The results presented here have documented striking differences in cell size distribution between adipocytes from obese, insulin resistant ZO rats and non-obese, insulin sensitive ZL rats, as well as delineate the adipogenic and inflammatory characteristics of small adipocytes that help to substantiate the observations we have made on insulin resistance, accumulation of small adipose cells, and adipose tissue gene expression in our human studies.11–13
The most notable difference between adipocytes from the ZO and ZL rats was in the cell size distribution. The ratio of small to large cells was increased 16-fold in adipocytes from ZO rats. Put in another way, small cells occupied more than half of the total number of cells in the ZO rat epididymal fat depot. By contrast, small cells comprised only 12% of the total adipose cell population in ZL rats. Moreover, the mean peak diameter of the large cells was significantly greater in ZO rats. Our ability to demonstrate this striking difference in cell size distribution was made possible by the use of updated Multisizer cell sizing methodology to characterize adipose tissue cellularity. Earlier Coulter Counter techniques were set to count cells above a certain plateau level that unintentionally overlooked smaller cells.5,6,16
Other methods such as microscopic or photographic visualization use a representative sample of cells that may not sufficiently account for adipose cell size spread. Histologic method to evaluate adipose cell diameter is dependent on the plane through which the adipocyte is sliced, and thereby subjected to the artifact of off-center sectioning. Through Multisizer technology we have been able to document the presence of an expanded population of small adipose cells in the epididymal adipose tissue of ZO rats, associated with a significant increase in the size of the large cells.
These findings are consistent with the hypothesis that adipose cells enlarge to a maximal state, followed by proliferation of small cells to accommodate further fat storage.17
Indeed, we have extended these observations further by assessing, in vivo, adipose tissue development in the ZO rat.18
By performing sequential micro-biopsies over a 21-week period, we observed that adipose tissue expansion exhibited a temporal periodicity of roughly 55 days during which cells increase in size and new, smaller adipose cells are recruited. This mechanism is triggered when the flux of lipid needing storage exceeds that of capacity for lipid uptake. In the present study, the preponderance of small adipose cells in the ZO rat fat pad indicates that the ability to recruit new cells is retained, although they appear to be dysfunctional in nature, as evidenced by their adipogenic and inflammatory characteristics.
The second main findings of our study are in the differential properties of small adipocytes. As compared to total cells, ZO rat small adipocytes exhibited significantly lower expression of adiponectin, an adipocytokine made exclusively by mature adipocytes, and whose plasma and adipose tissue levels are decreased in human insulin resistance, independent of obesity.11,19
That differential expression of adiponectin was robust despite comparison of small cells with the total mixed cell population intimates that the differences between small and large cells may, in fact, be greater. As compared to small adipocytes from ZL rats, small adipocytes from ZO rats also had decreased levels of adiponectin and PPARγ, highlighting not only that the small adipocytes from ZO rats have an impaired capacity for differentiation, but that they may be functionally disparate from the ZL rat small adipose cells. These findings help to substantiate our findings in human subcutaneous adipose tissue, in which accumulation of small adipose cells was associated with decreased adipose tissue expression of genes related to adipocyte differentiation in insulin resistance, independent of obesity.11
Taken together, it is reasonable to postulate that small adipose cells may contribute to systemic insulin resistance via their impaired capacity for fat storage.
The other defining properties of small adipocytes are their modest pro-inflammatory nature. Of the five inflammatory genes tested, visfatin and IL-6 were upregulated in small as compared with total adipocytes from ZO rats, suggesting that inflammation may serve as an additional mechanism by which small adipocytes may contribute to insulin resistance. Small adipocytes from ZO rats also expressed increased levels of IL-6 when compared with small cells from ZL rats. These findings are supportive of our previous data in humans, in which we showed that inflammatory activity was independently associated with increased proportion of small adipose cells in subcutaneous adipose tissue, as well as measures of insulin resistance.12,13
Thus, it seems plausible that the inflammatory properties of small adipose cells may contribute to development of insulin resistance at a systemic level, although causality remains to be elucidated.
It is valuable to consider results of the small adipocytes within the larger context of the adipose tissue and total adipocyte population as a whole. Isolated adipocytes from ZO as compared with ZL rats demonstrated evidence of impaired differentiation and greater inflammatory activity. Taken together with data from the small adipocytes, one may infer that these characteristics of the ZO rat adipose tissue depot as a whole are accounted for by the expanded presence of small adipocytes. While upregulated GLUT4 levels in ZO rat adipocytes as compared to their lean counterparts may appear to be an exception, these data are consistent with well-established evidence that regulation of adipose cell GLUT4 occurs in an age-dependent pattern in ZO rats;20,21
adipocytes of young ZO rats exhibit hyper-responsiveness to insulin-stimulated glucose transport (despite systemic hyper-insulinemia/insulin resistance), whereas adipocytes from older ZO rats exhibit impaired glucose uptake. Likewise, increased leptin in ZO as compared to ZL rat adipocytes is ascribed to the ZO rat genetic makeup.
Overall, these findings are consistent with previous studies from our group and others supporting the association of obesity-related insulin resistance with impaired adipogenesis and inflammatory states.11,12,15,22–25
How impaired differentiation, increased inflammation, and accumulation of small adipocytes may interrelate to promote insulin resistance remains more speculative. One possibility is that inflammatory cytokines secreted by small cells impair terminal adipocyte differentiation, leading to ineffective triacylglycerol storage and accumulation of further small cells. An alternate explanation is that non-adipose cell inflammatory mediators, i.e. macrophages or other myeloid-derived cells, residing in adipose tissue exert negative effects on adipocyte function.26–28
In this context, it is important to re-emphasize that the small cells we have detected in our studies are adipose cells, and not macrophages.11,13
This is an important distinction, for while it is known that macrophages can promote inflammatory activity in adipose tissue,29
our findings address the direct role small adipose cells may have in inflammation. Finally, as another hypothesis, it is also plausible that the primary defect lies in adipose cell differentiation, with development of an inflammatory phenotype in the small adipocytes as a secondary process.
This study is one of several studies published on the genetic or metabolic characteristics of small adipocytes, of which mixed results have been reported. One study showed that small and large adipose cells in mice fed a high-fat diet did not differ in various measures of insulin resistance.30
Immune-related genes were reported to be upregulated in large versus small human adipocytes.31
Results from another study were contrary to ours, namely, that pro-inflammatory factors correlated with increasing adipocyte size.32
Among studies that did not specifically separate or characterize small and/or large cells, several described positive relationships between adipose cell enlargement and insulin resistance.33, 34
On the other hand, Pasarica et al35
reported that patients with type 2 diabetes had an increased proportion of small adipose cells in the subcutaneous adipose tissue depot as compared with BMI-matched, non-diabetic individuals. It is also important to discuss briefly our recent findings that treatment with pioglitazone led to recruitment of small adipocytes in human abdominal subcutaneous adipose tissue, as well as redistribution of fat from visceral to subcutaneous depots.36
While these findings may appear to conflict with that of the present study, we have postulated that small adipocytes that accumulate in response to the adipogenic effect of thiazolidinedione therapy36
may differ from those found in insulin resistant, untreated individuals,11
or as in the present case, the hyper-insulinemic, ZO rat. Functional studies could help to elicit whether these two small adipose cell populations differ in their capacity for lipid storage.
The strengths of our approach lie in the use of updated cell size techniques to accurately assess adipose tissue cellularity, and separation of small adipocytes for characterization and comparison. Adipocyte isolation eliminated the potential confounding effects of macrophages on inflammatory changes in the fat depot. A limitation of our study is its cross-sectional design, which precludes assumptions on causality. In addition, comparison of total adipose cells with small cells in the ZO rats may have underestimated the true effect size, given that small cells contribute substantially to the total adipose cell population. Nonetheless, that we were able to elicit the reported gene expression differences suggest that these findings are real. It is also possible that gene and protein expression levels in the adipose cells may vary. Finally, while ZO rats are useful animal models of insulin resistance, these findings may be strain-specific. We recently reported that recruitment of small adipose cells was greater in an obesity-prone mouse as compared with an obesity-resistant mouse strain, an effect enhanced under high fat feeding conditions.37
That we have been able to document an expanded proportion of small adipose cells using two different rodent models of obesity helps to validate these findings. It would be important to perform future investigations in human subjects, as well as conduct functional studies in the small adipose cells to support the genetic characteristics found in the present study.
We had postulated based on our studies in humans that small, rather than large adipose cells, were closely associated with insulin resistance. Our results in ZO rats have shown that small adipocytes manifest evidence of impaired adipogenesis and increased inflammatory activity, two mechanisms by which small adipocytes may contribute to whole-body insulin resistance in the ZO rat. These small adipocytes in the ZO rat appear to be distinct from those of the ZL rat, underscoring the relevance of metabolic phenotype in evaluating small adipocyte functionality. These findings provide novel insight into the role small adipocytes may have in the development of insulin resistance.