Obese gene expression at in vivo levels by fat pads derived from s.c. implanted 3T3-F442A preadipocytes

Abstract

3T3-F442A preadipocytes implanted s.c. into athymic mice develop into fat pads that are indistinguishable from normal adipose tissue. Implanted preadipocytes harboring a beta-galactosidase transgene gave rise to fat pads in which almost all adipocytes expressed beta-galactosidase. This finding proved that the implanted 3T3-F442A preadipocytes, rather than endogenous preadipose cells, gave rise to the newly developed "adipose tissue." 3T3-F442A preadipocytes, when differentiated into adipocytes in cell culture, express the obese gene at an unexpectedly low level, i.e., </=1% the level in adipose tissue. However, adipose tissue derived from s.c. implanted 3T3-F442A preadipocytes expressed leptin mRNA at a level comparable to that in epididymal adipose tissue. These findings indicate that a factor(s) or condition, present in the tissue context and necessary for maximal obese gene expression, is lacking in cell culture. Furthermore, adipocytes derived from the implanted cells were hormonally responsive in that leptin mRNA levels were up-regulated 3- to 8-fold by glucocorticoid injection into the host animal. Thus, these findings indicate that adipose-specific promoter-reporter constructs, transfected into 3T3-F442A preadipocytes, can be tested in an in vivo context during and after development of these cells into adipose tissue. Furthermore, the effect of transgenes on the adipogenic development of the implanted preadipocytes can be assessed. Thus, this approach offers a faster and less costly alternative to the transgenic mouse method for assessing adipose gene function.

Figure 1

Development of fat pads from s.c. implanted 3T3-F442A preadipocytes. 3T3-F442A preadipocytes (3 × 10⁷ cells) harboring a β-galactosidase expression vector were harvested at near confluence and injected s.c. at the sternum of outbred Crl athymic mice. (A) A typical fat pad that developed at the site of injection 4 weeks after implantation. (B) Fat pads that developed at the site of implantation (Right) and epididymal fat pads (Left) were excised, fixed in buffered formalin, and immmunohistochemically stained for β-galactosidase with eosin as a counterstain.

Figure 2

Time course of adipose development from s.c. implanted 3T3-F442A preadipocytes. Outbred Crl athymic mice were injected s.c. with 3 × 10⁷ 3T3-F442A preadipocytes. Fat pads that developed from the implanted preadipocytes were excised, fixed, sectioned, and stained with hematoxylin and eosin at 1 (A), 2 (B), 3 (C), 5 (D), 8 (E), and 10 weeks (F) after implantation. (G) Epididymal adipose tissue from a mouse harboring implanted 3T3-F442A preadipocytes for 6 weeks was excised and treated in an identical manner.

Figure 3

Expression of leptin mRNA and other adipose-specific mRNAs during adipose development from s.c. implanted 3T3-F442A preadipocytes. The level of expression leptin mRNA and mRNAs encoding other adipocyte markers was determined by Northern blot analysis of total RNA isolated from fat pads derived from the implanted preadipocytes described in Fig. 2. (A) Northern blots of RNA isolated from adipose tissue derived from implanted 3T3-F442A preadipocytes, from epididymal white adipose tissue (eWAT), and from in vitro differentiated 3T3-F442A adipocytes. Approximately 10 μg of total RNA was loaded per lane. The blot was hybridized to a ³²P-labeled probe for leptin and then stripped and reused for hybridization to probes for C/EBPα, SCD1, 422/aP2, and 28S rRNA. (B) Plots of the results in A showing the relative levels of each transcript expressed as percentage of the level of transcript in RNA from epididymal adipose tissue. Hybridization signals on Northern blots (shown in A) were quantified using a phosphorimager and were normalized to the corresponding signals from the rRNA. Transcript levels are expressed as percentage of the mean transcript level of eWAT. Results are represented as the mean ± range (n = 2).

Figure 4

Hormonal responsiveness of the obese gene in adipose tissue derived from s.c. implanted 3T3-F442A preadipocytes and from epididymal fat pads. (A) Six weeks after the implantation of 3T3-F442A preadipocytes, four outbred Crl:NU/NU-nuBR athymic mice were injected i.p., two with dexamethasone [3.7 μg/g (body weight)] and two with vehicle. Similarly, four inbred BALB/cAnNCrl-nuBR athymic mice were injected with dexamethasone or vehicle. Six hours later, RNA was isolated from epididymal fat pads and fat pads derived from implanted 3T3-F442A preadipocytes and used for Northern blotting. Blots were analyzed as indicated in Fig. 3. (B) Plots showing the levels of expression of leptin mRNA quantitated from the results in A and from the the Northern blot of the RNA from the BALB/c mice (not shown). Transcript levels were normalized to the level of the adipose-specific 422/aP2 transcript and are expressed as percentage of the mean level in eWAT of untreated mice of the same strain.