Laser capture microdissection and quantitative-PCR analysis.

Laser capture microdissection (LCM) provides an efficient and precise method for the sampling of single cells or subgroups of cells in heterogeneous tissues such as the brain. We have recently applied LCM coupled with microarray analysis for the examination of gene expression in the hypothalamic arcuate nucleus of free fed versus fasted rats. We successfully used QPCR analysis to validate our findings and to confirm the regulation of several known neuropeptides. We show that the combination of LCM and QPCR analysis provides a reliable, fast, and efficient alternative to semiquantitative in situ hybridization analysis of gene expression.

Characterization of beta-cell mass and insulin resistance in diet-induced obese and diet-resistant rats.

The selectively bred diet-induced obese (DIO) and diet-resistant (DR) rats represent a polygenetic animal model mimicking most clinical variables characterizing the human metabolic syndrome. When fed a high-energy (HE) diet DIO rats develop visceral obesity, dyslipidemia, hyperinsulinemia, and insulin resistance but never frank diabetes. To improve our understanding of the underlying cause for the deteriorating glucose and insulin parameters, we have investigated possible adaptive responses in DIO and DR rats at the level of the insulin-producing beta-cells. At the time of weaning, DR rats were found to have a higher body weight and beta-cell mass compared to DIO rats, and elevated insulin and glucose responses to an oral glucose load. However, at 2.5 months of age, and for the remaining study period, the effect of genotype became evident: the chow-fed DIO rats steadily increased their body weight and beta-cell mass, as well as insulin and glucose levels compared to the DR rats. HE feeding affected both DIO and DR rats leading to an increased body weight and an increased beta-cell mass. Interestingly, although the beta-cell mass in DR rats and chow-fed DIO rats appeared to constantly increase with age, the beta-cell mass in the HE-fed DIO rats did not continue to do so. This might constitute part of an explanation for their reduced glucose tolerance. Collectively, the data support the use of HE-fed DIO rats as a model of human obesity and insulin resistance, and accentuate its relevance for studies examining the benefit of pharmaceutical compounds targeting this disease complex.

The imprinted gene neuronatin is regulated by metabolic status and associated with obesity.

Using restriction fragment differential display (RFDD) technology, we have identified the imprinted gene neuronatin (Nnat) as a hypothalamic target under the influence of leptin. Nnat mRNA expression is decreased in several key appetite regulatory hypothalamic nuclei in rodents with impaired leptin signaling and during fasting conditions. Furthermore, peripheral administration of leptin to ob/ob mice normalizes hypothalamic Nnat expression. Comparative immunohistochemical analysis of human and rat hypothalami demonstrates that NNAT protein is present in anatomically equivalent nuclei, suggesting human physiological relevance of the gene product(s). A putative role of Nnat in human energy homeostasis is further emphasized by a consistent association between single nucleotide polymorphisms (SNPs) in the human Nnat gene and severe childhood and adult obesity.

A comparison of glycemic control, water retention, and musculoskeletal effects of balaglitazone and pioglitazone in diet-induced obese rats.

Agonists of Perioxisome Proliferator-Activator Receptor gamma (PPARgamma), which work as insulin sensitizers, are approved for type 2 diabetes. However, adverse effects, such as oedemas, infarctions, and increased fracture rates, limit their applicability. We performed a head-to-head comparison of equipotent glucose lowering concentrations of the partial PPARgamma agonist balaglitazone and the full agonist pioglitazone in male diet-induced obese rats, to investigate effects on bone formation, fluid retention and fat accumulation. Sixty male dio induced obese rats were divided into five categories: vehicle, pioglitazone 10 mg/kg, pioglitazone 30 mg/kg, balaglitazone 5 mg/kg, balaglitazone 10 mg/kg. At day -7, 21 and 42 fasting serum samples were collected and whole body tissue composition was evaluated by MR scanning. Food intake and bodyweights were monitored during the study period. At day 42, an oral glucose tolerance test was performed to evaluate glucose homeostasis in the rats. During oral glucose tolerance test both pioglitazone and balaglitazone lowered baseline glucose and maintained the suppression during the oral glucose tolerance test. Both lowered basal insulin, peak insulin secretion and total insulin during oral glucose tolerance test. Both drugs increased bodyweight, although this was more pronounced in the pioglitazone 30 group. MR scans of body fat and water showed that all treatment groups increased their fat mass, whereas only the pioglitazone 30 group accumulated water. Pioglitazone treatment led to reduction of the bone formation marker osteocalcin, whereas balaglitazone treatment did not affect it. Balaglitazone is a novel PPARgamma agonist, which potently lowers glucose levels, while it neither affects fluid retention nor bone formation parameters.

Dissociation of antihyperglycaemic and adverse effects of partial perioxisome proliferator-activated receptor (PPAR-gamma) agonist balaglitazone.

Balaglitazone is a novel thiazolidinedione in clinical development for the treatment of type 2 diabetes. Common side effects associated with PPARgamma receptor agonists are weight gain, oedema and adipogenesis. Balaglitazone is a selective partial PPARgamma agonist and it has been speculated that such compounds have a more favourable safety margin than full agonists. We have compared impact of equi-efficacious antihyperglycaemic doses of balaglitazone with full PPARgamma agonist rosiglitazone on body fluid accumulation, cardiac enlargement, and adipogenesis. Equi-efficacious antihyperglycaemic doses (ED(90)) of balaglitazone (3 mg/kg/day) and rosiglitazone (6 mg/kg/day) were determined in male diabetic db/db mice. In adult male rats treated for up to 42 days, feeding, drinking, anthropometry, and plasma volumes were measured. Total plasma volume was measured with dye dilution technique. Compared to vehicle, rosiglitazone consistently increased food intake throughout the 42 day treatment period. In contrast, balaglitazone increased food intake in the last week of the experiment. However, both rosiglitazone and balaglitazone increased water intake. After 42 days, rosiglitazone treated rats displayed significantly elevated adiposity. Rosiglitazone increased total blood and plasma volumes throughout the treatment. Twenty-one days of balaglitazone treatment had no significant impact on blood or plasma volumes, whilst 42 days of balaglitazone increased plasma volume but to a significantly lesser extent than seen for rosiglitazone (vehicle: 46.1+/-1.5; balaglitazone: 50.8+/-1.21; rosiglitazone: 54.6+/-1.6 ml/kg). Heart weight was significantly elevated only in rosiglitazone treated animals. At doses inducing comparable antihyperglycaemic control, the full PPARgamma agonist, rosiglitazone, induces more pronounced body fluid retention and heart enlargement than seen for the partial PPARgamma agonist, balaglitazone. Thus, partial agonists may pose safer alternative to current anti-diabetic therapy with full PPARgamma agonist.

Insulin-like growth factor-1/insulin bypasses Pref-1/FA1-mediated inhibition of adipocyte differentiation.

Pref-1 is a highly glycosylated Delta-like transmembrane protein containing six epidermal growth factor-like repeats in the extracellular domain. Pref-1 is abundantly expressed in preadipocytes, but expression is down-regulated during adipocyte differentiation. Forced expression of Pref-1 in 3T3-L1 cells was reported to inhibit adipocyte differentiation. Here we show that efficient and regulated processing of Pref-1 occurs in 3T3-L1 preadipocytes releasing most of the extracellular domain as a 50-kDa heterogeneous protein, previously isolated and characterized as FA1. Unexpectedly, we found that forced expression of the soluble form, FA1, or full-length Pref-1 did not inhibit adipocyte differentiation of 3T3-L1 cells when differentiation was induced by standard treatment with methylisobutylxanthine, dexamethasone, and high concentrations of insulin. However, forced expression of either form of Pref-1/FA1 in 3T3-L1 or 3T3-F442A cells inhibited adipocyte differentiation when insulin or insulin-like growth factor-1 (IGF-1) was omitted from the differentiation mixture. We demonstrate that the level of the mature form of the IGF-1 receptor is reduced and that IGF-1-dependent activation of p42/p44 mitogen-activated protein kinases (MAPKs) is compromised in preadipocytes with forced expression of Pref-1. This is accompanied by suppression of clonal expansion and terminal differentiation. Accordingly, supplementation with insulin or IGF-1 rescued p42/p44 MAPK activation, clonal expansion, and adipocyte differentiation in a dose-dependent manner.

Genomic organization of the mouse peroxisome proliferator-activated receptor beta/delta gene: alternative promoter usage and splicing yield transcripts exhibiting differential translational efficiency.

Peroxisome proliferator-activated receptor (PPAR) beta/delta is ubiquitously expressed, but the level of expression differs markedly between different cell types. In order to determine the molecular mechanisms governing PPARbeta/delta gene expression, we have isolated and characterized the mouse gene encoding PPARbeta/delta. The gene spans approx. 41 kb and comprises 11 exons of which the six exons located in the 3'-end of the gene are included in all transcripts. Primer-extension and 5'-rapid amplification of cDNA ends experiments revealed the presence of multiple transcription start points and splice variants, originating from the use of at least four different promoters. One of these transcription start points was found to be used predominantly in all tissues examined. Initiation from this major transcription start point gives rise to a transcript with a 548 nt 5'-untranslated leader containing eight upstream AUG codons. We show that the presence of the 548 nt leader resulted in a low translational efficiency of the corresponding PPARbeta/delta mRNA and propose, based on structural features of the 5'-untranslated region, that translational initiation may be mediated via an internal ribosome entry site-dependent mechanism.

The gene encoding the Acyl-CoA-binding protein is activated by peroxisome proliferator-activated receptor gamma through an intronic response element functionally conserved between humans and rodents.

The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular protein that specifically binds acyl-CoA esters with high affinity and is structurally and functionally conserved from yeast to mammals. In vitro studies indicate that ACBP may regulate the availability of acyl-CoA esters for various metabolic and regulatory purposes. The protein is particularly abundant in cells with a high level of lipogenesis and de novo fatty acid synthesis and is significantly induced during adipocyte differentiation. However, the molecular mechanisms underlying the regulation of ACBP expression in mammalian cells have remained largely unknown. Here we report that ACBP is a novel peroxisome proliferator-activated receptor (PPAR)gamma target gene. The rat ACBP gene is directly activated by PPARgamma/retinoid X receptor alpha (RXRalpha) and PPARalpha/RXRalpha, but not by PPARdelta/RXRalpha, through a PPAR-response element in intron 1, which is functionally conserved in the human ACBP gene. The intronic PPAR-response element (PPRE) mediates induction by endogenous PPARgamma in murine adipocytes and confers responsiveness to the PPARgamma-selective ligand BRL49653. Finally, we have used chromatin immunoprecipitation to demonstrate that the intronic PPRE efficiently binds PPARgamma/RXR in its natural chromatin context in adipocytes. Thus, the PPRE in intron 1 of the ACBP gene is a bona fide PPARgamma-response element.