Researchers have discovered a previously unknown way that pancreatic cells decide how much insulin to secrete. It could provide a promising new target to develop drugs for boosting insulin production in people with Type 2 diabetes.
In a pair of papers recently published in Cell Metabolism, scientists from the University of Wisconsin-Madison and their colleagues point to an overlooked enzyme known as pyruvate kinase as the primary way pancreatic beta cells sense sugar levels and release the appropriate amount of insulin.
From several proof-of-concept experiments in rodents and on human pancreatic cells, the team found that drugs stimulating pyruvate kinase not only increase the secretion of insulin but have other metabolically protective effects in the liver, muscle and red blood cells.
The findings suggest that activating pyruvate kinase could be a new way to increase insulin secretion to counter Type 2 diabetes, but more research would be required before any new treatments were available.
“Too much insulin can lower blood sugar to dangerous levels, and too little insulin can lead to diabetes,” says Matthew Merrins, a professor of medicine at the UW School of Medicine and Public Health who led the work.
“The question we’re asking here is: How do nutrients like glucose and amino acids turn on beta cells in the pancreas to release just the right amount of insulin?”
The work was accomplished by carefully dissecting the paradoxical timing of key biochemical events in the prevailing understanding of how pancreatic beta cells respond to nutrients in the blood. The researchers point to a new, richer model to understand how this important process is controlled that resolves these inconsistencies.
For decades, scientists believed that mitochondria, the energy generators in cells, initiated insulin secretion. It was a natural explanation, because mitochondria produce the high-energy molecule ATP, in the process depleting ATP’s low-energy version, ADP.
The drop in ADP stimulates calcium – the ultimate trigger to release stored insulin.
But the timing didn’t make sense. Mitochondria are most active after insulin secretion has already begun, not before. Plus, mitochondria would stall out before exhausting enough ADP to trigger insulin secretion.
A clue to solving these apparent paradoxes came from studies in the 1980s on heart muscle cells. At the time, scientists found that the enzyme pyruvate kinase – which converts sugar into energy, independently of mitochondria – could also severely deplete ADP.
This process happens near ADP-sensing proteins involved in insulin release in the pancreas. Maybe, Merrins’ team thought, the pancreas took advantage of this proximity to fine-tune the release of insulin.
In initial experiments, the researchers supplied sugar and ADP to sections of pancreatic cells containing pyruvate kinase. The enzyme gobbled up both components, depleting ADP. Because pyruvate kinase was located near the ADP-sensing protein that triggers insulin secretion, it had a big effect.
“That’s one of the important concepts in our paper: the location of metabolism is critical to its function,” says Merrins.
Using mouse and human pancreatic islets, the clusters of cells that release insulin, the researchers tried stimulating pyruvate kinase activity.
Drugs that activate the enzyme quadrupled the release of insulin, but only when there was enough sugar around – a sign that pyruvate kinase can’t be forced to release too much insulin.
“Pyruvate kinase doesn’t change how much fuel comes into the cell, it just changes how that fuel is used,” says Merrins. “Drugs that active pyruvate kinase strongly boost insulin secretion without causing too much insulin release that can lead to hypoglycemia.”
In all, they discovered evidence of a more complex way in which pancreatic beta cells decide when and how much insulin to release, akin to a two-cycle engine.
In the first cycle, blood sugar gets processed by pyruvate kinase, depleting ADP. Mitochondria keep the process going by feeding pyruvate kinase even more material, which causes ADP levels to crash, ultimately stimulating enough calcium entry into the cell to release insulin.
In the second cycle, mitochondria switch from feeding pyruvate kinase with material to producing the high-energy molecule ATP, which is needed to fully release insulin. Then the process resets.
In the companion study, led by Merrins’ colleagues at Yale University, the researchers examined how pyruvate kinase activators affected metabolism in healthy and obese rats.
In a series of experiments, they found that activating pyruvate kinase increased both insulin secretion and insulin sensitivity while improving sugar metabolism in liver and red blood cells. Such treatments could be helpful for people with Type 2 diabetes, who don’t produce enough insulin and have dysfunctional sugar metabolism as a result.
“The therapeutic idea here is we could rewire metabolism to more efficiently trigger insulin secretion while improving the function of other organs at the same time,” says Merrins.
A characteristic feature of pancreatic β-cells is their ability to couple metabolic glucose sensing with appropriate insulin secretion.
The most widely-accepted description of the sensing mechanism involves the oxidation of glucose carbons in the mitochondria to generate a proton motive force that, through ATP synthase, sequentially raises the ATP/ADP ratio, closes KATP channels, and activates Ca2+ influx, which triggers insulin granule fusion with the plasma membrane (Prentki et al., 2013).
Many different components of the glucose-sensing apparatus are well-characterized. For example, glucokinase (GK) and KATP channels are genetically linked to insulin secretion through both gain and loss of function mutations in humans (Nichols, 2006).
However, several lines of evidence challenge one key component of the canonical mechanism – the exclusivity of coupling oxidative phosphorylation (OxPhos) to KATP channel closure.
A central tenet of mitochondrial respiratory control is that, in the presence of adequate O2 and substrate, mitochondrial OxPhos is dependent on ADP availability (Chance and Williams, 1955). Workload in the form of ATP hydrolysis is therefore the principal driver for OxPhos in many cells.
Although the β-cell ATP/ADP ratio is unusually substrate sensitive, a work dominated drive for OxPhos poses a challenge to the canonical β-cell model, since ADP privation is the physiological driver of KATP channel closure (Koster et al., 2005; Nicholls, 2016).
Consistent with ADP limitation, mitochondrial respiration is highest after membrane depolarization, rather than during the triggering phase when KATP channels close (Jung et al., 2000).
Furthermore, calcium influx precedes oxygen consumption during glucose-stimulated oscillations (Kennedy et al., 2002), implying that the canonical model does not hold at steady state.
If the dependence of KATP closure on OxPhos is to be questioned, is there an alternative ATP/ADP generator that is limited by glucose and functions prior to membrane depolarization?
One clue may be that anaplerotic flux through pyruvate carboxylase (PC) is more strongly correlated with insulin secretion than oxidative flux through pyruvate dehydrogenase (PDH) (Alves et al., 2015; Fransson et al., 2006; MacDonald et al., 2005; Prentki et al., 2013).
Glucose carbons that transit through PC generate 40% of cytosolic PEP through the cataplerotic mitochondrial PEP carboxykinase (PEPCK-M) reaction (Stark et al., 2009). This ‘PEP cycle’ has been linked to insulin secretion (Jesinkey et al., 2019; Stark et al., 2009) and provides a mechanism distinct from OxPhos for cytosolic ATP/ADP generation via pyruvate kinase (PK), which is allosterically activated by fructose 1,6-bisphosphate (FBP) prior to membrane depolarization (Merrins et al., 2013, 2016).
Here we provide evidence for a revised model of β-cell metabolism based on the ability of PK to initiate KATP channel closure at the plasma membrane. In this model, cytosolic ADP lowering by PK-driven PEP hydrolysis deprives mitochondria of ADP, at the same time creating antiphase OxPhos oscillations. Rather than triggering depolarization, mitochondrial OxPhos provides the energy to sustain membrane depolarization and insulin secretion.
Through a mechanism that would not be predicted by the canonical pathway, pharmacologic activation of PK amplifies the metabolic response without increasing glucose oxidation, providing a potential new therapeutic strategy for diabetes that does not inappropriately trigger insulin secretion at low glucose.
In a companion paper in this issue (Abulizi et al., 2019), we show that the control of glucose signal strength by PK is dependent on PEPCK-M, and demonstrate the in vivo relevance of small molecule PK activation as a therapeutic strategy in pre-clinical models of diabetes.
These data provide evidence that PEP hydrolysis by PK, rather than OxPhos, provides the energetic push required to raise ATP/ADP beyond the threshold for KATP channel closure. The functional linkage between PK and KATP in β-cells is consistent with prior studies in cardiac myocytes, where PK closes KATP channels (Weiss and Lamp, 1987; Dhar-Chowdhury et al., 2005).
Importantly, PEP can close KATP channels in isolated membrane patches despite the presence of the strong KATP channel-opener ADP, indicating that PK can influence the metabolic microenvironment near the channel.
The demonstration that PK is able to restrict OxPhos by locally depriving the mitochondria of ADP provides a second example of β-cell metabolic compartmentation in which ADP is the signal carrier, and solidifies the importance of glycolytic ATP/ADP regulation.
Our data do not undermine the previously described metabolic control exerted by GK that is limited to glycolysis (Matschinsky and Ellerman, 1968). Real-time imaging of calcium oscillations reaffirmed that the threshold for membrane depolarization is set by GK and observable in the duty cycle (Henquin, 2009), and identify a second mode of triggering based on mitochondrial PEP biosynthesis.
Based on the canonical OxPhos-only model, PK activation should not be able to increase GSIS since GK activity determines the glycolytic and, consequently, mitochondrial flux rates.
It is important to note that anaplerosis has been identified as an important component of metabolic coupling without a clear mechanism (Prentki et al., 2013). Islet perifusion studies revealed that, independently of initiating membrane depolarization (i.e. in the presence of KCl), the dynamically regulated, allosterically recruitable PK appears to play a dominant role in amplifying secretion.
This is reinforced by the requirement for an AcCoA source for triggering but an anaplerotic carbon source for metabolic amplification in human and mouse islets. Thus, a key finding here is that PK underlies a separate mode of β-cell glucose-sensing downstream of GK that may provide a mechanism dependent upon anaplerosis. While targeting OxPhos in β-cells has not been successful therapeutically, preclinical data suggest the anaplerotic mechanism may be of potential benefit (Abulizi et al., 2019).
Surprisingly, PK activation did not significantly increase time averaged mitochondrial metabolism. Instead, it increased the frequency rather than the duration of the calcium pulses. Human islet secretion measurements likewise indicated that PK acts as a fuel-dependent amplifier that does not significantly lower the threshold for insulin secretion.
The most straightforward explanation for metabolic amplification is that PK is limiting during periods of high glycolytic flux, such as the state induced by KCl and high calcium, when FBP levels are low and the recruitable PK isoforms are least active. PK activators stabilize the active enzyme and circumvent the need for FBP.
These data from intact cells corroborate prior studies showing that ADP and PEP (but not pyruvate) are sufficient for biphasic insulin secretion in membrane-permeabilized islets, including in the presence of rotenone (Pizarro-Delgado et al., 2016).
Incorporating the data herein, we propose a 2-state model of β-cell metabolism describing the relationship of PK to the intrinsic β-cell metabolic oscillations. In this model, ADP availability switches between a state where anaplerosis is high and one where OxPhos is high. Here, the importance of metabolic control exerted by ADP stems from the simple fact that ADP must be reduced to low μM concentrations for KATP channels to close (Tarasov et al., 2006) and that mitochondrial ATP synthase cannot run without ADP (Chance and Williams, 1955).
Transition between the 2 states is toggled by the large amount of ATP hydrolysis associated with membrane depolarization, pumps, and vesicle fusion (Nicholls, 2016; Affourtit et al., 2018). A key advantage of allosteric PK recruitment is the ability to reinforce metabolic oscillations in response to metabolic regulators like FBP (Merrins et al., 2013, 2016). Consequently, the apex of PK activity occurs just prior to membrane depolarization, at the nadir of OxPhos, which resumes following depolarization-initiated ATP hydrolysis.
This back and forth between an electrically silent triggering phase and an active oxidative secretory phase allow β-cell mitochondria to move between PEP biosynthesis and OxPhos. The mitochondrial contribution of PK substrate from the PEP cycle during the silent phase boosts PEP production beyond what is achievable by glycolysis alone to enhance the cytosolic ATP/ADP-generating capacity of PK in the triggering phase.
Paradoxically, PK recruitment occurs in parallel with the slowing of glycolytic flux. However, this recruitment occurs in the setting of continuous GK flux, when FBP levels rise and flux through PC increases, rerouting pyruvate flux through PEPCK-M (Jesinkey et al., 2019; Kibbey et al., 2007; Stark et al., 2009).
Prior estimations of PEP cycling showed rates could reach ∼40% of the total PK flux in islets (Stark et al., 2009). These estimations did not consider that oscillatory metabolism divides OxPhos and anaplerosis into separate phases. As such, mitochondrial PEP cycling could potentially explain nearly all of PK activity during the electrically silent phase.
The 2-state model recognizes that the PEP cycle primarily triggers via PK while OxPhos primarily sustains the active phase. As both processes generate ATP, their individual contributions may not be fully separable in either the time or compartment domains, and both processes are necessary to achieve maximal secretion.
However, the improper timing of OxPhos relative to membrane depolarization (Figure 7 and (Jung et al., 2000)) raises the question of whether it is bioenergetically feasible that mitochondria, even if strategically positioned at the plasma membrane, could lower ADP sufficiently to close KATP channels, as PK was demonstrated to do.
Our data are supported by observations of high ATP generation at the plasma membrane relative to the cytosol and mitochondria (Kennedy et al., 1999). It remains to be determined if there is a point at saturating glucose levels where, through either glycolysis and/or the PEP cycle, PK can locally deplete ADP sufficient to close KATP channels while other mitochondria at sites of active ATP hydrolysis have adequate ADP to conduct OxPhos simultaneously.
This work only shows that it is possible for PEP cycle-supported, PK-mediated mitochondrial and KATP channel ADP privation to occur.
Although many qualitative β-cell bioenergetics studies have shown that ADP supply and demand are critical for OxPhos and insulin secretion (Figure 6D and (Ainscow and Rutter, 2002; Doliba et al., 2003; Panten et al., 1986; Sweet et al., 2004; Affourtit et al., 2018)), quantitative metabolic control analyses in β-cells have remained lacking (Affourtit et al., 2018; Nicholls, 2016).
Since tools to quantify the phosphorylation potential and ATP flux currently rely on temporal averaging (Affourtit et al., 2018), it is not yet possible to calculate the relative contribution of PK and OxPhos to KATP closure. We also lack the means to determine how PK-driven ATP/ADP cycling bioenergetically supports increased exocytosis relative to OxPhos.
Approaches will need to be developed that can absolutely quantify adenine nucleotides and their sources in a subcompartment-specific and time-resolved way. However, reevaluation of such β-cell bioenergetics may have similar application to other metabolic cycles and pathways that have been proposed (Schuit et al., 1997; Farfari et al., 2000; Joseph et al., 2006; Jensen et al., 2008; Prentki et al., 2013) but are not addressed in this simplified 2-state model.
This new understanding of the native role of PK in β-cell metabolism has broad implications in non-native environments where it has been coopted, such as in cancer (Dayton et al., 2016; Israelsen et al., 2013). It may be particularly advantageous for cancer or other dividing cells to shut down oxidative phosphorylation in order for the mitochondria to synthesize, rather than oxidize, building blocks.
Time-resolved stable isotope measurements identify that OxPhos and anaplerosis are anti-phase with each other and suggests that mitochondria choose not to oxidize and synthesize simultaneously. In light of this, the phenomenon of PK-associated Warburg metabolism, while often considered for its ATP-generating capacity, may be even more important for its ADP lowering capacity (Harris and Fenton, 2019).
Just as in β-cells, ADP deprivation may shift mitochondrial activity from OxPhos into the biosynthesis of essential nutrients needed to support cell division, or the generation of reactive oxygen species used for signaling. Of note, cells with more robust PEP cycling have an increased mass of elongated mitochondria while those deficient have fewer more fragmented mitochondria (Jesinkey 2019).
The ability of PK isoforms to oscillate may also provide additional advantages not considered here, e.g. intermittently reducing membrane potential to reduce free radical damage that might occur if mitochondria were perpetually kept in a ‘State 4-like’ situation. In this context, activating PK may have some potential therapeutic benefits in certain situations where Warburg metabolism is observed.
Finally, the ability of pharmacologic PK activators to raise insulin secretion has broad conceptual implications for type 2 diabetes therapies. Not only is PK identified as a novel target for diabetes therapy, but we demonstrate that, for a given level of glucose, the secretory pathway can be internally remodeled to tune the glucose responsiveness of healthy and diseased human β-cells.
Currently available drugs that increase insulin secretion by stimulating glucose uptake (e.g. GK activators) elevate the metabolic workload on each β-cell (Porat et al., 2011) and can lead to glucotoxic-like damage (Nakamura and Terauchi, 2015). Drugs that increase insulin secretion by directly triggering membrane depolarization such as sulfonylureas, while invaluable for treating some forms of MODY (Kim, 2015), decouple β-cell nutrient sensing with insulin secretion and increase the risk of hypoglycemia. PK-dependent remodeling of the β-cell metabolic pathways could lead to a treatment for diabetes that avoids these pitfalls.
reference link : https://www.biorxiv.org/content/10.1101/2020.01.15.907790v1.full
More information: Sophie L. Lewandowski et al. Pyruvate Kinase Controls Signal Strength in the Insulin Secretory Pathway, Cell Metabolism (2020). DOI: 10.1016/j.cmet.2020.10.007
Multi-Tissue Acceleration of the Mitochondrial Phosphoenolpyruvate Cycle Improves Whole-Body Metabolic Health, Cell Metabolism (2020). DOI: 10.1016/j.cmet.2020.10.006 , www.cell.com/cell-metabolism/p … -4131(20)30539-8.pdf