CD14 deficiency impacts glucose homeostasis in mice through altered adrenal tone

Abstract

The toll-like receptors comprise one of the most conserved components of the innate immune system, signaling the presence of molecules of microbial origin. It has been proposed that signaling through TLR4, which requires CD14 to recognize bacterial lipopolysaccharide (LPS), may generate low-grade inflammation and thereby affect insulin sensitivity and glucose metabolism. To examine the long-term influence of partial innate immune signaling disruption on glucose homeostasis, we analyzed knockout mice deficient in CD14 backcrossed into the diabetes-prone C57BL6 background at 6 or 12 months of age. CD14-ko mice, fed either normal or high-fat diets, displayed significant glucose intolerance compared to wild type controls. They also displayed elevated norepinephrine urinary excretion and increased adrenal medullary volume, as well as an enhanced norepinephrine secretory response to insulin-induced hypoglycemia. These results point out a previously unappreciated crosstalk between innate immune- and sympathoadrenal- systems, which exerts a major long-term effect on glucose homeostasis.

Figure 1. Body weight, adiposity, and activity in wild-type and CD14-ko mice.

(A) Growth curves of mice on ND and HFD; NMR analysis of absolute lean mass (B) and fat mass (C) as a percentage of total dry mass. (D) Stereotypic movement determined by consecutive breaks in the same beam path in 26 week old mice on ND. (E) Ambulatory activity as estimated by sequential beam breaks in the x-y axis over a 72-hour period after 24-hour acclimatization in 26 week old mice on ND. Data presented in panels A–D are mean±SEM. (n = 8 mice per group, n = 4 mice for activity measurements). Data in E is a representative experiment which was reproduced 4 times with similar results.

Figure 2. Effects of disruption of the LPS signaling pathway on glucose tolerance.

Glucose (A,C) and insulin (B,D) tolerance curves for 26-week (A,B) and 52-week old (C,D) wild-type and CD14-ko mice on normal and HFD diets. Statistical differences between groups were determined by 2-way ANOVA, using Bonferroni multiple comparisons test. n = 8 mice per group. The values for basal glucose (mean and SEM in mg/dl, n = 8) for wild-type and CD14-ko were: 26 weeks on ND: 80±3 and 77±4; 26 weeks on HFD: 107±8 and 108±4; 52 weeks on ND: 92±1.5 and 104±4; 52 weeks on HFD: 115±5 and 132±5. None of the differences were significant as a function of phenotype.

Figure 3. Effects of disruption of the LPS signaling pathway on counter-regulatory response to hypoglycemia.

Norepinephrine levels in A) serum of wild type and CD14-ko 26 week old mice on ND following injection i.p. with insulin (1–1.5 U/kg body weight) *p<0.05, n = 6; B) urine collected over a 24 h period ***p<0.001, n = 8). C) Area under the curve of the glucose excursions for WT and CD14-ko mice following saline or propranolol (2 mg/kg of body weight) injection 30 minutes prior to insulin tolerance tests. **p<0.01 n = 4 for each group. Statistical differences were determined by student t-tests.

Figure 4. Effects of disruption of the LPS signaling pathway on adrenal gland morphology.

A) Adrenal glands were excised, fixed and serially sectioned, and representative sections of a gland from a WT and a CD14-ko mouse are shown. The medulla is outlined by the slashed line. B) Every 10th section was photographed, and in each image the entire gland was pseudocolored yellow, and the medulla was pseudocolored orange. The sum of the areas of all sections were used to estimate total (C) and medullary (D) adrenal volumes in the glands from each genotype. *p<0.05 determined by student t-test, n = 4 adrenal glands per group.

Figure 5. Effect of free fatty acids on peritoneal macrophages.

A) IL6 secretion from peritoneal macrophages obtained from wild type or CD14-ko mice incubated in the absence or presence of the indicated concentrations of BSA alone, BSA-conjugated fatty acids, or LPS in the presence of increasing concentrations of palmitic acid. B) Oxygen consumption by C2C12 cells incubated in the absence or presence of the indicated concentration of BSA-conjugated fatty acids, C) ATP levels in peritoneal macrophages obtained from wild type mice incubated in the absence or presence of the indicated concentrations of BSA alone or BSA-conjugated fatty acids. Shown are the means of duplicates from representative experiments which were repeated a minimum of three times with similar results.