDL-C < 40 mg/dL in men or < 50 mg/dL in women (or receiving drug therapy for reduced HDL-C) • Waist circumference >102 cm (40 in) in men or >88 cm (35 in) in women; if Asian American, >90 cm (35 in) in men or >80 cm (32 in) in women.

However, in the context of the invention, the phrase “metabolic syndrome” is used in its broadest sense, and encompasses having one, two, three, four or five of the above risk factors.

The term “diabetes” as used herein is synonymous with “type 2 diabetes” or “TIID”, which is well defined in the art and takes its normal meaning herein.

Methods for identifying targets of drugs

The leptin B deficient and/or leptin receptor deficient teleost model of the invention can advantageously be used in methods of identifying targets for known drugs for the treatment of diabetes and/or metabolic syndrome. Further, the leptin B deficient and/or leptin receptor deficient teleost model of the invention can advantageously be used in methods of identifying targets for test compounds (“putative drug(s)”) identified as being capable of reducing insulin resistance in accordance with the present invention.

Identifying the targets of known or putative drugs allows the skilled person to further understand the mechanism underlying the conditions to be treated and the generation of new drugs directed against the identified target.

In one aspect, the present invention provides a method of identifying a target of a drug for the treatment of diabetes and/or metabolic syndrome, said method comprising: a. modifying the gene or protein expression of a potential target in a leptin B deficient or a leptin receptor deficient teleost; b. contacting the teleost with the drug; and c. determining the effect of the drug on a basal glucose level in the teleost, or on glucose uptake in the teleost.

Suitably, the method described for identifying the potential target of a drug or putative drug for treating diabetes and/or metabolic syndrome may utilise one or more controls, as appropriate.

For example, a control may be added to confirm that the drug or putative drug is effective in reducing insulin resistance in a leptin B deficient and/or leptin receptor deficient teleost. Suitably, in the control all contacting steps and reaction conditions are identical to the test method, with the exception that step a) of the method is omitted (i.e. the gene and protein expression of the potential target is not modified in the teleost), such that a decrease in insulin resistance is observed.

Further, analysis may be performed to confirm that modification of the gene and/or protein expression of the potential target in step a) of the method has occurred. For example, where a morpholino has been used to reduce protein expression of the potential target, a Western blot may be conducted to confirm that the protein level of the potential target of interest has been reduced.

Hence, in the method of identifying a target of a drug for treating diabetes and/or metabolic syndrome, a decrease in glucose uptake compared to the control, or compared to a predetermined level, identifies the potential target as a target for the drug or putative drug.

As used herein, “predetermined level” refers to a previously calculated threshold of glucose uptake following administration of the drug or putative drug to a leptin B deficient and/or leptin receptor deficient teleost in accordance with the method, wherein the predetermined level is calculated based on a method in which the reaction steps and conditions are identical to the test method with the exception that step a) of the method is omitted (i.e. the gene and protein expression of the potential target is not modified in the teleost), such that a decrease in insulin resistance is observed.

The potential target may be a gene or may be an mRNA or protein encoded by a gene. The phrase “potential target gene” used herein refers to a gene that is itself the potential target of a drug, and a gene that encodes an mRNA or protein, wherein the encoded mRNA or protein is the potential target of the drug.

Suitably, the potential target gene in the teleost may silenced. Alternatively, the expression product (such as mRNA and/or protein) of a potential target gene may be reduced.

Any suitable means may be utilised to modify the potential target including the use of: zinc finger nucleases, TALEN, CRISPR silencing, morpholinos, or RNAi.

Suitably, the modification may result in a loss of function mutation to the potential target gene. Methods of generating loss of function mutations have been disclosed above in relation to generating leptin B deficient or leptin receptor deficient teleosts. It is a matter of routine to apply such methods to generate loss of function mutations of the potential target gene.

Suitably, the modifying step may involve inhibiting an expression product of the potential target. Inhibitors can include, for example, protein, peptides, small molecules, antagonist antibodies, etc. The inhibitor may lead to destabilization of the protein product of the teleost gene and/or inhibit the activity of the protein product of the teleost gene.

Suitably, the modifying step comprises using a morpholino for the potential target. Suitably, the morpholino targeting the potential drug target is injected at a very early stage into the teleost embryo (for example the morpholino may be injected into a single cell teleost embryo).

The morpholino may be at any suitable concentration. Suitably, the morpholino may be at a concentration of between about 0.01 mM and about 0.1 mM, suitably between about 0.05mM and about 0.1mM. Suitably, the morpholino may be at a concentration of about 0.08mM. Suitably, the morpholino may be dissolved in any suitable buffer such as one comprising one or more of: NaCI, KCI, Ca(NO3)2 and HEPES.

Suitably, the volume will be adjusted to the embryonic stage and the concentration of the morpholino. For example, when a morpholino is injected at a concentration of about 0.08mM into a single cell teleost a suitable volume may be about 1 nl.

Specificity of a potential morpholino against a specific target can readily be determined - see Juez et al. (2104), for example.

Suitably, the expression product (such as mRNA and/or protein) of the gene of the potential target may be increased. Various means of activating gene transcription and translation are known in the art.

Following modification of the gene or protein expression of a potential target in a leptin B deficient or a leptin receptor deficient teleost, the modified teleost may be contacted with the known drug for treating diabetes and/or metabolic syndrome (or putative drug which was been identified as modulating insulin resistance in a leptin B deficient and/or a leptin receptor deficient teleost in accordance with the invention).

The contacting step in this method may be in accordance with any step of contacting a test compound with the teleost model detailed in the contacting section above. Thus any of the features described in the contacting section above may equally be utilised for this method, provided that the terminology “test compound” is substituted with “drug” or “putative drug”.

Likewise, the determining step in this method may be in accordance with any step of determining basal glucose levels in the teleost, or determining glucose uptake in the teleost detailed in the determining section above. Thus, any of the features described in the determining section above may equally be utilised for this method, provided that the terminology “test compound” is substituted with “potential target”.

Suitably, the method of identifying a target of a drug for the treatment of diabetes and/or metabolic syndrome may comprise contacting the teleost with a glucose solution and measuring glucose uptake in the teleost, wherein a decrease in glucose uptake compared to a control or compared to a predetermined level identifies the potential target as a target for the drug.

Suitably, the method of identifying a target of a drug for the treatment of diabetes and/or metabolic syndrome may comprise contacting the teleost with insulin solution and subsequently measuring the effect on the basal glucose level in the teleost, wherein an increase in glucose level over time or compared to a control or a predetermined level, identifies the potential target as a target for the drug.

Methods for optimising the formulation of a drug for the treatment of diabetes and/or metabolic syndrome

The leptin B deficient and/or leptin receptor deficient teleost model of the invention can advantageously be used in methods of optimising the formulation of drugs for the treatment of diabetes and/or metabolic syndrome.

In one aspect, the present invention provides a method for optimising the formulation of a drug for the treatment of diabetes and/or metabolic syndrome, said method comprising: a. contacting a first formulation of the drug with a first leptin B deficient and/or a leptin receptor deficient teleost; b. determining the effect of the first formulation on a basal glucose level in the teleost, or on glucose uptake in the teleost; c. contacting a second formulation of the drug with a second leptin B deficient and/or a leptin receptor deficient teleost; d. determining the effect of the second formulation on a basal glucose level in the teleost, or on glucose uptake in the teleost; and e. comparing the basal glucose levels, or glucose uptake determined in steps b) and d), and selecting the formulation which results in a higher glucose uptake or a lower glucose basal level.

Suitably, the first leptin B deficient and/or a leptin receptor deficient teleost may be identical to second leptin B deficient and/or a leptin receptor deficient teleost.

Suitably, both the first teleost and the second teleost may be leptin B deficient.

Suitably, both the first teleost and the second teleost may be leptin receptor deficient.

Suitably, both the first teleost and the second teleost may be both leptin B deficient and leptin receptor deficient.

Suitably, the teleost may be a zebrafish.

Suitably, the first teleost and the second teleost are at the same time point post fertilisation. A teleost may be at any development stage in hpf or dpf as utilised in other methods of the invention and described elsewhere herein.

The two contacting steps (steps a) and c)) and the various conditions of these steps should preferably be identical, with the exception of the formulation that is being contacted with the teleost. Any means of contacting the teleost with the formulation can be employed. Each of the contacting steps/conditions in the “contacting” section above can be used for this method with the exception that “the test compound” is substituted with the “first formulation” for the contacting step in step a) and the “the test compound” is substituted with the “second formulation” for the contacting step in step c).

The two determining steps (steps b) and d)) and the various conditions of these steps should preferably be identical, with the exception of the formulation that is being contacted with the teleost. Any means of determining the effect of the formulation on a basal glucose level in the teleost, or on glucose uptake in the teleost can be employed. Each of the determining steps/conditions in the “determining” section above can be used for this method with the exception that “the test compound” is substituted with the “first formulation” for the determining step in step b) and the “the test compound” is substituted with the “second formulation” for the determining step in step d).

Suitably, the method comprises comparing the basal glucose levels, or glucose uptake determined in steps b) and d), and selecting the formulation which results in a higher glucose uptake or a lower glucose basal level. Thus the optimised formulation is selected.

Suitably, the optimised formulation selected in step e) can then undergo further optimisation. Hence, the process of optimisation can be repeated with the preferred formulation being compared to a new third formulation. The process for optimisation can then be repeated as many times as desired each time with the most preferred formulation being selected until the optimal formulation is arrived at.

Any desired parameter to be optimised in a drug formulation may be altered between a first and second (or further subsequent) formulation. Examples of parameters to be altered include: drug dose, release rate, buffers and drug concentration.

Uses

In one aspect, the present invention provides the use of a leptin B deficient and/or a leptin receptor deficient teleost embryo or larva as a model of diabetes or metabolic syndrome.

Various advantages of such teleosts as a model of diabetes or metabolic syndrome have been described herein, including: • Improved modelling for diabetes or metabolic syndrome compared with rodents due to a more representative circadian rhythm; • Cost-effectiveness and scalability of the model for high throughput analysis; • Automation of various uses for the model including drug screening and drug target screening; • Speed of use - no time wasted providing high fat diets, the model can be used early from e.g. from 4hpf; • Can be combined with screening such as VAST to identify where a drug is active; • The teleost grow and reproduce normally.

In another aspect, the present invention provides the use of a leptin B deficient and/or a leptin receptor deficient teleost to: a. screen test compounds for their ability to modulate insulin resistance; b. identify a compound for treating diabetes and/or metabolic syndrome; c. identify a target of a drug for treatment of diabetes and/or metabolic syndrome; d. optimise a drug formulation or regimen for the treatment of diabetes and/or metabolic syndrome; and/or e. identify the location(s) of drug activity in the teleost.

Suitably, the teleost may be an embryo or larva, preferably less than 3dpf. Suitably, the teleost may be as young as 4hpf.

Suitably, the teleost may be a zebrafish.

Further Model

In another aspect, the present invention provides an insulin resistant teleost model which may be utilised for all of the methods and uses of the present invention in replacement for leptin B deficient and/or leptin receptor deficient teleost described above. Suitably, said insulin resistant teleost model may be insulin resistant through sustained exposure to glucose.

The inventors have shown that immersing 4dpf wild-type (i.e. not leptin B or leptin receptor deficient) zebrafish larvae in egg water containing 250mM glucose concentration for two hours did not result in insulin resistance.

However, subsequent immersion for at least another 2 hours in egg water containing 250mM glucose concentration (following an intermediate washing step) resulted in hyperglycemia and insulin resistance.

Therefore, sustained exposure of a teleost to glucose at an early stage in development (e.g. up to 4dpf) may be used to generate an insulin resistant teleost model without the need to specifically target gene expression. Advantageously, this may provide a teleost model for diabetes and/or metabolic syndrome without injection procedures and stress inducing anesthetic treatment.

It would be a matter of routine for a skilled person to modify the glucose or glucose analog concentration and duration of exposure to achieve an insulin resistant model.

Potential drug

The methods for screening a test compound for insulin resistance modulating activity and methods of identifying a compound for treating diabetes and/or metabolic syndrome in accordance with the invention were tested using the positive control metformin. Furthermore, said methods were further tested on a potential drug NSC87877.

The formula for NSC87877 (8-Hydroxy-7-[(6-sulfo-2-napthyl)azo]-5-quinolinesulfonic acid) is represented below:

NSC87877 is known as an inhibitor of shp2 and shp1 protein tyrosine phosphatases. The present inventors postulated that it may have utility in the treatment or prevention of metabolic syndrome or type 2 diabetes.

The present inventors have now surprisingly found that this compound reduces the insulin resistance in a leptin B deficient teleost model and has utility in the treatment or prevention of diabetes and/or metabolic syndrome.

Accordingly, the present invention provides a method of treating or preventing diabetes and/or metabolic syndrome in a subject in need thereof, comprising administering a therapeutically effective amount of NSC87877 to said subject.

The present invention further provides a therapeutically effective amount of NSC87877 for use in treating or preventing diabetes and/or metabolic syndrome in a subject in need thereof.

The present invention also provides the use of a therapeutically effective amount of NSC87877 in the manufacture of a medicament for treating or preventing diabetes and/or metabolic syndrome in a subject in need thereof.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1 94); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

Aspects of the invention are demonstrated by the following non-limiting examples.

EXAMPLES

MATERIALS AND METHODS

Zebrafish husbandry

Zebrafish lines were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (zfin.org). The breeding of adult fish was approved by the local animal welfare committee (DEC) of the University of Leiden (license number: 10612) and adhered to the international guidelines specified by the EU Animal Protection

Directive 2010/63/EU. Adult zebrafish were not sacrificed for this study. All experiments in this study were performed on embryos/larvae before the free-feeding stage and did not fall under animal experimentation law according to the EU Animal Protection Directive 2010/63/EU.

Fish lines used in this work were the following: wild-type (wt) strain AB/TL, homozygous mutant (lepb—/—) and wt siblings (lepb+/+). Homozygous F1 carriers were outcrossed once against wt, and were subsequently incrossed, resulting in lepb—/— and lepb+/+ siblings that were used for experiments. For genotyping, genomic DNA was amplified using forward primer 5-GAGACTCTCCTGAGGACACTGG-3' (SEQ ID NO:1) and reverse primer 5-GCATGGCTTACACATTTCAGAG-3' (SEQ ID NO:2), amplifying a 201 base pair (bp) product containing the mutation, which can be detected using 2% agarose gel. Embryos were grown at 28.5°C in egg water (60 pg/ml sea salt, Sera marin, Heinsberg, Germany). For live-imaging or injection assays, larvae were anesthetized in egg water medium containing 0.02% buffered Tricaine (3-aminobenzoic acid ethyl ester; Sigma-Aldrich, St Louis, MO, USA).

Insulin injection

To inject PBS and human recombinant insulin (Sigma-Aldrich, the Netherlands), 1 nl was injected into the caudal aorta of 4 dpf zebrafish larvae using a glass capillary as described in Juez et al., 2014.

Glucose treatment

Zebrafish larvae at 4 dpf were placed in 12 well plates (10 embryo per well) and immersed for two hours in 4 mL egg water, containing 250 mM of glucose (Sigma, USA, CAS. No. 50-99-7). After immersion first group was washed three times with egg water and collected for measurements, the rest were exposed to clean egg medium. Samples were taken after 120 min and after 240 min of washing period. As a control, larvae were exposed to mannitol (250 mM; Sigma, USA, CAS No. 69-65-8), instead of glucose, under the same conditions.

In Vivo Glucose Uptake Assay

Controls and lepb mutants were injected at 8 hpf and at 24 hpf in the yolk with 2.5 mg/mL 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent glucose analog (Life Technologies), and incubated at 28.5 °C for 60 minutes. At the termination of the incubation period, seven embryos per condition from 1 day old group were anesthetized with 3-aminobenzoic acid ethyl ester methanesulfonate (Sigma-Aldrich), both groups were analyzed under a fluorescence stereomicroscope and confocal microscope.

Metformin treatment

Wild types and lepb mutants, used for the ELISA assays, were treated with 10uM Metformin Cayman Chemicals, USA), added to egg water contained DMSO from 3dpf for 24 hours, as the control group, larvae were incubated only in water with DMSO. Embryos used for the fluorescent glucose assay received 10uM/nL Metformin at 2 hpf under the egg chorion and the second dose together with fluorescent glucose injection at 24 hpf. NSC87877 treatment

Wilde types and lepb mutants, used for the ELISA assays, were treated with 10μΜ NSC87877 added to egg water contained DMSO from 3dpf for 24 hours, as the control group, larvae were incubated only in water with DMSO. Embryos used for the fluorescent glucose assay received 10pM/nL NSC87877 at 2 hpf under the egg chorion and the second dose together with fluorescent glucose injection into the yolk at 24 hpf and 8 hpf.

Glucose measurements

Quantitative analysis of glucose levels was performed from whole body lysates using a glucose assay kit (Cayman Chemical,USA). Briefly, 7 zebrafish larvae in each experimental group were sonicated in 30 pL Assay Buffer on ice. According to the instructions, standard curves were generated using glucose standard solution. A total of 25 pL assay Enzyme Mix (Cayman) was added and incubated for 10min at 37°C. Fluorescence (514 nm) was measured using a BioTek plate reader equipped with GEN 5 software (v.2.04, BioTek, Winooski, VT, USA).

Morpholino injections

For knockdown of ptpn6, a morpholino oligonucleotide (Gene Tools, LLC, Philomath, OR, USA) targeting ptpn6 was injected into 1-cell zebrafish embryo. The morpholino (5'-ACTCATTCCTTACCCGATGCGGAGC-3' (SEQ ID NO:3)) was diluted to a concentration of 0.08mM in 1x Danieau's buffer (58mM NaCI, 0.7mM KCI, 0.4mM MgSO4, 0.6mM Ca(NO3)2, and 5.0mM HEPES (pH 7.6)) and 1 nl was injected using a Femtojet injector (Eppendorf, Hamburg, Germany). Specificity of the morpholino was confirmed previously by Juez et al., 2014.

Imaging

Bright-field images were obtained using a Leica M165C stereomicroscope equipped with a DFC420C digital color camera (Leica Microsystems, Wetzlar, Germany). For fluorescent image acquisition, a Leica MZ16FA stereo fluorescence microscope equipped with a DFC420C digital color camera (Leica Microsystems) was used together with a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems).

Statistical analyses

Statistical differences were analysed with Prism 6.0 (GraphPad Software, San Diego, CA, USA) using t-test for comparisons between two groups and one-way ANOVA (with Tukey's post hoc test correction) for multiple group comparisons and considered to be significant at P<0.05.

RESULTS

General characterization of leptin b mutant zebrafish larvae

The CRISPR/cas9 gene editing tool was used to generate a lepb knock out zebrafish mutant line. A single-guide RNA (sgRNA) was designed to exon 2 of lepb, where the target site was located (Fig. 1A). Adult F0 fish from sgRNA injections were incrossed to obtain F1 generation, where germline transmission of mutant alleles was confirmed by genotyping of its offspring. After outcrossing with the wild type line, and two incrosses, the inventors generated a knock out mutant line which was used for this research. Two groups of lepb -/- and lepb +/+ larvae from the third generation were compared under normal embryo raising conditions to test for differences in unchallenged survival during development (Fig. 1B). Further development of lepb -/- was normal, with the larvae reaching adulthood in a normal time span, leading to adults with a normal fertility rate at the expected time period (data not shown). The inventors found that there was no obvious difference of the body size between mutant and wild type adults.

Lack of leptin b expression causes of insulin resistance

To study how the lepb zebrafish mutant responds to hyperinsulinemia, the inventors injected insulin into the caudal aorta at 4 days post fertilisation (dpf). A glucose measurement was performed at 0, 30, and 240 minutes after the injection (Fig. 2A). The results (Fig. 3A) show a significance downregulation of glucose level after insulin injection in wild type fish, whereas no effect of the injection was noticed in the mutant line. In contrast, glucose level increases after insulin administration. Moreover, glucose basal levels at the first time point were much higher than in the wild type controls. These results indicate that the lepB mutant is insulin resistant even prior to 4 days post fertilization.

The inventors used the diabetic phenotype of the lepb mutant to test a new method of rapidly testing glucose metabolism, without injection procedures and stress inducing anaesthetic treatment. In this method, 4 dpf zebrafish larvae were immersed in egg water containing 250mM glucose concentration for two hours. Afterwards the larvae were incubated for 4 hours in glucose free medium. The non-metabolisable compound mannitol was used as a control for osmotic effects. Samples were taken at 0, 120 and 240 minutes after washing by immediate homogenization of whole larvae in buffer (Fig. 2B). The results of the glucose measurement show that in the control group free glucose concentrations reach the basal level after 240 minutes post washing. In contrast, glucose levels remain at very high levels in the lepB mutant after the washing step. These results show that the rapid glucose bathing method is highly efficient in demonstrating the diabetic characteristics offish larvae at 4 days post fertilization (Fig. 3B). Moreover, the inventors confirmed that a longer exposure to a high concentration of glucose results in hyperglycemia and insulin resistance. To achieve this, zebrafish larvae after the washing steps were immersed again in the glucose containing medium. The samples taken after 120 and 240 min showed constant increasing of free glucose levels (Fig. 3C).

In order to further study glucose metabolism in the lepB larvae the inventors utilised a previously published method, based on the injection of 2-NBDG, a fluorescently labelled glucose analog, into the yolk of embryos 24 hours post fertilization (Marin Juez et al., 2015) (Fig. 2C). In agreement with this publication, the inventors observed that in wild type larvae the fluorescent glucose is rapidly transported into the tissues of the embryo, with the brain as the most prominent destination (Fig. 3D). In contrast, in lepb mutants there is no observable glucose uptake from injected yolk, where all the injected glucose remains. In conclusion, glucose transport in the lepB mutant is completely blocked already at 24 hours post fertilization.

Drug and morpholino treatments of the lepB mutant

The inventors have used these methods for monitoring glucose uptake to test the effect of the antidiabetic drug metformin. Metformin was added to control and lepB mutant fish at 3 dpf, and 24 hours later the glucose bathing assay was performed (Fig. 2D). The results show that Metformin at a concentration of 10 μΜ was highly effective in reverting the lepB diabetic phenotype to the wild type phenotype (Fig. 4A). However, using the fluorescent glucose injection method the inventors observed only a marginal effect of Metformin at 24 hours post fertilisation (hpf) (Fig. 4B, and Fig, 3E).

The inventors also tested other putative anti-diabetic drugs. Based on previous results, of the function of phosphatases in insulin resistance, the inventors tested NSC87877, a compound known to target non-receptor tyrosine phosphatases. Previous results demonstrated that zebrafish larvae can become insulin resistant when treated with a high dose of insulin (Marin-Juez et al., 2015). In WT fish, NSC87877 reversed the diabetic phenotype, induced by an administration of high dose of Human Recombinant Insulin. After the second injection, performed after 240 min, zebrafish WT larvae develop insulin resistance, hyper insulinemia and hyperglycemia, whereas NSC87877 treated larvae reaches their physiological glucose level after 30 min post the second injection (Fig. 4C). NSC878777 was also able to completely revert the diabetic phenotype of the lepB mutant a 4 dpf (Fig. 4D). Interestingly, NSC87877 was also able to significantly revert the glucose uptake capacity at 24 hpf in the fluorescent glucose assay (Fig. 4E and Fig. 4F).

Figure 5A shows, using the fluorescent glucose injection method, the results of Metformin treatment (representative pictures): the inventors did not observe the same Metformin effect at the earlier stages of the zebrafish development. The LepB mutant remains diabetic after Metformin treatment at early stages of development. Fig. 5B shows, using the fluorescent glucose injection method, the results of Metformin (representative pictures): the inventors did not observe the same effect at the earlier stages of zebrafish development. LepB mutant remains diabetic after Metformin treatment.

Challenged by these results, the inventors also tested the effect of NSC87877 at even earlier embryonic stages (Fig. 5C). Surprisingly, even at 8 hpf the leptin mutant already showed a glucose transport phenotype that was rescued by the treatment of NSC87877 for only two hours prior to the fluorescent measurement. Stereo fluorescence microscopy quantification was confirmed by confocal laser scanning microscopy imaging (Fig. 5D).

In order to get an indication on the target of NSC87877 responsible for rescuing the lepB phenotype, the inventors tested morpholino’s against the SHP-1 gene. This gene was a likely target considering the previous results showing its function in regulating insulin resistance in a wild larval test system. Using the fluorescent glucose assay, the inventors could demonstrate that knock down of the SHP-1 gene completely reverted the lepB mutant to the wild type phenotype (Fig. 6A and 6B). In contrast, knockdown of other putative targets of NSC87877, SHP-2a and SHP-2b, did not revert the lepB phenotype. This indicates that SHP-1 is the likely target of NSC87877 responsible for the reversion of the lepB phenotype. A high throughput robotic method fortesting anti-diabetic drugs.

On basis of the results for NSC87877 as an antidiabetic drug for 24 hpf embryos, the inventors designed a robotic assay that automates the procedure of testing uptake of fluorescent glucose. In this method the inventors robotically inject the antidiabetic compounds through the chorion of 4 hpf embryos. At 24 hpf ,the inventors dechorionated the embryos enzymatically and robotically injected fluorescent glucose. Subsequently, embryos were analysed using the published previously vertebrate automated screening technology (VAST) (Fig. 7). The fluorescence of the head regions of a large number of larvae was quantified using automated image analysis. The results replicated the results of the manual method showing the effect of NSC87877, but with better statistical P values.

CONCLUSIONS

The lepB mutant zebrafish line is diabetic at early stages of embryonic development. This diabetic phenotype is characterized by insulin resistance, and a block of glucose uptake at the systemic level along with particular organs such as the brain.

The diabetic phenotype can be reverted by 24 hours of treatment with Metformin at 3 days post fertilization. However, this does not work at earlier stages of development.

The phosphatase inhibitor NSC87877 can also revert the diabetic phenotype of the lepB mutant at 3 dPF, at much earlier stages of development then metformin.

Based on the positive treatment results, the inventors have designed a high throughput method for testing antidiabetic drugs.

DISCUSSION

Although DUSP26 has also been indicated as a target of NSC87877 (Song et al, BBRC 2009), the results presented herein indicate that the most likely target of NSC87877, resulting in an underlying anti-diabetic effect, is SHP-1

The early glucose transport phenotype of the lepB mutant indicates a function of insulin receptors at the early stages of embryogenesis. Indeed, one of the zebrafish insulin receptors (insrb) was reported to be expressed already at 18 somite stage, and both insulin receptors were maternally expressed in fertilized eggs (Toyoshima et at., Endocrinology 2008). In addition, two insulin genes have been described to be expressed during early zebrafish development. Of these two genes, insb was shown to be expressed at proliferating blastomeres at 3 and 4 hpf (Papasani etal., 2006).

Metformin did not work well at earlier larval stages using fluorescent glucose uptake studies. Several possible explanations include there being no uptake of Metformin through the skin (the mouth only opens later), or the effect observed at 4 dpf is entirely through gluconeogenesis. Indeed, this is the effect that would be predicted from rodent studies.

Considering that the signalling pathway of insulin is highly conserved within vertebrates, the model is highly useful as a model for anitdiabetic drugs. Glucose transporters are also conserved, although a homolog of glut4 has not been found in zebrafish.

The positive effect of Metformin shows that translation of mammalian test systems can translated to the zebrafish larval system. However, the zebrafish larval system is much simpler since it is not complicated by feeding. A feeding system is not only expensive but also can lead to differences between test systems and therefore variation of results.

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Claims (44)

A method for screening a test compound for insulin resistance modulating activity, the method comprising: a. Contacting the test compound with a leptin B deficient or a leptin receptor deficient teleost; and B. determining the effect of the test compound on basal glucose levels in the teleost or on glucose uptake in the teleost. The method of claim 1, wherein the teleost is an embryo or a larva. The method of claim 1 or claim 2, wherein the teleost is a zebrafish. A method according to any one of claims 1 to 3, wherein the contacting step is performed when the teleost is at a stage of less than three days after fertilization (dpf). A method according to any one of the preceding claims, wherein the contacting step comprises injecting the teleost with, or immersing the teleost in a solution comprising the test compound. The method of claim 5, wherein the contacting step comprises injecting the test compound through the teleost's egg web. A method according to any one of the following claims, wherein the determining step comprises contacting the teleost with a glucose solution, and measuring the glucose uptake in the teleost, wherein an increase in glucose uptake relative to a control, or when compared to a predetermined level, indicates that the test compound has the ability to reduce insulin resistance. The method of claim 7, wherein the teleost is stripped of the egg membrane and immersed in the glucose solution. The method of any one of claims 1 to 6, wherein the determining step comprises contacting the teleost with an insulin solution, and then measuring the effect on basal glucose levels in the teleost, wherein a decrease in the basal glucose level over time compared to the control or compared to a predetermined level indicates that the test compound has the ability to reduce insulin resistance. The method of any one of the preceding claims, wherein the glucose is labeled. The method of claim 10, wherein the glucose is fluorescently labeled. The method of any one of the following claims, wherein the method is an automated method. A method of identifying a compound for treating diabetes and / or metabolic syndrome, the method comprising determining the effect of a compound on insulin resistance, in accordance with any of claims 1 to 12, and the selecting a compound that possesses the ability to reduce insulin resistance. The method of claim 13, wherein the method is an automated method. A method according to any one of claims 12 to 14, wherein the connection by means of a robot is injected through the egg membrane of the teleost. A method according to any of claims 12 to 15, wherein the contacting step is performed when the teleost is at a stage of less than 22 hours after fertilization (hpf). The method of any one of claims 12 to 16, wherein the glucose is labeled, and wherein the teleost is analyzed by using an automated vertebrate screening technology. A method according to any one of claims 12 to 17, wherein the method is suitable for realizing a high processing capacity. A method of identifying a target of a medicament for the treatment of diabetes and / or metabolic syndrome, the method comprising: a. Modifying the gene or protein expression of a potential target in a leptin-B -deficient or a leptin-receptor deficient teleost; b. bringing the teleost into contact with the medicine; and c. determining the effect of the drug on a basal glucose level in the teleost or on glucose uptake in the teleost. The method of claim 19, wherein step (a) comprises the use of a morpholine for the potential purpose. A method according to claim 19 or claim 20, wherein the determining step comprises contacting the teleost with a glucose solution, and measuring the glucose uptake in the teleost, wherein a decrease in glucose uptake compared to a control or compared to a predetermined level, indicates that the potential goal is a goal for the drug. The method of claim 19 or claim 20, wherein the determining step comprises contacting the teleost with insulin solution, and then measuring the effect on the basal glucose level in the teleost, wherein an increase in the glucose level in the over time or in comparison with a control or with a predetermined level, indicates the potential target as a target for the drug. A method for optimizing the formulation of a medicament for the treatment of diabetes and / or metabolic syndrome, the method comprising: a. Contacting a first formulation of the medicament with a leptin B deficient or a leptin receptor deficient teleost; b. determining the effect of the first formulation on a basal glucose level in the teleost or on the glucose uptake in the teleost; c. contacting a second formulation of the drug with a second leptin B deficient or leptin receptor deficient teleost; d. determining the effect of the second formulation on a basal glucose level in the teleost or on the glucose uptake in the teleost; and e. comparing the basal glucose levels or glucose readings as determined in steps (b) and (d), and selecting the formulation that gives rise to a higher glucose uptake or to a lower basal glucose level. The method of claim 23, wherein one or more of the following parameters are changed in the second formulation relative to the first formulation: the dosage of the drug, the release rate, buffers. The method of any one of claims 19 to 24, wherein the teleost is an embryo or a larva. The method of any one of claims 19 to 25, wherein the teleost is a zebrafish. The method of any one of claims 19 to 26, wherein the step (s) of contacting is or are performed when the teleost is at a stage of less than 3 dpf. The method of any one of claims 19 to 27, wherein the contacting step (s) comprises injecting or comprising the teleost with, or immersing the teleost in a solution comprising the medicament. A method according to claim 28, wherein the contacting step (s) comprises injecting or comprising the formulation through the eggplant of the teleost. The method of any one of claims 23 to 29, wherein the steps of determining comprises contacting the teleost with a glucose solution, and measuring the glucose uptake in the teleost. A method according to claim 21 or claim 30, wherein the teleost is stripped of the egg membrane and immersed in a glucose solution. The method of any one of claims 23 to 31, wherein the determining steps include contacting the teleost with an insulin solution, and then measuring the effect on the basal glucose level in the teleost. The method of any one of claims 19 to 32, wherein the glucose is labeled. The method of claim 33, wherein the glucose is fluorescently labeled. The method of any one of claims 19 to 34, wherein the method is an automated method. A method as claimed in any one of claims 19 to 35, wherein the medicament is injected through the teleost of the teleost using a robot. The method of any one of claims 19 to 36, wherein the contacting step is performed when the teleost is at a stage of less than 22 hours after fertilization (hpf). The method of any one of claims 19 to 37, wherein the glucose is labeled, and wherein the teleost is analyzed by using an automated vertebrate screening technology. A method according to any one of claims 19 to 38, wherein the method is suitable for realizing a high processing capacity. 40. Use of a leptin B deficient or a leptin receptor deficient embryo or larva of a teleost as a model for diabetes or for metabolic syndrome. 41. Use of a leptin B deficient or leptin receptor deficient teleost to: a. Test test compounds for their ability to modulate insulin resistance, b. identify a compound for treating diabetes and / or metabolic syndrome; c. identifying a target of a medicament for the treatment of diabetes and / or metabolic syndrome; d. optimize a drug formulation or drug regimen for the treatment of diabetes and / or metabolic syndrome; and / or e. identify the location (s) of drug activity in the teleost. The use of claim 41, wherein the teleost is an embryo or a larva. The use of any one of claims 40 to 42, wherein the teleost is a zebrafish. The use of any one of claims one and 40 to 43, wherein the teleost is at a stage of less than 3 dpf.