A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein.

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.

Nesprins anchor kinesin-1 motors to the nucleus to drive nuclear distribution in muscle cells.

During skeletal muscle development, nuclei move dynamically through myotubes in a microtubule-dependent manner, driven by the microtubule motor protein kinesin-1. Loss of kinesin-1 leads to improperly positioned nuclei in culture and in vivo. Two models have been proposed to explain how kinesin-1 functions to move nuclei in myotubes. In the cargo model, kinesin-1 acts directly from the surface of the nucleus, whereas in an alternative model, kinesin-1 moves nuclei indirectly by sliding anti-parallel microtubules. Here, we test the hypothesis that an ensemble of Kif5B motors acts from the nuclear envelope to distribute nuclei throughout the length of syncytial myotubes. First, using an inducible dimerization system, we show that controlled recruitment of truncated, constitutively active kinesin-1 motors to the nuclear envelope is sufficient to prevent the nuclear aggregation resulting from depletion of endogenous kinesin-1. Second, we identify a conserved kinesin light chain (KLC)-binding motif in the nuclear envelope proteins nesprin-1 and nesprin-2, and show that recruitment of the motor complex to the nucleus via this LEWD motif is essential for nuclear distribution. Together, our findings demonstrate that the nucleus is a kinesin-1 cargo in myotubes and that nesprins function as nuclear cargo adaptors. The importance of achieving and maintaining proper nuclear position is not restricted to muscle fibers, suggesting that the nesprin-dependent recruitment of kinesin-1 to the nuclear envelope through the interaction of a conserved LEWD motif with kinesin light chain might be a general mechanism for cell-type-specific nuclear positioning during development.

TM6SF2 rs58542926 impacts lipid processing in liver and small intestine.

The transmembrane 6 superfamily member 2 (TM6SF2) loss-of-function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically associated with lower levels of hepatically derived triglyceride-rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites for triglyceride-rich lipoprotein biogenesis and export. In light of this, we hypothesized that TM6SF2 may exhibit analogous effects on both liver and intestine lipid homeostasis. To test this, we genotyped rs58542926 in 983 bariatric surgery patients from the Geisinger Medical Center for Nutrition and Weight Management, Geisinger Health System, in Pennsylvania and from 3,556 study participants enrolled in the Amish Complex Disease Research Program. Although these two cohorts have different metabolic profiles, carriers in both cohorts had improved fasting lipid profiles. Importantly, following a high-fat challenge, carriers in the Amish Complex Disease Research Program cohort exhibited significantly lower postprandial serum triglycerides, suggestive of a role for TM6SF2 in the small intestine. To gain further insight into this putative role, effects of TM6SF2 deficiency were studied in a zebrafish model and in cultured human Caco-2 enterocytes. In both systems TM6SF2 deficiency resulted in defects in small intestine metabolism in response to dietary lipids, including significantly increased lipid accumulation, decreased lipid clearance, and increased endoplasmic reticulum stress. CONCLUSIONS: These data strongly support a role of TM6SF2 in the regulation of postprandial lipemia, potentially through a similar function for TM6SF2 in the lipidation and/or export of both hepatically and intestinally derived triglyceride-rich lipoproteins. (Hepatology 2017;65:1526-1542).

An HPLC-CAD/fluorescence lipidomics platform using fluorescent fatty acids as metabolic tracers.

Fluorescent lipids are important tools for live imaging in cell culture and animal models, yet their metabolism has not been well-characterized. Here we describe a novel combined HPLC and LC-MS/MS method developed to characterize both total lipid profiles and the products of fluorescently labeled lipids. Using this approach, we found that lipids labeled with the fluorescent tags, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene [BODIPY(558/568)], and dipyrrometheneboron difluoride undecanoic acid (TopFluor) are all metabolized into varying arrays of polar and nonpolar fluorescent lipid products when they are fed to larval zebrafish. Quantitative metabolic labeling experiments performed in this system revealed significant effects of total dietary lipid composition on fluorescent lipid partitioning. We provide evidence that cholesterol metabolism in the intestine is important in determining the metabolic fates of dietary FAs. Using this method, we found that inhibitors of dietary cholesterol absorption and esterification both decreased incorporation of dietary fluorescent FAs into cholesterol esters (CEs), suggesting that CE synthesis in enterocytes is primarily responsive to the availability of dietary cholesterol. These results are the first to comprehensively characterize fluorescent FA metabolism and to demonstrate their utility as metabolic labeling reagents, effectively coupling quantitative biochemistry with live imaging studies.

Endoplasmic Reticulum Lipid Flux Influences Enterocyte Nuclear Morphology and Lipid-dependent Transcriptional Responses.

Responding to a high-fat meal requires an interplay between multiple digestive tissues, sympathetic response pathways, and the gut microbiome. The epithelial enterocytes of the intestine are responsible for absorbing dietary nutrients and preparing them for circulation to distal tissues, which requires significant changes in cellular activity, including both morphological and transcriptional responses. Following a high-fat meal, we observe morphological changes in the enterocytes of larval zebrafish, including elongation of mitochondria, formation and expansion of lipid droplets, and the rapid and transient ruffling of the nuclear periphery. Dietary and pharmacological manipulation of zebrafish larvae demonstrated that these subcellular changes are specific to triglyceride absorption. The transcriptional changes that occur simultaneously with these morphological changes were determined using RNA sequencing, revealing a cohort of up-regulated genes associated with lipid droplet formation and lipid transport via lipoprotein particles. Using a microsomal triglyceride transfer protein (MTP) inhibitor to block ß-lipoprotein particle formation, we demonstrate that the transcriptional response to a high-fat meal is associated with the transfer of ER triglyceride to nascent ß-lipoproteins, possibly through the activation of Creb3l3/cyclic AMP-responsive element-binding protein. These data suggest that a transient increase in ER lipids is the likely mediator of the initial physiological response of intestinal enterocytes to dietary lipid.

Opposing microtubule motors drive robust nuclear dynamics in developing muscle cells.

Dynamic interactions with the cytoskeleton drive the movement and positioning of nuclei in many cell types. During muscle cell development, myoblasts fuse to form syncytial myofibers with nuclei positioned regularly along the length of the cell. Nuclear translocation in developing myotubes requires microtubules, but the mechanisms involved have not been elucidated. We find that as nuclei actively translocate through the cell, they rotate in three dimensions. The nuclear envelope, nucleoli and chromocenters within the nucleus rotate together as a unit. Both translocation and rotation require an intact microtubule cytoskeleton, which forms a dynamic bipolar network around nuclei. The plus- and minus-end-directed microtubule motor proteins, kinesin-1 and dynein, localize to the nuclear envelope in myotubes. Kinesin-1 localization is mediated at least in part by interaction with klarsicht/ANC-1/Syne homology (KASH) proteins. Depletion of kinesin-1 abolishes nuclear rotation and significantly inhibits nuclear translocation, resulting in the abnormal aggregation of nuclei at the midline of the myotube. Dynein depletion also inhibits nuclear dynamics, but to a lesser extent, leading to altered spacing between adjacent nuclei. Thus, oppositely directed motors acting from the surface of the nucleus drive nuclear motility in myotubes. The variable dynamics observed for individual nuclei within a single myotube are likely to result from the stochastic activity of competing motors interacting with a complex bipolar microtubule cytoskeleton that is also continuously remodeled as the nuclei move. The three-dimensional rotation of myotube nuclei may facilitate their motility through the complex and crowded cellular environment of the developing muscle cell, allowing for proper myonuclear positioning.

Pla2g12b drives expansion of triglyceride-rich lipoproteins.

Vertebrates transport hydrophobic triglycerides through the circulatory system by packaging them within amphipathic particles called Triglyceride-Rich Lipoproteins. Yet, it remains largely unknown how triglycerides are loaded onto these particles. Mutations in Phospholipase A2 group 12B (PLA2G12B) are known to disrupt lipoprotein homeostasis, but its mechanistic role in this process remains unclear. Here we report that PLA2G12B channels lipids within the lumen of the endoplasmic reticulum into nascent lipoproteins. This activity promotes efficient lipid secretion while preventing excess accumulation of intracellular lipids. We characterize the functional domains, subcellular localization, and interacting partners of PLA2G12B, demonstrating that PLA2G12B is calcium-dependent and tightly associated with the membrane of the endoplasmic reticulum. We also detect profound resistance to atherosclerosis in PLA2G12B mutant mice, suggesting an evolutionary tradeoff between triglyceride transport and cardiovascular disease risk. Here we identify PLA2G12B as a key driver of triglyceride incorporation into vertebrate lipoproteins.

Capzb2 interacts with beta-tubulin to regulate growth cone morphology and neurite outgrowth.

Capping protein (CP) is a heterodimer that regulates actin assembly by binding to the barbed end of F-actin. In cultured nonneuronal cells, each CP subunit plays a critical role in the organization and dynamics of lamellipodia and filopodia. Mutations in either alpha or beta CP subunit result in retinal degeneration in Drosophila. However, the function of CP subunits in mammalian neurons remains unclear. Here, we investigate the role of the beta CP subunit expressed in the brain, Capzb2, in growth cone morphology and neurite outgrowth. We found that silencing Capzb2 in hippocampal neurons resulted in short neurites and misshapen growth cones in which microtubules overgrew into the periphery and completely overlapped with F-actin. In searching for the mechanisms underlying these cytoskeletal abnormalities, we identified beta-tubulin as a novel binding partner of Capzb2 and demonstrated that Capzb2 decreases the rate and the extent of tubulin polymerization in vitro. We mapped the region of Capzb2 that was required for the subunit to interact with beta-tubulin and inhibit microtubule polymerization. A mutant Capzb2 lacking this region was able to bind F-actin and form a CP heterodimer with alpha2-subunit. However, this mutant was unable to rescue the growth cone and neurite outgrowth phenotypes caused by Capzb2 knockdown. Together, these data suggest that Capzb2 plays an important role in growth cone formation and neurite outgrowth and that the underlying mechanism may involve direct interaction between Capzb2 and microtubules.

Imaging cytoplasmic lipid droplets in vivo with fluorescent perilipin 2 and perilipin 3 knock-in zebrafish.

Cytoplasmic lipid droplets are highly dynamic storage organelles that are critical for cellular lipid homeostasis. While the molecular details of lipid droplet dynamics are a very active area of investigation, this work has been primarily performed in cultured cells. Taking advantage of the powerful transgenic and in vivo imaging opportunities available in zebrafish, we built a suite of tools to study lipid droplets in real time from the subcellular to the whole organism level. Fluorescently tagging the lipid droplet-associated proteins, perilipin 2 and perilipin 3, in the endogenous loci permits visualization of lipid droplets in the intestine, liver, and adipose tissue. Using these tools, we found that perilipin 3 is rapidly loaded on intestinal lipid droplets following a high-fat meal and later replaced by perilipin 2. These powerful new tools will facilitate studies on the role of lipid droplets in different tissues, under different genetic and physiological manipulations, and in a variety of human disease models.

Aged men experience disturbances in recovery following submaximal exercise.

BACKGROUND: Physiological responses to exercise of moderate intensity and duration among aged compared to young adults have yet to be clearly defined. Further, the effects of aging on the rate and effectiveness of postexercise recovery are unknown. METHODS: Here, selected physiological responses during and following exercise of the same relative intensity were examined in untrained young and aged men. RESULTS: Generally, the two groups displayed similar responses during 30 minutes of exercise. During recovery, however, numerous age-related differences were manifested. Relative heart rate (% peak) was higher during recovery among the aged group. Postexercise lactate remained increased longer among aged men, and blood glucose regulation was impaired during recovery. This difference in circulating glucose was associated with insulin responses whereby young, but not aged men experienced a postexercise spike. Unlike that in young men, rectal temperature among aged men continued to increase through the entire recovery period. CONCLUSIONS: These data suggest that aged men encounter problems in recovering from submaximal exercise.

Methods for Assessing Nuclear Rotation and Nuclear Positioning in Developing Skeletal Muscle Cells.

Skeletal muscle cells are large syncytia, containing hundreds of nuclei positioned regularly along the length of the fiber. During development, nuclei are actively distributed throughout the myotube by the microtubule motor proteins, kinesin-1, and cytoplasmic dynein. Nuclear movement consists of translocation along the long axis of the cell concurrent with three-dimensional rotation of nuclei. In this chapter we describe methods for quantitatively assessing the speed of nuclear rotation in cultured myotubes using live-cell imaging techniques coupled with rigid body kinematic analyses. Additionally, we provide protocols for analyzing nuclear distribution in myotubes.

Bronchoconstriction during cross-country skiing: is there really a refractory period?

PURPOSE: The asthmatic airway responds to exercise by bronchodilation (BD) during and bronchoconstriction (BC) after exercise. A refractory period induced by an initial exercise challenge that provides protection against BC during a subsequent exercise bout has also been observed. However, no studies examining during-exercise response or refractoriness during long-duration field exercise by elite athletes have been performed. This study examined airway response and refractoriness during approximately 42-min cross-country ski time trial preceded by a 6- to 9-min 2.5-km high-intensity warm-up ski. METHODS: Eighteen elite athletes cross-country skied seven successive 2.5-km loops. Spirometry was performed pre- and at 5, 10, and 15 min post loop 1; loops 2-7 were treated as a race (XCR) with maneuvers performed within 20 s after loops 2-6 and serially for 15 min after lap 7. RESULTS: Nine of 18 subjects demonstrated a >or=10% fall from baseline in FEV(1) (EIB+): five after lap 1 and four during or after laps 2-7. FEV(1) for EIB+ athletes during XCR was not different from post lap 1 FEV. Only one EIB+ subject demonstrated significant refractoriness. Four EIB+ athletes had a less than 10% fall in FEV after the initial 2.5-km exercise challenge but developed EIB (>or=10% fall) during the subsequent 6 x 2.5 km XCR exercise challenge. FEF(25-75) falls mirrored FEV(1), but demonstrated greater BD during XCR. CONCLUSION: Bronchoconstriction occurs in athletes during prolonged exercise and may thus influence performance. Variability in bronchial hyperresponsiveness onset and the lack of significant refractoriness in our study cohort of athletes is consistent with an exercise bronchoconstrictive dysfunction that is different than frank asthma and is yet to be clearly defined.

Greater transport efficiencies of the membrane fatty acid transporters FAT/CD36 and FATP4 compared with FABPpm and FATP1 and differential effects on fatty acid esterification and oxidation in rat skeletal muscle.

In selected mammalian tissues, long chain fatty acid transporters (FABPpm, FAT/CD36, FATP1, and FATP4) are co-expressed. There is controversy as to whether they all function as membrane-bound transporters and whether they channel fatty acids to oxidation and/or esterification. Among skeletal muscles, the protein expression of FABPpm, FAT/CD36, and FATP4, but not FATP1, correlated highly with the capacities for oxidative metabolism (r>or=0.94), fatty acid oxidation (r>or=0.88), and triacylglycerol esterification (r>or=0.87). We overexpressed independently FABPpm, FAT/CD36, FATP1, and FATP4, within a normal physiologic range, in rat skeletal muscle, to determine the effects on fatty acid transport and metabolism. Independent overexpression of each fatty acid transporter occurred without altering either the expression or plasmalemmal content of other fatty acid transporters. All transporters increased fatty acid transport, but FAT/CD36 and FATP4 were 2.3- and 1.7-fold more effective than FABPpm and FATP1, respectively. Fatty acid transporters failed to alter the rates of fatty acid esterification into triacylglycerols. In contrast, all transporters increased the rates of long chain fatty acid oxidation, but the effects of FABPpm and FAT/CD36 were 3-fold greater than for FATP1 and FATP4. Thus, fatty acid transporters exhibit different capacities for fatty acid transport and metabolism. In vivo, FAT/CD36 and FATP4 are the most effective fatty acid transporters, whereas FABPpm and FAT/CD36 are key for stimulating fatty acid oxidation.

The neuromuscular junction: anatomical features and adaptations to various forms of increased, or decreased neuromuscular activity.

The neuromuscular junction (NMJ) allows communication between motor neurons and muscle fibers. During development, marked morphological changes occur as the functional NMJ is formed. During the postnatal period of rapid growth and muscle enlargement, endplate size concurrently increases. Even beyond this period of pronounced plasticity, the NMJ undergoes subtle morphological remodeling--expansion and retraction--although its overall dimensions remain stable. This natural, continual NMJ remodeling is amplified with alterations in neuromuscular activity. Increased activity, presented by exercise training, typically results in expansion of NMJ size. Disuse, brought about by neurotoxins, denervation, or spaceflight, also elicits substantial reconfiguring of the endplate.

Neuromuscular adaptations to spaceflight are specific to postural muscles.

The effects of microgravity were determined in muscles of differing function and myofiber-type composition. Rats were assigned either to a 10-day spaceflight mission or to ground-based control conditions. Following the experimental period, hindlimb muscles were obtained from both groups. Cytofluorescent techniques were used to examine neuromuscular junctions (NMJs) from both slow- and fast-twitch fibers. Histochemical procedures were employed to assess myofiber profiles (size and type). Results indicate that microgravity did not alter NMJ structure or myofiber profile in the tibialis anterior, a predominantly fast-twitch, nonpostural muscle. Similarly, the NMJs and myofibers of deep regions of the gastrocnemius, a locomotor muscle possessing a mixed fiber population, were unaffected by spaceflight. In contrast, both myofibers and NMJs of the soleus-a postural muscle-demonstrated significant (P < 0.05) plasticity following exposure to spaceflight. Moreover, NMJs of both fast- and slow-twitch myofibers displayed similar remodeling in that muscle. Our findings suggest that the deleterious effects of microgravity are most apparent among postural muscles, and are manifested both in myofibers and their synapses.

Age-related differences in synaptic plasticity following muscle unloading.

The objective of the present investigation was to determine the effects of muscle unloading-a form of subtotal disuse- on the morphology of the neuromuscular junction (NMJ) in younger and aged animals. Sixteen aged (22 months) and 16 young adult (8 months) male Fischer 344 rats were assigned to control and hindlimb suspension (HS) conditions (n=8/group). At the conclusion of the 4 week experimental period, soleus muscles were collected, and immunofluorescent procedures were used to visualize acetylcholine (ACh) vesicles and receptors, nerve terminal branching, as well as NCAM and NT-4 expression. Quantitative analyses revealed that aged controls displayed significant (p<0.05) reductions in area and perimeter length of ACh vesicle and receptor regions, without affecting nerve terminal branch number or length. In contrast to younger NMJs, which were resilient to the effects of unloading, NMJs of aged HS rats demonstrated significant expansion of ACh vesicle and receptor dimensions compared to aged controls. Qualitative analyses of NCAM staining indicated that aging alone somewhat increased this molecule's expression (aged controls>young controls). Among the four groups, however, the greatest amount of NCAM content was detected among aged HS muscles, matching the degree of synaptic plasticity exhibited in those muscles. Unlike NCAM, the expression of NT-4 did not appear to differ among the treatment groups. These data suggest that although young adult muscle maintains normal NMJ structure during prolonged exposure to unloading, aged NMJs experience significant adaptation to that stimulus.

Evaluation of cardiovascular demands of game play and practice in women's ice hockey.

Preparation for the physical demands of competition often involves game simulation during practice. This paradigm is thought to promote physiological adaptations that enhance maximal performance. However, a mismatch between practice intensity and actual competition intensity may not provide adequate training to achieve optimal game-play fitness. The purpose of this study was to evaluate the effectiveness of practice in meeting the cardiovascular demands of a women's ice hockey game. Heart rate (HR) data from 11 U.S. National Women's Ice Hockey team members were collected (5-second intervals) during a game and a typical practice session. Data was normalized to individual HRmax determined during Vo(2)max testing. Working time was defined as a game shift or practice-working interval. Mean working HR was greater during the game than the practice, 90 +/- 2% and 76 +/- 3% of HRmax, respectively (p < 0.05). Mean percent session time (game or practice) >90% HRmax was also longer during the game than the practice, 10.5 +/- 4.1% and 5.6 +/- 3.5% (p < 0.05), respectively. Mean session HR, percent time >80% HRmax, and mean resting HR were not different between game and practice (68 +/- 7% vs. 69 +/- 5%, 23.2 +/- 5.3% vs. 26.1 +/- 9.2%, and 59 +/- 8% vs. 56 +/- 5%, respectively). Elite women hockey players experience significantly greater cardiovascular load during game play than during practice. This mismatch in cardiovascular demand may prevent players from achieving "game shape," thus affecting competition play.