A search for candidate genes for lipodystrophy, obesity and diabetes via gene expression analysis of A-ZIP/F-1 mice

doi:10.1016/S0888-7543(03)00024-7

Genomics

Volume 81, Issue 4, April 2003, Pages 378-390

https://doi.org/10.1016/S0888-7543(03)00024-7 Get rights and content

Abstract

Genome scans for diabetes have identified many regions of the human genome that correlate with the disease state. To identify candidate genes for type 2 diabetes, we examined the transgenic A-ZIP/F-1 mouse. This mouse model has no white fat, resulting in abnormal levels of glucose, insulin, and leptin, making the A-ZIP/F-1 mice a good model for lipodystrophy and insulin resistance. We used cDNA-based microarrays to find differentially expressed genes in four tissues of these mice. We examined these results in the context of human linkage scans for lipodystrophy, obesity, and type 2 diabetes. We combined 199 known human orthologs of the misregulated mouse genes with 33 published human genome scans on a genome map. Integrating expression data with human linkage results permitted us to suggest and prioritize candidate genes for lipodystrophy and related disorders. These genes include a cluster of 3 S100A genes on chromosome 1 and SLPI1 on chromosome 20.

Section snippets

Microarray analysis

Gene expression levels in four tissues of 7-month-old male A-ZIP/F-1 mice were compared to sex, age, and FVB/N strain-matched mice. The cDNA microarrays contained 11,382 PCR products for 5,110 named genes and 6272 ESTs. Total RNA from A-ZIP/F-1 and wildtype tissues were converted to cDNA and labeled with either cyanine-3 (Cy3) or cyanine-5 (Cy5) fluorescent dye. cDNAs from A-ZIP/F-1 and wildtype tissue were hybridized to microarrays, fluorescence intensity measured, and the ratio of Cy3 to Cy5

Discussion

Most success using genome scans to define genes involved in diabetes (diabetogenes) has come from examining the relatively rare, autosomal dominant form of the disease, maturity-onset diabetes of the young, for which the genetic lesion is a single locus [19], [20], [21]. In contrast, most forms of type II diabetes appear to be polygenic and are much more difficult to dissect genetically [1]. Large family and population studies examining genetic susceptibility to type II diabetes have identified

RNA isolation

Tissues from four 7-month-old male, heterozygous A-ZIP/F-1 and four equivalent wildtype controls (all FVB/N background) were collected. Mice had free access to food and water for approximately 12 h before being sacrificed by cervical dislocation. Tissues were quickly rinsed in physiological saline and flash frozen in liquid nitrogen. Entire hearts and livers were collected. Entire muscle tissue from both hind limbs was collected. Whole brain minus the cerebellum was collected. Frozen tissues

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On day 12, all animals received a single injection of cocaine (10 mg/kg) and were placed in the same test boxes for 15 min while their locomotion was measured. The map was designed as described previously (Mir et al., 2003). The human orthologs of the 84 genes differentially expressed in AP-1 expressing animals 24 h after cocaine, were identified and their coordinates in the human genome were obtained from the UCSC Genome Database (http://genome.ucsc.edu, May 2004 version of the human genome).
We have expressed A-FOS, an inhibitor of activator protein-1 (AP-1) DNA binding, in adult mouse striatal neurons. We observed normal behavior including locomotion and exploratory activities. Following a single injection of cocaine, locomotion increased similarly in both the A-FOS expressing and littermate controls. However, following repeated injections of cocaine, the A-FOS expressing mice showed increased locomotion relative to littermate controls, an increase that persisted following a week of withdrawal and subsequent cocaine administration. These results indicate that AP-1 suppresses this behavioral response to cocaine. We analyzed mRNA from the striatum before and 4 and 24 h after a single cocaine injection in both A-FOS and control striata using Affymetrix microarrays (430 2.0 Array) to identify genes mis-regulated by A-FOS that may mediate the increased locomotor sensitization to cocaine. A-FOS expression did not change gene expression in the basal state or 4 h following cocaine treatment relative to controls. However, 24 h after an acute cocaine treatment, 84 genes were identified that were differentially expressed between the A-FOS and control mice. Fifty-six genes are down-regulated while 28 genes are up-regulated including previously identified candidates for addiction including brain-derived neurotrophic factor and period homolog 1. Using a random sample of identified genes, quantitative PCR was used to verify the microarray studies. The chromosomal location of these 84 genes was compared with human genome scans of addiction to identify potential genes in humans that are involved in addiction.
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After incubation with a HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA), luminescence was quantified using an Image Station 440 CF (Kodak, Rochester, NY). In a recent transcriptome analysis comparing day/night rhythms in gene expression between wild-type and A-ZIP/F-1 mice (http://neuroscience.nih.gov/Baler/CIG30ms/supplementaldata.pdf), the Elovl3 gene displayed the most robust amplitude among only 11 transcripts whose circadian oscillations remained largely unaffected by the severe liver decompensation characteristic of this transgenic model (Table 1 ) (15, 16). The level of Elovl3 expression had been reported previously to increase by ∼4.8-fold in the CLOCK mutant mouse at ZT8 (4).
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Insulin is an important hormone produced and secreted from pancreatic beta cells. It plays a central role in the coordinated metabolism of the major sources of energy for the body; glucose and fat. Variation in insulin secretion during the fed and fasted states ensures optimal oxidation of glucose and storage of fat (lipid) during the fed state, and oxidation of fat and conservation of glucose during the fasted state. If this system is disturbed, adverse effects on energy supply to tissues and on circulating blood glucose and lipid levels can occur. Insulin resistance is the clinical state in which a normal or increased insulin level produces a reduced/impaired biological response. The cellular mechanisms involved in the development of insulin resistance and the role of diet are yet to be fully elucidated. Recent research has identified effects of fatty acids on both insulin signaling and on insulin secretion, as well as on transcription factors involved in the regulation of cellular lipid and energy homeostasis, which provide new insight into the mechanisms by which high-fat diets, and disturbances in fatty acid metabolism in obesity, could impair insulin sensitivity. This chapter presents the evidence for a possible role of dietary fat in the development of insulin resistance and type 2 diabetes.
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Regular articleA search for candidate genes for lipodystrophy, obesity and diabetes via gene expression analysis of A-ZIP/F-1 mice

Abstract

Section snippets

Microarray analysis

Discussion

RNA isolation

J. Lipid Res.

J. Clin. Invest.

Mol. Cell

Proc. Natl. Acad. Sci. USA

Trends Genet.

Proc. Natl. Acad. Sci. USA

J. Biol. Chem.

Nat. Genet.

J. Biol. Chem.

Am. J. Hum. Genet.

Am. J. Hum. Genet.

Diabetes

Nat. Genet.

Am. J. Hum. Genet.

J. Clin. Endocrinol. Metab.

Nat. Genet.

Diabetologia

Am. J. Hum. Genet.

Nat. Genet.

Nat. Genet.

Genetics of non-insulin-dependent (type-II) diabetes mellitus

Annu. Rev. Med.

A genome-wide search for human non-insulin-dependent (type 2) diabetes genes reveals a major susceptibility locus on chromosome 2

Nat. Genet.

Life without white fata transgenic mouse

Genes Dev.

Targeted expression of a toxin gene to adipose tissuetransgenic mice resistant to obesity

Genes Dev.

Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissuemodel for congenital generalized lipodystrophy

Genes Dev.

Dimerization specificity of the leucine zipper-containing bZIP motif on DNA bindingprediction and rational design

Genes Dev.

Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice

J. Clin. Invest.

Regular article
A search for candidate genes for lipodystrophy, obesity and diabetes via gene expression analysis of A-ZIP/F-1 mice