Does lipopolysaccharide (LPS), also known as endotoxin, cause obesity? Reasons to think it might (in mice at least) come from a high-profile paper published in the journal Diabetes in 2007 and titled “Metabolic Endotoxemia Initiates Obesity and Insulin Resistance” (cited 3045 times according to Google Scholar, which is a lot).
LPS makes up a key part of the cell wall of many types of bacteria that live in the intestines and when these bacteria die the LPS is released into the gut. The bacteria in the gut contain a lot of LPS, which is okay as long as it stays in the gut. LPS triggers an inflammatory response if it gets into the body and is one of the main molecules that the body senses to detect the presence of bacteria.
Briefly, the authors of this study reported that feeding mice a high-fat diet* makes them fat and increases the amount of LPS leaking through into the blood from the gut. Mice with a gene knockout (CD14) that prevents the mice responding to LPS did not get as fat.
*”Mice were fed a control (A04, Villemoisson sur Orge, France) or a high-fat, carbohydrate-free diet for 2 or 4 weeks following protocols. The diet contained 72% fat (corn oil and lard), 28% protein, and <1% carbohydrate as energy content (17).”
The high-fat diet used in this study was a bit unusual but that is beyond this blog post.
This is quite a long and complex paper, but let’s focus on one of the key findings. Infusing LPS into mice over four weeks increased mouse body weight and body fat (highlighted in yellow). This seemed to confirm a direct role for LPS in making the mice fatter.
In a follow-up paper, the authors hypothesised that LPS might play a key role in the development of metabolic disorders.
Is this replicable?
While exciting findings make for big papers it is important to know whether these results have been replicated. This was not easy to find and it probably would have altered my PhD research had I found the following rather earlier.
I discovered, much later, that the original authors had repeated this LPS infusion study in a later 2013 paper titled “Chronic endocannabinoid system stimulation induces muscle macrophage and lipid accumulation in type 2 diabetic mice independently of metabolic endotoxaemia“. In this study, they infused LPS over six weeks into mice fed both a control diet and a high-fat diet.
It appeared that they could not replicate the effects seen in the first study, although the wording of the text does not make this obvious.
If you look at Table 2 there were no significant effects of LPS infusion on body fat or weight gain compared to control mice (CT). Neither were there any significant effects of LPS in high-fat diet fed mice.
While the highlighted text says that LPS increased weight gain by 44%, if you look at the figures in Table 2 LPS treatment only increased the body weight gain by 24%, which was not significant (this could be a typo in the paper). While LPS did “increased the adipose tissue weight compared with the control mice” this was only by 0.03 grams (30 milligrams), not a meaningful difference. Effects of LPS on blood glucose or insulin were also not statistically significant.
Has anyone else repeated these results?
For a long time after, I struggled to find other studies that had tried to repeat these findings. Less exciting results tend to be less emphasised in papers or get published in less prominent journals. I eventually found them by searching for the brand of osmotic minipump that was used to infuse the LPS into mice or rats. So what papers did I find?
Vila, 2014: Four weeks of LPS infusion…
“This treatment induced a 2-fold increase in LPS plasma level (Figure 7A), with no change in body weight (Figure S7A).”
Liang, 2014: Sixteen weeks of LPS infusion…
“Table 1 shows data of risk factors that are typically associated with NASH development in humans, for example, body weight, visceral fat mass, plasma lipids, fasting glucose and insulin, and ALAT. These risk factors were not affected by LPS.”
Iwashita, 2013: Four weeks of LPS infusion…
“Body weights of all mice rose slightly for 4 weeks and the differences between groups were not significant (Fig. 1A). While fasting plasma glucose levels differed slightly among the groups after 4 weeks of LPS or PBS infusion, the differences did not reach statistical significance (Fig. 1Bi). There were no significant differences in GTT results among the groups (Fig. 1Bii).”
“Subsequently, the effect of a higher dose (1.0 ng/g/h) of LPS for 4 weeks was also investigated. None of the groups showed alterations in body weight or fasting glucose concentrations (data not shown).”
Nøhr, 2016: Twenty-eight days of LPS infusion…
“In the first few days following implantation of osmotic mini-pumps, LPS mice regardless of resveratrol dropped (≈ 10%) in body weight (Fig 1B). After 28 days of treatment, no differences in body weight were seen between the groups (Fig 1C).”
I found one paper that seemed at first to support the original findings in mice.
Ahn, 2015: Four weeks of LPS infusion…
“However, body weight and relative weight of epididymal fat were increased in the LPS group compared with the NC group (Fig. 1B and C). The area of fat tissue shown by MRI also increased (Fig. 1D). Interestingly, as shown by the results of H&E staining, the epididymal fat tissue in the LPS group showed hypertrophy (Fig. 1E), whereas there were no significant differences in other tissues (Supplementary Fig. 2).”
However, when looking at the results of this study the differences between groups is small.
These differences were quite small and it is worth being cautious of data presented as percentage weight change without any details of the actual body weight.
Hsieh, 2008: Four weeks of LPS infusion…
“As shown in Table 2, there were no significant differences in body weight, mean arterial blood pressure, fasting plasma glucose and insulin, AST, ALT, and albumin levels between the two groups during the study period.”
Wu, 2012: Fourteen days of LPS infusion…
“On day 14 after IP infusion of the endotoxin, there is no significant change in body weight (266±3 vs. 260±6 g, P>0.05, n=16), body temperature (36±1 vs. 37±2°C, P>0.05, n=16), daily food (22±5 vs. 23±5 g/day, P>0.05, n=16), or water (47±5 vs. 50±7 mL/day, P>0.05, n=16) intake between the saline- and LPS-treated animals. Chronic.”
Fischer, 2015: Four weeks of LPS infusion…
“Our results show that LPS infusion in male rats for 4 weeks neither increased body weight nor affected insulin sensitivity.”
“Furthermore, there was no difference in fat mass between groups…”
La Serre, 2015: Six weeks of LPS infusion…
“…there was no significant difference in final body weight between LPS-treated and LF-fed rats.”
“Chronic LPS treatment led to a redistribution of adiposity characterized by a decrease in epidydimal fat (8.8 ± 0.2 vs. 10.7 ± 0.8 g, p < 0.05) and an increase in mesenteric fat (8.3 ± 0.4 vs. 7.0 ± 0.2 g, p < 0.05) (Fig. 2C).”
In La Serre, 2015 total body fat remained about the same but there were small differences in the weight of different body fat stores. However, the reduction in epididymal fat is contrary to that found by Ahn, 2015 above.
Out of the five mouse studies and four rat studies that had both infused LPS, only one showed any increase in body weight and body fat, and that was small. The others studies reported no increase in body weight or fat after LPS infusion. This suggests that LPS is not a reliable way to make rodents fat.
I think this is a good example of negative results being rather difficult to find in the scientific literature, even while the original paper which showed a positive result retains a high profile and remains highly cited. Less interesting results tend to be relegated to a sentence in the results section that easily goes unnoticed, while the papers themselves focus on some other aspect of the study.