Diana Hall

Peroxisomal and microsomal lipid pathways associated with resistance to hepatic steatosis and reduced pro-inflammatory state

Accumulation of fat in the liver increases the risk to develop fibrosis and cirrhosis and is associated with development of the metabolic syndrome. Here, to identify genes or gene pathways that may underlie the genetic susceptibility to fat accumulation in liver, we studied A/J and C57Bl/6 mice that are resistant and sensitive to diet-induced hepatosteatosis and obesity, respectively. We performed comparative transcriptomic and lipidomic analysis of the livers of both strains of mice fed a high fat diet for 2, 10, and 30 days. We found that resistance to steatosis in A/J mice was associated with the following: (i) a coordinated up-regulation of 10 genes controlling peroxisome biogenesis and β-oxidation; (ii) an increased expression of the elongase Elovl5 and desaturases Fads1 and Fads2. In agreement with these observations, peroxisomal β-oxidation was increased in livers of A/J mice, and lipidomic analysis showed increased concentrations of long chain fatty acid-containing triglycerides, arachidonic acid-containing lysophosphatidylcholine, and 2-arachidonylglycerol, a cannabinoid receptor agonist. We found that the anti-inflammatory CB2 receptor was the main hepatic cannabinoid receptor, which was highly expressed in Kupffer cells. We further found that A/J mice had a lower pro-inflammatory state as determined by lower plasma levels and IL-1β and granulocyte-CSF and reduced hepatic expression of their mRNAs, which were found only in Kupffer cells. This suggests that increased 2-arachidonylglycerol production may limit Kupffer cell activity. Collectively, our data suggest that genetic variations in the expression of peroxisomal β-oxidation genes and of genes controlling the production of an anti-inflammatory lipid may underlie the differential susceptibility to diet-induced hepatic steatosis and pro-inflammatory state.

Different Transcriptional Control of Metabolism and Extracellular Matrix in Visceral and Subcutaneous Fat of Obese and Rimonabant Treated Mice

Background The visceral (VAT) and subcutaneous (SCAT) adipose tissues play different roles in physiology and obesity. The molecular mechanisms underlying their expansion in obesity and following body weight reduction are poorly defined. Methodology C57Bl/6 mice fed a high fat diet (HFD) for 6 months developed low, medium, or high body weight as compared to normal chow fed mice. Mice from each groups were then treated with the cannabinoid receptor 1 antagonist rimonabant or vehicle for 24 days to normalize their body weight. Transcriptomic data for visceral and subcutaneous adipose tissues from each group of mice were obtained and analyzed to identify: i) genes regulated by HFD irrespective of body weight, ii) genes whose expression correlated with body weight, iii) the biological processes activated in each tissue using gene set enrichment analysis (GSEA), iv) the transcriptional programs affected by rimonabant. Principal Findings In VAT, “metabolic” genes encoding enzymes for lipid and steroid biosynthesis and glucose catabolism were down-regulated irrespective of body weight whereas “structure” genes controlling cell architecture and tissue remodeling had expression levels correlated with body weight. In SCAT, the identified “metabolic” and “structure” genes were mostly different from those identified in VAT and were regulated irrespective of body weight. GSEA indicated active adipogenesis in both tissues but a more prominent involvement of tissue stroma in VAT than in SCAT. Rimonabant treatment normalized most gene expression but further reduced oxidative phosphorylation gene expression in SCAT but not in VAT. Conclusion VAT and SCAT show strikingly different gene expression programs in response to high fat diet and rimonabant treatment. Our results may lead to identification of therapeutic targets acting on specific fat depots to control obesity.

DIP–STR: Highly Sensitive Markers for the Analysis of Unbalanced Genomic Mixtures

Samples containing highly unbalanced DNA mixtures from two individuals commonly occur both in forensic mixed stains and in peripheral blood DNA microchimerism induced by pregnancy or following organ transplant. Because of PCR amplification bias, the genetic identification of a DNA that contributes trace amounts to a mixed sample represents a tremendous challenge. This means that standard genetic markers, namely microsatellites, also referred as short tandem repeats (STR), and single‐nucleotide polymorphism (SNP) have limited power in addressing common questions of forensic and medical genetics. To address this issue, we developed a molecular marker, named DIP–STR that relies on pairing deletion–insertion polymorphisms (DIP) with STR. This novel analytical approach allows for the unambiguous genotyping of a minor component in the presence of a major component, where DIP–STR genotypes of the minor were successfully procured at ratios up to 1:1,000. The compound nature of this marker generates a high level of polymorphism that is suitable for identity testing. Here, we demonstrate the power of the DIP–STR approach on an initial set of nine markers surveyed in a Swiss population. Finally, we discuss the limitations and potential applications of our new system including preliminary tests on clinical samples and estimates of their performance on simulated DNA mixtures.

Oxidative Phosphorylation Flexibility in the Liver of Mice Resistant to High-Fat Diet–Induced Hepatic Steatosis

OBJECTIVE To identify metabolic pathways that may underlie susceptibility or resistance to high-fat diet–induced hepatic steatosis. RESEARCH DESIGN AND METHODS We performed comparative transcriptomic analysis of the livers of A/J and C57Bl/6 mice, which are, respectively, resistant and susceptible to high-fat diet–induced hepatosteatosis and obesity. Mice from both strains were fed a normal chow or a high-fat diet for 2, 10, and 30 days, and transcriptomic data were analyzed by time-dependent gene set enrichment analysis. Biochemical analysis of mitochondrial respiration was performed to confirm the transcriptomic analysis. RESULTS Time-dependent gene set enrichment analysis revealed a rapid, transient, and coordinate upregulation of 13 oxidative phosphorylation genes after initiation of high-fat diet feeding in the A/J, but not in the C57Bl/6, mouse livers. Biochemical analysis using liver mitochondria from both strains of mice confirmed a rapid increase by high-fat diet feeding of the respiration rate in A/J but not C57Bl/6 mice. Importantly, ATP production was the same in both types of mitochondria, indicating increased uncoupling of the A/J mitochondria. CONCLUSIONS Together with previous data showing increased expression of mitochondrial β-oxidation genes in C57Bl/6 but not A/J mouse livers, our present study suggests that an important aspect of the adaptation of livers to high-fat diet feeding is to increase the activity of the oxidative phosphorylation chain and its uncoupling to dissipate the excess of incoming metabolic energy and to reduce the production of reactive oxygen species. The flexibility in oxidative phosphorylation activity may thus participate in the protection of A/J mouse livers against the initial damages induced by high-fat diet feeding that may lead to hepatosteatosis.

A novel set of DIP-STR markers for improved analysis of challenging DNA mixtures

Currently available molecular biology tools allow forensic scientists to characterize DNA evidence found at crime scenes for a large variety of samples, including those of limited quantity and quality, and achieve high levels of individualization. Yet, standard forensic markers provide limited or no results when applied to mixed DNA samples where the contributors are present in very different proportions (unbalanced DNA mixtures). This becomes an issue mostly for the analysis of trace samples collected on the victim or from touched objects. To this end, we recently proposed an innovative type of genetic marker, named DIP-STR that relies on pairing deletion/insertion polymorphisms (DIP) with standard short tandem repeats (STR). This novel compound marker allows detection of the minor DNA contributor in a DNA mixture of any gender and cellular origin with unprecedented resolution (beyond a DNA ratio of 1:1000). To provide a novel analytical tool useful in practice to common forensic laboratories, this article describes the first set of 10 DIP-STR markers selected according to forensic technical standards. The novel DIP-STR regions are short (between 146 and 271 bp), include only highly polymorphic tri-, tetra- and pentanucleotide tandem repeats and are located on different chromosomes or chromosomal arms to provide statistically independent results. This novel set of DIP-STR can target the amplification of 0.03-0.1 ng of DNA when mixed with a 1000-fold excess of major DNA. DIP-STR relative allele frequencies are estimated based on a survey of 103 Swiss individuals. Finally, this study provides an estimate of the occurrence of informative alleles and a calculation of the corresponding random match probability of the detected minor DIP-STR genotype assessed across 10,506 pairwise conceptual mixtures.

Analysis of fetal DNA in maternal plasma with markers designed for forensic DNA mixture resolution

AbstractPurposeWith the description of circulating fetal DNA in maternal blood, noninvasive prenatal diagnostics became theoretically possible. As the presence of background maternal DNA interferes with the detection of fetal DNA, analytical methods require genetic markers capable of distinguishing by quantitative or targeted approaches the minor population of DNA molecules of the fetus. Here we evaluate the feasibility of analyzing fetal DNA with novel DIP-STR genetic markers, designed for the investigation of forensic mixed biological evidence.MethodsThe DIP-STR molecular approach is based on sequence-specific analysis of paternally inherited fetal alleles. These sequences are biallelic deletion/insertion polymorphisms (DIPs) located very close to short tandem repeat (STR) markers, for combined analysis. In this study, 48 women were tested with 28 DIP-STRs during the first, second, and third trimester of pregnancy.ResultsPositive results were obtained across markers, including longer ones (386 base-pairs) and with blood samples collected during early pregnancy, such as 10 weeks of gestational age.ConclusionThese data show that DIP-STR markers can be used to amplify specific genomic regions of circulating fetal DNA to obtain targeted genetic information. This method may contribute to developments in noninvasive prenatal paternity testing and diagnosis of certain genetic diseases.

Inferring biogeographic ancestry with compound markers of slow and fast evolving polymorphisms

Bio-geographic ancestry is an area of considerable interest in the medical genetics, anthropology and forensics. Although genome-wide panels are ideal as they provide dense genotyping data, small sets of ancestry informative marker provide a cost-effective way to investigate genetic ancestry and population structure. Here, we investigate the performance of a reduced marker set that combine different types of autosomal markers through haplotype analysis. In particular, recently described DIP-STR markers should offer the advantage of comprising both, low mutation rate Indels (DIPs), to study human history over longer time scale; and high mutation rate STRs, to trace relatively recent demographic events. In this study, we assessed the ability of an initial set of 23 DIP-STRs to distinguish major population groups using the HGDP-CEPH reference samples. The results obtained applying the STRUCTURE algorithm show that the discrimination capacity of the DIP-STRs is comparable to currently used small-scale ancestry informative markers by approaching seven major demographic groups. Yet, the DIP-STRs show an improved success rate in assigning individuals to populations of Europe and Middle East. These data show a remarkable ability of a preliminary set of 23 DIP-STR markers to infer major biogeographic origins. A novel set of DIP-STRs preselected to contain ancestry information should lead to further improvements.