diabetic embryopathy: a selective review of recent trends

f Diab Camp 1993; 7:204-212

SCHOLARLY REVIEW

Diabetic Embrvopathv: A Selective Review of Recent Tre6di

J

Lester Baker Ronald Piddington

INTRODUCTION

lthough not always thought of in the same

A league as diabetic retinopathy and diabetic nephropathy, diabetic embryopathy represents a major complication of diabetes.

Infants born to women with diabetes have a malformation risk which is two to three times greater than that seen in the general population.lJ A recent study reported that the relative risk for major malformations among infants of women with insulin-dependent diabetes was 7.9 (95% confidence interval, 1.9-33.5).3 In addition, these birth defects involve major organ sys- tems, and are thus more likely to be lethal.1*3,4 Indeed, congenital malformations now represent the major cause of deaths in infants of diabetic mothers.4 The congenital malformations seen in excess in infants of diabetic mothers, and thus most characteristic of the diabetic embryopathy syndrome, involve deformities of the central nervous system and the heart.z,3,5 Central nervous system defects in infants of diabetic mothers range from anencephaly to spina bifida,3 and result from abnormalities in the normal embryonic development of the neural folds. The fact that they appear so characteristic for the teratogenic diabetic insult, plus the fact that neural tube development takes place during the viable period of rodent embryo culture, has led to much animal work in which neural tube malformations have been used to study diabetic embryopathy. This review will focus on recent evidence delineat-

Division of EndoQinologylDiabetes, Children’s Hospital of Phila- delphia, Department of Pediatrics, Universitv of Pennsvlvania School of kfedkini (L.B.), and Deparknent of katomy ahd Histology, School of Dental Medicine, University of Pennsylvania (R.P.), Phila- delphia, Pennsvlvania, USA

tieprint requkts to be sent to: Dr. Lester Baker, Division of Endo- crinology/Diabetes, Children’s Hospital of Philadelphia, 34th & Civic Center Boulevard, Philadelphia, PA 19104.

0 1993 Journal of Diabetes and Its Gnnpiimtions

ing specific pathways which appear to play important rules in the mechanism of diabetic embryopathy.

INVOLVEMENT OF MYO-INOSITOL IN DIABETIC EMBRYOPATHY

Many factors have been proposed as important in the mechanism producing diabetic embryopathy. For the most part, these hypotheses have tended to remain separate from the abnormalities in intracellular myo- inositol and phosphoinositide metabolism, which have been implicated as a possible model for the mechanism of diabetic complications in other tissues.6 In oversimplified terms, this model postulates that elevated glucose levels result in tissue myo-inositol depletion. In turn, this affects phosphoinositide metabolism, a key intracellular signalling system that provides such important second messengers as the inositol phosphates, diacylglycerol and arachidonic acid. Recent evidence indicates that the myo-inositol pathway may be involved in the mechanism of diabetic embryopathy.

That myo-inositol is important for normal embryonic development was clearly delineated by Co&oft.’ In studies of the nutritional requirements of postimplan- tation rat embryos in culture, a specific need for myu- inositol was found. Rat embryos explanted at 9.0 and 9.5 days of gestation had an absolute requirement for fiyo-inositol (along with other “vitamins”) for normal development. Of particular interest is the fact that 50% (6 of 12) of the rat embryos explanted at 9.5 days and cultured in a myo-inositol-depleted medium demonstrated neural tube fusion defects.

No measurements of changes in embryo myo-inositol content were provided in the paper of Cockcroft, and no longitudinal studies of specific developmental changes in embryonic myo-inositol concentration have been reported. However, because either an elevated glucose concentration in the culture medium or diabe-

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J Diab Cmnp 1993; 7:204-212 DIABETIC EMBRYOPATHY: RECENT TRENDS 205

tes in the pregnant animal is associated with a diminu- tion of embryo myo-inositol concentration when compared to the normal, this suggests that a developmental increase in myo-inositol level had not taken place in the presence of a high glucose level. This thesis is supported by the demonstration of a developmental increase in myo-inositol level in neuroectodermal tissue by Sussman and Matschinsky.8 They studied ll- and 12-day-old rat embryos obtained from normal and diabetic pregnant rats. During the time coincident with normal neural tube fusion (days 10 and 12 in the rat), the myo-inositol content of neuroectodermal neural tissue in the embryos of normal rats showed a 190% increase (p < 0.001, day 12 versus day 11). In rat embryos of the same gestational age obtained from diabetic rats, the development change in myo-inositol was almost completely blunted. In the embryos obtained from diabetic pregnancies, the myo-inositol concentration of 12-day neural tissue was an insignificant 22% higher than that found at 11 days.

Other groups have used the in vitro rodent embryo culture technique to demonstrate that elevated glucose concentrations in the media are associated with a reduction in the myo-inositol content of the embryo. Hod and co-workers9 studied rat embryos explanted at 9.5 days and cultured for 48 h under control (6.7 mM) or high glucose (56.1 n&I) conditions. They found an almost 50% reduction in total conceptus myo-inositol (489 f 40 ng versus 209 f 5 ng, p < 0.01). Hashimoto et allo used a similar 48-h culture of rat embryos explanted at day 9.5. The control medium also contained 6.7 mM glucose, and the rrzyo-inositol content of the control embryo was 1612 f 154 r&I/g protein. Increas- ing concentrations of glucose in the medium resulted in parallel decreases of the myo-inositol concentration in the embryo. Significant decreases were found at 40 and 73.4 mM glucose, with the embryos incubated in these conditions of elevated glucose concentrations demonstrating myo-inositol concentrations of 918 f 183 and 570 f 94 nM/g protein respectively (p versus control < 0.01). The myo-inositol level in the extra- embryonic membranes showed a similar tendency to fall as the glucose concentration in the medium was raised, but the differences were not significant.

As in other tissues where an elevation in glucose level is associated with a decrease in the tissue myo- inositol content, the precise mechanism whereby this phenomenon occurs in a developing embryo is uncer- tain. Recently, Weigensberg and co-workers’l studied the uptake of myo-inositol in the lO.Sday rat embryo. In a 24-h incubation, it was found that myo-inositol uptake by the fetus was a low-affinity system which produced tissue concentrations only threefold higher than in the medium, a situation markedly different from that seen in many adult tissues. Glucose was a potent and specific competitor of this myo-inositol

transport system in the embryo. An increase in medium glucose concentration from control levels of 6.7 mM to 16.7 mM resulted in a significant decrease of almost one-third in the net myo-inositol uptake, as studied in a 3-h incubation (101.5 f 6.0 nglconceptus versus 68.0 f 3.7, p< 0.01). Incubation in the presence of two other hexoses (mannose and galactose) did not result in competitive inhibition of myo-inositol transport in the embryo.

Whatever the mediating mechanism(s), the central point is that an elevation of the glucose concentration either in vitro or in vivo is associated with a significant reduction in the embryo myo-inositol level. Moreover, the incidence of malformations induced by excess glucose levels can be reduced by supplementation with myo-inositol which restores the embryo myo-inositol concentration to normal values. The ability of myo- inositol to protect against neural tube lesions produced by excess glucose has been confirmed by several groups. Baker and co-workerPJ3 explanted mouse embryos on day 8 l/4, and cultured them for 48 h in the presence of control (L-glucose, 44 mM), D-glucose (44 n&i), and D-glucose (44 n&f) plus myo-inositol(2-9 n&l). Rostral neural tube fusion occurred significantly less fre- quently in embryos exposed to high glucose levels than in controls (p < 0.001). When myo-inositol at a concentration of 4 mM was added to the high-glucose medium, a significant protective effect (55% fusion versus 35%, p < 0.02) was seen. The lower (2 mM) and higher (9 m.M) concentrations of myo-inositol were ineffective in protecting against the inhibition of rostral neural tube closure caused by the elevated glucose levels. The protective effect of 4 mM myo-inositol was not complete, since the rate of normal neural tube fusion obtained in the high glucose plus myo-inositol- supplemented medium was still significantly different (p < 0.01) than found in the control embryos.

Hod et aI and Hashimoto and co-workers*0 also evaluated the protective effects of myo-inositol. In addition, specific measurements of myo-inositol concentration were made in embryos cultured in the presence of control conditions, high glucose, and high gl~ose plus myo-inositol supplementation. In both studies, the myo-inositol supplementation was documented to return the embryo myo-inositol levels back to those seen in the control situation. Myo-inositol supplementation was associated with complete protection against glucose-induced malformations. In the report of Hod and co-workers,9 the malformation rate was reduced from 33% in the high-glucose medium to 6% in the medium containing high glucose plus myo-inositol (p < 0.001). In the communication of Hashimoto and associates,10 the incidence of malformations was reduced from 22% in the high-glucose medium to 7.1% in the medium containing glucose plus myo-inositol (p < 0.002).

206 BAKER AND PIDDINGTON ] Diab Camp 1993; 7:2M-212

While these papers appear to confirm and comple- ment each other, it should be noted that there are some major discrepancies. For example, Baker et all3 used several concentrations of myo-inositol to evaluate the possible existence of a dose-response curve. Baker and colleagues found 4 mM myo-inositol supplementation to be partially protective, whereas myo-inositol added to a concentration of 9 mM was not protective at all. Hashimoto et al.‘O used a myo-inositol supplementation of 0.28 mM, where Hod et al9 added 8.25 mM myo-inositol. The embryo myo-inositol concentrations in these two experiments varied considerably. In the studies by Hod et al.,9 the control embryo myo-inositol concentration was approximately 10,000 r&i/g protein; this decreased to 4828 r&I/g protein in the presence of a high glucose concentration, but was returned to 11,265 nM/g protein in the presence of a medium containing high glucose plus myo-inositol supplementation. Hashimoto and colleagueslo found the control embryos to have a myo-inositol concentration of 1612 r&I/g protein; this was decreased to 570 r&I/g protein in the presence of a high glucose concentration, but was returned to normal levels (1567 nM/g protein) in the presence of the myo-inositol supplementation. The differences could relate to (a) a mathematical error in reporting; (b) differences in species (Baker et all3 used mice, whereas Hod et a1.9 and Hashimoto et al.‘O studied rat embryos); or (c) unknown factors, Even though these reports came to the same conclusions concerning the protective effects of myo-inositol, it is of concern to find the wide differences in the amount of myo-inositol supplementation found to be effective, and the major discrepancy in the embryo myo-inositol levels.

The protective effect of myo-inositol supplementation has also been shown in vivo. Baker et all2 supplemented the rat chow given pregnant diabetic rats with 1% myo-inositol. This resulted in maternal serum concentrations which were three- to fourfold higher than that seen in the non-myo-inositol-supplemented diabetic pregnant rats. The incidence of neural tube fusion defects was reduced from 9.7% in the embryos born to diabetic rats that received the control chow versus 3.7% in the embryos born to pregnant diabetic mothers that had received the 1% myo-inositol- supplemented chow. While these data support the significant protection afforded by myo-inositol against glucose-induced failure of neural tube fusion, the protection was again only partial, in that the incidence of neural tube fusion defects was still above that seen in the embryos born to nondiabetic control animals.

Similar data were obtained by Akashi and co- workers,14 who provided direct measurements of embryo myo-inositol content. The pregnant rats were either untreated, given insulin, or had chow supplemented with 2% myo-inositol. The insulin treatment was aggressive; the serum glucose levels and body

weight on day 11 of the insulin-treated diabetic rats were not different from that seen in the normal control pregnant animals. Maternal serum myo-inositol concentrations were similar in the control, the diabetic, and the diabetic-plus-insulin groups, but were elevated sevenfold in the rats that received the chow supplemented with 2% myo-inositol. The embryo myo- inositol content followed the pattern seen in the embryo culture model. There was a significant reduction of the myo-inositol concentration in the embryos of the diabetic animals as compared to controls (1247 f 88 nM/g protein versus 1966 f 140, respectively, p < 0.01). Both aggressive treatment with insulin and supplementation with myo-inositol restored the embryo myo-inositol concentration to the control levels. Despite the return to normal of the myo-inositol concentration in the embryo, there was not complete protection afforded. There was a clear and significant reduction of neural tube lesions in the embryos whose mothers had received myo-inositol supplementation (9.6% versus the 16.8% found in the untreated diabetic). However, this was still higher than that found in the embryos found to the nondiabetic controls (0.9%) or the insulin-treated group (2.5%).

Further evidence that there may be a causal relationship between myo-inositol depletion in the embryo and neural tube malformations comes from the novel experiments of Strieleman et alI5 They cultured rat embryos from day 9.5 to day 11.5 in the presence of increasing concentrations (0.06 to 33.3 mM) of scyllo- inositol, an isomer of myo-inositol which is transported into cells (scyZIo-inositol competes with myo-inositol for the myo-inositol transport system), but not metabo- lized further into phosphoinositides. These workers demonstrated that the addition of scyZlo-inositol to the culture medium was associated with a decrease in embryo myu-inositol concentration. The effects of .scy12~+ inositol on embryonic development were quite anal- ogous to that produced by excess glucose concentrations: increasing concentrations of scyflo-inositol produced dose-dependent increases in the malformation rate of the neural tube and the brain.

Unlike the situation in several other tissues affected by diabetic complications, the excess concentration of sorbitol seen in the presence of high-glucose levels does not appear to play an important role in the mechanism of teratogenesis. Studies have clearly documented a significant elevation of embryo sorbitol levels, which accompanies an increase in the medium glucose concentration and the previously noted decrease in the embryo myo-inositol level. For example, in the experiments of Hashimoto et aLTo using the rat embryo culture system, the sorbitol level rose from a control value of 307 f 54 r&I/g protein to 7000 f 1130 in the presence of 23.4 mM of glucose. In their in vivo studies, Akashi and co-workers” found the embryonic

J Diab Camp 1993; 7:204-212

sorbitol level to go from the nondiabetic 243 + 29 to 1836 f 388 r&I/g protein in the diabetic animals. The evidence that the elevated sorbitol concentration is not a key factor in the teratogenic mechanism comes in part from the fact that the protective effects of myo- inositol occur despite the presence of sorbitol levels which are unchanged from the elevated level seen in the diabetic state.l”,14 In addition, several groups have documented that aldose reductase inhibitors (Sorbinil, Statil, ON0 2235) lower sorbitol levels in the presence of hyperglycemia, but do not protect against the teratogenic effects of the elevated glucose concentration 10,16,17

Strieleman and colleagues15 recently provided the first evidence in the embryo that the lowered myo- inositol concentration after incubation in a medium containing high levels of glucose is associated with an alteration in phosphoinositide metabolism. Rat fe- tuses were explanted into medium on gestational day 9.5 and cultured for 24 h in the presence of glucose concentrations varying from the control 6.7 mM to 73.3 mM. With increased glucose levels, there was a dose- dependent significant decrease in phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate (PI, PIP, and PIPS). Further, the increases in medium-glucose concentration were paralleled by a stepwise reduction in the embryo pool of inositol phosphates (IPI, I& and I&). These data indicate major changes in the phosphoinositide intracellular signal transduction system in the developing embryo which could contribute to the mechanism of diabetic embryopathy.

A ROLE FOR THE ARACHIDONIC ACID CASCADE IN DIABETIC EMBRYOPATHY

The review of the literature thus far presented would strongly suggest that the “myo-inositol depletion hypothesis” implicated in the mechanism of other diabetic complications may also be involved in the mechanism of diabetic embryopathy. Several lines of evidence also indicate that the arachidonic acid pathway may play a role. This evidence includes the following:

1.

2.

3.

4.

arachidonic acid supplementation protects against hyperglycemia-induced teratogenesis, both in vivo and in vitro; the significant protective effects of myo-inositol supplementation against the teratogenic effects of a high glucose concentration on neural tube fusion can be reversed by indomethacin, an inhibitor of arachidonic acid metabolism; supplementation of prostaglandins, a product of arachidonic acid metabolism, to a high-glucose culture medium protects against embryonic malformations; and prostaglandin EZ levels in the mouse embryo are

DIABETIC EMBRYOPATHY: RECENT TRENDS 207

high around the time of neural tube fusion and then decrease. These developmental changes are blunted in embryos of diabetic mice.

Arachidonic Acid Supplementation is Protective. Using the mouse embryo culture technique, Goldman and associates16 reported that arachidonic supplementation (1 and 10 ug/mL) protected against the failure of neural tube fusion seen when the embryos were incubated in the presence of high (8 mg/mL) glucose concentration alone. These results were confirmed by Pinter and colleagues19 in the rat embryo culture system. Arachidonic acid supplementation at a concentration of 80 ug/mL gave complete protection against embryonic malformations seen in the presence of high glucose (9.5 mg/mL in their studies). Arachidonic acid supplementation at a dose of 20 ug/mL afforded significant protection as well, but lower levels of arachidonic acid (1 and 10 ug/mL as used by Goldman et al.18) were ineffective. It is not clear whether the doses of arachidonic acid required to provide protection against glucose-induced malformations in these two studies reflect simply the different animal models used.

Goldman et all8 also demonstrated the protective effects of arachidonic acid in vivo. Subcutaneous injec- tions of arachidonic acid were administered to pregnant diabetic rats. In the embryos born to the control diabetic animals, neural tube fusion defects were found in ll%, micrognathia in 7%, and cleft palate in 11%. Arachidonic acid given by injection to the mothers overall provided a significant (p < 0.01) reduction of these malformations to levels of 3.8%, 0.8%, and 4%, respectively, with better results seen in the animals injected with a higher dose (400 mglkg versus 200 mglkg), and over a longer period of time (days 5 to 10 versus days 9 to 12).

No information is available concerning developmental changes in the embryo in arachidonic acid content during the time of neural tube fusion. Pinter and colleague# examined the lipid profiles of embryos and yolk sacs incubated in the presence of media containing control levels of glucose, high glucose concentration, and high glucose plus arachidonic acid supplementation. They were not able to document differences in the concentration of arachidonic acid between these groups to support the theory of a deficiency of arachidonic acid. This is perhaps not surprising, since the arachidonic acid concentrations were measured only at the end of the 48-h culture period. Important but short-lived changes which might have occurred around the time of neural tube closure, such as is the case with myo-inositol* and prostaglandin levels,21 would have been missed.

Reversal of myo-Inositol Protection by Indomethacin. As discussed in the previous section, Baker and col-

208 BAKER AND PIDDINGTON J Did Cimp 1993; 7:2@4-222

league@ demonstrated in mouse embryo culture that supplemental myo-inositol (4 mM), when added to a high glucose-containing medium (44 mM), exerted significant, albeit incomplete, protection against failure of neural tube fusion. When indomethacin (40 PM) was added to the culture medium containing the high level of glucose plus a protective concentration of myo- inositol, the protection afforded by myo-inositol was completely reversed. l3 Indomethacin primarily acts to inhibit arachidonic acid metabolism, including its con- version to prostaglandins. This set of experiments therefore suggests that the mechanism of diabetic embryopathy involves both the myo-inositol and the arachidonic acid pathways. The exact nature of the linkage between the two remains speculative.

Prostaglandin Supplementation to the High Glucose Culture Medium Protects Against Embryonic Malfor- mations. The hypothesis that prostaglandins play a direct role in the pathway leading to diabetic embryopathy has been tested and confiied. Baker and co-workerP used the mouse embryo culture system to test PGEla, PGFza, and PG12 (prostacyclin), prostaglandins known to be present in embryonic rats and mice. They found prostaglandin Ez to be the most effective of the prostaglandms in reversing the inhibition of neural tube fusion induced by high (44 n&i) levels of glucose. PGEz at a concentration of 142 nM completely protected the embryo from the teratogenic effect,s of the high glucose concentration on neural tube fusion (p versus glucose < 0.02; p versus controls = NS).

Goto and colleagues” confirmed and extended these observations. They also used a mouse embryo culture system. Incubations were carried out in the presence of either control, high glucose (52.7 mM), and high glucose plus varying concentrations of PGEr (0.0028 nM to 283.7 r&I). In the presence of 52.7 mM glucose, 83% of the embryos demonstrated failure of neural tube fusion. While the lowest and highest doses of PGEz had no effect, supplementation of 0.028 nM of PGE2 to the high glucose medium reduced the incidence of neural tube defects to 55% (p < 0.01 versus high glucose alone), and doses of 0.28, 2.8, and 28.4 nM further reduced the incidence of defects (down to levels of 29%-35%, p < 0.001 versus high glucose alone).

There are several differences between these two studies. Baker and co-worker@ found complete protection afforded by PGE2 at a concentration of 142 nM. Goto and collaboratorz? found a significant reduction of the incidence of defects in the embryos cultured in the presence of high glucose and levels of prostaglan- dinEZbetween0.28 and 28.4nM. However, the protection was not complete, as the values found remained significantly different from the controls. It is unclear whether these differences reflect the different strains

of mice used, the different levels of glucose employed in the medium (52.7 versus 44 r&I), or the fact that Goto et al.” had no intermediate prostaglandin concentrations between 28.4 nM, which was partially protective, and 284 nM, which afforded no protection at all. Perhaps the problem relates merely to a dosage effect, as the optimal dose in the studies of Baker et alI3 was 142 nM. They, l3 along with Goto et al.,= found higher doses to have apparent teratogenic effects.

Goto and colleagues22 also examined the effects of prostaglandin EZ in embryos cultured in diabetic serum. The incidence of neural tube defects in mouse embryos cultured in serum obtained from diabetic rats was 81%, as compared to 5% in the embryos incubated in the presence of normal rat serum. Supplementation of PGEz at a concentration of 28.4 nM to the medium containing diabetic serum was protective, reducing the incidence of neural tube defects to 26%. While significantly different (p < 0.001) from the results obtained when the embryos were incubated in the presence of diabetic serum alone, the incidence of neural tube fusion defects was still higher than seen in the controls.

Developmental Changes in the Concentration of PGE2 in the Embryo. The possible role of PGEz in the pathway of diabetic embryopathy is also supported by recent preliminary in vivo data of Piddington and co- workers (21). They measured l?GE2 levels in embryos obtained from normal and diabetic mice on days 8,9, 10 and 11 of pregnancy. The results were then adjusted for somite number, since the development of embryos differs even among normal litter mates; this maneuver also permits the effect of maternal diabetes on delayed development to be differentiated from a teratogenic effect. In the normal embryos, a clear developmental pattern was seen. The prostaglandin EZ levels were highest in embryos obtained from normal control mice on day 8, at the O-8 somite stage; a consistent decrease from this high level was found over the next 3 days. In contrast, no clear developmental pattern was seen in embryos obtained from diabetic mice. In the embryos obtained from diabetic mice at the 2-8 somite stage, the PGEz level was significantly reduced when compared to the non-diabetic control, suggesting that the diabetic milieu interfered with the normal PGEz developmental pattern. This effect of diabetes on developmental changes is reminiscent of that found for myo-inositol in neuroectodermal tissue (8). In those studies, the increase in embryo neuroectodermal myo-

inositol content seen in the normal situation during neurulation was blunted by approximately 40% in the presence of maternal diabetes.

Despite some inconsistencies and methodological

J Diab Camp 2993; 7:204-212 DIABETIC EMBRYOPATHY: RECENT TRENDS 209

differences, the data thus far reviewed suggest that both the myo-inositol and the arachidonic acid/prostaglandin pathways are involved in the mechanism of diabetic embryopathy. The nature of the linkage between these two pathways is unclear, but it is conceivable that the phosphoinositide signaling system may control the level of activity of one of the phospholi- pases in the PLA2 system which, in turn, regulates the intracellular availability of free arachidonic acid for further metabolic purposes.

The study of Goto and colleagues,= cited above, is important in another way. As had been shown before,= serum from a diabetic animal is more terato- genie than can be accounted for on the basis of the glucose concentration alone. For example, the diabetic serum used in the study of Goto et al.= had a glucose concentration of 22.2 mM. Embryos cultured in the presence of this diabetic serum showed an incidence of neural tube defects of 81% (21126). When the control serum was adjusted to contain a similar glucose level of 22.2 mM, the incidence of neural tube defects was only 10% (2/21). This type of observation suggests that, while hyperglycemia may be key in the patho- genesis of diabetic embryopathy, other factors, presumably contained in diabetic serum, must be of importance in the pathogenetic mechanism. This ap- proach, termed the “multifactorial” hypothesis, has been primarily advanced by Sadler and co-workers.” Among the possible factors that might be involved are: elevations of &hydroxybutyrate;” presence of soma- tomedin inhibitors;26 alterations of glycosaminoglycan synthesis;n and deficiencies of trace metals.28 It is possible, but clearly not documented, that these factors could produce the observed abnormalities in the mya- inositol and arachidonic acidlprostaglandin pathways. It is also conceivable that they contribute to the diabetic teratogenic potential through other quite separate mechanisms.

POSSIBLE ROLE OF FREE OXYGEN RADICALS IN DIABETIC TERATOGENESIS

A novel hypothesis for the mechanism of diabetic embryopathy which may not apparently involve the myo- inositol-arachidonic acid/prostaglandin pathways has recently received attention by Eriksson and Borg.29,30 This hypothesis postulates that increased free oxygen radical formation in embryonic tissues is causally re- lated to malformations. The evidence for this model comes largely from the ability of free oxygen radical scavenging enzymes to protect against glucose-induced malformations. Citing the suggestion that several of the diabetic complications may result from increased production of free oxygen radicals, they in- vestigated this possibility in rat embryos cultured in the presence of media containing 10 mM glucose (control), 50 mM glucose, or 50 mM glucose plus either

citiolone (an inducer of free oxygen radical scavenging enzymes) or the oxygen radical scavenging enzymes superoxide dismutase, catalase, or glutathione peroxidase.29 As had been well documented previously, incubation of rat embryos in the presence of 50 mM glucose resulted in a major malformation rate of 81% (most of these malformations reflected failure of neural tube fusion). Addition of superoxide dismutase was completely protective in the presence of 50 mM glucose, returning the malformation rate to levels seen in the presence of 10 mM glucose. The addition of citiolone, catalase, or glutathione peroxidase to the medium containing 50 mM glucose resulted in significant protection, but the rate of major malformations was still elevated above that seen in the control situation.

Eriksson and Borg (30) followed up these observations with a series of innovative experiments. They incubated rat embryos in 48-h culture in the presence of 10 mM glucose, 50 mM glucose, 10 mM glucose plus 3 mM pyruvate, 10 mM glucose plus 10 mM fi-hydroxybutyrate, or 10 mM glucose plus 3 mM a-ketoisocaproic acid (a product of branched chain amino-acid metabolism). In addition, these culture conditions were repeated with the supplementation to them of 2.5 MUlL of superoxide dismutase or 400 PM of a-cyano4hydroxycinnamic acid (CHC), an inhibitor of pyruvate mitochondrial transport. As was seen in previous experiments, incubation in the presence of 50 mM glucose produced a major malformation rate of 92%. Incubation of embryos in the presence of 10 mM glucose with pyruvate (3 mM), B-hydroxybutyrate (10 mM) or a-ketoisocaproic acid (3 n&I) produced malformation rates of 59%, 78%, and 90%, all of which were significantly above the malformation rates seen in the presence of 10 n-&l glucose alone. Incubation in the presence of superoxide dismutase significantly protected against the major malformations induced by all of the above teratogenic conditions (50 mM glucose alone, 10 mM glucose plus 3 mM pyruvate, 10 mM glucose plus 10 mM b-hydroxybutyrate, or 10 mM glucose plus 3 mM a-ketoisocaproate). The addition of CHC to the culture media provided significant protection only against the major malformations induced by 50 mM glucose plus 3 mM pyruvate. No protection was seen when this agent was added to the cultures containing 10 mM glucose plus either 10 mM g-hydroxybutyrate or 3 mM a-ketoisocaproate. The authors interpret these experiments to show that free oxygen radical scavenging enzymes can protect against embryonic malformations caused by several different metabolites which have the potential for gen- erating free oxygen radicals. This represents an exten- sion of their previous work which showed that high glucose levels might affect the dysmorphogenic pro- cess by an enhanced production of free oxygen radi-

210 BAKER AND PIDDINGTON J Diab Camp 1993; 7:204-212

cal~.~ In addition, because it is known that B-hydroxybutyrate and a-ketoisocaproate enter the mitochondria without using the pyruvate carrier, the experiments utilizing CHC (a specific noncompetitive inhibitor of mitochondrial pyruvate uptake) suggest strongly that the free oxygen radicals which are important in teratogenesis are produced in the mitochondria. Embryonic mitochondria are relatively immature and may not be able to handle an excess of oxidative substrate entering mitochondrial metabolism. The embryo incubated in a diabetic milieu may thus be hit by a double insult: too much oxidative substrate and too little mitochondrial capacity to handle the increased load. This combina- tion might thus lead to the generation of enough free oxygen radicals to induce embryonic malformations. The “free oxygen radical teratogenesis” hypothesis is extremely challenging; it remains to be seen whether it will fit into the conceptual model of diabetic embryopathy involving the myo-inositol and arachidonic acidlprostaglandin pathways. As pointed out by Eriks- son and Borg, 2q,3o the most likely effect of increased free oxygen radical activity would be enhanced lipid peroxidation. In turn, this might lead to a possible imbalance in prostaglandin synthesis, thus explaining the protective effects of arachidonic acid and prostaglandins. A possible bridge to the myo-inositol pathway could be through the apparent role of phospholipase AZ activity in protecting membranes from oxidative injury. 31 The beneficial effect of myo-inositol supplementation to high glucose culture might be ex- plained by restoration of normal phosphoinositide signaling which results in normalized PLA2 activity, thereby enhancing its ability to deal with an oxidative stress.

CONCLUSION

These past few years have been a fruitful time in the area of diabetic embryopathy. Clinically, the field has moved to the point where many feel that the problem of congenital malformations in infants of diabetic mothers may be on the verge of elimination by the achievement of meticulous diabetic control during the preconceptional and early organogenesis (prior to the sixth to eighth gestational week) phases of pregnancy.32-M (It is worth a somewhat lengthy aside here to critically discuss the major paper which is said to undermine these claims, as it comes from the Diabetes in Early Pregnancy Group.% Despite its prestigious provenance, the title of the paper, “Lack of relation of increased malformation rates in infants of diabetic mothers to glycemic control during organogenesis,” is little short of outrageous, and completely unjustified by the body of data presented in the text. In addition to several methodologic problems, perhaps the most grievous error lies in the conclusion reached by the analysis of hemoglobin Ale levels and glucose profiles.

The study found that “women with malformed infants did not differ significantly from the others with respect to fasting, preprandrial, postprandrial, or bedtime glucose levels, percentage of glucose measurements in the hypoglycemic range, or glycosylated hemoglobin levels . . . during organogenesis.” This is the basis for the conclusion reflected in the title that glycemic control during organogenesis has no relation to the risk for malformation. The authors appear themselves not to believe this statement, as the article concludes with the clinical recommendation “that women and their physicians strive to attain stable, good diabetic control in the periconceptual period and throughout pregnancy.” The faulty logic which led to the title of this paper may best be seen if an analogy is used: in a randomized, controlled, prospective trial, investiga- tors find that drug A is significantly better than drug B. However, an analysis of treatment failures in the patients treated with drug A reveals that their drug levels were no different than in the patients who re- sponded. On the basis of the drug level analysis, is it permissible to then go back and conclude that drug A bears no relationship to the improved response seen in that group of patients?)

Significant progress has also been made in the research of diabetic embryopathy. The field has progressed from a state of “phenomenology” (the documentation of what levels of glucose, B-hydroxybutyrate, and other potential teratogens were required to produce malformations, or what combinations were most potent, and studies examining the most critical time for an insult to occur) to a stage where broad- based theories were put forth (such as “fuel mediated teratogenesis”).361n this review, attention has been placed on recent evidence concerning the delineation of several specific pathways which may play important roles in the mechanism of diabetic embryopathy. De- spite some inconsistencies, several groups have now shown that the “myo-inositol depletion” hypothesis appears to be involved in diabetic embryopathy, similar to what has been found for several of the other diabetic complications. Presumably, the deficiency of myo-inositol causes perturbations in the phosphoinositide system of intracellular signal transduction. Although the intermediary steps have not been de- fined, the disturbance in phosphoinositide signaling may then cause the abnormalities in the arachidonic acidlprostaglandin pathway documented by several groups. The recent observations concerning the possible role of free oxygen radicals in diabetic teratogenic- ity provide another potential part of the puzzle. The challenge for current researchers in diabetic embryopathy is to examine other possible factors, and to determine whether (and how) these several pathways converge at some point to produce the congenital malformations typical of diabetes.

1 Diab Camp 1993; 7:2oP212 DIABETIC EMBRYOPATHY: RBCBIUT TRENDS 211

ACKNOWLEDGMENT

The authors wish to thank Ms. J. Joyce for her technical help and Ms. E. Alicea-Cruz for her secretarial assistance. We are grateful to Dr. Ulf Eriksson for allowing us to quote his 1993 manuscript prior to actual publication. This work was supported by grants from the Juvenile Diabetes Foundation In- ternational (191850), the Samuel Lunenfeld Charitable Foun- dation, and the NIH (RROO24O).

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diabetic embryopathy: a selective review of recent trends

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