New approaches to gene discovery with animal models of obesity and diabetes

DNA-based approaches to the discovery of genes contributing to the development of type 2 diabetes have not been very successful despite substantial investments of time and money. The multiple gene-gene and gene-environment interactions that influence the development of type 2 diabetes mean that DNA approaches are not the ideal tool for defining the etiology of this complex disease. Gene expression-based technologies may prove to be a more rewarding strategy to identify diabetes candidate genes. There are a number of RNA-based technologies available to identify genes that are differentially expressed in various tissues in type 2 diabetes. These include differential display polymerase chain reaction (ddPCR), suppression subtractive hybridization (SSH), and cDNA microarrays. The power of new technologies to detect differential gene expression is ideally suited to studies utilizing appropriate animal models 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 be important in the disease process and provide novel therapeutic approaches. This paper will describe a new gene discovery, beacon, a novel gene linked with energy intake. As the functional characterization of novel genes discovered in our laboratory using this approach continues, it is anticipated that we will soon be able to compile a definitive list of genes that are important in the development of obesity and type 2 diabetes.

Novel genes in the liver of diabetic Psammomys obesus

Type 2 diabetes mellitus is a metabolic disease characterised by defects in insulin secretion and insulin action and disturbances in carbohydrate, fat and protein metabolism. Hepatic insulin resistance contributes to hyperglycemia and also leads to disturbances in fat metabolism in type 2 diabetes. Psammomys obesus is a unique poly genie animal model of type 2 diabetes and obesity, ideally suited for studies examining physiological and genetic aspects of these diseases. To identify metabolic abnormalities potentially contributing to the obesity and diabetes phenotype in P. obesus, indirect calorimetry was used to characterise whole body energy expenditure and substrate utilisation. Lean-NGT, obese-IGT and obese-diabetic animals were examined in fed and fasted states and following 14 days of dietary energy restriction. Energy expenditure and fat oxidation were elevated in the obese-IGT and obese-diabetic groups in proportion to body weight. Glucose oxidation was not different between groups. Obese-diabetic P. obesus displayed elevated nocturnal blood glucose levels and fat oxidation. Following 14 days of dietary energy restriction, body weight was reduced and plasma insulin and blood glucose levels were normalised in all groups. Glucose oxidation was reduced and fat oxidation was increased. After 24 hours of fasting, plasma insulin and blood glucose levels were normalised in all groups. Energy expenditure and glucose oxidation were greatly reduced and fat oxidation was increased. Following either dietary energy restriction or fasting, energy expenditure, glucose oxidation and fat oxidation were not different between groups of P. obesus. Energy expenditure and whole body substrate utilisation in P. obesus was similar to that seen in humans. P. obesus responded normally to short term fasting and dietary energy restriction. Elevated nocturnal fat oxidation rates and plasma glucose levels in obese-diabetic P. obesus may be an important factor in the pathogenesis of obesity and type 2 diabetes in these animals. These studies have further validated P. obesus as an ideal animal model of type 2 diabetes and obesity. It was hypothesised that many genes in the liver of P. obesus involved in glucose and fat metabolism would be differentially expressed between lean-NGT and obese-diabetic animals. These genes may represent significant factors in the pathophysiology of type 2 diabetes. Two gene discovery experiments were conducted using suppression subtractive hybridisation (SSH) to enrich a cDNA library for differentially expressed genes. Experiment 1 used cDNA dot blots to screen 576 clones with cDNA derived from lean-NGT and obese-diabetic animals. 6 clones were identified as overexpressed in lean-NGT animals and 6 were overexpressed in obese-diabetic animals. These 12 clones were sequenced and SYBR-Green PCR was used to confirm differential gene expression. 4 genes were overexpressed (≥1.5 fold) in lean-NGT animals and 4 genes were overexpressed (≥1.5 fold) in obese-diabetic animals. To explore the physiological role of these genes, hepatic gene expression was examined in several physiological conditions. One gene, encoding thyroxine binding globulin (TBG), was confirmed as overexpressed in lean-NGT P. obesus with ad libitum access to food, relative to both obese-IGT and obese-diabetic animals. TBG expression decreased with fasting in all animals. Fasting TBG expression remained greater in lean-NGT animals than obese-IGT and obese-diabetic animals. TBG expression was not significantly affected by dietary energy restriction. TBG is involved in thyroid metabolism and is potentially involved in the regulation of energy expenditure. Fasting increased hepatic site 1 protease (SIP) expression in lean-NGT animals but was not significantly affected in obese-IGT and obese-diabetic animals. SIP expression was not significantly affected by dietary energy restriction. SIP is involved in the proteolytic processing of steroid response element binding proteins (SREBP). SREBPs are insulin responsive and are known to be involved in lipid metabolism. Gene expression studies found TBG and SIP were associated with obesity and diabetes. Future research will determine whether TBG and SIP are important in the pathogenesis of these diseases. Experiment 2 used SSH and cDNA microarray to screen 8064 clones. 223 clones were identified as overexpressed in lean-NGT P. obesus and 274 clones were overexpressed in obese-diabetic P. obesus (p ≤0.05). The 9 most significantly differentially expressed clones identified from the microarray screen were sequenced (p ≤0.01). 7 novel genes were identified as well as; sulfotransferase related protein and albumin. These 2 genes have not previously been associated with either type 2 diabetes or obesity. It is unclear why hepatic expression of these genes may differ between lean-NGT and obese-diabetic groups of P. obesus. Subsequent studies will explore the potential role of these novel and known genes in the pathophysiology of type 2 diabetes.

Lichen flora of the Mount Donna Buang Scenic Reserve, Victoria

This report investigated the lichen flora of the Mt Donna Buang Scenic Reserve in Victoria, There were several aims: to describe the lichens of the region, to produce a pictorial key enabling field identification and to determine any distribution patterns. A floristic survey covering approximately 50 square km was undertaken to determine lichen diversity of the region generally. Lichens were sampled along roads, tracks, walking trails and in sections of bush, taking into account forest type and, particularly, areas that were lichen rich. Seventy-five lichen species in 43 genera and 27 families were identified and described from the region. An unknown, species H, also was described. Of the 76 lichen species, 22 were crastose and the remainder macrolichens. The best represented families were: Cladoniaceae (8 species), Hypogymniaceae (6), Lobariaceae (7), Lecideaceae (6), Pannariaceae (6) and Parmeliaccae (6). This study described 12 species (17%) which previously were not known for Victoria and which are a first record for the state. These include: Cladonia sarmentosa (J.D. Hook & Taylor) Dodge, Graphis librata Knight, Parmelinopsis neodamaziana (Elix & Johnston) Elix & Hale, Pertusaria novaezelandiae Szatala, Placopsis pardlina f. microphylla Lamb, Porina leptalea AX. Sm., Pseudocyphellaria ardesiaca Galloway, Trapeliopsis congregant (Zahlbr.) Brako, Menegazzia myriotrema (Mull. Arg.) P. James, Bunodophoron scrobiculatum (Church. Bab,) Wedin, Parmelia testacea Stirton and Menegazzia purpurascens S. Louwhoff sp. nov.. The last eight species are new to the mainland and, apart from Menegazzia purpurascens, previously were known only from Tasmania. Five main elements of distribution were identified for the lichen flora of the Mt Donna Buang Scenic Reserve: cosmopolitan, austral/australasian, paleotropical, pantropical and western pacific. The majority of species (68%) had austral/australasian distributions, eleven (16%) were endemic to Australia and nine (13%) occurred only in Tasmania , Victoria and New Zealand. A pictorial, dichotomous key was constructed for the lichen flora of the Mt Donna Buang Scenic Reserve. Previously, keys to the lichen flora of Tasmanian rainforests were suggested as appropriate to similar areas in Victoria, however, the Victorian forests include a significant sclerophyll element The key presented is specific for the study site but is appropriate to similar regions in Victoria and has been tested in a number of these areas. The key was designed to be ‘user-friendly’ so that the experienced and inexperienced alike are able to use it. A more detailed investigation of the lichen flora of the Mt Donna Buang Scenic Reserve was carried out in order to determine distribution. A total of 50 quadrats, each 20m x 20m in size, were sampled. Within each, the dominant vegetation type was determined and individuals were identified and location noted. The cover abundance of each lichen species on each individual tree was estimated using a modified Braun-Blanquet scale. A total of 710 trees, representing 13 different species, were examined. Nothofagus cunninghamii (Hook.) Oerst, Eucalyptus regnans R Mull., Acacia dealbata Link, A. melanoxylon R. Br., Hedycarya angustifolia A. Cunn. and Atherosperma moschatum Labill. were the six most common tree species encountered at the study site. Nothofagus cunninghamii supported the greatest lichen diversity (39 species), although most species occurred on less than 10% of the trees. The majority of lichens occurring on N. cunninghamii A. melanoxylon, A. dealbata and H. angustifolia were foliose or crustose, those on Ł. regnans fruticose and foliose and those on A moschatum crustose. Bunodophoron australe was the only lichen species at the study site to occur on one host, Nothofagus cunninghamiL Many occurred on a number of different hosts, but were most common on one particular tree species. The distribution of lichens at the study site was analysed with a rnultivariate statistical package (PATN) which dealt with ‘pattern analysis’. The program ‘SSH’ in PATN which uses the Bray-Curtis ordination technique, was used to create scatterplots displaying the degree of dissimilarity between quadrats in terms of presence/absence of lichen species. The program ‘TWAY’ in PATN was used to construct a two way table to display which lichen species occurred in each vegetation type. The pattern analysis revealed that the lichens of the Mt Donna Buang Scenic Reserve were not restricted to any particular forest type, but particular lichens, or groups of lichens, tended to predominate in certain vegetation communities. This concurs with work done by others in Tasmanian forests. Quadrats which were situated in cool temperate rainforest were grouped more closely with each other than with quadrats in other vegetation types. These also supported the greatest number of lichen species. This was not surprising since N. cunninghamii the dominant tree species in cool temperate rainforest, supported the greatest lichen diversity.