WO2011098757A1

WO2011098757A1 - Cell based bioassay

Info

Publication number: WO2011098757A1
Application number: PCT/GB2011/000170
Authority: WO
Inventors: Calum Sutherland
Original assignee: Tmri Limited
Priority date: 2010-02-10
Filing date: 2011-02-10
Publication date: 2011-08-18
Also published as: GB201002130D0

Abstract

We disclose a cell-based bioassay allowing the analysis of insulin resistance in subjects suffering from or susceptible to type 2 diabetes.

Description

Cell Based Bioassay

The invention relates to a cell-based bioassay and its use in the identification of insulin sensitizing agents and including a diagnostic assay for the determination of a subject's sensitivity to insulin and optionally to treat a subject either suffering from type 2 diabetes or wherein said subject has a predisposition to type 2 diabetes.

Insulin functions to regulate glucose homeostasis. In conditions of hyperglycemia [abnormally high levels of serum glucose] the pancreatic β cells of the Islets of Langerhans synthesize proinsulin which is enzymatically cleaved at its amino and carboxy-termini to produce insulin, a 51 amino acid polypeptide. Insulin is secreted and acts on target cells [e.g. liver, muscle, adipose tissue] that express insulin receptors. The activation of insulin receptors leads to a signal transduction cascade that results in expression of glucose transporters in muscle which remove excess glucose with subsequent conversion of glucose into glycogen for storage. In liver, insulin shuts off endogenous glucose production and hepatic glucose output. Once glucose levels return to normal insulin is degraded thus removing its biological effects. The insulin receptor is a tyrosine kinase and is a tetrameric transmembrane receptor comprising two a subunits and two β subunits. The a subunits are extracellular and bind insulin. The β subunits are transmembrane and include ATP and tyrosine kinase domains which become activated on insulin binding.

There are a number of pathological conditions that result in hyperglycaemia the most well known being diabetes mellitus. Diabetes mellitus can be of type 1 or type 2. Type 1 diabetes is an autoimmune disease resulting in destruction of the pancreatic β cells which means the subject is unable to manufacture any insulin. Type 2 diabetes is a more complicated condition and can result from a number of associated ailments but typically involves resistance to the metabolic actions of insulin. For example, type 2 diabetes is associated with age, obesity, a sedentary life style and insulin resistance. An associated condition is called Metabolic Syndrome which may predispose subjects to type 2 diabetes. The symptoms associated with this syndrome are high blood pressure, dyslipidemia, increased body fat deposition and cardiovascular disease.

Administration of insulin is an effective means to control conditions such as type 1 and type 2 diabetes. Insulin resistance is a common characteristic of type 2 diabetes mellitus and is one of the major independent risk factors for progression of the disease. The treatment of insulin resistance is either by substantial weight loss and exercise with a concomitant increase in insulin sensitivity or by administration of insulin sensitizing agents which restore insulin sensitivity to those subjects that present with insulin resistance. The current approved agents to control diabetes 2 that are proposed to have some insulin sensitizing activity are rosiglitazone or metformin which both have undesirable side effects. The identification of novel insulin sensitizing agents that have better disease control and side effect profile is highly desirable.

In order to design truly effective insulin sensitising agents targeted at the mechanism of disease development in response to obesity we have generated an obesity-related insulin resistant cell model. Rat hepatoma cells (H4IIE) are grown in the presence of serum isolated from obese rodents or obese human volunteers and the insulin sensitivity of the cells monitored over time by the repression of a key insulin responsive gene. Higher insulin concentrations are required to fully repress the gene in the cells grown in obese rodent serum compared with those grown in serum from lean rodents (almost a 10-fold shift in insulin sensitivity). This was reversed by restoring normal growth medium, while the insulin resistance could be prevented by rosiglitazone or metformin. Meanwhile, growth of cells in serum collected from obese human volunteers with diabetes also reduced the insulin sensitivity of the rat cells. High body mass index, glucose and leptin levels correlated with the ability of serum from an individual (even within the obese diabetic group) to generate insulin resistance in the model.

This disclosure relates to a novel insulin resistant cell model that can be used to study the molecular development of obesity-linked insulin resistance, screen for compounds which overcome obesity-related insulin resistance and sub-classify insulin resistant patients thereby influencing their treatment.

Statements of Invention

According to an aspect of the invention there is provided a screening method for the identification of an insulin sensitizing agent comprising the steps:

i) providing a cell wherein said cell is transfected with a nucleic acid molecule comprising an insulin responsive promoter wherein said promoter is operably linked to a nucleic acid molecule that encodes a reporter molecule; ii) forming a preparation comprising the cell in i) and an isolated biological sample obtained from a subject wherein said subject is either: a) obese; or

b) non-obese; and,

incubating said preparation under cell culture conditions conducive to the growth and maintenance of said cell in culture;

iii) addition of at least one an agent to be tested for insulin sensitizing activity to the preparation^] formed in ii); and

iv) analyzing the biological effect of said agentfs] on at least one biomarker of insulin activity and comparing said biomarker to a control cell population which has not be contacted with said agent.

In a preferred method of the invention said agent[s] have an effect on the activity of the insulin responsive promoter.

In a preferred method of the invention said promoter is the mammalian cytosolic soluble phosphoenolpyruvate [GTP] gene promoter, or regulatory part thereof.

Examples of PEPCK gene sequences, include UniGene Hs.1872 Homo sapiens (human) PCK1. UniGene Rn.104376 Rattus norvegicus (Norway rat) Pck1 or UniGene Mm.477474 Mus musculus (mouse) Pck1],

Obesity" is measured in relation to a scale based on Body Mass Index Body Mass Index (BMI) is an approximate measure of obesity, and is calculated by dividing the mass of the individual (in kilograms) by the height squared (in metres squared), i.e. BMI= Weight (kg)/Height² (m²). A healthy BMI range in UK is 18-25, overweight is 25-30 and obese is >30.

In a preferred method of the invention said cell is a mammalian cell adapted for expression of the human PEPCK promoter.

In a preferred method of the invention said cell is a human cell.

In an alternative preferred method of the invention said cell is a rodent cell, for example a rat or mouse cell. In a preferred method of the invention said cell is a cell that is naturally responsive to insulin.

In a preferred method of the invention said cell is selected from the group consisting of: a hepatocyte, an adipocyte or a muscle cell.

In a preferred method of the invention said cell is a hepatocyte.

In a preferred method of the invention said hepatocyte cell is the rat hepatocyte cell-line RH4IIE.

In a preferred method of the invention said biological sample is a serum sample, preferably a serum sample isolated from an obese human subject.

In a preferred method of the invention said reporter molecule is a fluorescence reporter.

The analysis of promoter activity can be conveniently monitored by fusing an insulin responsive promoter, such as the PEPCK promoter, to a nucleic acid that encodes a "reporter" protein or polypeptide. Examples are well known in the art and include enzymes such as β glucuronidase, chloramphenicol acetyltransferase or luciferase. Reporters that are proteinaceous fluorophores are also known in the art. Green fluorescent protein, GFP, is a spontaneously fluorescent protein isolated from coelenterates, such as the Pacific jellyfish, Aequoria victoria. Its role is to transduce, by energy transfer, the blue chemiluminescence of another protein, aequorin, into green fluorescent light. GFP can function as a protein tag, as it tolerates N- and C-terminal fusions to a broad variety of proteins many of which have been shown to retain native function. Most often it is used in the form of enhanced GFP in which codon usage is adapted to the human code. Other proteinaceous fluorophores include yellow, red and blue variant fluorescent proteins [e.g. BFP, CFP, GFP, YFP and RFP] These are commercially available from, for example Clontech (www. clontech. com) .

In a preferred method of the invention said fluorescence reporter is a fluorescent protein selected from the group comprising: BFP, CFP, GFP, YFP and RFP, and the mutants thereof. In a preferred method of the invention said method detects one, two, three, four, five, six or seven biomarkers indicative of insulin activity.

In a preferred method of the invention said at least one biomarker is selected from the group consisting of: leptin and/or LDL cholesterol and/or triglycerides.

According to a further aspect of the invention there is provided a cell array wherein said array comprises a plurality of identical assay preparations comprising a cell wherein said cell is transfected with a nucleic acid molecule comprising an insulin responsive promoter wherein said promoter is operably linked to a nucleic acid molecule that encodes a reporter molecule.

In a preferred embodiment of the invention said insulin responsive promoter is the PEPCK promoter, preferably the human PEPCK promoter.

The screening of large numbers of agents requires preparing arrays of cells for the handling of cells and the administration of agents. Assay devices, for example, include standard multiwell microtitre plates with formats such as 6, 12, 48, 96 and 384 wells which are typically used for compatibility with automated loading and robotic handling systems. Typically, high throughput screens use homogeneous mixtures of agents with an indicator compound which is either converted or modified resulting in the production of a signal. The signal is measured by suitable means (for example detection of fluorescence emission, optical density, or radioactivity) followed by integration of the signals from each well containing the cells, agent and indicator compound.

According to a further aspect of the invention there is provided a method for the high through put screening of insulin sensitizing agents comprising the steps:

i) providing an array according to the invention;

ii) contacting the array with a plurality of agents to be tested for insulin sensitizing activity;

iii) collating the activity data in (ii) above;

iv) converting the collated data into a data analysable form; and optionally v) providing an output for the analysed data.

A number of methods are known which image and extract information concerning the spatial and temporal changes occurring in cells or nuclei expressing, for example fluorescent molecules and other markers of gene expression (see Taylor et al Am. Scientist 80: 322-335, 1992). Moreover, US5, 989,835 and US09/031.271 , both of which are incorporated by reference, disclose optical systems for determining the distribution or activity of fluorescent reporter molecules in cells for screening large numbers of agents for biological activity. The systems disclosed in the above patents also describe a computerised method for processing, storing and displaying the data generated.

According to a further aspect of the invention there is provided a method to analyse a subject to determine whether said subject is or is not insulin resistant comprising the steps:

i) providing a cell wherein said cell is transfected with a nucleic acid molecule comprising an insulin responsive promoter wherein said promoter is operably linked to a nucleic acid molecule that encodes a reporter molecule;

ii) forming a preparation comprising the cell in i) and an isolated biological sample obtained from an obese subject, or a subject suspected of being susceptible to obesity, and incubating said preparation under cell culture conditions conducive to the growth and maintenance of said cell in culture;

iii) analyzing the biological effect of said isolated biological sample on at least one biomarker of insulin activity and comparing said biomarker to a control cell population which is obtained from a non-obese subject; and iv) determining whether the subject is or is not insulin resistant.

In a preferred method of the invention said isolated biological sample is analyzed for an effect on the activity of the insulin responsive promoter.

According to a further aspect of the invention there is provided a method to analyse and treat a human subject suspected of suffering from or having a predisposition to type 2 diabetes comprising the steps:

i) providing a cell wherein said cell is transfected with a nucleic acid molecule comprising an insulin responsive promoter wherein said promoter is operably linked to a nucleic acid molecule that encodes a reporter molecule; ii) forming a preparation comprising the cell in i) and an isolated biological sample obtained from an obese human subject, or a human subject suspected of being susceptible to obesity, and incubating said preparation under cell culture conditions conducive to the growth and maintenance of said cell in culture;

iii) analyzing the biological effect of said isolated biological sample on at least one biomarker of insulin activity and comparing said biomarker to a control cell population which is obtained from a non-obese subject;

iv) determining whether the subject contains a serum factor capable of generating hepatic insulin resistance; and

v) determining a treatment regime that will benefit the subject in controlling or preventing the onset of insulin resistance and hence type 2 diabetes.

In a preferred method of the invention said insulin responsive promoter is the PEPCK promoter, preferably the human PEPCK promoter.

In a preferred method of the invention said biological sample is a body fluid sample, for example a blood or serum sample.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does 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.

An embodiment of the invention will now be described by example only and with reference to the following figures: Figure 1 illustrates that the growth of cells in serum from obese rodents alters the insulin sensitivity of the PEPCK gene promoter. HL1 C cells (H4IIE cells with CAT reporter) were grown in serum isolated from obese rats that had been on a high fat diet for 3 months (HFS), or lean chow fed littermates (Con). After 9 days (A) or 24 days (B), cells were serum starved for 16h prior to exposure to 8CPT-CAMP (0.1 mM), dexamethasone (500n ) and insulin as indicated for 3h. Protein was harvested and CAT reporter assayed as a measure of PEPCK gene promoter activity as detailed under Methods. (C) A direct comparison of insulin regulation of PEPCK gene promoter in cells exposed to HFS for 1 , 2 or 3 weeks. *p<0.05, comparison of 3 weeks and control. (D) HL1 C cells were grown for 3 weeks in serum isolated from obese Fa/Fa or lean Fa/+ rats prior to analysis of insulin regulation of the PEPCK gene promoter as above. Data is presented relative to the CAT activity in the appropriate Dex/cAMP induced control;

Figure 2 illustrates that the growth of cells in serum from obese rodents alters insulin regulation of PEPCK mRNA. HL1C cells were grown for 3 weeks in serum isolated from obese Fa/Fa or lean Fa/+ rats prior to serum starvation and exposure to glucocorticoid (Dex), cAMP and insulin as in Fig.1. After exposure to hormones total RNA was isolated and PEPCK mRNA levels were quantified by RT-PCR. Data is presented as relative PEPCK mRNA (average ±SEM) after normalisation to cyclophilin mRNA in each sample. Percent inhibition by insulin is indicated in each case. *p<0.05;

Figure 3 illustrates that normal insulin sensitivity of cells is restored by returning the cells to standard serum. (A) H4IIE cells were grown for 3 weeks in 5% Fa/Fa serum. RNA was harvested from half of the cells following exposure to hormones as in Fig.1 , the remainder were placed in 5% FCS for 1 week prior to hormone exposure and RNA isolation, while RNA was also isolated from H4IIE cells grown for 3 weeks in Fa/+ lean serum followed by 1 week in FCS prior to hormone exposure. (B) Relative PEPCK mRNA (average ±SEM) for the samples isolated above are shown following normalisation for cyclophilin mRNA in each sample. Percent inhibition by insulin is indicated in each case. ^*p<0.05;

Figure 4 illustrates the generation of insulin resistance is prevented by pioglitazone and metformin. H4IIE cells were grown for 3 weeks in 5% Fa/Fa serum, in the presence or absence of pioglitazone or metformin as indicated. Cells were serum starved for 16h prior to 3h exposure to dexamethasone (500nM), 8CPT-CAMP (0.1 mM) ± insulin as indicated, and RNA was isolated. PEPCK mRNA levels (average ±SEM) are presented for each cell treatment after correction for cyclophilin mRNA. Percent inhibition by 1 n insulin in each condition is presented.

Figure 5 illustrates that the growth of cells in serum from obese humans with diabetes alters insulin regulation of PEPCK mRNA. H4IIE cells were grown for 3 weeks in 5% serum collected from 40 human volunteers, 20 who were obese and diabetic (Cases), 20 who were lean and non-diabetic (Controls). After 3 weeks in the serum the 40 different pools of cells were serum starved for 16h prior to 3h exposure to dexamethasone (500n ), 8CPT-cAMP (0.1 mM) ± insulin as indicated, and RNA was isolated. PEPCK mRNA levels (average ±SEM) are presented (n=20, assayed at least in duplicate for each condition) after correction for cyclophilin mRNA;

Figure 6 illustrates that there is variation in the degree of insulin resistance generated by growth of cells in serum from obese volunteers with diabetes. The response to 0.1 nM insulin for each individual pool of cells grown in the 20 serums collected from the obese diabetic volunteers is shown relative to the average Control response. The 10 serums that generated the greatest resistant to insulin are labelled insulin resistant and a comparison of clinical parameters of the insulin resistant group is presented in Table 2

Figure 7 is the nucleotide sequence of the rat PEPCK promoter.

Materials and Methods

Actrapid (human insulin) was from Novo Nordisk A/S (Bagsvaerd, Denmark). 2x Universal PCR Master Mix, No AmpErase UNG from Applied Biosystems, Complete™ protease inhibitor cocktail tablets from Roche, and 8-(4-chloro-phenylthio)-cAMP from Calbiochem. All primers and probes were synthesized and purified by Sigma-Aldrich. All other chemicals were of the highest grade obtainable.

Animals

Diets were purchased from research diets (HF diet D12331 and control diet D12328, http://www.researchdiets.com/pdf/Data-20Sheets/D12331.pdf) and Male Sprague Dawleys (starting at 10 weeks of age) were kept on the diet for up to 3 months prior to sacrifice. Serum was prepared immediately and all serum from each group combined, then aliquoted and snap frozen for storage at -80oC. Cell Culture

H4IIE cells, a rat hepatoma cell, and HL1 C (a H4IIE line with a stable insertion of a PEPCK promoter, chloramphenicol acetyl transferase gene construct (), were maintained in DMEM (Dulbecco's modified Eagle's medium) containing 1000 mg/l glucose, 1 % (v/v) penicillin/streptomycin, and 5 % (v/v) FBS, in a 37 °C 5 % C0₂ incubator.

Exposure of cells to experimental serum

Three different mix of sera were used: 1) 2.5 % foetal bovine serum plus 2.5 % rat serum obtained either from regularly fed rats (Control Serum) or from rats following a high fat diet (High Fat Serum); 2) 2.5 % foetal bovine serum plus 2.5 % serum from lean regular rats (Control Serum) or FaFa obese rats (Obese Serum); 3) 5 % human serum from either lean volunteers (Control Human Serum) or obese diabetic volunteers (CASE Human Serum).

In brief, cells were cultured and passaged as normal in the experimental sera. Cells were serum starved for 16 hours prior to measurement of insulin sensitivity. Hormones were added as described in figure legends, and after 3h protein (for CAT Assay, HL1C) or RNA (for Taqman, H4IIE) were harvested.

CAT Assay

Cells were isolated by trypsinisation, washed in PBS and pelleted by centrifugation at 1400 rpm for 5 min. Cell lysate was prepared by sonication in 0.25M TRIS, total protein assessed by BCA assay (Pierce), and general cellular protein removed by heating at 65oC for 10 min and centrifugation. Supernatants were tested for CAT activity as described previously (23), and data expressed as Units of CAT activity per mg of cellular protein.

RNA Isolation and Real-Time quantitative reverse transcriptase PCR

Following hormone treatments total cellular RNA was extracted from H4IIE cells using TRIreagent™ (Sigma) according to the manufacturer's instructions. cDNA was synthesized from total cellular RNA using Superscript™ II reverse transcriptase kit (Invitrogen). Real-time PCR analysis was carried out using ABIPrism7700 sequence detector (Applied Biosystems). Primer sequences were to rat PEPCK (forward- ACA GGC AAG GTC ATC ATG CA; reverse- TGC CGA AGT TGT AGC CAA AGA; probe- FAM-ACC CCT TCG CTA TGC GGC CC-TAMRA) and rat cyclophilin (forward- TTA CTA GGT CTG GCA GGA AGA TTA AAG; reverse- CTG CAT CTC TTG TCT CCA ATG TG; probe- FAM- AGA GGA CCA AGG CGT TAT CGA A-TAMRA). PCR reactions were carried out using the following cycling conditions - 50°C - 2min x1 , 95°C - 10min x1 followed by 40 cycles of 95°C - 15s, 60°C - 1 min.

Human Volunteers/Recruitment

Twenty control (BMI<25, Non-Diabetic) subjects were recruited from the general population along with twenty obese (BMI>30) type 2 diabetics recruited from the diabetes clinic. All subjects were male, non-smokers and taking no other medication known to influence insulin sensitivity (Table 1). They were relatively pharmaceutically naive subjects to reduce potential confounding effects of long term intervention, the exception being that all Cases were on a statin at time of visit. Volunteers fasted overnight prior to a visit to the Clinical Research Centre where 200ml of fasted whole blood was collected. Serum was prepared and stored at -80°C in 10ml_ aliquots. BMI, blood pressure and waist measurements were taken during the visit.

Clinical and Biochemical Measurements

TNFa, insulin, adiponectin, CRP and leptin were assayed in plasma or serum by ELISA in the Translational Medicine Research Institute. Other biochemical measurements were performed by the Clinical Biochemistry Department, Ninewells Hospital.

EXAMPLES

Diet induced obesity

15 male Sprague Dawley rats (10 weeks of age) were placed on a high fat (HF) diet for 12 weeks. 15 littermates were given normal chow (Con) for the same period. Serum was prepared from both the diet induced obese (DIO) and normal chow fed control animals, and this was aliquoted and stored at -80C until required. HL1C cells were grown in DMEM containing either 2.5% FCS + 2.5% DIO-derived serum, or 2.5% FCS + 2.5% Con serum. The cell morphology and growth rate during culture and passage (every 2 or 3 days) was similar in both cases (data not shown). After 9 or 24 days cells were serum starved for 3h, then exposed to hormones prior to measurement of PEPCK transcription. HL1C cells contain a CAT reporter with expression under the control of the PEPCK gene promoter (23; 24). In these cells CAT expression is normally low but can be induced 5- 10 fold by exposure to glucocorticoid and cAMP for 3h. However the induction can be completely blocked by including insulin in the culture medium (23). Repression by insulin is dose-dependent with an EC50 of approximately 0.1 nM, and >80% inhibition at 1 nM insulin (Fig.lA and (23; 25-27)). Growth in DIO-derived rat serum for 9 days did not produce any change in the insulin sensitivity of the HL1C cells (Fig.lA), however exposure to DIO-derived serum for 24 days resulted in a right shift of the insulin dose response curve (Fig.l B). The EC50 for insulin regulation of PEPCK-CAT rose to 1 nM although complete inhibition of the gene promoter was still achieved by 10nM insulin (Fig. l B). The experiment was repeated with fresh DIO-derived serum and the insulin sensitivity of the HL1C cells was reduced following 2 weeks of growth in the test serum (Fig.1C), but did not change between 2 and 3 weeks of exposure (Fig.lC). This suggested that there is a maximal effect of the serum on insulin sensitivity, and this occurs around 2 weeks of growth in DIO-derived serum.

Genetic predisposition to obesity

We next obtained serum from 12-14 week old FaFa rats (obese), and age matched Fa/+ (lean) rats. HL1C cells were grown in either 2.5% FaFa or 2.5% Fa/+ serum (supplemented with 2.5% FCS in each case), for 3 weeks prior to analysis of PEPCK gene promoter regulation (Fig 1 D). Once more insulin sensitivity of the HL1C cells was reduced in cells grown in serum from this leptin receptor deficient obesity and IR rat model, with the EC50 for repression of the PEPCK gene promoter again shifting from around 0.1 to 1 nM insulin (Fig.l D).

Endogenous Gene Regulation

The PEPCK-CAT reporter gene is more sensitive to insulin than the endogenous PEPCK gene promoter, with the EC50 for PEPCK mRNA repression by insulin closer to 1 nM (Fig.2). However, growth of the H4IIE cell line in serum from obese Fa/Fa rats also reduced the insulin sensitivity of the endogenous PEPCK gene promoter, with a significant difference between the response to 1 or 10nM insulin (Fig.2). The effect was more apparent when cells were grown in 5% serum (with no supplement of FCS) (Fig.2). The serum from Fa/Fa rats also made the PEPCK mRNA levels more sensitive to glucocorticoid and cAMP induction (Fig.2 and Fig.3). Reversibility

In order to establish whether normal insulin sensitivity could be restored to the 'resistant' cells, we generated insulin resistance by culturing cells for 3 weeks in 5% Fa/Fa serum, then transferring half of the cells to 5% FCS for 1 week, and comparing the insulin sensitivity of the two batches of cells and also cells grown throughout in 5% Fa/+ lean serum for an equivalent time (Fig.3). Removal of the Fa/Fa serum for 1 week restored the normal insulin sensitivity of the cells, showing that the resistance is reversible.

Effect of Insulin Sensitisers

Cells were incubated with Fa/Fa or Fa/+ serum ± 0.1 mM metformin, or ± 1 μΜ Pioglitazone. Drugs were replaced at every cell passage (along with fresh serum), and after 3 weeks the insulin sensitivity of the cells established (Fig.4). The presence of metformin reduced PEPCK gene transcription, irrespective of the growth conditions or hormone challenge. Indeed 3 weeks growth in the presence of metformin reduced basal PEPCK transcript, partly reduced the induction by glucocorticoids and cAMP, while restoring full repression by insulin (Fig.4). The same response was seen whether the cells had been grown in Fa/+ or FaFa serum, hence this drug prevented reduced insulin action on PEPCK transcription by the Fa/Fa serum, but appeared to do so by directly repressing the gene irrespective of the presence of insulin resistance.

In contrast, growth in Pioglitazone resulted in prevention of reduced insulin sensitivity of the cells exposed to Fa/Fa serum without any effect on cells grown in Fa/+ serum (Fig.4). Basal, induced and insulin-repressed levels of the cells in Fa/+ serum were almost identical ± Pioglitazone. Meanwhile, this drug restored insulin repressive actions in the Fa/Fa serum treated cells to that of the Fa/+ serum treated cells, indicating it improved insulin sensitivity rather than simply repressed PEPCK transcription.

This suggests that the cell model can identify agents likely to improve insulin sensitivity, including those that have no affect on insulin sensitivity in healthy cells, but also distinguish between insulin sensitising agents and drugs that simply repress the PEPCK gene in an insulin mimetic type manner. Analysis of human serum donated by obese diabetic volunteers

Serum was donated by twenty obese (BMI>30) diabetic volunteers (being treated by diet alone), and twenty lean healthy volunteers. Cells were then cultures for 3 weeks in all forty serum samples (5%) prior to analysis of insulin repression of PEPCK mRNA. The average response to 0.1 nM or 0.5n insulin of H4IIE cells cultured in serum from obese diabetics was significantly different from that of cells cultured in serum from lean non-diabetics (Figure 5). However there was no difference in the magnitude of gene induction by glucocorticoids/cAMP, or in the response to higher concentrations of insulin between controls and cases. There was a broad range of response across the individual samples from the obese diabetics (Figure 6). The Cases could be separated into two groups based on the insulin sensitivity of the cells following growth in the serum; most insulin resistant (Group 1 , lowest 10 responses to insulin) and most insulin sensitive (Group 2, highest 10 response, closest response to that seen in the average control). The levels of glucose, total and LDL cholesterol along with measurements of weight, waist size and waisthip ratio were significantly different between Groups 1 and 2 (Table 2). Differences in blood leptin concentration and B I approached significance (Table 2). The lower total and LDL cholesterol in the cases compared to controls were most probably due to the effects of HMG CoA reductase inhibitors which all of the Cases were taking. However it was surprising that there was a significant difference between cholesterol levels between the two obese diabetic groups, with a significantly higher cholesterol level in the resistant group, despite 19 out of the 20 Cases being on the same statin (Table 1).

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Table 1 : Comparison of clinical parameters of the insulin resistant and insulin sensitive

Cases.

Resistant Cases Sensitive Significance,

(n=10) Cases (n=10) students t-test (p=)

GLUCOSE (mmol/L) 9.2611.52 7.46+0.64 0.0537

HbAlc% 6.96±0.67 6.53+0.42 0.3048

INSULIN 17.25±7.2 15.61+2.65 0.6839

CRP 2676±11 15 3183+1055 0.2921

LEPTIN 19248±8239 1189213966 0.1389

ADIPONECTIN 4687±1431 4728+1606 0.9705

TNFa 1.2±0.3 1.06+0.1 0.41 16

T Choi mmol/L 4.74+0.44 4.13+0.41 0.0694

LDL mmol/L 2.62±0.41 2.1310.16 0.0787

HDL mmol/L 1.22±0.21 1.15+0.24 0.5688

Trigs mmol/L 2.34+0.83 1.97+0.94 0.5779

Age 57.7+1.65 55.7+2.74 0.2400

Height cm 176.6±4.95 172.8±3.16 0.2238

Weight Kg 1 14.46112.3 98.8714.33 0.0386*

BMI 36.7+3.58 33.2+2.2 0.1234

Waist:Hip 1.04+0.04 1.00+0.02 0.05

HOMA 2.44 2.18 0.6097

Claims

1 A screening method for the identification of an insulin sensitizing agent comprising the steps:

ii) forming a preparation comprising the cell in i) and an isolated biological sample obtained from a subject wherein said subject is either:

a) obese; or

b) non-obese; and,

iii) addition of at least one agent to be tested for insulin sensitizing activity to the preparations] formed in ii); and iv) analyzing the biological effect of said agent[s] on the activity of the insulin responsive promoter and at least one biomarker of insulin activity and comparing said insulin responsive promoter activity and biomarker to a control cell population which has not be contacted with said agent.

2. The method according to claim 1 wherein said promoter is the phosphoenolpyruvate, cytosolic [GTP] gene promoter [PEPCK].

3. The method according to claim 2 wherein said cell is a mammalian cell adapted for expression of the human PEPCK promoter or regulatory part thereof.

4. The method according to any of claims 1-3 wherein said cell is a human cell.

5. The method according to any of claims 1-3 wherein said cell is a rodent cell.

6. The method according to any of claims 1-5 wherein said cell is a cell that is naturally responsive to insulin.

7. The method according to claim 6 wherein said cell is selected from the group consisting of: a hepatocyte, an adipocyte or a muscle cell.

8. The method according to claim 7 wherein said cell is a hepatocyte.

9. The method according to claim 8 wherein said hepatocyte cell is the rat hepatocyte cell-line RH4IIE.

10. The method according to any of claims 1 -9 wherein said biological sample is a serum sample.

1 1. The method according to any of claims 1-10 wherein the sample is isolated from an obese human subject.

12. The method according to any of claims 1 -1 1 wherein said reporter molecule is a fluorescence reporter.

13. The method according to claim 12 wherein said fluorescence reporter is a fluorescent protein selected from the group comprising: BFP, CFP, GFP, YFP and RFP, and the mutants thereof.

14. The method according to any of claims 1-13 wherein said method detects at least one, two, three, four, five, six or seven biomarkers indicative of insulin activity.

15. The method according to claim 14 wherein said at least one biomarker is selected from the group consisting of: leptin and/or LDL cholesterol and/or triglycerides.

16. A cell array wherein said array comprises a plurality of identical assay preparations comprising a cell wherein said cell is transfected with a nucleic acid molecule comprising an insulin responsive promoter wherein said promoter is operably linked to a nucleic acid molecule that encodes a reporter molecule.

17. The array according to claim 16 wherein said insulin responsive promoter is the PEPCK promoter.

18. The array according to claim 17 wherein said PEPCK promoter is the human PEPCK promoter.

19. A method for the high through put screening of insulin sensitizing agents comprising the steps:

i) providing an array according to any of claims 16-18; ii) contacting the array with a plurality of agents to be tested for insulin sensitizing activity;

iii) collating the activity data in (ii) above;

iv) converting the collated data into a data analysable form; and optionally

v) providing an output for the analysed data.

20. A method to analyse a subject to determine whether said subject contains a serum factor that induces insulin resistance comprising the steps:

iii) analyzing the biological effect of said isolated biological sample on the activity of the insulin responsive promoter and at least one biomarker of insulin activity and comparing said insulin responsive promoter activity and biomarker to a control cell population which is obtained from a non-obese subject; and

iv) determining whether the subject is or is not insulin resistant.

21. A method to analyse and treat a human subject suspected of suffering from or having a predisposition to type 2 diabetes comprising the steps:

iii) analyzing the biological effect of said isolated biological sample on the activity of the insulin responsive promoter and at least one biomarker of insulin activity and comparing said insulin responsive promoter activity and biomarker to a control cell population which is obtained from a non-obese subject;

iv) determining whether the subject has a serum factor that induces insulin resistance; and

v) determining a treatment regime that will benefit the subject in controlling or preventing the onset of type 2 diabetes.

22. The method according to claim 20 or 21 wherein said insulin responsive promoter is the PEPCK promoter.

23. The method according to claim 22 wherein the PEPCK promoter is the human PEPCK promoter.

24. The method according to any of claims 20-23 wherein said biological sample is a body fluid sample.

25. The method according to claim 24 wherein said body fluid sample is a blood or serum sample.