Prolonged exercise shifts ventilatory parameters at the moderate-to-heavy intensity transition.

PURPOSE: To quantify the effects of prolonged cycling on the rate of ventilation ([Formula: see text]), frequency of respiration (FR), and tidal volume (VT) associated with the moderate-to-heavy intensity transition. METHODS: Fourteen endurance-trained cyclists and triathletes (one female) completed an assessment of the moderate-to-heavy intensity transition, determined as the first ventilatory threshold (VT1), before (PRE) and after (POST) two hours of moderate-intensity cycling. The power output, [Formula: see text], FR, and VT associated with VT1 were determined PRE and POST. RESULTS: As previously reported, power output at VT1 significantly decreased by ~ 10% from PRE to POST. The [Formula: see text] associated with VT1 was unchanged from PRE to POST (72 ± 12 vs. 69 ± 13 L.min-1, ∆ - 3 ± 5 L.min-1, ∆ - 4 ± 8%, P = 0.075), and relatively consistent (within-subject coefficient of variation, 5.4% [3.7, 8.0%]). The [Formula: see text] associated with VT1 was produced with increased FR (27.6 ± 5.8 vs. 31.9 ± 6.5 breaths.min-1, ∆ 4.3 ± 3.1 breaths.min-1, ∆ 16 ± 11%, P = 0.0002) and decreased VT (2.62 ± 0.43 vs. 2.19 ± 0.36 L.breath-1, ∆ - 0.44 ± 0.22 L.breath-1, ∆ - 16 ± 7%, P = 0.0002) in POST. CONCLUSION: These data suggest prolonged exercise shifts ventilatory parameters at the moderate-to-heavy intensity transition, but [Formula: see text] remains stable. Real-time monitoring of [Formula: see text] may be a useful means of assessing proximity to the moderate-to-heavy intensity transition during prolonged exercise and is worthy of further research.

Prolonged cycling reduces power output at the moderate-to-heavy intensity transition.

PURPOSE: To determine the effect of prolonged exercise on moderate-to-heavy intensity transition power output and heart rate. METHODS: Fourteen endurance-trained cyclists and triathletes took part in the present investigation (13 males, 1 female, V·O2peak 59.9 ± 6.8 mL.kg-1.min-1). Following a characterisation trial, participants undertook a five-stage incremental step test to determine the power output and heart rate at the moderate-to-heavy intensity transition before and after two hours of cycling at 90% of the estimated power output at first ventilatory threshold (VT1). RESULTS: Power output at the moderate-to-heavy intensity transition significantly decreased following acute prolonged exercise when determined using expired gases (VT1, 217 ± 42 W vs. 196 ± 42 W, P < 0.0001) and blood lactate concentrations (LoglogLT, 212 ± 47 W vs. 190 ± 47 W, P = 0.004). This was attributable to loss of efficiency (VT1, -8 ± 10 W; LoglogLT, - 7 ± 9 W) and rates of metabolic energy expenditure at the transition (VT1, - 14 ± 11 W; LoglogLT, - 15 ± 22 W). The heart rate associated with the moderate-to-heavy intensity transition increased following acute prolonged exercise (VT1, 142 ± 9 beats.min-1 vs. 151 ± 12 beats.min-1, P < 0.001; LoglogLT, 140 ± 13 beats.min-1 vs. 150 ± 15 beats.min-1, P = 0.006). CONCLUSION: These results demonstrate the external work output at the moderate-to-heavy intensity transition decreases during prolonged exercise due to decreased efficiency and rates of metabolic energy expenditure, but the associated heart rate increases. Therefore, individual assessments of athlete 'durability' are warranted.

Endoplasmic Reticulum-Associated Degradation and Lipid Homeostasis.

The endoplasmic reticulum is the port of entry for proteins into the secretory pathway and the site of synthesis for several important lipids, including cholesterol, triacylglycerol, and phospholipids. Protein production within the endoplasmic reticulum is tightly regulated by a cohort of resident machinery that coordinates the folding, modification, and deployment of secreted and integral membrane proteins. Proteins failing to attain their native conformation are degraded through the endoplasmic reticulum-associated degradation (ERAD) pathway via a series of tightly coupled steps: substrate recognition, dislocation, and ubiquitin-dependent proteasomal destruction. The same ERAD machinery also controls the flux through various metabolic pathways by coupling the turnover of metabolic enzymes to the levels of key metabolites. We review the current understanding and biological significance of ERAD-mediated regulation of lipid metabolism in mammalian cells.

The Regulatory Domain of Squalene Monooxygenase Contains a Re-entrant Loop and Senses Cholesterol via a Conformational Change.

Squalene monooxygenase (SM) is an important control point in cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA reductase. Although it is known to associate with the endoplasmic reticulum, its topology has not been determined. We have elucidated the membrane topology of the sterol-responsive domain of SM comprising the first 100 amino acids fused to GFP (SM N100-GFP) by determining the accessibility of 16 introduced cysteines to the cysteine-reactive, membrane-impermeable reagent PEG-maleimide. We have identified a region integrally associated with the endoplasmic reticulum membrane that is likely to interact with cholesterol or respond to cholesterol-induced membrane effects. By comparing cysteine accessibility with and without cholesterol treatment, we further present evidence to suggest that cholesterol induces a conformational change in SM N100-GFP. This change is likely to lead to its targeted degradation by the ubiquitin-proteasome system because degradation is blunted by treatment with the chemical chaperone glycerol, which retains SM N100-GFP in its native conformation. Furthermore, degradation can be disrupted by insertion of two N-terminal myc tags, implicating the N terminus in this process. Together, this information provides new molecular insights into the regulation of this critical control point in cholesterol synthesis.

Universal CG cloning of polymerase chain reaction products.

Single-insert cloning of DNA fragments without restriction enzymes has traditionally been achieved using TA cloning, with annealing of a polymerase chain reaction (PCR) fragment containing a single overhanging 3' A to a plasmid vector containing a 3' T. In this article, we show that the analogous "CG cloning" is faster and far more efficient, using AhdI to generate a C-vector. For an afternoon ligation, CG cloning achieved double the cloning efficiency and more than 4-fold the number of transformants compared with TA cloning. However, blunt-end ligation was markedly more efficient than both. CG cloning could prove to be extremely useful for single-copy high-throughput cloning.

Squalene mono-oxygenase, a key enzyme in cholesterol synthesis, is stabilized by unsaturated fatty acids.

SM (squalene mono-oxygenase) catalyses the first oxygenation step in cholesterol synthesis, immediately before the formation of the steroid backbone at lanosterol. SM is an important control point in the pathway, and is regulated at the post-translational level by accelerated cholesterol-dependent ubiquitination and proteasomal degradation, which is associated with the accumulation of squalene. Using model cell systems, we report that SM is stabilized by unsaturated fatty acids. Treatment with unsaturated fatty acids such as oleate, but not saturated fatty acids, increased protein levels of SM or SM-N100-GFP (the first 100 amino acids of SM fused to GFP) at the post-translational level and partially overcame cholesterol-dependent degradation, as well as reversing cholesterol-dependent squalene accumulation. Maximum stabilization required activation of fatty acids, but not triacylglycerol or phosphatidylcholine synthesis. The mechanism of oleate-mediated stabilization appeared to occur through reduced ubiquitination by the E3 ubiquitin ligase MARCH6. Stabilization of a cholesterol biosynthetic enzyme by unsaturated fatty acids may help maintain a constant cholesterol/phospholipid ratio.

Cholesterol through the looking glass: ability of its enantiomer also to elicit homeostatic responses.

How cholesterol is sensed to maintain homeostasis has been explained by direct binding to a specific protein, Scap, or through altering the physical properties of the membrane. The enantiomer of cholesterol (ent-cholesterol) is a valuable tool in distinguishing between these two models because it shares nonspecific membrane effects with native cholesterol (nat-cholesterol), but not specific binding interactions. This is the first study to compare ent- and nat-cholesterol directly on major molecular parameters of cholesterol homeostasis. We found that ent-cholesterol suppressed activation of the master transcriptional regulator of cholesterol metabolism, SREBP-2, almost as effectively as nat-cholesterol. Importantly, ent-cholesterol induced a conformational change in the cholesterol-sensing protein Scap in isolated membranes in vitro, even when steps were taken to eliminate potential confounding effects from endogenous cholesterol. Ent-cholesterol also accelerated proteasomal degradation of the key cholesterol biosynthetic enzyme, squalene monooxygenase. Together, these findings provide compelling evidence that cholesterol maintains its own homeostasis not only via direct protein interactions, but also by altering membrane properties.

Akt acutely activates the cholesterogenic transcription factor SREBP-2.

Akt is an essential protein kinase for cell growth, proliferation, and survival. Perturbed Akt regulation is associated with a number of human diseases, such as cancer and diabetes. Recently, evidence has emerged that Akt is involved in the regulation of the sterol-regulatory element binding proteins, which are master transcriptional regulators of lipid metabolism. This offers a means by which synthesis of new membrane can be coordinated with cell growth and proliferation. However, the link between Akt and sterol-regulatory element binding protein-2, the major isoform participating in cholesterol regulation, is relatively unexplored. In the present study, we employed insulin-like growth factor-1 as an inducer of Akt signalling, and showed that it increased sterol-regulatory element binding protein-2 activation acutely (within 1h). This insulin-like growth factor-1-induced sterol-regulatory element binding protein-2 activation was blunted when Akt was inhibited pharmacologically or molecularly with small interfering RNA. Furthermore, we employed a rapalog heterodimerisation system to specifically and rapidly activate Akt, and found that sterol-regulatory element binding protein-2 activation was increased in response to Akt activation. Together, this study provides compelling evidence that Akt contributes to the acute regulation of cholesterol metabolism through activating sterol-regulatory element binding protein-2.

Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation.

Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism.

How essential is cholesterol?

Cholesterol is an apparently indispensable lipid for numerous processes required for cell proliferation. Levels of this molecule are primarily regulated at the transcriptional level by the SREBPs (sterol-regulatory-element-binding proteins) and LXR (liver X receptor). In this issue of the Biochemical Journal, Rodríguez-Acebes et al. show that a cholesterol precursor, desmosterol, can support cell proliferation in the absence of cholesterol in a murine macrophage-like model (J774-D cells). These cells are defective in DHCR24 (sterol-Delta24-reductase, or 3beta-hydroxysterol Delta24-reductase), leading to desmosterol accumulation, and yet sterol homoeostasis appears to be normal with respect to SREBP processing and LXR activation. Other potentially cholesterol-dependent processes which were not the focus of this study are briefly discussed, such as lipid-raft-dependent cell signalling.

The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and affects 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and the cholesterol synthesis pathway.

The mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis, is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect was not established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increases cell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation, we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytes and model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulator of flux through the cholesterol synthesis pathway.

A practical comparison of ligation-independent cloning techniques.

The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. Here, we outline and optimise these techniques and identify important factors to guide cloning project design, including avoiding PCR artefacts such as primer-dimers and vector plasmid background. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.

Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase.

Exquisite control of cholesterol synthesis is crucial for maintaining homeostasis of this vital yet potentially toxic lipid. Squalene monooxygenase (SM) catalyzes the first oxygenation step in cholesterol synthesis, acting on squalene before cyclization into the basic steroid structure. Using model cell systems, we found that cholesterol caused the accumulation of the substrate squalene, suggesting that SM may serve as a flux-controlling enzyme beyond 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, considered as rate limiting). Cholesterol accelerated the proteasomal degradation of SM which required the N-terminal domain, partially conserved in vertebrates but not in lower organisms. Unlike HMGR, SM degradation is not mediated by Insig, 24,25-dihydrolanosterol, or side-chain oxysterols, but rather by cholesterol itself. Importantly, SM's N-terminal domain conferred cholesterol-regulated turnover on heterologous fusion proteins. Furthermore, proteasomal inhibition almost totally eliminated squalene accumulation, highlighting the importance of this degradation mechanism for the control of SM and suggesting this as a possible control point in cholesterol synthesis.