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Summary: Lactobacillus plantarum and Juvenile Growth

    The research paper entitled "Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition" was published by Schwarzer et al. in the journal Science on February 19th, 2016 (Schwarzer et al., 2016). This paper sought to elucidate the previously unknown role of the intestinal microbiota in juvenile growth. The researchers found that specific lactobacilli strains promote juvenile growth and protect against stunted growth caused by undernutrition. These findings have major implications on the potential prevention of stunted human growth in malnourished treatment by microbial intervention. This paper presented logical and thorough experiments to investigate its hypotheses. Each conclusion presented in the paper was well supported from the given data. Additionally, each figure was clearly presented with useful labels and simple designs. My major complaint is with the flow of the manuscript, which jumps from figure to figure instead of introducing the data sequentially. A rearrangement of the figures or the manuscript could have alleviated this problem and improved readability.

Figure 1

    To identify the general role of the microbiota in juvenile growth, wild-type (WT) and germ-free (GF) mice were fed a standard diet until 8 weeks of age (56 days after birth). Despite similar amounts of food consumption (as shown in a supplementary figure), WT animals exhibited greater body weights and body lengths than GF animals. Figure 1A displays body weight over time, and Figure 1C displays body length over time for both WT and GF mice. Figure 1B and 1D are quantifications of part A and C, respectively. The researchers observed statistically significant differences in both body weight and body length after weaning between WT and GF mice. Despite these differences, GF and WT animals demonstrated no significant differences in adiposity, cortical bone density, or leptin levels (from the supplementary figures). Figures 1E and 1F display representative femur bones of WT and GF mice. The researchers found that several bone growth parameters, including femur length and cortical thickness, were reduced in GF mice. Together, these data suggest that the microbiota is an active component in stimulating juvenile growth.


Figure 2

    Mammalian growth is by the somatotropic axis, a hormonal system in which growth hormone (GH) stimulates the liver and other tissues to secrete insulin-like growth factor-1 (IGF-1) which in turn stimulates growth. To determine whether the microbiota maintains the activity of the somatotropic axis, serum levels of GH, IGF-1, and IGFBP-3 (an IGF-1 carrier protein) were determined by ELISA (Enzyme-Linked Immunosorbant Assay). Expression levels of Igf-1 and Igfbp3 were determined in the liver by qRT-PCR.The researchers did not observe significant differences in serum GH between WT and GF mice (Figure 2A). However, GF mice exhibited a significant reduction in IGF-1 and IGFBP-3 at multiple time points (Figures 2B and 2C). Igf-1 and Igfbp3 transcript levels were also significantly reduced in GF mice (Figures 2D and 2E).

    Phosphorylation of AKT serves as a marker of IGF-1 receptor (IGF-1R) activity, a downstream marker of the somatotropic axis.The ratio of phosphorylated AKT (pAKT) to total AKT (tAKT) was determined by densiometric analysis of Western Blot data. Representative Western Blots of WT and GF animals are displayed in Figure 2F along with the ratios of pAKT to tAKT for the two groups. The researchers found increased phosphorylation of AKT in WT mice, indicating greater somatotropic axis activity in WT mice. Taken together, these data provide evidence that the microbiota modulates the activity of the somatotropic axis. This finding alludes to a potential mechanism of microbiota-induced juvenile growth, which is further explored  in a later figure.


Figure 3

    From here, the researchers characterized the contribution of the gut microbiota to juvenile growth under the condition of chronic undernutrition. For the undernutrition treatment, animals were fed a non-standard animal chow with low protein, fat, and vitamins. Figure 3A shows that after weaning at Day 21, mice receiving the nutritionally depleted diet demonstrated a dramatic reduction in body weight. GF mice receiving the nutritionally deficient diet demonstrated a greater reduction in body weight than WT mice. Figure 3C shows similar reductions in growth in response to the depleted diet as measured by body length. Once again, GF mice demonstrated a greater reduction in body length in response to undernutrition. Whole mice and femurs from WT and GF mice on the depleted diet are shown in Figures 3B and 3D. 

    Previous work from this group demonstrated that the introduction specific lactobacilli strains could rescue deficits in juvenile growth in GF Drosophila Melanogaster. As such, the researchers hypothesized that lactobacilli strains were responsible for the effects of the microbiota on juvenile growth and on the somatotropic axis. To test this hypothesis, the researchers generated two strains of monocolonized mice with one of two strains of Lactobacillus plantarum, dubbed Lp-WJL and Lp-NIZ02877.

    As Figure 3A,C, and D demonstrates, the monocolonized mouse strains grow faster (defined by body weight, body length, and femur length) than GF mice in the standard and depleted diet. Furthermore, Lp-WJL mice grew faster than Lp-NIZ02877 mice, indicating that juvenile growth depends on the specific Lactobacillus plantarum strain. Together, this figure demonstrates that specific lactobacilli support juvenile growth and protect against the effects of undernutrition.

  


Figure 4

    From here, the researchers investigated the effects of the microbiota on the somatotropic axis during undernutrition. As in Figure 2, serum levels of GH, IFG-1, and IGFBP-3 were quantified via ELISA (Figures 4A-C). IGF-1 and IGFBP-3 levels were significantly reduced in GF mice at 56 days after birth, which GH was enriched in GF serum at 28 days after birth. This data indicates reduced somatotropic axis activity in GF mice during undernutrition.

    In Figure 4D-G, WT mice were treated with picropodophyllin (PPP), a selective IGF-1R inhibitor. Dimethyl Sulfoxide (DMSO)  was used as a vehicle control. Weight gain, body length, and femur length were all significantly reduced in WT mice in response to PPP, indicating that IGF-1 signaling is necessary for juvenile growth. In the depleted diet group, PPP treatment reduced femur length in WT mice, indicating that IGF-1 signaling is necessary for growth even during undernutrition. 

    The two monocolonized mice strains were also tested for GH, IGF-1, and IGFBP-3 in both the standard diet and depleted diet (Figures 4A-C).


Reference
Schwarzer, M., Makki K., Storelli, G., et al. (2016). Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition. Science 315(6275): 854-857.

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