The VLCKD is increasingly promoted as a strategy to fight obesity. Although the VLCKD is effective for weight loss and weight control, a comprehensive determination of its relationship with biochemical and physiological changes, in particular with the adipokines produced by AT, is still largely unexplored. This is the first study that evaluated total adiponectin serum levels using an ELISA test and its oligomeric profile using Western blotting analysis. Furthermore, the Western blotting analysis is a semi-quantitative method to support the previous results obtained by the ELISA test.
In this study, anthropometric and biochemical parameters in VLCKD participants before and after eight weeks of diet intervention were investigated, focusing on the effects of this diet on total adiponectin and its oligomeric profile. The results of the present study showed that the eight-week intervention with the VLCKD produced significant weight loss in our participants, decreasing pro-inflammatory cytokine production, increasing adiponectin serum levels, and improving metabolic profile. To support our results, several studies reported beneficial effects of a ketogenic diet [
17,
18,
19,
20]. In addition, VLCKD is advantageous in increasing satiety despite a negative energy balance, and sustaining basal energy expenditure despite body weight loss due to a sparing of fat mass [
20,
21,
22]. Interestingly, as reported by Zhang et al., a ketogenic diet in combination with exercise reduced PPARγ and lipid synthetic genes, as well as enhancing the PPARα and lipid β-oxidation gene program in the liver compared to those in a ketogenic diet without exercise [
23]. On the contrary, some studies reported that any beneficial effects are only transient [
24]. In addition, Ellenbroek et al. supported that a long-term ketogenic diet (22 weeks) caused dyslipidemia, a pro-inflammatory state, signs of hepatic steatosis, glucose intolerance, and a reduction in beta and alpha cell mass, without weight loss in mice; however, the induction of ketosis and the response to ketosis in man and mouse are quite different, and 22 weeks is a very long period for a mouse that could be compared to several years in human beings [
23,
25]. The strength of this study is that, in a short period, the VLCKD changed the anthropometric and metabolic profile of our participants in a statistically significant manner. It is well known that VAT is dangerous to health, generating chronic low inflammation and leading to an imbalance in the function of adipose tissue; for these reasons, the inverse correlation between adiponectin and VAT and pro-inflammatory cytokines such as TNF-α is very important [
26,
27,
28]. Gustafson and colleagues reported that, among the fat storage compartments in the body, VAT was found to be an important source of pro-inflammatory adipokines such as TNF-α and IL-6, and it was associated with an increased risk for atherosclerosis, more so than subcutaneous fat [
27]. Moreover, significant changes in VAT, as well as in inflammatory and adipose tissue activity biomarkers, were reported, suggesting that the VLCKD, in the short term, can be considered very important in the deregulation of the balance between abdominal fat and the production of pro-inflammatory mediators. We also tested adiponectin, whose expression is strongly increased after eight weeks of VLCKD. Adiponectin is the most abundant circulating adipokine, and plasmatic levels, together with free fatty acids (FFA), are statistically inversely related to body fat, abdominal visceral fat, and glucose and lipid metabolism. This adipokine, through its HMW oligomers, which are the most biologically active, strongly increased in our participants after the diet intervention. Adiponectin exerts an anti-inflammatory effect and modulates insulin sensitivity by stimulating glucose utilization and fatty acid oxidation. On the contrary, elevated FFA was linked with the development of insulin resistance, defects in insulin secretion, nonalcoholic fatty liver disease, and metabolic syndrome [
28]. As reported by Nigro et al., serum adiponectin levels are reduced in obese and diabetic subjects and are considered as a marker of various metabolic diseases, as well as of improvement of metabolic activity [
29]. Furthermore, the results of the present study show a negative correlation between adiponectin and glycated hemoglobin; these findings, in agreement with Okoro et al., confirm the strong involvement of adiponectin in metabolic syndrome and in the establishment of type 2 diabetes [
29,
30,
31]. However, the molecular mechanism underlying the cause-and-effect relationship between hypoadiponectinemia and insulin resistance is not yet fully clear [
32]. Indeed, in vivo and in vitro studies suggested that adiponectin has an antidiabetic and hypoglycemic effect [
33], activating hepatic insulin receptor and promoting pancreatic beta-cell function [
33,
34,
35].
Data in the literature confirmed that the ketogenic diet is associated with increases in adiponectin in obese subjects [
31,
35]. Sherrier et al. hypothesized that the level of nutritional ketosis may be an important factor in the regulation of adiponectin expression, since ketones influence AMPK activity through adiponectin [
36]. Furthermore, it is worth mentioning that adipose tissue is the target of many metabolically active factors, many of which induce the secretion of adiponectin. [
36]. On the contrary, Garaulet et al. reported that adiponectin is related to protection against the metabolic syndrome but is not involved in the regulation of VLCD-induced improvement of insulin sensitivity [
37]. In a chronic inflammatory state, such as obesity, the physiologic status of the cells presents changes in adipose tissue altering the production of adipokines [
35]. In this scenario, another essential function of adiponectin and its HMW oligomers is their role in inflammatory and immune responses. We found a negative correlation between adiponectin and pro-inflammatory cytokines such as TNF-α; in fact, adiponectin is able to suppress nucleus NF-κB translocation and pro-inflammatory cytokine expression, including TNF-α, IL-1b, and IL-6 [
38,
39,
40]. In addition, as we demonstrated, adiponectin positively correlates with IL-10 serum levels; in fact, it increases the expression of anti-inflammatory mediators, such as IL-10, and induces the polarization of anti-inflammatory M2 macrophages [
41,
42]. Furthermore, adiponectin enhances cold-induced browning of subcutaneous adipose tissue through M2 macrophage proliferation and promotes cell proliferation via the activation of serine/threonine-specific protein kinase Akt, consequently leading to beige cell activation [
43,
44,
45]. Moreover, Tsuchida et al. suggested that adiponectin, through innate immune response-dependent mechanisms, can regulate insulin sensitivity and energy expenditure. On the other hand, adiponectin and its HMW oligomers are strongly involved not only in metabolic processes but also in inflammatory and immune responses [
45]. In light of this evidence, the negative correlations that we found between adiponectin and lipid profile, VAT, CPR, and TNF-α confirmed the profound involvement of adiponectin in many metabolic and inflammatory diseases and, in parallel, also confirmed the beneficial short-term effects of VLCKD intervention not only in the treatment of obesity but also in the establishment of obesity-correlated diseases. Indeed, for its anti-inflammatory properties, the ketogenic diet is used as an adjuvant treatment in the cancer therapy. As suggested by Weber et al., a ketogenic diet creates an unfavorable metabolic environment for cancer cells and, thus, can be regarded as a promising adjuvant as a patient-specific multifactorial therapy [
15]. The main limitation of this study is related to the small number of participants; contrariwise, the short time of the observation period can represent a strong point of the study. Indeed, the short period of VLCKD intervention has beneficial effects not only on metabolic rate but also on the inflammatory state, improving adiponectin and IL-10 levels, as well as reducing both TNF-α and IL-6 levels.