-

Perspectives in Diabetes Pancreatic P-Cell Glucokinase Closing the Gap Between Theoretical Concepts and Experimental Realities Franz M. Matschinsky, Benjamin Glaser, and Mark A. Magnuson

There remains a wide gap between theoretical concepts and experimental realities in the enzyme kinetics and biochemical genetics of the pancreatic P-cell gluco- kinase-glucose sensor. It is the goal of present efforts in many laboratories to bridge this gap. This perspective intends to provide a timely review of this crucial aspect of research in glucose homeostasis. It deals briefly with some fundamentals of glucokinase enzyme kinetics, offers some pertinent biochemical genetic considera- tions, takes stock of the current experimental data- base of the field by emphasizing human studies and referring to recent mouse studies, and ventures a few extrapolations into the future of this endeavor. Diabetes 47:307-315, 1998

A discussion of the glucokinase-glucose-sensor paradigm in the study of glucose homeostasis in health and disease requires a brief introduction to currently accepted views about regulation of

insulin secretion from pancreatic p-cells and about the cen- tral role of the liver in glucose metabolism (13).

Insulin secretion is primarily substrate controlled, and glu- cose serves as the preeminent secretagogue among nutrient molecules. Substrate-stimulated insulin release is, however, modified sigruficantly by endocrine and neural factors and may be influenced by certain drugs (Fig. 1). For an under- standing of the p-cell's role in glucose homeostasis, it is important to appreciate the concepts of the Pcell glucose sen- sor, of the glucose threshold for stimulation of insulin release, and of the glucose set point of the organism.

From the Department of Biochemistry and Biophysics and the Diabetes Research Center (EM.M.), University of Pennsylvania, Philadelphia, Penn- sylvania; the Department of Molecular Physiology and Biophysics (M.A.M.), Vanderbilt University, Nashville, Tennessee; and the Department of Endocrinology and Metabolism (B.G.), Hebrew University, Hadassah Medical School, Jerusalem, Israel.

Address correspondence and reprint requests to Dr. Franz Matschinsky, University of Pennsylvania Medical Center, Diabetes Research Center, 510 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104-6015.

Received for publication 4 September 1997 and accepted in revised form 3 November 1997.

gk , gene that encodes GK; GK, glucokinase enzyme; gk' and gk-, mutant gk encoding GK with altered activity; GK+ and GK-, GK with increased or decreased activity due to changes in Kc,, or So, or instability; GK-RP, gluco- kinase regulatory protein; GST, glutathione S-transferase; HI-GK, gluco- kinase-linked hyperinsulinemia; MODY-2, maturity-onset diabetes of the young (type 2); OGTT, oral glucose tolerance test.

To begin with the latter, the physiological set point of blood glucose in humans and many laboratory animals is close to 5 mrnoV1. It is precisely maintained by the interplay of several neural and endocrine glucostatic systems, all char- acterized by glucose-sensing devices and by an ability to gen- erate a hormonal or neural signal that contributes to the maintenance of the blood glucose level at the set point ideal for a particular organism. The p-cells and a-cells of the pan- creatic-islet glucostat are the outstanding examples of two opposing systems designed to keep the blood glucose con- stant. The pituitary-adrenal axis and the adrenergic and cholinergic limbs of the autonomic nervous system constitute the efferent pathways of feedback loop(s), with glucose- sensing cells yet to be characterized but participating sigrufi- cmtly in blood glucose control (4). New findings suggest that the leptin system may also be important (5).

The glucose threshold refers to the glucose level at which the different participating control systems in glucose homoeostasis are turned on or turned off. The p-cell's appar- ent glucose threshold for stimulation of insulin release is largely an expression of the electrophysiology and the secre- tory machinery of the cell, subject to modification by various factors, including the nutritional state and the endocrine or neural input. Physiologically, the threshold is precisely acljusted to secrete the hormone at a rate and glucose level that are appropriate for maintaining the blood glucose close to its set point of 5 mmoV1.

The term glucose sensor refers to the molecular device that allows rapid and precise quantitation of the ambient glucose level. Glucohase serves as glucose sensor in the case of the p-cells (1). The glucose sensors of other glucostatic cells are not well characterized, but glucokinase is expressed in sev- eral rare neuroendocrine cell types and thus may play this role at least in some, if not all, of them (6). The kinetic properties of glucokinase are ideally suited for this purpose (1,7). Most importantly, the enzyme's low affinity for glucose and its sig- moidal glucose dependency guarantee optimal responsiveness at physiological glucose levels. The So, for glucose is 8.4 rntnoM, and its inflection point is 3.9 mmoV1, as a manifesta- tion of its Hill coefficient n, of 1.7, which makes the enzyme most sensitive to glucose level changes close to the physio- logical threshold for stimulated insulin release.

Glucokinase is ratelimiting for p-cell glucose usage (1). It governs glucose oxidation and ATP generation and, therefore, the energy potential of the cell. Because the energy potential expressed as the ATPIADP ratio controls insulin release at

DIABETES, VOL. 47, MARCH 1998

Insulin Epinephrine

potenrintors

Bay K 8644 Inhibitors

Nifedipine

FIG. 1. Minimal model of stimulus secretion coupling in pancreatic p-cells stimulated by glucose. GK refers to the glucokinase glucose sensor. The glucose transporters (referring to GLUT1 and GLUTZ), the ATP-sensitive potassium channel, its associated sulfonylurea receptor, and the voltage-dependent calcium channel are schematically depicted as cell membrane components. The PLC-PKC (phospholipase C-protein kinase C) and the CAMP-PKA (cyclic AMP-protein kinase A) systems modify insulin secretion cooperatively with free cytosolic calcium. G,, G,, and G,, associated with their respective receptors, refer to specific trimeric G proteins. Examples of physiological potentiators and inhibitors and of specific drugs affecting insulin secretion and their sites of action are given. ATPases linked to exocytosis are not depicted for reasons of clarity but should be imagined as part of the minimal model. AC, adenylate cyclase; ACh, acetylcholine; DAG, diacylglycerol; GLP-1, glucagon- like peptide 1; IP,, inositol triphosphate; PKC, protein kinase C; PLC, phospholipase C; SUR, sulfonylurea receptor.

multiple sites, glucokinase also governs the hormone secre- tion process. Glucokinase exerts such a unique regulatory role in pcell metabohm because it is virtually irreversible and not influenced by feedback inhibition, in contrast to the other hex- okinases (I and 11), which determine glucose usage in most if not all cell types. Its activity and the levels of the two sub- strates glucose and MgATP" wholly determine flux. Meta- bolic coupling factors-molecules that link intermediary metabolism with membrane events-include ATP, ADP, and malonyl-CoA. They are generated as a function of gluco- kinase rate and activate the secretory machinery. Critical among the molecular devices initiating and sustaining secre- tion are the ATP-sensitive K+-channel and the voltage-sensi- tive Ca2+-channel, which control the membrane potential and Ca2+ influx from outside the cell (8). The ATPases that ener- gize exocytosis are also critically dependent on the energy potential (9). Together, these processes determine the thresh- old and the rate for insulin secretion. The glucokinase glucose sensor has, therefore, a high control strength in the process of substrate-induced insulin release.

The control strength of any enzymatic process for a given pathway can be defined by a simple expression:

Enzyme control strength = relative change of pathway flux relative change of e m m e activity

The control strength of the glucokinase (or hexokinase IV) enzyme (GK) for metabolic flux through glycolysis has been thoroughly studied and is close to unity (10). This implies that changes of its total p-cell activity by a small fraction (e.g.,

25%) alter the rate of glucose metabohm precisely that much and, hence, alter the rate of insulin release when glucose is the sole or the permissive stimulus. It is to be recalled that the rel- ative changes of insulin release are much larger than the rel- ative changes of glucokinase and glycolysis resulting from an intracellular system of gain control.

The ion channels and associated regulatory proteins (e.g., sulfonylurea receptor-1 [SUR-11) also have a very strong impact on p-cell function and may dramatically change the threshold level at which glucose is perceived a s stimulus. They could be altered so that substrate control is altogether elim- inated (11,12). However, metabolic control theory has not been systematically applied to ion channels and exocytosis- related ATPases. They can therefore be described in qualita- tive terms only.

Because of their biochemical and biophysical design fea- tures, whch are briefly sketched here, it is not surprising that the glucokinase glucose sensor and critical ion channels have moved to center stage of p-cell physiology and patho- physiology.

Hepatic glucokinase plays an essential role complemen- tary to p-cell glucokinase in the maintenance of glucose homeostasis (2,3). It controls the hepatic extraction of portal glucose and mediates the modification by glucose of hor- mone induction of Ltype pyruvate kinase, fatty acid synthase, the glucose transporter GLUT2, and glucose6phosphatase (3). Glycogenesis and lipogenesis from lactate, pyruvate, alanine, and glutamine are glucosedependent processes and may thus be regulated by glucokinase (2). Because hepatic glucokinase gene expression is regulated largely by insulin and glucose and

DIABETES, VOL. 47, MARCH 1998

- - -

F M MATSCHINSKY, B GLASER, A N D M.A. MAGNUSON

b .- - g 150

g $ I 1 2 5 c = 0 '- L loo 8 2 8 ' 75 6 E U Q 50 0 - 0

25

heterozygous gk knock-out

wild-type

GK gene copy number

FIG. 2. Impact of the hepatoinsular glucokinase feedback loop on the glucose set point of transgenic mice. The influence of a fourfold range of the glucokinase gene dosage on average blood glucose of transgenic mice is shown. Modified with permission from Niswender et al. (3).

Pcell glucokinase levels are governed by glucose, the concept of a hepatoinsular glucokinase feedback loop (2,3,13) becomes increasingly attractive. The high relevance of this control system is demonstrated by recent data on glucokinase gene locus transgenic mice and heterozygous glucokinase null knock-out mice, which cany anywhere from one to four functional glucokinase gene copies. Analysis of these mice clearly demonstrates that blood glucose concentration is a function of glucokinase gene dosage (Fig. 2). This experi- ment shows that the hepatoinsular glucokinase feedback loop is a dominant control system in glucose homeostasis and can change the physiological set point of the organism in either direction. It also demonstrates that attempting to estimate the relative importance of hepatic or P-cell glucokinase in blood glucose regulation is a difficult undertaking, given the cyclic interdependence of the two (Fig. 2).

BASIC CONCEPTS OF GK KINETICS The physiological and pathological relevance of the gluco- kinase glucose-sensor concept as it applies to both Pcells and hepatocytes can be fathomed only if the basics of GK kinet- ics are appreciated (1,7,14). In particular, the recent molec- ular genetic and metabolic clinical findings in patients with maturity-onset diabetes of the young (type 2) (MODY-2) and HI-GK (glucokinase-linked hyperinsulinemia) cannot be understood without a grasp of these concepts (15,16).

The kinetics of glucose phosphorylation by glucokinase are best described by the Hill equation (7,14):

where v = the specific activity or turnover of GK at a given glu- cose level S (IUlmg protein or s-I), S = the glucose concen- tration (nunolll), V = the maximal specific activity or K, of GK (IUImg protein or s-I), So, = the glucose level at the half-max- imal rate (mmolll), and the exponent n = the Hill coefficient or nH as a measure of the sigmoidicity of the glucose depen- dency (which is unitless). Glucose phosphorylation is conve- niently monitored in the spectrophotometer or fluorometer using an indicator reaction that generates or uses NAD(P)H

O 20 40 60 80 100

Glucose, mM

Glucose. mM

FIG. 3. A: Nonlinear fit of concentration dependency of glucose phos- phorylation by human wild-type recombinant GST-glucokinase. B: Hanes-Woolf transformation of data presented in A . The apparent So,, is indicated. Careful inspection reveals that the plot is slightly non- linear even at the high glucose levels used here to estimate Vm,, and So,, and that errors of data interpretations may readily occur.

(7,14). However, great care must be taken that the pH, the ionic strength, the K+ and ~ g ' concentrations, the level of the sec- ond substrate M~ATP~-, and the sulphydryl reagent (usually mercaptoethanol or dithiothreitol) are precisely controlled. For example, lowering the pH from 8.0 to 6.8 increases the So,, value nearly twofold, and the M~ATP level greatly influences the glucose So,, value and cooperativity. The temperature is usually kept at S30°C because the enzyme is unstable. The indi- cator systems must not be rate-limiting. tinder such conditions and using reasonable glucokinase levels, reaction progress curves are proportionate to glucokinase amount and are lin- ear for 5-10 rnin. A plot of Sversus v is clearly sigmoidal (Fig. 3A). It shows an So,, of 8.4 mmolll, and saturation is accom- plished at 30-50 mrnolll glucose. The n, is obtained by non- linear curve fitting and is usually - 1.7. Sometimes, particularly in studies of crude tissue extracts from pancreatic islets or liver, it is not unreasonable to use a linear transformation of the Michaelis-Menten equation (i.e., where n = 1 in Eq. I), and in that manner obtain approximate So,, or V,,, values by extrapolations. The Hanes-Woolf plot of S versus S/Vis most suitable for this purpose (Fig. 3B), with the error usually being small. While much of the fundamental work about the role of glucokinase in liver and islet glucose metabolism was done with these theoretically wrong kinetic methods (1,7), the use of nonlinear kinetics is obligatory for work with purified native or recombinant wild-type and mutant GK because

DIABETES, VOL. 47, MARCH 1998

errors due to unorthodox kinetics may be large, and thus results may be misleading (7,14). For characterizing wild-type and mutant enzymes, it is also mandatory to assess the well- defined relative affmities to other sugar substrates, e.g., man- nose and fructose. Glucokinase is inhibited by glucokinase reg- ulatory protein (GK-RP) and by longchain acyl-CoA (17). The effectiveness of these inhibitors provides another informative measure of the enzyme's structure and function. The effects of GK-RP are probably physiologically irrelevant for the pcell, since regulatory proteins may not be present but are crucial for understanding GK function in hepatocytes. Table 1 lists best approximations of established kinetic constants of human islet GK under standard conditions obtained with nonlinear analysis of data. A simple thennolability test may provide the first lead in characterizing an instability mutant (14).

EXTRAPOLATIONS OF ENZYME KINETICS TO CLINICAL METABOLIC AND BIOCHEMICAL GENETICS STUDIES

Because of its high control strength over glycolysis, gluco- kinase determines the rate and concentration dependency of glucose-stimulated insulin release. The postabsorptive thresh- old for stimulation of release (i.e., when the p-cell is not mod- ified by the vagus and gut hormones) is reached physiologically at 2W0% of the maximal rate of P-cell glucose metabolism or p-cell GK turnover (Fig. 4). Half-maximal secretion is achieved at 50-60% of these parameters. This information can be used to predict the impact of glucokinase changes on the glucose levels at threshold and half-maximal stimulation of insulin release. The rate of glucose phosphorylation in P- cells at threshold (calculated here for the upper limit at 300h of Kcat) is defined by the following expression:

100

8 m 2 75 2 c .- - 2 9 50 $

25 ........ * ....... Insulin release

-A- GK Activity 0

TABLE 1 Kinetic characteristics of human wild-type recombinant gluco- kinase

Kinetic parameter Close approximation

Kcat

S,) 5 (glucose) K", (ATPI n~ Inflection point I,, , stearyl-CoA* I,,, GK-RPt

*At the inflection point of glucose. ?At the inflection point of glucose and using 1 pg/ml of GST-GK.

A factor of 2 is used because glucokinase expression in the islet organ is controlled by both alleles of the gene. Using the established Kcat of 50 s-', n, of 1.7, and glucose threshold of 5 mrnolll, the equation may be solved.

An analogous computation may be carried out for half- maximal glucose stimulation at 12-14 mmol~l. Vat So,, of 13 mmoH is 68 s-' in terms of Kc,. The impact of any kinetic changes of GK on insulin release, caused by mutations of the gene, for example, may then be calculated with the assump- tion that all other functions of the pcell glucostat remain con- stant. Within limits, this assumption appears to be very rea- sonable and is borne out by clinical observations (15,lG). The model calculations are made with the hypothetical situ-

20 30 40

Mannosc, mM

FIG. 4. The threshold for stimulation of secretion due t o glucose or mannose. Insulin secretion from freshly isolated rat islets was studied with a perifusion system. Islets were first perifused in the absence of substrate, and starting at 30 min they were stimulated with a hexose ramp of 0.8 mmol . I-' . min-' to determine apparent threshold and half-maximal stimulant levels. Hexose phosphorylation was measured spec- trophotometrically, and glucokinase Kc,, values were obtained. Relative data are presented. A shows data with glucose, and B shows results with mannose. Relative K,, values (s-') for the hexose threshold (25-30%) and hexose levels resulting in half-maximal (50-60%) stimulation of insulin release may be obtained from these graphs.

DIABETES, VOL. 47, MARCH 1998

ation where only one allele is modified and the other is kept constant because of the autosomal dominant nature of the rel- evant clinical entities (Fig. 5). So,, or Kc,, of one allele is changed here beyond physiologically relevant ranges to fully characterize the nature of the system. The impact of P-cell GK alterations is striking. Plots of So,, or l/Kcat versus the glucose threshold of the P-cell represent hyperbolic functions (Fig. 5A). Normoglycemic wild-type subjects (GWGK) exhibit a P- cell glucose threshold of 5 mmol/l. Heterozygotes with loss- of-function mutations in 50% of the enzyme (GKIGK-), because of either increased S,, or lowered Kc,, show pro- gressive elevations of the threshold to a maximal 8-9 mmol/l. Quite remarkably, even a singular twofold alteration in one allele of the So,, or the K,, raises the apparent threshold to -7.0 mmol/l. Three- to fourfold changes in either parameter show near-maximal effects. Clinically, therefore, it may not matter whether the glucose So,, increases to 30 or 300 mmol~l or whether the Kcat drops to one-third or to zero. In contrast, heterozygotes with a gain-of-function mutation in 50% of the enzyrne exhibit a lowering of the glucose threshold (16). A 50% decrease of the So,, or a doubling of the Kcat of only one GK allele results in a change of the glucose threshold from 5 to -3.5 nunol/l. Even lower thresholds would be reached if the mutations were more dramatic. Using a factor of 10 for So,, or Kc,, the glucose threshold extrapolates to - 1 mmol/l. An analogous graph can be constructed for the glucose level that is needed to achieve the physiologically equally relevant half-maxin~al insulin release (Fig. 5B). The impact of affinity and activity mutants of the enzyme on half-nlaximal release may be modeled on an average glucose So, for stimulated insulin release of 13 mrnol/l (Fig. 4) and the kinetic constants of GK (Table I). Glucose levels required for half-maximal rates of secretion increase almost linearly from 1 to 50 mn~ol/l as the glucose So,, of the mutant enzyme increases 70- fold. A fourfold change of Kcat of the mutant enzyme from 25 to 100 s-' lowers the So,, for secretion almost linearly from 30 to 5 mrnol/l glucose. When the K, falls to less than half of con- trol, very high glucose levels would be required to achieve half-maximal rates of release, or glucose phosphorylation rates may never be sufficient to reach the critical value of 68 s-'. This is indicated by an exponential increase of glucose So,, values for insulin release as a function of l/Kcar Guided by these modeling data, the thresholds and So,, levels of glu- cose for insulin release may be determined in subjects with MODY-2 and HI-GK by carefully designed clinical tests.

The hepatoinsular glucokinase feedback loop implies that the impact of glucokinase mutations may be self-limiting, at least to some extent. As the blood glucose rises or falls, it will increase or decrease the expression or stability of the GK in hepatocytes and P-cells alike such that the phenotype may be less abnormal than one might predict if such compensatory mechanisms were ignored (3). Recent results with MODY-2 (18) and with GK gene locus transgenic mice (3,19,20) show that such adaptations do indeed take place.

Some basic genetic considerations are probably in order at this juncture to help assess the practical relevance of these model studies for a population in equilibrium. It is assumed, with good reason, that glucokinase gene expression in the P- cell is determined largely by gene dosage. A wild-type allele frequency of 1.0 would result in a p-cell glucokinase activity defined as the sum of the enzyme turnover numbers of the two alleles 2 X Kc, or 100 s-' (Eq. 2), and the aggregate KcaJSo,,

10- A 50.5 mutants z E P s

L 5 g 5 = 8 - ul - - t m

10 20 30 40 of mutant GK allele, mM

0 0.02 0.04 0.06 0.08

lIKcat of mutant GK allele, sec

- GK 1 GK

U) mutants

t X

20 40 60 80 S0.5 of mutant GK allele, mM

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18

lIKcat of mutant GK allele, sec

FIG. 5. Dominance of glucokinase in glucose-stimulated insulin release. Equation 2 is used, but one normal (Wt) and one mutant (Mt) enzyme are considered such that

snwt X K Wt rat SnMt K C s t M t " = -- - -

nwt + S"Mt + ~ 0 , 5 D M t s-I snwt + So,,

Glucose thresholds and So., values for insulin secretion were computed as a function of the glucose So,, of the dominant mutant glucokinase forms (GKf or GK-), with the other kept normal (GK) at 8.4 mmoyl. Calculations with Kcat as the variable (normally 50 s-' and plotted here as the reciprocal value lIK,,,) were similarly d0ne.A shows the glucose threshold data, and B the half-maximal insulin release data.

(an expression of the glucose phosphorylation efficiency) would be 11.9 s-' X mmol/l-'. With autosomal-dominant inheritance of mutations, predictable from the high control strength of the glucokinase glucose sensor, and assuming, for the sake of argument and simplicity, an incidence for the affected phenotype of 1:1,000 subjects, a wild-type allele fre- quency of 0.999 and a mutant allele frequency of 0.001 can then be calculated. (Estimates indicate that the frequency of MODY-2 may be as high as 1:2,000 subjects (28), but it is too early to make any reahtic estimate about the frequency of HI- GK.) The aggregate P-cell glucohase activity is predicted to change depending on the type of mutation. Chances for detecting an affected homozygote @k-lgk- or gk'lgk') would be extremely low, i.e., 1:4,000,000. If the incidence were 1:10,000, the chance for homozygocity would drop further by a factor of 100. Consanguinity would, of course, increase the odds dramatically. Depending on the nature of the mutants, homozygocity might not necessarily be lethal (Fig. 5). These

DIABETES, VOL. 47, MARCH 1998 311

purely theoretical predictions emanate from the classical Hardy-Weinberg model (2 1).

THE DATABASE FOR ENZYME KINETICS OF THE P-CELL GLUCOKINASE GLUCOSE SENSOR Kinetic studies of glucokinase of islet tissue extracts usually apply Michaelis-Menten kinetic analysis for practical rea- sons. In islet tissue homogenates of rats, mice, hamsters, or humans, glucokinase often accounts for only one-fourth of the total glucose phosphorylation capacity (22). The high-affin- ity hexokinase(s) contribute(s) the major fraction, usually pre- cluding the application of sophisticated kinetic analysis because of the error structure of the data. It is not surprising then that the reported data on specific activities of the enzyme (usually expressed in terms of micromoles of glucose phosphorylated per minute [ILJ] on a protein or DNA basis) vary greatly. However, when measurements are done com- petently, a basal activity of 0.1-0.3 mU/pg islet DNA is repro- ducibly reported, and apparent So,, values, frequently referred to as K,, usually fall within a range of 8-15 rnmoH. Even though the kinetic analysis of these data is, strictly speaking, unorthodox, their wide application has uncovered a wealth of important information. The data provided, for example, the experimental basis for the glucokinase glucose sensor hypothesis in rodents and h m ~ a n s (1,2,22) and for the concept that the pcell enzyme is directly inducible by glucose, which contrasts with the situation in the liver, where the enzynle is induced by insulin (23). The latter results are con- sistent with the molecular genetic analyses of glucokinase gene expression in liver and pancreatic islets, which have revealed a single gene with two different cell type-specific promoters controlling glucokinase biosynthesis (24,25).

Analytical methods are available that are sufficiently pre- cise and reproducible for conducting kinetic studies of crude pancreatic islets extracts that may include accurate deter- mination of specific activity, glucose affinity, and even coop- erativity (26). But the expense of such precise measurements would be relatively high compared with the fluorimetric method commonly used and may be justifiable only rarely, considering what may be gained. Any radiometric procedure that assures that hexokinases other than glucokinase are strongly inhibited is suitable. This may be accomplished by including high levels of glucose-6-P or glucose-1,6-P, in the assay mixture.

It might be expected that available kinetic data about puri- fied or recombinant islet glucokinase are more precise and reliable. Surprisingly, this is far from true. There are several reasons for this state of affairs, but a few typical examples will suffice for a general alert and perspective. One reason for dis- crepancies is the uncritical application of enzyme kinetic methods to the study of a sigmoidal enzyme like GK (7,14). Many reports are based on the use of linear transformation of Michaelis-Menten plots, whose limitations were discussed (7,14). It is noteworthy that the error introduced by using the Hames-Woolf plot (the most suitable linearization for enzymes with nonlinear kinetics of the kind considered here) is actually not >25% for the So,, value and even less pro- nounced for the Kc, of the enzyme, provided the full range of substrate levels (0.3-100 mmoM for the wild type and as high as 0.5-1.0 moH for mutants) is chosen for the assay (7,14). If the selection of substrate levels is in any way biased, very large errors may ensue (Fig. 3). Another reason for the wide

discrepancies in the literature stems from the use of human recombinant glucokinase cDNA that may be incidentally modified by an artifact of unknown origin (27). This problem is vividly illustrated by reports of several studies with a human recombinant islet glucokinase, which carried an ini- tially unrecognized base change resulting in an aspartic acid to alanine substitution at position 158. Apparent K, or So,, val- ues for this enzyme were reported to be 6-8 mmoH in a series of studies from one group (28-31) but were found to be 3 4 mmoH in two other publications with overlapping authorship (27,32). Such differences are critical in view of the high control strength of glucohase in p-cell metabolism (Fig. 5). Kinetic studies with D158A enzyme (mistaken for wild-type glucolunase) indicated that the well-established hexose specificity of glucokinase, characterized by a twofold preference for the glucose molecule compared with the man- nose molecule, may not be preserved in the hunlan recom- binant enzyme preparation (32,33). These data-accepted at face value-were used as an argument against one of the tenets for the glucokinase-glucose-sensor paradigm, which is based on strict parallelism of the structure-activity relation- ship between hexose phosphorylation by glucokinase, the usage of the different hexoses by intact islets, and their potency to cause insulin release. It is of course possible that the D158A conversion does indeed change hexose speci- ficity-an observation requiring independent confirmation- but such data have no bearing on the problem of hexose specificity of stimulated insulin release by the normal p-cell.

The interpretation of kinetic data in studies of anomeric specificity of human recombinant glucokinase and several mutants of the enzyme is also open to criticism (32). Because most of the recombinant proteins (with the exception of mutant enzyme N166R), including the so-called "wild type," had the unrecognized D158A substitution, all other mutants were actually double mutants. Thus, much data for S151C, N204S, K56A, N204A, and E209A is put into question. It was reported that the maximal velocity of four of the six mutants was higher with the a- than with the p-anomer of glucose, in contrast to what was found with the wild type. The differences of the activity ratios were small, considering that statistics were not given, i.e., 1.17, 1.14, 1.54, and 1.56 in the affected group compared with 0.73 and 0.73 in the nonaffected group of mutants. The So,, values for a-D-glucose were, however, lower than the So, values for p- glucose in all but one of seven different enzyme forms, demonstrating that the a- anomeric preference at low glucose levels was relatively well maintained. It is the ability of glucokinase to discriminate between a- and p-anomers at relatively low D-glucose levels that explains the higher potency of the a-anomer as stimula- tor of release. From these data, it was concluded that the higher insulinotropic efficiency of a- than (3-glucose cannot be ascribed to the intrinsic catalytic properties of p-cell glucokinase. This conclusion is false. Studies like these betray a total lack of quantitative thinking in dealing with enzyme kinetic data related to p-cell function.

As disclosed by Veiga da Cunha et al. in 1996 (27), the great majority of the literature, only partly reviewed here, on which biochemical genetics considerations of MODY-2 are based is flawed by the apparently inadvertent inclusion of the D158A substitution in wild-type and mutant enzymes alike. The possibility that the artificial D158A substitution did influ- ence the kinetic constants in studies of various MODY-2

DLABETES, VOL. 47, MARCH 1998

mutants cannot be taken lightly. Much of the extensive work done with these double mutants published so far needs to be reevaluated.

One additional aspect of enzyme kinetics of human mutant glucokinases that is often ignored is the structural instability of the enzyme (14,34). The preparation of a recombinant enzyme by lengthy purification schemes may result in inac- tivation of a kinetically normal but unstable protein, such that the purified end product shows little resemblance to the mol- ecule in situ. Application of the glutathione S-transferase (GST) (14) or polyhistidyl (35) fusion approach combined with single-step affmity chromatography offers a state-of- the-art technique to minin~ize interference by inactivation of inherently unstable glucokinase mutants, as clearly docu- mented for E300K (14,34).

Because of this difficulty in identlfylng and characterizing instability mutants, we have used the GST fusion-protein technique generically for the purification of human wild- type and mutant recombinant glucokinase (14,34). The wild- type GST enzyme is kinetically indistinguishable from the native glucokinase, and there is good reason to believe that the kinetic characteristics of mutants are also well pre- served in the fusion protein (14,34). It remains to be seen how this database obtained with the genetically correct wild-type and mutant fusion proteins compares with corre- sponding enzymes purified by a classical procedure or using an alternative fusion-protein design. Such work is currently in progress.

It is also highly advisable that kinetic analysis of mutant glucokinase includes a quantitative assessment of the effects that physiological inhibitors have on enzyme kinetics. The inhibition by GK-RP and by long-chain acyl-CoA should be part of a battery of standard tests for GK mutants (7,17). These tests should be performed because they may provide specific information on molecular structure and function of the enzyme and because alterations of the efficacy of these inhibitors may explain aspects of the clinical phenotypes in glucokinase-related disease states. In selected cases, this has been accomplished. The double mutant D158AlV203A showed a significant decrease of affmity (>1.5-fold) for the GK-RP (27). It remains to be confirmed whether this affinity change persists in the pure MODY-2 mutant V203A. It was observed that the V455M mutation, which causes hyperinsu- linemia, does not change its sensitivity to GK-RP (16). In addition, this same mutant was equally sensitive to stearyl-

CoA as the wild type. It must be noted that such studies with physiological inhibitors need to be performed at equivalent glucose levels, i.e., considering the So,, of the particular enzyme forms, because GK-RP and acyl-CoA inhibit GK com- petitively with glucose (16,27).

BIOCHEMICAL GENETICS OF MODY-2 AND HI-GK

The discovery in 1992 of glucokinase mutants in MODY-2 patients, and more recently (1997) in subjects with HI-GK, is clearly the culmination of the explorations that established the glucokinase glucose sensor concept as essential for understanding glucose homeostasis in many laboratory ani- mals and humans (16,36). Froguel et al. (36) were the first to document the linkage between MODY-2 and glucokinase. More recently, Glaser et al. (16) complemented these findings by documenting hypoglycemia caused by a glucokinase mutation. Work in many laboratories in many parts of the world have identified as many as 44 mutations in 260 affected individuals that cause a mild MODY-2 form of diabetes (15), and, so far, one mutation that caused hypoglycemia in five individuals of a family with a three-generation pedigree (16). Figure 6 summarizes the locations of all known mutations.

Individuals with MODY-2 usually present with mild hyper- glycemia that is discovered by routine examination (15). On average, the fasting blood glucose is 7.0 + 1.1 mmol/l com- pared with 5.0 + 0.5 mmom in control subjects, and the 2-h value during an oral glucose tolerance test (OGTT) is 9.4 + 3.0 mmol/l, contrasting with 5.1 * 1.3 in normal subjects. Fasting serum insulins are comparable (60 + 48 and 66 + 42 pmol/l). The 2-h serum insulins during the OG?T are also similar (198 + 180 vs. 174 + 150 pmol/l). The insulinogenic indexes (insulin/glucose) readily calculated from these data indicate a secretory deficit of P-cells during fasting and after an oral glucose load. These data and the results of more sophisticated clinical studies demonstrate that the average glucose thresh- old for stimulation of insulin release from the P-cells is increased from 5 to -7 mmoM and imply an average reduc- tion of the K,, or increase of its So,, of one GK form minimally by 50%. Interestingly, the reported changes in kinetic constants of mutated MODY-2 glucokinase are often far more pro- nounced than might be expected from the phenotypes (18,37). This apparent discrepancy has been explained by compensation from the remaining normal allele due to some induction of both islet and hepatic glucokinase as a conse- quence of the mild hyperglycemia. This may be a partial

8- cell promoter E70K

Liver promoter

FIG. 6. Currently known glucokinase mutants causing MODY-2 or HI-GK. Modified from Velho e t al. (15).

DIABETES, VOL. 47, MARCH 1998

explanation. In addition, many concerns expressed in this dis- cussion of kinetic studies of mutant enzyme make it likely that, in many cases, the available lunetic data may be an unreliable basis for sophisticated quantitative analysis of clinical metabolic observations. Attempts at computer mod- eling of glucose-induced insulin release by Sturis et al. (18) have the shortcoming of ignoring sigrnoidicity as a critical fac- tor in the kinetic analysis. As a result, modeled glucose dose- dependency curves of release may not realistically represent the situation in vivo. Given the uncerbhties of the kinetic data as discussed above, it is not surprising that discrepancies of modeled and clinical data remain substantial.

Some of these problems were probably avoided in a recent biochemical genetic study of HI-GK because of the V455M GK mutant (16). The HI-GK syndrome is characterized clinically by hyperinsulinemia and associated hypoglycemia that is sometimes severe enough to cause seizures. Insulin secretion ceases when the blood glucose falls below 2 mmoVI, i.e., it is glucose regulated. Hypoglycemia may be controlled by fre- quent meals and diazoxide. The model used in this study accurately predicted the observed clinical data. The singular kinetic change of the mutant enzyme causing HI-GK was a lowering of the So,, from 8.4 to 2.9 mmoM. A threshold of 2.0-2.5 mmoM glucose was predicted close to the observed data of -2 mmoM. This success may have been helped by the fact that, in the case of the hypoglycemia syndromes, data fall in the steep, and therefore sensitive, range of the gluco- kinase-insulin secretion diagram (Fig. 5), contrasting with the relatively flat, and therefore less sensitive, portion of this relationship that applies to the MODY-2 cases. The discovery of any gain-of-function glucokinase mutants that cause a less marked hypoglycemia, e.g., with a p-cell glucose threshold of 3.5 vs. 5 rnmoM, may nevertheless be very difficult.

PERSPECTIVE It is a great challenge for clinical investigators to recognize and dlagnose even milder forms of MODY-2 or HI-GK than those currently known and to team up with molecular geneti- cists and biochemists for characterizing, with great care, the causative mutations of such cases. Natural mutants of g lum lunase in humans have unsurpassed heuristic sigruficance because molecular structure and function and phenotypical expression are present in one. A second challenge is to reassess and complete the existing molecular genetic, enzyme kinetic, and metabolic database for the 44 known glucokinase mutations in MODY-2 such that an internally consistent and comprehensive disease identity may be defined. The existence of HI-GK and the theoretical possibility of a conlpound heterozygote gkflgk- may argue for an over- arching definition of a glucokinase glucose sensor disease entity. This could be a genetically and pathophysiologically useful concept. The quest of developing cell and animal mod- els for testing the glucokinase glucose sensor concept, as it presents itself from the wide variety of experimental and clinical observations, needs to be vigorously continued because many fundamental questions remain to be answered. Glucokinase turnover in p-cells is one of them. The cell-specific effects of glucokinase, e.g., in liver and brain, are another. Crystallization of glucokinase or of the gluco- kinase1GK-RP complex also remains an m e t challenge. A better understanding of glucose homeostasis promises to come from such endeavors. A full comprehension of the cm-

cial role of glucokinase in glucose homeostasis clearly iden- tifies the enzyme as a potential target of drug discovery and, given success in such an undertaking, there may even arise some therapeutic benefit from the fundamental studies that now extend over a period of 3 decades (38,39).

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