Glucose and fatty acid metabolism in a 3 tissue in-vitro model challenged with normo- and hyperglycaemia

Abstract

Nutrient balance in the human body is maintained through systemic signaling between different cells and tissues. Breaking down this circuitry to its most basic elements and reconstructing the metabolic network in-vitro provides a systematic method to gain a better understanding of how cross-talk between the organs contributes to the whole body metabolic profile and of the specific role of each different cell type. To this end, a 3-way connected culture of hepatocytes, adipose tissue and endothelial cells representing a simplified model of energetic substrate metabolism in the visceral region was developed. The 3-way culture was shown to maintain glucose and fatty acid homeostasis in-vitro. Subsequently it was challenged with insulin and high glucose concentrations to simulate hyperglycaemia. The aim was to study the capacity of the 3-way culture to maintain or restore normal circulating glucose concentrations in response to insulin and to investigate the effects these conditions on other metabolites involved in glucose and lipid metabolism. The results show that the system's metabolic profile changes dramatically in the presence of high concentrations of glucose, and that these changes are modulated by the presence of insulin. Furthermore, we observed an increase in E-selectin levels in hyperglycaemic conditions and increased IL-6 concentrations in insulin-free-hyperglycaemic conditions, indicating, respectively, endothelial injury and proinflammatory stress in the challenged 3-way system.

Figure 1. Schematic of the 3-way connected culture.

QV is the low shear Quasi-Vivo chamber for hepatocytes and adipose tissue and LFC is the high shear laminar flow chamber for endothelial cells. The flow rate used was 250 µL/min. Total circuit volume is 15 mL, with 3 mL of priming volume per chamber and associated tubing and 6 mL in the mixing chamber and pump.

Figure 2. Star plot of measured metabolites in fasting state conditions at 48 H.

A) Fractional variation in metabolite concentrations for 1-way dynamic cultures of AT, EC and HEP (data published in ref. [18]). B) Fractional variation in metabolite concentrations for 2-way (AT+EC) connected culture and 3-way (AT+EC+HEP) connected culture. GLU (glucose), GLY (glycerol), LAC (lactate), ALA (L-alanine), E-SEL (E-selectin) (data partially published in ref [12]).

Figure 3. Glucose, FFA and glycerol in the 3-way cultures in FS, PARS, D1 and D2 conditions.

A) Glucose variations over time. B) Changes in medium glucose concentration with respect to initial media concentration at 48 H (* = p<0.05 vs. D1). C) FFA variations over time. D) Changes in medium FFA concentration with respect to initial media concentration at 48 H (* = p<0.05 vs. FS; ** = p<0.05 vs. D1). E) Glycerol variations over time. F) Changes in medium glycerol concentration with respect to initial media concentration at 48 H (* = p<0.05 vs. D1). In all cases time 0 represents the levels of metabolites in fresh media. Data are expressed as means ± SD (3≤n≤6). Error bars represent the standard deviation.

Figure 4. Medium lactate and L-alanine in the 3-way cultures in FS, PARS, D1 and D2 conditions.

A) Lactate variations over time. B) Changes in medium lactate concentration with respect to initial media concentration at 48 H (* = p<0.05 vs. FS; ** = p<0.05 vs. D1). C) L-alanine variations over time. D) Changes in medium L-alanine concentration with respect to initial media concentration at 48 H (* = p<0.01 vs. D1). In all cases time 0 represents the levels of metabolites in fresh media. Data are expressed as means ± SD (3≤n≤6). Error bars represent the standard deviation.

Figure 5. Medium IL-6 and E-selectin

in the 3-way cultures in FS, PARS, D1 and D2 conditions. A) IL-6 variations over time. B) Summary of the changes in Il-6 concentrations over 48 H (* = p<0.05 vs. D1). C) E-selectin variations over time. D) Summary of the changes in E-selectin concentrations over 48 H (* = p<0.05 vs. FS, ** = p<0.05 vs. D1). Time 0 represents the levels of markers in fresh media. Data are expressed as means ± SD (3≤n≤6). Error bars represent the standard deviation.

Figure 6. Star plot of measured metabolites in the 3-way connected cultures.

Fractional variation in GLU (glucose), FFA, GLY (glycerol), LAC (lactate), ALA (L-alanine), IL-6 and E-SEL (E-selectin) at 48 H in fasting, post-adsorptive resting state, diabetes 1 and diabetes 2 simulating conditions in the 3-way connected culture.