Proliferation and differentiation are two important aspects in fat tissue development. In our experiment, we have shown that berberine has a growth-factor-like role on human preadipocytes, promoting cell proliferation. Our result is consistent with previous studies showing that berberine can promote the proliferation of mouse 3T3-L1 preadipocyte [22
]. Adult obesity is mainly due to the increased volume of fat cells which are abnormally hypertrophic [23
]. Our in vitro
experiments showed that berberine significantly inhibited the omental preadipocytes to become mature adipocytes judging by their morphology or lipid-specific Oil-Red-O staining. Therefore, berberine has potential clinical application in reducing visceral fat and controlling central obesity. It has been reported that fat tissue composed of a higher amount of small fat cells is more sensitive to insulin compared with fat tissue with the same lipid content composed of a small number of large fat cells, and also the former has very little inflammatory responses [24
]. Our results showed that berberine can promote human fat cell proliferation and inhibit fat cell enlargement, indicating that it may be able to reduce inflammation responses, improve insulin sensitivity of visceral adipose tissue, and reduce or eliminate the visceral adipose tissue. Moreover, our in vivo
study also showed that, after taking berberine for three month, patients with metabolic syndrome were found to reduce their waist circumferences and BMI to varying degrees. This positive result therefore seems in good agreement with the in vitro
The nuclear receptor PPARγ
and members of the C/EBP family take important roles in adipogenesis [25
], and the major players are PPARγ
] and C/EBPα
]. Many studies have showed that berberine inhibited the mRNA and protein levels of adipogenesis-related transcription factors PPARγ
2 and C/EBPα
]. We studied the effect and transcriptional impact of berberine on human preadipocyte differentiation. Our result showed that the berberine can inhibit PPARγ
2 and C/EBPα
mRNA expression simultaneously during the human preadipocyte differentiation process. Recently, the transcription factors GATA binding protein 2 and 3 (GATA-2 and GATA-3) have been shown to be important gate keepers of the differentiation process [30
]. Studies from HU et al. showed that berberine increases expression of GATA-2 and GATA-3 during inhibition of adipocyte differentiation in both murine cell lines 3T3-L1 and human white preadipocytes cell line. But contradictorily, they also found that the differentiation inhibition mechanisms of berberine appeared to be independent of PPARγ
2 and C/EBPα
in that human white preadipocyte cell line while being dependent on decreasing of PPARγ
2 and C/EBPα
gene expression in 3T3-L1 lines [32
]. Those results also seem to contrast with our findings from human primarily cultured preadipocytes. Since there is still lack of well-characterized human preadipocyte cell lines, further studies in human primarily cultures are particularly needed to clarify the results from those cell lines.
Lipoprotein lipase is a kind of glycoprotein synthesized and secreted by fat cells. Current understandings of lipoprotein lipase's physiological functions are to decompose chylomicrons and triglycerides into very low-density lipoproteins and to promote the lipoprotein transfer between triglyceride phospholipids and apolipoproteins and so forth. We found that berberine reduced lipoprotein lipase mRNA expression in human fat cells which is consistent with the recent study by Choi et al. showing that berberine reduces mouse 3T3-L1 lipoprotein lipase mRNA expression [9
]. As discussed by Kong et al. [34
], oral administration of berberine in 32 hypercholesterolemia patients for 3 months reduced serum cholesterol by 29%, triglycerides by 35%, and LDL cholesterol by 25%. Our clinical study of the effects of berberine on total cholesterol, triglycerides, and LDL cholesterol along with analysis of liver and kidney adverse reactions also indicates that berberine could be a cheap, efficient, and safe lipid-lowering drug in metabolic syndrome patients.
Leptin and adiponectin have been shown to play an important role in insulin resistance. During the process of preadipocytes differentiation, secretion of leptin and adiponectin showed different kinetics: in the first phase low levels of leptin secretion can be detected in fat cells, but no adiponectin secretion can be detected. The amount of leptin secretion continued to increase in a synchronized fashion with the differentiation process, while the secretion of adiponectin was only observed when preadipocytes gradually mature and their cytoplasm began to be filled with lipid droplets. This result suggests that only mature fat cells can secrete adiponectin which can be used as a specific marker to determine the maturity of fat cells. In the midstages of differentiation, secretion of leptin and adiponectin increased with an increased number of fat cells; however, the rate of increase in adiponectin was more obvious. In the late stages of differentiation (at day 17–21), a morphological change of the cells showed that the majority of cells differentiated into fat cells with their cytoplasm filled with large lipid droplets. Oil-Red-O staining showed that lipid content in the cytoplasm remained at a steady high level. At this point, leptin secretion remained at high levels, while adiponectin secretion was seen to show a clearly downward trend. This difference suggests that fat cells in different fat-storing states secrete leptin and adiponectin differently, which may also reflect on their functional differences. The differentiation stages of fat cells from clinically obese patients may be different from normal people. In obese patients, the fat cells may show a high-leptin and low-adiponectin secretion pattern. In our differentiation experiment, berberine not only inhibited the differentiation and maturation of preadipocytes but also suppressed leptin and adiponectin secretion. This suggests to us that, in the visceral adipose tissue accumulation process, berberine has a role in endocrine function regulation and can promote the reversion of the initial process of fat storing. Our clinical observation showed that in patients with newly diagnosed metabolic syndrome the level of leptin dropped significantly with berberine treatment after 3 months. This is different from results shown in rat studies and may reflect species differences. This may suggest that berberine has the potential for anticentral obesity and regulating obesity-related endocrine dysfunctions, thus achieving a balance between fat cell factors. Studies have reported that the ratio of leptin and adiponectin reflects the body's insulin resistance [35
]. We have confirmed that berberine reduces HOMA-IR and the ratio of leptin and adiponectin. As it inhibits PPARγ
2 mRNA expression and has more effects on weight loss and reducing leptin levels, berberine regulates insulin sensitivity with a mechanism different from the insulin sensitizer, thiazolidinediones. Thus, berberine provides an additional way for clinical treatment of metabolic syndrome and obesity-related diseases. However, our experiment is only a preliminary study on the mechanisms of effects of berberine on serum adipokines. A large-scale clinical study is ideally required to include different population groups and more experiments concerning the mechanism details.
In conclusion, in order to explore the mechanism of berberine's role in improving insulin sensitivity, we used human adipose tissue as material, focusing on the proliferation, differentiation, and adipokine secretion of human preadipocytes. We tried to find clues from the in vitro
experiments and then verified them in clinical trial. Our clinical study has some limitations relative to the randomized, placebo-controlled clinical design. However, our result is in agreement with the findings from previous large sample, well-designed clinical studies [23
], indicating that berberine improves glucose and lipid metabolism disorders. More particularly, we find that berberine can improve insulin sensitivity by adjusting adipokine secretion both in primarily cultured preadipocytes as well as in metabolic syndrome patients, and this was not well characterized in previous human studies.