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3T3-L1
3T3-L1 is a preadipocyte cell line that is commonly used in cell biology research to study adipogenesis, adipokine signaling, and the cellular mechanisms of obesity. The 3T3-L1 cell line was selected for from mouse embryonic 3T3 cells for their intrinsic ability to synthesize and store triglyceride fat. Like 3T3 cells, 3T3-L1 cells originally have fibroblastic morphology but differentiate in culture to take on adipocytic characteristics making them ideal candidates for research on adipose tissue.

Ever since the 3T3-L1 cell line was solidified in the mid-1970s, using these cells as an in-vitro model has become a staple in all forms of adipose research. Although the majority of the research done with this cell line uses 3T3-L1 as a mode for studying adipocyte metabolism, a substantial amount of literature has been published regarding the characterization of the cell line itself as well as perfecting the differentiation process from fibroblasts to adipocytes.

History
The derivation of the 3T3-L1 cell line was first recorded by name in 1974 by Howard Green and Mark Meuth when they published “An Established Pre-adipose Cell Line and its Differentiation in Culture” in the journal Cell. The selection of this cell line came nearly a decade after Howard Green and George Todaro isolated the parent cell line, 3T3, from a Swiss mouse embryo. Among working with the original 3T3 cell line, Green and colleagues realized that certain 3T3 clones had a predisposition to accumulate lipid droplets when the cells reached a resting state in culture. Observing this characteristic, the researchers began selecting and isolating the cells that seemed to display an adipocyte-like phenotype; after repeating this process with multiple subclones, Green's laboratory arrived at the 3T3-L1 cell line. After the cell line was isolated and stabilized, it has been used in extensive amounts of research and is now commercially available from multiple biological research companies such as American Type Culture Collection (ATCC), Sigma-Aldrich, and Zen-Bio.

Differentiation
Although the 3T3-L1 was established and characterized for its ability to spontaneously differentiate from fibroblasts to adipocytes, methods on how to do this most effectively have been perfected over the course of decades. When Green’s lab first isolated the cell line, it was noted that the differentiation process would occur in basal medium soon after the cells reached confluency in culture but not all the cells in the culture would differentiate. They also found that bromodeoxyuridine would arrest the differentiation process in cells that were grown to confluency without affecting collagen synthesis. In the years following the origin of the cell line, Green began testing the effects of lipolytic and lipogenic hormones on the differentiation process and found that many of them increased the differentiation yield, most notably insulin.

Testing of various chemicals as potential differentiating agents continued after Green and a standard differentiation protocol for 3T3-L1 cells was soon developed. Fibroblast cells reach confluency in approximately two days in basal growth medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1.5g/L of sodium bicarbonate, 10% bovine calf serum, and antibiotics. The basal medium is then replaced with a cocktail of agents inducing adipocyte differentiation:  insulin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone. 1 μg/mL of insulin is typically added to induce glucose uptake through increased GLUT1 activity. 0.5mM of 3-isobutyl-1-methylxanthine (IBMX) is used to stimulate the cAMP-dependent protein kinase pathway which is assumed to regulate a variety of genes relating to lipolysis and thermogenesis. While 0.25μM of dexamethasone is added as a glucocorticoid receptor pathway stimulant which also activates a variety of genes related to adipocyte metabolism. Despite preadipocytes only being exposed to these agents for around 48 hours, maximum differentiation is not observed until up to two weeks after. Differentiation is typically observed using Oil Red O staining due to it's ability to bind to lipids within the cells.

Although the three chemicals mentioned above are the standard protocol for adipocyte differentiation, complete differentiation of 3T3-L1 fibroblasts is not always observed and its efficacy significantly decreases with increased passaging. As a result, various research has suggested alterations to the standard protocol by either supplementation with other agents or altering part of the current model. For example, the addition of rosiglitazone, a PPARγ agonist, at a 2μM concentration yields near complete adipocyte differentiation. Additionally, replacing IBMX with troglitazone, an insulin sensitizing agent similar to rosiglitazone, yields adipocyte differentiation in fewer days.

Characterization
Despite the 3T3-L1 cell line being used as a staple for studying adipocytes for nearly half a century, debate regarding the exact type of adipocytes 3T3-L1 cells represent when differentiated is still being had. In developing the cell line, Green and Meuth said little regarding the classification of 3T3-L1 other than that the cells looked as if they represented brown adipocytes or developing white adipocytes. This observation by the founders of the cell line was based solely on the morphology of the multilocular lipid droplets present in the cells. While white adipocytes are known for their large unilocular lipid droplet, brown adipocytes are characterized by having multiple, smaller lipid droplets in the cytosol as the originators observed in the differentiated 3T3-L1 cells.

Decades after the founders’ observations, the discussion surrounding the characterization of the cell line has gotten heavier. Multiple studies have been published questioning and debating reasons as to why differentiated 3T3-L1 cells represent more white or brown adipocytic characteristics based on morphology, genomics, and metabolic processes. When looking at the genome and bioenergetics of 3T3-L1 cells that were differentiated using the standard protocol, it seems as if they represent white adipocytes more so than brown adipocytes. However, arguments regarding the proteins involved in the uptake of glucose by adipocytes say otherwise. All types of adipocytes absorb glucose in similar fashion:  insulin binds to receptor tyrosine kinases (RTK) triggering the phosphatidylinositol 3-kinase (PI3K)/Akt pathway which mediates the transcription and exocytosis of GLUT4 to the plasma membrane, GLUT4 then allows glucose to flow into the cell for storage and energy purposes. In the case of differing adipocyte phenotypes, special attention has been placed on PI3K/Akt pathway’s reliance on a specific Ras GTPase, RalA. In brown adipocytes, but not white adipocytes, glucose uptake is dependent upon RalA signaling in the PI3K/Akt pathway. Evidence suggests that differentiated 3T3-L1 cells are also dependent upon RalA as well, supporting Green’s original observation that the cell line most resembles brown adipocytes.

Browning
Despite the opposing viewpoints, all research attempting to classify 3T3-L1 adipocytes have pointed to one conclusion:  the line between brown and white adipocytes is not as thick as it once seemed. In-vivo, white and brown adipocytes are derived from two separate progenitor cell lineages, myogenic factor 5 (Myf5) negative and positive respectively. Until research done with adipose cell lines, such as 3T3-L1, it seemed as if Myf5 determined the fate of adipocytes. Nevertheless, 3T3-L1 research has indicated that adipocytes may have more plasticity. Depending on the culture conditions, 3T3-L1 cells can acquire more brown adipocyte-like metabolism through a process called browning. This browning process mainly deals with the up-regulation of certain proteins that are observed in higher levels in brown adipocytes such as uncoupled protein receptor 1 (UCP1) and PGC-1α. UCP1 is an inner mitochondrial membrane protein that can serve as a proton uniporter and symporter, while PGC-1α acts as a coactivator for a variety of transcription factors which activate genes involved in brown adipocyte metabolism (notably the PPAR family of genes). Proteins like these, especially UCP1, allow adipocytes to use a proton gradient and fatty acids to produce heat through adaptive thermogenesis. Though adaptive thermogenesis is a process that typically only takes place in brown adipocytes, the discovered ability for white-like adipocytes to be induced into adaptive thermogenesis gave rise to another type of adipocyte, beige adipocytes. Being seen as a hybrid of white and brown adipocytes, beige adipocytes have multilocular lipid droplets, can participate in adaptive thermogenesis, but store more energy in the form of triacylglycerol than purely brown adipose cells.

Research Uses
The vast majority of research done with 3T3-L1 cells relate to adipocyte metabolism with respect to obesity such as the effects of environmental conditions on insulin resistance. Given that obesity primarily arises from excess white adipose tissue (WAT) and differentiated 3T3-L1 cells embody many characteristics of white adipocytes, they provide a great medium to study the metabolic complications of the disease in vitro. While embodying many WAT qualities, the ability for differentiated 3T3-L1 cells to acquire more thermogenic qualities under certain conditions has suggested the possibility of this phenomenon being able to occur in-vivo as well. Being able to induce browning in human WAT would yield a possible therapeutic mechanism for those suffering from obesity as thermogenic adipocytes dissipate more so than store fat. Thus, many studies involving 3T3-L1 cells involve exposing the differentiated cell line to ingestible chemicals found in foods to observe the effect on their metabolic state and if browning is induced. Studies of this sort have suggested compounds found in a variety of foods, from strawberries to green tea, can induce the browning phenomenon in 3T3-L1 cells.

An additional use of 3T3-L1 cells is the study of adipokine signaling and its effect on the immune system, specifically with regards to the low-grade inflammation that is typically observed in obesity. Models using 3T3-L1 paired with macrophage cell lines, such as RAW 264.7, have allowed researchers to study the inflammatory effects of adipocyte signaling in-vitro.