Previous studies conducted in 3T3-L1 adipocytes revealed the induction of mitochondrial biogenesis during the process of differentiation, suggesting an important role for mitochondrial mass in white adipocyte function. Furthermore, exposure of fully differentiated 3T3-L1 adipocytes to rosiglitazone led to pronounced changes in mitochondrial morphology and to a small but significant increase in mitochondrial mass. We searched for evidence for mitochondrial plasticity in vivo in white adipocytes isolated during the development of obesity and in response to rosiglitazone in ob/ob mice.
Age-matched C57BL/6J and ob/ob mice were used in this study. The effectiveness of rosiglitazone treatment was evidenced by the blood glucose concentration of ob/ob mice, which decreased to that seen in age-matched C57BL/6J mice (Figure , left). During the 2 weeks of treatment of ob/ob mice with rosiglitazone, a small but statistically significant increase in body weight was detected (Figure , middle). Interestingly, while epididymal fat pad mass increased considerably between 4 and 26 weeks of age in ob/ob animals, no statistically significant difference was detected in fat pad mass after rosiglitazone treatment (Figure , right).
Figure 1 Effects of rosiglitazone on ob/ob mice. Fasting blood glucose concentrations, whole-body weight, and isolated fat pad weight were obtained in control animals or animals treated with rosiglitazone (Rosi) for 14 days. Data were derived from 10 independent (more ...)
Isolated adipocytes were obtained from epididymal fat pads from 26-week-old ob/ob
mice and age-matched C57BL/6J mice and were analyzed by fluorescence microscopy with an antibody against the mitochondrial chaperone mHsp70 (Figure ). Cells obtained from 26-week-old ob/ob
mice were significantly larger than cells from age-matched C57BL/6J animals. In both cases, mitochondrial staining with anti-mHsp70 appeared as a punctate pattern distributed throughout the cytoplasm of the cell, with an increase in concentration near the perinuclear region, where the cytoplasmic volume is greatest. The average fluorescence intensities per cell from projected three-dimensional image stacks were 0.53, 1.05, and 1.58 units for C57BL6/J and ob/ob
mice and for ob/ob
mice after rosiglitazone treatment. However, cells from ob/ob
mice had approximately twice the diameter of, and thus a surface area 4 times and volume 8 times larger than, cells from C57BL6/J mice. Assuming a requirement for mitochondrial mass proportional to cell size, these results indicate a deficit in mHsp70 staining in cells from ob/ob
mice relative to that of controls. Because average cell size did not differ between ob/ob
and rosiglitazone-treated ob/ob
mice, these results indicate an increase of approximately 50% in staining in cells from rosiglitazone-treated mice. The staining pattern of cells from rosiglitazone-treated animals differed markedly from that of cells from untreated ob/ob
mice in that it revealed a fine reticular pattern, also distributed throughout the cytoplasmic volume but significantly more concentrated in the perinuclear region (Figure ). In addition, dense mitochondrial staining was observed surrounding small, droplet-like structures found in many cells from rosiglitazone-treated mice. The changes in mitochondrial density and morphology were also seen in cells stained with anti-mHsp60 (Figure ); cells from rosiglitazone-treated animals contained almost twice the amount of mHsp60 per cell relative to cells from untreated animals. It is relevant that the staining pattern found in adipocytes from rosiglitazone-treated animals was seen in cells of a size equal to those from 26-week-old untreated animals, a finding that suggests that not only small cells (8
) but also large cells within the adipose tissue are susceptible to the actions of rosiglitazone.
Figure 2 Immunofluorescence analysis of mitochondrial Hsp70. Adipocytes were isolated from the epididymal fat pads of 26-week-old C57BL/6J, ob/ob, and rosiglitazone-treated ob/ob (ob/ob + Rosi) mice. Adipocytes were fixed and stained and image stacks were (more ...)
Figure 3 Immunofluorescence analysis of mitochondrial Hsp60. Isolated adipocytes from age-matched untreated and rosiglitazone-treated ob/ob mice were fixed, stained, and imaged with a conventional wide-field microscope fitted with a 40× or 100× (more ...)
To rule out the possibility that the changes described above were due to rosiglitazone-induced alterations in mitochondrial chaperones, rather than to changes in true mitochondrial mass, we imaged live isolated adipocytes after incubating them with a specific indicator of mitochondrial mass, MitoTracker green. Mitochondria in age-matched C57BL/6J mice and 26-week-old ob/ob animals appeared as discrete punctate structures, while mitochondria in cells from ob/ob mice treated with rosiglitazone were reticular and surrounded what appeared to be small lipid droplets present in many cells (Figure ). Thus, rosiglitazone treatment in vivo results in pronounced changes in global mitochondrial mass and morphology. The presence of what appeared to be small lipid droplets surrounded by mitochondria suggests that considerable rearrangements of the lipid-metabolizing machinery of the white adipocyte occur in response to PPARγ stimulation by rosiglitazone.
Figure 4 Live cell imaging of mitochondria with MitoTracker Green FM. Adipocytes were isolated from epididymal fat pads of 26-week-old C57BL/6J or ob/ob mice that were either left untreated or were treated with rosiglitazone for 14 days. Cells were incubated with (more ...)
To probe for the molecular basis of these morphological alterations, we surveyed a database of RNA expression levels obtained using Affymetrix chips (Figure ). Of the approximately 36,000 genes and expressed sequence tag clusters in the murine genome U74v2 probe set, 11,949 were detected as present in cells from 4- and 26-week-old ob/ob mice. The level of expression of 4,560 (38%) of these genes differed between samples from 4- and 26-week-old mice. Of these, 2,245 (about 19%) were increased in transcript level and 2,315 (about 19%) were decreased. The database was then “queried” using a list of 700 probe sets previously annotated as “mitochondrial” in the Affymetrix database. Of these probe sets, 373 detected genes present in cells from 4- and 26-week-old ob/ob mice. Approximately 204 (55%) of these were significantly altered by the onset of obesity, with 23 (6%) of genes found to be increased and 181 (49%) found to be decreased. These results suggested that genes related to mitochondrial structure and function are overrepresented in the group of genes that decrease in expression level during the onset of obesity.
Figure 5 Analysis of gene expression changes in adipocytes from ob/ob mice. Total RNA was extracted from isolated adipocytes from 4-week-old (4 week) untreated, 26-week-old (26 week) untreated, and 26-week-old rosiglitazone-treated ob/ob mice and was used to probe (more ...)
To determine how specifically this overrepresentation relates to genes of the mitochondria compared with those of other organelles, we used two additional lists of probe sets to “query” the database. One set consisted of 513 probe sets annotated with the term “endoplasmic reticulum” and another set consisted of 802 probe sets annotated with the term “nuclear.” The “endoplasmic reticulum” probe set detected 249 genes present in cells from 4- and 26-week-old ob/ob mice. Of these, 99 (40%) differed between samples from 4- and 26- week-old mice, with 47 (19%) increased and 52 (21%) decreased. The “nuclear” probe set detected 353 genes, of which 123 (35%) differed between samples from 4- and 26-week-old mice, with 57 (16%) increased and 66 (19%) decreased. This similar frequency of genes displaying increased or decreased expression levels in the “endoplasmic reticulum” and “nuclear” probe set is similar to that seen for the “all-gene” probe set, but different from that seen for the “mitochondrial” set, for which genes displaying decreases in expression levels were 7–8 times more frequent than those displaying increases in expression levels.
The changes occurring after treatment with rosiglitazone in 26-week-old mice were also analyzed (Figure ). In the U75 probe set, 12,808 genes were detected as being present in cells from 26-week-old ob/ob mice. The levels of expression of 1,539 (12%) of these were altered by exposure to rosiglitazone. The expression levels of 1,102 (9%) were increased and 437 (3.4%) were decreased. The database was then “queried” with the same organelle-specific probe set lists described above. Of the probe sets annotated as “mitochondrial,” 403 detected genes expressed in cells from 26-week-old ob/ob animals. Of these, 131 (32%) were altered by rosiglitazone treatment, with 130 (32%) displaying increased expression levels and only 1 (0.2%) displaying a decreased expression level. Thus, genes associated with mitochondria are disproportionately affected by rosiglitazone treatment, with a strong bias toward an increase in gene expression. In contrast, other organelle-related genes displayed changes similar to those displayed by the total gene pool. Of the “endoplasmic reticulum” genes, 255 were detected in cells from 26-week-old animals. Of these, 28 (11%) were altered by rosiglitazone treatment, with 18 (7%) displaying increased expression and 10 (4%) displaying decreased expression. Similarly, 391 “nuclear” genes were detected in cells from 26-week-old animals. Of these, 53 (14%) were altered by rosiglitazone treatment, with 41 (11%) displaying increased expression and 12 (3%) displaying decreased expression.
In addition to causing a disproportionate increase in the expression of existing mRNAs for mitochondrial genes, rosiglitazone induced the expression of approximately 100 genes that were not detected in white adipocytes from 26-week-old ob/ob
mice. Among these are mitochondrial genes that would be expected to strongly influence energy metabolism and fatty acid oxidation and that are typically present in brown but not white adipocytes (Table ). These include UCP-1, Cidea (9
), and the M isoform of carnitine palmitoyltransferase I (10
Rosiglitazone-induced mitochondrial gene expression in ob/ob mouse adipocytes
To survey the degree of concordance between the changes in mRNA levels and changes at the protein level, we purified mitochondria from isolated adipocytes, solubilized them, and analyzed them by sucrose gradient centrifugation and SDS-PAGE. When normalized to the initial protein content of the cell homogenates from which mitochondria were isolated, the yield of mitochondrial protein was approximately double from cells from rosiglitazone-treated ob/ob mice compared with untreated age-matched controls. This increase yield is consistent with the doubling of mitochondrial mass on a per-cell basis deduced by mitochondrial staining (Figure ). The more abundant proteins that displayed changes in response to rosiglitazone were excised and identified by mass spectrometry (Figure ). We then “queried” the Affymetrix dataset to determine the expression pattern of the mRNAs for these specific proteins (Table ). As expected, concordant increases in protein and mRNA levels in response to rosiglitazone were observed for many of the proteins identified by mass spectrometry. However, the increases in protein abundance exceeded the increased level of expression detected by the Affymetrix chip analysis in several instances. For example, a 2- to 3-fold increase in protein levels of malate dehydrogenase (Mor1) corresponded to a 1.45-fold increase in mRNA abundance. More strikingly, the mitochondrial chaperones mHsp70 and mHsp60 were not “scored” as present in mouse adipose cells in the Affymetrix chip despite being abundant mitochondrial proteins, as judged by mass spectrometry. Moreover, the rosiglitazone-induced increase in the level of chaperone expression, which was evident both by mass spectrometry (Figure ) and Western blotting (Figure ), was not detected by the Affymetrix chip analysis. This discordance suggests that mitochondrial protein expression may be particularly subject to regulation at the post-translational level and that some of the actions of rosiglitazone on mitochondrial structure/function may take place through translational control.
Figure 6 Comparative analysis of mitochondrial protein expression in untreated and rosiglitazone-treated adipocytes. Mitochondria isolated from adipocytes from untreated (–) and rosiglitazone-treated (+) ob/ob mice were analyzed by sucrose gradient (more ...)
The mRNA expression levels of proteins identified by mass spectrometry
Figure 7 Western blotting of mitochondrial proteins. Isolated adipocytes were lysed in sample buffer containing 1% SDS. Equal concentrations of total protein (20 μg) were analyzed by SDS-PAGE and Western blotting using antibodies against mitochondrial (more ...)
Among the proteins increased in response to rosiglitazone are several key proteins of the fatty acid β-oxidation pathway. Notably, the protein levels of short-chain, medium-chain, long-chain, and very-long-chain acyl-CoA dehydrogenases, which catalyze the key reaction in the β-oxidation of fatty acids, were found to be increased by rosiglitazone. Reducing equivalents from these dehydrogenases are transported to the main respiratory chain by the electron transfer flavoprotein, composed of two subunits, Etfa
, and by the electron transfer flavoprotein dehydrogenase Etfdh
). The levels of all three of these proteins were detected to be increased by mass spectrometry and Affymetrix chip analysis. These results suggest that β-oxidation of fatty acids might be increased in response to rosiglitazone. To directly test this hypothesis, we measured the conversion of [14C]palmitate to [14C]O2
by isolated adipocytes derived from control and ob/ob
mice. A significant increase in palmitate oxidation was detected in cells from rosiglitazone-treated animals (Figure ).
Figure 8 Fatty acid oxidation in isolated adipocytes. Adipocytes were isolated from 26-week-old untreated or rosiglitazone-treated ob/ob mice. Palmitate oxidation was estimated by the measurement of [14C]O2 release over a 2-hour period as described (more ...)
Additional evidence for the functional relevance of the observed alterations in mitochondrial mass and composition in obesity is the marked depression in oxygen consumption by isolated adipocytes from ob/ob mice compared with that of cells from 4-week-old ob/ob animals (Figure ) or age-matched C57BL/6J mice (data not shown). This depressed oxygen consumption rate was almost completely reversed in cells from rosiglitazone-treated mice (Figure ).
Figure 9 Oxygen consumption in isolated adipocytes. Adipocytes were isolated from 4-week-old, 26-week-old untreated, or 26-week-old rosiglitazone-treated ob/ob mice. Equal volumes of packed cells were separated into aliquots in wells of a 96-well BD Oxygen Biosensor (more ...)
To investigate the potential mechanisms involved in the observed changes in mitochondrial mass and function in response to rosiglitazone, we analyzed the levels of PGC-1α (Figure ). While the transcript levels for this coactivator were below the limits of detection in the Affymetrix chip, direct Northern blotting revealed a signal for PGC-1α, which was increased significantly in response to rosiglitazone.
Figure 10 PGC-1α expression in epididymal fat. Total RNA was prepared from fat pads from ob/ob mice before (–) and after (+) rosiglitazone treatment and was subjected to Northern blotting with a 32P-labeled mouse PGC-1 cDNA fragment as a (more ...)