3.1 Effects of piperine on body weight, mesenteric fat accumulation, dietary intake and Lee’s index
To explore the effect of piperine on the established obesity, the body weight, mesenteric fat accumulation, Lee’s index, glycolipid metabolism and insulin sensitivity were assessed in MSG-obese insulin resistant mice upon piperine treatments. The results show that MSG caused more mesenteric fat accumulation and body weight gain in MSG-obese insulin resistant mice (Model: 70.2 ± 2.54) than normal mice (Normal 48.09 ±1.43, P<0.0001). In contrast, the piperine treatment relieved mesenteric fat accumulation and body weight gain (Fig. 1A-B). It was found that from the 4th week onwards, the body weight of the piperine-treated mice began to decrease and eventually reached 53.0 ± 2.88g, significantly different from that of the MSG-obese mice (70.2 ± 2.54g, P<0.001). Additionally, the body weight of metformin-treated mice declined to 58.6 ± 4.18 g. The effect of a 40 mg/kg B.W dose of piperine was better than metformin. No significant differences of daily food intake and Lee’s index were observed between the model and piperine-treated groups, metformin also has no significant effect on these two indicators (Fig. 1C-D).
3.2 Effects of piperine on visceral organ indexes
The relative weights of the abdominal fat, pancreas, liver, and kidney were calculated after the mice were sacrificed. Compared to the normal group (visceral index of abdominal adipose: 2.58 ± 0.24%; Pancreas index: 0.566 ± 0.016%; Kidney index:1.242 ± 0.041%; Liver index: 4.198 ± 0.095%), the results show that the MSG-obese mice had a higher visceral index of abdominal adipose(8.11 ± 0.56%) and lower pancreas(0.282 ± 0.0126%), kidney (0.724 ± 0.028%), liver index (2.709 ± 0.130%), the differences between groups were significant (P < 0.001). As expected, the piperine treatment completely reduced the visceral index of the abdominal adipose(6.273 ± 0.606%, P<0.05) (Fig. 2A) and increased the visceral index of the pancreas(0.3393 ± 0.014%, P<0.05) in the MSG obese mice (Fig. 2B). The data suggest that piperine may protect the pancreas to a certain extent and restore the relative weight of the pancreas. Besides, we found that the piperine treatment did not change the relative weight of kidney (Fig. 2C) and liver (Fig. 2D) (P>0.05). We also found that metformin reduced index of the abdominal adipose (6.149 ± 0.358%, P<0.05) in MSG mice, the effect was quantitatively similar to piperine. Furthermore, metformin significantly increased liver index(3.13 ± 0.12%, P<0.05), but there was no changes on pancreas and kidney indices (P>0.05).
3.3 Effects of piperine on the changes of glycolipid metabolism parameters
Obesity is a main factor causing glycolipid metabolism disorders. The regulatory effect of piperine on glycolipid metabolism was examined in this section. As assumed, the obese mice had higher levels of FBG (Model: 6.45 ± 0.409; Normal: 4.32 ± 0.276, P<0.01), serum TC (Model: 5.663 ± 0.657; Normal:3.668 ± 0.197, P<0.01) and TG (Model: 1.407 ± 0.082; Normal: 1.117 ± 0.047, P<0.05). In contrast, the piperine treatment dramatically reduced the FBG (4.717 ± 0.441, 27.0% reduction compared with the model mice, P<0.01), serum TC (3.554 ± 0.299, 37.3% reduction, P<0.01) and TG levels (0.935 ± 0.0534, 33.6% reduction, P<0.001) (Fig. 3A-C). Additionally, the MSG mice showed obvious hyperinsulinemia (Model:4.498 ± 0.865; Normal: 1.276 ± 0.172, P<0.01). Piperine administration also had a certain relieving effect on the serum insulin level (3.373 ± 0.934). However, it is not statistically different (Fig. 3D) (P>0.05). Metformin also significantly lowered FBG (4.488 ± 0.292, 30.5% reduction, P<0.01), TC (3.747 ± 0.280, 33.9% reduction, P<0.01), and TG (1.091 ± 0.063, 22.1% reduction, P<0.01). The results suggest that the effect of piperine on glucose metabolism is similar to metformin, but piperine may have a better effect on lipid metabolism compared with metformin.
3.4 Effects of piperine on oral glucose tolerance test 、hyperglycemic clamp experiment and insulin tolerance test
The effects of piperine on insulin sensitivity was evaluated using the insulin tolerance test (ITT). Oral glucose tolerance test (OGTT) and hyperglycemic clamp experiment were used to assess the effects of piperine on glucose tolerance. Administration of MSG led to significant insulin resistance and glucose intolerance in the ICR mice, which were markedly attenuated in the piperine-treated MSG mice.
Glucose tolerance results are summarized in Fig. 4A. The results show that the glucose level in the MSG mice significantly increased in the first 30 min after glucose load (9.91 ± 0.63 mM vs. 12.4 ± 1.46 mM), however, the glucose levels were 25% lower in the piperine treated mice compared with that of the control mice (9.28 ± 0.65 mM vs. 12.4 ± 1.46 mM). The results also showed that the integrated glucose level was greatly lowered in the piperine and metformin treated mice compared with that of the control mice (Fig. 4B). The glucose level in the MSG mice could be completely recovered by piperine at 40 mg/kg B.W compared with metformin, the effect of piperine was similar.
The result of hyperglycemic clamp experiment showed that the GIR of MSG mice was significantly lower than that of the normal group (Model: 6.564 ± 0.391; Normal: 17.49 ± 0.505, P<0.0001), indicating that there was significant glucose intolerance in MSG-obese mice. After 10 weeks of administration, compared with the model group, both piperine and metformin treatment increased the GIR of MSG mice by 45.1% (11.95 ± 0.486, P<0.0001) and 57.6% (15.49 ± 0.408, P<0.0001), respectively, suggesting that piperine is beneficial to improve the sensitivity of islet β cells to glucose stimulation in obese mice, that is, it can improve the function of islet β cells（Fig. 4C）.
The ITT results show that after 40 min of insulin injection, blood glucose in the piperine group decreased by 33.02%, which is significantly higher than that in the MSG group (15.26%), indicating that the piperine treatment improved systemic insulin sensitivity in the MSG-obese mice (Fig. 4D-E).
3.5 Effects of piperine on pathological changes in the abdominal adipose and liver
Heavy accumulation of fat in the liver was observed by histomorphological analysis, indicating there were severe pathological changes of nonalcoholic fatty live disease (NAFLD) in the MSG mice. The livers of the mice in the model group showed heavy hepatic steatosis, whereas, in the MSG mice, the steatosis was partially relieved by the piperine treatment (Fig. 5A). In some obese patients, insulin resistance occurs due to the accumulation of “dysfunctional” adipose tissues, which are characterized by “large” lipid-laden adipocytes. Our results show that the adipocyte size was greatly increased by MSG, while the hypertrophic adipocyte was ameliorated by piperine treatment. These effects of 40 mg/kg B.W of piperine were better than those observed with the metformin group (Fig. 5B-C). These data indicate that piperine plays a vital role on regulating lipid metabolism in the abdominal adipose and liver, both are the main targets of insulin.
3.6 Effect of piperine on improving systemic inflammation
The routine blood test results showed that WBC, Lymphocyte, and Monocyte in the piperine-treated group were significantly lower than the mice in the model group (Table 2). Besides, we found that serum pro-inflammatory cytokines such as LPS ( Model: 413.2 ± 19.59; Normal: 250.2 ± 12.16, P<0.001) , IL-1β ( Model: 28.78 ± 0.495; Normal: 15.24 ± 1.259, P<0.01) and Gal-3 ( Model: 2.527 ± 0.070; Normal: 1.115 ± 0.058, P<0.0001) were elevated in the MSG mice compared with the normal mice. At the end of the 10-week period, the serum level of LPS ( 311.2 ± 11.01, P<0.001), IL-1β ( 22.62 ± 0.877, P<0.01) and Gal-3 ( 1.479 ± 0.068, P<0.0001) were significantly reduced in the piperine-treated mice compared with that in the control mice (Fig. 6A-C). Additionally, although the serum anti-inflammatory cytokine IL-10 in the model mice ( 20.6 ± 0.782) was lower than that in the normal mice ( 29.61 ± 1.345, P<0.01) , the administration of piperine did not restore this indicator (19.6 ± 1.286, P>0.05) (Fig. 6D). These effects of piperine were similar to those observed with the metformin group, so above results demonstrated that the piperine treatment suppressed the obesity-enhanced inflammatory responses in obese mice.
3.7 Effects of piperine on inflammatory mediator gene and protein expressions in the adipose tissue
In order to detect the inflammatory status of adipose tissue in each group of mice, we examined the expression of M1-like macrophage marker CD11c and related inflammatory cytokines at the mRNA level. qRT-PCR showed that the mRNA level of CD11c, IL-1β, Gal-3 and TNF-a were significantly increased in the adipose tissue in MSG obese mice. In contrast, these genes were markedly decreased in the piperine-treated group (Fig. 7A-D). The results also demonstrated that significant reductions in IL-1β and Gal-3 were observed in the metformin-treated group compared to the model group.
We simultaneously measured the protein expression of CD11c and Gal-3. Immunohistochemistry results showed that both CD11c and Gal-3 were over-expressed in the adipose tissue of the MSG group. Piperine treatment reduced the level of both key proteins, CD11c and Gal-3, in the adipose tissue (Fig. 8A-D). Together, these results indicated that piperine alleviated obesity enhanced M1-like macrophage polarization and the secretion of pro-inflammatory cytokines in the abdominal adipose tissue, which is consistent with serum pro-inflammatory cytokine levels. In fact, M1-like macrophage polarization in visceral adipose tissue is the source of systemic inflammation.
3.8 Effect of piperine on in vitro macrophage polarization
The inhibitory effect of piperine on M1 macrophage polarization was evaluated using an inflammatory cell culture model. As expected, LPS treatment increased mRNA expressions of TNF-α, IL-1β and M1 marker CD11c, while piperine inhibits LPS-induced TNF-α, IL-1β and CD11c expression in a concentration-dependent manner in RAW 264.7 cells (Fig. 9A-C). We also found the LPS-stimulated IL-1β production was inhibited by piperine and also their combination (Fig. 9D). Furthermore, we examined the TLR-4, CD11c and IL-1β with a Western blot analysis. The results showed that piperine (20, 40, and 80μM) inhibited the expression of TLR-4, IL-1β, and CD11c in the RAW264.7 cells after LPS treatment (Fig. 10A-B).