Loss of pericytes

Loss of pericytes is one of the earliest and most specific signs of diabetic retinopathy. This finding was first described by Cogan, Kuwabara and coworkers after examining trypsin-digested retinal vasculature flat mounts from diabetic human subjects.^4–6 Since their initial report, their findings have been confirmed by various investigators.^2,7,8 In humans and canines, trypsin digestion of retinal vasculature flat mounts reveals the loss of pericytes as evidenced by the development of pericyte ghosts, empty aneurysmal spaces bulging from the capillary walls that lack the darkly staining nucleus of a viable pericyte (Fig. 46.1A). Pericytes are normally identifiable in these spaces by their nuclei which stain darkly and are spaced regularly along the capillary wall, producing the appearance of “bumps on a log” (Fig. 46.1B).

Fig. 46.1 (A) Pericyte ghosts (G) in a trypsin digest preparation from a 42-year-old woman with background diabetic retinopathy who died of a dissecting aneurysm of the aorta. The ghost represents the vacant space in the capillary basement membrane, formerly occupied by an intramural pericyte nucleus that has degenerated. Normal pericyte (P) and endothelial cell (E) nuclei are also shown. The preparation was stained with periodic acid–Schiff (PAS) reagent and hematoxylin. Magnification ×575. (B) A trypsin digest preparation from the retina of a nondiabetic, 55-year-old man who died of a myocardial infarction. Note the regular array of pericyte (P) and endothelial cell (E) nuclei. The preparation was stained with PAS reagent and hematoxylin. Magnification ×450.

Pericytes are contractile cells that play an important role in microvascular autoregulation.⁹ Loss of pericytes leads to alterations of vascular intercellular contacts and impairment of the inner blood–retina barrier. These effects result in the venous dilation and beading that is visible clinically. Loss of intercellular contacts also appears to promote endothelial cell proliferation resulting in the development of microaneurysms.¹⁰ Loss of pericytes appears to be especially significant in the development of diabetic retinopathy, although pericyte loss has also been reported in diabetic peripheral neuropathy leading to neuronal ischemia.¹¹ The mechanism by which hyperglycemia leads to pericyte degeneration remains largely unknown. The two leading hypotheses implicate the aldose reductase pathway and platelet-derived growth factor-beta (PDGF-β).

Akagi et al. reported the localization of the enzyme aldose reductase in retinal capillary pericytes but not in endothelial cells in human specimens using immunohistochemistry techniques.¹² These findings would be consistent with the specific loss of capillary pericytes observed in diabetic retinopathy and other microvascular complications of diabetes.¹³ Two other groups, however, were unable to identify the presence of aldose reductase in rodent and canine retinal capillaries using immunohistochemistry techniques.^14,15 In addition, a third group reported the presence of aldose reductase activity in cultured bovine retinal capillary pericytes as well as retinal capillary endothelial cells. They also found aldose reductase activity in cultured monkey retinal pericytes.¹⁶ These conflicting reports probably result from species-specific differences in aldose reductase expression and highlight the need to use caution when interpreting animal models of disease.

PDGF-β has been found to be critical in the recruitment of pericytes in the vasculature of various tissues and organs.¹⁷ It is well documented that endothelial cells express PDGF-β,^18–21 and in vitro pericytes are known to express PDGF-β receptors and respond to PDGF-β.^22,23 Lindahl et al. reported that in the PDGF-β-deficient mouse model, pericytes fail to develop in developing capillaries during angiogenesis.²⁴ Subsequent studies using PDGF-β- and PDGF receptor-β (PDGFR-β)-deficient mice found that while PDGF-β/PDGFR-β-independent induction of pericyte precursors may occur, the expansion of pericytes is dependent on an intact PDGF-β/PDGFR-β paracrine signaling pathway.²⁵ Ablation of either PDGF-β or PDGFR-β led to identical phenotypes in these mice.²⁶ These studies suggest that endothelial cell-derived PDGF-β promotes the co-migration of PDGFR-β-expressing pericytes along sprouting new vessels, and the interruption of the PDGF-β/PDGFR-β results in the observed deficiency of pericytes in capillaries. Since PDGF-β has been found to be critical in the recruitment of pericytes during angiogenesis, it has been suggested that PDGF-β may play an important role in maintaining pericyte viability in mature vasculature, although no studies have confirmed this hypothesis.

Capillary basement membrane thickening

Thickening of capillary basement membranes is a well-documented lesion of diabetic retinopathy, visible on electron microscopy. Additional electron microscopic findings include deposition of fibrillar collagen and “Swiss cheese” vacuolization of the otherwise homogenous pattern of basement membrane collagen. The biochemical mechanism leading to basement membrane thickening remains unknown but studies suggest a role for the aldose reductase and the sorbitol pathway.^27–30 Nondiabetic rats fed a galactose-rich diet for prolonged periods of time develop retinal capillary basement membrane thickening, fibrillar collagen deposition, and Swiss cheese vacuolization. By contrast, rats fed a control diet or a galactose-rich diet along with the aldose reductase inhibitor sorbinil do not develop basement membrane thickening.^27,30 However, the observation that basement membrane thickening of renal glomeruli in diabetic and galactosemic rats is not inhibited by aldose reductase inhibitors casts doubt that the aldose reductase pathway is the primary pathway and suggests that basement membrane thickening may be a secondary nonspecific response.³¹

Glycation of basement membrane collagen by enzymatic³² and nonenzymatic processes³³ may be another mechanism that plays a role in basement membrane thickening. Besides type IV collagen, the predominant type of collagen present in the basement membrane, other collagen types, and noncollagen macromolecules, such as laminin,³⁴ entactin, heparan sulfate proteoglycan,^35,36 and basement membrane-bound growth factors are also present.³⁵ Glycation appears to alter the structure of the basement membrane by changing the chemical composition and relative amounts of these components. For example, Shimomura/Spiro and Spiro/Spiro reported decreased heparan sulfate proteoglycan in renal glomeruli from diabetic human subjects.^37,38 Quantitative electron microscopic immunocytochemical studies have found that retinal and renal glomeruli basement membrane thickening in galactosemic rats is associated with a relative increase in the levels of type IV collagen and laminin, while the relative levels of heparan sulfate proteoglycan remain unchanged.^29,31

Microaneurysms

Although pericyte loss is the earliest sign of diabetic retinopathy, it is only observable histologically. The earliest clinically visible sign of diabetic retinopathy is the microaneurysm.³⁹ Microaneurysms appear as grape-like or spindle-shaped dilations of retinal capillaries on light microscopy.⁴ They can be either hypercellular or acellular. By ophthalmoscopic examination, microaneurysms appear as tiny, intraretinal red dots located in the inner retina. By fluorescein angiography, they appear as punctate hyperfluorescent dots with variable amounts of fluorescein leakage.

Pericyte cell death and loss of vascular intercellular contacts may lead to endothelial cell proliferation and microaneurysm development.^24,40,41 Pericytes appear to exert an antiproliferative effect and pericyte loss may explain the development of hypercellular microaneurysms. However, this mechanism does not account for acellular microaneurysms. Acellular microaneurysms may develop from hypercellular microaneurysms that become acellular from endothelial cell and pericyte apoptosis.⁴²

Pericyte loss may also result in weakening of the capillary wall, promoting the development of microaneurysms at the structural weak points. Pericytes contain myofibrils with contractile properties and may act as smooth muscle cells of larger vessels, exerting tone to the vessel wall in order to counteract the transmural pressure. Loss of pericyte tone may result in focal dilation of the vessel wall leading to the development of a microaneurysm. However, the transmural pressure of the capillary bed is low relative to the arterial circulation, and retinal capillary microaneurysms can develop in other diseases in which pericyte loss is not observed.^43,44

Capillary acellularity

Complete loss of the cellular elements of the retinal capillary network can be seen as a more advanced microvascular lesion in diabetic retinopathy and other microvascular retinopathies. In clinicopathological correlations with fluorescein angiograms performed shortly before the eye was enucleated for therapeutic purposes or because the patient died and the eye was removed during autopsy, the retinal vascular digests of the enucleated eye showed that acellular capillaries are nonfunctional since they appeared as regions of nonperfusion on angiography.⁴⁵ The mechanism by which capillaries become acellular is unknown and can be seen in diabetes, other retinal microvascular diseases, or in experimental diabetes or galactosemia in animal models. Since capillary acellularity is not unique to diabetes, a variety of pathogenic mechanisms may result in this nonspecific finding.

Breakdown of blood–retina barrier

Breakdown of the blood–retina barrier is an important pathophysiologic feature of diabetic retinopathy that leads to the development of macular edema, the leading cause of vision loss in diabetic patients. One mechanism by which the function of this barrier becomes altered involves opening of the tight junctions between vascular endothelial cell processes.^46,47 These tight junctions, also known as zonula occludens, appear as a pentalaminar structure on electron microscopy, consisting of two outer and one central electron-dense layer sandwiching two electron-lucent layers, giving the appearance of two “fused” plasma membranes. Using tracers such as lanthanum chloride or horseradish peroxidase, electron microscopy can be used to demonstrate that these molecules cannot pass in the presence of an intact tight junction. However, when the tight junctions are open, they become permeable to these tracer molecules.⁴⁷ Several important proteins are involved with the formation and function of tight junctions, with ZO-1 (zonula occludens) and occludin being the best characterized. In the presence of histamine, the expression of ZO-1 in cultured retinal endothelial cells is reduced in a dose-dependent manner.⁴⁸ Culturing in astrocyte-conditioned medium increases expression of ZO-1 while high glucose decreases expression of ZO-1.⁴⁹ These in vitro results have also been supported by in vivo studies. Reduced expression and anatomic distribution of occludin was found in experimental diabetes.⁵⁰ Likewise, in experimentally diabetic rats and in diabetic humans, antihistamines reduce leakage of fluorescein into the vitreous.^51,52

Vascular endothelial growth factor (VEGF) has been found to be an important mediator leading to the breakdown of the inner blood–retina barrier. Before the discovery of its well-documented role in promoting neovascularization, VEGF was found to increase the permeability of vessels, leading to its alternative name “vascular permeability factor”.⁵³ The mechanism by which VEGF leads to the breakdown of the inner blood–retina barrier appears to involve alteration of endothelial cell tight junctions. Intravitreal injection of VEGF in rats increased the production of the free radical nitric oxide and led to phosphorylation of ZO-1^54,55

Another important factor promoting retinal vascular permeability involves the kallikrein–kinin system. Proteonomic studies of the vitreous from patients with advanced diabetic retinopathy have identified components of the kallikrein kinin system, including plasma kallikrein, factor XII, and kininogen.^56,57 In rodent models, activation of plasma kallikrein in the vitreous has been found to increase retinal vascular permeability.⁵⁸ Likewise, inhibition of the kallikrein kinin system reduces retinal vascular leakage caused by diabetes and hypertension.^58,59 The mechanism by which the kallikrein kinin system promotes vascular permeability probably involves bradykinin. Bradykinin, via nitric oxide, induces vasorelaxation of retinal arterioles.⁶⁰ Intravenous infusion of bradykinin results in dilation of retinal arterioles and venules, an effect that is reduced by indomethacin and the cyclooxygenase-2 selective inhibitor nimesulide.⁶¹ Bradykinin is also a neuropeptide with a direct effect on glia and neurons, which can release vasoactive factors that affect blood flow and vascular permeability.⁶²

The aldose reductase theory

Elevation of intracellular glucose levels can cause increased activation of the aldose reductase pathway. Also known as the polyol pathway or the sorbitol pathway, the aldose reductase pathway is a series of intracellular reactions that involves the enzymes aldose reductase and sorbitol dehydrogenase.^69,70 Aldose reductase uses the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor to reduce many aldose sugars into their respective sugar alcohols. Glucose is reduced to sorbitol, which is then oxidized into fructose by sorbitol dehydrogenase. However, sorbitol may build up to high intracellular levels because the sorbitol dehydrogenase reaction is slow and the accumulating sorbitol does not easily cross the plasma membrane into the extracellular space. In normoglycemic conditions, the aldose reductase pathway is nonoperative because glucose is a poor substrate for aldose reductase due to its high binding constant (kM). However, in the setting of hyperglycemia as seen with uncontrolled diabetes, the aldose reductase pathway becomes activated once the other enzymatic pathways of glucose metabolism become saturated. Lens epithelium expresses high levels of aldose reductase and accumulation of sorbitol is believed to lead to the development of a cataract in diabetes.⁷¹ Osmotic stress has been proposed as the mechanism by which elevated intracellular sorbitol leads to the pathologic changes seen in diabetes.⁷² However, the levels of sorbitol in vascular cells are in the nanomolar range, which is orders of magnitude less than other glucose metabolites, which have ranges in the micromolar and millimolar range.⁷³

A different mechanism that may account for the role of aldose reductase involves the cellular redox balance. Increases in the utilization of aldose reductase in the hyperglycemic state of diabetes will result in a decline in intracellular NADPH that alters the cellular redox balance. Reduction of intracellular NADPH may also decrease the production of nitric oxide in endothelial cells.⁷⁴ Similarly, the increased use of sorbitol dehydrogenase can lead to an increase in the NADH/NAD⁺ ratio that alters the cellular redox balance and may lead to oxidative stress and cellular damage.⁷⁵

Of interest to animal models of diabetes, galactose is reduced by aldose reductase into galactilol. However, sorbitol dehydrogenase cannot oxidize galactilol, resulting in the rapid intracellular accumulation of galactilol. In the setting of chronic galactosemia, diabetic-like vascular basement membrane changes,^27,28 and pericyte loss, development of microaneurysms, and capillary acellularity⁷⁶ have been reported. When these experiments were repeated and the animals were also treated with aldose reductase inhibitors, it was reported that the development of diabetic-like retinopathy changes was slowed, although most animals developed some degree of changes.^30,77–79 While aldose reductase inhibitors were reported to slow or prevent some of the pathologic changes in animal models of diabetes, the aldose reductase inhibitor sorbinil was found not to be effective in humans in the Sorbinil Retinopathy trial.⁸⁰ The lack of efficacy of aldose reductase inhibitors in human trials may, however, reflect dose-limiting side-effects of the drug that may have precluded it from achieving therapeutic levels in the tissues of interest.

Advanced glycation endproduct (AGE) theory

Accelerated aging by nonenzymatic glycation and crosslinking of proteins has been proposed as a mechanism to explain the complications of diabetes.⁸¹ Advanced glycation endproducts (AGEs) is the collective name given to proteins, lipids, and nucleic acids that undergo irreversible modification by reducing sugars or sugar-derived products. The series of chemical reactions that lead to the formation of AGEs is called the Maillard reaction. The Maillard reaction is responsible for the “browning” of tissue seen with aging as well as the “browning” of food during cooking. The initial chemical reaction is known as early glycation and involves reversible nonenzymatic binding of a sugar to amino acid groups on proteins, lipids, or nucleic acids. They form Schiff bases which can undergo rearrangement to form more stable Amadori products. Glycosylated hemoglobin (HbA1c) and fructosamine are well-known examples of Amadori products used clinically as markers of glycemic control. Although they are not AGEs, they can undergo further reactions to eventually lead to the formation of AGEs. Formation of AGEs may directly damage cells by impairing the function of a variety of proteins,⁸² including both extracellular proteins like collagen⁸³ and intracellular proteins.^84,85

The cellular effect of AGEs is also mediated by its binding to receptors, namely receptor for AGE (RAGE). RAGE is a multiligand transmembrane receptor that is part of the immunoglobulin superfamily of proteins.^86,87 When bound to AGEs, it initiates a cascade of signal transduction involving at least p21^ras, p44/p42 mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and protein kinase C (PKC).^88–93 Activation of these intracellular kinases can subsequently lead to cell dysfunction.⁹⁴ Other receptors have also been reported to bind to AGEs, such as the macrophage scavenger receptor, P60, P90, and galectin-3.^94–96 Aminoguanidine is an inhibitor of AGE formation and has been reported to block the development of many of the microvascular complications of diabetes in animal models.^97–100 The effects of aminoguanidine cannot be automatically attributed to the blockade of the AGE pathway, however, since aminoguanidine also has parallel action as an inhibitor of inducible nitric oxide synthase and oxidants.¹⁰¹ Owing to limits secondary to toxicity, clinical trials in humans have been inconclusive; in animal models, however, the use of soluble RAGE to block binding of AGEs to RAGE has been found to prevent many of the effects of hyperglycemia.¹⁰²

Reactive oxygen intermediates (ROI) theory

One of the oldest theories proposes that chronic hyperglycemia leads to the complications of diabetes by increasing oxidative stress. The usual metabolic pathway of glucose is through glycolysis and the tricarboxylic acid cycle, which takes place in the mitochondria and yields reducing equivalents used to drive the synthesis of adenosine triphosphate via oxidative phosphorylation. However, byproducts of oxidative phosphorylation include free radicals, such as superoxide anion, whose production is increased by high levels of glucose.¹⁰³ Free radicals can damage mitochondrial DNA¹⁰⁴ as well as cellular proteins¹⁰⁵ and are also produced by the autoxidation of glucose. Elevated oxidative stress also reduces nitric oxide levels,^106,107 promotes leukocyte adhesion to the endothelium and decreases the barrier function of endothelial cells,¹⁰⁸ and damages cellular proteins.¹⁰⁹ In diabetic mice that overexpress Cu²⁺/Zn²⁺ superoxide dismutase, they develop less mesangial expansion compared to wild-type diabetic mice, suggesting that oxidative stress promotes at least some complications of diabetes.¹¹⁰ Oxidative stress can also activate PKC by increasing the formation of diacylglycerol (DAG).¹¹¹

There is some evidence of increased oxidative stress in diabetic patients. It has been reported that diabetic patients have lower levels of antioxidants, such as vitamin C, vitamin E, and glutathione,^112–114 although these results have not been unequivocally reproduced by other researchers.¹¹⁵ However, other markers of oxidative stress, such as oxidized low-density lipoprotein¹¹⁶ and urinary isoprostanes, are elevated in diabetic patients.¹¹⁷ In animal models of diabetes, the use of antioxidants has blocked the development of some of the microvascular complications of diabetes.^118–122 One clinical trial reported that high doses of vitamin E (>1000 IU/day) and lipoic acid improved retinal blood flow and creatinine clearance in diabetic patients.¹²³ However, most studies evaluating antioxidants to prevent the complications of diabetes in people have been unsuccessful.¹²⁴

Protein kinase C (PKC) theory

PKC is a ubiquitous enzyme that appears to promote the development of many of the complications of diabetes without the involvement of the aldose reductase pathway. It has been observed that diabetes and galactosemia can produce elevation of DAG within cells of the retina and aorta in dogs despite treatment with the aldose reductase inhibitor sorbinil.^125,126 Activation of PKC occurs through the activation of phospholipase C, which leads to an increase in intracellular Ca²⁺ and DAG, which in turn results in the activation of PKC.¹²⁷ Hyperglycemia can result in pathological activation of PKC. Elevated glucose levels result in activation of the glycolytic pathway and lead to increased levels of intracellular glyceraldehyde-3-phosphate. Glyceraldehyde-3-phosphate can promote the de novo synthesis of DAG through glycerol-3-phosphate, which in turn activates PKC.¹²⁸ Activation of PKC can also be mediated by AGE⁹² and ROI.¹¹¹

Elevated levels of DAG and PKC activity has been detected in the tissues of animals with diabetes.¹²⁹ The pathologic effects of PKC activation that cause vascular damage are mediated through increased vascular permeability,¹³⁰ disruption of nitric oxide regulation,^131,132 increased leukocyte adhesion to vessel walls,¹³³ and changes in blood flow.¹³⁴ In fact, the effects of PKC on retinal blood flow as measured by fluorescein video angiography, as well as glomerular filtration rate and albumin excretion rate, were improved in a dose-dependent fashion with ruboxistaurin (LY333531), a PKC-β inhibitor.¹³⁵ VEGF¹³⁶ and endothelin¹³⁷ can also activate PKC, which in turn can promote the expression of growth factors like VEGF¹³⁸ and transforming growth factor-beta (TGF-β).¹³⁹ PKC activation can influence other signally pathways, such as MAPK or NF-κB.¹⁴⁰

PKC inhibition by ruboxistaurin has been reported to block many of the vascular abnormalities in endothelial cells and contractile cells from the retina, arteries, and renal glomeruli.¹⁴¹ In animal models of diabetes, ruboxistaurin protected against or reversed many of the early vascular changes seen with retinopathy, nephropathy, and neuropathy.^{135,136,142,143} However, a prospective clinical trial of ruboxistaurin did not meet its primary outcome (progression to sight-threatening diabetic macular edema or application of focal/grid photocoagulation for diabetic macular edema) at 30 months although there was a significant reduction of progression to sight-threatening diabetic macular edema when considered alone.¹⁴⁴ An open-label extension of the Protein Kinase C Diabetic Retinopathy Study 2 (PKCDRS-2) reported that over a 6-year study period patients with the greatest ruboxistaurin exposure (~5 years) had less sustained moderate vision loss (>15-letter decline) compared to those in the original placebo group (~2 years of ruboxistaurin use).¹⁴⁵

Insulin receptors and glucose transporters

In certain types of cells, such as adipocytes and skeletal muscle cells, insulin is required to transport glucose from the extracellular fluid across the plasma membrane into the cytoplasm. This action requires a specific receptor for insulin on the plasma membrane. Although it has been commonly stated that the microvascular complications of diabetes do not occur in tissues in which insulin is required for the transport of glucose into cells, insulin receptors have been reported on the pericytes and endothelial cells of the retinal microvessels.¹⁴⁶ There is no evidence, however, that the retinal microvascular insulin receptors are required for glucose transport, although insulin does enhance glycogen synthesis from radiolabeled glucose in retinal microvascular pericytes and endothelial cells and aortic smooth-muscle cells, but not in aortic endothelial cells.¹⁴⁶ Insulin in physiologic concentrations (as low as 10 ng/ml) stimulated [3H]-thymidine incorporation into retinal microvascular pericytes and endothelial cells and aortic smooth-muscle cells but not aortic endothelial cells.¹⁴⁶ It is noteworthy that, in these experiments, such low concentrations of insulin produced an effect, because unphysiologically high (e.g., 1 mg/ml) concentrations of insulin will stimulate proliferation of many types of cultured cells. However, since microvascular endothelial cells and pericytes do not normally proliferate in the mature retina,¹⁴⁷ the importance of these results for normal retinal vascular physiology is unclear. Additionally, these results indicate that there are metabolic differences between microvascular endothelial cells and the endothelial cells of larger vessels, so that translation of results from one type of vascular endothelial cell to another must be done with great caution.

There are at least five different types of facilitated cell membrane glucose transporters, designated GLUT1, GLUT2, GLUT3, GLUT4, and GLUT5, that appear to be most important for the intracellular transport of glucose in tissues like the retina that do not require insulin. Of these, GLUT1 appears to be the most prevalent in the retina,^148–150 occurring in microvascular and macrovascular endothelial cells and on RPE cells, as well as in the Müller cells. GLUT2 localization has been reported by immunocytochemistry at the apical ends of the Müller cells of the rat retina, facing the interphotoreceptor matrix,¹⁵¹ while GLUT3 has been reported by similar techniques to be localized to the plexiform layers of the rat¹⁵² and human¹⁵⁰ retina. An initial report using light microscopic immunocytochemistry in human eyes¹⁴⁸ also reported GLUT1 in the nerve fiber layer of the retina and in photoreceptor cell bodies, but GLUT1 was absent from retinal neovascular proliferations in the eyes of diabetic subjects. Subsequently, these investigators used quantitative immunogold electron microscopic immunocytochemistry to examine GLUT1 localization in the eyes of two nondiabetic subjects and three diabetic subjects with little or no retinopathy.¹⁴⁹ In approximately half of the retinal microvessels from the diabetic subjects there was no quantitative difference in GLUT1 immunoreactivity by comparison with microvessels from the two normal individuals. However, in the other half of the retinal microvessels studied from diabetic individuals, these investigators reported an increase of GLUT1 immunoreactivity of about 18-fold over normal on the luminal plasma membranes. If these findings can be confirmed in a much larger number of eyes, such upregulation could be a mechanism that initiates glucose-mediated cellular damage by permitting a much greater influx of glucose into cells.

Whether galactose also enters cells by one or more of these facilitated glucose transporters has not been directly tested. However, the fact that rats fed a 50% galactose diet – which also contains the normal amount of glucose – double their food intake by comparison with normal rats, but nevertheless gain weight at only 60–70% of the rate of the normal animals,¹⁵³ suggests that the excessive amount of galactose competes with glucose for the transport sites, thereby limiting the entry of glucose into cells and diminishing glucose-requiring cellular energy metabolism. However, galactose can participate in other cellular pathways along with glucose, including protein glycation/advanced glycation endproduct formation and synthesis of DAG to activate PKC.¹²⁶ Whether glucose or galactose can upregulate the mRNAs governing synthesis of any of the GLUT proteins in a fashion such that this upregulation persists long after cessation of the hyperglycemic or galactosemic state has not yet been explored.