Recent studies show that caloric restriction (CR) modulates energy expenditure and body fat metabolism, it also regulates telomere length, which is associated with longevity in some experimental animals [1–3]. Other studies suggest that certain dietary components found in spices may exert a beneficial effect on metabolism. For example, curcumin (Cur, Fig. 1), the major polyphenol present in the spice turmeric, possess anti-obesity and anti-inflammatory properties [4–6]. Piperine (Pip, Fig. 1), a bioactive alkaloid in pepper, has been suggested to increase Cur’s absorption and bioavailability [7]. In addition, Cur and Pip have been suggested to attenuate inflammation [8, 9]. Our previous study sought to evaluate whether Cur and/or Pip could potentiate CR’s effect on body weight reduction in mice made obese by feeding a high-fat diet (HFD) [10]. CR favorably impacted metabolic profiles of obesity (i.e., lower body weight, plasma glucose and plasma insulin) in obese mice, but the addition of Cur and/or Pip into the CR diet resulted in no further measurable benefit on total body weight.

Telomeres are important component of cell division that protect the genome DNA against cellular senescence and promote chromosomal stability. Shortening of the telomeres and DNA damage is associated with age-related disorders, obesity, immune dysregulation, cardiovascular disease and cancer [11–13]. Several studies reported an inverse association of telomere length with obesity [3, 14, 15]. The effect of Cur and/ or Pip on telomere length in obese mice undergoing CR is not known. In the present study, we further investigated whether Cur and/or Pip could improve metabolic profiles in obese mice subjected to CR on a HFD. To this end, we measured the changes in body weight, area under the curve (AUC) of percent total body fat, telomere length and several markers of inflammation.

CR, known to modulate energy balance, is an effective way to reduce the risk of metabolic syndrome and obesity in humans [1, 2], and great interest exists to accelerate this process in order to reach an ideal body weight. To this end, Cur has been shown to have the potential to prevent chronic inflammatory diseases and has recently emerged as a food component with the potential to suppress body weight gain [4]. In our earlier studies, we demonstrated that Cur may have anti-obesity properties [5]. However, its low bioavailability appears to be a hindrance to its health benefits. To overcome this drawback, Pip has been shown to increase absorption of Cur from the GI tract and increase its bioavailability [7]. We found that during Phase 2 of our study (10% CR), the average bodyweight of mice stabilized at approximately 50 g. At the end of Phase 3 (20% CR), the average bodyweight had dropped to nearly 40–45 g. Supplementing HFD with Cur, Pip or Cur + Pip had no effect on the rate of weight loss. Interestingly, the percentage of body fat loss during Phase 3 was significantly affected by supplementation with Cur + Pip. This observation indicates that when mice on a HFD are subjected to 20% CR, the initial loss of total body fat occurs with a minimal loss of lean body mass. The percent of body fat, which changed over 20 weeks, is better depicted as AUC in Fig. 3. Using AUC, we could perform statistical analysis and demonstrate the significant impact of Cur + Pip on modulation of total body fat. Supplementing HFD with Cur or Pip alone could not suppress adiposity beyond CR’s effect. As previously noted, plasma glucose and insulin levels were not affected by Cur or Pip alone or Cur + Pip beyond CR effect [8]. These results indicate that the impact of 20% CR on total body weight reduction of HFD-fed mice was high, and Cur + Pip supplementation was effective at further reducing of body fat.

One of the deleterious effects of elevated total body fat is the presence of low grade chronic inflammation, which can lead to metabolic syndrome and its associated diseases including cancer, diabetes, cardiovascular disease, inflammatory bowel disease, neurodegenerative disease, pancreatitis and psoriasis [4]. Therefore, we measured a battery of inflammatory cytokines in the plasma of the 4 CR groups treated with HFD with and without Cur (Groups 1–3 and 5). Daily intake of curcumin reduced several inflammatory cytokines in HFD-fed mice [5, 6, 9], we did not see a significant effect of Cur alone on plasma levels of inflammatory cytokines (Fig. 4). However, we found that CR mice fed HFD and supplemented with Cur + Pip had significantly lower IL-1β and KC/GRO concentrations in plasma compared to HFD ad libitum. In general, CR reduces expression of inflammatory cytokines [25]. Other inflammatory cytokines (IFN-γ, IL-10, IL-12 p70 and IL-6) were not affected by including CR, CUR and Pip in the HFD, which can be due to the presence of very low background levels in this study. On the other hand, Pip alone might be worked as antioxidant in Cur + Pip administrated group. To prove this, it would be needed for the confirmation of Pip concentration in the body. We analyzed Cur metabolites by HPLC-MS/MS to investigate whether decreased levels of IL-1β and KC/GRO depend on the alteration of Cur metabolism by Pip (Fig. 5). Analysis revealed that most of the Cur in plasma was conjugated as CurG. Other conjugates (CurS, CurDG, CurDS and CurGS) and Cur were found at extremely low levels in plasma. In general, almost of orally administrated Cur are discharged into feces. In the small intestine, Cur is subjected to conjugation reaction with UDP-glucuronosyltransferase or sulfotransferase by the second phase reaction (phase 2 metabolism) to provide CurG. Then, small amount of Cur and CurG are absorbed from the small intestine, transported to the liver via the portal vein, and undergoes a further conjugation reaction by the phase 2 metabolism. After that, it circulates in the blood and reaches various organs [18, 26]. These data support our previous observations that CurG is the major metabolite found in rodent plasma after administration of Cur [18]. We found no significant difference in CurG levels between groups supplemented with Cur alone or combined with Pip. This indicates that Pip did not alter Cur’s metabolism under the present experimental conditions.

It is reported that Cur conjugation (e.g., glucuronidation) occurs in the liver and intestines [26, 27]. Pip reportedly inhibits liver and intestinal glucuronidation activity possibly due to the presence of a conjugated double bond in the Pip molecule [28]. Shoba et al. performed a single oral dose study (2 g Cur/kg and 20 mg Pip/kg body weight) and found that Pip increased absorption and bioavailability of Cur by 200% in rats [7]. As far as we know, this report is the only one report that showed quantitative relationship between CUR and Pip intake. Compared to our study (9 mg/kg), they used higher amount of Pip (20 mg/kg). It needs to clarify the effective amount of Pip dose on Cur metabolism. Hlavačková et al. performed a daily administration study (100 mg Cur + 20 mg Pip/kg body weight for 6 weeks) and reported that Cur + Pip administration favorably impacted N-nitro-L-arginine-methylester-induced hypertension in rats [29]. In our study, the daily dose of Pip was approximately 9 mg/kg body weight [30]. Our study dose may be too low to increase Cur’s absorption and bioavailability substantially. Thus, we can deduce that regulation of telomere length and the decrease of cytokines (IL-1β and KC/GRO) have no connection with the potentially modifying effect of Pip on Cur metabolism. On the other hand, Pip is considered to have anti-inflammatory properties itself [31]. Hence, it is possible to deduce that decrease of these cytokines and fat loss would be enhanced by anti-oxidative activity of Pip and Cur. Also, further study will need to clarify the anti-inflammatory effects of Pip.

A study by Kanitkar et al. reported that daily Cur intake (7.5 mg/kg body weight) reduced IL-1β in the serum of STZ-induced diabetic mice [32]. In the present study, we found a negative correlation between plasma CurG and IL-1β (Table 1), which could possibly be attributed to the antioxidant activity of CurG. This outcome warrants future investigation.

Telomere length is associated with oxidative stress and aging [20]. CR and dietary bioactive antioxidants such as Cur and grape seed extract have been shown to be effective in protecting and maintaining telomere length [33]. Therefore, we sought to determine the impact of 20% CR in combination with the antioxidant activities of Cur + Pip on maintenance of liver telomere (Fig. 6). As a result, minor modulation of telomere length was observed. The possibility is that including CR in HFD-fed mice might have induced oxidative/lipid stress resulting in a shorter telomere [24]. However, Cur and Pip attenuated oxidative stress and affected telomere lengths to some extent because both Cur and Pip have anti oxidative activity. It might appear that stress of CR in HFD fed mice induces an oxidative stress condition which results in more shortening of the relative telomere length. Several researches showed sudden calorie restriction had a potential of oxidative stress. [34] In the present study, we mainly focused to diet. However, it has a potential of age-related changes in the metabolism [35]. Because, our study period undertaken for 53 weeks and it had a potential of metabolic alternation within the study. In the future, it needs to confirm the effect of aging on CUR and Pip with the obesity. Further studies are needed to clarify the impact of Cur + Pip on attenuating oxidative stress under CR and telomere length.

We found that supplementing HFD of mice undergoing 20% CR with a combination of Cur + Pip may accelerate CR induced loss of total body fat. This combination also appears to be an effective intervention at suppressing HFD induced inflammation. In summary, combination of Cur and Pip appear to have the potential to enhance CR effects for the prevention of metabolic syndrome. Both of which are associated with good health status.