Lipopolysaccharide Potentiates Insulin-Driven Hypoglycemic Shock

Abstract

Critically ill patients typically present with hyperglycemia. Treatment with conventional insulin therapy (targeting 144-180 mg/dl) improves patient survival; however, intensive insulin therapy (IIT) targeting normal blood glucose levels (81-108 mg/dl) increases the incidence of moderate and severe hypoglycemia, and increases mortality. Septic patients are especially prone to IIT-induced hypoglycemia, but the mechanism remains unknown. Here, we show that codelivery of insulin with otherwise sublethal doses of LPS induced hypoglycemic shock in mice within 1-2 h. LPS impaired clearance of insulin, which amplified insulin receptor signaling. These effects were mediated by caspase-11, TLR4, and complement, each of which trigger eicosanoid production that potentiates insulin signaling. Finally, in an animal model of sepsis, we observed that Salmonella typhimurium-infected mice exhibited simultaneous impaired insulin clearance coexisting with insulin resistance. Our results raise the possibility that septic patients have impaired insulin clearance, which could increase their susceptibility to hypoglycemia during IIT, contraindicating its use.

Fig. 1. TLR4 and caspase-11 each contribute to lethality in primed mice challenged with LPS

Survival of poly(I:C) primed mice challenged with LPS (54,000 μg/kg) in DPBS (without insulin). Data are pooled from 2 experiments.

Fig. 2. LPS delivered in OptiMEM rapidly induces shock

(A)Survival of mice challenged with LPS (400 μg/kg) in DPBS, DMEM, or OptiMEM. (B)Survival of mice challenged with OptiMEM with or without LPS (400 μg/kg). (C and D) Core temperatures (C) and blood glucose concentrations (D) at 1h of mice after challenge with LPS in DBPS or OptiMEM as described in (A). Survival data were pooled from 2 experiments. Temperature and glucose data are representative of 2 independent experiments. Error bars, mean ± SD. Statistically significant differences were determined by a 2-tailed unpaired t-test; * P ≤ 0.05.

Fig. 3. Co-delivery of LPS and insulin drives hypoglycemic shock

(A) Survival of mice challenged with LPS (400 μg/kg), insulin, or both. (B and C) Core temperatures (B) and blood glucose concentrations (C) of mice 1 h after challenge as in (A). (D) Survival of mice challenged with Pam₃CSK_{4 (}1 mg/kg) with or without insulin. (E) Survival of mice after challenge with LPS (400 μg/kg) and insulin and subsequent control or glucose injection. (A - E) Pooled data from 2 independent experiments. Error bars, mean ± SD. Statistically significant differences were determined by ANOVA and Tukey's multiple comparisons test; * P ≤ 0.05.

Fig. 4. LPS impairs insulin clearance and amplifies insulin receptor signaling

(A and B) Plasma TNF and IL-6 concentrations from mice 1 h after DBPS injection or challenge with LPS (400 μg/kg), insulin, or both. Data pooled from 2 experiments. (C - H) AKT and insulin receptor phosphorylation observed at 1 h by Western blot in mice challenged as in (A). Western blots are representative of 3 or more (liver and quadricep AKT, liver IR) or 2 (quad IR) independent experiments. Densitometry data are pooled from 3 or more (D, E, G) or 2 (H) experiments. (I) Plasma insulin and C-peptide concentrations at 1 h in mice challenged with insulin and C-peptide with or without LPS. Pooled data from 2 experiments. Error bars, mean ± SD. Statistically significant differences were determined by a 2-tailed unpaired t-test; * P ≤ 0.05.

Fig. 5. LPS enhances insulin signaling via caspase-11, TLR4, and complement

(A) Blood glucose concentrations over time in mice treated with insulin. Pooled data from 2 experiments. (B and C) Survival of mice after challenge with insulin and LPS (B, 2.5 μg/kg; C, 40 μg/kg). Pooled data from 2 experiments. (D - G) Phosphorylation of liver AKT (D, E) and insulin receptor (F, G) determined by Western blot 1 h after challenge as in (B). Densitometry data are pooled from 3 experiments. (H, I) Survival of control or CVF treated mice after challenge with LPS (40 μg/kg) and insulin. Pooled data from 2 experiments. Error bars, mean ± SD. Statistically significant differences were determined by a 2-tailed unpaired t-test; * P ≤ 0.05.

Fig. 6. Eicosanoid signaling acts downstream of LPS to amplify insulin activity

(A) Survival of DMSO or indomethacin treated mice after challenge with LPS (2.5 μg/kg) and insulin. (B and C) Core temperatures (B) and blood glucose concentrations (C) of mice 1 h after challenge as described in (A). (D - G) Phosphorylation of liver AKT (D, F) and insulin receptor (E, G) determined by Western blot 1 h after challenge as described in (A). (H) Plasma insulin concentrations of mice 1 h after treatment as in (B). (I) Plasma prostaglandin concentrations in mice 1h after challenge with DBPS, LPS (400 μg/kg), insulin, or LPS and insulin. (J) P values for the indicated comparisons of data from (J) as determined by ANOVA and Tukey's multiple comparisons test. (K) Survival of mice challenged with insulin and LPS (10 μg/kg). Data are pooled from at 3 (A, K) or 2 experiments (B – I). Error bars, mean ± SD. Statistically significant differences were determined by a 2-tailed unpaired t-test; * P ≤ 0.05.

Fig. 7. Septic mice exhibit simultaneous insulin resistance and impaired insulin clearance

Mice were injected with PBS or S. Typhimurium. After 5 days they were challenged with insulin. (A) Change in blood glucose of control and infected mice before and 1h after insulin challenge. (B – E) Liver AKT and insulin receptor phosphorylation observed 1 h after insulin challenge in mice by Western blot. Western blots are representative of 2 independent experiments. Densitometry data are pooled from 2 experiments. (F) Plasma insulin concentrations 1 h after insulin challenge. Pooled data from 2 experiments. Error bars, mean ± SD. Statistically significant differences were determined by a 2-tailed unpaired t-test; * P ≤ 0.05.