Outcomes of the experiments conducted in the present investigation demonstrate that fenugreek seed does indeed exhibit insulin-sensitizing effects in tissues. Evidence for this conclusion is based on observations that glucose infusion rate (GIR) values significantly increased in participating subjects, following ten-day treatment periods with fenugreek seed preparations (
p = 0.001327). The largest increases in GIR, corresponding to an insulin sensitizing effect of 97%, occurred in two patients with the lowest GIR values in the test group, measured before initiation of treatment. GIR increases were also observed in the other six volunteers treated with fenugreek capsules, however, to a much lesser extent than in the two subjects with the lowest measured GIRs. In contrast, placebo-treated subjects, failed to exhibit significant change in GIR values. According to the abovementioned findings, we may conclude that the TFG-treatment is more effective when the patient is insulin-resistant, and that it does not have influence on glucose homeostasis under normal, physiological conditions. Additionally, no significant placebo-associated changes were observed in MCRI, QUICKI, ISI, HOMA-IR, and HOMA-B, serum glucose, plasma insulin and serum lipids. Interpretation of these data is, nevertheless, subject to the caveat that the present study is a preliminary analysis of fenugreek seed effects and not intended as a definitive analysis of T2DM pathomechanism. Here, the sample ‘n’ for test subjects is low, and it is anticipated that more comprehensive future studies will involve participation of larger groups of volunteers. Nevertheless, results of this small pilot study are encouraging, since safety and efficacy of fenugreek seeds have been proven in human subjects, with parallel changes in the plasma level of MCH, a mediator that may play an important role in glucose-homeostasis. Future clinical and basic lab analyses of this biomaterial, planned by the authors, will extend the experiments described here into detailed characterization of signaling processes contributing to insulin sensitization that are sensitive to modulation by fenugreek and other natural products. Such investigations will be conducted in tandem with evaluations of currently available insulin-sensitizing drugs to determine the capability of natural substances, to potentiate their pharmacological value and reduce occurrence of side effects. Haines et al. have previously conducted product development research that achieved these objectives [
48]. A major outcome of the present study included an observation that following ten days of fenugreek seed ingestion, serum MCH levels were significantly reduced (
p = 0.0464), while no such reduction was observed in the placebo-treated group. These results demonstrate that fenugreek-mediated augmentation of insulin sensitization and improved glucose metabolism correlate with and may be substantially dependent on decrease of MCH levels. The MCH is involved in several physiological functions in vertebrates. Evidence suggests its crucial role in energy homeostasis, food intake, obesity and T2DM. The increased levels of MCH is correlated with hyperphagia, body weight gain, increased white adipose tissue and liver mass, reduce brown adipose tissue function, moreover, elevated MCH may produce hyperglycemia, hyperinsulinemia, and hyperleptinemia [
49,
50,
51,
52,
53,
54]. On the contrary, MCH-knockout mice are lean and resistant to obesity, furthermore, resistant to age-related glucose intolerance [
55]. In the periphery, MCH was detected in several tissues, e.g., in the beta cells of the pancreatic islets, adipose tissues and in the duodenum. MCH induces islet hyperplasia, which suggest that it may regulate pancreatic islet secretory function and beta cell dynamics [
35,
36,
56,
57]. Moreover, Pereira-da-Silva et al. suggested that MCH may act as the central modulator of insulin activity, as it could modify glucose metabolism [
45]. MCH-containing neurons and MCH receptors have a critical role in energy homeostasis. These (second order and downstream) neurons regulated by POMC (proopio-melanocortin) and NPY/AGRP (neuropeptide Y/agouti-related peptide) (first-order) neurons and modulate food intake. First order neurons can process the signals arriving from the periphery like ghrelin, insulin and leptin. NPY/AGRP will stimulate and POMC will inhibit MCH-containing neurons. Ghrelin act as a positive modulator on NPY/AGRP; thus, if the MCH levels will increase, food intake will be enhanced. Effects of insulin and leptin are antagonistic, whereby POMC neurons will be stimulated by these hormones, thus, MCH-containing neurons will be inhibited and food intake will decrease. MCH-containing neurons are negatively affected by autoregulation as well [
58]. Furthermore, MCH-containing neurons not only secrete MCH, but also produce GABA (gamma-amino butyric acid), CART (cocaine- and amphetamine-regulated transcript) and nesfatin. These neurons can be directly activated by orexinergic neurons, and indirectly by glutamate release from excitatory fibers. In contrast, MCH inhibits orexinergic neurons and the neighboring GABAergic fibers. Furthermore, MCH-containing neurons may be inhibited by MCH, GABA, NE (norepinephrine, mediated by alpha-2 receptors), serotonin, acetylcholine (muscarinic), neuropeptide Y and histamine [
59]. Interestingly, Hausen et al. found that insulin can act directly on MCH-containing neurons, which resulted in impairment of locomotor activity and insulin sensitivity [
60]. This is a reasonable interpretation of the data, since elevated levels of the hormone are associated with increase in the food intake and, thus, body weight, and with an increased rate of preadipocyte differentiation [
61].
To investigate the correlation between the MCH and glucose, as well as lipid metabolism, we measured the blood levels of the correspondent parameters. Although literature data presents an important decrease in the aforementioned blood parameters after fenugreek treatment, it is important to highlight the major differences between these study designs and our experimental concept. Accordingly, numerous experimental and clinical reports are available, but all enrolled patients with impaired glucose tolerance, prediabetes or manifest T2DM, while in our study, only healthy volunteers were examined [
8,
62,
63]. Another important difference was that our volunteers were treated with a lower daily dose of TFG, 3 g/day instead of 10 g/day used by others [
8,
62,
63]. Finally, we treated the volunteers only for ten days, in particular interest to short-term effects of TFG-treatment [
8,
62,
63]. Probably due to these differences, we failed to find significant changes in fasting glucose, insulin or serum lipid concentrations. However, to our knowledge, this is the first study to demonstrate that fenugreek seeds may contribute to the regulation of glucose metabolism by restoring the insulin sensitivity of peripheral tissues, shown by the most sensitive method (HEGC) on human volunteers. Moreover, we observed that the fasting insulin level is higher in the TFG-treated group at the end of the study, probably due to the insulinotropic effect of the fenugreek seeds [
64,
65,
66].