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KEY POINTS: We have recently shown that a high-fat, high-calorie (HFHC) diet decreases whole body glucose clearance without impairing skeletal muscle insulin signalling, in healthy lean individuals. These diets are also known to increase skeletal muscle IMTG stores, but the effect on lipid metabolites leading to skeletal muscle insulin resistance has not been investigated. This study measured the effect of 7 days' HFHC diet on (1) skeletal muscle concentration of lipid metabolites, and (2) potential changes in the perilipin (PLIN) content of the lipid droplets storing intramuscular triglyceride (IMTG). The HFHC diet increased PLIN3 protein expression and redistributed PLIN2 to lipid droplet stores in type I fibres. The HFHC diet increased IMTG content in type I fibres, while lipid metabolite concentrations remained the same. The data suggest that the increases in IMTG stores assists in reducing the accumulation of lipid metabolites known to contribute to skeletal muscle insulin resistance. ABSTRACT: A high-fat, high-calorie (HFHC) diet reduces whole body glucose clearance without impairing skeletal muscle insulin signalling in healthy lean individuals. HFHC diets also increase skeletal muscle lipid stores. However, unlike certain lipid metabolites, intramuscular triglyceride (IMTG) stored within lipid droplets (LDs) does not directly contribute to skeletal muscle insulin resistance. Increased expression of perilipin (PLIN) proteins and colocalisation to LDs has been shown to assist in IMTG storage. We aimed to test the hypothesis that 7 days on a HFHC diet increases IMTG content while minimising accumulation of lipid metabolites known to disrupt skeletal muscle insulin signalling in sedentary and obese individuals. We also aimed to identify changes in expression and subcellular distribution of proteins involved in IMTG storage. Muscle biopsies were obtained from the m. vastus lateralis of 13 (11 males, 2 females) healthy lean individuals (age: 23 ± 2.5 years; body mass index: 24.5 ± 2.4 kg m-2 ), following an overnight fast, before and after consuming a high-fat (64% energy), high-calorie (+47% kcal) diet for 7 days. After the HFHC diet, IMTG content increased in type I fibres only (+101%; P < 0.001), whereas there was no change in the concentration of either total diacylglycerol (P = 0.123) or total ceramides (P = 0.150). Of the PLINs investigated, only PLIN3 content increased (+50%; P < 0.01) solely in type I fibres. LDs labelled with PLIN2 increased (+80%; P < 0.01), also in type I fibres only. We propose that these adaptations of LDs support IMTG storage and minimise accumulation of lipid metabolites to protect skeletal muscle insulin signalling following 7 days' HFHC diet.

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Sample preparation is central to acquiring meaningful molecule-specific images with SIMS, especially when submicron lateral resolution is involved. The issue is to maintain the distribution of target molecules while attempting to introduce biological cells or tissue into the high vacuum environment of the mass spectrometer. Here we compare freeze-drying, freeze-etching, freeze-fracture and trehalose vitrification as possible strategies for these experiments. The results show that the prospects for successful imaging experiments are greatly improved with all of these methods when using cluster ion bombardment, particularly C(60) (+) ions, not only due to increased sensitivity of this projectiles, but also since it removes contamination overlayers without insult to the underlying chemistry. The emergence of 3-dimensional imaging capabilities also suggests that sample preparation should not perturb the 3-dimensional morphology of the cell, a situation not generally possible during freeze-drying. Hence, sample preparation and projectile type are strongly coupled parameters for bioimaging with mass spectrometry.

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Phagocytosis is a major component of the animal immune system where apoptotic cellular material, metabolites, and waste are safely processed. Further, efficient phagocytosis by macrophages is key to maintaining healthy vascular systems and preventing atherosclerosis. Single-cell images of macrophage phagocytosis of red blood cells, RBCs, and polystyrene microspheres have been chemically mapped with TOF-SIMS. We demonstrate here cholesterol and phosphocholine localizations as relative to time and activity.

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We report the use of SIMS imaging to quantify the relative difference in the amount of lipid between two sections, the plasma membrane and the cytoplasm, of single cells from two different populations. Cells were each labeled with lipophillic dyes, frozen, fractured and analyzed in a ToF-SIMS mass spectrometer equipped with a 40 keV C(60) (+) ion source. In addition to identifying cells from separate populations, the lipophilic dyes can be used as a marker for the outer leaflet of the cell membrane and therefore as a depth finder. Here, we show that it is possible to compare the amount of lipids with particular headgroups in the cell membrane of a treated cell to the membrane of a control cell. Following erosion of the cell membranes, the amount of the two specific lipid head groups in the cytoplasm of the treated cell can be compared to those lipids in a control cell. Here we take the first step in this experimental design and display the ability to analyze multiple sections of frozen cells following a single fracture.

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Forty-eight patients with perinatal static encephalopathy were treated with unilateral varus rotational femoral osteotomy for hip subluxation. Twenty-one of the 48 also had concomitant pelvic osteotomy. Twenty-seven of the patients were nonambulators. Bilateral soft-tissue releases had been performed previously in 20 patients. At the time of varus rotational osteotomy, the mean age was 8 years, 11 months. The indication for a varus rotational osteotomy was femoral head subluxation or dislocation in all cases. The preoperative center-edge angle ranged from -8 degrees to 18 degrees (mean, 4 degrees), and on the contralateral side, the center-edge angle ranged from 0 degree to 32 degrees (mean, 19 degrees). The center-edge angle on the operative side at follow-up averaged 25 degrees (-10 degrees to 78 degrees), an improvement of 21 degrees, and on the contralateral side averaged 27 degrees (range, 9 degrees-40 degrees), an improvement of 8 degrees. Forty-seven patients have had no subsequent surgery on the contralateral hip. One patient had a varus rotational osteotomy on the contralateral hip 5 years postoperatively; however, this hip was subluxated at the time of the index procedure. Twelve patients have had no surgery at any time on the contralateral side. None of these patients had deterioration of the contralateral hip. Surgery on one hip should not, in itself, be an indication for surgery on the contralateral concentrically reduced hip with adequate abduction in patients with acetabular dysplasia secondary to perinatal static encephalopathy.

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Frequently, a patient undergoing anterior cruciate ligament (ACL) reconstruction 4 to 6 weeks following the acute injury will be noted to have decreased anterior translation during a Lachman and anterior drawer testing at the time of surgery compared with their examination at the time of their acute injury. Orthopedic surgeons frequently attribute this decreased anterior translation to scarring of the ACL to the posterior cruciate ligament (PCL). We undertook a cadaver study to evaluate this. Four cadaveric lower extremity specimens underwent a midline posterior approach to the knee followed by arthrotomy. The ACL then was cut from its origin on the lateral femoral condyle. Following its release, the ACL was sutured tightly to the PCL. Prior to dissection, the specimens underwent baseline KT-1000 knee arthrometer testing. Following the arthrotomy but prior to ACL release, the KT-1000 test was repeated. KT-1000 testing then was performed after ACL release and repeated after suturing the ACL to the PCL. The results revealed no change in KT-1000 testing following arthrotomy. Anterior translation increased an average of 5 mm following release of the ACL and after suturing to the PCL. No specimen showed a significant decrease in anterior translation following suturing of the ACL to the PCL. These results indicate that while decreased anterior translation from the time of injury to the time of reconstruction may occur, this cannot be explained based on scarring of the ACL stump to the PCL.

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