Endotoxins are associated with visceral fat mass in type 1 diabetes

Bacterial lipopolysaccharides (LPS), potent inducers of inflammation, have been associated with chronic metabolic disturbances. Obesity is linked to dyslipidemia, increased body adiposity, and endotoxemia. We investigated the cross-sectional relationships between serum LPS activity and body adiposity as well as inflammation in 242 subjects with type 1 diabetes. Body fat distribution was measured by DXA and serum LPS activity by the limulus amebocyte lysate end-point assay. Since no interaction between visceral fat mass and sex was observed, data were pooled for the subsequent analyses. LPS was independently associated with visceral fat mass, when adjusted for traditional risk factors (age, sex, kidney status, hsCRP, insulin sensitivity). In the multivariate analysis, serum LPS activity and triglyceride concentrations had a joint effect on visceral fat mass, independent of these factors alone. A combination of high LPS and high hsCRP concentrations was also observed in those with the largest visceral fat mass. In conclusion, high serum LPS activity levels were associated with visceral fat mass in subjects with type 1 diabetes strengthening its role in the development of central obesity, inflammation and insulin resistance.


Subjects and Methods
Data from individuals with type 1 diabetes were collected during the Finnish Diabetic Nephropathy (FinnDiane) Study visits taking place in Helsinki between years 2011 and 2016. In the current analyses, we included data from all individuals with completed LPS and body composition measurements, and either normal urinary albumin excretion rate (T1D-normo, n = 156), microalbuminuria (T1D-micro, n = 42) or macroalbuminuria (T1D-macro, n = 46). Renal status was assessed by the albumin excretion rate (AER) in two out of three consecutive timed urine collections using the following criteria: normal AER < 20 μ g/min or < 30 mg/24 h, and macroalbuminuria ≥ 200 μ g/min or ≥ 300 mg/24 h. Kidney transplant recipients and those undergoing dialysis were excluded from the analysis. Type 1 diabetes was defined as disease onset before the age of 40 and the initiation of insulin injections within one year of diagnosis.
Body composition was measured by DXA (Lunar iDXA by GE Healthcare, Scanex Medical Systems, Finland). The iDXA software calculates the body fat distribution including visceral-, android-, and gynoid fat mass. BMI was calculated (weight/height 2 ). Insulin sensitivity was assessed calculating the estimated glucose disposal rate (eGDR): eGDR = 24.4 − 12.97 × WHR − 3.39 × AHT − 0.60 × A1C, where WHR stands for waist-to-hip ratio and AHT for antihypertensive treatment and/or blood pressure ≥ 140/90 mmHg (yes = 1, no = 0). Daily insulin dose was self-reported. Serum lipid concentrations were analyzed centrally by automated enzymatic methods (Hoffmann-La Roche, Basel, Switzerland). Serum high-sensitive C-reactive protein (hsCRP) concentration was measured by immunoassay (Modular analyzer, Roche). LPS activity was measured by the Limulus amebocyte lysate (LAL) assay from 1:5 diluted fasting serum samples (Hycult Biotechnology, Uden, the Netherlands). For the LAL assay, inter-and intra-assay coefficients of variation were 16.1% and 4.5%, respectively. The sensitivity limit for the assay was 0.02 EU/ml. Statistical analyses. Normality of variable distribution was assessed with the Kolmogorov-Smirnov test.
Normally distributed variables are reported as mean ± standard deviation, non-normally distributed variables as median [25 th -75 th quartile]. Correlation coefficients were calculated by Spearman or Pearson's correlation test as appropriate. Differences in frequencies were assessed by Pearson's Chi squared analysis. Differences in variation between groups were calculated using Mann-Whitney U-test, Kruskal-Wallis test and ANOVA, as appropriate. The population was divided into tertiles based on serum LPS activity, triglyceride-, or hsCRP concentrations to further explore the role of endotoxins, dyslipidemia, and inflammation on visceral fat mass.
The interaction between LPS and gender on visceral fat mass was assessed using a generalized linear model. Since no interaction was evident, all data were analyzed together. The interaction between LPS and triglyceride concentration on visceral fat mass was analyzed similarly. As LPS and triglyceride concentration showed significant interaction, the interaction term was included in the multivariable model. Because LPS and triglycerides are highly correlated, we furthermore visualized their relative relationship using the generalized additive modeling (GAM) without a priori assumptions of the shape of the relation. The GAM modelling allows the inclusion of non-parametric smoothing functions to identify a potential non-linearity in the relationship between the independent and the dependent variables 14,15 . The generalized cross-validation function (GCV) was used as a criterion

Ethics. The study protocol was approved by the Ethics Committee of the Hospital District of Helsinki and
Uusimaa. The study was carried out in accordance with the approved guidelines. Participants gave their written informed consent prior to participating in the study.

Results
Patient characteristics divided by renal status are shown in Table 1. The proportion of men was highest among T1D-macro. The diabetes duration and visceral fat mass increased with worsening renal status. Insulin sensitivity (eGDR) was highest in T1D-normo. No differences were observed in HbA 1c , HDL cholesterol concentration, LPS activity, insulin dose, use of insulin pump, BMI or hsCRP concentration amongst the three groups. Serum LPS activity correlated positively with BMI (r = 0.274, p < 0.001), visceral fat mass (r = 0.248, p < 0.001), android to gynoid fat ratio (r = 0.231, p < 0.001), serum triglyceride concentration (r = 0.477, p < 0.001), and hsCRP concentration (r = 0.276, p < 0.001). Negative correlation was observed between insulin dose and eGDR (r = − 0.205, p = 0.002). Supplementary Figure S1a and S1b show that triglyceride concentration seems to overrun the effect of LPS on the visceral fat mass. However, the relationship between LPS and triglycerides is complicated and interaction between LPS and triglyceride concentration was found, indicating that LPS has an effect on certain levels of triglyceride concentration. Therefore, the effect of serum LPS activity and triglyceride concentration on visceral fat mass were further explored in LPS and triglyceride tertiles (low/medium/high). Subjects in the high triglyceride tertile presented the highest visceral fat mass. Notably, within each LPS tertile, the visceral fat mass was observed to increase towards the higher TG tertiles (p < 0.001, all) (Fig. 1A). Moreover, the visceral fat mass among those with high LPS activity and high triglyceride level was higher compared to the group with both low LPS activity and low triglyceride level (high/high vs. low/low, median  (Fig. 1B).
The independent association between LPS activity and visceral fat mass was analyzed in a number of multivariable models ( Table 2)

Discussion
Inflammation and insulin resistance are intertwined processes that are strongly linked to overweight and intra-abdominal obesity. We observed that serum LPS activity is associated with visceral fat mass in type 1 diabetes, independent of the traditional risk factors such as age, insulin sensitivity, and inflammation.
High serum LPS activity has been associated with central obesity and insulin resistance, and it has also been shown to predict the development of type 2 diabetes 6,17 . Intestinal and oral microbial communities are the most likely sources of bacterial endotoxins in humans. Frequent use of antibiotics may have adverse effects on gut homeostasis accompanied by dysbiosis and increased intestinal permeability of bacterial compounds. Childhood hospitalizations related to infections have been associated with an increased risk of obesity and metabolic syndrome in adulthood 18 . In conditions of intestinal inflammation, such as Crohn's disease, visceral fat accumulation is evident 19 . Dietary fat intake may increase systemic LPS activity, driving adipose inflammation 20 . In a recent study, circulating LPS activity levels correlated with intra-abdominal fat volumes, but only modestly with the subcutaneous fat volumes in patients undergoing bariatric surgery. Notably, a significant decrease in serum endotoxin levels was still evident one year after the operation 21 . Higher CRP levels may arise through a local inflammation of the adipose tissue associated with an increased bacterial burden 19 .
Serum triglycerides and LPS are strongly correlated 9 . Indeed, we observed an interaction between LPS and triglycerides on visceral fat mass. Importantly, the interaction term was independent of the individual factors alone, indicating that the combination of high serum LPS and triglycerides have additive effects on visceral fat mass. This observation is in concordance with previous rat studies, showing that a low dose intraperitoneal LPS injection increases liver triglyceride production, while high doses decrease triglyceride-rich lipoprotein clearance 22 . Moreover, in healthy volunteers, a small intravenous dose of LPS acutely increased serum triglyceride levels 23 , while in mice a subcutaneous infusion of LPS for four weeks significantly increased visceral fat mass 4 . Cross-sectional nature and small study population are potential limitations of the current study. Moreover, due to the exclusion of individuals with end-stage renal disease, the results may not be generalizable to these individuals. Prospective studies are needed to establish the causalities related to the observations.
In conclusion, independent of traditional risk factors, serum LPS activity is associated with visceral fat mass in type 1 diabetes. The LPS-association with visceral fat is pronounced at high triglyceride concentrations. This strengthens the hypothesis that both bacterial endotoxins and dyslipidemia contribute to the development of central obesity, inflammation and insulin resistance and thus may subsequently be associated with adverse cardio-metabolic outcomes.