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Paul Thornalley’s blog – insights from deliberations, investigations and reflections

Hello. This is the start of a blog by Paul J Thornalley. I would like to discuss the simple principle of unscheduled glycolysis and glycolytic overload that underlies the pathogenesis of high glucose concentration in insulin resistance, beta-cell glucotoxicity, development of type 2 diabetes and vascular complications of diabetes and the impaired incretin effect. I then discuss how this can be alleviated by novel targeted therapy.

Pathogenesis of hyperglycemia mediated through glycolytic overload and its alleviation by precision dietary supplement, GlucoRegulate

The damaging effects of hyperglycemia are a consequence of hexokinase-gated increased flux of glucose metabolism without increased glycolytic enzyme activities to accommodate it. We will explore what these means in a moment but overlooking the key role of hexokinases in controlling flux of glucose metabolism for the last 25 years has impaired advances in understanding of the pathogenesis of hyperglycemia and its alleviation. So, let’s see what’s been missing.

Normally when there is increased flux of glucose metabolism into cells – such as skeletal muscle cells in the absorptive period, the activities of glycolytic enzymes downstream of hexokinases 1 and 2 are increased such that steady-state levels of glycolytic intermediates are kept within tolerable levels. It is when increased flux of glucose metabolism – initiated by stabilization of hexokinase-2 to proteolysis by high cellular glucose concentration at sites of vascular complications, for example – occurs without increased activities of glycolytic enzymes downstream that glycolytic intermediates increase to levels that activate multiple pathways of pathogenesis. We described this in our paper of last year in detail – Hexokinase-linked glycolytic overload and unscheduled glycolysis in hyperglycemia-induced pathogenesis of insulin resistance, beta-cell glucotoxicity, and diabetic vascular complications, Rabbani & Thornalley, Frontiers in Endocrinology, 14: 1268308, 2024.

The difference between normal glucose metabolism and glycolytic overload can be likened to that of a dam holding back water with minimal overflow (normal glucose metabolism).

In contrast, glycolytic overload may be likened to where the dam is breached by excessive water input, overtopping the dam wall and causing flooding into expanded areas with resulting damage through excessive water going where it normally doesn’t.

A visual analogy of glycolytic overload.

Paul J Thornalley 12th August 2025

Hexokinases 1 and 2 – normally protective against damaging effects of high glucose concentration but protection is subverted for hexokinase-2 in prolonged hyperglycemia

Hexokinase-1 (HK1) has K_M(Glucose) of 61 μM and Hexokinase-2 (HK2) has K_M(Glucose) = 340 μM, so they are normally highly saturated with glucose under physiological conditions. This means in the absorptive phase (after a meal), blood glucose concentration increases but the rate of glucose metabolism in tissues where HK1 and HK2 control entry of glucose into metabolism stays the same. This protects against glycolytic overload the brain and heart – mostly HK1, and vascular tissues – HK1 and HK2 (including kidney retina and peripheral nerve). It also means there is glucose ready and waiting to be metabolized in these tissues. So, no need to worry about neurons of the brain running short on glucose!

When there is a prolonged period of increased cellular glucose, something happens to HK2. It is stabilized to proteolysis and thereby HK2 protein and activity increase without other increase of other glycolytic enzymes. The half-life of HK2 is normally 8 – 10 h and approximately doubles when exposed to high glucose concentration. This increases the concentration of HK2 approximately 2-fold. Where HK2 contributes significantly to total hexokinase activity, it increase flux of glucose metabolism by 2 – 3 fold without increase in activity of other glycolytic enzymes, initiating glycolytic overload. Where is HK2 a major player in hexokinase activity? Vascular cells, renal cells, retina, Schwann cells of peripheral nerve and early-stage embryo – all vulnerable to glycolytic overload in prolonged periods of high hyperglycemia. HK2-linked glycolytic overload is now seen as a key initiator of hyperglycemia-induced pathogenesis in diabetic vascular disease – including endothelial dysfunction and increase risk of coronary artery disease, diabetic kidney disease, diabetic retinopathy, diabetic neuropathy and diabetic embryopathy (Rabbani & Thornalley, Frontiers in Endocrinology, 14: 1268308, 2024).

Paul J Thornalley 13th August 2025

Unscheduled glycolysis in peripheral insulin resistance

Hexokinase-2 is also a major hexokinase in skeletal muscle and adipose tissue. These are sites where glucose is taken up at increased rates in the absorptive phase by insulin-stimulated recruitment of GLUT4 glucose transporters to the plasma membrane and where glucose uptake in the fasting phase is mediated by GLUT1 glucose transporter. HK2 operates in situ at 40% – 60% saturation. Here it is increased glucose uptake and increased in situ activity of HK2 in the fasting phase driven by mass action effects of increased fasting glucose concentration that stimulates unscheduled glycolysis. This activates processes decreasing insulin-stimulated glucose uptake in the absorptive phase or peripheral insulin resistance. The key determinant of increased fasting plasma glucose or impaired fasting glucose is hepatic insulin resistance giving rise to hypersecretion of glucose from the liver during the fasting phase (Rabbani & Thornalley, Frontiers in Endocrinology, 14: 1268308, 2024).

Paul J Thornalley 14th August 2025

Initiation of hexokinase-2 linked glycolytic overload – initiated by glucose-induced stabilization of hexokinase 2 to proteolysis

It is critical to know the mechanism of initiation of metabolic dysfunction in hyperglycemia as this is the key regulatory point to appreciate how increase and duration of increase in glucose concentration affects regulation of glucose metabolism. The initiation step also has multiple downstream pathways and only by correcting the key initiating step can all of the multiple downstream pathways be alleviated too. The key initiating step is glucose-induced stabilization of hexokinase 2 (HK2) to proteolysis with consequent increased HK2 protein and total hexokinase activity

High glucose-induced, HK2-linked responses are:

1. Stabilization of HK2 to proteolysis by high cellular glucose concentration
2. Increased flux of glucose metabolism without increased activity of glycolytic enzymes → wave of increased glycolytic intermediates. “Unscheduled glycolysis”
3. This stimulates hexosamine, PKC and dicarbonyl stress pathways
4. Increased G-6-P → detachment of HK2 from mitochondria, mitochondrial membrane polarization and increased reactive oxygen species (ROS)
5. HK2 detached from mitochondria produces increased glycogen synthesis by metabolic channeling

Abbreviations: G-6-P, glucose-6-phosphate; HK1 & HK2, hexokinase-1 & -2; VDAC, voltage-dependent anion channel. References: Irshad et al., Sci Rep 9, 7889, 2019; Ashour et al., BMJ Open Diabetes Research & Care. 8, e001458, 2020; Rabbani & Thornalley Frontiers in Endocrinol 14, 1268308, 2024

Paul J Thornalley 15th August 2025

Scheduled and unscheduled glycolysis

Normally when there is increased rate of glucose metabolism in cells – such as in skeletal muscle in the absorptive phase, insulin and transcriptional signaling through Mondo A increase expression and and activity of glycolytic enzymes, enhancing the onward metabolism of glycolytic intermediates and keeping the steady-state concentrations within the normal range. This is scheduled glycolysis. When increased flux of glucose metabolism occurs by increased hexokinase activity without increase in other early-stage glycolytic enzymes – glucose-6-phosphate isomerase (GPI), phosphofructokinase (PFK), aldolase (ALDO), triosephosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GA3PD), concentrations of glycolytic intermediates increase to abnormally high levels. Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (F16BP), dihydroxyacetonephosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P) all increase. This is unscheduled glycolysis. It occurs because for enzymes operating below saturation, flux v ≈ k_cat/K_M x [Enzyme][Substrate]. So, with [Enzyme], k_cat and K_M all unchanged, when flux v increases so does the steady-state substrate concentration [Substrate] correspondingly. For abnormally increased G6P, F6P, DHAP and GA3P, pathogenic mechanisms are activated.

I made a mathematical model of early-stage glycolysis with normal fasting plasma glucose of 5mM (FPG5), high fasting plasma glucose of 20 mM with not provision for stabilization of hexokinase-2 (HK2) to proteolysis by the high glucose concentration (FPG20) and high fasting plasma glucose of 20 mM with stabilization of HK2 to proteolysis inducing unscheduled glycolysis (FPG20_UG). Only in the latter case with increased HK2, glycolytic intermediates increased in high fasting plasma glucose – shown for G6P, F6P and rate of formation of methylglyoxal (MG) which forms spontaneously at trace levels from DHAP and GA3P. This is the mechanistic origin of metabolic dysfunction in hyperglycemia (Rabbani & Thornalley, Frontiers in Endocrinology, 14: 1268308, 2024). Presented at EASD 2022.

Paul J Thornalley 19th August 2025