Aging results in paradoxical susceptibility of fat cell progenitors to lipotoxicity
Abstract
Aging is associated with metabolic syndrome, tissue damage by cytotoxic lipids, and altered fatty acid handling. Fat tissue dysfunction may contribute to these processes. This could result, in part, from age-related changes in preadipocytes, since they give rise to new fat cells throughout life. To test this hypothesis, preadipocytes cultured from rats of different ages were exposed to oleic acid, the most abundant fatty acyl moiety in fat tissue and the diet. At fatty acid concentrations at which preadipocytes from young animals remained viable, cells from old animals accumulated lipid in multiple small lipid droplets and died, with increased apoptotic index, caspase activity, BAX, and p53. Rather than inducing apoptosis, oleic acid promoted adipogenesis in preadipocytes from young animals, with appearance of large lipid droplets. CCAAT/enhancer-binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor-γ (PPARγ) increased to a greater extent in cells from young than old animals after oleate exposure. Oleic acid, but not glucose, oxidation was impaired in preadipocytes and fat cells from old animals. Expression of carnitine palmitoyltransferase (CPT)-1, which catalyzes the rate-limiting step in fatty acid β-oxidation, was not reduced in preadipocytes from old animals. At lower fatty acid levels, constitutively active CPT I expression enhanced β-oxidation. At higher levels, CPT I was not as effective in enhancing β-oxidation in preadipocytes from old as young animals, suggesting that mitochondrial dysfunction may contribute. Consistent with this, medium-chain acyl-CoA dehydrogenase expression was reduced in preadipocytes from old animals. Thus preadipocyte fatty acid handling changes with aging, with increased susceptibly to lipotoxicity and impaired fatty acid-induced adipogenesis and β-oxidation.
the principal function of adipose tissue is to store energy. Lipids are a particularly efficient form in which to store energy because of their high caloric density. However, lipids are cytotoxic. Fat tissue protects nonadipose tissues from lipotoxicity by storing fatty acids as less cytotoxic neutral triglycerides (18, 65). Because nonadipose tissues have limited capacity to store lipids and exogenous fatty acids can cause apoptosis within hours in cultured cells (5, 51), if fat tissue function becomes dysregulated, lipotoxicity in other tissues can ensue. Indeed, lipotoxicity may be among the mechanisms contributing to development of the metabolic syndrome, which is more prevalent in older than younger individuals, affecting from 24 to 40% of individuals in their 60s and 70s (62).
Fat cells and preadipocytes are remarkably resistant to cytotoxic effects of fatty acids. Although direct measurements of fatty acid concentrations adjacent to fat cells are not available and would be technically challenging to do, these concentrations are most likely very high, particularly during lipolysis. When cells are exposed to fatty acids, their intracellular pH drops since unionized, hydrophobic fatty acids that have diffused across the cell membrane are reionized (13). The decrease in intracellular pH that accompanies fatty acid transfer across fat cell membranes following induction of lipolysis is as high as that which occurs when cells are exposed to 65 μM oleic acid in the absence of albumin (see Figs. 1 and 2 in Ref. 13). These levels are lethal to most types of cells, other than fat cells (53).
Fig. 1.Oleic acid induces death of preadipocytes from old animals. Epididymal preadipocytes from 3-mo (3M)- and 30-mo (30M)-old rats were treated with basal medium containing 1, 1.5, or 2 mM oleate (5:1 with BSA) for 24 h after confluence. At 1 and 1.5 mM oleate, the proportion of cells with large lipid droplets was higher in cells from young than old animals. At 2 mM oleate, cells from young animals remained viable and accumulated lipid droplets, whereas cells from old animals died. Bars represent 20 μm in these phase-contrast photomicrographs. Data are representative of 7 experiments.
Fig. 2.Apoptosis resulting from oleic acid increases with aging. A: epididymal preadipocytes from a 30-mo-old rat were exposed to 0.8 mM oleic acid for 24 h and then strained with bisbenzamide. White arrows, cells undergoing apoptosis; green arrows, dead cells. Bar indicates 20 μm. B: apoptotic index was higher in confluent undifferentiated epididymal preadipocytes from 30- than 3-mo-old rats exposed to 0.8 mM oleic acid in α-minimal essential medium (αMEM) containing 10% FBS for 24 h (n = 6 animals/age group; P < 0.005; t-test). C: caspase-3 activity was higher in preadipocytes from 30- than 7-mo-old rats (n = 8 animals/age group; P < 0.005; t-test). p53 (D) and BAX (E) levels were higher in preadipocytes from old than young animals following exposure to fatty acid. Epididymal preadipocytes from 4- and 25-mo-old rats were exposed to 1 mM oleic acid (5:1 with albumin) for 24 h, when p53 and BAX were measured by PCR (n = 3; *P < 0.05; t-test).
Dramatic changes in fat depot size, fat tissue distribution, and function occur throughout the life span (22, 40, 41). The sizes of fat depots increase through middle age, then decline in advanced old age. Although total body fat may decrease in old age (60), percent body fat declines very little, and may even remain constant or increase (35). This occurs because fat is redistributed from fat depots to other sites and because body weight declines in concert with total body fat content. This decline in fat within traditional fat depots in old age is accompanied by accumulation of fat in muscle, bone marrow, and other sites outside fat depots. Thus, in old age, there is less fat where it should be and more fat where it should not be, with clinical consequences (46). For example, lower trunk muscle fat infiltration is associated with decreased functional capacity in elderly subjects (29).
New fat cells arise from preadipocytes throughout the life span, since new fat cells continue to appear during adulthood and preadipocytes are present in fat depots at all ages (6, 30, 45, 47, 56, 64). Preadipocytes account for 15–50% of cells in fat tissue (45). Lipid accumulation, increases in lipogenic enzyme activities, and changes in differentiation-dependent gene expression during differentiation decrease with age in preadipocytes cultured under identical conditions originating from individuals of various ages (8, 20, 21, 28, 42–44, 70). This could contribute to the decreased fat cell and fat depot sizes that occur in advanced old age (33, 42, 46). Thus preadipocyte function becomes impaired with aging due, in part, to inherent processes.
We therefore hypothesized that handling of fatty acids changes with aging in preadipocytes, predisposing preadipocytes to become susceptible to lipotoxicity. To test this hypothesis, we cultured preadipocytes under identical conditions from animals of different ages, exposed them to fatty acids, and determined the impact on fatty acid handling and cell survival. Oleate was studied since it is the most abundant fatty acid in the diet and in rat and human fat tissue triglyceride (48, 52). By studying oleate instead of palmitate, confounding effects of ceramide generation from palmitate were minimized. We found that preadipocytes from old animals underwent apoptosis on exposure to fatty acid concentrations that were not cytotoxic to preadipocytes from young animals. Preadipocytes from old animals were resistant to adipogenesis in response to fatty acids, impeding this defense against lipotoxicity. Preadipocyte fatty acid β-oxidation decreased with aging. Thus preadipocyte fatty acid handling becomes dysfunctional with aging, making these cells paradoxically susceptible to lipotoxicity, potentially contributing to fat tissue dysfunction and systemic metabolic consequences.
METHODS
Preadipocyte cultures.
Preadipocytes were isolated from specific pathogen-free, male, Brown Norway rats aged 3–4 (young), 17 (middle aged), and 24–30 (old) mo, obtained from the Harlan Sprague Dawley (Indianapolis, IN) colony maintained under contract by the NIA (median survival 26 mo, maximum survival 32 mo). Epididymal fat depots were removed under sterile conditions. Fat tissue was minced into fragments, digested in 1 mg/ml collagenase for 60 min at 37°C, and filtered as previously described (42, 44, 45, 70). After the digests were centrifuged, the pellets were resuspended in a basal medium [α-minimal essential medium (αMEM) containing 10% FBS and antibiotics] and plated. After 12 h, a period during which no replication occurs (21), the adherent preadipocytes were washed, trypsinized, and replated at a density of 4 × 104 cells/cm2. We found that replating reduces endothelial cell, multipotent mesenchymal progenitor cell, and macrophage contamination and results in accurate plating densities (plating density affects capacity of preadipocytes to differentiate; see Ref. 73). Medium was changed every 2 days. We have previously demonstrated that, using this method, preadipocyte recoveries are similar among age groups and that our isolation procedures result in >90% pure populations of preadipocytes, irrespective of animal age (42). Markers of macrophages [F4/80 (emr1), adam8, scya4, cd11β, cd45 (ptprc)], endothelial cells [vegfr (flk1), vegfr2 (kdr), cd46 (mcp), and von Willebrand], and multipotent mesenchymal progenitors (cd34, connexin 43, sox2, smad4) indicate that age-related shifts in abundance of these cell types in preadipocyte cultures do not account for our results. Brown fat preadipocytes are not present in cultures prepared using our methods (44). All experiments were conducted with preadipocytes cultured from different age groups in parallel.
Incubation with fatty acids.
Cells were incubated for 24 h with 0.8–2 mM oleic or linoleic acids (NuCHEK Prep, Elysian, MN) complexed to fatty acid-free Fraction V BSA (Sigma, St. Louis, MO) at a 5:1 molar ratio in basal medium (αMEM + 10% FBS).
CO2 collection and analysis.
Preadipocytes were incubated with labeled substrates ([1-14C]oleate, [1-14C]linoleate, or [U-14C]glucose) in basal medium for 2 h. 14CO2 released from cells was then determined using a modification of a previously described method (55). Briefly, 25-cm2 culture flasks were sealed with a rubber septum with a plastic well attached. A folded 1-cm2 filter paper was placed inside the well. Fatty acid-derived CO2 was released by injection of 0.5 ml 9 M H2SO4 in the flasks and trapped by 0.2 ml 10 N NaOH injected on the filter paper inside the attached wells for 48 h. The filter papers together with the center wells that contained the absorbed 14CO2 were removed for scintillation counting.
Determination of the fatty acid composition of triglyceride.
Triglyceride components in the cellular lipid fraction obtained by Folch extraction were separated by TLC (hexane-ethyl ether-acetic acid 70:30:1) and methylated in a BF3-methanol solution (14% vol/vol BF3 in methanol) at 90°C for 30 min. Gas-liquid chromatography (GLC) analysis was performed as described previously (26).
NMR studies.
To measure fatty acid incorporation in cellular lipids, a solution containing 13C=O-labeled fatty acid (10 mM, 5 mol fatty acid/mol BSA) was added to culture medium during a regular medium change to achieve a 1.0 mM fatty acid concentration. After incubation for 24 h, cellular lipid extracts were analyzed by NMR as described previously (25, 27).
Scanning electron microscopy.
Scanning electron microscopic analysis was accomplished using a method that permits examination of nondehydrated, intact cells in multiple planes (66). Confluent, undifferentiated epididymal preadipocytes isolated from 3- and 30-mo-old rats were plated on Cell-Tak-coated (BD Biosciences, Bedford, MA) EI membranes (Quantomix, Rehovot, Israel) at a density of 2,000 cells/well. These membranes are transparent to electrons and protect hydrated samples from the vacuum. Cells were treated with fatty acids or control solution for 24 h. The cells were then fixed in paraformaldehyde and stained with osmium tetroxide and uranyl acetate. Cells were imaged by detecting back-scattered electrons using a JEOL JSM-6060-LV scanning electron microscope.
Apoptotic index.
Cells were stained with bisbenzamide and examined by fluorescence microscopy by observers unaware of the depot origin or treatment of the cells. Cells were classified as apoptotic if they exhibited irregular nuclear condensation (16). The apoptotic index was the percent of such nuclei as a function of all of the nuclei in a field.
Caspase-3 assay.
Caspase-3 activity was assessed in extracts of confluent epididymal preadipocytes using a kit (EnzChek Caspase-3 Assay Kit no. 1; Molecular Probes, Eugene, OR) by measuring the fluorescence signal of the cleaved product by the enzyme (31). Activity was expressed as a function of cell number.
mRNA analysis.
Total RNA was extracted from preadipocytes using the guanidinium thiocyanate-phenol method (12). Total RNA (1 μg) was reverse transcribed with Moloney murine leukemia virus RT (Invitrogen, Carlsbad, CA) and random oligonucleotides (Amersham, Piscataway, NJ). cDNA (5 μl) was used for the PCR reaction, in which mRNAs being analyzed were coamplified with an internal 18S rRNA control sequence (58). Analysis of mRNA expression was carried out during the exponential phase of amplification using the following primers: perilipin, 5′-gaccctgctggatggagac-3′ and 5′-cagctgcaggactctctgg-3′ (based on sequence NM 013094); C/EBPα, 5′-aggtgctggagttgaccagt-3′ and 5′-cagcctagagatccagcgac-3′ (based on sequence NM 007678); peroxisome proliferator-activated receptor-γ (PPARγ), 5′-ccagagcatggtgccttcgct-3′ and 5′-cagcaaccattgggtcagctc-3′ (based on sequence Y12882); muscle carnitine palmitoyltransferase (CPT) I, 5′-gctacagcacctctcagaga-3′ and 5′-ttatagcagttgccgtgcag-3′ (based on sequence AF029875); liver CPT I, 5′-tgcctctatgtggtgtccaa-3′ and 5′-ggcttgtctcaagtgcttcc-3′ (based on sequence NM 031559); p53, 5′-tttgaggttcgtgtttgtgc-3′ and 5′-ccacggatcttaagggtgaa-3′ (based on sequence NM 030989); BAX, 5′-ctgcagaggatgattgctga-3′ and 5′-gatcagctcgggcactttag-3′ (based on sequence AF235993); Bcl-2, 5′-caaactctgttggggcattt-3′ and 5′-ttgcatggctagaacacagc-3′ (based on sequence NM 080888); and medium-chain acyl-CoA dehydrogenase (MCAD), 5′-aactaaacatgggtcagcgg-3′ and 5′-ctccttggtgctccactagc-3′ (based on sequence NM 016986). The ratios of intensity of target to internal control bands were used to indicate the relative abundance of message in the samples. 18S rRNA amplification was titrated to match that of mRNAs being analyzed by adding competitive primers (Ambion, Austin, TX) that modulate extension of 18S cDNA.
Adenoviral infection.
Adenovirus encoding a CPT I mutant protein, M593S, was constructed (57). Adenovirus encoding human enhanced green fluorescent protein was obtained from GeneCore at the University of Iowa (Ames, IA). Confluent preadipocytes were washed two times with PBS, and growth medium was replaced with αMEM containing 1% FBS. Adenoviral particles (4 × 109) were diluted in 100 μl PBS containing 0.4 mM polyethyleneimine (PEI) and incubated for 20 min at room temperature. After incubation, the viral-PEI complex was added to preadipocytes for 18 h. After infection, cells were washed two times with PBS and incubated with fatty acid. Infection efficiency was assessed by determining the percentage of cells expressing green fluorescent protein by fluorescence microscopy.
Statistical analysis.
For each experiment, preadipocytes from one or more rats within each age group were pooled so that n represents the minimum number of rats studied. Results of densitometric analyses are expressed as a function of cell number. Means ± SE are shown. t-Tests or ANOVA with post hoc comparisons using Duncan's multiple-range test were used as appropriate (32, 39).
RESULTS
Fatty acids induce apoptosis in preadipocytes from old animals.
We noted distinct effects of different concentrations of oleic acid on preadipocytes from young and old animals (Fig. 1). Confluent, undifferentiated preadipocytes were exposed to 1–2 mM oleate in the presence of albumin (the molar ratio of oleate to BSA was 5:1) for 24 h. These concentrations are close to those in the circulation during fasting or in diabetes and have been previously used in lipotoxicity studies (23, 71). At this ratio of oleate to albumin, these concentrations are equivalent to ∼12.5 μM of unbound oleate in the exogenous medium, as calculated based on the first five high-affinity binding coefficients of albumin (63). Varying oleate concentrations while keeping albumin at the same 5:1 oleate-to-BSA molar ratio does not affect exogenous unbound oleate concentration but does alter fatty acid availability to cells. Use of oleate at low millimolar concentrations allows efficient delivery of fatty acids to cells over time without significantly altering the apparent fatty acid-to-BSA ratio in the medium. Under our incubation conditions, preadipocytes from both old and young animals accumulated lipid droplets. Exposure to 2 mM oleate in the presence of albumin caused extensive death of preadipocytes from old animals, whereas cells from young animals remained viable (Fig. 1). At lower oleate concentrations (0.8 mM) in the presence of albumin, nuclei exhibiting changes characteristic of apoptosis were apparent in preadipocytes from old animals (Fig. 2A). The percentage of cells with apoptotic nuclei was higher in preadipocytes from old than young animals (Fig. 2B). Caspase-3 activity was higher in preadipocytes from 30- than 3-mo-old animals. Abundance of message for the proapoptotic factors, p53 and BAX, was higher in preadipocytes from old than young rats following exposure to oleic acid (Fig. 2, D and E). Expression of the anti-apoptotic factor, Bcl-2, was similar in preadipocytes from young and old animals (data not shown). Thus apoptosis occurs to a greater extent in preadipocytes from old than young animals following exposure to oleic acid.
Incorporation of [1-13C]oleate.
To define mechanisms responsible for increased susceptibility of preadipocytes from old animals to cytotoxic effects of fatty acids, we first examined how aging affects fatty acid handling. Preadipocytes from young, middle-aged, and old rats were exposed to 1.0 mM [1-13C]oleate. Incorporation into cellular lipids by 13C-NMR was assayed as described previously (25–27). A typical 13C-NMR spectrum of lipid extracted from cells so treated is shown in Fig. 3A. From these NMR data, it was evident that most of the [1-13C]oleate in lipids was incorporated into triglyceride, whereas only a small amount was incorporated into phospholipid. This was true regardless of age. We perturbed the system by adding the essential fatty acid, linoleate, for the following reasons. Linoleate competes with oleate for the sn-2 position in triglyceride (TG; see Ref. 25). Linoleate is more extensively oxidized than oleate in rat liver mitochondria (7) and might compete with oleate for oxidation. Linoleate is potentially relevant to aging since reduced levels have been found in tissues from old individuals (48, 69). Incorporation of oleate in the sn-1 and -3 (sn1,3) and sn-2 positions of glycerol was assessed by the relative peak intensity ratio [TG(1,3)/TG(2)], as described previously (25, 27). In the absence of linoleate, [1-13C]oleate was distributed nearly equally among the sn-1,3 and sn-2 positions [TG(1,3)/TG(2) ∼2.0]. Replacing 33% of the added oleate with linoleate produced an increase in the TG(1,3)/TG(2) ratio (P < 0.01; n = 7; Duncan's multiple-range test; Fig. 3B). This indicates preferential incorporation of [1-13C]oleate in the sn-1,3 positions, possibly as a consequence of competition between oleate and linoleate for the sn-2 position (25). Although the TG(1,3)/TG(2) ratio tended to be lower in cells from young than old animals, this trend was not statistically significant.
Fig. 3.Fatty acid handling in preadipocytes from young and old animals. The 13C NMR spectrum of cellular lipid extracts was determined in preadipocytes incubated with 1.0 mM [1-13C]oleate in basal medium for 24 h (A). Peak assignments were performed as described previously (27). Intensity ratios of [1-13C]oleate esterified to the sn-1,3 and sn-2 positions were determined (B). Preadipocytes from 3 (young)-, 17 (middle-aged)-, and 24 (old)-mo-old rats were treated with 1.0 mM [1-13C]oleate (open bars) or 0.6 mM [1-13C]oleate plus 0.3 mM linoleate (filled bars) for 24 h. Linoleate caused a shift of oleate from the sn-2 to the sn-1,3 positions of glycerol; this was not affected significantly by aging. Fatty acyl composition of cellular triglyceride in preadipocytes from young (C) and old (D) rats was similar in 6 experiments.
We determined the acyl-chain composition of stored fatty acids in the triglyceride fractions of preadipocytes from young and old animals following incubation with oleate or oleate and linoleate by GLC. Acyl-chain composition was essentially the same in preadipocytes from both young (Fig. 3C) and old (Fig. 3D) rats, as well as in middle-aged animals (data not shown). In all age groups, oleate was the dominant moiety (86–91%) in cells that were not treated with linoleate. Palmitate (16:0), the precursor of ceramide, was no more abundant in preadipocytes from old than young animals. Replacement of 33% of exogenous oleate by linoleate resulted in a replacement of only 22% of oleate by linoleate in the triglyceride pool. This agrees with a previous report indicating that oleate is more readily esterified than linoleate in vivo (50). Thus fatty acid composition of cellular lipids is similar in preadipocytes from young and old rats following fatty acid exposure. Hence differences in stored lipid composition, such as increased de novo synthesis and accumulation of palmitate, do not likely account for the increased lipotoxicity we observed in preadipocytes from old animals.
Adipogenesis induced by fatty acids declines with aging.
The capacity of preadipocytes for adipogenesis may protect these cells from lipotoxicity by increasing abundance of fatty acid-binding proteins, pathways for storing fatty acids as triglycerides, and other mechanisms, such as increased expression of anti-apoptotic proteins (54). As noted above, preadipocytes from old animals store exogenous lipids in small droplets rather than the large lipid droplets that form in preadipocytes from younger animals (Fig. 1). To test if adipogenesis in response to exogenous fatty acids decreases with aging, preadipocytes cultured from young and old animals were exposed to 1 mM oleate for 24 h. Control cells from both young and old animals did not accumulate lipid droplets detectable by phase-contrast microscopy. More cells with large lipid droplets were evident in cultures from young than old animals after treatment with oleate for 24 h (Fig. 4). These cultures were examined by observers who were not aware of the ages of animals from which the preadipocytes had been isolated. The percentage, 9.2 ± 1.8%, of the preadipocytes from young animals developed from one to five large droplets during the 24-h treatment with oleate, whereas 4.3 ± 1.2% of cells from old animals contained such large droplets [P < 0.0005; n = 7; t-test; we elected to count cells with 1–5 large, doubly refractile droplets since their abundance correlates with cellular triglyceride (42), and they are readily distinguishable from other less-differentiated cells]. Although fewer of these cells were observed in cultures from old animals, preadipocytes from old animals contained more very small droplets than cells from young animals. This was not associated with differences in perilipin message: perilipin mRNA abundance by real-time PCR in preadipocytes treated with 1 mM oleic acid and 5:1 albumin for 24 h from young and old rats was 0.80 ± 0.21 and 0.83 ± 0.40 (arbitrary units; n = 3), respectively. However, expression of the adipogenic transcription factors, PPARγ2 and C/EBPα, was higher in preadipocytes from young than old animals after fatty acid treatment (Fig. 5, A and B). Thus preadipocytes from young animals respond to exogenous fatty acids by initiating adipogenesis, which would be anticipated to confer protection from lipotoxicity over time both to the differentiating preadipocytes and the whole organism.
Fig. 4.Adipogenesis induced by fatty acids declines with aging. More large lipid droplets form in preadipocytes from young than old animals after fatty acid treatment. Confluent epididymal undifferentiated preadipocytes isolated from 3- and 30-mo-old rats were incubated with 1 mM oleic acid and 0.33 mM BSA for 24 h, fixed in paraformaldehyde, and stained with osmium tetroxide and uranyl acetate. Scanning electron microscopic images were made by detecting backscattered electrons (magnification × 2,000). Data are representative of 3 experiments.
Fig. 5.Expression of the adipogenic transcription factors, peroxisome proliferator-activated receptor (PPAR)γ2 (A) and C/EBPα (B), is higher in preadipocytes from young than old animals following fatty acid treatment. Epididymal preadipocytes from 3- and 24-mo-old rats were treated with 1.0 mM oleic acid and 0.33 mM BSA or a control medium for 24 h. PPARγ2 and C/EBPα levels relative to 18S rRNA were determined by PCR. A representative experiment is shown at top, and means ± SE of 3 experiments are shown on bottom. *P < 0.05.
Preadipocyte and fat cell β-oxidation decline with aging.
Fatty acid β-oxidation declines in a number of cell types with aging (65). To test if this alteration in fatty acid handling also occurs in preadipocytes, cells from young and old rats were treated with 1-14C-labeled fatty acids, and 14CO2 production was determined acutely (2-h incubations). Because linoleate may compete with oleate for β-oxidation, we replaced 33% of the oleate with linoleate during incubations. This did not have a significant effect on CO2 production from [1-14C]oleate in preadipocytes from young animals (Fig. 6A). However, CO2 production from oleate was suppressed ∼20% in preadipocytes from old rats in the presence of linoleate (Fig. 6A).
Fig. 6.Aging is associated with reduced CO2 generation from exogenous oleate. A: 1.0 mM [1-14C]oleate (open bars) or 0.6 mM [1-14C]oleate and 0.6 mM unlabeled linoleate (filled bars) were added for 2 h to confluent epididymal preadipocytes isolated from young and old rats in 6 experiments. More 14CO2 was generated by cells from young than old rats (P < 0.02; ANOVA, n = 6). Addition of linoleate resulted in a greater reduction of CO2 produced from exogenous oleate by cells from old than young animals (P < 0.01; Duncan's multiple-range test, n = 6). The endogenous contribution of stored fatty acids to fatty acid oxidation was insignificant since cellular triglyceride content was negligible before and after the incubations began. B: 1.0 mM [1-14C]linoleate (filled bars) or 0.3 mM [1-14C]linoleate and 0.6 mM unlabeled oleate (open bars) were added for 2 h to confluent epididymal preadipocytes isolated from young and old rats in 5 experiments. Unlike the changes with age in CO2 generated from oleate shown in A, there was no significant effect of age on CO2 generation from linoleate (P = 0.59; ANOVA, n = 5). More CO2 was generated from linoleate than oleate (P < 0.001; ANOVA).
Slightly more CO2 was produced from linoleate (Fig. 6B) than from oleate (Fig. 6A). Oleate caused a substantial reduction in CO2 production from [1-14C]linoleate compared with preadipocytes exposed only to linoleate. Aging did not have a significant effect on the amount of CO2 produced from exogenous linoleate, unlike the effects of aging on CO2 production from oleate.
CO2 production from glucose does not decrease with aging.
With the use of conditions similar to those in the above experiments, cells were incubated with 5.5 mM [U-14C]glucose without exogenous fatty acids for 2 h. In six experiments, preadipocytes from young animals released 10.4 ± 0.6 pmol CO2/106 cells, whereas cells from old rats released slightly more CO2 [12.6 ± 1.0 pmol/106 cells (P < 0.05)]. Hence, aging is not associated with a decline, but rather an increase, in the capacity of preadipocytes to generate CO2 from glucose under these conditions. In 13C NMR studies, we found that preadipocytes from old rats incorporated 27 ± 5% more [1-13C]oleate in triglyceride in the presence of 5 mM glucose than cells from young animals (P < 0.005). This is consistent with the modest increase in glucose oxidation with aging. The extent of CO2 production from glucose was at least fourfold greater than the extent of fatty acid conversion to CO2 in the preceding experiments. This indicates that flux through those components of the fatty acid oxidation pathway that are shared with the glucose oxidation pathway was not limiting under these experimental conditions.
Fat cell oleate β-oxidation declines with aging.
To test if the decline in fatty acid β-oxidation in cultured preadipocytes also occurs in freshly isolated fat cells, epididymal adipocytes were prepared from fat depots of young and old rats by collagenase digestion (Fig. 7). Cells were prelabeled with [9,10-3H]oleate [5 μCi/ml, ∼10 nM, in Krebs-Ringer bicarbonate (KRB) buffer, 5 mM glucose] for 30 min and then washed. Under these conditions, >99% of the labeled fatty acid were esterified into cellular triglycerides. The specificity of isotope-labeled fatty acid was calculated as β-counts per nanogram endogenous triglyceride. Cells were then incubated in KRB solution for 2 h without exogenous fatty acid. The release of 3H2O was measured as an index of β-oxidation from endogenously released fatty acid (72). In parallel, cells were incubated with 5.5 mM [14C]glucose for 2 h, and release of 14CO2 was measured as above. There was essentially no difference in glucose oxidation between adipocytes from young and old animals, as in the case of undifferentiated preadipocytes.
Fig. 7.Fat cell oleate oxidation declines with aging, whereas glucose oxidation undergoes little change. Fat cells were isolated from epididymal fat depots of 6- and 24-mo-old rats by collagenase digestion and prelabeled with [9, 10-3H]oleate for 30 min. 3H2O production over the next 2 h was measured. Parallel cultures were incubated with [U-14C]glucose (5 mM) in a Krebs-Ringer bicarbonate (KRB) solution for 2 h, and release of 14CO2 was measured directly. More 3H2O (filled bars) was generated from oleate by fat cells from young than old rats (n = 3; P < 0.005; t-test), whereas CO2 (open bars) generated from glucose was similar between age groups (P = 0.7).
CPT I message does not decline substantially with aging.
CPT I-mediated transfer of acyl-CoA fatty acids into mitochondria is generally the rate-limiting step for β-oxidation of fatty acids (17). Both preadipocytes and fat cells express CPT I, although at different levels and different isoforms (11). L-CPT I (liver isoform) mRNA did not decline substantially with aging (Fig. 8), nor did the muscle isoform (data not shown).
Fig. 8.Carnitine palmitoyltransferase-1 (CPT I) does not decline substantially with aging. Confluent epididymal preadipocytes from 3- and 30-mo-old rats were treated with a 1 mM oleate (with 5:1 albumin) or control solution for 24 h. CPT I (liver isoform; L-CPT I) mRNA was then assayed by competitive PCR. A representative experiment is shown on top, and mean ± SE L-CPT I levels (as a function of 18S rRNA) are shown on bottom (n = 6; P = 0.3; t-test).
Overexpression of a malonyl-CoA-resistant CPT I mutant enhances fatty acid β-oxidation in preadipocytes from young and old animals.
CPT I activity is regulated by malonyl-CoA and other intracellular factors, rendering message, protein, and in vitro activity measurements difficult to interpret. For example, CPT I mRNA decreases during fasting, but β-oxidation increases (74). To test if the decline in fatty acid β-oxidation may be at or downstream from the level of intracellular CPT I activity, we overexpressed a constitutively active form of L-CPT I, M593S, by infecting preadipocytes from young and old animals with L-CPT I M593S adenovirus or a GFP-expressing adenovirus. Infection efficiency was ∼80% in both young and old preadipocytes. At low oleic acid concentrations, the constitutively active CPT I mutant increased β-oxidation in preadipocytes from both young and old animals to approximately the same levels (Fig. 9A). Under these conditions, basal fatty acid β-oxidation was similar in preadipocytes from young and old rats. However, at higher fatty acid concentrations (in the presence albumin), impaired β-oxidation in preadipocytes from the old animals was apparent (Fig. 9B; P < 0.01). The constitutively active CPT I mutant M593S virus increased β-oxidation compared with the control virus in preadipocytes from both young and old animals (P < 0.01; Duncan's multiple-range test). However, β-oxidation was lower in the M593S-infected preadipocytes from old than young animals (P < 0.01), as was the case in the control preadipocytes. This occurred despite the same infection conditions as in the preceding experiments in which the M593S virus equalized β-oxidation in preadipocytes from old and young animals after treatment with oleic acid at 100 μM. Thus, although the CPT I pathway appears able to handle lower fatty acid fluxes in the β-oxidation pathway in preadipocytes from old rats, mitochondrial dysfunction may limit fatty acid β-oxidation with aging in preadipocytes at higher fatty acid concentrations.
Fig. 9.CPT I overexpression is less effective in enhancing β-oxidation at high concentrations of fatty acids in preadipocytes from old than young animals. A: a constitutively active CPT I mutant increased β-oxidation at lower fatty acid concentrations in preadipocytes from both young and old animals to approximately the same levels. Preadipocytes from 3- and 30-mo-old rats infected with an adenovirus expressing L-CPT I M593S or a control green fluorescent protein (GFP) adenovirus were incubated with 100 μM oleate (with 5:1 albumin and 5 mM glucose) in parallel for 3 h (n = 3 separate experiments). β-Oxidation is expressed as a percentage of total values within each experiment. B: the constitutively active CPT I mutant was less effective in equalizing β-oxidation in the presence of high concentrations of fatty acid in preadipocytes from old and young animals. Preadipocytes from 3- and 30-mo-old rats infected with the L-CPT I M593S or GFP adenovirus were incubated with 1.0 mM oleate (5:1 albumin and 5 mM glucose) for 3 h (n = 3 separate experiments).
MCAD expression is decreased in preadipocytes from old rats.
Because β-oxidation in preadipocytes from old animals was restricted compared with cells from young animals exposed to high oleic acid concentrations, we tested the hypothesis that expression of MCAD, a key enzyme in the intramitochondrial long-chain fatty acid β-oxidation pathway (38), declines with aging. MCAD mRNA was lower in confluent epididymal preadipocytes from old than young animals treated with 1 mM oleate in the presence of albumin for 24 h (Fig. 10). Reduced MCAD expression is consistent with the possibility that mitochondrial dysfunction could contribute to restricted preadipocyte β-oxidation with aging.
Fig. 10.Medium-chain acyl-CoA dehydrogenase (MCAD) expression declines with aging. Confluent epididymal preadipocytes from 3- and 30-mo-old rats were treated with 1 mM oleate (with 5:1 albumin) or control solution for 24 h. MCAD mRNA was then assayed by competitive PCR. A representative experiment is shown at top, and mean ± SE MCAD levels (as a function of 18S rRNA) are shown on bottom (n = 3; a > b > c > d, P < 0.05; Duncan's multiple-range test).
DISCUSSION
Fat tissue becomes dysfunctional in old age. Depot and fat cell sizes change, fat is redistributed, insulin responsiveness decreases, response to lipolytic agents declines, and inflammatory cytokine production by fat tissue increases (15, 24, 41, 46). These changes are accompanied by increased prevalence of diabetes, hyperlipidemia, hypertension, and atherosclerosis, together with lipid accumulation and lipotoxicity in tissues other than fat. By way of analogy, defective adipose tissue in lipodystrophic syndromes is also associated with lipotoxicity in peripheral tissues and insulin resistance (46, 49, 62, 67, 68). Here we found that the preadipocytes from which new fat cells develop throughout the life span and that reside in a high fatty acid microenvironment are themselves increasingly subject to lipotoxicity. Decreased adipogenesis in response to fatty acids may contribute to this. Increased susceptibility of preadipocytes from old animals to fatty acids may exacerbate systemic lipotoxicity, since preadipocytes normally defend against lipotoxicity by becoming fat cells that sequester excess fatty acids. Furthermore, preadipocytes are an important cell type in their own right, since they comprise 15–50% of cells in fat tissue and have profiles of secreted proteins distinct from those of fat cells (46). Thus compromised preadipocyte fatty acid handling may have wide-ranging ramifications. As in other cell types, fatty acid β-oxidation becomes impaired with aging in both preadipocytes and fat cells, also indicating that broad changes in fatty acid handling occur in fat tissue with aging.
High fatty acid concentrations may play a role in the defensive function of fat tissue by removing dysfunctional cells and suppressing invading microorganisms [such as Helicobacter pylori (59), pneumococcus (14), and Mycobacteria (1, 2)]. Additionally, adipose tissue fatty acids may defend against overshoot effects of chronically high insulin levels by promoting insulin resistance. In younger individuals, preadipocytes and fat cells are resistant to lipotoxicity. Treatment of collagenase-isolated rat epididymal fat cells for up to 24 h with 1.5 mM oleate or palmitate (at a free fatty acid-to-albumin ratio of 2.5:1) has no significant impact on lipolysis or insulin action (53). In the current study, we found that primary rat preadipocytes isolated from young animals can resist 2.0 mM oleate at 5:1 with BSA (for a free oleate concentration ∼12.5 μM) for 24 h. However, preadipocytes from old animals treated with oleic acid accumulated multiple small lipid droplets more characteristic of lipotoxicity than differentiation, were more likely to die, had more nuclei with morphological changes compatible with apoptosis, had higher caspase-3 activity, and expressed higher levels of pro-apoptotic factors than cells from young animals. Thus preadipocytes become paradoxically sensitive to lipotoxicity with aging. The potentially beneficial effects of the high fatty acid concentrations in fat tissue early in life appear to be deleterious later in life.
Accumulation of toxic metabolites of palmitate or decreased adiponectin or leptin do not appear to have accounted for increased lipotoxicity with aging under our experimental conditions. Ceramide, a toxic metabolite of palmitate that has been implicated in lipotoxicity in pancreatic β-cells (10, 65), is not generated directly from oleate, which, on its own, induced lipotoxicity in preadipocytes from old animals. Palmitate levels were low in preadipocytes from young rats under the conditions of our experiments and tended to be even lower in cells from old animals (palmitate is 16:0 in Fig. 3, C and D). Thus accumulation of toxic metabolites of palmitate is unlikely to explain the increased lipotoxicity we found in response to oleic acid. Leptin and adiponectin can enhance β-oxidation and protect against lipotoxicity in other cell types (65). However, differences in these adipokines do not explain the decreased β-oxidation and increased lipotoxicity we observed in isolated preadipocytes with aging, since undifferentiated preadipocytes do not produce adiponectin or leptin and preadipocytes from young and old animals were grown in the same culture media. This supports the contention that inherent, possibly cell-autonomous mechanisms contribute to increased susceptibility to apoptosis as well as decreased β-oxidation with aging, in addition to any effects that age-related changes in these adipokines may have in vivo.
Preadipocytes contribute to protection from excess systemic lipids by differentiating into fat cells that sequester lipid. The preadipocytes used in this study were maintained in a basal medium that does not promote differentiation. Fatty acids were added to this basal medium without extra differentiation-promoting agents, such as insulin, thiazolidinediones, glucocorticoids, or isobutyl methylxanthine. Fatty acids induced adipogenesis in the primary preadipocytes from young rats, with formation of large lipid droplets and increased PPARγ and C/EBPα expression within 24 h. Fatty acids also induce differentiation in preadipocyte cell lines (3, 4, 19). Fewer cells from the old animals treated with fatty acids developed large lipid inclusions, and these cells expressed less PPARγ and C/EBPα than cells from young animals. Reduced adipogenesis in cells from old animals treated with fatty acids is compatible with previous reports that adipogenesis in response to differentiation-inducing media declines with aging (8, 20, 21, 28, 33, 42–44, 70). This occurs despite isolation procedures that guarantee that >90% of the cells isolated from animals of all ages are preadipocytes (42, 70).
Mechanisms contributing to decreased preadipocyte differentiation with aging appear to include processes occurring at or before the increase in adipogenic transcription factor expression during differentiation (33, 34). In our current study, PPARγ and C/EBPα abundance was lower after fatty acid exposure in preadipocytes from old than young animals. This is consistent with the finding that the increase in these adipogenic transcription factors following treatment with differentiation-inducing medium is blunted in preadipocytes from old compared with young animals (33). Activation of cellular stress response pathways could be among the mechanisms impeding preadipocyte differentiation with aging (34, 46). The lipotoxicity induced by fatty acid exposure might contribute to this. Reduced capacity to differentiate and express proteins that bind cytotoxic fatty acids and convert them into less toxic triglycerides may, in turn, be a cause of reduced long-term protection from lipotoxicity, setting up a vicious cycle.
Ability to metabolize fatty acids by β-oxidation was compromised in both preadipocytes and the fat cells that develop from them in old animals. Alternatively, the fat cells might have developed earlier in life, with the functional changes occurring with aging being independent of the changes in preadipocytes. Impaired β-oxidation occurs in other cell types with aging and may contribute to increased intramyocellular lipid and lipotoxicity in obesity and diabetes (36, 37). Glucose and fatty acid oxidation share the final steps of flux through the tricarboxylic acid cycle and oxidative phosphorylation. CO2 generation from glucose did not decrease with aging in either preadipocytes or fat cells under the conditions employed in these experiments. Hence it is unlikely that limitations in tricarboxylic acid cycle function or oxidative phosphorylation are the cause of reduced CO2 generation from oleate. Aging was not associated with reduced CO2 production from linoleate. CO2 generation from oleate declined with aging, particularly at high fluxes (1 mM oleate at 5:1 BSA-oleate), especially when oleate and linoleate were present in combination. This selective effect of aging on preadipocyte fatty acid utilization may be related to oleate being a better substrate than linoleate for esterification (50), although linoleate is a better substrate than oleate for β-oxidation and so competed with oleate for β-oxidation. Indeed, linoleate decreases mitochondrial membrane potential, thereby stimulating increased fatty acid oxidation (9, 61). Our results suggest that this may occur to a greater extent in cells from young than old animals. Because increasing CPT I activity under conditions of high fatty acid flux did not increase oleate β-oxidation (while doing so at lower flux did) and because MCAD expression was reduced with aging, mitochondrial dysfunction and/or dysfunction of specific intramitochondrial enzymes, rather than transfer of lipid into mitochondria, may be a key mechanism contributing to impaired fatty acid β-oxidation with aging.
Preadipocyte defenses against lipotoxicity become impaired with aging, possibly because of inherent aging processes such as DNA damage, effects of reactive oxygen species, mitochondrial dysfunction, cellular stress response pathway activation, or other fundamental aging processes. Increased preadipocyte susceptibility to lipotoxicity may ultimately contribute to fat tissue dysfunction, compromising capacity of fat tissue to protect other tissues from lipid accumulation and lipotoxicity. Thus preadipocytes become paradoxically susceptible to lipotoxicity. This could set up a cycle of lipotoxicity in fat tissue, with fatty acids contributing to preadipocyte dysfunction, impeding adipogenesis with failure to store fatty acid as neutral triglyceride, leading to further increases in fatty acids, and compounding fat tissue dysfunction. Decreased capacity to store excess fatty acid in fat could contribute to lipotoxicity in nonadipose tissues, such as muscle and pancreatic β-cells, in old age.
GRANTS
This work was supported by National Institutes of Health Grants AG/DK-13925 (J. L. Kirkland), DK-59261 (W. Guo), and DK-46200 (B. E. Corkey and J. L. Kirkland).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We acknowledge the technical support by G. Chan, assistance with electron microscopy by Alon Sabban (Quantomix), and administrative support by J. Armstrong.
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