New Approaches to Gene Discovery with Animal Models of Obesity and Diabetes

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Ann. N.Y. Acad. Sci. 967: 403–413 (2002). © 2002 New York Academy of Sciences.

New Approaches to Gene Discovery with Animal Models of Obesity and Diabetes

GREG COLLIER,a,b KEN WALDER,a ANDREA DE SILVA,a JANETTE TENNE-BROWN,a ANDREW SANIGORSKI,a DAVID SEGAL,a LAKSHMI KANTHAM,a AND GUY AUGERTc

aMetabolic Research Unit, School of Health Sciences, Deakin University, Geelong, AustraliabAutogen Limited, Melbourne, Victoria, AustraliacMerck-Lipha, Lyon, France

ABSTRACT: DNA-based approaches to the discovery of genes contributing to thedevelopment of type 2 diabetes have not been very successful despite substantialinvestments of time and money. The multiple gene-gene and gene-environmentinteractions that influence the development of type 2 diabetes mean that DNAapproaches are not the ideal tool for defining the etiology of this complexdisease. Gene expression–based technologies may prove to be a more rewardingstrategy to identify diabetes candidate genes. There are a number of RNA-based technologies available to identify genes that are differentially expressedin various tissues in type 2 diabetes. These include differential displaypolymerase chain reaction (ddPCR), suppression subtractive hybridization(SSH), and cDNA microarrays. The power of new technologies to detect differ-ential gene expression is ideally suited to studies utilizing appropriate animalmodels of human disease. We have shown that the gene expression approach,in combination with an excellent animal model such as the Israeli sand rat(Psammomys obesus), can provide novel genes and pathways that may beimportant in the disease process and provide novel therapeutic approaches.This paper will describe a new gene discovery, beacon, a novel gene linked withenergy intake. As the functional characterization of novel genes discovered inour laboratory using this approach continues, it is anticipated that we will soonbe able to compile a definitive list of genes that are important in the developmentof obesity and type 2 diabetes.

KEYWORDS: animal model of obesity and diabetes; gene discovery; RNAapproaches to gene discovery

DNA-based approaches for the discovery of genes that contribute to the developmentof type 2 diabetes have not been very successful despite substantial investment oftime and money. The etiology of a complex disease such as type 2 diabetes is noteasily solved using current DNA-based approaches due to the multiple gene-geneand gene-environment interactions. Large numbers and/or variable combinations of

Address for correspondence: Greg Collier, Metabolic Research Unit, School of HealthSciences, Deakin University, Geelong, Australia. Voice: (613) 5227 2547; fax: (613) 5227 2170.

[email protected]

404 ANNALS NEW YORK ACADEMY OF SCIENCES

small gene defects leading to the final disease state also complicate these analysesand make identification of important genes difficult. However, there are alternativestrategies in the field of gene discovery in type 2 diabetes, such as RNA- (geneexpression) or proteomics-based approaches. Gene expression–based technologiesmay prove to be a more rewarding approach to identify diabetes candidate genes and,in combination with appropriate animal models, will be a powerful tool in under-standing the underlying mechanisms of human polygenic diseases such as diabetes.

A number of RNA-based technologies are available to identify genes that aredifferentially expressed in various tissues. These include differential display poly-merase chain reaction (ddPCR), suppression subtractive hybridization (SSH), andcDNA microarrays. Both ddPCR and SSH have been successfully used to identifynovel genes involved in energy metabolism. For example, in our laboratory, ddPCRwas used to identify beacon, a novel polypeptide involved in the regulation of energybalance, which is differentially expressed in the hypothalamus of obese/diabetic andlean/nondiabetic Israeli sand rats (Psammomys obesus).1 Recently, Steppan andcolleagues2 used the SSH technique to identify genes differentially expressed inadipocytes exposed to rosiglitazone, a PPARγ agonist used to treat type 2 diabetes.One of the identified genes, resistin, is a soluble molecule that may provide a linkbetween obesity and type 2 diabetes.

Representational difference analysis (RDA) is a powerful RNA-based techniquethat allows the comparison of two populations of mRNA and obtains clones of genesthat are expressed in one population and not in the other. RDA has the advantage ofanalyzing gene differences in the 5′-portion of cDNAs, allowing alternatively splicedvariants and importantly coding regions to be detected. Unfortunately, several roundsof subtraction are necessary to isolate rare transcripts. To overcome the technicallimitations of traditional subtractive methods, a new method has been developedcalled suppression subtractive hybridization (SSH).3 The key advantage of SSH isits ability, via second-order hybridization kinetics, to exponentially amplify andequalize both rare and abundant differentially expressed transcripts while suppressingsequences that are common in both populations. The generated cDNAs can then bedirectly inserted into a variety of cloning strategies such as a T/A cloning vector. Afterpicking, either the clones can be screened using cDNA dot blots to eliminate falsepositives and then sequenced or alternatively clones can be used to generate sub-tracted cDNA libraries for future microarray analysis. Generating cDNA librariesusing SSH is relatively quick and, more importantly, it is species- and tissue-specific.

Techniques such as SSH and ddPCR can be used to generate target genes usingstandard laboratory equipment and at a reasonable cost. The major disadvantages ofthese methods are that they are labor-intensive and they identify only small numbersof differentially expressed genes. A further limitation is the binary nature of thecomparisons using these techniques, which makes them unsuited to complex experi-ments such as time-course or dose-response studies. However, cDNA microarrayexperiments can provide gene expression data for thousands of genes and from largenumbers of experimental samples. This technology is ideally suited to complex,multivariate analyses that generate detailed expression profiles, which in turn havethe potential to increase our understanding of the underlying mechanisms associatedwith a disease. Current analysis techniques tend to focus on clustering algorithmsthat identify genes exhibiting similar expression patterns.4 Recent advances in the

405COLLIER et al.: RNA APPROACHES TO NOVEL GENE DISCOVERY

analysis of microarray data have led to the construction of models to identify groupsof genes involved in selected physiological processes.5 Current progress in this fieldsuggests that cDNA microarray technology will facilitate the identification of keygenes and pathways involved in the pathogenesis of type 2 diabetes.

The power of these new technologies to detect differential gene expression isideally suited to studies utilizing appropriate animal models of human diseasebecause very few polygenic, outbred rodent models of obesity and type 2 diabetesexist. One such model, Psammomys obesus (Israeli sand rat) has been studiedextensively in our laboratory.1,6–12 Psammomys obesus are gerbil-like rodents foundin the desert areas of the Middle East and North Africa. They remain lean and freefrom diabetes in their native habitat, subsisting on a diet composed mainly of saltbush (Atriplex halimus).13 When taken into the laboratory, however, and allowedfree access to standard rodent chow, varying degrees of obesity, insulin resistance,and type 2 diabetes develop.6,14 Adult Psammomys obesus have a wide range ofbody weight and body fat content that forms a continuous distribution. It is theheterogeneous response to a relatively energy-dense diet that makes Psammomysobesus more analogous to the pattern of human obesity and type 2 diabetes thanhomogenous single-gene animal models.

A number of metabolic disturbances have been identified in obese, diabeticPsammomys obesus relative to their lean littermates: hyperglycemia, insulin resis-tance, hyperphagia, obesity, and dyslipidemia.6–8,12 Hepatic insulin resistance15 anda defective insulin receptor signaling pathway have been reported in diabeticPsammomys obesus.16,17 These animals also have hyperproinsulinemia, reducedpancreatic insulin storage capacity, and beta cell apoptosis.18,19 Increased expres-sion of protein kinase (PKC epsilon) in skeletal muscle of Psammomys obesus hasrecently been reported. This overexpression of PKC epsilon may be causally relatedto the development of insulin resistance in these animals, possibly by increasing thedegradation of insulin receptors.20 Elevated levels of leptin concentrations in obesediabetic animals have been reported,10 with resistance to the effects of peripheral(intraperitoneal) leptin administration in obese, but not lean, animals.11 As a species,Psammomys obesus appear to be relatively insensitive to the effects of leptin admin-istration and it is possible that the leptin resistance exhibited in this study may becontributing to the development of obesity.

The body weight distribution in Psammomys obesus approximates a normal distri-bution and closely resembles that observed in human populations. Animals above the75th percentile for body weight have increased body fat content and a greater risk ofdeveloping diabetes. Increased visceral fat content was also associated with elevatedblood glucose and plasma insulin concentrations.12 Cross-sectional analysis of theanimal population of Psammomys obesus reveals heterogenous distributions of bloodglucose, plasma insulin, and body weight.6,12 It is this aspect of the development ofdiabetes and obesity in Psammomys obesus that is of most importance because thesedistributions are almost identical to the patterns observed in cross-sectional studies ofhuman populations, including the inverted U-shaped relationship between bloodglucose and insulin concentrations termed “Starling’s curve of the pancreas”.21,22

Moreover, we have demonstrated that genetic factors account for 51% of the variationin body weight and 23–26% of the variation in blood glucose and plasma insulinconcentrations in Psammomys obesus.12 Recently, during the sequential development


of insulin resistance and diabetes in Psammomys obesus, it has been shown thatvarious disturbances occur in plasma lipid profile and lipoprotein composition, as wellas in liver cholesterol metabolism.23 Thus, Psammomys obesus represents an excellentanimal model of obesity and type 2 diabetes that exhibits a phenotypic pattern closelyresembling that observed in human population studies.

In our laboratory, the use of modern technologies to detect differential geneexpression, in combination with an excellent animal model such as Psammomysobesus, has resulted in the identification of novel genes important in the develop-ment of type 2 diabetes. Using ddPCR, the beacon gene was identified as a geneproduct overexpressed in the hypothalamus of obese, diabetic animals. Furtherstudies in a larger group of animals demonstrated that the beacon gene wasexpressed in the hypothalamus in direct proportion to the body fat content.1 Beaconis expressed ubiquitously throughout the body and encodes a small protein of 73amino acids. The human beacon gene consists of 2194 nucleotides arranged into5 exons and 4 introns and has been mapped to chromosome 19. The Psammomysobesus beacon gene is composed of 4 exons, has a shorter 5′-untranslated region, andconsequently lacks the first exon present in the human gene1 (Genbank Accession:AF318186).

Candidate gene identification is only the first step in determining the gene’simportance in the development of diabetes. It is necessary to determine the physio-logical function of the protein produced by the novel gene and to validate the poten-tial of this protein as a therapeutic target. Recent years have seen the addition ofvarious new tools to the existing classical biochemical techniques for understandingthe role of proteins of unknown function. The research tools and strategies for func-tional studies that have been applied in our own and various other laboratories areoutlined in FIGURE 1 and will be discussed here in the context of functional analysisof the beacon protein.

Bioinformatic tools can assist in the recognition of certain structural elements andconsensus sequence motifs present in the putative amino acid sequence. For example,it is possible to predict the regions corresponding to transmembrane domains, hydro-philic and hydrophobic regions, and sequence motifs for posttranslational modifica-tions such as phosphorylation, glycosylation, isoprenylation, and geranylation. Whenpresent, the consensus targeting sequences for secretion or those that direct the newlysynthesized proteins to different subcellular compartments such as mitochondria,peroxisomes, endoplasmic reticulum, and nucleus can also be detected using a num-ber of commercially or publicly available software tools. It is rare, however, that thebioinformatics of a protein will provide sufficient clues to indicate the function of aprotein. Implementation of hypothesis-based experimental strategies are oftennecessary to arrive at a deeper understanding of the precise function of a gene product.

In our laboratory, a research strategy is designed for each selected target based onthe information obtained from bioinformatics and the physiological context in whichthe gene is discovered. The functional studies are directed to understand the functionat a molecular level with isolated proteins or at a cellular level by examining thealtered biochemical or metabolic functions of cells as a consequence of inhibition oroverexpression of the gene of interest. Where possible, we apply in vivo studieseither by direct administration of the protein or by gene delivery into the animalsusing viral expression vectors.


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It is helpful to have the purified protein available not only as a reagent for in vivoand in vitro experiments, but also for raising antibodies. It is relatively more simpleto genetically engineer bacterial strains and produce recombinant proteins than toisolate native proteins from tissue sources. Beacon was predicted to be a soluble pro-tein with no obvious targeting signals and transmembrane domains. We have clonedbeacon into a bacterial expression vector designed for expression of GST fusion(Glutathione S-transferase) proteins with a built-in cleavage site for subsequentremoval of the GST tag with thrombin. The expressed GST-beacon fusion proteinwas separated from the rest of the proteins in the bacterial lysate by affinity bindingof the GST tag to Glutathione Sepharose beads. Under optimized conditions of ex-pression and purification, we have been able to produce 25 mg of pure homogeneousbeacon protein per liter of culture.

Monoclonal and polyclonal antibodies make valuable tools for several functionalstudies such as immunoblotting, immunohistochemistry, and immunoprecipitation.Preparation of monoclonal antibodies is labor-intensive; however, once the clonesare established, new stocks can be easily prepared. A monoclonal antibody specificfor beacon was produced and used in immunohistochemical localization studies inbrain sections. Beacon was highly expressed in a region of the hypothalamus calledthe retrochiasmatic nucleus,1 an area implicated in the regulation of energy balance.

Work involved in producing polyclonal antibodies is less than for monoclonalantibodies and, as multiple epitopes are recognized, is in general more effective inrecognizing antigens present in native or denatured conformations and for immuno-precipitation of antigens from dilute test samples. It is known that small and highlyconserved proteins make weak antigens and therefore we chose to immunize rabbitswith beacon conjugated to a much larger diphtheria toxoid protein. The conjugatedbeacon aggregates produced a good immune response. To obtain pure beacon-specificantibodies, we bound beacon protein to chemically activated “Aminolink” solidbeads and passed the sera through the matrix followed by elution of bound beacon-specific antibodies. The sera purified on the affinity matrix were highly enriched andbeacon-specific. On Western blots, beacon was detectable in several of thePsammomys obesus tissues such as brain, liver, kidney, skeletal muscle, and adiposetissue. The functional role of beacon in peripheral tissues is yet to be assessed.

A variety of eukaryotic plasmid and viral (retroviral or adenoviral) expressionvectors were employed to overexpress genes of interest in insulin responsive celllines such as differentiated 3T3-L1 (adipocyte), C2C12 or L6 (muscle), HepG2 orH4IIE (hepatocyte), or min6 (pancreatic). As an alternative, carefully designed anti-sense oligonucleotides are used to inhibit endogenous expression of genes of interest.Studies can be conducted in which the gene of interest is overexpressed or inhibited,and the cell’s response to insulin, such as changes in glucose uptake, glycogensynthesis, or lipogenesis, is examined. This information can reveal the nature ofmetabolic pathways in which the gene of interest is involved. When the studies incell culture models are completed, our plan is to apply the same vectors and anti-sense oligonucleotides that produce an effect for in vivo studies in live animals andvalidate the observed effects.

Almost all proteins fulfill their functional role via interaction with other proteins.Identification of interacting proteins and any knowledge of their functional propertiescan also provide clues for positioning the candidate protein in a defined signal trans-


FIGURE 2. Animals were treated with 3, 15, or 30 µg beacon per day for 7 days.Cumulative food intake (A) and change in body weight (B) are shown. *p < 0.05 vs. salinegroup.


FIGURE 3. Animals were treated with 15 µg of beacon or 15 µg of both beacon andNPY per day for 7 days. Cumulative food intake (A) and change in body weight (B) areshown. *p < 0.05 vs. saline group; #p < 0.05 vs. NPY group; **p < 0.05 vs. NPY, beacon,and saline groups.


duction pathway. Protein-protein interactions can be identified using coexpressiontechnologies such as the yeast two-hybrid system24–27 or biophysical techniquessuch as Fluorescence and Bioluminescence Resonance Energy Transfer28,29 orBiacore.30 All of these techniques aim to identify interactions between two proteins.Using the yeast two-hybrid method, we have identified a novel kinase that interactswith beacon. The events of the intracellular kinase cascade play a significant role inthe signaling pathway downstream to binding of insulin to its receptor. The novelkinase identified in our laboratory may thus provide a novel candidate pathway inthe pathogenesis of obesity and type 2 diabetes. Importantly, this pathway nowprovides a target for the development of chemical interventions to decrease beaconaction, which can then be developed further as a potential therapeutic agent in thetreatment of obesity.

The physiological function of beacon in the regulation of energy balance wasconfirmed by direct administration in vivo in Psammomys obesus. Intracerebro-ventricular (ICV) administration of beacon for 7 days resulted in a dose-dependentincrease in food intake and body weight gain (FIG. 2) and in a 2-fold increase inhypothalamic expression of NPY.1 In addition, coadministration of beacon (15 µg/day) and NPY (15 µg/day) had a synergistic orexigenic effect, resulting in a dramaticincrease in food intake and body weight gain that was significantly greater than thesum of the responses seen when the peptides were administered separately (FIG. 3).1

The substantial body weight gain after ICV administration of beacon was due toincreased fat accumulation.31 Indirect calorimetry was used to investigate effects ofICV beacon administration on energy expenditure, physical activity, and substrateutilization. Beacon treatment had no effect on these parameters in Psammomysobesus and did not affect feed efficiency.31 Therefore, the actions of beacon in thehypothalamus result in increased food intake, which in turn causes accumulation ofbody fat leading to excessive body weight gain. The observation of dramatic physio-logical effects of beacon in vivo strongly supports the hypothesis that beacon playsa major role in the regulation of energy balance in Psammomys obesus. Thus, beaconrepresents an interesting new target with great potential for the design of therapeuticagents for obesity and type 2 diabetes.

In summary, with the combination of an excellent animal model for the study ofdiabetes in Psammomys obesus and RNA-based technologies such as ddPCR, wediscovered a novel gene, beacon. After establishing that the beacon gene wasexpressed in the hypothalamus in direct proportion to body fat content, it wasnecessary to determine the physiological function of the gene product. Using a raftof technologies, the functional role of the gene protein was established and inter-action with other proteins examined. Confirmation of the function of the novel pro-tein occurred by direct application in vivo in an animal model. Finally, with detailsof the pathway of beacon action defined, it is hoped that this information will be usedfor high-throughput screening in the quest for a new therapeutic approach to obesityand diabetes.

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New Approaches to Gene Discovery with Animal Models of Obesity and Diabetes

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