Effects of glycemic control on glucose utilization and mitochondrial respiration during resuscitated murine septic shock

Abstract

Background: This study aims to test the hypothesis whether lowering glycemia improves mitochondrial function and thereby attenuates apoptotic cell death during resuscitated murine septic shock.

Methods: Immediately and 6 h after cecal ligation and puncture (CLP), mice randomly received either vehicle or the anti-diabetic drug EMD008 (100 μg · g(-1)). At 15 h post CLP, mice were anesthetized, mechanically ventilated, instrumented and rendered normo- or hyperglycemic (target glycemia 100 ± 20 and 180 ± 50 mg · dL(-1), respectively) by infusing stable, non-radioactive isotope-labeled (13)C6-glucose. Target hemodynamics was achieved by colloid fluid resuscitation and continuous i.v. noradrenaline, and mechanical ventilation was titrated according to blood gases and pulmonary compliance measurements. Gluconeogenesis and glucose oxidation were derived from blood and expiratory glucose and (13)CO2 isotope enrichments, respectively; mathematical modeling allowed analyzing isotope data for glucose uptake as a function of glycemia. Postmortem liver tissue was analyzed for HO-1, AMPK, caspase-3, and Bax (western blotting) expression as well as for mitochondrial respiratory activity (high-resolution respirometry).

Results: Hyperglycemia lowered mitochondrial respiratory capacity; EMD008 treatment was associated with increased mitochondrial respiration. Hyperglycemia decreased AMPK phosphorylation, and EMD008 attenuated both this effect as well as the expression of activated caspase-3 and Bax. During hyperglycemia EMD008 increased HO-1 expression. During hyperglycemia, maximal mitochondrial oxidative phosphorylation rate was directly related to HO-1 expression, while it was unrelated to AMPK activation. According to the mathematical modeling, EMD008 increased the slope of glucose uptake plotted as a function of glycemia.

Conclusions: During resuscitated, polymicrobial, murine septic shock, glycemic control either by reducing glucose infusion rates or EMD008 improved glucose uptake and thereby liver tissue mitochondrial respiratory activity. EMD008 effects were more pronounced during hyperglycemia and coincided with attenuated markers of apoptosis. The effects of glucose control were at least in part due to the up-regulation of HO-1 and activation of AMPK.

Figure 1

Relationships between maximal mitochondrial oxidative phosphorylation, HO-1 expression, and AMPK activation. Maximal oxidative phosphorylation plotted as a function of HO-1 expression (A) and of AMPK activation (B), and HO-1 expression plotted as a function of AMPK activation (C). Hyperglycemic animals (squares) are shown on the left, normoglycemic mice (circles) on the right panels each; vehicle-treated mice are represented by open symbols and EMD008-treated animals by black symbols. Overall correlation between mitochondrial oxidative phosphorylation, HO-1 expression, and AMPK activation was r = 0.15 (p = 0.47) and r = 0.2 (p = 0.31), respectively. Overall correlation between HO-1 expression and AMPK activation was r = 0.33 (p = 0.098). Due to technical difficulties, the number of observations was reduced in the normoglycemic groups, which limits the statistical reliability of the evaluation.

Figure 2

Results of mathematical modeling of isotope enrichment data presented as glucose uptake. Lines represent mean and 95% confidence intervals for EMD008- (straight lines) and vehicle-treated (broken lines) animals. There is a loose correlation between AMPK activation values and glycemia and hence some of the effects of AMPK activation can be carried over to the glucose variable, when the impact of AMPK activation is ignored in Equation 2. (A) Regression lines obtained under these conditions and when different parameters were used for the EMD008 and for control groups. At comparable glucose concentrations, the predicted uptake is higher for the EMD008 group. When the AMPK effect is considered and one set of coefficients is used for all groups, then a significant effect of AMPK on glucose uptake can be established. Since a dependency on two factors is difficult to visualize, the actual values for AMPK used in the term k ₂ AMPK in Equation 2 are expressed by a glycemia-dependent term derived from a linear approximate relation between AMPK activation and glycemia, which allows to express glucose uptake as a function of glycemia alone. (B) An approximate replacement which demonstrates that EMD008 increased the slope of the relation between glucose disposal and glycemia [28].