## 4 Discussion
In this study, LPS was used to mimic the obesity-induced inflammation and insulin resistance in RAW 264.7 macrophages and 3T3-L1 adipocytes, respectively. LPS also stimulates the inflammation process via inducing IKK-NF\(\kappa\)B signaling through Toll-like receptors [3; 29]. In our study, when compared with untreated cells, LPS induced inflammation in RAW 264.7 macrophages by increasing the level of NO. Moreover, the CCA extract significantly inhibits the LPS-induced NO production in macrophages. Our findings confirm a previous study that described that the _C. carandas_ fruit juice down-regulated the induction of inflammatory inducible nitric oxide synthase and COX-2 in LPS-stimulated macrophages [12]. Our in vitro study utilized LPS to stimulate insulin resistance. As mentioned above, LPS activates the expression and secretion of pro-inflammatory cytokines, such as TNF-\(\alpha\), from either macrophages or adipocytes [3; 29]. These inflammatory cytokines stimulate lipolysis through the actions of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), which are responsible for the degradation of triglycerides into glycerol and free fatty acids [29]. It has been proven that LPS infusion causes increased lipolysis rates and insulin resistance [30]. The released FFAs and cytokines stimulate adipose tissue inflammation through IKK-NF\(\kappa\)B signaling and impair insulin action in insulin-sensitive cells. Impairment in the insulin signaling cascade leads to impaired GLUT4 translocation in skeletal muscles and adipose cells [29; 31]. The present study found that LPS induced insulin resistance in 3T3-L1 adipocytes by decreasing the cellular glucose uptake and increasing the free glycerol released content. This observation supports a previous study that LPS can block insulin signaling by disrupting the IRS-1 phosphorylation, resulting in the inhibition of the GLUT-4 translocation through the Akt pathway [29; 32]. Importantly, the treatment of the CCA extract significantly inhibited insulin resistance in the LPS-treated adipocytes, as it induced insulin to stimulate glucose uptake by approximately 20% and inhibit lipolysis by approximately 20%. These indicated that the CCA extract could improve insulin sensitivity in LPS-treated adipocytes. Rats with non-genetic type 2 diabetes induced by a combination of a high-fat diet followed by STZ injection are widely used in research because these rats show insulin resistance and hyperglycemia without hypoinsulina, which are the hallmarks of T2 DM [33]. The low dose of the STZ injection (35 mg/kg BW) in the high-fat-diet-fed rats caused the absence of hypoinsulina, which normally appears with hyperglycemia. This is shown by no differences in circulating insulin levels between diabetic and normal rats, consistent with the partial destruction of pancreatic \(\beta\)-cells. Therefore, the combination of a high-fat diet and low-dose STZ injection was used to induce T2 DM rats in this study. In the in vivo study, hyperglycemia, dyslipidemia, and insulin resistance were seen in rats that received an HFD plus STZ injection, suggesting that T2 DM was successfully established as in previous studies [33]. Antihyperglycemic benefits of CCA extract supplements were found in diabetic rats. The metabolic parameters indicated that a 12-week treatment of the CCA extract at 200 and 400 mg/kg BW successfully reduced the fasting plasma glucose and improved glucose tolerance, showing its antihyperglycemic benefit in both fasting and post-prandial states. Consistently, the antihyperglycemic property of _C. carandas_ unripe fruit was also demonstrated in previous reports using different diabetic models. This suggests that secondary metabolites, such as polyphenics present in _C. carandas_ fruit, may play an important role in its antidiabetic potential [8; 9]. Hyperglycemia in T2 DM results either from the inadequacy of insulin secretion by pancreatic \(\beta\)-cells or the inefficiency of cells to use insulin. Both conditions can affect glucose uptake or disposal by cells [34].