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 (Heydemann, 2016; Reed & Scribner 1990). Leptin has been extensively studied in obesity and TIID and has been shown to play a key role in whole-body energy homeostasis, however, its function in insulin signaling is still poorly understood. Leptin is a cytokine produced mainly by mature adipocytes in white adipose tissue. In the brain it regulates food intake, appetite behaviors and energy expenditure. Leptin mutations in rodents lead to hyper obesity and other abnormalities, which are described as main factors that influence development of diabetic phenotype symptoms (Wang et al., 2014). The molecular mechanisms by which leptin controls insulin resistance in various target tissues are largely unknown (Zhou and Rui, 2013). Leptin's function is correlated with protein tyrosine phosphatases that are key regulatory factors in many signal transduction pathways underlying vertebrate development (Ullrich and Schlessinger, 1990). Protein tyrosine phosphatase 1 B (PTP1 B) has emerged as a novel promising therapeutic target for the treatment of type 2 diabetes, as it plays an important role in the negative regulation of insulin signal transduction pathways (Tamrakar et al., 2014). Moreover, the expression of hypothalamic PTP1 B is up regulated in leptin resistant animals (White et al., 2009). It was recently shown that inhibition of low-molecular-weight tyrosine phosphatase (LMPTP) in rodents results in attenuation of high-fat diet-induced diabetes (Stanford et al., 2017). In adult zebrafish the leptin receptor and leptin genes have a conserved role in glucose homeostasis but, do not appear to play a role in adipose tissue homeostasis (Michel et al, 2016). Zebrafish models have been proposed as alternative test systems for studying insulin resistance and TIID that give several opportunities to explore metabolic diseases, using numerous transgenic and knockout lines (Gut et al., 2017). Although there are already established diabetic adult zebrafish models, which are based on a high fat feeding system (Michel et al., 2016; Zang et al., 2017), there is still a lack of alternative early stage larval models, which provide the opportunity to perform fast and large-scale screening assays, shortly after fertilization. As shown by Marin Juez et al. (2014) zebrafish larvae are highly suited to study insulin resistance and are therefore a promising model system to study TIID in a non-feeding situation. Marin Juez ef al., identified Shp1 , also called ptpn6, 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.

In a further aspect the 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.

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

In a further aspect the invention provides 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. In a further aspect the invention provides use of a leptin B deficient or a leptin receptor deficient non-human mammalian embryo as a model of diabetes or metabolic syndrome.

In a further aspect the invention provides a use of a leptin B deficient or a leptin receptor deficient non-human mammalian to:

c. screen test compounds for their ability to modulate insulin resistance;

d. identify a compound for treating diabetes and/or metabolic syndrome;

e. identify a target of a drug for treatment of diabetes and/or metabolic syndrome; f. optimise a drug formulation or regimen for the treatment of diabetes and/or metabolic syndrome; and/or

g. identify the location(s) of drug activity in the embryo. Suitably, the non-human mammalian embryo is a mouse embryo.

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 1 B 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.

Figure 8 shows a schematic overview of methods used for measuring glucose transport, (a) Method 1 - insulin injection: Zebrafish larvae at 4 dpf receive 1 nl_ of human recombinant insulin or PBS, into the caudal vein. The samples are collected at 0, 30 and 240 min post injections, to measure free insulin glucose level in the body, using ELISA Glucose Assay Kit. (b) Method 2 - glucose immersion: Zebrafish larvae at 4dpf are immersed in a medium containing 250 mM 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 ELISA Glucose Assay Kit. It is an alternative and less invasive method of testing insulin resistance in zebrafish larvae, (c) Method 3 - fluorescent glucose injection at 24 hpf: Zebrafish embryos at 24 hpf are injected into the yolk with fluorescent labelled glucose. One hour after injections, accumulation of glucose in the brain of WT and mutant zebrafish larvae can be observed with stereo fluorescence microscopy and has been quantified for the brain area using a custom written script (d) Method 4 - fluorescent glucose injection at 4 or 8 hpf: Drugs have been injected into the yolk of zebrafish embryos at early stages of embryogenesis (2 hpf). In the case of the 8 hpf analyses the inventors made use of CLSM to distinguish the zygotic cells from the yolk. At 4 hpf stereo fluorescence microscopy was sufficient to discern the yolk from the zygotic cell mass.

Figure 9 shows the insulin sensitivity and glucose levels of lepb mutant larvae at 4 dpf. (a) Results of human recombinant insulin (INS) injection using method 1 (Fig. 8) (b) Glucose levels determined after immersion in glucose using method 2 (Fig. 8) (c) The effect of metformin on glucose levels using method 2 (Fig. 8) (d) The effect of NSC87877 on glucose levels using method 2 (Fig. 8). Data (mean ±S.EM.) is combined from five biological replicates (n=10 larvae/group), significance was measure between lepb+/+ and lepb-/- groups unless indicated differently. *P<0.05, **P<0.01 , ***P<0.001. Figure 10 shows quantification of glucose distribution at different stage embryos. Images were obtained by stereo fluorescence microscopy unless indicated otherwise, (panel a, c) Injection of 2-NBDG in 24 hours embryos according to method 3 (Fig. 8c). (Panel b and d) Early stages (8 hpf) 2-NBDG injections according to method 4 (Fig. 8d). NSC87877 was injected through the chorion or into the yolk sac at 2 hpf. Use was made of CLSM to discern the yolk form the zygotic cell mass. (Panel e and f) Early stages (4 hpf) 2-NBDG injections according to method 4 (Fig. 8d). NSC87877 or recombinant human leptin protein (HRL) was injected into the yolk sac at 2 hpf. Data (mean ±S.EM.) is combined from three biological replicates (n=10 larvae/group). *P<0.05, **P<0.01 , ***P<0.001.

Figure 11 shows the effect of injection of human recombinant insulin into the yolk of 4 hpf zebrafish embryos. Injections with 1 nl_ of glucose (200mg/ml) in the presence of 2-NBDG results in accumulation of fluorescence in the yolk of 4 hpf embryos. 100 nM human recombinant insulin (INS) was co-injected with the 2-NBDG. Data (mean ±S.EM.) is combined from three biological replicates (n=10 larvae/group). *P<0.05, **P<0.01 , ***P<0.001. Figure 12 shows gene knockdown studies in zebrafish and Xenopus laevis embryos, (panel a and d) Knockdown of gene expression with morpholino's against lepb, lepa and lepr and imaging of glucose distribution at 5 hpf using method 4 (Fig. 8d). (panel e and f). (Panel e and f) Rescue of the lepb glucose transport deficiency by ptpn6 morpholino at 48 hpf. Glucose distribution was measured by injection of fluorescent glucose using method 3 (Fig. 8). Morpholino knock down of both the leptin S and leptin L genes of Xenopus laevis embryos. 2- NBDG was injected at 48 hpf distribution was quantified using a custom script. Data (mean ±S.EM.) is combined from three biological replicates (n=10 larvae/group). *P<0.05, **P<0.01 , ***P<0.001. Figure 13 shows (a) Method 3 (yolk injections glucose): In WT larvae fluorescent glucose diffuse via yolk barrier and accumulates in the brain, whereas in the lepb larvae glucose remains in the yolk. The inventors observe only a marginal effect of metformin in the lepb larvae at 24 hpf. (b) Method 3: Effect of metformin (Quantification). After quantification of the fluorescent signal in the brain, there is no significant difference between lepb and metformin treated embryos. Cell to yolk fluorescent ratio, based on the confocal pictures, shows the difference in glucose uptake from the yolk between the three groups.

Figure 14 shows that at the earlier stages glucose transport was not leptin dependent in the lepb mutation.

Figure 15 shows in (a) and (b) that it is possible to rescue lepb morpholino phenotype with injections of human recombinant leptin, but not in leptin receptor knockdowns.

Figure 16 shows that the knockdown of Shp-2a and Shp-2b did not restore glucose transport in the lepb mutant. 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 ef 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 PTPN 11 ) is ubiquitously expressed, and is also expressed in the cells that express SHP-1 (Feng ef 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 1 B (PTP1 B) 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 ef al. 2014). Furthermore, expression of hypothalamic PTP1 B is up regulated in leptin resistant animals (White ef 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 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 a/., 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 41 1 : 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) or a 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 1 μΜ 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 a/., 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 et a/., 2012 and Yu et al., 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 et al. (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 et al., 2012 and Yu et al., 2017). It is noted that there are also various commercial blood glucose measurement devices available for testing 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 et al., 2014; Guo et al., 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 et al 2014 and Guo et al 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 0.1 mM. 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(N0 ₃ ) 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 I

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.

Further Model II

Insulin signalling in vertebrates is highly conserved and therefore the inventors' findings in zebrafish and Xenopus are translatable to non-human mammalian organisms. Based on the inventors' test system in early embryogenesis using fluorescent glucose analogs the inventors also devised a test system in early mice embryos.

Accordingly, in another aspect, the present invention provides an insulin resistant non-human mammalian embryo 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 non-human mammalian embryo model may be insulin resistant through sustained exposure to glucose.

As used herein, the terms "mammal" and "mammalian" refer to any vertebrate animal, including monotremes, marsupials and placental, that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals). Examples of non-human mammalian species include primates (e.g., monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs) and ruminents (e.g., cows, pigs, horses). Suitably, the non-human mammalian embryo is a rodent embryo, more suitably a mouse embryo.

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 shpl 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 at 4 hpf and at 24 hpf were injected 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 30-60 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 analysed under a fluorescence stereomicroscope and a confocal microscope.

Metformin treatment

Wild types and lepb mutants, used for the ELISA assays, were treated with 10μΜ 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 10μΜ 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 Ι ΟμΜ/nL NSC87877 at 2 hpf under the egg chorion and the second dose together with fluorescent glucose injection into the yolk at 24 hpf, 4hpf 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 particular genes (ie. ptpn6), morpholino oligonucleotides (Gene Tools, LLC, Philomath, OR, USA) were injected into 1 -cell zebrafish embryo. The morpholinos (see Table 1 for sequences) was diluted to a concentration of 0.08 mM in 1 * Danieau's buffer (58 mM NaCI, 0.7mM KCI, 0.4mM MgS04, 0.6mM Ca(N03)2, and 5.0mM HEPES (pH 7.6)) and 1 nL was injected using a Femtojet injector (Eppendorf, Hamburg, Germany) (see Figure 15). Specificity of the ptpn6 morpholino was confirmed previously by Juez et al., 2014.

Table 1 : Sequence of morpholinos for particular genes probed in this study.

Frog husbandry and microinjection

All frog (Xenopus laevis) procedures and care were approved by the animal experiments committee (dierexperimentencommissie, DEC) of Leiden university. Frog embryos were collected by natural mating of wild-type females with males. For morpholino injection, morpholinos against the long-form (MO: ) and the short-form (MO:) transcripts of leptin were mixed together. 30ng of each was then injected into the blastomeres of the embryo at 2-cell or 4-cell stage. After injection, the embryos were cultured to stage 26 (staged according to Nieuwkoop and Faber) for glucose injection. For rescue experiments, 10μΜ NSC87877 was co-injected with the two morpholinos at 2-cell or 4-cell stage. Images quantification

Bright-field images were obtained using a Leica M165C stereomicroscope equipped with a DFC420C digital colour camera (Leica Microsystems, Wetzlar, Germany). For fluorescent image acquisition, a Leica MZ16FA stereo fluorescence microscope equipped with a DFC420C digital colour camera (Leica Microsystems) was used with GFP filter settings or together with a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems). For confocal laser scanning microscopy (CLSM) the inventors used a Leica TCS SPE (Leica Microsystems). For each larva a bright field image and a fluorescent channel image were obtained. In 24 hours post fertilization analyses of the glucose values first the overall shape of the larva was extracted from the bright field channel - this gives the total area. The yolk and yolk-extension are extracted from the fluorescent channel as these have the highest fluorescence, from this the surface areas for yolk and yolk-extension are established. The body of the larva can be found by excluding the area of yolk and yolk-extension and within the body the otic vesicle is taken as a boundary for the head. In this manner the body is divided in areas for each of which the surface area is computed, and which sum to the total surface. The area of the head is determined from the bright field image and is used as a mask in the fluorescent channel image to obtain the fluorescence for only the head area. This is expressed as a numerical density, that is, the sum of the total fluorescence in the head area divided by the surface area of the head. The images and computations are corrected for aspecific background fluorescence.

To estimate the fluorescence ratio between zygotic cell mass and yolk in early zebrafish embryos using wide field stereo microscopy, a square of 4 micrometer in center of these two parts of the embryos was quantified for fluorescence intensity. Ratio between the two blocks was measured using pixel counting software as described in Stoop et al. (201 1). In Xenopus laevis embryos the inventors compared the total fluorescence in the larvae to the fluorescence at the injection site that was arbitrarily defined as a square of 10 micrometer. To measure the fluorescence ratio between cell mass and yolk in early zebrafish embryos with CLSM the inventors measured the total volume of these parts of the embryo and quantified the fluorescence in these parts using FIJI software.

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. EXAMPLE 1

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. 1 B). 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 of fish 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 ef a/., 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 a/., 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 for testing 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 al., 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 et al., 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.

EXAMPLE 2

General characterization of leptin b mutant zebrafish larvae

The zebrafish genome contains two leptin genes, lepa and lepb, and one leptin receptor gene (Gorissen et a/., 2009) which have been previously studied by gene knock down and knock out studies (Liu et al 2012, and Michel et al, 2016). Since pilot morpholino studies indicated a possible function of lepb in glucose transport of zebrafish larvae ( Fig. 1) the CRISPR/CAS9 gene editing tool was used to generate a lepb knock out zebrafish mutant line. The sgRNA was designed to target exon 2 of the lepb gene were the target site was located ( Fig. 1A). Adult F0 fish from sgRNA injections were incrossed to obtain the 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 selected knock out mutant lines which were used for this research. Two groups of lepb ^1' and lepb ^+/+ larvae from the third generation were compared under normal embryo raising conditions to test for differences in unchallenged survival during development (Fig. 1 B). Further development of lepb ^'1' 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).

Mutation of the lepb gene causes insulin resistance in the larval stage

To study how the lepb ^1' zebrafish mutant responds to hyperinsulinemia, the inventors injected insulin into the caudal aorta of a zebrafish larvae at 4 dpf. Glucose measurements were performed at 0, 30, and 240 minutes after the injection (Fig. 8A). The results (Fig. 9A) show a significant decrease in glucose level after insulin injection in wild type fish, whereas the glucose level rather increases after insulin administration in the mutant. Moreover, glucose basal levels at the first time point were much higher than in the wild type controls. These results indicate that the lepb ^1' mutant is insulin resistant even prior to 4 days post fertilization.

The inventors used the diabetic phenotype of a lepb ^'1' mutant, to establish a method that corresponds to Oral Glucose Tolerance Test (OGTT) used in mice (Nagy and Einwallne 2018), to rapidly analyse glucose metabolism without injection procedures and stress-inducing anesthetic treatment. In this method, similar to the glucose tolerance test applied in mice, 4 dpf zebrafish larvae were immersed in egg water containing a 250 mM 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 the buffer (Fig. 8B). 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 to demonstrate the diabetic characteristics of fish larvae at 4 days post fertilization (Fig. 9B).

In order to further study glucose metabolism in the lepb ^'1' larvae the inventors used a previously published method based on the injection of 2-NBDG, a fluorescently labeled glucose analog in the yolk at 24 hours post fertilization embryos (Marin Juez et a/., 2015) (Fig.

8C). 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. 10A). In contrast, in lepb ^'1' mutants there is no observable glucose uptake from injected yolk, where all the injected glucose remains. In conclusion, glucose transport in the lepb ^'1' mutant is completely blocked already at 24 hours post fertilization (Fig. 10A and 10B).

Drug treatments of the lepb ^'1' mutant.

The inventors have used the developed methods described herein for measuring glucose uptake to test the effect of the antidiabetic drug metformin. Metformin was added to control and lepb ^'1' fish at 3 dpf and the glucose bathing assay was performed 24 hours later (Fig. 8D). 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. 9C). However, using the fluorescent glucose injection method the inventors observed only a marginal effect of metformin at 24 hpf ( Fig. 13). The inventors also tested other putative anti-diabetic drugs (manuscript in preparation). One of the compounds, NSC87877 that targets non-receptor tyrosine phosphatases, was selected based on previous results on the function of phosphatases in insulin resistance. NSC87877 showed to be able to completely revert the diabetic phenotype of the lepb ^'1' at 4 dpf (Fig. 9D). Interestingly, NSC87877 was also able to significantly revert the glucose uptake deficiency at 24 hpf in the fluorescent glucose assay (Fig. 10A and 10B). lepb controls glucose transport and insulin resistance during the early embryonic stages. The inventors then tested the function of the lepb gene during early embryonic stages. The inventors found that knockout of lepb completely inhibits glucose transport between yolk and the developing zygotic cells even at very early stages, namely after 4 and 8hpf. However, at earlier stages than 64 cell stage glucose transport was not influenced by the lepb mutation. Moreover, the inventors found NSC 87877, injected under the chorion or in the yolk (Fig. 8D), partially reverses glucose transport inhibition at 4 and 8 hpf (Fig. 10C, 10D. 10E and 10F). Interestingly, recombinant human leptin was also able to rescue glucose transport in the mutant. In order to test whether the defect in glucose transport in early embryogenesis was related to insulin resistance the inventors developed an assay for testing the effect of insulin at 4 hpf. This assay is based on the injection in the yolk of 1 nl_ glucose solution of 200 mg/ml in the yolk in the presence of the standard concentration of 2-NBDG. In wild type embryos transport of the fluorescence glucose derivative is no longer observed due to competition with unlabeled glucose. The apparent limitation of the glucose transport capacity at this glucose concentration could be overcome by the co-injection of human recombinant insulin showing the sensitivity of early embryos to insulin. In contrast glucose transport in the lepb ^'1' mutant was not significantly affected by injection of insulin, indicating insulin resistance (Fig. 1 1A and 1 1 B).

Gene knockdown studies for leptin signaling pathway analysis and translational studies in Xenopus laevis

The inventors have used morpholino anti sense technology to further study the signal transduction pathways underlying the identified function of leptin in early embryogenesis. Firstly, the inventors showed that the phenotype of knockdown of the lepb gene is glucose transport in early embryogenesis is also observed after injection of morpholino's against lepb (Fig. 12C and 12D). Subsequently the inventors also tested morpholino's against the leptin receptor (lepr) and the second leptin gene (/epa). The results (Fig. 12C and 12D) show that lepr phenocopies accurately the effect of the lepb morpholino treatment. In contrast, lepa showed no significant effect on glucose transport in early embryogenesis (Fig. 12C and 12D). The inventors were able to partially rescue the knockdown effect of lepb by injection of human recombinant leptin, but as expected not the lepr knockdown (Fig. 14). These results show that a /ep6-/eprsignaling pathway is functionally similar to the function of leptin in humans. In order to get an indication on the target of NSC87877 that is responsible for rescuing the lepb phenotype the inventors tested morpholino's against the most likely targets of this inhibitor (Chen et al, 2006; Song et al, 2009). Using the fluorescent glucose assay, the inventors could demonstrate that knock down of the ptpn6 gene completely reverted the lepb ^'1' mutant to the wild type phenotype (Fig. 12A and 12B). In contrast, knock down of other possible targets of NSC87877, the closely related phosphatases ptpn11a (Shp2a) and ptpn11b (Shp2b), or the dual specificity phosphatase DUSP26 did not restore glucose transport ( Fig. 16). This indicates that ptpn6 is the likely target of NSC87877 responsible for the reversion of the lepb ^' phenotype. The inventors used Xenopus laevis ,that has been used classically for embryogenic studies to show that the function of lepb in glucose transport is also relevant in embryos of other vertebrate organisms. Two morpholino's against the two leptin genes of X. laevis were designed and tested simultaneously as described in the material and methods. Leptin knockdown results in inhibition of glucose transport after 24hpf. Showing that lack of leptin expression in X.laevis leads to a similar glucose transport inhibition as in the zebrafish larvae. Moreover, morphant larvae show developmental abnormalities comparing to the control group. Importantly, injections with NSC87877 together with morpholino not only rescue the developmental phenotype, but also glucose transport in the morphants (Fig. 12). These results clearly show that leptin plays a crucial role in glucose transport during early embryogenesis also in X.laevis.

CONCLUSIONS

In this study, the inventors show that leptin deficient zebrafish mutant embryos and larvae appear to be totally insulin resistant and show a diabetic phenotype at all stages of embryogenesis and larval development. This phenotype can be reversed by the injection of human leptin. Both metformin and the phosphatase inhibitor NSC87877 are able to reverse this diabetic phenotype of the zebrafish larvae at the larval stage. In contrast to metformin, NSC87877 was also active at early embryonic stages. Gene knockdown studies in the leptin mutant background indicate that the results are translatable to Xenopus laevis embryos and without wishing to be bound to a particular theory, that the small non-receptor tyrosine phosphatase Ptpn6 is the most likely target responsible for the antidiabetic effect of NSC87877.

In this study the inventors analysed the function of leptin and ptpn6 in insulin resistance in zebrafish larvae. The inventors have developed a novel high throughput method to test antidiabetic drugs based on the fact that leptin deficient zebrafish larvae are totally insulin resistant and show as a result a diabetic phenotype already at very early stages of embryonic development. The inventors show that metformin is highly effective for treating this diabetic phenotype in 4 days old zebrafish larvae, and using their high throughput test system the inventors have identified also the phosphatase inhibitor NSC87877 is an alternative antidiabetic drug that shows anti-diabetic effects at much earlier time points of development. Gene knockdown studies in the leptin mutant background indicate that, without wishing to be bound to a particular theory, Shp-1 is the most likely target responsible for the antidiabetic effect of NSC8787.

DISCUSSION

The inventors show that lepb ^'1' mutant zebrafish line is diabetic during larval development. This diabetic phenotype is characterized by insulin resistance and subsequent inhibition of glucose uptake at both systemic level and peripheral organs such as the brain. The inventors also demonstrate that leptin b is essential for transport of glucose in the early stages of embryogenesis. Gene knockdown studies show that the leptin receptor is equally important to lepb but that lepa doesn't seem to play an important function. Knockdown of the lepb gene could be rescued by injection of human recombinant leptin even though this protein has only 18 percent of identity with the zebrafish leptin protein, showing the relevance of the results in the developed zebrafish test system for the function of mammalian leptin. In order to show that the findings observed are indeed relevant for the function of leptin in embryogenesis of other vertebrates models the inventors tested the function of leptin in Xenopus laevis which is one of the few other animal models in which embryos can be easily handled. The results show that the two X. laevis leptins have a function in glucose transport during embryogenesis. Since the X.laevis leptins are distantly related in sequence to the zebrafish leptins, this indicated that the function of leptin in glucose transport is translatable to all vertebrates. The essential function of leptin in glucose transport during embryogenesis is surprising since in rodent models leptin was thought to have a complex function in insulin resistance that involves systemic signaling via the blood stream (Wang et al, 2014), whereas at 4 hpf and organ system has not yet developed. This function of leptin b indicates a function of insulin receptors at the very early stages of embryogenesis that is confirmed by the effects of human recombinant insulin injected into the yolk sac. In previous work one of the zebrafish insulin receptors (Insrb) was reported to be expressed at 18 somite stage and both insulin receptors were maternally expressed in fertilized eggs (Toyoshima et al., 2008). In addition, two insulins 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 et al., 2006). However, there is no knowledge which glucose transporters could be involved in glucose transport during embryogenesis. Considering the fact that glucose transport up to 64 cells stage was not dependent on leptin shows that such transporters and their control by leptin develops after the syncytial stage of embryogenesis. The results described herein demonstrate that also in adults leptin is directly involved in glucose homeostasis, in line with the study of Michel et al, (2016) who reported a diabetic phenotype in lepr knockdown zebrafish. This supports the notion that the leptin signaling pathway can be used for therapeutic purposes as reviewed by Coppari and Bjorbaek (2012). Although leptin has mainly been reported to be produced by adipocytes, and few other tissues such as the intestinal epithelium mainly during inflammatory conditions (Azuma et al, 2001 ; Mackey-Lawrence and Petri 2012) there is also evidence that leptin is produced by human skeletal muscle in adults (Wolsk et al, 2012). In a recent publication by Kang et al. (2016) enhancer elements controlling lepb expression in zebrafish were shown to be triggered in injured tissues. Considering that these enhancer elements were also functional in mice tissue during wounding suggests a conserved function of leptin in wound repair. These and many other results indicate that in adults the function of leptin is much broader than the canonical adipocyte-brain axis (Faggioni et al, 2001 ; Fantuzzi et al, 2000; Lago et al, 2008; Poeggeler et al, 2009). It has also been shown that in human placenta there is an abundant production of the leptin protein (Zhao et al, 2004). Considering that leptin in mammalian cells can be transported by transcytosis to neighboring cells or tissues (Cammisotto et al, 2010; Tu et al, 2010), it is indicative that in mammalian embryogenesis placental leptin plays a role in glucose transport in the zygote. In analogy with adult mammalian diabetes studies, the diabetic phenotype of zebrafish larvae can be reverted by external treatment with metformin at 3 days post fertilization. However, such treatment with metformin was not effective at earlier stages of development. The phosphatase inhibitor NSC87877 can also revert the diabetic phenotype of the lepb ^'1' mutant at 3 dpf, and even at much earlier stages of development. The fact that metformin was not active at earlier larval stages using fluorescent glucose uptake studies can be explained in several ways. An explanation is that there is no uptake of metformin through the skin and the observed effect a 4 dpf is through oral uptake when the mouth of the larvae has opened possibly because it only functions via ingestion in the intestinal track as observed in mammals (Bailey et al. 1996). In this respect NSC87877 that is active after external treatment at 24 hpf, as it is taken up through the skin and therefore could have applications in non-oral dosing systems.

Based on the reversal of the lepb ^'1' mutant by gene knockdown of ptpn6, without wishing to be bound to a particular theory, the product of this gene is the most likely target of NSC87877 underlying the anti-diabetic effect. Knock down of other possible targets of NSC87877, ptpn11a and ptpn11b and dusp26 could not rescue the lepb ^'1' phenotype. In vitro inhibition studies showed that NSC87877 has a similar inhibitory effect on truncated human PTPN6 and PTPN11 proteins at a five-time higher IC50 than PTP1 B (Chen etal., 2006). Itwas later shown that NSC87877 has a more potent inhibitory effect on the dual phosphatase DUSP26 than on full length human PTPN6 protein (Song et al, 2009). The expression pattern of dusp26 that is restricted to neuroendocrine tissues in zebrafish larvae (Yang et al., 2017) supports the negative results observed in the rescue assay. The direct effect of metformin and NSC87877 in reversal of insulin resistance caused by a genetic defect shows the powerful action of these drugs at the level of insulin resistance. An effect of metformin on restoration of zebrafish larval beta cell development in a lepr mutant was previously demonstrated by Michel et al (2016). The drug metformin is already used for many years as first choice anti-diabetic drugs (Powers, 2012), however, its targets are multiple and therefore its function is still poorly understood (Foretz et al, 2014; Florez 2017). In particular, the effect of metformin on insulin resistance, the hallmark of type 2 diabetes, needs further study (Natali et al., 2006; Fullerton et al., 2013). The signaling pathway of insulin is highly conserved within all vertebrates. The positive effect of metformin in the zebrafish larval system shows that the results of mammalian diabetic test systems can be translated to lower vertebrate test systems. Considering that for treatment of Til D it is required that the very basic effect of insulin resistance needs to be overcome, indicates that the high throughput zebrafish model described herein will be useful to identify new potentially antidiabetic drugs that can optionally be further tested in mammalian studies.

Example 3

In another aspect, the present invention provides an insulin resistant mouse embryo model. Insulin signalling in vertebrates is highly conserved and therefore the inventors' findings in zebrafish and Xenopus are translatable to mammalian organisms. Based on the inventors' test system in early embryogenesis using fluorescent glucose analogs the inventors also devised a test system in early mice embryos.

The inventors used fertilized eggs from mice and used the same fluorescent glucose derivative as used in zebrafish and Xenopus. However, since mice embryos are very small it is difficult to inject the glucose inside the embryos without destroying them. Therefore, the inventors tested whether glucose might not be taken up by external addition. The inventors added 1 to 5 μΙ_ of fluorescent glucose (same concentration as used for injection in zebrafish) to mouse embryos in small containers with a small volume of oocyte growth medium (50 to 200 μΙ_) at several embryonic stages from morula to blastocyst stage. Then after a few minutes of incubation the medium was washed briefly a few time and embryos were subjected to confocal laser scanning microscopy. The results showed that fluorescent glucose was detectable inside several cells of the cell mass. However not all cells contained the fluorescent glucose showing that the glucose was not present inside the cells as a result of free diffusion but rather due to active transport. Therefore, microinjection of fluorescent glucose into mouse embryos is not needed in order to study active transport of glucose. This makes it possible to test fertilized eggs from mutant mouse for testing glucose transport. It is also possible to inject CRISPR/CAS construct in the fertilized eggs and after several hours, e.g. 4 days until they reach blastocyst stage, and then test glucose transport using the inventors' assay.

The latter has the advantage that also other mammalian systems can be used for which there are fewer mutants available. The fertilized eggs can also be frozen using standard protocols, embryos from many different mouse strains The company Janvier sells. The company Janvier sells both embryos that derive from a cross of homozygous leptin deficient (ob/ob minus/minus) parents or from a cross of heterozygous parents. The former is preferred although they are more expensive (because of the need to for in vitro fertilization since the homozygous mice are infertile) since there is no chance of a maternal rescue of the phenotype in homozygous offspring. Based on the inventors' results in zebrafish the leptin deficient mouse embryos are expected to be deficient in glucose transport. We can use this expected phenotype to test drugs that are added to the medium of the mice embryos. If the drug reverts the glucose transport phenotype in a similar way as in the zebrafish embryos this shows an antidiabetic effect of the compound. This assay is very fast and with the use of confocal laser scanning microscopy technology not disturbed by fluorescent background of the medium. Since detection of fluorescence is quantitative also minor effects on glucose transport are detectable. Just as the inventors have done for the zebrafish embryo test system, this system can also be used to identify the targets of a drug by inactivating the putative target gene in the leptin deficient mouse embryos using CRISPR/CAS technology. The use of this technology for transient inactivation of mouse genes has been described in literature and would be a matter of routine for a skilled person. The aforementioned screening system will make the mouse screening system more efficient. In the first place the mouse embryos are very small and easy to lose during manipulation. It is therefore useful to keep them in small containers that can also be directly used in confocal microscopy. Since mouse embryos are very sensitive to temperature changes they should be kept in a temperature-controlled environment. The added compounds are advised be warmed up to the growth temperature. Instead of using a confocal laser scanning microscope to detect transport of fluorescent glucose also other means of fluorescence detection methods are possible (E.g. vertebrate automated screening system, or fluorescent tomography microscopy).

REFERENCES

Azuma T, Suto H, Ito Y, Ohtani M, Dojo M, Kuriyama M, Kato T. Gastric leptin and

Helicobacter pylori infection. Gut. 2001 Sep;49(3):324-9. Alvin C. Diabetes Mellitus, Powers in Harrison's Principles of Internal Medicine, 18th edition, Chapter 345, ISBN 978-0071748896. Dennis Kasper, Anthony Fauci, Stephen Hauser, Dan Longo, J. Larry Jameson, Joseph Loscalzo

Bailey CJ, Turner RC. Metformin. N Engl J Med. 1996 Feb 29;334(9):574-9.

Cammisotto PG, Bendayan M, Sane A, Dominguez M, Garofalo C, Levy E. Receptor-

Mediated Transcytosis of Leptin through Human Intestinal Cells In Vitro. Int J Cell Biol. 2010;2010:928169. doi: 10.1 155/2010/928169. Epub 2010 Apr 29.

Chen L, Sung SS, Yip ML, Lawrence HR, Ren Y, Guida WC, Sebti SM, Lawrence NJ, Wu J. 2006. Discovery of a novel shp2 protein tyrosine phosphatase inhibitor. Mol Pharmacol. ;70(2):562-70.

Chen Y, Ma H, Zhu D, Zhao G, Wang L, Fu X, Chen W. 2017. Discovery of Novel Insulin Sensitizers: Promising Approaches and Targets. PPAR Res. 2017:8360919.

expression of two insulins in zebrafish (Danio reno). Physiol Genomics 27 :79 - 85

Coppari R, Bj0rbask C. Leptin revisited: its mechanism of action and potential for treating diabetes. Nat Rev Drug Discov. 2012 Sep;1 1 (9):692-708. doi: 10.1038/nrd3757. Review.

Faggioni R, Feingold KR, Grunfeld C. Leptin regulation of the immune response and the immunodeficiency of malnutrition. FASEB J. 2001 ; 15(14):2565-2571. [PubMed: 1 1726531]

Fantuzzi G, Faggioni R. Leptin in the regulation of immunity, inflammation, and hematopoiesis. J Leukoc Biol. 2000; 68(4): 437-446. [PubMed: 1 1037963]

Feng GS, Hui CC, Pawson T. 1993. SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases. Science 259: 1607-161 1

Guo Y, Veneman WJ, Spaink HP, Verbeek FJ.Biomed Opt Express. 2017 Apr 26;8(5):261 1-2634. doi: 10.1364/BOE.8.002611. eCollection 2017 May 1

Florez JC. 2017. The pharmacogenetics of metformin. Diabetologia. 60:1648-1655

Foretz M, Guigas B, Bertrand L, Pollak M, Viollet B. Metformin: from mechanisms of action to therapies. Cell Metab. 2014 Dec 2;20(6):953-66.

Fullerton MD, Galic S, Marcinko K, Sikkema S, Pulinilkunnil T, Chen ZP, O'Neill HM, Ford RJ, Palanivel R, O'Brien M, Hardie DG, Macaulay SL, Schertzer JD, Dyck JR, van Denderen BJ, Kemp BE, Steinberg GR. 2013. Nat Med.; 19(12): 1649-54.

Gorissen M, Bernier NJ, Nabuurs SB, Flik G, Huising MO. 2009. Two divergent leptin paralogues in zebrafish (Danio rerio) that originate early in teleostean evolution. J Endocrinol. 2009 Jun;201 (3):329-39.

Gut P, Reischauer S, Stainier DYR, Arnaout R. 2017. Little fish, big data: zebrafish as a model for cardiovascular and metabolic disease. Physiol Rev. 97(3):889-938. Heydemann A, Gonzalez-Vega M, Berhanu TK, Mull AJ, Sharma R, Holley-Cuthrell J. Hepatic Adaptations to a High Fat Diet in the MRL Mouse Strain are Associated with an Inefficient Oxidative Phosphorylation System. Jacobs J Diabetes Endocrinol. 2016 Dec;2(1). pii: 013. Epub 2016 Oct 25.

Hu FB. 2011. Globalization of Diabetes. Diabetes Care 34 1249-1257. (doi:

10.2337/dc1 1-0442)

Kang J, Hu J, Karra R, Dickson AL, Tornini VA, Nachtrab G, Gemberling M, Goldman JA, Black BL, Poss KD. Modulation of tissue repair by regeneration enhancer elements. Nature. 2016 Apr 14;532(7598):201-6. doi: 10. 1038/nature17644. Epub 2016 Apr 6.

Lago R, Gomez R, Lago F, Gomez-Reino J, Gualillo O. Leptin beyond body weight regulation-current concepts concerning its role in immune function and inflammation. Cell Immunol. 2008; 252(1-2):139-145. [PubMed: 18289518]

Liu Q, Dalman M, Chen Y, Akhter M, Brahmandam S, Patel Y, Lowe J, Thakkar M, Gregory AV, Phelps D, Riley C, Londraville RL. 2012. Knockdown of leptin A expression dramatically alters zebrafish development. Gen Comp Endocrinol. 2012 Sep 15; 178(3): 562- 72. doi: 10.1016/j.ygcen.2012.07.011. Epub 2012 Jul 25.

Mackey-Lawrence NM, Petri WA Jr. Leptin and mucosal immunity. Mucosal Immunol. 2012 Sep;5(5):472-9. doi: 10.1038/mi.2012.40. Epub 2012 Jun 13. Review.

Man ^' n-Juez R, Jong-Raadsen S, Yang S, Spaink HP. 2014. Hyperinsulinemia induces insulin resistance and immune suppression via Ptpn6/Shp1 in zebrafish. J Endocrinol. 2014 Aug; 222 : 229-241.

Marin-Juez R, Rovira M, Crespo D, van der Vaart M, Spaink HP, Planas JV. 2015.

GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish. J Cereb Blood Flow Metab. 35(1):74-85.

Matthews RJ, Bowne DB, Flores E, Thomas ML. 1992. Characterization of hematopoietic intracellular protein tyrosine phosphatases: description of a phosphatase containing an SH2 domain and another enriched in proline-, glutamic acid-, serine-, and threonine-rich sequences. Mol Cell Biol. 12(5):2396-405.

Michel M, Page-McCaw PS, Chen W, Cone RD. 2016. Leptin signaling regulates glucose homeostasis, but not adipostasis, in the zebrafish. Proc Natl Acad Sci U S A. 113

(1 1):3084-9.

Mori H, Hanada R, Hanada T, Aki D, Mashima R, Nishinakamura H, Torisu T, Chien KR, Yasukawa H, Yoshimura A. 2004. Socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to diet-induced obesity. Nat Med. 10(7):739-43.

Nagy C, Einwallner E. 2018. Study of In Vivo Glucose Metabolism in High-fat Diet-fed

Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). J Vis Exp. 2018 Jan 7;(131). Natali A, Ferrannini E. 2006. Effects of metformin and thiazolidinediones on suppression of hepatic glucose production and stimulation of glucose uptake in type 2 diabetes: a systematic review. Diabetologia. 49(3):434-41.

Ng M, Fleming T, Robinson M, Thomson, Graetz N, Margono C, Mullany EC, Biryukov S, et al. 2014 Global, regional and national prevalence of overweight and obesity in children and adults 1980-2013: A systematic analysis. The Lancet 384766 - 781. (doi : 10.1016/S0140- 6736(14)60460-8)

Papasani MR, Robison BD, Hardy RW, Hill RA. 2006. Early developmental

Poeggeler B, Schulz C, Pappolla MA, Bodo E, Tiede S, Lehnert H, Paus R. Leptin and the skin: a new frontier. Exp Dermatol. 2010 Jan; 19(1):12-8. doi: 10.11 11/j.1600- 0625.2009.00930.x. Epub 2009 Jul 8. Review

Powers, Alvin C. "Chapter 344. Diabetes Mellitus." Harrison's Principles of Internal Medicine, 18e Eds. Dan L. Longo, et al. New York, NY: McGraw-Hill, 2012,

Reed & Scribner 1990. In-vivo and in-vitro models of type 2 diabetes in pharmaceutical drug discovery. Diabetes Obes Metab.;1 (2):75-86.

Stanford SM, Aleshin AE, Zhang V, Ardecky RJ, Hedrick MP, Zou J, Ganji SR, Bliss MR, Yamamoto F, Bobkov AA, Kiselar J, Liu Y, Cadwell GW, Khare S, Yu J, Barquilla A, Chung TDY, Mustelin T, Schenk S, Bankston LA, Liddington RC, Pinkerton AB, Bottini N. Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase. Nat Chem Biol. 13(6):624-632

Song M, Park JE, Park SG, Lee DH, Choi HK, Park BC, Ryu SE, Kim JH, Cho S. NSC- 87877, inhibitor of SHP-1/2 PTPs, inhibits dual-specificity phosphatase 26 (DUSP26). Biochem Biophys Res Commun. 2009 Apr 17;381 (4):491-5. doi: 10.1016/j.bbrc.2009.02.069. Epub 2009 Feb 20.

Tamrakar AK, Maurya CK, Rai AK. 2014. PTP1B inhibitors for type 2 diabetes treatment: a patent review (2011 - 2014). Expert Opin Ther Pat. 24(10): 1101-15.

Toyoshima Y, Monson C, Duan C, Wu Y, Gao C, Yakar S, Sadler KC, LeRoith D. 2008. The role of insulin receptor signaling in zebrafish embryogenesis. Endocrinology. 149(12):5996-6005.

Tu H, Hsuchou H, Kastin AJ, Wu X, Pan W. Unique leptin trafficking by a tailless receptor. FASEB J. 2010 Jul;24(7):2281-91. doi: 10.1096/fj.09-143487. Epub 2010 Mar 1 1.

Ullrich, A. and Schlessinger, J. 1990. Signal transduction by receptors with tyrosine kinase activity. Cell 61 , 203-212

Wang B, Chandrasekera PC, Pippin JJ. 2014. Leptin- and leptin receptor-deficient rodent models: relevance for human type 2 diabetes. Curr Diabetes Rev. 10 (2): 131 -45. White CL, Whittington A, Barnes MJ, Wang Z, Bray GA, Morrison CD. 2009. HF diets increase hypothalamic PTP1B and induce leptin resistance through both leptin-dependent and -independent mechanisms. Am J Physiol Endocrinol Metab. 296(2): E291 -9.

Wolsk E, Mygind H, Grandahl TS, Pedersen BK, van Hall G. Human skeletal muscle releases leptin in vivo. Cytokine. 2012 Dec;60(3):667-73.

Yang CH, Yeh YJ, Wang JY, Liu YW, Chen YL, Cheng HW, Cheng CM, Chuang YJ, Yuh CH, Chen YR. 2017. NEAP/DUSP26 suppresses receptor tyrosine kinases and regulates neuronal development in zebrafish. Sci Rep. ;7(1):5241.

Zang L, Shimada Y, Nishimura N. 2017. Development of a Novel Zebrafish Model for Type 2 Diabetes Mellitus. Sci Rep. 7 (1): 1461.

Zhou Y, Rui L.2013. Leptin signaling and leptin resistance. Front Med. 2013 Jun; 7(2): 207-222.

J Vis Exp. 2014 Jun 26;(88):e51649. Establishment and optimization of a high throughput setup to study Staphylococcus epidermidis and Mycobacterium marinum infection as a model for drug discovery. Veneman WJ, Man ^' n-Juez R, de Sonneville , Ordas A, Jong- Raadsen S, Meijer AH, Spaink HP.

Veetil JV, Jin S, Ye K.; J Diabetes Sci Technol. 2012 Nov 1 ;6(6): 1276-85.

Fluorescence lifetime imaging microscopy of intracellular glucose dynamics.

Yu M, Zhao K, Zhu X, Tang S, Nie Z, Huang Y, Zhao P, Yao S.

Biosens Bioelectron. 2017 Sep 15;95:41-47. Development of near-infrared ratiometric fluorescent probe based on cationic conjugated polymer and CdTe/CdS QDs for label-free determination of glucose in human body fluids.