Kinetic modelling of β-cell metabolism reveals control points in the insulin-regulating pyruvate cycling pathways

Abstract

Insulin, a key hormone in the regulation of glucose homoeostasis, is secreted by pancreatic β-cells in response to elevated glucose levels. Insulin is released in a biphasic manner in response to glucose metabolism in β-cells. The first phase of insulin secretion is triggered by an increase in the ATP:ADP ratio; the second phase occurs in response to both a rise in ATP:ADP and other key metabolic signals, including a rise in the NADPH:NADP⁺ ratio. Experimental evidence indicates that pyruvate-cycling pathways play an important role in the elevation of the NADPH:NADP⁺ ratio in response to glucose. The authors developed a kinetic model for the tricarboxylic acid cycle and pyruvate cycling pathways. The authors successfully validated the model against experimental observations and performed a sensitivity analysis to identify key regulatory interactions in the system. The model predicts that the dicarboxylate carrier and the pyruvate transporter are the most important regulators of pyruvate cycling and NADPH production. In contrast, the analysis showed that variation in the pyruvate carboxylase flux was compensated by a response in the activity of mitochondrial isocitrate dehydrogenase (ICD_m ) resulting in minimal effect on overall pyruvate cycling flux. The model predictions suggest starting points for further experimental investigation, as well as potential drug targets for the treatment of type 2 diabetes.

Keywords: beta-cell metabolism; global sensitivity analysis; mathematical model; type 2 diabetes.

FIGURE 1

Pyruvate cycling pathways in β‐cells: Shared reactions (brown): Pyruvate is converted to oxaloacetate by PC. Pyruvate is transported between the cytosol and mitochondria through the pyruvate transporter (PYC). In the cytosol, malate is converted to pyruvate by malic enzyme (MEc). Pyruvate/malate shuttle (orange): Oxaloacetate is converted to malate, while malate is converted to pyruvate through mitochondrial malic enzyme or exported to the cytosol through the dicarboxylate carrier (DIC), where cytosolic malic enzyme converts malate to pyruvate. Pyruvate/citrate shuttle (violet): oxaloacetate is converted to citrate, then transported into the cytosol, where citrate lyase (CL) converts citrate to oxaloacetate, which is then converted to malate by cytosolic malate dehydrogenase (MDH_c). Finally, pyruvate is formed. Pyruvate/isocitrate shuttle (green): Oxaloacetate is converted to citrate and then to isocitrate, both of which are transported into the cytosol. Citrate is converted to isocitrate by ACO_c. Isocitrate can be converted to α‐ketoglutarate (α‐KG) via ICD_c. Isocitrate is then converted to α‐KG by ICD_c. Then α‐KG can enter the mitochondria for conversion to malate by tricarboxylic acid (TCA) cycle enzymes, and subsequent conversion to pyruvate by ME_m or ME_c, thus completing the pyruvate cycle. NADPH producing reaction steps are labelled in blue. The kinetic parameters of the six‐step influx glycolysis model (boxed) are excluded from the sensitivity analysis below.

FIGURE 2

Best‐fit model steady‐state model predictions compared to data from Ronnebaum et al. [11]. (a) Low glucose, control. (b) High glucose, control. (c) Low glucose, ICD_c knock‐down by 39.1%. (d) High glucose, ICD_c knock‐down by 39.1%. NADPH inset is on a different scale.

FIGURE 3

Model simulations of the NADPH:NADP⁺, MAL:PYR, and ICIT:AKG ratios over a range of glucose concentrations.

FIGURE 4

Parametric sensitivity ranking of the pyruvate cycling ratio. (a) Extended Fourier amplitude sensitivity test (eFAST) total effect. (b) eFAST first order. (c) Partial rank correlation coefficient (PRCC). (d) One‐at‐a‐time Sensitivity.

FIGURE 5

Parametric sensitivity ranking for the NADPH concentration. Panel (a) extended Fourier amplitude sensitivity test (eFAST) total effect. Panel (b) eFAST first order. Panel (c) Partial rank correlation coefficient (PRCC). Panel (d) One‐at‐a‐time sensitivity.

FIGURE 6

Predicted effect of perturbation in the V_max of pyruvate carboxylase (PC) on reaction fluxes. The five most sensitive fluxes and the five least sensitive fluxes are plotted. (a) One‐at‐a‐time sensitivity. (b) Partial rank correlation coefficient (PRCC) ranking, (c) extended Fourier amplitude sensitivity test (eFAST) total effect and (d) eFAST first order. For flux descriptions, refer to the supplementary text.