Adipogenesis & Oil Red O staining
Figure 1. 3T3-L1 cells are seeded into the plate (D-2) and wait until full confluency(D-0). BCS media is changed into DMI media(D-0) in 48 to72 hours (D-3). Media is changed into FI media for 48hours(D-5) after that change normal FBS media for 48hours(D-7).
Figure 2. Schematic figures of Oil Red O staining of differentiated 3T3-L1 adipocyte. Lipid accumulations in DMI treated mature adipocytes were clear compared to the undifferentiated cells, suggesting Oil Red O staining is reasonable method to quantify lipid accumulation with colorimetry.
Figure 3. Oil red O stained 3T3-L1 adipocyte in 6-well plates
We validated our proof of concept by inducing lipid accumulation in 3T3-L1 adipocytes using a well-established chemical differentiation cocktail. Following differentiation, intracellular lipids were visualized through Oil Red O staining, and cell morphology was captured via light microscopy. To quantify lipid content, the bound dye was extracted with isopropanol and absorbance was measured at 518 nm using a microplate reader in triplicate.
Measurement of DBTL1(Raw Bacteria)
Figure 4. Co-culture of mature adipocytes and Lactobacillus delbrueckii subsp. Lactis shows about 25% reduction in lipid accumulation and the difference between the coculture groups and the negative control were statistically significant. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001). However, degree of lipid reduction was not MOI dependent.
Lactobacillus lactis shows 25% inhibition of lipid accumulation.
Figure 5. Co-culture of mature adipocytes and Lactobacillus casei shows about 25% reduction in lipid accumulation. The difference between the co-culture groups and the negative control were statistically significant except the MOI 1 sample. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus casei shows 25% inhibition of lipid accumulation.
Figure 6. Co-culture of mature adipocytes and Lactobacillus crispatus shows about 30% reduction in lipid accumulation. The difference between the co-culture groups and the negative control were statistically significant, with MOI 100 being most efficient in lowering lipid accumulation. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus crispatus shows 30% inhibition of lipid accumulation.
Figure 7. Co-culture of mature adipocytes and Lactobacillus acidophilus was not effective in reducing lipid accumulation. The difference between the co-culture groups and the negative control were not statistically significant. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus acidophilus shows no inhibition of lipid accumulation.
Figure 8. Co-culture of mature adipocytes and Lactobacillus rhamnosus shows about 30% reduction in lipid accumulation. The difference between the co-culture groups and the negative control were statistically significant, with MOI 100 being most efficient in lowering lipid accumulation. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus rhamnosus shows 30% inhibition of lipid accumulation.
Figure 9. Co-culture of mature adipocytes and Lactobacillus gaseri shows about 25% reduction in lipid accumulation. The difference between the co-culture groups and the negative control were statistically significant, with MOI 100 being most efficient in lowering lipid accumulation. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus gasseri shows 25% inhibition of lipid accumulation.
Figure 10. Most Lactobacillus sub species except Lactobacillus acidophilus shows inhibition in lipid accumulation when co-cultured with DMI induced mature adipocytes. These in vitro data suggest that Lactobacillus sub-species are potential research targets for obesity treatments.
Most of Lactobacillus spp. shows inhibition effect to lipid accumulation. Our in vitro results reflect the facts that Lactobacillus spp. could regulate fat accumulation.
Measurement of DBTL2(Supernatant)
Figure 11. Treatment Lactobacillus delbrueckii subsp. Lactis supernatant on mature adipocytes shows no reduction in lipid accumulation, different from the co-culture experiment which resulted in reduced lipid content. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus lactis supernatant shows no inhibition of lipid accumulation.
Figure 12. Treatment Lactobacillus casei supernatant on mature adipocytes shows no reduction in lipid accumulation, different from the co-culture experiment which resulted in reduced lipid content. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus casei supernatant shows no inhibition of lipid accumulation.
Figure 13. Treatment of mature adipocytes with Lactobacillus casei supernatant resulted in a significant 50% increase in lipid accumulation, in contrast to the co-culture experiment which resulted in reduced lipid content. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus crispatus shows 50% increase of lipid accumulation.
Figure 14. Treatment of mature adipocytes with Lactobacillus acidophilus supernatant resulted in a significant 25% increase in lipid accumulation. In the co-culture experiment of Lactobacillus acidophilus, there was no significant change in the lipid storage in the mature adipocytes. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus acidophilus shows 25% increase of lipid accumulation.
Figure 15. Treatment of mature adipocytes with Lactobacillus rhamnosus supernatant resulted in a significant 45% decrease in lipid accumulation, consistent with the coculture experiment. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus rhamnosus shows 45% inhibition of lipid accumulation.
Figure 16. Treatment of mature adipocytes with Lactobacillus gaseri supernatant shows no reduction in lipid accumulation, different from the co-culture experiment which resulted in reduced lipid content. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus gasseri shows no inhibition of lipid accumulation.
Figure 17. Lipid accumulation of Lactobacillus spp. supernatant
Only supernatant from Lactobacillus rhamnosus shows inhibition of lipid accumulation.
Other strains shows no inhibition or even increase of lipid accumulation.
Therefore, specifically extracellular materials from Lactobacillus rhamnosus contains the substance which regulate the fat accumulation rather than other species.
Measurement of DBTL3 (Exosome)
Figure 18. Treatment of mature adipocytes with exosomes isolated from Lactobacillus delbrueckii subsp. Lactis supernatant shows no reduction in lipid accumulation. In fact, 60% increase of lipid accumulation was observed in the high exosome concentration. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus lactis shows 60% increase of lipid accumulation.
Figure 19. Treatment of mature adipocytes with exosomes isolated from Lactobacillus casei supernatant shows no reduction in lipid accumulation. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus casei shows no inhibition of lipid accumulation.
Figure 20. Treatment of mature adipocytes with exosomes isolated from Lactobacillus crispatus supernatant resulted in increase of lipid accumulation about 30%. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus crispatus shows 30% increase of lipid accumulation.
Figure 21. Treatment of mature adipocytes with exosomes isolated from Lactobacillus acidophilus supernatant shows no reduction in lipid accumulation. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus acidophilus shows no inhibition of lipid accumulation.
Figure 22. Treatment of mature adipocytes with exosomes isolated from Lactobacillus rhamnosus supernatant resulted in a remarkable 80% reduction in lipid accumulation, which was greater than the 45% reduction observed with the supernatant treatment. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus rhamnosus shows 80% inhibition of lipid accumulation.
Figure 23. Treatment of mature adipocytes with exosomes isolated from Lactobacillus gaseri supernatant shows no reduction in lipid accumulation. (ns p≥0.05, * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001).
Lactobacillus gasseri shows no inhibition of lipid accumulation.
Figure 24. Exosomes isolated from Lactobacillus rhamnosus caused an 80% reduction in lipid accumulation, whereas exosomes from other Lactobacillus strains did not reduce lipid storage. These results suggest that only exosomes from L. rhamnosus may contain specific factors capable of regulating lipid accumulation and adipogenesis-related genes.
Exosome isolated from Lactobacillus rhamnosus shows 80% reduction of lipid accumulation rather than other strains.
Therefore, we assume that potential substance in exosome of Lactobacillus rhamnosus specifically regulate lipid accumulation and whether exosome controls adipogenesis genes.
Figure 25. mRNA expression levels measured by RT-qPCR represented that exosomes isolated from Lactobacillus rhamnosus supernatant downregulate the representative gene of adipogenesis (PPARγ and C/EBPα) by inducing AMPK genes
Exosome isolated from Lactobacillus rhamnosus supernatant downregulate the representative gene of adipogenesis(lipogenesis) by inducing AMPK genes.
Figure 26. Western blot analysis representing correlations between adipogenesis-related proteins and Lactobacillus rhamnosus exosome concentrations. The master regulators of adipogenesis (PPARγ and C/EBPα) were significantly inhibited in a concentrationdependent manner, while AMPK expression was upregulated. β-Actin was used as a housekeeping protein, and the similar β-Actin band intensities indicate that the observed differences in PPARγ, C/EBPα, and AMPK expression were not due to unequal protein loading in SDS-PAGE.
Exosome isolated from Lactobacillus rhamnosus supernatant downregulate the representative gene of adipogenesis(lipogenesis) by inducing AMPK genes in protein level.
Measurement of DBTL4 (hisF)
Figure 27. mRNA expression levels measured by RT-qPCR showed that engineered hisF proteins successfully inhibited the master regulators of adipogenesis and lipid accumulation (PPARγ and C/EBPα). At the same time, AMPK expression was upregulated in a concentration-dependent manner with increasing hisF.
Engineered hisF alone treating to adipocyte during adipogenesis lead to 50% inhibition of lipid accumulation.
Figure 28. mRNA expression levels measured by RT-qPCR showed that engineered hisF proteins successfully inhibited the master regulators of adipogenesis and lipid accumulation (PPARγ and C/EBPα). At the same time, AMPK expression was upregulated in a concentration-dependent manner with increasing hisF.
Engineered hisF alone inhibit the master regulator of adipogenesis genes by upregulating AMPK genes significantly.
Figure 29. Western blot analysis showing different levels of protein expression in engineered hisF-treated adipocytes compared with the negative control. The master regulators of adipogenesis (PPARγ and C/EBPα) were significantly inhibited, while AMPK expression was upregulated. β-Actin was used as a housekeeping protein, and the similar β-Actin band intensities indicate that the observed differences in PPARγ, C/EBPα, and AMPK expression were not due to unequal protein loading in SDS-PAGE.
Engineered hisF alone inhibit the master regulator of adipogenesis genes by upregulating AMPK genes significantly in protein level.
Conclusion
Our project provides a robust proof of concept that the hisF protein, isolated from the exosomes of Lactobacillus rhamnosus, effectively inhibits adipogenesis in vitro.
The main goal of our project was to move from a general observation that certain probiotics can influence body weight to identifying a specific, functional biomolecule responsible for this effect. We have successfully demonstrated this concept through a series of logical and systematic experiments.
Key attributions
- Specificity: We demonstrated that the anti-adipogenic effect is not a general property of all Lactobacillus species. Through our DBTL cycles, we narrowed down the effect from six strains to a single one: Lactobacillus rhamnosus.
- Mechanism Identification: We pinpointed the location of the active component to the exosomes secreted by L. rhamnosus.
- Molecular Validation: Through proteomic analysis, we identified a single protein, hisF, as the active agent.
- Functional Confirmation: Critically, we cloned, expressed, and purified the hisF protein. When this purified protein was applied to differentiating fat cells, it replicated the inhibitory effect observed in earlier stages, reducing lipid accumulation by up to 50%.
- Pathway Elucidation: We further demonstrated that the hisF protein functions by up-regulating the master metabolic regulator AMPK, which in turn down-regulates the key adipogenic transcription factors Ppary and C/ebpa.
This final experiment, using a single, purified, and synthetically produced protein to achieve the desired biological outcome, serves as a definitive proof of concept. It validates our initial hypothesis and establishes the hisF protein as a promising candidate for the development of new therapeutic agents to combat obesity.