Male castration increases adiposity via small intestinal microbial alterations

Abstract

Castration of young males is widely used in the cattle industry to improve meat quality, but the mechanism linking hypogonadism and host metabolism is not clear. Here, we use metataxonomic and metabolomic approaches to evaluate the intestinal microbiota and host metabolism in male, castrated male (CtM), and female cattle. After pubescence, the CtM cattle harbor distinct ileal microbiota dominated by the family Peptostreptococcaceae and exhibit distinct serum and muscle amino acid profiles (i.e., highly abundant branched-chain amino acids), with increased extra- and intramuscular fat storage. We also evaluate the causative factor(s) that underpin the alteration of the intestinal microbiota and host metabolic phenotype in response to hypogonadism. Castration of male mice phenocopies both the intestinal microbial alterations and obese-prone metabolism observed in cattle. Antibiotic treatment and fecal microbiota transplantation experiments in a mouse model confirm that the intestinal microbial alterations associated with hypogonadism are a key contributor to the obese phenotype in the CtM animals. Collectively, targeting the gut microbiota is a potential therapeutic strategy for the treatment of both hypogonadism and obesity.

Keywords: Peptostreptococcaceae; branched-chain amino acids; hypogonadism; ileal microbiota; intramuscular fat.

Figure 1. The rectal microbial and serum metabolome profiles of the post‐pubescent Hanwoo

1. A–C

   Determination of the body weight (A), and serum testosterone (B) and 17β‐estradiol (C) levels, in post‐pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group).

2. D

   PCoA of the rectal bacterial 16S rRNA gene sequences based on the weighted UniFrac distance matrix.

3. E

   Discriminant taxa of the male and CtM rectal microbiota, as determined by the linear discriminant analysis effect size (LEfSe). The different abundances are represented by LDA scores.

4. F

   The serum metabolome profiles were analyzed using GC‐TOF‐MS and clustered by PCoA based on the Bray–Curtis dissimilarity matrix.

5. G

   The relative abundances of serum metabolites displayed as bar graphs (n = 5 in each group).

6. H

   The BCAA serum levels quantified enzymatically, as described in the Materials and Methods section.

Data information: The data were analyzed by using ANOVA followed by Tukey’s post hoc test (A to C) and the Mann–Whitney U‐test (G and H). The lines, boxes, and whiskers in the box plot diagrams represent the median, first, and third quartiles, and min‐to‐max distribution of replicate values, respectively. The scattered dots in the bar graphs/box plot diagrams represent the individual replicates. *P < 0.05, **P < 0.01, and ***P < 0.001. CtM, castrated male.

Figure 2. Microbial metataxonomic profiles of different compartments of the intestinal tract of the adult male and castrated male (CtM) Hanwoo

1. A

   Serum testosterone levels in the adult male and CtM Hanwoo (n = 10 in each group). The scattered dots in the bar graph represent the individual replicates.

2. B, C

   PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the rumen, ileum, and colon, shown for the different segments of the gastrointestinal tract (B) and combined data (C).

3. D

   Microbial dissimilarity (calculated based on the weighted UniFrac distance matrix, 100 values in each group) between the male and CtM groups in the different intestinal compartments. The lines, boxes, and whiskers in the box plot diagrams represent the median, first, and third quartiles, and min‐to‐max distribution of replicate values, respectively.

4. E

   The relative abundances of abundant bacterial taxa (> 0.5% of the mean abundance).

Data information: The data were analyzed by using the Mann–Whitney U‐test (A), PERMANOVA with 999 permutations (B), or ANOVA followed by Tukey’s post hoc test (D). *P < 0.05, **P < 0.01, and ***P < 0.001. CtM, castrated male.

Figure 3. Correlation analysis of the intestinal microbial taxa and metabolites from the castrated male (CtM) Hanwoo

1. A

   The discriminant microbial taxa of the ileum, cecum, and colon of ten adult CtM Hanwoo are shown, determined to the genus level by the linear discriminant analysis (LDA) effect size (LEfSe) method. The LDA scores represent the degree of consistent difference in the relative abundance between groups. For orphan sequences (i.e., unassigned at the genus level), a high‐rank lineage is provided (f, family; o, order).

2. B

   The intestinal branched‐chain amino acid (BCAA) levels in the adult male and CtM Hanwoo (n = 10 in each group). BCAA levels in the luminal contents of the ileum, cecum, and colon were quantified by an enzymatic method using a BCAA assay kit. The lines, boxes, and whiskers in the box plot diagrams represent the median, first, and third quartiles, and min‐to‐max distribution of replicate values, respectively. The scattered dots in the box plot diagrams represent the individual replicates.

Data information: (A) Spearman’s rank correlation coefficients and the corresponding P values were calculated based on comparisons of the relative abundances of 30 discriminant microbial taxa and 32 intestinal metabolites. Holm–Sidak correction was used for multiple comparisons. (B) The data were analyzed by the Mann–Whitney U‐test. *P < 0.05, **P < 0.01, ***P < 0.001. OA, organic acids; S and SA, sugars and sugar alcohols; FA and L, fatty acids and lipids.

Figure 4. Intramuscular metabolomic profiles of the adult male and castrated male (CtM) Hanwoo

1. A

   Representative images of dressed bodies of the adult male and CtM Hanwoo (n = 10 in each group).

2. B

   Dressed body weight, thickness of the dorsal subcutaneous fat, total weight of the extramuscular fat (posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat), and serum levels of the branched‐chain amino acids (BCAAs).

3. C

   Representative images of fresh striploin muscle from the adult male and CtM carcasses (n = 5 in each group).

4. D

   The intramuscular fat area in size‐normalized muscle.

5. E

   The intramuscular metabolome profiles analyzed using GC‐TOF‐MS and clustered by PCoA based on the Bray–Curtis dissimilarity matrix

6. F, G

   The relative abundances of intramuscular metabolites (F) and β‐hydroxybutyrate (3‐HB) (G) in the male and CtM samples.

7. H

   The serum and muscle ketone body levels, quantified enzymatically, as described in the Materials and Methods section (n = 10 in each group).

Data information: The data were analyzed by using the Mann–Whitney U‐test (B, D, F, G, and H). The lines, boxes, and whiskers in the box plot diagrams represent the median, first, and third quartiles, and min‐to‐max distribution of replicate values, respectively. The scattered dots in the box plot diagrams represent the individual replicates. *P < 0.05, **P < 0.01, and ***P < 0.001. CtM, castrated male; BCAAs, branched‐chain amino acids; 3‐HB, β‐hydroxybutyrate.

Figure 5. The intestinal microbiota and metabolic phenotypes of the male and castrated male (CtM) mice

1. A

   Schematic design of the hypogonadism experiments in the mouse model (n = 6 in each group).

2. B

   Serum testosterone levels.

3. C

   PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon, shown according to the different diets and antibiotic treatment.

4. D

   The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plots.

5. E

   The serum branched‐chain amino acid (BCAA) levels quantified enzymatically, as described in the Materials and Methods section.

6. F

   The total weight of the extramuscular fat, including posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat.

7. G, H

   Representative images (G) and weight (H) of the hindlimb intermuscular fat. The images are from a longitudinal section of the hind leg. The fat weight data are presented as a percentage of the body weight.

8. I

   Serum and hindlimb muscle ketone body levels quantified enzymatically.

Data information: The data were analyzed by using the unpaired Student’s t‐test (B, D to F, H, and I) or PERMANOVA with 999 permutations (C). *Comparison of the Sham and CtM mice; #comparison of the LFD and HFD mice; and †comparison of the HFD and HFD + ABX mice. The lines, boxes, and whiskers in the box plot diagrams represent the median, first, and third quartiles, and min‐to‐max distribution of replicate values, respectively. The scattered dots in the box plot diagrams represent the individual replicates. *P < 0.05, **P < 0.01, and ***P < 0.001. LFD, low‐fat diet; HFD, high‐fat diet; ABX, antibiotics; CtM, castrated male; BCAAs, branched‐chain amino acids; 3‐HB, β‐hydroxybutyrate.

Figure 6. The intestinal microbiota and metabolic phenotypes of male mice in response to fecal microbiota transplantation (FMT)

1. A

   Schematic design for the mouse FMT experiments (n = 4–5 in each group).

2. B

   The serum testosterone levels in the Sham‐R and castrated male (CtM)‐R mice.

3. C

   PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon.

4. D

   The discriminant microbial taxa were determined by using the LEfSe and presented using the LDA score.

5. E

   The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plots.

6. F

   Weights of the posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat in the Sham‐R and CtM‐R mice.

7. G, H

   Representative images (G) and weight (H) of the hindlimb intermuscular fat in the Sham‐R and CtM‐R mice. Images were obtained from a longitudinal section of the hind leg. The fat weight data are presented as a percentage of the body weight.

8. I

   Serum and hindlimb muscle ketone body levels, quantified enzymatically.

Data information: The data were analyzed by using the unpaired Student’s t‐test (B, E, F, H, and I) or PERMANOVA with 999 permutations (C). The lines, boxes, and whiskers in the box plot diagrams represent the median, first, and third quartiles, and min‐to‐max distribution of replicate values, respectively. The scattered dots in the box plot diagrams represent the individual replicates. *P < 0.05 and **P < 0.01. LFD, low‐fat diet; HFD, high‐fat diet; ABX, antibiotics; FMT, fecal microbiota transplantation; CtM, castrated male; 3‐HB, β‐hydroxybutyrate.

Figure 7. The metabolic phenotypes and intestinal microbiota of male mice fed branched‐chain amino acid (BCAA)‐supplemented diets

1. A

   Schematic design of the chronic BCAA feeding experiments (n = 6 in each group).

2. B

   Body weight gain in response to the different levels of dietary BCAAs.

3. C

   Representative images of gross anatomy.

4. D

   The total weight of the extramuscular fat (posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat) in the mice fed high‐fat diet (HFD) with or without BCAAs.

5. E

   The serum BCAA levels in the mice fed HFD with or without BCAAs.

6. F

   PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon are shown. The body weight gain data are presented as a percentage of the initial body weight. The fat weight data are presented as a percentage of the body weight.

7. G

   Serum and hindlimb muscle ketone body levels, quantified enzymatically.

Data information: The data were analyzed by using the unpaired Student’s t‐test (B, D, E, and G). *Comparison of the 0% BCAA and 3% BCAA or 5% BCAA‐fed mice. The lines, boxes, and whiskers in the box plot diagrams represent the median, first, and third quartiles, and min‐to‐max distribution of replicate values, respectively. The scattered dots in the box plot diagrams represent the individual replicates. *P < 0.05, **P < 0.01, and ***P < 0.001. HFD, high‐fat diet; BCAA, branched‐chain amino acid; 3‐HB, β‐hydroxybutyrate.

Figure 8

A model for the increased level of body fat storage by intestinal microbial alteration in the castrated cattle.