Insulin, Muscle Glucose Uptake, and Hexokinase: Revisiting the Road Not Taken

Abstract

Research conducted over the last 50 yr has provided insight into the mechanisms by which insulin stimulates glucose transport across the skeletal muscle cell membrane Transport alone, however, does not result in net glucose uptake as free glucose equilibrates across the cell membrane and is not metabolized. Glucose uptake requires that glucose is phosphorylated by hexokinases. Phosphorylated glucose cannot leave the cell and is the substrate for metabolism. It is indisputable that glucose phosphorylation is essential for glucose uptake. Major advances have been made in defining the regulation of the insulin-stimulated glucose transporter (GLUT4) in skeletal muscle. By contrast, the insulin-regulated hexokinase (hexokinase II) parallels Robert Frost’s “The Road Not Taken.” Here the case is made that an understanding of glucose phosphorylation by hexokinase II is necessary to define the regulation of skeletal muscle glucose uptake in health and insulin resistance. Results of studies from different physiological disciplines that have elegantly described how hexokinase II can be regulated are summarized to provide a framework for potential application to skeletal muscle. Mechanisms by which hexokinase II is regulated in skeletal muscle await rigorous examination.

Introduction

Skeletal muscle glucose uptake requires that glucose be delivered by the circulation to myofibers, transported into myofibers, and irreversibly phosphorylated within myofibers (FIGURE 1). Each of these three steps are necessary. The relative regulatory importance of these three steps under different physiological and disease conditions is unresolved. The specific emphasis of this review is on the roles and regulation of muscle glucose uptake within the myofiber by glucose transport and phosphorylation. In much of the literature, muscle glucose membrane transport is treated as analogous to muscle glucose uptake. Net glucose uptake does not occur without phosphorylation. Recent reviews of muscle insulin action largely dismiss glucose phosphorylation (3–6). These reviews are an accurate reflection of research emphasis in this area. A Pubmed search (July 2021) of “skeletal muscle” and “glucose transport” returned 1,850 published papers, while a search of “skeletal muscle” and “glucose phosphorylation” returned only 91 papers. A search of “skeletal muscle” and the insulin-regulated glucose transporter “GLUT4” returned 2,811 published papers. A search of “skeletal muscle” and the glucose phosphorylation isozyme “hexokinase II” and “hexokinase 2” returned only 199 published papers. The case is made that glucose phosphorylation by hexokinase II is an important determinant of the rate of insulin-stimulated muscle glucose uptake in vivo and contributes to insulin resistance. Studies of hexokinase II conducted in a variety of cell systems in other biological disciplines has demonstrated the complexity with which this enzyme is regulated. Despite these advances how this enzyme is regulated in skeletal muscle remains largely unknown particularly in vivo. Considering that skeletal muscle is the bulk of insulin-sensitive tissue and a major site of insulin resistance there is a need to 1) first recognize that muscle glucose phosphorylation by a hexokinase is fundamental to muscle glucose uptake; 2) define the importance of glucose phosphorylation by hexokinase II in health and insulin resistance; and 3) describe the means by which hexokinases are regulated in skeletal muscle.

FIGURE 1.

Muscle glucose uptake requires that glucose be delivered to the muscle membrane, transported across the membrane, and phosphorylated within the cells The transparent intracellular phosphorylation gear reflects the sparse literature on control of skeletal muscle phosphorylation and the failure to consider the processes involved in interpretation of muscle glucose uptake. Modified from Refs. 1 and 2, with permission from Advances in Experimental Medicine and Biology and Journal of Applied Physiology, respectively.

Whence We Came

The 20th century saw great advancements in understanding insulin action and carbohydrate metabolism. F. G. Banting presented “The internal secretion of the pancreas” at the 1921 meeting of the American Physiological Society in New Haven. Attendees heard the first report of a pancreatic extract with blood glucose lowering effects. Insulin sequence (7–11) and structure (12, 13) were subsequently defined. There continue to be gains in how β-cells of the pancreatic islets synthesize and secrete insulin (14–16). In convergence with the discovery of insulin and the advances that followed was research defining pathways for skeletal muscle carbohydrate metabolism. Hill (17, 18), Cori and Cori (19, 20), and Krebs (21–23) defined anaerobic and aerobic glycolysis, glycogen metabolism, and the tricarboxylic acid cycle. The effects of insulin on carbohydrate metabolism were then defined. Insulin was shown to increase muscle permeability to glucose (24) by activating translocation of glucose transporters to the cell membrane (25–27). The insulin-regulatable glucose transporter GLUT4 was identified and the gene that encodes it was cloned (28). GLUT4 translocation has also been shown to be regulated by exercise, hypoxia and cold exposure (29). GLUT1 is also expressed in skeletal muscle but is insensitive to insulin and other acute stimuli. It was also recognized that insulin increases the expression and alters the compartmentation of glucose phosphorylation by hexokinase (30). The product of the hexokinase reaction glucose-6-phosphate (G6P) “locks” glucose in the cell and is a substrate for glycogen storage (31) and glycolysis (32).

Insulin binds to a homodimeric membrane receptor (33), which is then autophosphorylated (34), resulting in activation of downstream signaling (35–43) leading to the actions of insulin. Defining the insulin signaling pathway opened the door for advancements in insulin action. Insulin binding to its receptor activates the phosphoinositol 3-kinase (PI3K)-Akt pathway in muscle and increases GLUT4 translocation to the cell membrane. Canonical insulin signaling involves Akt-activated phosphorylation of TBC1D4 (44, 45), which activates a Rab GTPase that tethers GLUT4 storage vesicles to F-actin for translocation to the cell membrane. SNARE and SNARE-associated proteins guide vesicle exocytosis. Noncanonical insulin-signaling involves PI3K activation of Rac1 and p21-activated kinase 1 (46–48) leading to translocation events culminating in GLUT4 storage vesicle exocytosis (49, 50). Reduced insulin stimulation results in GLUT4 endocytosis and reconstitution into GLUT4 storage vesicles, which are either recycled or stored.

Glucose transport is bidirectional. Glucose uptake requires irreversible glucose phosphorylation by a hexokinase. Hexokinases I and hexokinase II are the two prominent hexokinase isozymes in skeletal muscle. Both are ubiquitously expressed, with hexokinase II most abundant in skeletal muscle. Hexokinases I and II result from the duplication and fusion of single glucose phosphorylating domains (51). They share common features but also have distinct regulatory characteristics. A key difference in the context of glucose uptake is that only hexokinase II expression and function are regulated by insulin.

A Case for Glucose Phosphorylation in the Regulation of Insulin-Stimulated Muscle Glucose Uptake

Functionally distinguishing glucose transport and glucose phosphorylation has been challenging. Muscle glucose transport in isolated muscle is too low even at suprapharmacological insulin levels to test the role of the low K_m hexokinases (24). Studies in vivo are required for physiological rates of muscle glucose uptake. The hypothesis is that if glucose phosphorylation poses a limitation to control of muscle glucose uptake intracellular glucose would increase. This has been difficult to test since intracellular glucose is not directly measurable. One study attempted to calculate intracellular glucose as the difference between whole muscle glucose measured by surface scanning nuclear magnetic resonance (NMR) in human subjects and interstitial glucose estimated using microdialysis in a separate cohort (52). This paper contains several concerns. A full discussion of these concerns is beyond the scope of this review. Nevertheless, it is necessary to briefly address this paper as it is often cited as the basis for dismissing a regulatory role for glucose phosphorylation in vivo. This study reports that muscle interstitial glucose is equivalent to arterialized plasma glucose during a hyperinsulinemic-glucose clamp (52). In this (52) and every other paper (53–68), glucose concentration in the plasma water will be ∼6% higher than the reported plasma glucose concentration due to the presence of plasma proteins. This will create a small plasma water-to-interstitial glucose gradient where it did not exist (52) and expand the plasma to interstitial glucose gradient where it does (53–68).

A representative interstitial glucose will be between arterial and venous glucose concentrations. It is possible for interstitial glucose to be lower than venous glucose, but it cannot be higher than arterial glucose (unless the muscle is producing glucose). The data of Cline et al. (52) is possible if glucose extraction is not appreciably increased by insulin. Insulin does appreciably increase glucose extraction, albeit less so in insulin resistance and diabetes. Other studies (53–68) show that muscle interstitial glucose is between ∼60 and 80% of arterialized plasma glucose during a hyperinsulinemic-glucose clamp. This range is compatible with typical arterial to venous glucose concentrations. Concerns with measurements of interstitial glucose are compounded by those of tissue glucose measurements using surface ¹³C-NMR. These have been discussed in detail elsewhere (69–72). Two other experiments have used circulating glucose and not interstitial glucose to calculate intramyocellular glucose (73, 74). These studies also concluded that there was no glucose inside the muscle cell. Again, had the lower glucose typical of interstitial fluid in the presence of hyperinsulinemia been used glucose would have been present in the muscle cell.

Even if intracellular glucose were feasible to measure directly a more significant measure may be local compartmentation of glucose and the hexokinase II product/inhibitor G6P within the cell. It is predictable that free glucose is highest on the inner surface of the cell membrane where it enters the cytosol in insulin-stimulated muscle and G6P would be highest in the intracellular region where hexokinases synthesize it. Because of the difficulty in measuring intracellular glucose, no less glucose and G6P compartmentation within the cell, functional readouts are a more effective means to measure sites of control of muscle glucose uptake.

Experiments have used alternative approaches to overcome the inability to measure intramyocellular glucose and estimate glucose phosphorylation capacity. These studies used radioactive glucose analogs in humans (70, 71, 75–79) and rats (80, 81) to determine whether control of muscle glucose uptake is distributed between glucose membrane transport and phosphorylation. These approaches, while innovative, are assumption dependent and have their own caveats. Despite the assumptions inherent in these approaches, all give the same general finding. Control of muscle glucose uptake is dominated by membrane transport in the sedentary, fasted state, but shifts so that control by glucose phosphorylation is increased during insulin stimulation. These studies show that intracellular glucose is increased by insulin stimulation implicating glucose phosphorylation as a limitation to the rate of glucose uptake (71, 76–78). Moreover, these studies show that the rate constant for glucose phosphorylation is increased suggesting that insulin stimulates hexokinase II activity (71, 77–81). Studies in rats used isotopic methods with a modeling approach to estimate glucose on the inner and outer surfaces of the cell membrane. It was found that insulin narrows the gradient from the outer to inner surface of the cell membrane in the presence of accelerated muscle glucose uptake. This approach showed that the muscle can become so permeable to glucose during insulin stimulation that it is no longer a barrier to glucose uptake with the result being that intramyocellular control shifts to glucose phosphorylation (81, 82).

Genetic mouse models provide an independent means to examine whether glucose uptake is best defined by membrane transport as a rate-limiting step or by the distributed control paradigm. A basic tenet of metabolic control analysis is the relationship of a change in a pathway reaction to the change in flux rate through the pathway (83). An index of glucose uptake (R_g) can be calculated using the rate that injected isotopic 2-deoxyglucose is phosphorylated in tissue (84). Mouse models were used to study mice during an insulin infusion rate that results in an increase in glucose disappearance that is ∼50% of the insulin-stimulated maximum (85). Mice fasted for 5-h studies have muscle glycogen concentrations of roughly 1.5–5.5 mg/g. This is ∼10 to 30% of muscle glycogen in overnight-fasted humans. It is possible that reduced muscle glycogen in the mouse could affect the relative control of glucose uptake by glucose transport and phosphorylation. In the basal state an increase in gastrocnemius GLUT4 resulted in a nearly equal increase in R_g (86). This is supported by studies that show a 50% genetic reduction in GLUT4 leads to a 50% reduction in muscle glycogen (87). The rate-limiting role of membrane transport for basal muscle glucose uptake is supported by the demonstration that neither overexpression nor a 50% genetic reduction of hexokinase II affects basal R_g (88). Taken together these studies show that glucose uptake in muscle of sedentary, postabsorptive mice is best defined by glucose transport as the dominant rate-limiting step.

The paradigm changes dramatically with insulin-stimulation (FIGURE 2). The robust translocation of GLUT4 to the cell surface in response to insulin increases permeability to glucose increasing the contribution of hexokinase II to control of muscle glucose uptake. Chang and colleagues (89) were the first to show that hexokinase II overexpression increased muscle glucose uptake of radioactive 2-deoxyglucose in response to insulin stimulation. Subsequent studies used the hyperinsulinemic-glucose clamp in mice overexpressing either GLUT4 or hexokinase II (84, 86–88). These studies support results of isotopic modeling approaches showing a greater role of glucose phosphorylation in muscle glucose uptake (75–78, 80, 81). GLUT4 overexpression does not increase insulin-stimulated muscle glucose uptake (86) or whole body glucose disappearance (90) during a short-term fast, while comparable overexpression of hexokinase II causes a twofold increase in R_g (86, 91). The fluid nature of distributed control of muscle glucose uptake is apparent as heterozygous deletion of GLUT4 does not affect insulin-stimulated muscle R_g in wild-type mice but prevents the accelerated muscle R_g in mice overexpressing hexokinase II (92). This illustrates that increased capacity for glucose phosphorylation capacity redistributes the onus of control during insulin stimulation so that there is a greater emphasis on glucose transport.

FIGURE 2.

Myofibers in the fasted, sedentary state have low permeability to glucose creating little challenge to low K_m myofiber hexokinases Insulin-stimulated muscle can become highly permeable to glucose as the glucose transporter GLUT4 is translocated to the cell membrane. The increased entry of glucose into the cytoplasm shifts control of glucose uptake to phosphorylation. HK, insulin-sensitive hexokinase isozyme.

The case for a functional role of hexokinase II is supported by strong “circumstantial evidence.” There is tight coordination between appearance of insulin sensitivity and hexokinase II and GLUT4 transporter expression during ontogenesis of skeletal muscle (93), and there is close correlation between GLUT4 and hexokinase II activities in single oxidative and nonoxidative muscle fibers. Insulin stimulation results in a striking increase in hexokinase II expression in humans (94–97), rodents (93, 98), and cultured cells (51, 99, 100). Although GLUT4 (101, 102) and hexokinase II (103, 104) gene expression both increase with various stimuli, the responsiveness of the hexokinase II gene is more robust particularly after exercise (103). The responsiveness of hexokinase II expression to insulin and exercise infers, but does not itself prove, that the rate of glucose phosphorylation is a limitation. The concept of symmorphosis put forth by Weibel and Taylor (105, 106) is that biological systems maintain an “economy of design” and presumes that nothing is expressed or synthesized without cause. If the system adheres to symmorphosis, the cell would not expend energy to make new hexokinase unless there is a need to do so.

Evidence

Insulin-stimulated GLUT4 translocation is so robust that it results in an increase in membrane glucose transport that shifts control of muscle glucose uptake to glucose phosphorylation by hexokinase.

Exercise creates a situation similar to insulin stimulation in that GLUT4 translocation makes the muscle cell membrane highly permeable to glucose (107). However, during exercise glucose phosphorylation by hexokinases can become an even greater barrier to muscle glucose uptake (82). Muscle glycogenolysis is increased leading to a greater formation of the hexokinase inhibitor G6P (108). Although insulin causes hyperemia (109), the hyperemia of exercise is much greater (110). Hyperemia sustains extramyocellular delivery of glucose further shifting control of glucose uptake to phosphorylation (111). Exercise has in some studies been shown to increase whole muscle free glucose (82, 112–115). This would suggest a shift in control from membrane transport to phosphorylation. Factors such as muscle fiber type, nutritional state, exercise duration, and exercise intensity may also influence the control of muscle glucose uptake due to accumulation of muscle G6P and, possibly, fatty acid availability. For example, during prolonged exercise, a condition that is characterized by a decrease in glycogen stores and if long enough a decline in circulating glucose, there is no increase in skeletal muscle free glucose and very likely no increase in muscle intracellular glucose.

A Case for Glucose Phosphorylation as a Contributor to Muscle Insulin Resistance

Compelling evidence of impairments in both glucose transport and phosphorylation exist in insulin-resistant states (70, 71, 77, 79). In addition, there is evidence that microcirculatory impairments cause impaired glucose delivery to the muscle membrane and contributes to insulin resistance (77, 116). Studies applying isotopic glucose analogs in obese (70, 77, 79) and type 2 diabetic (70, 71, 77, 79) subjects show that in the setting of insulin resistance, the rate constants for glucose transport and phosphorylation decrease (70, 71, 77, 79). It is important to note that deficits in phosphorylation are observed in the range of insulin concentrations that occur in daily living (78, 79). The relative contributions of glucose transport and phosphorylation may also depend on the severity of the disease state (77, 117). Studies applying a combination of isotopic glucose analogs and positron emission tomography show the distribution of control of glucose uptake in obese and type 2 diabetic subjects is dependent on the specific muscle. Surface scanners placed over the mixed fibers of the thigh show control by phosphorylation in obese subjects and type 2 diabetics (79) is greater than when scanners are placed on the oxidative soleus muscle (77). The surface ¹³C-NMR approach that was used in healthy subjects (described above) was also used to measure total muscle glucose in obese and type 2 diabetics (52). The difficulties in the interpretation of this study were discussed previously. It is referenced because of the frequency with which it is cited and to emphasize the need for a more critical examination.

While dysregulation of glucose transport alone is unlikely to be the sole cause of muscle insulin resistance, it is clearly a major contributor (70, 71, 77, 79, 117, 118). Studies in genetic mouse models illustrate the contrasting sites of control in lean and insulin-resistant states. Although hexokinase II overexpression in lean mice results in increased insulin-stimulated gastrocnemius R_g, hexokinase II overexpression does not improve the insulin resistance caused by diet-induced obesity (119). This suggests that in obese mice the vascular delivery of glucose and/or membrane transport of glucose preempt glucose phosphorylation as the primary cause of the insulin resistance in this model (77, 120). The importance of steps proximal to skeletal muscle hexokinase is evident by the demonstration that GLUT4 overexpression (121–123) generally but not in every case (124) improves insulin action in insulin-resistant mice and treatment with vasodilators significantly corrects insulin resistance in rodents (125–127). The former is further supported by a recent study that showed that implantation of myoblasts engineered to overexpress GLUT4 in abdominal muscle of an immunodeficient, insulin-resistant mouse model improves glucose tolerance (118). The latter emphasizes the significance of extramyocellular barriers to muscle glucose uptake (109, 111). Even though hexokinase II overexpression does not correct insulin resistance in obese mice, it is notable that a genetic 50% reduction in hexokinase II decreases insulin-stimulated muscle R_g in diet-induce obese mice (88).

Studies in humans and mice emphasize the importance of defective glucose transport in the insulin-resistant and diabetic states, but also suggest that correction of glucose membrane transport alone will not completely reverse muscle insulin resistance as defects in vascular blood flow to muscle (109) and glucose phosphorylation (70, 71, 77, 79) are also present. These findings suggest that the most effective therapy for insulin resistance will improve muscle vascularity, glucose membrane transport and glucose phosphorylation. It might be expected that the distribution of control will vary with severity, pathogenesis, and duration of the insulin-resistant state. Considerably more work is necessary to understand muscle glucose uptake in health and insulin resistance in the context of distributed control.

Evidence

While membrane transport of glucose is a major site of insulin resistance, glucose phosphorylation is also a significant component of the insulin resistance of muscle glucose uptake.

Hexokinase II Regulation: Implications to Muscle Glucose Uptake

The regulation of hexokinase II in skeletal muscle has received so little attention that there is little consensus on the regulation of this key enzyme. Measurement of total skeletal muscle hexokinase activity (128) and hexokinase I and II (117) activities has been shown to be unaffected in insulin-resistant human subjects, while in other studies hexokinase II activity (129, 130), mRNA (130), and protein (130) have been shown to be decreased in similar populations. Diabetic and insulin-resistant animal models have also shown both no change in hexokinase activity (131–133), as well as decreased skeletal muscle hexokinase II protein (134), mRNA (134), and activity (98, 134–137).

Hexokinase II, like GLUT4, is regulated by how its compartmentalized. In contrast to GLUT4 which has been studied extensively, the regulation of skeletal muscle hexokinase II has received little attention. What follows is a compilation of regulatory mechanisms compiled from studies in various cell systems used to study cancer, signaling pathways, nutrient trafficking, and bioenergetics. A great unknown is how findings from cell systems translate to skeletal muscle physiology and metabolism. Studies in cell lines show mechanisms by which hexokinase II is 1) expressed by the gene that encodes it; 2) compartmentalized; 3) controlled by insulin signaling; and 4) regulated by metabolites such as glucose, G6P, inorganic phosphate, divalent cations, and ATP.

Structural differences between hexokinase I and II lead to functional differences. The amino- and carboxyl-terminal hemidomains of hexokinase II possess intrinsic catalytic activity, while the catalytic activity of hexokinase I is restricted to its carboxyl-terminal hemidomain (138). The K_m for hexokinases I and II reflect their high affinity for glucose (∼30 and ∼300 μM, respectively). G6P has a K_i for hexokinase I and II of only ∼20 μM (138). While both hexokinases I and II are sensitive to feedback inhibition by G6P, only hexokinase II is inhibited by inorganic phosphate (139). Other key structural differences are that hexokinase II, but not hexokinase I, have Akt and mechanistic target of rapamycin complex 1 (mTORC1) binding sites (140). These differences result in very different roles for hexokinase II compared to hexokinase I during insulin stimulation and under other conditions.

Hexokinases are bound to the mitochondria and are present in the cytosol (141, 142). Mitochondria-bound hexokinase results from binding to the voltage-dependent anion channel 1 (VDAC1) (143, 144). VDAC1 spans the outer mitochondrial membrane, forming a complex with adenine nucleotide translocase (ANT), which spans the inner mitochondrial membrane. These proteins complex to form components of the mitochondrial permeability transition pore (mPTP) (145). The binding of hexokinases to VDAC1 on the outer mitochondrial membrane couples ATP produced in the mitochondria to glucose phosphorylation and allows for efficient return of ADP to the mitochondrial matrix for mitochondrial ATP resynthesis (141). Hexokinase binding to VDAC1 not only accelerates carbon flux through the glycolytic pathway to form pyruvate that is oxidized by the mitochondria but by recycling ADP to the mitochondrial matrix allows for efficient electron transfer through the respiratory complexes.

Hexokinase binding to VDAC1 reduces VDAC1 conductance across the outer mitochondrial membrane (146). This lower VDAC1 conductance protects the electrochemical gradient within the mitochondria. At the same time, VDAC1 conductance must be adequate to permit ATP and ADP to transit the outer mitochondrial membrane. Regulation of VDAC1 conductance by hexokinases is important in control of the release of reactive oxygen species (ROS) from the mitochondria. A decrease in VDAC1 conductance by hexokinase prevents excess ROS from entering the cytosol but is adequate to prevent ROS accumulation and toxicity within the mitochondria (147).

The effect of hexokinases on the mPTP is difficult to elucidate since the composition of the mPTP remains controversial. It is difficult to distinguish proteins that are the structural core of mPTP and those that act as regulators. In addition to VDAC1 and ANT, proteins implicated in mPTP structure or regulation include tubulins, translocator protein (TPSO), creatine kinase (CK), phosphate carrier protein, cyclophilin, and F-ATPase. VDAC1 interacts with tubulins and TPSO on the outer mitochondrial membrane to control compartmentation of ATP, ADP, and inorganic phosphate (147, 148). It is unknown whether hexokinase effects on VDAC1 conductance involves interaction with these proteins. ANT is central to mPTP function as it is the dominant controller of oxidative flux in mitochondria isolated from human muscle at physiological ADP concentrations (149). CK contributes to energetics of the mPTP by interactions with ANT and VDAC1. The VDAC1-CK-ANT complex allows for creatine phosphate coupling to the phosphorylation and dephosphorylation of adenine nucleotides (150). Inorganic phosphate carrier protein also interacts with ANT and by doing so regulates inorganic phosphate transport across the inner mitochondrial membrane (151). It has been postulated that ANT interaction with cyclophilin and F-ATPase in the mitochondrial matrix directly couples the mPTP to oxidative phosphorylation (152). It may be postulated that hexokinase control of VDAC1 conductance alters effectiveness of components and regulators of the mPTP (146, 147, 150).

It is notable that mitochondrial bioenergetics are also coupled to hexokinase II by a UCP3-mediated increase in hexokinase II binding to VDAC1. This has been shown to promote efficient aerobic metabolism, increase glucose uptake, and reduce mitochondrial ROS emissions in mice and myoblasts exposed to high glucose (153).

It is significant that mitochondria-bound hexokinases I and II are more effective in driving glycolysis than the cytosolic hexokinases (154). Hexokinase I is primarily mitochondria-bound, whereas hexokinase II is characterized by dynamic movement between mitochondrial and cytosolic compartments (139). G6P and inorganic phosphate at physiological levels inhibit and cause disassociation of hexokinase II from the mitochondria (145, 155–157), while divalent cations (e.g., Mg²⁺ and Ca²⁺) and glucose deprivation enhance the association to mitochondria (67, 107, 137). Overall, studies in cell systems show that a greater fraction of hexokinase II is mitochondria-bound when the need for energy is high.

Hexokinase II, as the insulin-sensitive and predominant skeletal muscle isoform, is of particular significance in metabolic disease. Insulin receptor binding leads to Akt activation (FIGURE 3). Activated Akt can be translocated to the mitochondria where it directly phosphorylates hexokinase II (155, 158, 159). Phosphorylated hexokinase II binds to mitochondria with greater affinity. Akt activation can also increase hexokinase II binding to mitochondria by phosphorylation of glycogen synthase kinase 3β (GSK3β) (160). Like Akt, GSK3β can be translocated to mitochondria. Activated GSK3β phosphorylates VDAC1 decreasing hexokinase II binding to mitochondria (160). This is a second mechanism by which Akt promotes hexokinase II binding to mitochondria. Other kinases have also been implicated in the regulation of the distribution of hexokinase II between mitochondria and cytosol through mechanisms that may be GSK3β-dependent or GSK3β-independent (138, 140, 145). Akt-stimulated mTORC1 activation increases expression of the hypoxia-inducible factor 1α (HIF-1α) transcription factor. HIF-1α induces expression of glycolytic enzymes, including hexokinase II (140). In summary, Akt-mediated phosphorylation of hexokinase II and GSK3β increase mitochondrial-bound hexokinase II. While the effects of Akt/mTORC1/HIF-1α increases the abundance of hexokinase II.

FIGURE 3.

Potential mechanisms by which insulin may regulate hexokinase II compartmentation and activity in skeletal muscleMechanism 1: Akt phosphorylation of hexokinase II increases its binding affinity to VDAC1, while Akt phosphorylation of GSK3β decreases its kinase activity reducing VDAC1 phosphorylation thereby increasing binding affinity to hexokinase II. Mechanism 2: hexokinase II activity is inhibited by feedback from G6P and PO₄, as well as divalent cations and other allosteric regulators. Allosteric inhibition of hexokinase II activity leads to disassociation from VDAC1. Mechanism 3: Akt leads to activation of mTORC1, which induces increased hexokinase II expression. HK II, insulin-sensitive hexokinase isozyme. ETC, electron transport chain; TCA, tricarboxylic acid cycle; VDAC1, voltage-dependent anion channel 1; ANT, adenine nucleotide transporter; mTORC1, mechanistic target of rapamycin complex 1; IRS-1, insulin receptor substrate-1; PI3K, phosphoinositol 3-kinase; G6P, glucose-6-phosphate; GSK3β, glycogen synthase kinase 3β.

Increases in hexokinase II binding to mitochondria are opposed by G6P-mediated feedback and by inorganic phosphate. Together this allows for sensitive on/off regulation of hexokinase II binding with mitochondria in accordance with metabolic status. Incubation of diaphragm with Insulin results in a 70% increase in hexokinase bound to mitochondria within 10 min (161). This increase is transient as hexokinase progressively disassociates from mitochondria as glycolytic intermediates rise. This is consistent with results of insulin stimulation in human muscle. A short-term insulin infusion of 30 min leads to an increase in mitochondrial-bound hexokinase II (97), while a prolonged insulin infusion of >4 h leads to disassociation of hexokinase II from mitochondria and an increase in cytosolic hexokinase II (95, 96). Cytosolic hexokinase II may be translocated to the cell membrane where it can be coupled to glucose transport (162). In this regard hexokinase II co-precipitates with myc-tagged GLUT4 stably expressed in L6 myotubes (163, 164). Hexokinase II interacts with the cytosolic loop of GLUT4 (164) creating potential for efficient coupling of glucose transport to phosphorylation. Regulation of interaction of GLUT4 and hexokinase II is decreased by the glycolytic enzyme GAPDH, which also interacts with the cytosolic loop of GLUT4 and ATP (164). Intuitively it may seem that the interaction of GLUT4 with hexokinase II would couple the accelerated insulin-stimulated glucose transport to the essential phosphorylation of glucose. Studies in L6 cells have shown that insulin stimulation causes disassociation of hexokinase II from GLUT4 (164). This at first glance is surprising. It may be that association of hexokinase II with the outer surface of the mitochondria is greater than the need to couple glucose transport to phosphorylation.

There are data to support that the fate of G6P is dependent on whether it is produced from hexokinase that is mitochondrial-bound or cytosolic (140, 145, 156, 157). Hexokinase II bound to mitochondria channels G6P preferentially into the glycolytic pathway (139), increases mitochondrial respiration possibly through UCP3 (153), and confers a bioenergetic advantage (30). A consequence of the disassociation of hexokinase II from its highly glycolytic mitochondrial-bound state is that a greater fraction of the glucose that is phosphorylated is channeled to glycogen stores (157). G6P formed during glycogen breakdown provides a mechanism to limit glucose uptake and storage into glycogen by inhibiting hexokinase during conditions in which glycogenolysis is high (e.g., exercise) or when glycogen stores are near capacity.

GLUT4 and hexokinase II compartmentation are important to the regulation of insulin-stimulated muscle glucose uptake. Insulin exerts control of cytoskeletal activity and is important to the compartmentation of these proteins. A major influence of the cytoskeletal activity is the integrin signaling pathway which senses changes in the physical or biochemical environment surrounding cells. It has been shown that disrupting the integrin signaling system in insulin-resistant mice that have increased extracellular matrix increases insulin action (165–168).

Evidence

Results from studies in isolated cell systems show that insulin signaling proteins compartmentalize hexokinase II to its more glycolytic mitochondrial-bound form while feedback from G6P and other metabolites inhibit hexokinase II and cause its disassociation from mitochondria.

Much More to Hexokinases than Glucose Phosphorylation

Hexokinases have been referred to as “a critical nexus of integration among energy production, preservation of mitochondrial integrity and cell viability, as well as energy conservation” (140) or as “the guardian of the mitochondria” (169). The protective effects of hexokinases have been described in cell systems, but again study in skeletal muscle is lacking. The role of hexokinase in cell survival and autophagy is reviewed elsewhere (170–174) and is only briefly summarized.

Hexokinase I and hexokinase II both have prosurvival properties. Hexokinase II is of added interest in the context of this review since it is preferentially upregulated by insulin, growth factors, and other physiological stimuli. The expression of hexokinase II is upregulated by prosurvival/stress response pathways, which are necessarily activated for cancer survival and growth. Increased hexokinase II expression is likely key in tumor cells that exhibit prominent Warburg effects. It is noteworthy that Akt activation and other signaling molecules that increase the affinity of hexokinase II binding to VDAC1 and the mPTP are also antiapoptotic and have prosurvival effects (140).

A primary mechanism by which mitochondrial-bound hexokinases exert protective effects is by preventing mitochondrial death pathways. A major mitochondrial death pathway is elicited by apoptotic Bcl-2 family proteins such as Bax and Bak effects by interaction with VDAC1 (169, 175–177). Activated Bax/Bak forms a pore at the mitochondrial outer membrane resulting in a release of apoptotic factors from the intramembrane space (177, 178). Mitochondrial-bound hexokinase II antagonizes Bcl-2 proteins from binding to mitochondria (145, 179, 180) by competitively inhibiting Bax binding to mitochondria (179, 181), antagonizing truncated Bid-induced Bax/Bak-mediated apoptosis (182), and decreasing reactive oxygen species generation (153, 158, 183, 184).

Cytosolic hexokinase II is proautophagic. Hexokinase II serves as a switch between energy production and energy conservation pathways. mTORC1 stimulates growth and proliferation and negatively regulates autophagy (185, 186). Hexokinase II binds to mTORC1 in the absence of glucose, inhibiting mTORC1 activity (187). This interaction is mediated by a well-conserved mTOR signaling motif on hexokinase II (171). This signaling motif is not present in hexokinase I. PI3K/Akt/mTORC1 activation stimulates hexokinase II expression under growth conditions, while accumulation of hexokinase II inhibits mTORC1 during starvation resulting in autophagy. This regulatory loop between hexokinase II and mTORC1 provides an adaptive mechanism controlling cell metabolic status.

Evidence

Hexokinase II compartmentation is pivotal to whether cells are in an energy producing growth and proliferative mode or whether they are autophagic.

Skeletal Muscle Hexokinase II: Unresolved and Uninterrogated

The case is made that the intramyocellular control of insulin-stimulated glucose uptake is distributed between glucose transport by GLUT4 and phosphorylation by hexokinase II. The paradigm where glucose transport is the rate-limiting step describes the sedentary, fasted state. Insulin stimulation increases GLUT4 translocation to the membrane. This increases permeability to glucose causing an increase in intracellular glucose and increased demand on glucose phosphorylation through hexokinase II. An appreciation that insulin-stimulated glucose transport does not equal insulin-stimulated glucose uptake opens the door for research to expand current understanding of muscle glucose uptake. This shift in control of muscle glucose uptake to glucose phosphorylation by hexokinase not only applies to insulin stimulation but also applies to exercise.

A case has also been made that insulin resistance and type 2 diabetes are not due to a defect in glucose membrane transport alone. In addition to impaired membrane transport, evidence exists for dysregulation in the rates that glucose is delivered to muscle and glucose is phosphorylated in muscle. The dominant myocellular impairment generally appears to be glucose transport but may under some conditions be glucose phosphorylation. Nevertheless, both participate in insulin resistance. Impairments in both insulin-stimulated glucose transport and phosphorylation in insulin-resistant and diabetic states suggest the most effective treatments will be those that correct or circumvents defects in both processes (Table 1).

Table 1.

Skeletal muscle hexokinase II and glucose uptake: unresolved and uninterrogated

Unresolved and Uninterrogated Questions
Under what circumstances does hexokinase II contribute to control?
Are there conditions when the ability to phosphorylate glucose is the major limitation?
Is it necessary to correct deficits in glucose phosphorylation to fully protect against insulin resistance?
How do mechanisms for regulation of hexokinase II and glucose phosphorylation demonstrated in cell systems translate to regulation in vivo?

Skeletal muscle hexokinase II exists in dynamic movement between the outer mitochondrial membrane and the cytosol. Little is otherwise known about regulation of hexokinase II in skeletal muscle. Drawing primarily from the cancer field it is evident that control of hexokinase II is complex. Hexokinase II binding affinity to mitochondria channels G6P into glycolysis, promotes nutrient oxidation, and maintains mitochondrial integrity. Insulin signaling proteins Akt and GSK3β increase hexokinase II binding affinity to mitochondria. Feedback inhibition of hexokinase II by G6P and other allosteric regulators cause disassociation of hexokinase II from mitochondria thereby opposing the actions of these signaling proteins. Insulin activation of Akt increases mTORC1 activity which promotes expression of genes for enzymes of energy producing pathways, including hexokinase II. Cytosolic hexokinase II provides another layer of control by inhibiting mTORC1 during energy deprivation creating autophagic conditions.

Studies in a range of cells show that hexokinase II is pivotal in determining glucose phosphorylation, glycolysis, and overall cell metabolic status. How studies of hexokinase II conducted in cell systems primarily intended to model cancer and other stressful conditions translate to skeletal muscle physiology remains to be determined. This is an important avenue for future studies. Understanding the role and regulation of hexokinase II is important to fully understand skeletal muscle glucose uptake. Moreover, it may add insight into mechanisms of muscle growth and atrophy.