WO2012000058A1

WO2012000058A1 - Inheritance of metabolic dysfunction

Info

Publication number: WO2012000058A1
Application number: PCT/AU2011/000830
Authority: WO
Inventors: Margaret Morris; Sheaufang Ng
Original assignee: Newsouth Innovations Pty Limited
Priority date: 2010-07-02
Filing date: 2011-07-01
Publication date: 2012-01-05

Abstract

The invention relates to determining risk factors for metabolic disease, especially diabetes and related diseases, to screening compounds for therapeutic activity, and to therapeutic or dietetic intervention in metabolic disease, especially diabetes and related disease.

Description

Inheritance of metabolic dysfunction

Field of the invention

Background of the invention

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

The global prevalence of obesity is increasing across most ages in both sexes. This is contributing to the early emergence of type 2 diabetes (T2DM and its related epidemic^1,2. Having either parent obese is an independent risk factor for childhood obesity³. While detrimental impacts of diet-induced maternal obesity on adiposity and metabolism in offspring are well established³, the extent of any contribution of obese fathers is unclear, particularly the role of non-genetic factors in the causal pathway.

While some studies indicate a biological role of fathers in obesity of their offspring^4,5, most human obesity appears related to complex gene-environment interactions⁶. Further, while some alleles associated with obesity are inherited solely from fathers^7,8, parental environmental exposures can also affect offspring phenotype⁹, with potential to contribute to the rapid expansion of obesity. Susceptibility of metabolic phenotype to environmentally initiated change also extends into early life through developmental plasticity⁹. To date, in humans, it has been difficult to separate effects of paternal genetic makeup from those of the father's environmental exposures on offspring, including variations in paternal nutrition, metabolic and hormonal status, or obesity itself¹⁰. In mice however, males whose mothers consumed a high fat diet (HFD) were heavier, diabetic and insulin resistant, and produced second generation offspring who were insulin resistant, although not obese". Whether this is a consequence of paternal in utero exposure or their adult sequelae of obesity and diabetes is unclear. Further, it is not known whether fathers can initiate intergenerational transmission of obesity/metabolic diseases, induced indirectly or directly, such as through exposure to a HFD.

It would be advantageous to provide for the assessment of risk of impairment in glucose tolerance and/or insulin production, or conditions associated with same, especially in pre-adult life, thereby rnmimising the risk or severity of these conditions at this time, or later in adult life.

It would be advantageous to provide for a system or process for screening for compounds or compositions that can be used to minimise impairment in glucose tolerance and/or insulin production, or conditions associated with same, or risk of developing these conditions, especially for screening for compounds that can be used to restore or improve metabolic function by minimising or improving impairments in insulin production or secretion.

It would be advantageous to provide for methods of therapeutic and/or dietetic intervention in individuals having, or at risk of impairment in glucose tolerance and/or insulin production, thereby minimising the risk or severity of these conditions during early or adult life.

Summary of the invention In one embodiment there is provided a method for determining whether an individual has developed, or is at risk of developing impaired glucose tolerance or a condition associated with same including the following steps:

- providing a test sample from an individual for whom impaired glucose tolerance or risk of developing same, or a condition associated with same is to be determined; - assessing the test sample for the level of expression of a target gene in pancreatic islet tissue of the individual, thereby forming a test sample gene expression profile;

- providing a control gene expression profile containing data on the level of expression of the target gene in pancreatic islet tissue from an individual not having impaired glucose tolerance; - comparing the test sample gene expression profile with the control gene expression profile to identify a difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile; determining that the individual has developed, or is at risk of developing impaired glucose tolerance, or a condition associated with same where there is a difference between the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile. In another embodiment there is provided a method for detennining the likelihood of a male individual transmitting genetic material to offspring that is capable of forming an impaired glucose tolerance phenotype in offspring including the step of:

- assessing whether the male individual has one or more indications associated with a high fat diet, wherein an assessment that the male individual has one or more indications associated with a high fat diet deteraiines that the male individual is likely to transmit genetic material to offspring that is capable of forming an impaired glucose tolerance phenotype in offspring.

In another embodiment there is provided a method for determining whether an individual has a risk factor for impaired glucose tolerance or condition associated with same including the step of:

- assessing whether prior to conception of the individual the father of the individual had one or more indications associated with a high fat diet; wherein an assessment that prior to conception the father had one or more of the indications determines that individual has a risk factor for impaired glucose tolerance or condition associated with same.

In another embodiment there is provided a system, kit or article of manufacture for use in implementing an above described method.

In another embodiment there is provided a method for determining whether a compound is useful for minimising impaired glucose tolerance or risk of developing same in an individual, or for minimising the severity of a condition associated with impaired glucose tolerance in an individual including the following steps: - providing a test sample having a level of expression of a target gene as observed in pancreatic islet tissue from an individual having impaired glucose tolerance or risk of developing same;

- providing a test compound for which a use in minimising impaired glucose tolerance or risk of developing same in an individual, or for minimising the severity of a conditions associated with impaired glucose tolerance in an individual is to be determined;

- contacting the test sample with the test compound;

- assessing the test sample for the level of expression of the target gene, thereby forming a test sample gene expression profile; - providing a control gene expression profile containing data on the level of expression of the target gene in pancreatic islet tissue from an individual not having impaired glucose tolerance;

- comparing the test sample gene expression profile with the control gene expression profile to identify a difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile; determining that the test compound is a compound useful for minimising impaired glucose tolerance or risk of developing same in an individual, or for minimising the severity of a condition associated with impaired glucose tolerance in an individual where there is no difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile.

In certain embodiments, the compound is a small molecule, polynucleotide, or peptide. In other embodiments, the assay is performed in vivo, in a cell, or in a tissue sample. In yet other embodiments, the assay is a biochemical assay performed in vitro. Assays particularly well suited for use in the present invention are well known in the art. In a further embodiment there is provided a method for minimising the risk of an individual developing impaired glucose tolerance or condition associated with same including the following steps:

- selecting an individual having a risk factor for impaired glucose tolerance; - providing clinical and/or dietetic intervention to the individual, thereby minimising impaired glucose tolerance in an individual, or risk of developing same or condition associated with same; wherein said risk factor is a paternal parent having one or more indications of a high fat diet prior to conception of the individual.

In another embodiment there is provided a system, kit or article of manufacture including written instructions for implementing an above described method. In one particular embodiment, the kit comprises reagents for quantitative amplification of the selected biomarkers. Alternatively, the kit may comprise a microarray. In another particular embodiment, the kit comprises 2 or more antibodies. In some embodiments the kit comprises 1 or more probes. In other embodiments, the kits may contain 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 200, 500 or more probes.

The present invention provides therapeutic molecules for the treatment or prevention of impaired glucose tolerance. In one embodiment, the therapeutic molecules comprise antibodies or immunogenic fragments of antibodies. In other embodiments, the molecules comprise antisense oligonucleotides, siRNAs, microRNAs, or other nucleic acids or nucleic acid analogues well known in the art. In particular embodiments, the therapeutic molecules specifically hybridize or immunogenically bind to a biomarker selected from the group consisting of those listed in Table 3 and any other marker shown herein to be differentially expressed in individuals having or at risk of impaired glucose tolerance.

Brief description of the drawings / figures

Figure 1 High fat diet (HFD) led to adiposity/ glucose intolerance/ insulin resistance in fathers a, Body weight trajectories (control, HFD; n = 8, 9). b, Cumulative energy intake (control, HFD; n = 4, 5). c, Total energy intake (control, HFD; n = 4, 5). d, Blood glucose during glucose tolerance test (GTT) (2 g.kg^"1) (control, HFD; n = 8, 9; AUC, area under curve), e, Plasma insulin during GTT (2 g.kg^_1) (control, HFD; n = 7, 9). f, Blood glucose during insulin tolerance test (ITT) (1 U.kg^"') (control, HFD; n = 7, 9). Data expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ****P < 0.0005, versus control. P values for significant differences between paternal groups in repeated measure analysis are shown at top of panel. Figure 2 Female offspring demonstrate impaired glucose tolerance/ insulin secretion to a glucose challenge a, Body weight trajectories (control, HFD; n = 8, 9). b, Specific growth rate (SGR) (control, HFD; n = 8, 9). c, Cumulative energy intake (control, HFD; n = 9, 7). d, Blood glucose during GTT (2 g.kg^_1) at 6 weeks (control, HFD; n = 8, 8; AUC, area under curve), e, Plasma insulin during GTT (2 g-kg^"1) at 6 weeks (control, HFD; n = 8, 8). f, Blood glucose during GTT (2 g-kg^"1) at 12 weeks (control, HFD; n = 5, 7). g, Plasma insulin during GTT (2 g.kg^"1) at 12 weeks (control, HFD; n = 5, 7). h, Insulin resistance index (IRI = glucose concentration (mM) x insulin concentration (ng-mT¹) x 0.0417 ÷ 22.5) at 12 weeks, i, Blood glucose during ΠΤ (0.5 U.kg^"') at 11 weeks (control, HF; n = 8, 9). Data expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, versus control. P values for significant differences between paternal groups in repeated measure analysis are shown at top of panel.

Supplementary Figure 2. Molecular Network 1 of differentially expressed islet genes in HFD female offspring, P < 0.01 (Supplementary Table 3). This includes members of the enriched Jak-STAT signalling pathway. Molecules are represented as nodes, the biological relationship between two nodes is represented as an edge (line) and changed expression as up- (green) or down- (red) regulation.

Supplementary Figure 3. Molecular Network 2 of differentially expressed islet genes in HFD female offspring, P < 0.01 (Supplementary Table 3). This includes members of the enriched MAPK signalling pathway, Pik3c3, shown to be epigenetically modified by a paternal HFD and other molecules related to insulin action. Molecules are represented as nodes, the biological relationship as an edge (line) and changed expression as up-(green) or down-(red) regulation.

Supplementary Figure 4. Molecular Network 1 of differentially expressed islet genes of HFD female offspring, P < 0.05 (Supplementary Table 3). This includes tnf (MAPK signalling and apoptosis pathways); and Il13ra2 (Jak-STAT pathway), shown to be epigenetically modified by a paternal HFD. Molecules are represented as nodes, the biological relationship as an edge (line) and changed expression as up-(green) or down-(red) regulation.

Detailed description of the embodiments

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

The inventors have identified a population including individuals having impaired glucose tolerance in fasting conditions and individuals at risk of developing this impairment. These individuals may be characterised in terms of a newly identified risk factor for impaired glucose tolerance, and especially that each individual has been identified as having a male parent who, at the time of conception of the individual, had one or more indications of exposure to a high fat diet.

A comparison with individuals not having the risk factor has identified differences in gene expression in pancreatic islet tissue. These differences in gene expression ostensibly form one or more biomarkers for impaired glucose tolerance arising from impaired insulin production and/or secretion. It follows that these biomarkers can be used to identify individuals having impaired glucose tolerance or at risk of developing same.

Critically, the invention identifies that the population of individuals may not have characteristics normally associated with individuals having impaired glucose tolerance. For example, the individuals may have normal insulin sensitivity, normal body weight and normal adiposity. It follows that prior to this invention, the risk profile of these individuals for glucose tolerance impairments and conditions associated with same was not understood and the risk of developing these conditions could not be routinely detected. In this context the invention has highly significant application. Further, given the genetic predisposition according to this invention, it follows that conditions normally associated with impairments in glucose tolerance are likely to emerge much earlier in these individuals. In this context, the screening and intervention methods according to this invention are highly significant for rriinimising the risk of an individual developing an impairment in glucose tolerance in pre adult life and for minimising the severity of related conditions that but for the invention, may arise in adult life.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps. In one embodiment there is provided a method for determining whether an individual has developed, or is at risk of developing impaired glucose tolerance or a condition associated with same including the following steps:

- providing a control gene expression profile containing data on the level of expression of the target gene in pancreatic islet tissue from an individual not having impaired glucose tolerance; - comparing the test sample gene expression profile with the control gene expression profile to identify a difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile; determining that the individual has developed, or is at risk of developing impaired glucose tolerance, or a condition associated with same where there is a difference between the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile. In one embodiment there is provided a method for detennining whether an individual has impaired insulin production, or is at risk of developing impaired insulin production, the method including the following steps:

- providing a test sample from an individual for whom impaired insulin production or risk of developing same is to be determined;

- assessing the test sample for the level of expression of a target gene in pancreatic islet tissue of the individual, thereby forming a test sample gene expression profile;

- providing a control gene expression profile containing data on the level of expression of the target gene in pancreatic islet tissue from an individual not having impaired insulin production;

- comparing the test sample gene expression profile with the control gene expression profile to identify a difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile; detennining that the individual has developed, or is at risk of developing impaired insulin production where there is a difference between the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile.

In another embocliment, there is provided a method for determining whether an individual has impaired insulin secretion, or is at risk of developing impaired insulin secretion, the method including the following steps: - providing a test sample from an individual for whom impaired insulin secretion or risk of developing same is to be determined;

- providing a control gene expression profile containing data on the level of expression of the target gene in pancreatic islet tissue from an individual not having impaired insulin secretion; - comparing the test sample gene expression profile with the control gene expression profile to identify a difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile; determining that the individual has developed, or is at risk of developing impaired insulin secretion where there is a difference between the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile.

In another embodiment there is provided a method for determining whether an individual has impaired insulin granule exocytosis, or is at risk for developing impaired insulin granule exocytosis, the method including the following steps: - providing a test sample from an individual for whom impaired insulin granule exocytosis or risk of developing same is to be determined;

- providing a control gene expression profile containing data on the level of expression of the target gene in pancreatic islet tissue from an individual not having impaired insulin granule exocytosis;

- comparing the test sample gene expression profile with the control gene expression profile to identify a difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile; detennining that the individual has developed, or is at risk of developing impaired insulin granule exocytosis where there is a difference between the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile.

In one embodiment there is provided a method for detennining whether an individual has an insufficient insulin reserve in response to glucose stimulation, or is at risk for developing an insufficient insulin reserve in response to glucose stimulation, the method including the following steps:

- providing a test sample from an individual for whom sufficiency of insulin reserve is to be determined; - assessing the test sample for the level of expression of a target gene in pancreatic islet tissue of the individual, thereby forming a test sample gene expression profile;

- providing a control gene expression profile containing data on the level of expression of the target gene in pancreatic islet tissue from an individual having sufficient insulin reserve; - comparing the test sample gene expression profile with the control gene expression profile to identify a difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile; determining that the individual has developed, or is at risk of developing insufficient reserve where there is a difference between the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile.

In one embodiment there is provided a method for detennining whether an individual has a reduced mass of pancreatic beta cell tissue or a reduction of pancreatic beta cells having a large size, or is at risk for developing a reduced mass of pancreatic beta cell tissue, or is at risk for developing a reduction of pancreatic beta cells having a large size, the method including the following steps:

- providing a test sample from an individual for whom mass or size of beta cell tissue is to be determined;

- assessing the test sample for the level of expression of a target gene in pancreatic islet tissue of the individual, thereby forming a test sample gene expression profile; - providing a control gene expression profile containing data on the level of expression of the target gene in pancreatic islet tissue from an individual having a normal mass or size of beta cell tissue; ·

- comparing the test sample gene expression profile with the control gene expression profile to identify a difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile; determining that the individual has developed, or is at risk of developing a reduced mass of pancreatic beta cell tissue or a predominance of pancreatic beta cells having a reduced size, where there is a difference between the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile.

In one embodiment, the individual may not have impaired fasting blood glucose.

In another embodiment, the individual may have normal plasma insulin. In another embodiment the individual may be insulin sensitive. In this embodiment, the individual may have a normal insulin resistance index or normal insulin response in an insulin tolerance test.

In one embodiment, the individual may not be obese.

In another embodiment, the individual may have normal adiposity. In another embodiment, the individual may have normal muscle mass.

In another embodiment, the individual may have a normal fasting plasma leptin.

In another embodiment, the individual may have a normal triglyceride and/or NEFA concentration.

In another embodiment, the individual may be male or female, preferably female. In another embodiment the individual may be pre -adult, i.e an infant, child, juvenile, pre-adolescent or adolescent.

In one embodiment the above described methods are useful for determining whether an individual is at risk of developing a cardiovascular condition including micro and macroangiopathy. In one embodiment the test sample may be a tissue sample or a sample of body fluid.

Preferably the test sample is a sample of tissue, such as mesodermal, endodermal or ectodermal tissue. In one embodiment the tissue is muscle, fat (for example as obtained from liposuction) or liver tissue. More preferably the tissue sample is pancreatic islet tissue. More preferably the tissue sample contains pancreatic beta cells. Where the tissue is body fluid it may be whole blood or fractions thereof including buffy coat (thin layer of white blood cells that forms when blood is spun in a centrifuge), peripheral blood lymphocytes, serum or plasma. Other body fluids include urine, CNS, pancreatic juice (for example, as obtained from endoscopic retrograde cholangiopancreatography), duodenal juice (for example, as obtained using oesophagoduodenogastroscppy) and tear fluid.

In one embodiment the test sample may be assessed for an increase in the level of expression of a target gene.

In another embodiment, the test sample may be assessed for a decrease in the level of expression of a target gene. In one embodiment the target gene is involved in the calcium signalling pathway as defined by EGG 04020 attached herewith.

In one embodiment the target gene is involved in the MAPK signalling pathway as defined by KEGG04010 attached herewith.

In one embodiment the target gene is involved in the Wnt signalling pathway as defined by KEGG04310 attached herewith.

In one embodiment the target gene is involved in apoptosis as defined by KEGG04210 attached herewith.

In one embodiment the target gene is involved in the cell cycle as defined by KEGG04110 attached herewith. In one embodiment the target gene is involved in the Jak/STAT signalling pathway as defined by KEGG04630 attached herewith.

In one embodiment the target gene is involved in olfactory transduction as defined by KEGG04740 attached herewith.

In one embodiment the target gene is involved in the exocytosis of insulin granules. In one embodiment, the target gene is selected from the group of genes shown in any of the figures or tables included herein. In one embodiment, 2 or more, or all target genes are selected from the group of genes shown in any of the figures or tables included herein.

In one embodiment the target gene is M3ra2, Pik3c3 or Casp3.

In one embodiment, the target gene is:

H13ra2 (Accession number NM_133538) wherein level of expression is increased or up regulated relative to control;

Mrll (Accession number NM OO 11276) wherein level of expression is increased or up regulated relative to control;

Fos (Accession number NM_0221 7) wherein level of expression is decreased or down regulated relative to control;

Pdelc (Accession number NM_031078) wherein level of expression is increased or up regulated relative to control;

Pdelb (Accession number NM 022710) wherein level of expression is decreased or down regulated relative to control;

Irs2 (Accession number ENSRNOTOOO) wherein level of expression is decreased or down regulated relative to control;

Wnt9a (Accession number NM_0011057) wherein level of expression is decreased or down regulated relative to control;

Wnt9b (Accession number NM_0011070) wherein level of expression is decreased or down regulated relative to control;

Kiflc (Accession number NM_145877) wherein level of expression is increased or up regulated relative to control;

Pik3c3 (Accession number NM_022958) wherein level of expression is increased or up regulated relative to control; kbke (Accession number NM 0011088) wherein level of expression is increased or up regulated relative to control;

Albg (Accession number NM 022258) wherein level of expression is decreased or down regulated relative to control;

Egrl (Accession number NM 012551) wherein level of expression is decreased or down regulated relative to control;

Npas4 (Accession number NM_153626) wherein level of expression is decreased or down regulated relative to control;

Foxo6 (Accession number ENSRNOTOOO) wherein level of expression is decreased or down regulated relative to control;

1123a (Accession number NM_130410) wherein level of expression is decreased or down regulated relative to control;

Bcl2ll (Accession number NM_031535) wherein level of expression is decreased or down regulated relative to control;

Tnf (Accession number NM_012675) wherein level of expression is decreased or down regulated relative to control;

Cpa3 (Accession number NM_019300) wherein level of expression is increased or up regulated relative to control;

Fosb (Accession number ENSRNOTOOO) wherein level of expression is decreased or down regulated relative to control;

Rgs2 (Accession number NM_053453) wherein level of expression is decreased or down regulated relative to control; or

Ghrl (Accession number NM_021669) wherein level of expression is decreased or down regulated relative to control. In another embodiment, the level of expression of a combination of target genes is assessed. In this embodiment, one or more of the following combinations of target genes may be assessed.

Albg, Npas4, Pdelc; Albg, Cpa3, Egrl, Fos, Fosb, Ikbke, Jll3ra2, Ulrll, Rgs2; Egrl, Fos, Rgs2;

Cpa3, Egrl, Fos, Fosb, Ikbke, I113ra2, ttlrll, Npas4, Rgs2; Casp3, Foxo6, Irs2, Pik3c3;

Irs2, Ill3ra2, Tnf, Fos, Tllrll, BcUll, Wnt9a, Wnt9b; Irs2, ni3ra2, Tnf, Fos, Mrll, Bcttll, Wnt9a, Wnt9b, Pdelb, Pdelc; and III 3ra2, Tn Fos, Mrll, Bcl2ll, Wnt9a, Wnt9b.

In another embodiment, the one or more combinations of target genes are as shown in Table 3 herein.

The degree of change in expression of any of the genes or gene combinations disclosed herein can be in the order of 1.01 fold or greater. It will be appreciated by those skilled in the art that a relatively small change in the expression of a particvdar gene can have a significant physiological impact. It will also be appreciated that where an increase in gene expression is observed, the fold change will be a number greater than 1 (such that a fold change of 2 equates to a 2-fold increase in the level of gene transcription). Similarly, it will be appreciated that where there is a decrease in gene expression, the fold change will be a number less than 1 (such that a 0.5 fold change equates to a 2-fold decrease in the level of gene transcription). Exemplary levels of expression of each of the genes useful in the method are defined in the attached tables and figures.

In one embodiment, the target gene is methylated on a cytosine in the test sample but not methylated on the same cytosine in the control. In one embodiment, the level of expression of the target gene may be assessed by measuring the level of RNA transcribed from target gene. Preferably the RNA is transcribed in pancreatic islet tissue or in a pancreatic beta cell. The RNA may be pre-mRNA, mRNA, or micro RNA. In one embodiment, the level of expression of the target gene may be assessed by measuring the level of protein translated from the target gene. Preferably the protein is translated in pancreatic islet tissue or in a pancreatic beta cell.

In one embodiment, the indication associated with a high fat diet may be increased body weight, for example obesity.

In one embodiment, the indication associated with high fat diet may be increased liver mass. In one embodiment, the indication associated with high fat diet may be increased energy intake.

In one embodiment, the indication associated with high fat diet may be increased adiposity.

In one embodiment, the indication associated with high fat diet may be increased plasma leptin.

In one embodiment, the indication associated with high fat diet may be impaired glucose tolerance. In one embodiment, the indication associated with high fat diet may be impaired insulin sensitivity, or insulin resistance.

In another embodiment there is provided a method for detennining the likelihood of a male individual transmitting genetic material to offspring that is capable of forming an impaired glucose tolerance phenotype in offspring including the step of:

- assessing whether the male individual has one or more indications associated with a high fat diet, wherein an assessment that the male individual has one or more indications associated with a high fat diet determines that the male individual is likely to transmit genetic material to offspring that is capable of forming an impaired glucose tolerance phenotype in offspring.

In one embodiment, the mdication associated with high fat diet may be increased liver mass. In one embodiment, the mdication associated with high fat diet may be increased energy intake.

In one embodiment, the indication associated with high fat diet may be impaired glucose tolerance.

In one embodiment, the indication associated with high fat diet may be impaired insulin sensitivity, or insulin resistance. In another embodiment there is provided a method for determining the likelihood of a male individual transmitting genetic material to offspring that is capable of fonriing an impaired glucose tolerance phenotype in offspring including the steps of:

- providing a test sample from a male individual; - assessing the test sample to determine the methylation status of a target gene in the test sample, thereby forming a test sample methylation status profile;

- providing a control methylation status profile containing data on the methylation status of the target gene in a sample from a male individual known not to have produced offspring having an impaired glucose tolerance phenotype; - comparing the test sample methylation status profile with the control methylation status profile to identify a difference in the methylation status of the target gene as between the test sample methylation status profile and the control methylation status profile; deterrnining that the individual is likely to transmit genetic material to offspring that is capable of forming an impaired glucose tolerance phenotype in offspring where there is a difference between the methylation status of the target gene as between the test sample methylation status profile and the control methylation status profile.

In one embodiment the target gene is involved in the calcium signalling pathway as defined by KEGG 04020 attached herewith.

In one embodiment the target gene is involved in apoptosis as defined by KEGG04210 attached herewith. In one embodiment the target gene is involved in the cell cycle as defined by

KEGG04110 attached herewith. In one embodiment the target gene is involved in the Jak/STAT signalling pathway as defined by KEGG04630 attached herewith.

In one embodiment the target gene is involved in olfactory transduction as defined by KEGG04740 attached herewith. In one embodiment the target gene is involved in the exocytosis of insulin granules.

In one embodiment, the target gene is selected from the group of genes shown in any of the figures or tables included herein.

In one embodiment, 2 or more, or all target genes are selected from the group of genes shown in any of the figures or tables included herein. In one embodiment the target gene is M3ra2, Pik3c3 or Casp3.

In one embodiment, the target gene is:

IJ13ra2 (Accession number NM_133538) wherein level of expression is increased or up regulated relative to control;

Illrll (Accession number NM_0011276) wherein level of expression is increased or up regulated relative to control;

Fos (Accession number NM_022197) wherein level of expression is decreased or down regulated relative to control;

Pdelc (Accession number NM_031078) wherein level of expression is increased or up regulated relative to control; Pdelb (Accession number NM_022710) wherein level of expression is decreased or down regulated relative to control;

Wnt9a (Accession number NM_0011057) wherein level of expression is decreased or down regulated relative to control; Wnt9b (Accession number NM 0011070) wherein level of expression is decreased or down regulated relative to control;

Pik3c3 (Accession number NM_022958) wherein level of expression is increased or up regulated relative to control;

Ikbke (Accession number NM_0011088) wherein level of expression is increased or up regulated relative to control;

Albg (Accession number NM_022258) wherein level of expression is decreased or down regulated relative to control;

Egrl (Accession number NM_012551) wherein level of expression is decreased or down regulated relative to control;

Npas4 (Accession number NM l 53626) wherein level of expression is decreased or down regulated relative to control;

1123a (Accession number NM 130410) wherein level of expression is decreased or down regulated relative to control;

cUll (Accession number NM_031535) wherein level of expression is decreased or down regulated relative to control;

Cpa3 (Accession number NM 019300) wherein level of expression is increased or up regulated relative to control;

Fosb (Accession number ENSRNOTOOO) wherein level of expression is decreased or down regulated relative to control; Rgs2 (Accession number NM 053453) wherein level of expression is decreased or down regulated relative to control; or

Ghrl (Accession number NM 021669) wherein level of expression is decreased or down regulated relative to control. In another embodiment, the level of expression of a combination of target genes is assessed. In this embodiment, one or more of the following combinations of target genes may be assessed.

Albg, Npas4, Pdelc;

Albg, Cpa3, Egrl, Fos, Fosb, Ikbke, H13ra2, Mrll, Rgs2; Egrl, Fos, Rgs2;

Cpa3, Egrl, Fos, Fosb, Ikbke, Ill3ra2, Illrll, Npas4, Rgs2; Casp3, Foxo6, Irs2, Pik3c3;

Irs2, ni3ra2, Tnf, Fos, Illrll, BcUll, Wnt9a, Wnt9b;

Irs2, ni3ra2, Tnf Fos, Illrll, BcUll, Wnt9a, Wnt9b, Pdelb, Pdelc; and Ill3ra2, Tnf Fos, Illrll, BcUll, Wnt9a, Wnt9b.

It is understood that the invention includes homologs of the biomarkers disclosed in the specification and tables such that the invention- provides for detection, measurement and targetting of gene homologs in other organisms. In a further embodiment, the biomarkers may include variants of genes disclosed herein, such as splice variants and single nucleotide polymorphisms.

In one embodiment, in the above described methods, the target gene may be directly detected. This is otherwise known as a "direct detection" of the target gene to measure the level of expression of the target gene. In other embodiments, the level of expression of a molecule, the expression of which is modulated in accordance with the expression of the target gene is measured. This is otherwise known as an "indirect detection" of the target gene to measure the level of expression of the target gene. In another embodiment there is provided a system, kit or article of manufacture for use in implementing the above described methods.

The system, kit or article of manufacture may include a nucleic acid for hybridising to the target gene or complementary strand of the target gene by Watson-Crick base pairing. The nucleic acid may be an oligonucleotide or like probe having from about 8 to 100 or more nucleotides.

Preferably the nucleic acid has a sequence sharing at least 70% nucleotide sequence identity, preferably 75% identity, preferably 80% identity, preferably 85% identity, preferably 90% identity, preferably 95% identity, preferably 98 or 99 % identity with the sequence of the target gene. Percent sequence identity is determined by conventional methods, by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) as disclosed in Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453, which is hereby incorporated by reference in its entirety. GAP is used with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

The system, kit or article of manufacture may include reagents for use in the amplification or digestion or other manipulation of nucleic acids including an enzyme, such as a polymerase or a restriction endonuclease. The system, kit or article of manufacture may include reagents for quantitative Northern and Southern blotting, and microarray and PCR.

The system, kit or article of manufacture may include an antibody for detecting a gene product produced by a target gene. The system, kit or article of manufacture may include reagents for use in assays including immunoassays, chromatography and mass spectrometry. One example of an immunoassay that is particular preferred is FACS.

Various assays that can be used to detect the presence of a target protein in a sample include enzyme linked immunosorbent assay (ELISA), Western blot, Radio-immunoassay (RIA) and Immunohistochemical analysis.

In a preferred embodiment, the system, kit or article of manufacture may form a point-of- care or point-of-use diagnostic test. Point-of-care testing (POCT) is defined as diagnostic testing at or near the site of patient care or tissue or body fluid. The motivation behind POCT is to bring the test conveniently and immediately to the patient, which in turn increases the likelihood that the patient will receive the results in a timely manner. Therefore, treatment can immediately follow diagnosis.

A non-limiting example of a point-of-care test is a lateral flow test. Lateral flow tests, also known as lateral flow rmmunochromatographic assays are a simple device intended to detect the presence (or absence) of a target protein, peptide or fragment thereof in sample. Lateral flow tests are a form of immunoassay in which the test sample flows along a solid substrate, for example paper strip, via capillary action. After the sample is applied to the test it encounters a coloured reagent which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with an antibody or antigen. Depending upon the analytes present in the sample the coloured reagent can become bound at the test line or zone. One result of the antibody-antigen binding is that it can release some material that has been pre-bound to the antibody, such as gold or colloid nanoparticles. The colloid in turn may produce a visible line on the substrate which can be detected either by the naked eye or imaging device such as silicon photodiode or CCD device. Lateral flow tests can operate as either competitive or sandwich assays. In principle any coloured particle can be used, however in a preferred embodiment either latex (blue colour) or nanometer sized particles of gold (red colour) are used. The gold particles are red in colour due to localised surface plasmon resonance. Fluorescent or magnetic labelled particles can also be used in combination with an electronic reader to assess the test result. In the case of sandwich assay format the sample first encounters coloured particles which are labelled with antibodies raised to the target protein, peptide or fragment thereof. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the target protein, peptide or fragment thereof. The test line will show as a coloured band in positive samples. However, for competitive assays the sample first encounters coloured particles which are labelled with the target protein, peptide or fragment thereof or an analogue. The test line contains antibodies to the target/its analogue. Unlabelled target protein, peptide or fragment thereof in the sample will block the binding sites on the antibodies preventing uptake of the coloured particles and the test line will show as a coloured band in negative samples.

In another embodiment there is provided a method for detenmning whether a compound is useful for minimising impaired glucose tolerance or risk of developing same in an individual, or for minimising the severity of a condition associated with impaired glucose tolerance in an individual including the following steps:

- providing a test sample having a level of expression of a target gene as observed in pancreatic islet tissue from an individual having impaired glucose tolerance or risk of developing same;

- contacting the test sample with the test compound; - assessing the test sample for the level of expression of the target gene, thereby forming a test sample gene expression profile;

- providing a control gene expression profile containing data on the level of expression of the target gene in pancreatic islet tissue from an individual not having impaired glucose tolerance; - comparing the test sample gene expression profile with the control gene expression profile to identify a difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile; detennining that the test compound is a compound useful for minimising impaired glucose tolerance or risk of developing same in an individual, or for minimising the severity_, of a condition associated with impaired glucose tolerance in an individual where there is no difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile.

In a preferred embodiment of the invention, the initial test sample for identifying compounds useful for minimising impaired glucose tolerance or risk of developing the same, is a rat pancreatic islet cell culture model.

Isolation of primary islets is known in the art.'² Briefly, primary islets are isolated immediately post-sacrifice from the pancreas of 12 week old male Sprague Dawley rats. The pancreas is excised and placed in cold (4 °C) coUagenase (type XI, 1 mg/ml, Sigma, MO). The excised pancreas is digested by incubation at 37 °C for 15 min with Hank's Balanced Salt Solution (HBSS, Sigma) containing 20 mM HEPES buffer, then washed (3x) with cold HBSS (20 mM HEPES, 2 g 1 BSA). Undigested tissue is removed by filtration through a nylon mesh (pore size 500 urn, Lomb Scientific). The islet tissue is washed by centrifugation (4 x 10 sec, 1,100 rpm, 4 °C). Islets are purified using a Ficoll-Paque gradient (GE Healthcare, Chalfont St. Giles, UK) and handpicked under a low magnification microscope. The islets are plated in 12- well cell-bind tissue culture plates (Corning Life Sciences, 10 islets/well), and maintained at 37 °C in Krebs-Ringer Bicarbonate HEPES buffer (0.1% (w/v) BSA, 2.8 mM D-glucose) In further embodiments of the invention, the test sample is a cell culture representing any tissue system. For example, a cell-line which has been immortalised, a primary cell culture derived from live tissue biopsy or surgery. In further examples, the cell culture is derived from body fluid (eg, blood) such as peripheral blood mononuclear cells. The cell can human, rat, rabbit or mouse or other mammalian cell culture model common to the art. The cells in culture are contacted with a number of candidate test compounds as provided below. It is preferable that the cells are contacted with candidate test compounds no earlier than 24 hours after plating and at a time such that the confluency of the cells in the culture plate is such that cells are viable.

A variety of methods may be used to identify compounds that prevent or treat impaired glucose tolerance. Typically, an assay that provides a readily measured parameter is adapted to be performed in the wells of multi-well plates in order to facilitate the screening of members of a library of test compounds as described herein. Thus, in one embodiment, an appropriate number of cells can be plated into the cells of a multi-well plate, and the effect of a test compound on the expression of a biomarker can be determined. It is preferable that the test compound is provided to the cells in a suitable carrier solution. For example, an insoluble compound would be initially dissolved in a solution of 70 % EtOH prior to diluting to an appropriate concentration in wanned cell culture medium. The appropriate control for each test drug is therefore the carrier solution without the drug (in order to rule out any influence on gene expression from the carrier solution). The test compound is provided to the cells at a concentration that is equivalent to that known to have a physiological affect, as determined from the art. Ideally, a range of concentrations for each test compound will be used but in the initial screening test, a maximum dose (as determined by reference to the prior art) will be utilised.

The test compound is therefore provided to the cells at an appropriate concentration and in an appropriate carrier. Following addition of the test compound to the culture medium, the cells are incubated under normal conditions. Cells are harvested at a number of time points in order to capture transient, rapid, or slow changes to expression of the target genes. For example, 0, 1, 4, 6 12, 16, 20, 24, 36 and 48 hour time points may be appropriate.

The compounds to be tested can be any small chemical compound, or a macromolecule, such as a protein, sugar, nucleic acid or lipid. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a test compound in this aspect of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.

In one embodiment, high throughput screening methods are used which involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds. Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. In this instance, such compounds are screened for their ability to reduce or increase the expression of the biomarkers of the invention.

A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

Preparation and screening of combinatorial chemical libraries are well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides, vinylogous polypeptides, nonpeptidal peptidomimetics with glucose scaffolding, analogous organic syntheses of small compound libraries, nucleic acid libraries, peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see PCT/US96/10287), carbohydrate libraries (U.S. Pat. No. 5,593,853), small organic molecule libraries (U.S. Pat No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288, 14, and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.)

In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 96 modulators. If 1536 well plates are used, then a single plate can easily assay from about 100-about 1500 different compounds. It is possible to assay many plates per day; assay screens for up to about 6,000, 20,000, 50,000, or 100,000 or more different compounds is possible using the integrated systems of the invention.

The term "test compound" or "drug candidate" or "modulator" or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, peptide, circular peptide, lipid, fatty acid, siRNA, polynucleotide, oligonucleotide, etc., to be tested for the capacity to directly or indirectly modulate endometriosis biomarkers. The test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.

A "small organic molecule" refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons. The test compound can be, but is not limited to being from the class of drugs including sulphonylureas and rapid-acting secretagogues/insulinotropics (e.g., glibenclamide, glipizide, rapaglinide), biguanides (e.g., metformin), a-glucosidase inhibitors (e.g., acarbose), thiazolidinediones (e.g.,pioglitazone, rosiglitazon), gut hormones (e.g. ghrelin) In a further embodiment, the test compound can be from a class of drugs which include anti-inflammatory drugs, anti-oxidants, drugs affecting apoptosis, agents affecting fatty acid metabolism, DNA and chromatin modifying agents, for example histone deactylase inhibiting agents, histone methylase and DNA methylase modifying drugs.

In another embodiment of the invention, the test compounds .identified through in vitro methods as outlined above, are further tested for their utility in an animal model. In a preferred embodiment, the initial animal model tested is a model representing poor glucose tolerance and insulin resistance. For example, the animal can be but is not limited the group including the ob/ob mouse, db/db mouse, the streptozotocin diabetic mouse, the streptozotocin diabetic rat, rats that are rendered obese and diabetic through high fat diet, the Zucker diabetic fatty rat, the sand rat and the obese rhesus monkey. The compounds will be tested for their ability to improve glucose tolerance or return expression of the biomarkers disclosed herein to levels the observed in individuals with no impairement glucose tolerance.

The test compounds can be tested indirectly using Min6 cells (a pancreatic Islet cell line) or primary islets derived from the animals described above. For example, in short term studies, cells are incubated for 1 h at 37°C in 24- well plates under basal glucose (2.8 mmol/l) or high- glucose (25 mmol/l) conditions. Insulin secretion is assessed by radioimmunoassay (RIA) of the concentration of insulin in the culture medium. In a further embodiment of the invention, the effects of long term exposure to the compounds of interest can be assessed by overnight incubation with the compound in the presence and absence of high glucose conditions, after equilibrating cells under low glucose conditions. ^12,13

The test compounds can also be tested directly in the animal models of impaired glucose tolerance as described above. Glucose tolerance tests (GTT), and insulin secretion during the GTT can be measured. GTT is performed after a 15 h overnight fast; glucose (2 g-kg^'1 body weight) is injected intraperitoneally and samples collected for 2 hours. Insulin tolerance can de deterrnined by injecting insulin (Actrapid, Novo Nordisk; 1 U.kg^"') and following blood glucose concentrations over time. Further measures may include a hyperglycemic clamp (to assess insulin secretion capacity) whereby plasma glucose concentration is acutely raised above basal levels by a continuous infusion of glucose. This hyperglycemic plateau is maintained by a variable glucose infusion, based on the rate of insulin secretion and glucose metabolism. The glucose infusion rate is an index of insulin secretion and glucose metabolism. Following treatment with the test compounds, the pancreatic tissue from the animals can also be excised and stained using conventional histological methodologies. For example, detection of glucagon, insulin and for various markers including inflammatory and apoptotic markers can be performed to give an indication of the success of treatment with the test compound. In a further embodiment there is provided a method for mmimising the risk of an individual developing impaired glucose tolerance or condition associated with same including the following steps:

- selecting an individual having a risk factor for impaired glucose tolerance;

- providing clinical and/or dietetic intervention to the individual, thereby minimising impaired glucose tolerance in an individual, or risk of developing same or condition associated with same; wherein said risk factor is a paternal parent having one or more indications of a high fat diet prior to conception of the individual.

Examples

Example 1

We mated male Sprague Dawley founders fed either HFD/control diet (Table 1), with females consuming control diet (Supplementary Table 2). As expected, HFD males had increased body weight, energy intake, adiposity and plasma leptin, and liver mass (Fig. la-c, Table 1), but reduced skeletal muscle mass relative to body weight (P = 0.017). The HFD males were also glucose intolerant and insulin resistant, displaying elevated blood glucose and plasma insulin at fasting and during glucose tolerance test (GTT; Fig. ld-e). Homeostasis model assessment of insulin resistance index (HOMA-IR; Table 1) was increased and insulin tolerance test (ITT) response blunted (Fig. If). Paternal HFD diet did not alter litter size or sex ratios. Day-1 body weight of female offspring of HFD fathers tended to be reduced (6.61 ± 0.15 vs 7.08 ± 0.26g control; n = 9, 8; P = 0.07); males (7.40 ± 0.21 vs 7.30 ± 0.20g control; n = 9, 8; respectively P= 0.74), suggesting restricted fetal growth and a paternal programming effect of common etiology. We further assessed females after weaning onto a control diet. A paternal HFD did not alter body weight, specific growth rate, energy intake (Fig. 2a-c) or energy efficiency (not shown) in female offspring.

Paternal HFD did not alter adiposity, muscle mass, fasting plasma leptin, triglyceride or NEFA concentrations in adult female offspring (Table 1). Either obesity may emerge later or may not progress through the paternal lineage in rodents. We next assessed glucose tolerance and its two key determinants, insulin secretion and sensitivity. A paternal HFD did not alter fasting blood glucose (Fig. 2d, f) or plasma insulin (Fig. 2e,g) in female offspring, but increased the blood glucose rise (peak 13.6 ± 0.3 vs 12.3 ± 0.4 mM; P = 0.043) and reduced insulin secretion (peak 1.4 ± 0.3 vs 2.7 ± 0.4 ngjnl^'1; P = 0.016) during 6 week GTT (Fig. 2d-e). A similar pattern was observed at 12 weeks, but with further impairment of glucose tolerance, evidenced by a greater glucose peak (+10% to +23% vs control) and increased AUC_glucose (+9% to +19%; Fig. 2f-g) in paternal HFD offspring. Insulin secretion during the first 30 min after glucose (insulinogenic index¹⁵, AUC_{insuline(0-30)} ÷ AUC_{glucose(0-30min)}) was halved in offspring of HFD fathers (38.7 ± 5.8 vs 86.8 ± 7.3 ng-mmol^-1; P = 0.004); but their insulin resistance index and response during ITT were unaltered (Fig. 2h-i). We then examined islet/ -cell abundance, and performed genome-wide microarray analysis of isolated islets to explore the mechanisms of impaired insulin secretion. A paternal HFD reduced relative islet area (-23%; P = 0.04), mainly due to reduced large islets (-18%; P = 0.031) and tended to reduce β-cell area (P = 0.09 Table 1) in offspring, implying impaired β-cell replication. We also observed an increase in small islets (+6%; P = 0.034; Table 1), in offspring of HFD fathers, suggesting a compensatory response to maintain normal β-cell mass. While not wanting to be bound by hypothesis, we propose that limited β-cell reserve in female offspring of HFD fathers is sufficient to maintain normal fasting glucose and insulin levels, but inadequate to preserve glucose-stimulated insulin secretion and glucose tolerance.

A paternal HFD altered the expression of 77 genes (21 up-, 56 down-regulated, P < 0.001; Supplementary Table 1) in adult female offspring; 642 genes at P < 0.01 had enriched Gene Ontology (GO) terms belonging to regulatory pathways associated with insulin and glucose metabolism i.e., cation and ATP binding, cytostructure and intracellular transport. Broader KEGG pathway analysis of 2492 genes (P < 0.05) revealed involvement of calcium-, MAPK-, Wnt-signalling, apoptosis and cell cycle (Table 2). Molecular networks were also identified, including direct interactions between members of Jak-STAT signalling (Supplementary Fig. 2 & Supplementary Table 3) and other functionally enriched pathways (Supplementary Table 3). Overall these findings are consistent with the pancreas morphology and, while not wanting to be bound by hypothesis, may suggest impaired insulin granule exocytosis¹⁵'¹⁶. The greatest fold difference in gene expression was observed in Ill3ra2, part of the Jak-STAT signalling pathway (Table 2). To determine if epigenetic mechanisms are involved, we performed bisulfite sequencing of a region proximal to the transcription start site. Methylation at cytosine -960 was reduced in HFD offspring (8.9 ± 2.2 %) compared to controls (33.6 ± 4.0 %, P < 0.001). Methylation of Pik3c3 was altered on cytosines -190, -167, -74 and - 69 (P < 0.05). Both epigenetically modified genes are part of key molecular networks (Supplementary table 3, Supplementary Figs. 3, 4). Collectively, and while not wanting to be bound by hypothesis, these data suggest that the epigenome of offspring of HFD fathers is altered.

Our findings show that paternal exposure to HFD can induce a phenotype in offspring whereby under fasting conditions, insulin secretion and glucose tolerance in response to a glucose challenge is impaired, identifying an influential pathway. Notably, the impaired glucose tolerance and insulin secretion, in the absence of obesity, in these female offspring suggests that a paternal HFD acts to particularly target the endocrine pancreas and β-cells early in offspring. Our findings are distinct from other work which has shown impact of HFD on insulin resistance.

To our knowledge, this is the first direct demonstration in any species that a paternal environmental exposure, HFD consumption, can induce fetal growth restriction, coupled with intergenerational transmission of impaired glucose-insulin homeostasis in their offspring. While not wanting to be bound by hypothesis, the underlying mechanisms appear to include epigenetic modifications. These findings extend the concept of developmental and adaptive plasticity to include a paternal role in the early life origins of disease and amplification of the diabetes epidemic. Example 2

Combined analysis of Fat and islet array data

For fat analysis total RNA was extracted from Retroperitoneal WAT using a miRNeasy Mini kit (Qiagen Pty Ltd, Cat# 217004), according to the manufacturer's protocol. RNA concentration and purity were assessed spectrophotometrically (Shimadzu BioSpec-nano; Kyoto, Japan) and integrity ascertained by RNA gel electrophoresis.

Analysis of Fat and islet array data

In adipose tissue, a paternal HFD altered the expression of 218 genes (59 up-, 159 down- regulated, P < 0.05; in adult female offspring. Gene enrichment analysis for the fat tissue (P < 0.01 ) showed alterations to expression of a number of genes which also had altered expression in islet tissue (data not shown). This included Pik3c3 and Casp3.

METHOD SUMMARY

Animal experiments. Litters from 8 control and 9 HFD fathers were included; one animal per litter was used for each test. Experimental protocols were approved by the Animal Care and Ethics Committee, University of New South Wales, Australia.

Microarray gene expression analysis. Total islet mRNA was extracted using miRNeasy Mini kits (Qiagen). Samples from 6 control and 5 HFD offspring, each from different fathers, with RNA integrity number (RIN, Agilent >7.5) were selected for transcriptomics using Affymetrix GeneChip® Rat Gene ST 1.0 arrays. Statistical analyses. Phenotype data were analyzed using SPSS 16.0 after log- transformation, or square root-transformation unless raw data were normally distributed. Single time measurements were analyzed by two-tailed Student's t-test or Mann Whitney U test, and time-courses by repeated-measures ANOVA. References

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2 Pinhas-Hamiel, O. & Zeitler, P. The global spread of type 2 diabetes mellitus in children and adolescents. J Pediatr 146, 693-700, doi:S002234760401217X [pii] 10.1016/j.jpeds.2004.12.042 (2005).

3 Morris, M. Early life influences on obesity risk: maternal overnutrition and programming of obesity. Expert Review Endocrinol Metab 4, 625-637 (2009).

4 Tarquini, B., Tarquini, R., Perfetto, F., Cornelissen, G. & Halberg, F. Genetic and environmental influences on human cord blood leptin concentration. Pediatrics 103, 998- 1006 (1999).

5 Power, C, Li, L., Manor, O. & Davey Smith, G. Combination of low birth weight and high adult body mass index: at what age is it established and what are its determinants? J Epidemiol Community Health 57, 969-973 (2003).

6 Bouchard, C. Childhood obesity: are genetic differences involved? Am J Clin Nutr 89, 1494S-1501S, doi:ajcn.2009.271l3C [pii] 10.3945/ajcn.2009.27113C (2009).

7 Guo, Y. F. et al. Assessment of genetic linkage and parent-of-origin effects on obesity. J Clin Endocrinol Metab 91, 4001-4005 (2006).

8 Le Stunff, C, Fallin, D. & Bougneres, P. Paternal transmission of the very common class I INS VNTR alleles predisposes to childhood obesity. Nat Genet 29, 96-99 (2001).

9 Gluckman, P. D. et al. Towards a new developmental synthesis: adaptive developmental plasticity and human disease. Lancet 373, 1654-1657, doi:S0140-6736(09)60234-8 [pii] 10.1016/S0140-6736(09)60234-8 (2009).

10 Li, L., Law, C, Lo Conte, R. & Power, C. Intergenerational influences on childhood body mass index: the effect of parental body mass index trajectories. Am J Clin Nutr 89, 551-557, doi:ajcn.2008.26759 [pii] 10.3945/ajcn.2008.26759 (2009). Dunn, G. A. & Bale, T. L. Maternal high-fat diet promotes body length increases and insulin insensitivity in second-generation mice. Endocrinology 150, 4999-5009, doi:en.2009-0500 [pii] 10.1210/en.2009-0500 (2009). MA. Fryirs, PJ. Baiter, M Appavoo, BE. Tuch, F Tabet, AK. Heather, -A Rye. Effects of high- density lipoproteins on pancreatic beta-cell insulin secretion Arterioscler Thromb Vase Biol. 2010;30:1642-1648. M. Szabat & J. D. Johnson & J. M. Piret Reciprocal modulation of adult beta cell maturity by activin A and follistatin Diabetologia (2010) 53:1680-1689 Sone, H. & Kagawa, Y. Pancreatic beta cell senescence contributes to the pathogenesis of type 2 diabetes in high-fat diet-induced diabetic mice. Diabetologia 48, 58-67, doi:10.1007/s00125-004-1605-2 (2005). Henquin, J. C, Nenquin, M., Ravier, M. A. & Szollosi, A. Shortcomings of current models of glucose-induced insulin secretion. Diabetes Obes Metab 11 Suppl 4, 168-179, doi:DOMl 109 [pii] 10.1111/j.1463-1326.2009.01109.x (2009). Wang, Z. & Thurmond, D. C. Mechanisms of biphasic insulin-granule exocytosis - roles of the cytoskeleton, small GTPases and SNARE proteins. J Cell Sci 122, 893-903, doi:122/7/893 [pii] 10.1242/jcs.034355 (2009).

Supplementary Table 1

54e

Claims

1. A method for detennining whether an individual has developed, or is at risk of developing impaired glucose tolerance or a condition associated with same including the following steps: - providing a test sample from an individual for whom impaired glucose tolerance or risk of developing same, or a condition associated with same is to be determined;

- assessing the. test sample for the level of expression of a target gene in pancreatic islet tissue of the individual, thereby forming a test sample gene expression profile;

- providing a control gene expression profile containing data on the level of expression of the target gene in pancreatic islet tissue from an individual not having impaired glucose tolerance;

- comparing the test sample gene expression profile with the control gene expression profile to identify a difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile; determining that the individual has developed, or is at risk of developing impaired glucose tolerance, or a condition associated with same where there is a difference between the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile.

2. The method of claim 1 wherein the test sample is a sample of body fluid or tissue.

3. The method of claim 2 wherein the body fluid is one selected from the group consisting of: whole blood or a fraction thereof, peripheral blood lymphocytes, serum, plasma., urine, CSF, pancreatic juice, duodenal juice and tear fluid.

4. The method of claim 2 wherein the tissue is derived from mesodermal, endodermal or ectodermal tissue.

5. The method of claim 4 wherein the tissue is one selected from the group consisting of: muscle, fat, liver tissue or pancreatic islet tissue.

6. The method of claim 1 wherein the target gene is one selected from the group consisting of Pik3c3, Casp3, Ill3ra2, Mrll, Fos, Pdelc, Pdelb, Irs2, Wnt9a, Wnt9b, Kiflc, Pik3c3, Ikbke, Albg, Egrl, Npas4, Foxo6, 1123a, Bcl2U, Tnf, Cpa3, Fosb, Rgs2, Ghrl.

7. A method for detennining whether a compound is useful for minimising impaired glucose tolerance or risk of developing same in an individual, or for minimising the severity of a condition associated with impaired glucose tolerance in an individual including the following steps:

- providing a control gene expression profile containing data on the level of expression of the target gene in pancreatic islet tissue from an individual not having impaired glucose tolerance; - comparing the test sample gene expression profile with the control gene expression profile to identify a difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile; determining that the test compound is a compound useful for minimising impaired glucose tolerance or risk of developing same in an individual, or for minimising the severity of a condition associated with impaired glucose tolerance in an individual where there is no difference in the level of expression of the target gene as between the test sample gene expression profile and the control gene expression profile.

8. The method of claim 7 wherein the target gene is one selected from the group consisting of Pik3c3, Casp3, Π13τα2, ttlrll, Fos, Pdelc, Pdelb, Irs2, Wnt9a, Wnt9b, Kiflc, Pik3c3, Ikbke, Albg, Egrl, Npas4, Foxo6, 1123a, BcUll, Tnf, Cpa3, Fosb, Rgs2, Ghrl.