Calorie restriction (CR) delays aging and extends lifespan in numerous organisms, including mice. Down-regulation of the somatotropic axis, including a reduction in insulin-like growth factor-1 (IGF-1), likely plays an important role in CR-induced lifespan extension, possibly by reducing cell proliferation rates, thereby delaying replicative senescence and inhibiting tumor promotion. Accordingly, elucidating the mechanism(s) by which IGF-1 is reduced in response to CR holds therapeutic potential in the fight against age-related diseases. Up-regulation of fibroblast growth factor 21 (FGF21) is one possible mechanism given that FGF21 expression is induced in response to nutritional deprivation and has been implicated as a negative regulator of IGF-1 expression. Here we investigated alterations in hepatic growth hormone (GH)-mediated IGF-1 production and signaling as well as the role of FGF21 in the regulation of IGF-1 levels and cell proliferation rates in response to moderate CR in adult mice. We found that in response to moderate CR, circulating GH and hepatic janus kinase 2 (JAK2) phosphorylation levels are unchanged but that hepatic signal transducer and activator of transcription 5 (STAT5) phosphorylation levels are reduced, identifying STAT5 phosphorylation as a potential key site of CR action within the somatotropic axis. Circadian measurements revealed that the relative level of FGF21 expression is both higher and lower in CR vs. ad libitum (AL)-fed mice, depending on the time of measurement. Employing FGF21-knockout mice, we determined that FGF21 is not required for the reduction in IGF-1 levels or cell proliferation rates in response to moderate CR. However, compared to AL-fed WT mice, AL-fed FGF21-knockout mice exhibited higher basal rates of cell proliferation, suggesting anti-mitotic effects of FGF21. This work provides insights into both GH-mediated IGF-1 production in the context of CR and the complex network that regulates FGF21 and IGF-1 expression and cell proliferation rates in response to nutritional status.
Increased protein synthesis is proposed as a mechanism of life-span extension during caloric restriction (CR). We hypothesized that CR does not increase protein synthesis in all tissues and protein fractions and that any increased protein synthesis with CR would be due to an increased anabolic effect of feeding. We used short- (4 hours) and long-term (6 weeks) methods to measure in vivo protein synthesis in lifelong ad libitum (AL) and CR mice. We did not detect an acute effect of feeding on protein synthesis while liver mitochondrial protein synthesis was lower in CR mice versus AL mice. Mammalian target of rapamycin (mTOR) signaling was repressed in liver and heart from CR mice indicative of energetic stress and suppression of growth. Our main findings were that CR did not increase rates of mixed protein synthesis over the long term or in response to acute feeding, and protein synthesis was maintained despite decreased mTOR signaling.
Aging; Stable isotope; Protein metabolism.
We recently validated in cross-sectional studies a new method to determine total body creatine pool size and skeletal muscle mass based on D3-creatine dilution from an oral dose and detection of urinary creatinine enrichment by isotope ratio mass spectrometry (IRMS). Routine clinical use of the method in aging and disease will require repeated application of the method, with a more widely available technology than IRMS, to enable determination of change in skeletal muscle mass in longitudinal studies. We therefore adapted the method to liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology, and sought to establish proof of concept for the repeated application of the method in a longitudinal study. Because the turnover of creatine is slow, it was also critical to determine the impact of background enrichment from an initial dose of oral D3-creatine on subsequent, longitudinal measurements of change in muscle mass.
Rats were given an oral tracer dose of D3-creatine (1.0 mg/kg body weight) at 10 and 17 weeks of age. LC-MS/MS was used to determine urinary D3-creatine, and urinary D3-creatinine enrichment, at time intervals after D3-creatine administration. Total body creatine pool size was calculated from urinary D3-creatinine enrichment at isotopic steady state 72 h after administration of D3-creatine tracer.
At 10 weeks of age, rat lean body mass (LBM) measured by quantitative magnetic resonance correlated with creatine pool size (r = 0.92, P = 0.0002). Over the next 7 weeks, the decline in urinary D3-creatinine enrichment was slow and linear, with a rate constant of 2.73 ± 0.06 %/day. Subtracting background urinary D3-creatinine enrichment from the elevated enrichment following a second dose of D3-creatine at 17 weeks permitted repeat calculations of creatine pool size. As at 10 weeks, 17-week LBM correlated with creatine pool size (r = 0.98, P <0.0001). In addition, the change in creatine pool size was correlated with the change in LBM during the 7 weeks of rat growth between measurements (r = 0.96, P <0.0001).
The LC-MS/MS-based D3-creatine dilution method can be applied repeatedly to measure total body creatine skeletal muscle mass change in longitudinal study.
Body composition; Total body skeletal muscle mass; Total body creatine pool size; Stable isotope dilution
Obesity develops due to altered energy homeostasis favoring fat storage. Here we describe a novel transcription co-regulator for adiposity and energy metabolism, TRIP-Br2 (also called SERTAD2). TRIP-Br2 null mice are resistant to obesity and obesity-related insulin resistance. Adipocytes of the knockout (KO) mice exhibited greater stimulated lipolysis secondary to enhanced expression of hormone sensitive lipase (HSL) and β3-adrenergic (Adrb3) receptors. The KOs also exhibit higher energy expenditure due to increased adipocyte thermogenesis and oxidative metabolism by up-regulating key enzymes in respective processes. Our data show for the first time that a cell cycle transcriptional co-regulator, TRIP-Br2, modulates fat storage through simultaneous regulation of lipolysis, thermogenesis and oxidative metabolism. These data together with the observation that TRIP-BR2 expression is selectively elevated in visceral fat in obese humans suggests that this transcriptional co-regulator is a novel therapeutic target for counteracting the development of obesity, insulin resistance and hyperlipidemia.
Low-fat, high-carbohydrate (LF/HC) diets commonly elevate plasma triglyceride (TG) concentrations, but the kinetic mechanisms responsible for this effect remain uncertain. Subjects with low TG (normolipidemic [NL]) and those with moderately elevated TG (hypertriglyceridemic [HTG]) were studied on both a control and an LF/HC diet. We measured VLDL particle and TG transport rates, plasma nonesterified fatty acid (NEFA) flux, and sources of fatty acids used for the assembly of VLDL-TG. The LF/HC diet resulted in a 60% elevation in TG, a 37% reduction in VLDL-TG clearance, and an 18% reduction in whole-body fat oxidation, but no significant change in VLDL-apo B or VLDL-TG secretion rates. Significant elevations in fasting apo B-48 concentrations were observed on the LF/HC in HTG subjects. In both groups, fasting de novo lipogenesis was low regardless of diet. The NEFA pool contributed the great majority of fatty acids to VLDL-TG in NL subjects on both diets, whereas in HTG subjects, the contribution of NEFA was somewhat lower overall and was reduced further in individuals on the LF/HC diet. Between 13% and 29% of VLDL-TG fatty acids remained unaccounted for by the sum of de novo lipogenesis and plasma NEFA input in HTG subjects. We conclude that (a) whole-food LF/HC diets reduce VLDL-TG clearance and do not increase VLDL-TG secretion or de novo lipogenesis; (b) sources of fatty acids for assembly of VLDL-TG differ between HTG and NL subjects and are further affected by diet composition; (c) the presence of chylomicron remnants in the fasting state on LF/HC diets may contribute to elevated TG levels by competing for VLDL-TG lipolysis and by providing a source of fatty acids for hepatic VLDL-TG synthesis; and (d) the assembly, production, and clearance of elevated plasma VLDL-TG in response to LF/HC diets therefore differ from those for elevated TG on higher-fat diets.
Previous studies of epidermal kinetics in psoriasis have relied on invasive biopsy procedures or the use of radioactive labels. We previously developed a non-invasive method for measuring keratin synthesis in human skin using deuterated water labeling, serial collection of tape strips and measurement of deuterium enrichment in protein by mass spectrometry. This powerful method can be applied to measure other skin proteins and lipids collected by tape stripping. Here, for the first time, we apply this technique to investigate the epidermal kinetics of psoriasis, the first step in defining a kinetic profile for normal skin versus activated or quiescent psoriatic skin.
Psoriatic subjects were given 2H2O orally as twice-daily doses for 16–38 days. Affected and unaffected skin was sampled by tape stripping and washing (modified Pachtman method). Proteins were isolated from the tape strips by a method that enriches for keratin. Turnover times were determined by gas chromatography/mass spectrometry. Kinetic data were compared to transepidermal water loss (TEWL).
Deuterium-labeled protein from lesional psoriatic skin appeared at the skin surface within 3–8 days of label administration, whereas labeled protein from non-lesional skin requires 10–20 days to appear. Psoriatic skin had similar rate of growth despite varying anatomic location. Proteins recovered from tape strips were identified by nanoscale liquid chromatography/tandem mass spectrometry. Isolated peptides were >98% from keratin in uninvolved skin and >72% keratin in psoriatic skin. Revealing that one-quarter of all newly synthesized proteins in psoriatic skin are antimicrobial defense and other immune-related proteins. TEWL values were greater in lesional than non-lesional skin, suggesting barrier compromise in psoriatic skin despite increased clinical thickness.
This simple, elegant, and non-invasive method for measuring epidermal protein synthesis, which can also be adapted to measure epidermal lipids, provides a metric that may reveal new insights into the mechanisms and dynamic processes underlying psoriasis and may also provide an objective scale for determining response to therapeutic agents in pre-clinical and clinical trials. This opens a pathway to the non-invasive study of kinetics of protein formation in psoriasis or other skin diseases.
Psoriasis; Kinetics; Keratin; Skin; Stable isotopes
Impairment of adipogenesis contributes to the development of obesity-related insulin resistance. The current in vitro approaches for its assessment represent crude estimates of the adipogenic potential because of the disruption of the in vivo microenvironment. A novel assessment of in vivo adipogenesis using the incorporation of the stable isotope deuterium (2H) into the DNA of isolated adipocytes and stroma-vascular fraction from adipose tissue has been developed. In the current study, we have refined this technique by purifying the adipocytes via a negative immune selection and sorting the plastic adherent stroma-vascular (aSV) subfraction (using 3 h culture) that contains mostly adipocyte progenitor cells and ∼10% of small adipocytes. Using a 3-week 8% 2H2O ingestion with a high-fat diet (HFD) or HFD plus pioglitazone (HFD-P), we demonstrate that the fractions of new aSV cells (faSV) and immunopurified adipocytes (fAD) (the ratio of their 2H-enrichment of DNA to the maximal 2H-enrichment of DNA of bone marrow reference cells) recapitulate the known hyperplastic mechanism of weight gain with pioglitazone treatment. We conclude that faSV and fAD are reliable indices of in vivo adipogenesis. The proposed method represents a valuable tool for studying the effect of interventions (drugs, diets, and exercise) on in vivo adipogenesis.
Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.
Progress in neurodegenerative disease research is hampered by the lack of biomarkers of neuronal dysfunction. We here identified a class of cerebrospinal fluid–based (CSF-based) kinetic biomarkers that reflect altered neuronal transport of protein cargo, a common feature of neurodegeneration. After a pulse administration of heavy water (2H2O), distinct, newly synthesized 2H-labeled neuronal proteins were transported to nerve terminals and secreted, and then appeared in CSF. In 3 mouse models of neurodegeneration, distinct 2H-cargo proteins displayed delayed appearance and disappearance kinetics in the CSF, suggestive of aberrant transport kinetics. Microtubule-modulating pharmacotherapy normalized CSF-based kinetics of affected 2H-cargo proteins and ameliorated neurodegenerative symptoms in mice. After 2H2O labeling, similar neuronal transport deficits were observed in CSF of patients with Parkinson’s disease (PD) compared with non-PD control subjects, which indicates that these biomarkers are translatable and relevant to human disease. Measurement of transport kinetics may provide a sensitive method to monitor progression of neurodegeneration and treatment effects.
While fatty acids (FAs) released by white adipose tissue (WAT) provide energy for other organs, lipolysis is also critical in brown adipose tissue (BAT), generating FAs for oxidation and UCP-1 activation for thermogenesis. Here, we show that adipose-specific ablation of desnutrin/ATGL in mice converts BAT to a WAT-like tissue. These mice exhibit severely impaired thermogenesis with increased expression of WAT-enriched genes but decreased BAT genes including UCP-1 with lower PPARα binding to its promoter, revealing the requirement of desnutrin-catalyzed lipolysis for maintaining BAT phenotype. We also show that desnutrin is phosphorylated by AMPK at S406, increasing TAG hydrolase activity, and provide evidence for increased lipolysis by AMPK phosphorylation of desnutrin in adipocytes and in vivo. Despite adiposity and impaired BAT function, desnutrin-ASKO mice have improved hepatic insulin sensitivity with lower DAG levels. Overall, desnutrin is phosphorylated/activated by AMPK to increase lipolysis and brings FA oxidation and UCP-1 induction for thermogenesis.
The failure of chemotherapeutic regimens to eradicate cancers often results from the outgrowth of minor subclones with more dangerous genomic abnormalities or with self-renewing capacity. To explore such intratumor complexities in B-cell chronic lymphocytic leukemia (CLL), we measured B-cell kinetics in vivo by quantifying deuterium (2H)-labeled cells as an indicator of a cell that had divided. Separating CLL clones on the basis of reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and cluster designation 5 (CD5) revealed that the CXCR4dimCD5bright (proliferative) fraction contained more 2H-labeled DNA and hence divided cells than the CXCR4brightCD5dim (resting) fraction. This enrichment was confirmed by the relative expression of two cell cycle–associated molecules in the same fractions, Ki-67 and minichromosome maintenance protein 6 (MCM6). Comparisons of global gene expression between the CXCR4dimCD5bright and CXCR4brightCD5dim fractions indicated higher levels of pro-proliferation and antiapoptotic genes and genes involved in oxidative injury in the proliferative fraction. An extended immunophenotype was also defined, providing a wider range of surface molecules characteristic of each fraction. These intraclonal analyses suggest a model of CLL cell biology in which the leukemic clone contains a spectrum of cells from the proliferative fraction, enriched in recently divided robust cells that are lymphoid tissue emigrants, to the resting fraction enriched in older, less vital cells that need to immigrate to lymphoid tissue or die. The model also suggests several targets preferentially expressed in the two populations amenable for therapeutic attack. Finally, the study lays the groundwork for future analyses that might provide a more robust understanding of the development and clonal evolution of this currently incurable disease.
Measurements of cell proliferation and matrix synthesis in cartilage explants have identified regulatory factors (e.g., interleukin 1, IL-1) that contribute to osteoarthritis and anabolic mediators (e.g., BMP-7) that may have therapeutic potential. The objective of this study was to develop a robust method for measuring cell proliferation and glycosaminoglycan synthesis in articular cartilage that could be applied in vivo.
A stable isotope-mass spectrometry approach was validated by measuring the metabolic effects of IL-1 and BMP-7 in cultures of mature and immature bovine cartilage explants. The method was also applied in vivo to quantify physiologic turnover rates of matrix and cells in the articular cartilage of normal rats. Heavy water was administered to explants in the culture medium and to rats via drinking water, and cartilage was analyzed for labeling of chondroitin sulfate (CS), hyaluronic acid (HA) and DNA.
As expected, IL-1 inhibited the synthesis of DNA and CS in cartilage explants. However, IL-1 inhibited HA synthesis only in immature cartilage. Futhermore, BMP-7 was generally stimulatory, but immature cartilage was significantly more responsive than mature cartilage, particularly in terms of HA and DNA synthesis. In vivo, labeling of CS and DNA in normal rats for up to a year indicated half-lives of 22 and 862 days, respectively, in the joint.
We describe a method by which deuterium from heavy water is traced into multiple metabolites from a single cartilage specimen to profile its metabolic activity. This method was demonstrated in tissue culture and rodents but may have significant clinical applications.
A main function of white adipose tissue is to release fatty acids from triacylglycerol for other tissues to use as an energy source. While endocrine regulation of lipolysis has been extensively studied, autocrine/paracrine regulation is not well understood. Here, we describe the role of AdPLA, the newly identified major adipocyte phospholipase A2, in the regulation of lipolysis and adiposity. AdPLA null mice have a markedly higher rate of lipolysis, due to increased cAMP levels arising from the marked reduction in adipose PGE2 that binds the Gαi-coupled receptor, EP3. AdPLA null mice have drastically reduced adipose tissue mass and triglyceride content, with normal adipogenesis. They also have higher energy expenditure with higher fatty acid oxidation within adipocytes. AdPLA deficient ob/ob mice remain hyperphagic but lean, with increased energy expenditure, yet have ectopic triglyceride storage and insulin resistance. AdPLA is a major regulator of adipocyte lipolysis and critical for the development of obesity.
To investigate the role of desnutrin in adipose tissue triacylglycerol (TAG) and fatty acid metabolism.
RESEARCH DESIGN AND METHODS
We generated transgenic mice overexpressing desnutrin (also called adipose triglyceride lipase [ATGL]) in adipocytes (aP2-desnutrin) and also performed adenoviral-mediated overexpression of desnutrin in 3T3-L1CARΔ1 adipocytes.
aP2-desnutrin mice were leaner with decreased adipose tissue TAG content and smaller adipocyte size. Overexpression of desnutrin increased lipolysis but did not result in increased serum nonesterified fatty acid levels or ectopic TAG storage. We found increased cycling between diacylglycerol (DAG) and TAG and increased fatty acid oxidation in adipocytes from these mice, as well as improved insulin sensitivity.
We show that by increasing lipolysis, desnutrin overexpression causes reduced adipocyte TAG content and attenuation of diet-induced obesity. Desnutrin-mediated lipolysis promotes fatty acid oxidation and re-esterification within adipocytes.
Angiogenesis is critical in the progression of prostate cancer. However, the interplay between the proliferation kinetics of tumor endothelial cells (angiogenesis) and tumor cells has not been investigated. Also, protein kinase C (PKC) regulates various aspects of tumor cell growth but its role in prostate cancer has not been investigated in detail. Here, we found that the proliferation rates of endothelial and tumor cells oscillate asynchronously during the growth of human prostate cancer xenografts. Furthermore, our analyses suggest that PKCβII was activated during increased angiogenesis and that PKCβII plays a key role in the proliferation of endothelial cells and tumor cells in human prostate cancer; treatment with a PKCβII-selective inhibitor, βIIV5-3, reduced angiogenesis and tumor cell proliferation. We also find a unique effect of PKCβII inhibition on normalizing pericentrin (a protein regulating cytokinesis), especially in endothelial cells as well as in tumor cells. PKCβII inhibition reduced the level and mislocalization of pericentrin and normalized microtubule organization in the tumor endothelial cells. Although pericentrin has been known to be upregulated in epithelial cells of prostate cancers, its level in tumor endothelium has not been studied in detail. We found that pericentrin is upregulated in human tumor endothelium compared with endothelium adjacent to normal glands in tissues from prostate cancer patients. Our results suggest that a PKCβII inhibitor such as βIIV5-3 may be used to reduce prostate cancer growth by targeting both angiogenesis and tumor cell growth.
Progressive HIV disease has been associated with loss of memory T cell responses to antigen. To better characterize and quantify long-lived memory T cells in vivo, we have refined an in vivo labeling technique to study the kinetics of phenotypically distinct, low frequency CD8+ T cell subpopulations in humans. HIV-negative subjects and antiretroviral-untreated HIV-infected subjects in varying stages of HIV disease were studied. After labeling the DNA of dividing cells with deuterated water (2H2O), 2H-label incorporation and die-away kinetics were quantified using a highly sensitive FACS/mass spectrometric method. Two different populations of long-lived memory CD8+ T cells were identified in HIV-negative subjects: CD8+CD45RA-CCR7+CD28+ central memory (TCM) cells expressing IL-7Rα and CD8+CD45RA+CCR7-CD28- RA effector memory (TEmra) cells expressing CD57. In pilot studies in HIV-infected subjects, TCm cells were found to have a shorter half-life and reduced abundance, particularly in those with high viral loads; TEmra cells, by contrast, retained a long half-life and accumulated in the face of progressive HIV disease. These data are consistent with the hypothesis that IL-7Rα+ TCm cells represent “true” memory CD8+ T cells, the loss of which may be responsible in part for the progressive loss of T cell memory function during progressive HIV infection.
Human; T cells; AIDS; Cell proliferation; Memory
Studies in animals have documented that, compared with glucose, dietary fructose induces dyslipidemia and insulin resistance. To assess the relative effects of these dietary sugars during sustained consumption in humans, overweight and obese subjects consumed glucose- or fructose-sweetened beverages providing 25% of energy requirements for 10 weeks. Although both groups exhibited similar weight gain during the intervention, visceral adipose volume was significantly increased only in subjects consuming fructose. Fasting plasma triglyceride concentrations increased by approximately 10% during 10 weeks of glucose consumption but not after fructose consumption. In contrast, hepatic de novo lipogenesis (DNL) and the 23-hour postprandial triglyceride AUC were increased specifically during fructose consumption. Similarly, markers of altered lipid metabolism and lipoprotein remodeling, including fasting apoB, LDL, small dense LDL, oxidized LDL, and postprandial concentrations of remnant-like particle–triglyceride and –cholesterol significantly increased during fructose but not glucose consumption. In addition, fasting plasma glucose and insulin levels increased and insulin sensitivity decreased in subjects consuming fructose but not in those consuming glucose. These data suggest that dietary fructose specifically increases DNL, promotes dyslipidemia, decreases insulin sensitivity, and increases visceral adiposity in overweight/obese adults.
Hepatic de novo lipogenesis (DNL) is markedly stimulated in humans by low-fat diets enriched in simple sugars. However, the dietary responsiveness of the key enzyme controlling DNL in human adipose tissue, fatty acid synthase (FAS), is uncertain.
Adipose tissue mRNA for FAS is increased in lean and obese subjects when hepatic DNL is elevated by a eucaloric, low-fat, high-sugar diet.
Twelve lean and 7 obese volunteers were given 2 eucaloric diets (10% vs. 30% fat, 75% vs. 55% carbohydrate, sugar/starch 60/40) each for 2 weeks by a random-order, cross-over design. FAS mRNA in abdominal and gluteal adipose tissue was compared to hepatic DNL measured in serum by isotopic and non-isotopic methods. Adipose tissue mRNA for TNF alpha and IL-6, inflammatory cytokines that modulate DNL, were also assayed.
The low-fat, high-sugar diet induced a 4 fold increase in maximum hepatic DNL (P<0.001) but only a 1.3 fold increase in adipose tissue FAS mRNA (P=0.029) and no change in cytokine mRNA. There was a borderline significant positive correlation between changes in FAS mRNA and hepatic DNL (P=0.039). Compared to lean subjects, obese subjects had lower levels of FAS mRNA and higher levels of cytokine mRNA (P<0.001).
The results suggest that key elements of human adipose tissue DNL are less responsive to dietary carbohydrate than is hepatic DNL and may be regulated by diet-independent factors. Irrespective of diet, there is reduced expression of the FAS gene and increased expression of cytokine genes in adipose tissue of obese subjects.
lipogenesis; gene expression; VLDL; triacylglycerol; palmitic acid; MIDA
Due to its relatively slow clinical progression, B cell chronic lymphocytic leukemia (B-CLL) is classically described as a disease of accumulation rather than proliferation. However, evidence for various forms of clonal evolution suggests that B-CLL clones may be more dynamic than previously assumed. We used a nonradioactive, stable isotopic labeling method to measure B-CLL cell kinetics in vivo. Nineteen patients drank an aliquot of deuterated water (2H2O) daily for 84 days, and 2H incorporation into the deoxyribose moiety of DNA of newly divided B-CLL cells was measured by gas chromatography/mass spectrometry, during and after the labeling period. Birth rates were calculated from the kinetic profiles. Death rates were defined as the difference between calculated birth and growth rates. These analyses demonstrated that the leukemic cells of each patient had definable and often substantial birth rates, varying from 0.1% to greater than 1.0% of the entire clone per day. Those patients with birth rates greater than 0.35% per day were much more likely to exhibit active or to develop progressive disease than those with lower birth rates Thus, B-CLL is not a static disease that results simply from accumulation of long-lived lymphocytes. Rather, it is a dynamic process composed also of cells that proliferate and die, often at appreciable levels. The extent to which this turnover occurs has not been previously appreciated. A correlation between birth rates and disease activity and progression appears to exist, which may help identify patients at risk for worsening disease in advance of clinical deterioration.
Antigenic stimulation of T cells gives rise to short-lived effector cells and long-lived memory cells. We used two stable isotope-labeling techniques to identify kinetically distinct subpopulations of T cells and to determine the effect of advanced infection with HIV-1. Long-term deuterated water (2H2O) incorporation into DNA demonstrated biphasic accrual of total and of memory/effector (m/e)–phenotype but not naive-phenotype T cells, consistent with the presence of short-lived and longer-lived subpopulations within the m/e-phenotype T cell pool. These results were mirrored by biphasic die-away kinetics in m/e- but not naive-phenotype T cells after short-term 2H-glucose labeling. Persistent label retention was observed in a subset of m/e-phenotype T cells (presumably memory T cells), confirming the presence of T cells with very different life spans in humans. In advanced HIV-1 infection, much higher proportions of T cells were short-lived, compared to healthy controls. Effective long-term anti-retroviral therapy restored values to normal. These results provide the first quantitative evidence that long-lived and quiescent T cells do indeed predominate in the T cell pool in humans and determine T cell pool size, as in rodents. The greatest impact of advanced HIV-1 infection is to reduce the generation of long-lived, potential progenitor T cells.
The mechanism of CD4+ T cell depletion in human immunodeficiency virus (HIV)-1 infection remains controversial. Using deuterated glucose to label the DNA of proliferating cells in vivo, we studied T cell dynamics in four normal subjects and seven HIV-1–infected patients naive to antiretroviral drugs. The results were analyzed using a newly developed mathematical model to determine fractional rates of lymphocyte proliferation and death. In CD4+ T cells, mean proliferation and death rates were elevated by 6.3- and 2.9-fold, respectively, in infected patients compared with normal controls. In CD8+ T cells, the mean proliferation rate was 7.7-fold higher in HIV-1 infection, but the mean death rate was not significantly increased. Five of the infected patients underwent subsequent deuterated glucose labeling studies after initiating antiretroviral therapy. The lymphocyte proliferation and death rates in both CD4+ and CD8+ cell populations were substantially reduced by 5–11 weeks and nearly normal by one year. Taken together, these new findings strongly indicate that CD4+ lymphocyte depletion seen in AIDS is primarily a consequence of increased cellular destruction, not decreased cellular production.
deuterated glucose; longitudinal study; mathematical model; apoptosis; mechanisms of CD4+ T cell depletion
Apo-E–deficient apo-B100–only mice (Apoe–/–Apob100/100) and LDL receptor–deficient apo-B100–only mice (Ldlr–/–Apob100/100) have similar total plasma cholesterol levels, but nearly all of the plasma cholesterol in the former animals is packaged in VLDL particles, whereas, in the latter, plasma cholesterol is found in smaller LDL particles. We compared the apo-B100–containing lipoprotein populations in these mice to determine their relation to susceptibility to atherosclerosis. The median size of the apo-B100–containing lipoprotein particles in Apoe–/–Apob100/100 plasma was 53.4 nm versus only 22.1 nm in Ldlr–/–Apob100/100 plasma. The plasma levels of apo-B100 were three- to fourfold higher in Ldlr–/–Apob100/100 mice than in Apoe–/–Apob100/100 mice. After 40 weeks on a chow diet, the Ldlr–/–Apob100/100 mice had more extensive atherosclerotic lesions than Apoe–/–Apob100/100 mice. The aortic DNA synthesis rate and the aortic free and esterified cholesterol contents were also higher in the Ldlr–/–Apob100/100 mice. These findings challenge the notion that all non-HDL lipoproteins are equally atherogenic and suggest that at a given cholesterol level, large numbers of small apo-B100–containing lipoproteins are more atherogenic than lower numbers of large apo-B100–containing lipoproteins.