1. Introduction
One of the major challenges in the near future is to cover the growing demand for food of the rising world population despite the increasing scarcity of natural resources (e.g., arable land and water) required for agricultural production. At the same time, the environmental burden from agricultural production, such as greenhouse gas (GHG) emissions, must be reduced in order to reach the global climate protection goals and contribute towards carbon neutrality. Against this background, agricultural production must be intensified in a sustainable way, so that more food is produced with fewer resources and fewer emissions. In this regard, the bioconversion of organic agro-industrial side streams into edible protein-rich biomass by insects has been recognized as a suitable strategy to face this challenge [
1,
2]. This is attributed to the fact that large-scale mass production of insect biomass in a circular food system allows resource-efficient recycling of regionally available agro-industrial co- and by-products, and is associated with lower usage of natural resources (space, water, energy) and lower environmental impact (e.g. less GHG emissions) than required for the production of conventional protein sources [
3,
4]. Amongst several insect species approved by the European Commission for insect-based food and feed, two species,
Tenebrio molitor (TM) and
Hermetia illucens (HI), have emerged as the most promising for resource-efficient mass production of protein-rich insect biomass such as insect larvae meal [
5,
6].
Apart from serving as a source of essential amino acids, which are required as building blocks for protein synthesis in both humans and monogastric farm animals, insect larvae meal might be interesting especially in humans due to pleiotropic health-related effects. For instance, dietary TM larvae meal was shown to cause antiadipogenic effects in high fat diet (HFD)-induced obese mice and to inhibit lipogenesis in cultured adipocytes [
7]. In addition, several studies demonstrated strong liver and plasma lipid-lowering effects and a pronounced inhibition of hepatic lipogenesis of partially defatted TM larvae meal in the obese Zucker rat [
8,
9,
10], an established model of liver steatosis, obesity, diabetes and metabolic syndrome. Likewise, Lee et al. [
11] reported that TM larvae meal decreases lipid accumulation and lipogenesis in the liver of HFD-induced obese mice. Although the bioactive compounds responsible for the inhibitory effect of TM larvae meal on lipogenesis remain to be identified, different constituents of TM larvae meal may be likely candidates for its lipid-lowering action. One is chitin, an intrinsic constituent of the insect’s exoskeleton, which makes up 9–13% of dry matter in TM larvae meal [
8,
12], and has been demonstrated to cause lipid-lowering effects [
13]. Other possible candidates are biologically active peptide sequences, which have been found in different dietary proteins including milk protein [
14], egg protein [
15], soybean protein [
16,
17] and peanut protein [
18]. Such bioactive peptides, which have been also identified in TM larvae protein [
19,
20], are known to be released during protein digestion and enter the circulation in intact form to mediate different biological effects, such as lipid-lowering or hypotensive activities. Since all of the abovementioned health-related effects have been observed with the use of TM larvae meal, the question arises of whether such effects can be also observed with the use of HI larvae meal, which also contains chitin (6.8% [
21]) and presumably also bioactive peptides. This, however, is currently unknown from the available literature. In order to close this gap of knowledge, the hypothesis was tested that dietary HI larvae meal attenuates liver steatosis development and hyperlipidemia in the obese Zucker rat, which is an established rodent model of liver steatosis and hyperlipidemia.
4. Discussion
In the present study, the hypothesis was tested that dietary HI larvae meal attenuates the development of liver steatosis and hyperlipidemia in the obese Zucker rat. In order to investigate this hypothesis, the protein from casein was partially replaced with protein from HI larvae meal at two different replacement levels (25% and 50%). Replacement of protein from casein with HI larvae meal at higher levels was not possible, because the protein content of the HI larvae meal was markedly lower than that of casein. Thus, only the effect of partial but not complete replacement of casein by HI larvae meal could be investigated. Although the protein concentration of the diets calculated using the standard N-to-protein conversion factor of 6.25 indicated that replacement of casein with HI larvae meal in the diets was not isoproteinogenous, it has to be considered that the protein concentration in the two HI larvae meal-containing diets was overestimated due to the presence of the nonprotein N-compound chitin. This was obvious when the calculated protein concentration in diets HI25 and HI50 was corrected for the amount of chitin present in the HI larvae meal. In line with this, calculating the sum of total dietary amino acids from the analyzed amounts of all individual amino acids revealed that the dietary protein concentration was comparable across the three diets. Considering this, partial replacement of casein by HI larvae meal was isoproteinogenous. In addition, the amount of fat and the fatty acid composition of the dietary fat was adjusted across the diets by using mixtures of different fats. Owing to this, the biological effects induced by replacing casein with HI larvae meal cannot be ascribed to specific fatty acids of the HI larvae meal.
The results of this study convincingly demonstrate that liver triglyceride and cholesterol concentrations, plasma cholesterol concentration and hepatic mRNA levels and/or activities of cholesterogenic and lipogenic genes—which are mainly regulated at the transcriptional level
via sterol regulatory element-binding proteins [
34]—were markedly reduced in rats fed HI larvae meal. In addition, our study showed that hepatic concentrations of fatty acids originating from
de novo-lipogenesis, such as C16:0, C16:1 n-7 and C18:1 n-9, were strongly reduced in both groups of rats fed HI larvae meal. Since these fatty acids contributed to almost 90% of total hepatic fatty acids, the sum of all individual fatty acids in the liver were also reduced in rats fed HI larvae meal. Moreover, the calculation of fatty acid desaturation indices from hepatic fatty acid concentrations revealed that ∆9, ∆6 and ∆5 desaturation pathways, which are responsible for the synthesis of monounsaturated (∆9) and polyunsaturated (∆6, ∆5) fatty acids, were clearly lowered in rats fed HI larvae meal. Our observation that the mRNA level of
Cyp7a1 did not differ across the obese groups of rats indicates that an increased hepatic bile acid synthesis from cholesterol did not contribute to the lowering of hepatic cholesterol concentration in rats fed the HI larvae meal. According to these results, the hypothesis of our study could be clearly verified that HI larvae meal, like TM larvae meal [
8,
9,
10,
11], attenuates liver steatosis and dyslipidemia in obese Zucker rats. In addition, the observations from our study indicate that the lipid-lowering effect of HI larvae meal is mainly caused by a profound inhibition of hepatic fatty acid, triglyceride and cholesterol synthesis. Although the liver lipid concentrations were not statistically different between the two groups of rats fed HI larvae meal, the observation that liver lipid concentrations were numerically lower and hepatic concentrations of C16:0, C16:1 n-7 and total fatty acids and hepatic mRNA levels of several lipogenic genes (
Fads1,
Fads2,
G6pd) were significantly lower in group O-HI50 than in group O-HI25, respectively, suggests that the inhibitory effect of HI larvae meal on lipid synthesis is dose-dependent. We are confident that all these findings are indicative of a marked inhibition of hepatic lipid synthesis by HI larvae meal, but not of a reduced bioavailability of fatty acids from the HI larvae meal-containing diets. In the latter case, body weight gain would have been reduced due to a reduced intake of digestible energy. This, however, was not the case. In addition, in a recent study with obese Zucker rats fed a diet with TM larvae meal, the apparent total tract digestibility of ether extract was reported to be 96% [
9]. This indicates that the digestibility of fatty acids from insect meal in rats is very high and a reduced bioavailability of fatty acids from HI larvae meal is likely not causative. Moreover, the main part of dietary fat in diet HI50 was derived from other fat sources, such as soybean oil and coconut fat, which are also highly digestible in rats. Furthermore, the marked repression of lipogenic genes in the liver, the strong inhibition of lipogenic enzyme activities and the strong reduction of fatty acids in the liver derived from
de novo-fatty acid synthesis in group O-HI50 does not support the assumption that an enhanced lipid mobilization from the liver was the main reason for the strong antisteatotic effect of HI larvae meal. However, future studies are warranted to clarify this issue. In the present study, adipose tissues weights were not weighed or histopathologically examined. However, the unaltered body weights, organ weights and feed intake across the obese groups did not suggest that adipose tissue weights or lipid deposition in adipose tissue were affected by casein replacement with HI larvae meal.
Regarding the heterogenous composition of the HI larvae meal, various bioactive compounds might be responsible for its lipid-lowering activity. One possible candidate is chitin, a structural polysaccharide consisting of β-(1–4)-
N-acetyl-D-glucosamine monomers which largely serves as a fermentation substrate in the large intestine of monogastric animals. According to the chitin content analyzed in the HI larvae meal (13% of FM), the diets HI25 and HI50 contained approximately 1.5% and 3.0% chitin, respectively. Several reports exist in the literature showing that feeding chitosan—the deacetylation product of chitin—and chitosan oligosaccharides at comparable chitin levels as in our study exerts potent antisteatotic and lipid-lowering effects in different rat models of fatty liver and obesity [
35,
36,
37,
38]. According to these studies, different mechanisms including the inhibition of hepatic lipid synthesis [
39] and a reduction of systemic and hepatic inflammation [
38,
40], which is known to promote hepatic lipid synthesis through stimulating NF-κB signaling pathway [
41], have been identified as important antisteatotic effects of chitosan. In a recent study with obese Zucker rats, feeding diets enriched with chitin
via supplementation of insects´ cuticles was also found to cause a pronounced inhibition of hepatic lipid accumulation and hepatic lipid concentrations [
42]. In addition, this study showed that feeding the chitin-containing diets modified the gut microbiota community structure in a favorable manner with increased abundances of bacterial families [
42], which are known to strengthen the gut barrier, decrease systemic and hepatic inflammation and attenuate hepatic steatosis in various rodent models of obesity, fatty liver and metabolic syndrome [
43,
44,
45,
46,
47]. Although we did not analyze the gut microbiota structure of the rats, it is not unlikely that feeding of the HI larvae meal-diets has also modified the gut microbiota composition of the obese rats in a beneficial manner, thereby contributing to the strong antisteatotic effects observed. In line with this, several studies with broilers and pigs have demonstrated that feeding of different insect larvae meals affects the gut microbiota composition [
12,
48,
49]. However, in order to clarify this speculative issue, future studies have to demonstrate that HI larvae meal alters the gut microbiota composition in a favorable manner in the obese Zucker rat. In addition, future studies have to demonstrate that the chronic systemic inflammatory condition and the impaired liver function, which is known to develop in obese Zucker rats [
42], is attenuated by partial replacement of casein by HI larvae meal. Moreover, considering recent indications that casein promotes pro-inflammatory activities, disturbs liver lipid metabolism and induces liver steatosis in obese Zucker rats when compared to soy protein isolate [
50,
51], it has to be clarified whether the markedly higher liver lipid accumulation in group O-C compared to groups O-HI25 and O-HI50 was caused by either a pro-inflammatory effect of casein or an anti-inflammatory effect of HI larvae meal. However, considering the relative liver weight and the body weight gain throughout the experiment, both of which did not differ across the obese groups of rats, no evidence for a negative effect of casein compared to HI larvae meal has been gained in the present study.
Apart from chitin, the protein fraction from HI larvae meal might be also a source of bioactive compounds with lipid-lowering activities. Indeed, cholesterol-lowering peptide sequences, which were identified in soybean protein [
16], have been made responsible for the cholesterol-lowering activities of different dietary proteins, such as soybean and other legume proteins [
52,
53,
54]. Despite the current lack of evidence for the presence of bioactive peptides with lipid-lowering activities in HI larvae protein, the occurrence of bioactive peptides with other biological activities, such as ACE-inhibitory peptides, has been reported for insect protein in several studies [
19,
20]. Based on this, it appears not unlikely that peptides with lipid-lowering actions are also present in HI larvae proteins. Future studies are required to address this issue. A further fraction of the HI larvae meal that was present in significant amounts is fat, despite the HI larvae meal having been partially defatted by the producer. However, we exclude the possibility that specific fatty acids in the HI larvae meal are responsible for its lipid-lowering activity, because both the amount of fat and the fatty acid composition of the dietary fat were adjusted across the diets by using mixtures of different fats.
A further factor that could account for the biological activity of HI larvae meal is the amino acid composition. Regarding TM larvae meal, one characteristic feature of its amino acid composition is a low concentration of methionine when compared to casein. This is relevant with regard to the lipid-lowering activity of TM larvae meal, because methionine restriction was reported to cause a marked inhibition of hepatic
de novo-lipogenesis and cholesterogenesis and the attenuation of liver steatosis [
55,
56,
57,
58,
59]. Based on this, we have recently raised the hypothesis that the lipid-lowering effect of isoproteinogenic replacement of casein by TM larvae meal is due to methionine restriction [
9]. However, in a subsequent study, in which the TM meal-containing diet was supplemented with methionine to a similar level as in the casein diet, the lipid-lowering effect of TM larvae meal was still observed [
10]. In addition, an increased supply of cysteine to the TM larvae meal-containing diet or the adjustment of essential amino acid levels between the TM larvae meal diet and the casein diet did not reverse the lipid-lowering effect of TM larvae meal [
10]. This clearly indicated that a divergent amino acid composition between TM larvae meal and casein, a low methionine level in TM larvae meal and a decreased cysteine synthesis as a consequence of a reduced methionine availability resulting from feeding TM larvae meal are not responsible for the lipid-lowering action of TM larvae meal. In the present study, the concentration of methionine in diets HI25 and HI50 was 10 and 20%, respectively—lower than in diet C (5.3 g/kg diet)—but the dietary methionine concentration in diets HI25 (4.8 g/kg diet) and HI50 (4.3 g/kg diet) was clearly higher than the maintenance requirement of methionine for rats (2.3 g methionine + cysteine/kg diet, from which cysteine may supply up to 50%; [
22]). Thus, we propose that differences in the dietary concentration of methionine between groups are not causative for the lipid-lowering effect of HI larvae meal.