Stearoyl-CoA desaturase 1 deficiency increases CTP:choline cytidylyltransferase translocation into the membrane and enhances phosphatidylcholine synthesis in liver

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in monounsaturated fatty acid synthesis. Previously, we showed that Scd1 deficiency reduces liver triglyceride accumulation and considerably decreases synthesis of very low density lipoprotein and its secretion in both lean and obese mice. In the present study, we found that Scd1 deficiency significantly modulates hepatic glycerophospholipid profile. The content of phosphatidylcholine (PC) was increased by 40% and the activities of CTP:choline cytidylyltransferase (CCT), the rate-limiting enzyme in de novo PC synthesis, and choline phosphotransferase were increased by 64 and 53%, respectively, in liver of Scd1-/- mice. In contrast, the protein level of phosphatidylethanolamine N-methyltransferase, an enzyme involved in PC synthesis via methylation of phosphatidylethanolamine, was decreased by 80% in the liver of Scd1-/- mice. Membrane translocation of CCT is required for its activation. Immunoblot analyses demonstrated that twice as much CCTalpha was associated with plasma membrane in livers of Scd1-/- compared with wild type mice, suggesting that Scd1 mutation leads to an increase in CCT membrane affinity. The incorporation of [(3)H]glycerol into PC was increased by 2.5-fold in Scd1-/- primary hepatocytes compared with those of wild type mice. Furthermore, mitochondrial glycerol-3-phosphate acyltransferase activity was reduced by 42% in liver of Scd1-/- mice; however, the activities of microsomal glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase, and ethanolamine phosphotransferase were not affected by Scd1 mutation. Our study revealed that SCD1 deficiency specifically increases CCT activity by promoting its translocation into membrane and enhances PC biosynthesis in liver.     

Membrane topology of mouse stearoyl-CoA desaturase 1

Stearoyl-CoA desaturase (SCD) is an integral membrane protein anchored in the endoplasmic reticulum. It catalyzes the biosynthesis of monounsaturated fatty acids that are required for the synthesis of triglycerides, cholesteryl esters, and phospholipids. Four mouse isoforms of SCD (SCD1-4) and two human isoforms have been characterized. In the current study, we characterize the topology of the mouse SCD1 isoform. Hydropathy analysis of the 355-amino acid mouse SCD1 protein predicts that the protein contains four transmembrane domains (TMDs) and three loops connecting the membrane-spanning domains. To define the topology of the protein, recombinant SCD1 constructs containing epitope tags were transiently expressed in HeLa cells and analyzed by indirect immunofluorescence and cysteine derivatization. Our data provide evidence that the N and C termini of SCD1 are oriented toward the cytosol with four transmembrane domains separated by two very short hydrophilic loops in the ER lumen and one large hydrophilic loop in the cytosol. In addition, based on the previous observation that SCD is a thiol enzyme, we sought to investigate whether the cysteine residues were essential for enzyme activity through mutagenesis studies, and our data suggest that the cysteines in SCD are not catalytically essential.     

Restoration of the CD4 T cell compartment after long-term highly active antiretroviral therapy without phenotypical signs of accelerated immunological aging

It remains uncertain whether full T cell reconstitution can be established in HIV-infected children and adults with long-term sustained virological control by highly active antiretroviral therapy (HAART). In this study, we comprehensively analyzed various phenotypical markers of CD4 T cell recovery. In addition to measuring T cell activation and proliferation markers, CD4 T cell generation and aging of the CD4 T cell compartment were assessed by measuring TCR excision circles and the fraction of CD31-expressing naive CD4 T cells. In all children and in adults with relatively high CD4 T cell counts at start of therapy (>200 cells/microl), total CD4 T cell numbers normalized within 1 year of therapy. After long-term HAART (4.4-9.6 years), naive CD4 T cell counts had normalized in both groups. Although in adults with low baseline CD4 T cell counts (<200 cells/microl) total CD4 T cell numbers normalized eventually after at least 7 years of HAART, naive CD4 T cell counts had still not recovered. TCR excision circle data showed that thymic T cell production contributed to naive T cell recovery at all ages. The fraction of CD31-expressing naive CD4 T cells was found to be normal, suggesting that the CD4 T cell repertoire was diverse after long-term HAART. Hence, under sustained viral suppression during long-term HAART, the T cell compartment has the potential to fully recover by generating new naive T cells both in children and in adults with high baseline CD4 T cells counts. Irrespective of baseline CD4 T cell counts, reconstitution occurred without a significant effect on T cell aging as reflected by markers for replicative history.     

Reconciling Longitudinal Naive T-Cell and TREC Dynamics during HIV-1 Infection

Naive T cells in untreated HIV-1 infected individuals have a reduced T-cell receptor excision circle (TREC) content. Previous mathematical models have suggested that this is due to increased naive T-cell division. It remains unclear, however, how reduced naive TREC contents can be reconciled with a gradual loss of naive T cells in HIV-1 infection. We performed longitudinal analyses in humans before and after HIV-1 seroconversion, and used a mathematical model to investigate which processes could explain the observed changes in naive T-cell numbers and TRECs during untreated HIV-1 disease progression. Both CD4⁺ and CD8⁺ naive T-cell TREC contents declined biphasically, with a rapid loss during the first year and a much slower loss during the chronic phase of infection. While naive CD8⁺ T-cell numbers hardly changed during follow-up, naive CD4⁺ T-cell counts continually declined. We show that a fine balance between increased T-cell division and loss in the peripheral naive T-cell pool can explain the observed short- and long-term changes in TRECs and naive T-cell numbers, especially if T-cell turnover during the acute phase is more increased than during the chronic phase of infection. Loss of thymic output, on the other hand, does not help to explain the biphasic loss of TRECs in HIV infection. The observed longitudinal changes in TRECs and naive T-cell numbers in HIV-infected individuals are most likely explained by a tight balance between increased T-cell division and death, suggesting that these changes are intrinsically linked in HIV infection.     

Focus on Quality: Investigating Residents’ Learning Climate Perceptions

Background

A department’s learning climate is known to contribute to the quality of postgraduate medical education and, as such, to the quality of patient care provided by residents. However, it is unclear how the learning climate is perceived over time.

Objectives

This study investigated whether the learning climate perceptions of residents changed over time.

Methods

The context for this study was residency training in the Netherlands. Between January 2012 and December 2014, residents from 223 training programs in 39 hospitals filled out the web-based Dutch Residency Educational Climate Test (D-RECT) to evaluate their clinical department’s learning climate. Residents had to fill out 35 validated questions using a five point Likert-scale. We analyzed data using generalized linear mixed (growth) models.

Results

Overall, 3982 D-RECT evaluations were available to investigate our aim. The overall mean D-RECT score was 3.9 (SD = 0.3). The growth model showed an increase in D-RECT scores over time (b = 0.03; 95% CI: 0.01–0.06; p < 0.05).

Conclusions

The observed increase in D-RECT scores implied that residents perceived an improvement in the learning climate over time. Future research could focus on factors that facilitate or hinder learning climate improvement, and investigate the roles that hospital governing committees play in safeguarding and improving the learning climate.     

The genetic structure of Australasian green turtles (Chelonia mydas): exploring the geographical scale of genetic exchange

Ecological and genetic studies of marine turtles generally support the hypothesis of natal homing, but leave open the question of the geographical scale of genetic exchange and the capacity of turtles to shift breeding sites. Here we combine analyses of mitochondrial DNA (mtDNA) variation and recapture data to assess the geographical scale of individual breeding populations and the distribution of such populations through Australasia. We conducted multiscale assessments of mtDNA variation among 714 samples from 27 green turtle rookeries and of adult female dispersal among nesting sites in eastern Australia. Many of these rookeries are on shelves that were flooded by rising sea levels less than 10 000 years (c. 450 generations) ago. Analyses of sequence variation among the mtDNA control region revealed 25 haplotypes, and their frequency distributions indicated 17 genetically distinct breeding stocks (Management Units) consisting either of individual rookeries or groups of rookeries in general that are separated by more than 500 km. The population structure inferred from mtDNA was consistent with the scale of movements observed in long-term mark-recapture studies of east Australian rookeries. Phylogenetic analysis of the haplotypes revealed five clades with significant partitioning of sequence diversity (Phi = 68.4) between Pacific Ocean and Southeast Asian/Indian Ocean rookeries. Isolation by distance was indicated for rookeries separated by up to 2000 km but explained only 12% of the genetic structure. The emerging general picture is one of dynamic population structure influenced by the capacity of females to relocate among proximal breeding sites, although this may be conditional on large population sizes as existed historically across this region.     

Optimization of human immunodeficiency virus type 1 envelope glycoproteins with V1/V2 deleted, using virus evolution

The human immunodeficiency virus type 1 envelope glycoprotein (Env) complex is the principal focus of neutralizing antibody-based vaccines. The functional Env complex is a trimer consisting of six individual subunits: three gp120 molecules and three gp41 molecules. The individual subunits have proven unsuccessful as vaccines presumably because they do not resemble the functional Env complex. Variable domains and carbohydrates shield vulnerable neutralization epitopes on the functional Env complex. The deletion of variable loops has been shown to improve gp120's immunogenicity; however, problems have been encountered when introducing such modifications in stabilized Env trimer constructs. To address these issues, we have created a set of V1/V2 and V3 loop deletion variants in the context of complete virus to allow optimization by forced virus evolution. Compensatory second-site substitutions included the addition and/or removal of specific carbohydrates, changes in the disulfide-bonded architecture of the V1/V2 stem, the replacement of hydrophobic residues by hydrophilic and charged residues, and changes in distal parts of gp120 and gp41. These viruses displayed increased sensitivity to neutralizing antibodies, demonstrating the improved exposure of conserved domains. The results show that we can select for functionally improved Env variants with loop deletions through forced virus evolution. Selected evolved Env variants were transferred to stabilized Env trimer constructs and were shown to improve trimer expression and secretion. Based on these findings, we can make recommendations on how to delete the V1/V2 domain from recombinant Env trimers for vaccine and X-ray crystallography studies. In general, virus evolution may provide a powerful tool to optimize Env vaccine antigens.     

Skin-specific Deletion of Stearoyl-CoA Desaturase-1 Alters Skin Lipid Composition and Protects Mice from High Fat Diet-induced Obesity

Stearoyl-CoA desaturase-1 (SCD1) catalyzes the synthesis of monounsaturated fatty acids and is an important regulator of whole body energy homeostasis. Severe cutaneous changes in mice globally deficient in SCD1 also indicate a role for SCD1 in maintaining skin lipids. We have generated mice with a skin-specific deletion of SCD1 (SKO) and report here that SKO mice display marked sebaceous gland hypoplasia and depletion of sebaceous lipids. In addition, SKO mice have significantly increased energy expenditure and are protected from high fat diet-induced obesity, thereby recapitulating the hypermetabolic phenotype of global SCD1 deficiency. Genes of fat oxidation, lipolysis, and thermogenesis, including uncoupling proteins and peroxisome proliferator-activated receptor-γ co-activator-1α, are up-regulated in peripheral tissues of SKO mice. However, unlike mice globally deficient in SCD1, SKO mice have an intact hepatic lipogenic response to acute high carbohydrate feeding. Despite increased basal thermogenesis, SKO mice display severe cold intolerance because of rapid depletion of fuel substrates, including hepatic glycogen, to maintain core body temperature. These data collectively indicate that SKO mice have increased cold perception because of loss of insulating factors in the skin. This results in up-regulation of thermogenic processes for temperature maintenance at the expense of fuel economy, illustrating cross-talk between the skin and peripheral tissues in maintaining energy homeostasis.Obesity is a multifactorial disease stemming from a combination of genetic, dietary, and lifestyle factors and the interaction between these components (1–3). The microsomal enzyme, stearoyl-CoA desaturase-1 (SCD1),³ is a critical control point in the development of metabolic diseases, including obesity and insulin resistance. SCD1 catalyzes the conversion of saturated fatty acids, such as palmitate (16:0) and stearate (18:0), into their Δ-9 monounsaturated products, palmitoleate (16:1 n-7) and oleate (18:1 n-9), respectively. Mice lacking the SCD1 enzyme because of a global deletion of the Scd1 gene (GKO) are lean and protected from diet-induced and leptin deficiency-induced obesity. These mice have a marked increase in energy expenditure and almost complete protection from high fat diet-induced weight gain and glucose intolerance (4–10).Because SCD1 is expressed in multiple tissues, including liver, brown and white adipose tissue, skeletal muscle, and skin, it has been difficult to determine the relative contributions of these tissues to the dramatically altered metabolic phenotypes of GKO mice. Studies using antisense oligonucleotide-mediated approaches to knock down Scd1 expression have reported protection from diet-induced weight gain and hepatic insulin resistance upon hepatic SCD1 inhibition (11–13). However, whereas the liver is a major target of these antisense oligonucleotides, they have also been reported to affect expression of target genes in adipose tissue (13, 14) and possibly other organs (15). Using Cre recombinase-mediated inhibition of hepatic Scd1, we recently reported that chronic deletion of SCD1 specifically in liver does not protect mice from high fat diet-induced obesity (16), suggesting that extra-hepatic tissues may play a more prominent role in the increased energy expenditure phenotype of global SCD1 deficiency (16).In addition to their hypermetabolic phenotype, global SCD1 deficiency also elicits marked cutaneous phenotypes, including dry skin, alopecia, and sebocyte hypoplasia (7, 17, 18). Given the severity of this skin phenotype in GKO mice, we sought to establish a specific role for SCD1 in the skin. In this study, we used the Cre-lox system to generate mice with a skin-specific deletion of SCD1 (SKO). We report here that SKO mice have a severe paucity of lipid-enriched sebocytes in the skin, resulting in dry skin, alopecia, and marked alterations in levels of key skin lipids. Unlike mice with global or liver-specific deletion of SCD1 (7, 16), SKO have an intact hepatic lipogenic response to dietary stimuli. However, deletion of skin SCD1 completely recapitulates the increased energy expenditure phenotype of GKO mice (7) and protects SKO mice from high fat diet-induced obesity, hepatic steatosis, and glucose intolerance. Elevation of genes encoding for cold-inducible factors, including peroxisome proliferator-activated receptor γ co-activator-1α (Pgc-1α) and uncoupling proteins (Ucps) in brown and white adipose tissue and skeletal muscle of SKO mice, suggests up-regulation of thermogenic processes for maintenance of core body temperature in SKO mice. Furthermore, the hypermetabolic phenotype of SKO mice, coupled with the loss of insulating factors in the skin, results in severe cold intolerance in SKO mice that is ameliorated by prior feeding with a high fat diet. To the best of our knowledge, this study represents the first example of skin-specific deletion of a lipogenic enzyme resulting in profound changes in systemic energy metabolism. These data elucidate an as yet under-appreciated role for skin SCD1 in triggering the altered metabolic phenotypes caused by global SCD1 deletion.