Abstract Human immunodeficiency virus–seronegative men aged 15–22 years who lost bone mineral density (BMD) during tenofovir disoproxil fumarate/emtricitabine preexposure prophylaxis (PrEP) showed BMD recovery 48 weeks following PrEP discontinuation. Lumbar spine and whole body BMD z-scores remained below baseline 48 weeks off PrEP in participants aged 15–19 years. Clinical Trials Registration. NCT01772823 (ATN 110) and NCT01769456 (ATN 113).
Objective To determine whether treatment with growth hormone (GH) enhances thymopoiesis in individuals infected with HIV-1. Methods Five HIV-1-infected adults were treated with GH for 6–12 months in a prospective open-label study. Immunological analyses were performed before GH treatment and repeated at 3 month intervals after GH initiation. Thymic mass was analysed using computed tomography with quantitative density and volume analysis. Analysis of circulating lymphocytes, including naive and memory T cell subsets, was performed using multiparameter flow cytometry. Results GH treatment was associated with a marked increase in thymic mass in all GH recipients. Circulating naive CD4 T cells also increased significantly in all patients during GH therapy, suggesting an enhancement of thymopoiesis. Conclusion GH has significant effects on the human immune system, including the reversal of thymic atrophy in HIV-1-infected adults. De-novo T cell production may thus be inducible in immunodeficient adults.
Interest in subspecialty training remains high for dermatology residency graduates. Fellowship program directors (FPDs) are largely responsible for fellowship organization and development. Here, we study the dermatology fellowship leadership landscape and identify notable differences in the characteristics of current dermatopathology, paediatric dermatology, and Mohs micrographic surgery and dermatologic oncology FPDs.
Abstract Introduction Gynecologic malignancies are an increasingly common proportion of central nervous system metastatic disease. As genetic sequencing technology improves and becomes more accessible, mutations associated with CNS metastasis are easier to elucidate. The aims of this case series and systematic literature review are to describe the patient population with CNS metastatic disease from a gynecologic primary, and to investigate why the proportion of CNS metastasis from gynecologic malignancies is increasing. Ultimately, we hope to improve understanding of this subset of metastatic CNS malignancies and improve management strategies. Methods A literature review of articles describing patients from 1990–2020 who were diagnosed with CNS metastasis from a known gynecologic primary malignancy was performed. Demographics, cancer type, mutation characteristics, management for metastatic disease, progression free survival, number of CNS metastases, and location of metastatic disease were assessed. Inclusion criteria were age>18 years, diagnosis of primary ovarian, uterine, or cervical cancer with confirmed metastatic disease to the CNS, including brain parenchyma, leptomeninges, or intradural spinal cord or dural metastases. Exclusion criteria included pediatric population and bony metastases (e.g., bony spine metastases without evidence of meningeal/parenchymal invasion). Results Our review showed that patients with gynecological metastasis to the CNS generally have worse outcomes regarding overall survival, progression free survival, and quality of life than patients without CNS metastasis. Discussion Our results infer that the reported increase in incidence of CNS metastasis from gynecologic malignancies is a reflection of improvement of detection given advances in technology, improved patient follow up, and increased overall survival of patients with gynecologic malignancies. Further characterization of mutations from gynecologic malignancies associated with brain metastasis could result in development of more treatment options for patients in the future and help determine factors that contribute to developing metastasis to the CNS of various degrees, thus, potentially inform treatment strategies.
Energy intake and expenditure in women runners and non-runners were assessed by weighed food records, evaluation of minute-by-minute activity diaries, and indirect calorimetry. All participants were adapted to their stated activity levels for at least 6 months and maintained a constant body-weight throughout their participation. Calculated daily energy intake equalled calculated expenditure in non-runners (7300 (SD 1536) v. 7476 (SD 872) kJ/d), but calculated energy expenditure in women running about 54 km/week was found to exceed intake by more than 2700 kJ/d (8259 (SD 1466) v. 10963 (SD 1367), P < 0.01). The runners showed no evidence of compensating for the increased energy expenditure associated with running by engaging in lower-intensity activities during non-running time. Further, runners did not decrease energy expended at various activities. The findings suggest that women adapted to high levels of activity may possess mechanisms to maintain body-weight without significantly increasing energy intake.
Background The pathogenesis of insulin resistance in the absence of obesity is unknown. In obesity, multiple stress kinases have been identified that impair the insulin signaling pathway via serine phosphorylation of key second messenger proteins. These stress kinases are activated through various mechanisms related to lipid oversupply locally in insulin target tissues and in various adipose depots. Methodology/Principal Findings To explore whether specific stress kinases that have been implicated in the insulin resistance of obesity are potentially contributing to insulin resistance in non-obese individuals, twenty healthy, non-obese, normoglycemic subjects identified as insulin sensitive or resistant were studied. Vastus lateralis muscle biopsies obtained during euglycemic, hyperinsulinemic clamp were evaluated for insulin signaling and for activation of stress kinase pathways. Total and regional adipose stores and intramyocellular lipids (IMCL) were assessed by DXA, MRI and 1H-MRS. In muscle of resistant subjects, phosphorylation of JNK was increased (1.36±0.23 vs. 0.78±0.10 OD units, P<0.05), while there was no evidence for activation of p38 MAPK or IKKβ. IRS-1 serine phosphorylation was increased (1.30±0.09 vs. 0.22±0.03 OD units, P<0.005) while insulin-stimulated tyrosine phosphorylation decreased (10.97±0.95 vs. 0.89±0.50 OD units, P<0.005). IMCL levels were twice as high in insulin resistant subjects (3.26±0.48 vs. 1.58±0.35% H2O peak, P<0.05), who also displayed increased total fat and abdominal fat when compared to insulin sensitive controls. Conclusions This is the first report demonstrating that insulin resistance in non-obese, normoglycemic subjects is associated with activation of the JNK pathway related to increased IMCL and higher total body and abdominal adipose stores. While JNK activation is consistent with a primary impact of muscle lipid accumulation on metabolic stress, further work is necessary to determine the relative contributions of the various mediators of impaired insulin signaling in this population.
Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis. However, guidelines for treatment are defined solely by fasting lipid levels, even though postprandial lipids may be more informative. In the postprandial state, circulating lipids consist of dietary fat transported from the intestine in chylomicrons (CMs; containing ApoB48) and fat transported from the liver in VLDL (containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because of the difficulty in separating these particles by ultracentrifugation. CM fractions have considerable contamination from VLDL (purity, 10%). To separate CMs from VLDL, we produced polyclonal antibodies against ApoB100 and generated immunoaffinity columns. TRLs isolated by ultracentrifugation of plasma were applied to these columns, and highly purified CMs were collected (purity, 90–94%). Overall eight healthy unmedicated adult volunteers (BMI, 27.2 ± 1.4 kg/m2; fasting triacylglycerol, 102.6 ± 19.5 mg/dl) participated in a feeding study, which contained an oral stable-isotope tracer (1-13C acetate). We then used this technique on plasma samples freshly collected during an 8 h human feeding study from a subset of four subjects. We analyzed fractionated lipoproteins by Western blot, isolated and derivatized triacylglycerols, and calculated fractional de novo lipogenesis. The results demonstrated effective separation of postprandial lipoproteins and substantially improved purity compared with ultracentrifugation protocols, using the immunoaffinity method. This method can be used to better delineate the role of dietary sugar and fat on postprandial lipids in cardiovascular risk and explore the potential role of CM remnants in atherosclerosis. Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis. However, guidelines for treatment are defined solely by fasting lipid levels, even though postprandial lipids may be more informative. In the postprandial state, circulating lipids consist of dietary fat transported from the intestine in chylomicrons (CMs; containing ApoB48) and fat transported from the liver in VLDL (containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because of the difficulty in separating these particles by ultracentrifugation. CM fractions have considerable contamination from VLDL (purity, 10%). To separate CMs from VLDL, we produced polyclonal antibodies against ApoB100 and generated immunoaffinity columns. TRLs isolated by ultracentrifugation of plasma were applied to these columns, and highly purified CMs were collected (purity, 90–94%). Overall eight healthy unmedicated adult volunteers (BMI, 27.2 ± 1.4 kg/m2; fasting triacylglycerol, 102.6 ± 19.5 mg/dl) participated in a feeding study, which contained an oral stable-isotope tracer (1-13C acetate). We then used this technique on plasma samples freshly collected during an 8 h human feeding study from a subset of four subjects. We analyzed fractionated lipoproteins by Western blot, isolated and derivatized triacylglycerols, and calculated fractional de novo lipogenesis. The results demonstrated effective separation of postprandial lipoproteins and substantially improved purity compared with ultracentrifugation protocols, using the immunoaffinity method. This method can be used to better delineate the role of dietary sugar and fat on postprandial lipids in cardiovascular risk and explore the potential role of CM remnants in atherosclerosis. Elevated levels of fasting plasma triacylglycerol (TAG) have been recognized as a risk factor for coronary heart disease, which, in turn, increases the risk of myocardial infarction (1Grundy S.M. Hypertriglyceridemia, atherogenic dyslipidemia, and the metabolic syndrome.Am. J. Cardiol. 1998; 81: 18B-25BAbstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, 2Austin M.A. Epidemiology of hypertriglyceridemia and cardiovascular disease.Am. J. Cardiol. 1999; 83: 13F-16FAbstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 3Austin M.A. Plasma triglyceride as a risk factor for coronary heart disease. The epidemiologic evidence and beyond.Am. J. Epidemiol. 1989; 129: 249-259Crossref PubMed Scopus (304) Google Scholar). In the 1970s, Zilversmit (4Zilversmit D.B. Atherogenesis: a postprandial phenomenon.Circulation. 1979; 60: 473-485Crossref PubMed Scopus (1436) Google Scholar) postulated that processes that cause atherosclerosis might occur during the postprandial period and that dietary lipids transported by chylomicrons (CMs) were an important contributor to cardiovascular risks. While Parks et al. (5Parks E.J. Krauss R.M. Christiansen M.P. Neese R.A. Hellerstein M.K. Effects of a low-fat, high-carbohydrate diet on VLDL-triglyceride assembly, production, and clearance.J. Clin. Invest. 1999; 104: 1087-1096Crossref PubMed Scopus (260) Google Scholar) were able to demonstrate that the flux of VLDL is greater during fasting versus the postprandial state, the role of CMs has yet to be elucidated. More recently, dietary sugars, specifically fructose, have been shown to increase TAG with supportive evidence that hepatic conversion of dietary sugars to fat [de novo lipogenesis (DNL)] could also be an important contributor to postprandial VLDL levels (6Schwarz J.M. Noworolski S.M. Wen M.J. Dyachenko A. Prior J.L. Weinberg M.E. Herraiz L.A. Tai V.W. Bergeron N. Bersot T.P. et al.Effect of a high-fructose weight-maintaining diet on lipogenesis and liver fat.J. Clin. Endocrinol. Metab. 2015; 100: 2434-2442Crossref PubMed Scopus (161) Google Scholar, 7Stanhope K.L. Schwarz J.M. Keim N.L. Griffen S.C. Bremer A.A. Graham J.L. Hatcher B. Cox C.L. Dyachenko A. Zhang W. et al.Consuming fructose-sweetened, not glucose-sweetened, beverages increases visceral adiposity and lipids and decreases insulin sensitivity in overweight/obese humans.J. Clin. Invest. 2009; 119: 1322-1334Crossref PubMed Scopus (1269) Google Scholar). Additionally, DNL and other metabolic markers, such as LDL-cholesterol and TAG, were significantly reduced in children with metabolic syndrome that underwent a 9 day fructose restriction diet (8Schwarz J.M. Noworolski S.M. Erkin-Cakmak A. Korn N.J. Wen M.J. Tai V.W. Jones G.M. Palii S.P. Velasco-Alin M. Pan K. et al.Effects of dietary fructose restriction on liver fat, de novo lipogenesis, and insulin kinetics in children with obesity.Gastroenterology. 2017; 153: 743-752Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 9Lustig R.H. Mulligan K. Noworolski S.M. Tai V.W. Wen M.J. Erkin-Cakmak A. Gugliucci A. Schwarz J.M. Isocaloric fructose restriction and metabolic improvement in children with obesity and metabolic syndrome.Obesity (Silver Spring). 2016; 24: 453-460Crossref PubMed Scopus (119) Google Scholar). Postprandial plasma TAGs are derived from two sources. First, dietary TAGs are hydrolyzed into monoacylglycerols and fatty acids are absorbed by the intestine, where they are re-esterified into TAGs, packaged into CMs, and secreted into the lymph before reaching the general circulation (10Harvey R.A. Ferrier D.R. Lippincott's Illustrated Reviews: Biochemistry. 5th edition. Wolters Kluwer Health, Philadelphia2011Google Scholar). Second, hepatic TAGs are synthesized from either free fatty acids that are released by the adipose tissue or synthesized de novo or derived from dietary CM remnants taken up by the liver. These hepatic TAGs are repackaged into VLDL and secreted into the bloodstream (11Taskinen M.R. Boren J. New insights into the pathophysiology of dyslipidemia in type 2 diabetes.Atherosclerosis. 2015; 239: 483-495Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Research into the relative roles and abundances of CM and VLDL particles in the postprandial state has been hindered because of limitations in laboratory methods used to separate these particles. Ultracentrifugation can achieve separation of triglyceride-rich lipoproteins (TRLs) and partial separation of CMs from VLDL, but cannot fully separate particles of overlapping densities, such as small CMs or CM remnants from large VLDL particles and VLDL remnants (12Lieberman M. Marks A.D. Peet A. Marks' Basic Medical Biochemistry: A Clinical Approach. 4th edition. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia2013Google Scholar). This technical barrier has limited investigations that aim to characterize: 1) the respective contribution of TAG from the intestines and from the liver to postprandial lipid metabolism; 2) the kinetic aspects, namely the respective turnover rates of CMs versus VLDL particles; 3) the differential clearance rates of TAG; or 4) the impact of dietary factors, such as carbohydrates, sugar, and branched-chain amino acids, among others, on postprandial lipid metabolism and cardiovascular risk. To overcome the aforementioned limitations of ultracentrifugation separation, we developed an immunoaffinity method to separate CMs from VLDL (Fig. 1). While alternative immunoaffinity methods exist (13Heath R.B. Karpe F. Milne R.W. Burdge G.C. Wootton S.A. Frayn K.N. Selective partitioning of dietary fatty acids into the VLDL TG pool in the early postprandial period.J. Lipid Res. 2003; 44: 2065-2072Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 14Sun F. Stolinski M. Shojaee-Moradie F. Lou S. Ma Y. Hovorka R. Umpleby A.M. A novel method for measuring intestinal and hepatic triacylglycerol kinetics.Am. J. Physiol. Endocrinol. Metab. 2013; 305: E1041-E1047Crossref PubMed Scopus (8) Google Scholar), we needed to obtain and document purer CMs as well as a robust method that would allow for the majority of intact CM particles to be recovered, as they are quite labile. Intact particles contain information regarding the tissue of origin and might allow for the investigation of human intestinal DNL, as has long been predicted in the literature. We generated polyclonal antibodies directed to a C-terminal sequence in ApoB100, which is absent in the ApoB48 sequence. This allowed for the separation of CM and VLDL particles. Antibodies were cross-linked to a protein G resin to create an anti-ApoB100 resin that allowed the isolation of purified CM particles. In the first part of this study, we applied postprandial TRL (<δ 1.0063 g/ml, 17 h spin) and impure CM fractions isolated by ultracentrifugation (CMU; <δ 1.0063 g/ml, 0.5 h spin) to the ApoB100-specific resin to generate purified CMs. In the second part of this study, we further characterized these purified particles; and to illustrate the application of our method, we measured fractional DNL (15Hellerstein M.K. Neese R.A. Schwarz J.M. Model for measuring absolute rates of hepatic de novo lipogenesis and reesterification of free fatty acids.Am. J. Physiol. 1993; 265: E814-E820PubMed Google Scholar) in the purified CM fractions using GC/MS and mass isotopomer distribution analysis (MIDA) (16Hellerstein M.K. Schwarz J.M. Neese R.A. Regulation of hepatic de novo lipogenesis in humans.Annu. Rev. Nutr. 1996; 16: 523-557Crossref PubMed Scopus (281) Google Scholar, 17Hellerstein M.K. Neese R.A. Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations.Am. J. Physiol. 1999; 276: E1146-E1170Crossref PubMed Google Scholar, 18Hellerstein M.K. Christiansen M. Kaempfer S. Kletke C. Wu K. Reid J.S. Mulligan K. Hellerstein N.S. Shackleton C.H. Measurement of de novo hepatic lipogenesis in humans using stable isotopes.J. Clin. Invest. 1991; 87: 1841-1852Crossref PubMed Scopus (296) Google Scholar, 19Hellerstein M.K. Kletke C. Kaempfer S. Wu K. Shackleton C.H. Use of mass isotopomer distributions in secreted lipids to sample lipogenic acetyl-CoA pool in vivo in humans.Am. J. Physiol. 1991; 261: E479-E486PubMed Google Scholar) from samples collected from human volunteers during an 8 h feeding study. We generated ApoB100 immunoaffinity columns to separate CMs from VLDL using a goat antibody raised against ApoB100-specific epitopes purified on an LDL column. Using the pATH20 vector, we produced a trpE fusion protein, which was used as an antigen for the production of α-ApoB100 antibodies in a goat (20Pullinger C.R. Zysow B.R. Hennessy L.K. Frost P.H. Malloy M.J. Kane J.P. Molecular cloning and characteristics of a new apolipoprotein C-II mutant identified in three unrelated individuals with hypercholesterolemia and hypertriglyceridemia.Hum. Mol. Genet. 1993; 2: 69-74Crossref PubMed Scopus (12) Google Scholar). A 1,862 bp APOB fragment was amplified using human genomic DNA (primers: 5′-ctcaccatattcaaaactagagttagaggg-3′ and 5′-atttagttcctcctcccccaagtttagc-3′). The product was digested with PstI and HindIII. A 784 bp band (APOB codons 4,108–4,369) was purified and ligated into pATH20 (21Mercy M.R. Troncoso J.C. Monteiro M.J. A new series of trpE vectors that enable high expression of nonfusion proteins in bacteria.Protein Expr. Purif. 1992; 3: 57-64Crossref PubMed Scopus (5) Google Scholar). This was used to transform competent Escherichia coli RR1 cells. Cells from positive colonies were grown at 37°C to mid log phase in tryptophan-supplemented medium in 10 ml starter cultures. These were diluted to 500 ml in tryptophan-free medium. After a 2 h incubation, indoleacrylic acid was added to induce expression. After an overnight incubation, the cells were pelleted and protein dissolved in urea/Tris/NaCl buffer. The 66 kDa-fusion protein was purified on preparative 12% SDS-PAGE gels and used to inoculate a goat. A naïve adult goat was given an initial immunization followed by four boosters over a 70 day period. Anti-serum was harvested on day −49, −63, −77, and −84. Goat anti-serum was diluted 2-fold and buffered with 100 mM Tris HCl (pH 8.0). Antibodies were precipitated on ice using saturated ammonium sulfate, up to 50%, stirring constantly for 6 h, after which samples were centrifuged at 3,000 g for 30 min at 4°C. The supernatant was removed and the pellet was washed twice and resuspended in PBS. The immunoglobulin solution was then dialyzed in PBS overnight at 4°C. The procedure employed a modified form of the Amino-Link coupling resin protocol (Thermo Fisher Scientific, Waltham, MA). The immobilized protein for the α-ApoB100 affinity column was LDL (ApoB100, δ 1.019–1.063 g/ml) purified from human serum by sequential ultracentrifugation. Four milliliters of LDL (1 mg protein/ml) in 10 mM PBS containing 20% sucrose was diluted 1:2 with 20% sucrose, 100 mM NaHCO3 (pH 9.0), 500 mM NaCl, and 1 mM EDTA. The final volume of the coupling load was 8 ml. At each step, protein content in the nonbound fractions was analyzed by Coomassie Plus protein assay (Thermo Fisher Scientific) to determine coupling efficiency. Amino Link Plus NaCNBH3 (sodium cyanoborohydride)-activated resin (Thermo Fisher Scientific) was equilibrated with 10 column volumes of coupling buffer. The coupling load was added to the resin and mixed for 2 h at room temperature, after which the columns were drained and washed with 5 column volumes of coupling buffer, followed by 3 column volumes of 100 mM PBS (pH 7.2). Approximately 50 mM of sodium cyanoborohydride were added to the column and mixed for 2 h at room temperature. The column was drained and washed with 5 column volumes of 1 M Tris-HCl (pH 8.0) to quench the reaction. To reduce the remaining amino groups, 1 M Tris-HCl (pH 8.0) and 50 mM NaCNBH3 were added to the column and mixed for 2 h at room temperature, then drained. The column was washed with 5 column volumes of 1 M NaCl, drained, then washed with 250 ml 1× PBS (pH 7.6), 1 mM EDTA, and 0.01% sodium azide and stored at 4°C. Three 5 ml columns were made and pooled. The binding efficiencies were consistently >90% and the columns bound approximately 0.56 mg/ml LDL. The LDL ApoB100 resin prepared as described above (15 ml) was equilibrated with PBS and 1 mM EDTA and added to the immunoglobulin solution and mixed for 48 h at 4°C. Eighty percent of the supernatant was decanted through a 20 ml low pressure column; the other 20% was mixed with the resin and pipetted onto the column and allowed to drain. The column was washed with PBS, 1 M NaCl, and 1 mM EDTA (5 vol) and then with 10 vol of PBS and 1 mM EDTA. Ten milliliters of 100 mM glycine (pH 2.5) were added to the column and incubated for 15 min to elute the antibody. The eluate was collected and the column neutralized with 1 M Tris-Base. The column was then stripped with elution buffer [0.1 M glycine (pH 1.9)] and neutralized with 5 column volumes of 0.1 M phosphate buffer (pH 8.0) and washed with 250 ml of PBS with 1 mM EDTA. The collected eluate was analyzed by Coomassie Plus protein assay to determine purification efficiency and antibody concentration. The flow-through (FT) fraction was reapplied and the procedure above followed; an equal amount of antibody was recovered. The antibody fraction from both preparations was pooled and concentrated for binding to Protein G UltraLink (Thermo Fisher Scientific) using Ultracel 10 kDa MWCO centrifugal filters (Amicon, EMD Millipore, Millipore-Sigma, Billerica, MA). Coomassie Plus protein assay was used to analyze the retentate and filtrate. The combined eluate was 28 ml at a concentration of 0.189 mg/ml or 5.3 mg; the filtrate was 0.00 mg/ml; the retentate was 2 ml at a concentration of 2.83 mg/ml or 5.6 mg, and recovery was ∼100%. The coupling and cross-linking of the purified α-ApoB100 antibody to Protein G Ultra Link resin (Thermo Fisher Scientific) were done according to the protocols provided by Thermo Fisher Scientific. The resin was prepared in a 4 ml Bio-Rad Econo column (Bio-Rad, Hercules, CA); samples of each step were taken and checked by the Coomassie Plus protein assay to determine coupling efficiency. Briefly, the protein G resin was equilibrated with 10 column volumes of PBS (pH 7.6). The α-ApoB100 antibody, purified as previously described, was added to the resin (4.5 mg in 2 ml of resin) and mixed overnight at 4°C. A solution of 25 mM disuccinimidyl suberate as a cross-linker was prepared in dimethylformamide and added to the column, which was allowed to come up to room temperature for 30 min. The column was drained and quenched with 2 column volumes of 40 mM Tris (pH 7.4) and then washed with 5 column volumes of each of the following buffers: PBS; 1 M NaCl (pH 7.6); 0.1 M glycine (pH 1.9); PBS and 0.001% NaN3 (pH 7.6). The coupling efficiency was determined to be 100% and there was no loss of antibody from the cross-linking reaction. To determine whether our immunoaffinity method (n = 4) was superior to traditional ultracentrifugation separation (n = 8), we used fasting and postprandial blood samples collected throughout a 1-13C acetate stable-isotope tracer study. From collected samples, we compared fractionated lipoproteins by using Western blots to observe apolipoproteins and by measuring fractional DNL. Fresh never-frozen TRL (δ < 1.0063 g/ml, >17 h spin at 40,000 rpm) or CMU (δ < 1.0063 g/ml, 0.5 h spin at 33,000 rpm) fractions were applied to the protein G-α-ApoB100 affinity resin and incubated overnight (16–24 h) at 4°C with continuous mixing for two consecutive passes (Fig. 1). Freezing samples leads to the degradation of CM particles. The volumes used were in a 1:2 ratio, 200 μl of equilibrated resin to 400 μl of sample in a spin column (Thermo Fisher Scientific). The FT fractions (containing ApoB48 particles) were removed by centrifugation and stored on ice to be reapplied to the same resin after it had been washed, eluted, and re-equilibrated. The resin from pass #1 was washed with 12 column volumes of PBS [1 mM EDTA, 0.5 M NaCl (pH 7.5)] and 12 column volumes of PBS [1 mM EDTA (pH 7.5)]. The resin was eluted with 0.1 M glycine (pH 1.9); VLDLs were eluted twice from columns after a 20 min incubation at room temperature. After the elution, the column was neutralized with 5 column volumes of 0.1 M phosphate buffer (pH 8.0), washed with 10 column volumes of PBS (pH 7.5), and equilibrated with 0.9% NaCl. After equilibration, the FT fraction was reapplied for pass #2. The FT fraction and the elution fractions ("E"; containing ApoB100 particles) were taken at each step for evaluation by Western blot. After the second pass and final elution, the protein G-α-ApoB100 resin was equilibrated with 10 column volumes of 0.9% NaCl and stored at 4°C. We found that two passes were needed for optimal results. Purified CMs obtained from TRLs will be referred to as CMIA and purified CMs obtained from CMU will be referred to as CMU+IA. Eight healthy volunteers (Table 1) with no history of chronic disease and currently taking no medications were recruited to take part in this study. The study was approved by the Institutional Review Board of Touro University California, Vallejo, CA, which adhered to the World Medical Association Declaration of Helsinki principles. Informed written consent was obtained from all participants before the start of the study.TABLE 1Anthropometric dataSubjectSexBMITAGCholHDL-CCalc. LDL-Ckg/m2mg/dlmg/dlmg/dlmg/dl1M29.590155331042M2497209351553F34.03111137684M30.089184411255M24.48015755876F21.06513350707F27.4170178401058F27.019918231111TAG, plasma triacylglycerol; Chol, plasma total cholesterol; Calc. LDL-C, calculated plasma LDL-C. LDL-C was calculated using the Friedwald formula, [(Chol – HDL-C) – (TAGs/5)], units arer milligrams per deciliter. Cohort #1, subjects 1–4 and cohort #2, subjects 5–8. Samples from cohort #1 underwent ultracentrifugation separation only, whereas samples from cohort #2 underwent separation by both ultracentrifugation and immunoaffinity. Open table in a new tab TAG, plasma triacylglycerol; Chol, plasma total cholesterol; Calc. LDL-C, calculated plasma LDL-C. LDL-C was calculated using the Friedwald formula, [(Chol – HDL-C) – (TAGs/5)], units arer milligrams per deciliter. Cohort #1, subjects 1–4 and cohort #2, subjects 5–8. Samples from cohort #1 underwent ultracentrifugation separation only, whereas samples from cohort #2 underwent separation by both ultracentrifugation and immunoaffinity. After a 10–12 h fast, participants underwent an 8 h feeding study to achieve a postprandial state, during which 67% of their estimated daily energy requirement was consumed (containing 15% of calories as protein, 35% as fat, and 50% as carbohydrate). For each participant, 1 liter of regular Coca-Cola was mixed with 3.5 g of 1-13C sodium acetate (Cambridge Isotopes, Tewksbury, MA) and evenly divided into 17 drinks. In addition, an 18 inch long commercially prepared tuna sandwich was divided into 9. This allowed for the constant oral administration of stable-isotope tracer in this study. After an initial double-sized meal, one meal (one sandwich portion) was consumed every hour for 7 h (Fig. 2). After an initial double-sized drink, one drink was consumed every 30 min. Blood was drawn at baseline and every hour for 7 h after the initial test meal. Specimens were collected in K2EDTA tubes with added preservatives (gentamicin sulfate, chloramphenicol sodium succinate, aprotinin, sodium azide, trolox, and benzamidine) and kept on ice until they were centrifuged within 30 min of collection (500 g for 10 min at 4°C) to separate plasma, which was stored at 4°C until the end of the study day (Fig. 2). Processing of samples, which were at no time frozen, began at the end of the study day. To minimize proteolysis of ApoB, samples were kept at a temperature of between 4°C and 12°C with chelating (EDTA) and protease (aprotinin and benzamidine) inhibitors present throughout the procedures. Plasma was collected as described above (n = 8). Four milliliters of plasma were overlaid with a δ 1.0063 g/ml saline solution and centrifuged (Beckman L8-80) at 134,934 g in a TfT45.6 rotor for ≥17 h at 12°C. The top layer of this fraction, ∼1.8 ml, was removed by tube slicing for further analysis and will be referred to as TRL. A second 4 ml aliquot of plasma was overlaid with a δ 1.0063 saline solution, placed in a TfT45.6 rotor, and centrifuged (Beckman L8-80) at 91,839 g for 30 min at 12°C (n = 8). Approximately 1.5 ml of the sample were removed by tube slicing and referred to as CMU (CM from ultracentrifugation). The remaining volume was transferred to a fresh tube, overlaid with a δ 1.0063 saline solution, placed in a TfT45.6 rotor, and centrifuged (Beckman L8-80) at 134,934 g for ≥17 h at 12°C. Tube slicing isolated the top 1.8 ml, the VLDL fraction. SDS-PAGE gels were transferred to PVDF Immobilon-P transfer membrane using the Transblot SD Semidry transfer cell (Bio-Rad) at 25 volts for 20 min per gel. Membranes were blocked using 5% nonfat dried milk (NFDM) in TTBS (0.5% Tween 20 in TBS), pH 7.5, and incubated overnight at 4°C. ApoB100 and ApoB48 (differentiated by their molecular weights) were detected by the anti-ApoB antibody-HRP (ab27622; Abcam, Cambridge, MA) (22Gugliucci A. Numaguchi M. Caccavello R. Kimura S. Small-dense low-density lipoproteins are the predominant apoB-100-containing lipoproteins in cord blood.Clin. Biochem. 2014; 47: 475-477Crossref PubMed Scopus (6) Google Scholar) at 1:18,000 in 5% NFDM in TTBS for 2 h at room temperature. ApoB100 was detected by the goat polyclonal antibody generated for this study at 1:2,000 or 0.23 μg/ml in 5% NFDM in TTBS overnight at 4°C. ApoB100 and ApoB48 were quantified using National Institutes of Health Image J software, and the recovery and purity of each fraction were calculated relative to TRL or CMU. Fractional DNL was determined by measuring biosynthetic rates of palmitate, isolated from fractionated plasma, using stable-isotope tracer methodology via GC/MS and MIDA (17Hellerstein M.K. Neese R.A. Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations.Am. J. Physiol. 1999; 276: E1146-E1170Crossref PubMed Google Scholar, 18Hellerstein M.K. Christiansen M. Kaempfer S. Kletke C. Wu K. Reid J.S. Mulligan K. Hellerstein N.S. Shackleton C.H. Measurement of de novo hepatic lipogenesis in humans using stable isotopes.J. Clin. Invest. 1991; 87: 1841-1852Crossref PubMed Scopus (296) Google Scholar, 19Hellerstein M.K. Kletke C. Kaempfer S. Wu K. Shackleton C.H. Use of mass isotopomer distributions in secreted lipids to sample lipogenic acetyl-CoA pool in vivo in humans.Am. J. Physiol. 1991; 261: E479-E486PubMed Google Scholar). In these experiments, palmitate from TAG was analyzed as a methyl-palmitate derivative (m/z 270, 271, and 272). Briefly, chloroform and methanolic-HCl were added to the TAG samples isolated by thin-layer chromatography and incubated at 37°C overnight. Next, hexane and 5% NaCl were added to the samples to extract fatty acid methyl esters. The resulting samples were then transferred to autosampler vials for GC/MS analysis. Methyl-palmitate isotopomers were analyzed by an electron ionization GC/MS instrument, Agilent-5973 (Agilent, Santa Clara, CA), operated in positive-ion mode of detection and equipped with a RESTEK Rxi-5ms capillary column (RESTEK, Bellefonte, PA). Data were analyzed using JMP software (SAS, Cary, NC). Differences in mean fractional DNL area under the curve (AUC) were analyzed using two-tailed t-test, and differences with P < 0.05 were considered statistically significant. TRL and CMU samples were applied to the α-ApoB100 columns and purified CMs collected (Fig. 1). Western blots were used to determine the ability of the traditional ultracentrifugation and immunoaffinity methods to purify CMs (Fig. 3). The CM lane demonstrates that the traditional ultracentrifugation method does not separate ApoB48- and ApoB100-containing particles (Fig. 3, blue boxes). Figure 3, red boxes, shows the absence of ApoB100 in CM fractions purified from either TRL or CMU using the immunoaffinity method, CMIA, and CMU+IA, respectively. Using this technique, we recovered 80–88% of VLDL and 57–90% of CMs, purity was 100% for VLDL and between 90% and 94% for CMs. To assess previously reported intestinal DNL in CMU and to understand the contribution of contamination by VLDL, we measured and compared fractional DNL in the CMU, TRL, and VLDL obtained by ultracentrifugation (VLDLU) fractions, which demonstrates the inability of ultracentrifugation to purify CMs from VLDL and supports the use our immunoaffinity method to possibly quantify enteral DNL. MIDA allows for the calculation of fractional DNL by measuring the relative distribution of isotopomers of palmitate (M+0, M+1, and M+2; incorporation of 0, 1, or 2 1-13C acetyl-CoA, respectively) (23Hellerstein M.K. Neese R.A. Mass isotopomer distribution analysis: a technique for measuring biosynthesis and turnover of polymers.Am. J. Physiol. 1992; 263: E988-E1001Crossref PubMed Google Scholar), where acetyl-CoA is the monomer and palmitate is the polymer. We found that the average DNL during the feeding study rose up to ∼11% in TRL, up to ∼9% in CMU, and up to ∼16% in VLDLU (Fig. 4). The mean VLDLU-DNL AUC over 5 h (study time 3–8 h) was greater than the mean TRL-DNL AUC (73.2 ± 11.5 × 5 h versus 53.9 ± 6.8 × 5 h, P = 0.012), while there was a smaller yet significant difference between the mean TRL-DNL AUC versus the CMU-DNL AUC (53.9 ± 6.8 × 5 h versus 46.6 ± 6.6 × 5 h, P = 0.0009). While statistical analyses show that there is a significant difference between the DNL AUC of fractionated samples and TRL, the Western blot analysis (Fig. 3, blue boxes) demonstrated that the traditional ultracentrifugation method resulted in CMs that were heavily contaminated by VLDL, consistent with the CMU fractional DNL measurements. TAG from the purified CM fractions, CMIA (7 samples per subject, totaling 28 samples), and CMU+IA (7 samples per subject, totaling 28 samples), were isolated, fro
Background: Therapy with HIV protease inhibitors (PI) has been shown to worsen glucose and lipid metabolism, but whether these changes are caused by direct drug effects, changes in disease status, or body composition is unclear. Therefore, we tested the effects of the PI combination lopinavir and ritonavir on glucose and lipid metabolism in HIV-negative subjects. Methods: A dose of 400 mg lopinavir/100 mg ritonavir was given twice a day to 10 HIV-negative men. Fasting glucose and insulin, lipid and lipoprotein profiles, oral glucose tolerance, insulin sensitivity by euglycemic hyperinsulinemic clamp, and body composition were determined before and after lopinavir/ritonavir treatment for 4 weeks. Results: On lopinavir/ritonavir, there was an increase in fasting triglyceride (0.89 ± 0.15 versus 1.63 ± 0.36 mmol/l; P = 0.007), free fatty acid (FFA; 0.33 ± 0.04 versus 0.43 ± 0.06 mmol/l; P = 0.001), and VLDL cholesterol (15.1 ± 2.6 versus 20 ± 3.3 mg/dl; P = 0.05) levels. There were no changes in fasting LDL, HDL, IDL, lipoprotein (a), or total cholesterol levels. Fasting glucose, insulin, and insulin-mediated glucose disposal were unchanged, but on a 2 h oral glucose tolerance test glucose and insulin increased. There were no changes in weight, body fat, or abdominal adipose tissue by computed tomography. Conclusion: Treatment with 4 weeks of lopinavir/ritonavir in HIV-negative men causes an increase in triglyceride levels, VLDL cholesterol, and FFA levels. Lopinavir/ritonavir leads to a deterioration in glucose tolerance at 2 h, but there is no significant change in insulin-mediated glucose disposal rate by euglycemic hyperinsulinemic clamp.
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