Fasting enriches liver triacylglycerol with n-3 polyunsaturated fatty acids: implications for understanding the adipose–liver axis in serum docosahexaenoic acid regulation

We investigated the effect of short-term fasting on coordinate changes in the fatty acid composition of adipose triacylglycerol (TAG), serum non-esterified fatty acids (NEFA), liver TAG, and serum TAG and phospholipids in mice fed ad libitum or fasted for 16 h overnight. In contrast to previous reports under conditions of maximal lipolysis, adipose tissue TAG was not preferentially depleted of n-3 PUFA or any specific fatty acids, nor were there any striking changes in the serum NEFA composition. Short-term fasting did, however, increase the hepatic proportion of n-3 PUFA, and almost all individual species of n-3 PUFA showed relative and absolute increases. The relative proportion of n-6 PUFA in liver TAG also increased but to a lesser extent, resulting in a significant decrease in the n-6:n-3 PUFA ratio (from 14.3 ± 2.54 to 9.6 ± 1.20), while the proportion of MUFA decreased significantly and SFA proportion did not change. Examination of genes involved in PUFA synthesis suggested that hepatic changes in the elongation and desaturation of precursor lipids could not explain this effect. Rather, an increase in the expression of fatty acid transporters specific for 22:6n-3 and other long-chain n-3 and n-6 PUFA likely mediated the observed hepatic enrichment. Analysis of serum phospholipids indicated a specific increase in the concentration of 22:6n-3 and 16:0, suggesting increased specific synthesis of DHA-enriched phospholipid by the liver for recirculation. Given the importance of blood phospholipid in distributing DHA to neural tissue, these findings have implications for understanding the adipose–liver–brain axis in n-3 PUFA metabolism.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0490-2) contains supplementary material, which is available to authorized users.     

Protective Effects of Soluble Collagen during Ultraviolet-A Crosslinking on Enzyme-Mediated Corneal Ectatic Models

Collagen crosslinking is a relatively new treatment for structural disorders of corneal ectasia, such as keratoconus. However, there is a lack of animal models of keratoconus, which has been an obstacle for carefully analyzing the mechanisms of crosslinking and evaluating new therapies. In this study, we treated rabbit eyes with collagenase and chondroitinase enzymes to generate ex vivo corneal ectatic models that simulate the structural disorder of keratoconus. The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system. After enzyme treatment, the eyes were exposed to riboflavin/UVA crosslinking with and without soluble type I collagen. Corneal morphology, collagen ultrastructure, and thermal stability were evaluated before and after crosslinking. Enzyme treatments resulted in corneal curvature changes, collagen ultrastructural damage, decreased swelling resistance and thermal stability, which are similar to what is observed in keratoconus eyes. UVA crosslinking restored swelling resistance and thermal stability, but ultrastructural damage were found in the crosslinked ectatic corneas. Adding soluble collagen during crosslinking provided ultrastructural protection and further enhanced the swelling resistance. Therefore, UVA crosslinking on the ectatic model mimicked typical clinical treatment for keratoconus, suggesting that this model replicates aspects of human keratoconus and could be used for investigating experimental therapies and treatments prior to translation.     

The interaction between CtIP and BRCA1 is not essential for resection-mediated DNA repair or tumor suppression

The CtIP protein facilitates homology-directed repair (HDR) of double-strand DNA breaks (DSBs) by initiating DNA resection, a process in which DSB ends are converted into 3′-ssDNA overhangs. The BRCA1 tumor suppressor, which interacts with CtIP in a phospho-dependent manner, has also been implicated in DSB repair through the HDR pathway. It was recently reported that the BRCA1–CtIP interaction is essential for HDR in chicken DT40 cells. To examine the role of this interaction in mammalian cells, we generated cells and mice that express Ctip polypeptides (Ctip-S326A) that fail to bind BRCA1. Surprisingly, isogenic lines of Ctip-S326A mutant and wild-type cells displayed comparable levels of HDR function and chromosomal stability. Although Ctip-S326A mutant cells were modestly sensitive to topoisomerase inhibitors, mice expressing Ctip-S326A polypeptides developed normally and did not exhibit a predisposition to cancer. Thus, in mammals, the phospho-dependent BRCA1–CtIP interaction is not essential for HDR-mediated DSB repair or for tumor suppression.     

Automated correlation of single particle tilt pairs for Random Conical Tilt and Orthogonal Tilt Reconstructions

One of the major methodological challenges in single particle electron microscopy is obtaining initial reconstructions which represent the structural heterogeneity of the dataset. Random Conical Tilt and Orthogonal Tilt Reconstruction techniques in combination with 3D alignment and classification can be used to obtain initial low-resolution reconstructions which represent the full range of structural heterogeneity of the dataset. In order to achieve statistical significance, however, a large number of 3D reconstructions, and, in turn, a large number of tilted image pairs are required. The extraction of single particle tilted image pairs from micrographs can be tedious and time-consuming, as it requires intensive user input even for semi-automated approaches. To overcome the bottleneck of manual selection of a large number of tilt pairs, we developed an algorithm for the correlation of single particle images from tilted image pairs in a fully automated and user-independent manner. The algorithm reliably correlates correct pairs even from noisy micrographs. We further demonstrate the applicability of the algorithm by using it to obtain initial references both from negative stain and unstained cryo datasets.     

Synthesis and evaluation of novel ligands for the histamine H4 receptor based on a pyrrolo[2,3-d]pyrimidine scaffold

Starting from a known H₄R ligand based on a pyrimidine skeleton, a series of novel analogues based on a pyrrolo[2,3-d]pyrimidine scaffold have been prepared. Whereas the original pyrimidine congener shows good affinity at hH₄R (K_i = 0.5 μM), its lacks selectivity with a K_i value for the hH₃R of 1 μM. Within the newly synthesized pyrrolo[2,3-d]pyrimidines, several congeners show K_i values of less than 1 μM at the hH₄R and show a much improved selectivity profile. Therefore, these series represent an interesting starting point for the discovery of novel hH₄R ligands.     

HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response

The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G₂/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.     

Two fatty acid‐binding proteins expressed in the intestine interact differently with endocannabinoids

Two different members of the fatty acid‐binding protein (FABP) family are found in enterocyte cells of the gastrointestinal system, namely liver‐type and intestinal fatty acid‐binding proteins (LFABP and IFABP, also called FABP1 and FABP2, respectively). Striking phenotypic differences have been observed in knockout mice for either protein, for example, high fat‐fed IFABP‐null mice remained lean, whereas LFABP‐null mice were obese, correlating with differences in food intake. This finding prompted us to investigate the role each protein plays in directing the specificity of binding to ligands involved in appetite regulation, such as fatty acid ethanolamides and related endocannabinoids. We determined the binding affinities for nine structurally related ligands using a fluorescence competition assay, revealing tighter binding to IFABP than LFABP for all ligands tested. We found that the head group of the ligand had more impact on binding affinity than the alkyl chain, with the strongest binding observed for the carboxyl group, followed by the amide, and then the glycerol ester. These trends were confirmed using two‐dimensional ¹H–¹⁵N nuclear magnetic resonance (NMR) to monitor chemical shift perturbation of the protein backbone resonances upon titration with ligand. Interestingly, the NMR data revealed that different residues of IFABP were involved in the coordination of endocannabinoids than those implicated for fatty acids, whereas the same residues of LFABP were involved for both classes of ligand. In addition, we identified residues that are uniquely affected by binding of all types of ligand to IFABP, suggesting a rationale for its tighter binding affinity compared with LFABP.     

Functional maturation of the human corticotropin releasing factor-adrenocorticotropic hormone system during intrauterine life. An in vitro study

Radioimmunoassay was used to determine ACTH secretion by cultured hypophyses of human fetuses from the 6th to the 30th week of intrauterine life and their responsiveness to hypothalamic extracts obtained from adult animals. CRF-like activity in the human hypothalamus was measured within the 6th to the 32nd week of prenatal development from changes in ACTH release by cultured cells of the adult rat hypophysis. It was established that starting from weeks 6-7 of embryogenesis, the human fetal hypophysis is capable of synthesizing and secreting immuno-reactive ACTH in vitro. The human fetus hypothalamus of the first trimester of gestation contained no CRF-like substance. The fetus hypothalamus of the second and third trimesters of pregnancy manifested a considerable amount of CRF-like substance. It is suggested that CRF appears at the end of the first trimester of pregnancy.