Effect of replacing soybean meal with Hermetia illucens meal on cecal microbiota, liver transcriptome, and plasma metabolome of broilers.

Despite the existence of a number of studies investigating the effect of insect meal on the growth performance of broilers, knowledge about the metabolic effects of insect meal in broilers is still scarce. Thus, the present study investigated the effect of partial replacement of soybean meal with Hermetia illucens (HI) larvae meal on the liver transcriptome, the plasma metabolome, and the cecal microbiota in broilers. For the study, 72 male one-day-old Cobb 500 broilers were divided into three groups and fed 3 different diets with either 0% (HI0), 7.5% (HI7.5), or 15% (HI15) defatted HI meal for 35 d. Each group consisted of 6 cages (replicates) with 4 broilers/cage. While body weight (BW) gain, feed intake, and feed:gain ratio did not differ between groups, breast muscle weight, carcass yield, and apparent ileal digestibility (AID) of 5 amino acids were higher in group HI15 than in group HI0 (P < 0.05). Indicators of α-diversity (Chao1 and Observed) in the cecal digesta were higher in groups HI15 and HI7.5 than in group HI0 (P < 0.05). The abundance of 5 families and 18 genera, all of which belonged to the Firmicutes phylum, in the cecal digesta differed among groups (P < 0.05). Concentrations of butyric acid, valeric acid, and isobutyric acid in the cecal digesta were lower in group HI15 than in the other 2 groups (P < 0.05), whereas those of total and other short-chain fatty acids were not different between groups. Liver transcriptomics revealed a total of 70 and 61 differentially expressed transcripts between groups HI15 vs. HI0 and between groups HI7.5 vs. HI0, respectively, (P < 0.05). Targeted metabolomics identified 138 metabolites, most of which were triglyceride species, being different between the 3 groups (FDR < 0.05). According to this study, dietary inclusion of HI larvae meal has no detrimental impact but increases breast muscle weight and carcass weight in broilers suggesting that HI larvae meal can be recommended as a sustainable alternative protein source for broilers.

Fat from Hermetia illucens Alters the Cecal Gut Microbiome and Lowers Hepatic Triglyceride Concentration in Comparison to Palm Oil in Obese Zucker Rats.

BACKGROUND: Palm oil (PO) is the most widely utilized plant oil for food production. Owing to the great ecologic problems associated with PO production, sustainably produced fats, such as insect fat, might be a suitable alternative. OBJECTIVES: The hypothesis was tested that fat from Hermetia illucens larvae (HF) compared with PO and soybean oil (SO) has no adverse effects on hepatic lipid metabolism, plasma metabolome, and cecal microbiome in obese Zucker rats. METHODS: Thirty male obese Zucker rats were randomly assigned to 3 groups (SO, PO, HF; n = 10 rats/group) and fed 3 different semisynthetic diets containing either SO, PO, or HF as the main fat source for 4 wk. The effects were evaluated by measurement of liver and plasma lipid concentrations, liver transcriptomics, targeted plasma metabolomics, and cecal microbiomics. RESULTS: Supplementation of HF reduced hepatic triglyceride concentration and messenger ribonucleic acid concentrations of selected genes involved in fatty acid and triglyceride synthesis in comparison to PO (P < 0.05). Pairwise comparison of the Simpson index and Jaccard index showed a higher cecal microbial α- and ß-diversity in rats fed the HF diet than in rats fed the PO diet (P = 0.015 and P = 0.027), but no difference between rats fed the diets with SO or PO. Taxonomic analysis of the cecal microbial community revealed a lower abundance of Clostridium_sensu_stricto_1 and a higher abundance of Blautia, Mucispirillum, Anaerotruncus, Harryflintia, and Peptococcus in rats supplemented with HF than in rats supplemented with PO (P < 0.05). CONCLUSIONS: HF, compared with PO, has liver lipid-lowering effects in obese Zucker rats, which may be caused by a shift in the gut microbial community. Thus, HF might serve as a sustainably produced fat alternative to PO for food production.

Effect of Hermetia illucens Fat, Compared with That of Soybean Oil and Palm Oil, on Hepatic Lipid Metabolism and Plasma Metabolome in Healthy Rats.

Palm oil (PO) is currently the most widely used fat source for food production, but insect fat from Hermetia illucens larvae (HF) might be a suitable alternative fat source, because its production is less harmful to the environment. The present study investigated the effect of HF, as compared to PO and soybean oil (SO), on the hepatic lipid metabolism and the plasma metabolome of healthy rats, which were randomly assigned to three groups (n = 10 rats/group), and fed three different semi-synthetic diets containing either SO, PO, or HF as the main fat source for 4 weeks. Feed intake, body weight gain, liver and plasma lipid concentrations, and the hepatic mRNA levels of genes involved in lipid metabolism and inflammation did not differ between groups. Targeted plasma metabolomics revealed 294 out of 630 metabolites analyzed to be different between groups. Principal component analysis showed a clear separation of the plasma metabolomes of the SO group and the other two groups, but no separation of those of the PO and the HF groups. The present study shows that HF exerts no adverse metabolic effects in healthy rats, compared to PO or SO, indicating that HF is a safe alternative fat source to PO for food production.

A High-Phosphorus Diet Moderately Alters the Lipidome and Transcriptome in the Skeletal Muscle of Adult Mice.

A high phosphorus intake has been associated with various metabolic disorders, including chronic kidney disease, cardiovascular disease, and osteoporosis. Recent studies have demonstrated the effects of dietary phosphorus on lipid and glucose metabolism. This study investigated the impact of a high-phosphorus diet on mouse skeletal muscle lipid composition and gene transcription. Adult male mice (n = 12/group) received either a diet with an adequate (0.3%) or a high (1.2%) phosphorus concentration for 6 weeks. The lipidome analysis showed that among the 17 analyzed lipid classes, the concentrations of three classes were reduced in the high phosphorus group compared to the adequate phosphorus group. These classes were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and lysophosphatidylcholine (LPC) (p < 0.05). Out of the three hundred and twenty-three individual lipid species analyzed, forty-nine showed reduced concentrations, while three showed increased concentrations in the high phosphorus group compared to the adequate phosphorus group. The muscle transcriptome analysis identified 142 up- and 222 down-regulated transcripts in the high phosphorus group compared to the adequate phosphorus group. Gene set enrichment analysis identified that genes that were up-regulated in the high phosphorus group were linked to the gene ontology terms "mitochondria" and "Notch signaling pathway", whereas genes that were down-regulated were linked to the "PI3K-AKT pathway". Overall, the effects of the high-phosphorus diet on the muscle lipidome and transcriptome were relatively modest, but consistently indicated an impact on lipid metabolism.

Effect of supplementation of vitamin D3 or vitamin D2 on serum concentrations of free and total 25-hydroxyvitamin D and the expression of genes involved in immune function in peripheral blood mononuclear cells of weaned pigs.

The present study aimed to compare the effects of vitamin D2 and vitamin D3 supplementation on concentrations of total and free 25(OH)D in plasma and the expression of genes involved in the innate immune system in peripheral blood mononuclear cells (PBMC) in weaned pigs. Five groups of pigs (with an initial body weight of around 9 kg) received basal diets supplemented with either 500 (control group), 1000 or 2000 IU vitamin D3/kg diet or 1000 or 2000 IU vitamin D2/kg diet for a period of 4 weeks. Vitamin D supplementation did not influence feed intake, body weight gain, feed conversion ratio, apparent total tract digestibility of calcium and phosphorus, and serum concentrations of calcium, inorganic phosphate and parathyroid hormone. Supplementation of vitamin D3 led to a dose-dependent increase of the concentrations of total and free 25(OH)D in serum. In contrast, pigs supplemented with 1000 or 2000 IU vitamin D2/kg diet did not have higher concentrations of total and free 25(OH)D in serum than the control group. The ratio of free/total 25(OH)D in serum was not influenced by vitamin D3 supplementation, whereas the group supplemented with 2000 IU vitamin D2/kg diet had a higher free/total 25(OH)D ratio than the groups supplemented with 1000 or 2000 IU vitamin D3/kg diet. Genes involved in vitamin D signalling (CYP27B1, VDR), as well as pro-inflammatory and immune regulatory genes (TLR4, TNF, IL1B and TGFB1) and genes encoding porcine protegrins (NPG1, NPG4), proteins belonging to the group of antimicrobial peptides, in PBMC were not different among groups supplemented with vitamin D3 or vitamin D2 and the control group. Therefore, the study indicates that supplementation of vitamin D2 causes much lower levels of total 25(OH)D than supplementation of vitamin D3 and that supplementation of vitamins D2 or D3 at moderate levels does not have an impact on the innate immune function in healthy pigs.

Replacement of soybean oil by Hermetia illucens larvae fat in broiler diets alters the breast muscle lipidome and reduces lipid oxidation of the breast muscle during heat-processing.

Replacement of soybean oil by insect fat from Hermetia illucens (HI) has been reported to increase the proportions of saturated fatty acids (SFA) and decrease those of polyunsaturated fatty acids (PUFA) in total lipids of breast and thigh meat in broilers. Since the susceptibility of meat to oxidation is strongly dependent on its PUFA content, the present study hypothesised that replacement of soybean oil by HI larvae fat in broiler diets reduces the formation of lipid oxidation products, including oxidation products of cholesterol and phytosterols, in heat-processed breast muscle of broilers. To test this hypothesis, 100 male, 1-day-old Cobb 500 broilers were assigned to three groups and fed three different nutrient adequate diets, which varied only in the fat source (group HI-0: 0% HI larvae fat and 5% soybean oil; group HI-2.5: 2.5% HI larvae fat and 2.5% soybean oil; group HI-5.0: 5.0% HI larvae fat and 0% soybean oil), in a three-phase feeding system for 35 days. While the growth performance of the broilers was not different, the absolute and relative breast muscle weights were higher in group HI-5.0 than in group HI-0 (p < 0.05). The proportions of C12:0, C14:0, C14:1, C16:0, C16:1 and total SFA were higher and those of C18:1, C18:2 n-6, C18:3 n-3 and total PUFA were lower in breast muscle total lipids of group HI-5.0 than in groups HI-2.5 and HI-0 (p < 0.05). Lipidomic analysis of breast muscle revealed that the concentration of triacylglycerols was 46% and 53% lower in groups HI-2.5 and HI-5.0, respectively, than in group HI-0 (p < 0.05), whereas all other lipid classes detected did not differ among groups. Concentrations of thiobarbituric acid-reactive substances, 7α-hydroxycholesterol, 7ß-hydroxycholesterol and total cholesterol oxidation products in heat-processed breast muscle were lower in group HI-5.0 than in group HI-0 (p < 0.05). Concentrations of oxidation products of phytosterols in heat-processed breast muscle were generally much lower than those of cholesterol oxidation products and did not differ between the three groups of broilers. In conclusion, complete replacement of soybean oil with HI larvae fat in broiler diets strongly alters the fatty acid composition of breast muscle total lipids and reduce lipid oxidation of the breast muscle during heat-processing.

Effect of replacement of soybean oil by Hermetia illucens fat on performance, digestibility, cecal microbiome, liver transcriptome and liver and plasma lipidomes of broilers.

BACKGROUND: In contrast to protein-rich insect meal, the feed potential of insect fat is generally less explored and knowledge about the suitability of insect fat as a fat source specifically in broiler diets is still limited. In view of this, the present study aimed to comprehensively investigate the effect of partial (50%) and complete replacement of soybean oil with insect fat from Hermetia illucens (HI) larvae in broiler diets on performance, fat digestibility, cecal microbiome, liver transcriptome and liver and plasma lipidomes. Thus, 100 male, 1-day-old Cobb 500 broilers were randomly assigned to three groups and fed three different diets with either 0 (group HI-0, n = 30), 2.5% (group HI-2.5, n = 35) or 5.0% (HI-5.0, n = 35) Hermetia illucens (HI) larvae fat for 35 d. RESULTS: Body weight gain, final body weight, feed intake, and feed:gain ratio during the whole period and apparent ileal digestibility coefficient for ether extract were not different between groups. Cecal microbial diversity did not differ between groups and taxonomic analysis revealed differences in the abundance of only four low-abundance bacterial taxa among groups; the abundances of phylum Actinobacteriota, class Coriobacteriia, order Coriobacteriales and family Eggerthellaceae were lower in group HI-5.0 compared to group HI-2.5 (P < 0.05). Concentrations of total and individual short-chain fatty acids in the cecal digesta were not different between the three groups. Liver transcriptomics revealed a total of 55 and 25 transcripts to be differentially expressed between groups HI-5.0 vs. HI-0 and groups HI-2.5 vs. HI-0, respectively (P < 0.05). The concentrations of most lipid classes, with the exception of phosphatidylethanolamine, phosphatidylglycerol and lysophosphatidylcholine in the liver and cholesterylester and ceramide in plasma (P < 0.05), and of the sum of all lipid classes were not different between groups. CONCLUSIONS: Partial and complete replacement of soybean oil with HI larvae fat in broiler diets had no effect on growth performance and only modest, but no adverse effects on the cecal microbiome and the metabolic health of broilers. This suggests that HI larvae fat can be used as an alternative fat source in broiler diets, thereby, making broiler production more sustainable.

Feeding of Hermetia illucens Larvae Meal Attenuates Hepatic Lipid Synthesis and Fatty Liver Development in Obese Zucker Rats.

The present study tested the hypothesis that dietary insect meal from Hermetia illucens (HI) larvae attenuates the development of liver steatosis and hyperlipidemia in the obese Zucker rat. To test the hypothesis, a 4-week trial with male, obese Zucker rats (n = 30) and male, lean Zucker rats (n = 10) was performed. The obese rats were assigned to three obese groups (group O-C, group O-HI25, group O-HI50) of 10 rats each. The lean rats served as a lean control group (L-C). Group L-C and group O-C were fed a control diet with 20% casein as protein source, whereas 25% and 50% of the protein from casein was replaced with protein from HI larvae meal in the diets of group O-HI25 and O-HI50, respectively. The staining of liver sections with Oil red O revealed an excessive lipid accumulation in the liver of group O-C compared to group L-C, whereas liver lipid accumulation in group O-HI25 and O-HI50 was markedly reduced compared to group O-C. Hepatic concentrations of triglycerides, cholesterol, C14:0, C16:0, C16:1, C18:0, C18:1, the sum of total fatty acids and hepatic mRNA levels of several genes associated with lipid synthesis and plasma concentration of cholesterol were markedly higher in group O-C than in group L-C, but lower in group O-HI50 than in group O-C (p < 0.05). In conclusion, partial replacement of casein by HI larvae meal attenuates liver steatosis and dyslipidemia in obese Zucker rats. This suggests that HI larvae meal serves as a functional food protecting from obesity-induced metabolic disorders.

Vitamin D in dairy cows: metabolism, status and functions in the immune system.

The function of vitamin D in calcium homoeostasis in dairy cows, such as in other vertebrates, is known for many years. In recent years, new and interesting, non-classical functions of vitamin D have been elucidated, including effects on the immune system. The major aim of this review is to provide an overview of effects of vitamin D or its metabolites on the immune system in dairy cows. The first part of the review provides an overview of vitamin D metabolism, with particular reference to the role of various proteins (25- and 1-hydroxylases, vitamin D binding protein, vitamin D receptor) in vitamin D signalling. The second part deals with the role of the concentration of 25-hydroxyvitamin D [25(OH)D] in plasma as an indicator of the vitamin D status in dairy cows, and its dependence on sunlight exposure and dietary vitamin D supplementation. In this part also the "free hormone hypothesis" is discussed, indicating that the concentration of free 25(OH)D might be a more valid indicator of the vitamin D status than the concentration of total 25(OH)D. The third part deals with classical and the non-classical functions of vitamin D. Among the non-classical functions which are based on an autocrine vitamin D signalling, particular reference is given to the effects of vitamin D and vitamin D metabolites on the immune system in bovine immune cells and in dairy cows. Recent findings provide some indication that vitamin D or its metabolite 25(OH)D could enhance the immune function in dairy cows and be useful for the prevention and therapy of mastitis. However, the number of studies reported so far in this respect is very limited. Thus, much more research is required to yield clear concepts for an optimised usage of vitamin D to improve the immune system and prevent infectious diseases in dairy cows.

Feeding of cuticles from Tenebrio molitor larvae modulates the gut microbiota and attenuates hepatic steatosis in obese Zucker rats.

Insect biomass obtained from large-scale mass-rearing of insect larvae has gained considerable attention in recent years as an alternative and sustainable source of food and feed. A byproduct from mass-rearing of insect larvae is the shed cuticles - the most external components of insects which are a relevant source of the polysaccharide chitin. While it has been shown that chitin modulates the gut microbiota and ameliorates lipid metabolic disorders in obese rodent models, feeding studies dealing with isolated insects' cuticles are completely lacking. Thus, the present study tested the hypothesis that dietary insects' cuticles modulate the gut microbiome and improve hepatic lipid metabolism in obese Zucker rats. To test this hypothesis, three groups of obese Zucker rats were fed a nutrient-adequate, semisynthetic basal diet which was supplemented with either 0% (group O), 1.5% (group O1.5) or 3.0% (group O3.0) Tenebrio molitor cuticles at the expense of cellulose. Oil red O-stained liver sections showed a marked lipid accumulation, but lipid accumulation was clearly less in group O3.0 than in groups O and O1.5. In line with this, hepatic lipid concentrations were 30% lower in group O3.0 than in group O (p < 0.05). No differences were observed across the obese groups regarding liver concentrations of methionine, S-adenosylmethionine and homocysteine. Analysis of cecal microbial community at the family level revealed that the relative abundances of Bifidobacteriaceae, Coriobacteriaceae Erysipelotrichaceae, Lactobacillaceae, Prevotellaceae, Sutterellaceae, unknown Deltaproteobacteria and unknown Firmicutes were higher and those of Anaeroplasmataceae, Desulfovibrionaceae, Eubacteriaceae, Ruminococcaceae, Saccharibacteria and unknown Clostridiales were lower in group O3.0 compared to group O (p < 0.05). Cecal digesta concentrations of total short-chain fatty acids, acetate and butyrate were higher in group O3.0 than in group O (p < 0.05). Targeted plasma metabolomics revealed 53 metabolites differing between groups, amongst which two indole metabolites, indole-3-propionic acid and 3-indoxylsulfate, were markedly elevated in group O3.0 compared to groups O1.5 and O. Regarding that increased abundances of bacteria of the Actinobacteria phylum and Lactobacillaceae family in the gut have been reported to be associated with antisteatotic, hepatoprotective and antiinflammatory effects, the pronounced increases of Bifidobacteriaceae and Coriobacteriaceae (both Actinobacteria), and of Lactobacillaceae in group O3.0 might have contributed to the amelioration of fatty liver.

Limited Impact of Pivalate-Induced Secondary Carnitine Deficiency on Hepatic Transcriptome and Hepatic and Plasma Metabolome in Nursery Pigs.

Administration of pivalate has been demonstrated to be suitable for the induction of secondary carnitine deficiency (CD) in pigs, as model objects for humans. In order to comprehensively characterize the metabolic effects of secondary CD in the liver of pigs, the present study aimed to carry out comparative analysis of the hepatic transcriptome and hepatic and plasma metabolome of a total of 12 male 5-week-old pigs administered either pivalate (group PIV, n = 6) or vehicle (group CON, n = 6) for 28 days. Pigs of group PIV had approximately 40-60% lower concentrations of free carnitine and acetylcarnitine in plasma, liver and different skeletal muscles than pigs of group CON (p < 0.05). Transcript profiling of the liver revealed 140 differentially expressed genes (DEGs) between group PIV and group CON (fold change > 1.2 or <-1.2, p-value < 0.05). Biological process terms dealing with the innate immune response were found to be enriched with the DEGs (p < 0.05). Using a targeted metabolomics approach for the simultaneous quantification of 630 metabolites, 9 liver metabolites and 18 plasma metabolites were identified to be different between group PIV and group CON (p < 0.05). Considering the limited alterations of the hepatic transcriptome and of the liver and plasma metabolome, it can be concluded that pivalate-induced secondary CD is not associated with significant hepatic metabolism changes in pigs.

Effect of Ecdysterone on the Hepatic Transcriptome and Lipid Metabolism in Lean and Obese Zucker Rats.

Conflicting reports exist with regard to the effect of ecdysterone, the predominating representative of steroid hormones in insects and plants, on hepatic and plasma lipid concentrations in different rodent models of obesity, fatty liver, and diabetes, indicating that the effect is dependent on the rodent model used. Here, the hypothesis was tested for the first time that ecdysterone causes lipid-lowering effects in genetically obese Zucker rats. To test this hypothesis, two groups of male obese Zucker rats (n = 8) were fed a nutrient-adequate diet supplemented without or with 0.5 g ecdysterone per kg diet. To study further if ecdysterone is capable of alleviating the strong lipid-synthetic activity in the liver of obese Zucker rats, the study included also two groups of male lean Zucker rats (n = 8) which also received either the ecdysterone-supplemented or the non-supplemented diet. While hepatic and plasma concentrations of triglycerides and cholesterol were markedly higher in the obese compared to the lean rats (p < 0.05), hepatic and plasma triglyceride and cholesterol concentrations did not differ between rats of the same genotype fed the diets without or with ecdysterone. In conclusion, the present study clearly shows that ecdysterone supplementation does not exhibit lipid-lowering actions in the liver and plasma of lean and obese Zucker rats.

High-phosphorus diets reduce aortic lesions and cardiomyocyte size and modify lipid metabolism in Ldl receptor knockout mice.

The consumption of phosphorus in Western populations largely exceeds the recommended intake, while vitamin D supply is often insufficient. Both situations are linked to an increased cardiovascular risk. A 17-week two-factorial study with Ldl receptor-/- mice was conducted to investigate the cardiovascular impact of dietary phosphorus [adequate (0.3%; P0.3) vs. high (1.5%; P1.5)] in combination with a low (50 IU/kg; D50) or adequate vitamin D diet (1000 IU/kg; D1000). The data demonstrate that mice fed the P1.5 vs. P0.3 diets developed smaller vascular lesions (p = 0.013) and cardiac hypotrophy (p = 0.011), which were accompanied by diminished IGF1 and insulin signalling activity in their hearts. Vitamin D showed no independent effect on atherogenesis and heart morphology. Feeding P1.5 vs. P0.3 diets resulted in markedly reduced serum triacylglycerols (p < 0.0001) and cholesterol (p < 0.0001), higher faecal lipid excretion (p < 0.0001) and a reduced mRNA abundance of hepatic sterol exporters and lipoprotein receptors. Minor hypocholesterolaemic and hypotriglyceridaemic effects were also found in mice fed the D1000 vs. D50 diets (p = 0.048, p = 0.026). To conclude, a high phosphorus intake strongly affected the formation of vascular lesions, cardiac morphology, and lipid metabolism, although these changes are not indicative of an increased cardiovascular risk.

Vitamin D Receptor Deficiency Does Not Affect Blood Pressure and Heart Function.

Vitamin D is thought to play a role in blood pressure regulation, which in turn can influence cardiovascular risk. Several meta-analyses of cohort studies found low serum levels of 25-hydroxyvitamin D to be associated with increased blood pressure or increased cardiovascular morbidity and mortality in the general population. Active vitamin D mediates its function via the vitamin D receptor (Vdr), which is a ligand-activated transcription factor. A suitable model to examine the causal role of vitamin D in blood pressure regulation and heart function is the Vdr knockout (Vdr-/-) mouse. To elucidate the role of vitamin D on blood pressure, heart function, and cardiac myocyte size, we conducted a long-term study using Vdr-/- mice and well-defined diets. Group 1 comprised Vdr-/- mice that received a high-calcium, high-phosphorus rescue diet to prevent hypocalcemia and a rickets phenotype. Groups 2 and 3 included Vdr+/+ mice that were fed either the rescue diet or a control diet containing normal amounts of these minerals. As Vdr is a nuclear factor that regulates transcription, we analyzed the renal mRNA expression and serum concentration of renin and found that the Vdr-/- group had an almost 50% higher renin mRNA expression in the kidney compared to both groups of Vdr+/+ mice. Additionally, serum concentration of renin in Vdr-/- mice was significantly higher than that of Vdr+/+ mice that received the rescue or control diet (+ 17%,+ 32%; P < 0.05). In contrast, renin activity was lower in Vdr-/- mice than in both groups of Vdr+/+ mice (P < 0.05). However, blood pressure, heart rate, cardiac myocyte sizes, and the expression of renal renin receptor, hepatic angiotensinogen and angiotensin II receptor, type 1, in kidney, liver and heart, did not differ between the three groups of mice. Additionally, data from transthoracic echocardiography did not indicate the role of Vdr on heart function, as the left ventricular ejection fraction, fractional shortening, and velocity of blood flow were comparable between the three groups. To conclude, the roles of Vdr and therefore most probably of vitamin D, in blood pressure regulation and heart function, were not confirmed by our findings.

Vitamin D Does Not Play a Functional Role in Adipose Tissue Development in Rodent Models.

SCOPE: Several studies have proposed a role of vitamin D in adipogenesis. Here, we sought to study the impact of the vitamin D receptor (Vdr) on adipocyte size in young and old mice and the effect of maternal vitamin D deficiency on fetal adipogenesis. METHODS AND RESULTS: Histological analysis of adipose tissues shows that Vdr knockout (KO) mice have smaller adipocytes than wild-type (WT) mice. Next, we compare young and old Vdr-KO and WT mice and find no differences in adipocyte sizes between weaned Vdr-KO and WT mice. However, 1-year-old Vdr-KO mice, suffering from alopecia, have smaller-sized adipocytes than WT mice, although they consume more food. To elucidate whether vitamin D can directly impact adipocyte development at a critical stage of adipogenesis, we feed rat dams a vitamin D deficient (0 IU kg-1 ) or vitamin D adequate (1000 IU kg-1 ) diet. Neither DNA microarray analysis of the adipose tissues of the newborn rats nor the adipocyte sizes of 21-day-old offspring show significant differences between the two groups. CONCLUSION: Data indicate that vitamin D does not play a fundamental role in adipogenesis because vitamin D does not affect fetal adipogenesis. Moreover, the smaller adipocytes observed in adult Vdr-KO mice are presumably caused by an increased energy expenditure due to alopecia.

The High Calcium, High Phosphorus Rescue Diet Is Not Suitable to Prevent Secondary Hyperparathyroidism in Vitamin D Receptor Deficient Mice.

The vitamin D receptor (VDR) knockout (KO) mouse is a common model to unravel novel metabolic functions of vitamin D. It is recommended to feed these mice a high calcium (2%), high phosphorus (1.25%) diet, termed rescue diet (RD) to prevent hypocalcaemia and secondary hyperparathyroidism. First, we characterized the individual response of VDR KO mice to feeding a RD and found that the RD was not capable of normalizing the parathyroid hormone (PTH) concentrations in each VDR KO mouse. In a second study, we aimed to study whether RD with additional 1 and 2% calcium (in total 3 and 4% of the diet) is able to prevent secondary hyperparathyroidism in the VDR KO mice. Wild type (WT) mice and VDR KO mice that received a normal calcium and phosphorus diet (ND) served as controls. Data demonstrated that the RD was no more efficient than the ND in normalizing PTH levels. An excessive dietary calcium concentration of 4% was required to reduce serum PTH concentrations in the VDR KO mice to PTH levels measured in WT mice. This diet, however, resulted in higher concentrations of circulating intact fibroblast growth factor 23 (iFGF23). To conclude, the commonly used RD is not suitable to normalize the serum PTH in VDR KO mice. Extremely high dietary calcium concentrations are necessary to prevent secondary hyperthyroidism in these mice, with the consequence that iFGF23 concentrations are being raised. Considering that PTH and iFGF23 exert numerous VDR independent effects, data obtained from VDR KO mice cannot be attributed solely to vitamin D.

Vitamin D receptor knockout mice exhibit elongated intestinal microvilli and increased ezrin expression.

In addition to its principle function as a calcium regulator, vitamin D can affect cell and tissue morphology. The intestine is an important target tissue of vitamin D, as it must ensure the efficient transport of nutrients across the epithelium while excluding the passage of harmful molecules and bacteria into the organism. These functions require a highly organized morphology, which may be modified by vitamin D deficiency. To elucidate the role of vitamin D in gut morphology and barrier function, we compared the enterocyte microstructures, gut permeability, and cytoskeletal and cell junction protein expression in vitamin D receptor (VDR) knockout (KO) and wild-type (WT) mice. We found that the duodenal epithelial cells in the VDR-KO mice had longer microvilli (+19%) than those of the WT mice (P < .05). Interestingly, microvilli elongation in the VDR-KO mice was associated with higher messenger RNA and protein expression of ezrin, which is involved in the regulation of microvillus morphogenesis. Intestinal tight junction width and permeability were assessed by measuring the fluorescein isothiocyanate dextran concentrations in plasma; the concentrations were comparable between the 2 groups of mice. We further observed a decrease in the messenger RNA and protein expression of the calcium-transporting tight junction protein claudin-2 in the VDR-KO mice compared with the WT mice (P < .05). In conclusion, the mice lacking VDR had longer enterocyte microvilli, likely as a result of increased ezrin expression. However, the morphology of the tight junctions and the intestinal permeability for large molecules were not affected.