This web page was produced as an assignment for an undergraduate course at Davidson College.

Assignment #2: Summary of Schwarzer et al., 2016

"Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition"

Schwarzer et al. (2016) characterized the effect of gut microbiota on juvenile mouse growth during both normal breeding and depleted diets. The researchers used wild-type and germ-free mice experiments and measured juvenile weight, body length, and femur length as markers of infant growth. For both diet types, wild-type mice with intact microbiota displayed more juvenile growth than germ-free mice. Additionally, the researchers examined the growth effects of two specific strains of Lactobacillus plantarum, Lp^WJL and Lp^NIZO2877, and found that only monocolonized Lp^WJL mice grew like wild-type mice under both diet conditions. The researchers attributed the growth benefits of the gut microbiota to increased somatotropic axis activity, and demonstrated this by finding increased expression level of major somatotropic axis genes in both wild-type and Lp^WJL mice compared to germ-free mice, even on depleted diets. Therefore, gut microbiota and Lp^WJL alone promotes juvenile growth even during chronic malnutrition through increasing the activity of the somatotropic axis. These findings highlight the importance of intact gut microbiota on infant growth, especially in cases of chronic malnutrition.

I think this is a well-written, thorough, and logical paper. The researchers’ hypotheses and experimental questions are easy to understand and follow as a reader. I believe that the researchers provide ample evidence to demonstrate that the gut microbiota promotes juvenile growth through enhancing the somatotropic axis, and this effect persists even during a depleted diet. However, I did not think a few figure panels were necessary. For instance, I believe it was a waste of “real estate” to include Figure 1F, because they only include this phenotype measurement once, and Figure 3B, because Figure 3C provides the same and more information. The authors addressed numerous interesting and important supplementary figures, and could have included them in this article if they did not use some of the redundant figure panels. Additionally, it would strengthen the researchers’ argument to include statistical analysis for Figure 3A and 3C to discriminate between the very small differences that they claim are substantial, such as the body lengths of mice on the breeding diet.

To determine the effect of gut microbiota on infant mice health, the researchers analyzed the weight, body length, and femur bones of wild-type and germ-free juvenile mice fed a normal breeding diet. Germ-free mice significantly weighed less (A), gained less weight per day (B), had shorter bodies (C), and grew in length less per day (D) than wild-type mice with intact gut microbiota. Additionally, germ-free mice had shorter femur bones (E) with reduced cortical thickness (F). Therefore, even in infant mice fed a normal breeding diet, gut microbiota promote juvenile growth.

The somatotropic axis is known to drive postnatal growth through the pituitary gland secreting GH, GH increasing expression of IGF-1, and IGF-1 and IGFBP-3 binding to stimulate somatic growth. Therefore, the researchers hypothesized that the gut microbiota promotes somatotropic activity to support infant growth. They measured the three main somatotropic genes’ protein levels in the serum (A-C) and mRNA expression in the liver (D, E) of wild-type and germ-free mice fed a normal breeding diet. Gene mRNA expression was normalized to Tbp (TATA box binding protein) to control for expression activity differences. Germ-free mice had significantly less IGF-1 and IGFBP-3 mRNA expression and protein levels at least 28 days after birth. Finally, they measured phosphorylation of a specific AKT, which is a marker for IGF-1 binding to its receptor, and found that germ-free mice had significantly less AKT phosphorylation. In all, this figure supports their hypothesis that the gut microbiota supports infant growth through increasing the activity of the somatotropic axis, specifically by increasing IGF-1 and IGFBP-3 expression and IGF-1 binding to its receptor.  

The researchers addressed three questions in this figure: (1) does the gut microbiota promote juvenile growth during chronic malnutrition, (2) does the bacterium Lactobacillus plantarum specifically protect juvenile growth, and (3) is L. plantarum’s protective effects strain specific? The researchers chose to investigate L. plantarum (Lp^WJL and Lp^NIZO2877) because they previously determined that the bacterium promotes growth in Drosphilia. The researchers analyzed wild-type, germ-free, monocolonized Lp^WJL, and monocolonized Lp^NIZO2877 mice on either breeding or depleted diets. They obtained monoclonized infant mice by exposing germ-free adults to the respective bacterial strain for 20 days and then mating the monocolonized adults. The researchers measured infant mouse weight, body length, and femur length. Wild-type and Lp^WJL mice had increased growth in all growth categories compared to germ-free and Lp^NIZO2877 mice within diet categories. All mice fed the breeding diets had more growth than those with depleted diets. The differences in infant growth are not apparent until after weaning, because infants are then reliant on solid food. Ultimately, this figure demonstrates that the gut microbiota is protective during a depleted diet and Lp^WJL is more protective than Lp^NIZO2877, so the growth effect of Lp in mice is strain specific.

Figure 4 A-C compares the protein levels of the three main somatotropic genes in the serum of wild-type, germ-free, Lp^WJL, and Lp^NIZO2877 mice fed the depleted diet after weaning. The protein levels in Lp^WJL mice mostly matched those in wild-type levels, which were both significantly greater than those in germ-free mice, and the protein levels in Lp^NIZO2877 and germ-free mice were similar. Thus, wild-type and Lp^WJL mice promote the somatotropic axis even during malnutrition. Additionally, Lp^WJL alone is nearly sufficient to rescue promotion of the somatotropic axis and therefore juvenile growth, whereas Lp^NIZO2877 has no effect on somatotropic activity. To further confirm that the somatotropic axis directly influences juvenile growth, the researchers treated wild-type mice with PPP, a specific inhibitor of the IGF-1 receptor, for 10 days (D-G). They found that PPP-injected mice have significantly less overall growth than the vehicle control (DMSO) mice when the mice are on a breeding diet, and that PPP-injected mice have significantly smaller femurs than the control on a depleted diet. These results support that the gut microbiota upregulates the somatotropic axis even during chronic undernutrition and that the somatotropic axis directly promotes juvenile mouse growth.