Proc. Natl. Acad. Sci. USAVol. 86, pp. 5141-5145, July 1989Medical Sciences

Activation of protein kinase C by elevation of glucoseconcentration: Proposal for a mechanism in the development ofdiabetic vascular complications

(inositol phospholipids/diacylglycerol/phosphatidic add/glucose metabolism)

TIAN-SHING LEE, KIRSTIE A. SALTSMAN, HIROMI OHASHI, AND GEORGE L. KING*Research Division, Joslin Diabetes Center, Department of Medicine, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02215

Communicated by Rachmiel Levine, April 10, 1989

ABSTRACT Hyperglycemia is believed to be the majorcause of diabetic vascular complications involving both mi-crovessels and arteries as in the retina, renal glomeruli, andaorta. It is unclear by which mechanism hyperglycemia isaltering the metabolism and functions of vascular cells, al-though changes in nonenzymatic protein glycosylation andincreases in cellular sorbitol levels have been postulated to beinvolved. Previously, we have reported that the elevation ofextracellular glucose levels with cultured bovine retinal capil-lary endothelial cells causes an increase in protein kinase C(PKC) activity of the membranous pool with a parallel decreasein the cytosol without alteration of its total activity. Now wedemonstrate that the mechanism for the activation of PKC isdue to an enhanced de novo synthesis of diacylglycerol asindicated by a 2-fold increase of ['4Cldiacylglycerol labelingfrom [14C]glucose. The elevated diacylglycerol de novo synthe-sis is secondarily due to increased formation of precursorsderived from glucose metabolism; this formation is enhancedby hyperglycemia as substantiated by elevated [3H1glucoseconversion into water. This effect of hyperglycemia on PKC isalso observed in cultured aortic smooth muscle and endothelialcells and the retina and kidney of diabetic rats, but not in thebrain. Since PKC in vascular cells has been shown to modulatehormone receptor turnover, neovascularization in vitro, andcell growth, we propose that this mechanism of enhancing themembranous PKC activities by hyperglycemia plays an impor-tant role in the development of diabetic vascular complications.

Hyperglycemia is probably an important etiologic factor inthe development of vascular complications in diabetic pa-tients, such as retinopathy (1), nephropathy (2), and accel-erated atherosclerosis (3). Nonenzymatic glycosylation (4, 5)of protein and accumulation of intracellular sorbitol withreduction of myo-inositol levels (6, 7) have been proposed tobe involved in the development of these vascular changes.

Protein kinase C (PKC), the Ca2+/phospholipid-dependentprotein kinase, appears to be involved in a variety of cellularfunctions such as signal transduction of cellular responses tohormones, growth factors, neurotransmitters, and drugs (8,9). In vascular cells, PKC has been shown to modulategrowth rate (10), DNA synthesis (11), hormone and growthfactor receptor turnover (12), smooth muscle contraction(13-17), and cAMP responses to different hormones (18, 19)and to stimulate neovascularization in vitro. Using culturedbovine retinal capillary endothelial cells, a cell type promi-nently involved in diabetic retinopathy (21), we have previ-ously reported that the membranous pool of PKC activitywas increased 100% by elevation of glucose level (22) but notby adding mannitol to the medium. This effect appears to bemostly an increase in the translocation of PKC from cytosol

to the membrane since the total PKC activity is not alteredand the maximally stimulated membranous PKC activities byphorbol 12-myristate 13-acetate is not changed by elevationof glucose levels (22). In the present study, we have char-acterized this observation and determined the mechanism forthe PKC activation by the elevation of glucose levels.

Cellular PKC activities are regulated by diacylglycerol(DAG) and inositol phosphate levels (23). DAG can bederived from the cleavage of phosphatidylinositol 4,5-bis-phosphate (23) by a phosphodiesterase or, alternatively,DAG can be formed de novo from the glycolytic intermedi-ates dihydroxyacetone phosphate and glycerol 3-phosphateafter stepwise acylation to lysophosphatidic acid and phos-phatidic acid (PA) (24, 25) (see Fig. 2). Since previously wehave reported that under hyperglycemic conditions inositolphosphate levels were not altered, the possibility ofincreasedde novo synthesis is examined and the mechanism for theincreased de novo synthesis is discussed. Portions of thesedata have been presented (26).

MATERIALS AND METHODSCell Culture. Bovine retinal capillary endothelial cells were

isolated by a series of homogenization and filtration stepsdescribed previously (27) and subsequently cultured withDulbecco's modified Eagle's medium (DMEM) with 10o(vol/vol) plasma-derived equine serum (HyClone). Bovineaortic smooth muscle and endothelial cells were obtained, bymethods previously described (11, 27), from calf aorta pur-chased from a local slaughterhouse.

Animals. Male Sprague-Dawley rats weighing 250-270 gwere induced to a diabetic state by a single intravenousinjection of streptozotocin (40 mg/kg of body weight). Sevendays after hyperglycemia was detected, rats were sacrificedunder ether anesthesia. Brain and retina were dissected forPKC purification and measurement.

Partial Purification of PKC. PKC was partially purifiedfrom cultured cells as described (22). Briefly, culture disheswere washed twice with Ca2+/Mg2+-free phosphate-bufferedsaline and twice with buffer A (20 mM Tris-HCI/2 mMEDTA/0.5 mM EGTA/0.33 M sucrose). Cells were scrapedand homogenized 30 strokes with a glass Dounce homoge-nizer with a glass pestle. After centrifugation at 2500 X g for10 min, the supernatant was centrifuged at 100,000 x g for 30min, and the resulting supernatant was retained as thecytosolic fraction. After ultracentrifugation, the pellets werewashed with buffer B (buffer A without sucrose), resus-pended in buffer B with 1% Triton X-100, and homogenizedagain. After a 60-min incubation, the homogenate was cen-

Abbreviations: PKC, protein kinase C; DAG, diacylglycerol; PA,phosphatidic acid.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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trifuged at 100,000 X g for 30 min, and the supernatant thusobtained was collected as the membranous fraction.Both membranous and cytosolic fractions were then

passed through 1-ml DE-52 columns, washed twice with 5 mlofbuffer B, and finally eluted with 1 ml of buffer B containing200 mM NaCl. Rat brain and retina were homogenized afterwashing with phosphate-buffered saline and were processedsimilarly.

Protein Kinase Assay. The activity of PKC was measuredby its ability to transfer 32p from [y-32P]ATP into histone H1in the presence or absence of diolein and phosphatidylserine(Avanti Polar Lipids) as described (22).PKC activities were calculated by subtracting the nonspe-

cific kinase activity (cpm obtained in the absence ofCa2+ andlipids) from the cpm obtained in the presence of Ca2' andlipids. After correction for protein content, the percentchanges were calculated. Protein determination was per-formed according to the method of Bradford (28).Measurement of Incorporation of 4[U-"4C]Glucose into

Lipids. Retinal capillary endothelial cells were grown toconfluence and then DMEM with 5.6 or 22.2mM glucose wasgiven for 5 days. On the day of labeling, P-60 dishes of cellswere washed and D-[U-14C]glucose was added to a finalconcentration of 33 mCi/mmol (1 Ci = 37 GBq). After a 2-hrincubation, cellular lipids were extracted as described byBligh and Dyer (29). DAG was separated on silica gel TLCplates developed together with standards in hexane/diethylether/acetic acid, 60:40:1 (vol/vol) (30). PA, phosphatidyl-inositol, phosphatidylinositol 4-phosphate, and phosphati-dylinositol 4,5-bisphosphate were resolved by TLC togetherwith standards by using a solvent system of chloroform/methanol/acetone/acetic acid/water, 60:20:23:18:12 (vol/vol) (31). Spots of lipids were visualized by either autoradi-ography or charring with H2SO4; individual spots, chosenaccording to the standards, were scraped into vials; and theradioactivity was determined by liquid scintillation spectrom-etry. All the lipid standards were from Avanti Polar Lipids.Measurement of Incorporation of D[5-3HIGlucose into Wa-

ter. After reaching confluence, retinal capillary cells wereincubated in DMEM containing 5.6 or 22.2 mM glucose forthe indicated length of time. D-[5-3H]Glucose was then addedat a final concentration of 0.36 mCi/mmol. After a 3-hrincubation at 37°C, the supernatant was removed from eachdish and filtered through a 0.22-Am filter (Millipore). Fourhundred microliters ofeach filtrate was placed in plastic wellswith the addition of 50 Al of concentrated H2SO4. Each

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plastic well was suspended in a tightly sealed scintillation vialcontaining 1 ml of double-distilled water. After a 20-hrincubation at 37°C, the plastic wells were removed, and theradioactivity of each vial was determined by liquid scintilla-tion spectrometry (32).

Analysis of Total DAG Content. Total cellular DAG levelswere measured by DAG kinase assays as described by Priesset al. (33) with DAG kinase purchased from Lipidex (West-field, NJ). A standard curve showed a linear slope from 50 to600 pmol of DAG.

RESULTSEffect of Elevated Glucose on PKC Activity. In all the

experiments conducted to examine the effect of glucose onPKC activity in tissue culture, cells were first grown toconfluency, and then control or elevated glucose concentra-tions were administered with concomitant lowering of theserum concentration in the medium to 2% in order to keep thecells in the quiescent state. In retinal capillary endothelialcells, after elevating the glucose concentration from 5.6 to22.2 mM in the medium, PKC activity in the membranouspool was unchanged initially but was elevated by 100 ± 10%(mean ± SD; P < 0.001) after 4 to 5 days of incubation. Itremained increased as long as glucose levels were keptelevated, even after 10 days (Fig. 1), without evidence ofdown-regulation. This was accompanied by a concomitant15% decrease in the cytosolic PKC activity. The apparent Kmof membranous PKC for histone H1 was determined as 0.44,M in cells exposed to 5.6 mM glucose. It was not signifi-cantly changed in cells exposed to 22.2 mM glucose, indi-cating that the elevated membranous PKC activity is due toan increase of Vmax and also suggesting an increase of PKCtranslocation from the cytosol to the membrane. After a 24-hrexposure, however, there was a depression of cytosolic PKCactivity by 45 ± 4% (P < 0.001) without an increase in themembranous pool (Fig. 1).The elevation of glucose levels increased membranous

PKC activities by 56% (P < 0.001) and 16% (P < 0.05) inaortic smooth muscle cells and endothelial cells, respec-tively, with a parallel decrease in the cytosolic pool by 24%(P < 0.01) and 3% (not significant) (Table 1).

In diabetic animals 7 days after diabetes was induced bystreptozotocin, PKC activity in rat retina was measured, andit showed a 43 ± 6% (P < 0.01) increase in the membranouspool, where mean plasma glucose level was 25.0 ± 1.2 mM

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FIG. 1. Effect of elevated glucose level on cellular PKC activities. (Left) Twenty-two and two-tenths millimolar glucose results in atime-dependent increase of membranous PKC activity and an acute depression of cytosolic PKC activity in cultured bovine retinal capillaryendothelial cells. After reaching confluence, medium was changed to DMEM containing 22.2 mM glucose or 5.6 mM glucose (as the control).After the indicated length of time, cells were harvested for PKC activity measurement. n = 4 for each point. (Right) Hyperglycemia caused a43% increase in membranous PKC activity in retina but not in brain of diabetic rats. Rats were induced to a diabetic state by an injection ofstreptozotocin and sacrificed 7 days after hyperglycemia had been detected. *, P < 0.01. n = 3 for retina; n = 4 for brain.

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Table 1. Distribution of PKC activities between membranous and cytosolic fractions in bovineaortic vascular cells and in Sprague-Dawley rat brains

PKC activity, pmol Of 32p incorporated per min per mg of protein

Bovine aortic smooth Bovine aorticmuscle cells endothelial cells

5.6 mM 22.2 mM 5.6 mM 22.2 mM Rat brainsFraction glucose glucose glucose glucose Control Diabetic

Membrane 588 ± 43 917 ± 68* 647 ± 52 754±37t 8880 ± 1420 9680 ± 1260Cytosol 580 ± 52 427 ± 27t 508 ± 25 495±58 2990 ± 320 3480 ± 470

Results are the means ± SD. Bovine aortic smooth muscle and endothelial cells were grown toconfluence and then kept in DMEM containing the indicated concentration of glucose for 5 days beforePKC activity measurement. Sprague-Dawley rats were treated as described in the Materials andMethods. For bovine aortic smooth muscle, n = 5; for bovine endothelial cells, n = 4; and for rat brains,n = 3.*, P < 0.001; t, P - 0.05; t, P < 0.01.

(n = 25) compared to 2.78 + 0.03 mM among nondiabetic rats(Fig. 1). An elevation of membranous PKC activities in thekidney of diabetic rats has also been observed (data notshown). In contrast, PKC activities isolated from brainshowed no difference between diabetic and nondiabetic rats(Table 1).

Incorporation of D-[U-14C]Glucose into DAG and Phospho-lipids. To characterize the mechanism by which elevatedglucose levels activate the membranous PKC activity invascular cells, we determined the effects of elevated glucoselevels on the de novo synthesis of DAG, a key regulator ofPKC activity. DAG can be derived from the cleavage ofphosphatidylinositol 4,5-bisphosphate by a phosphodi-esterase (23) or, alternatively, DAG can be formed de novofrom the glycolytic intermediates dihydroxyacetone phos-phate and glycerol 3-phosphate after stepwise acylation tolysophosphatidic acid and PA (24, 25) (Fig. 2). Since previ-ously we have reported that under hyperglycemic conditionsinositol phosphate levels were not altered, the possibility ofincreased de novo synthesis was examined directly by mea-suring the incorporation ofglucose metabolites into DAG andphospholipids. The transfer of 14C from D-[U-'4C]glucose toDAG was increased 150 + 13% (P < 0.001) after a 5-day

elevation of glucose concentration from 5.6 to 22.2 mM (Fig.3). The incubation of cells with [14C]glucose lasted only 2 hrbefore lipids were extracted, suggesting that glycolytic inter-mediates appear very rapidly in DAG. This result, togetherwith the finding that [14C]glucose labeling of PA is increasedby 96 ± 8% (P < 0.001), confirms that the de novo synthesisofPA andDAG is accelerated by elevated glucose levels (Fig.3). Further, we found a 70 to 100% increase in ['4C]glucoselabeling of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate underthe same conditions (Fig. 3). Labeling of all these phospho-lipids increased to a similar extent, suggesting that the labelappearing in PA, both a precursor and a metabolite of inositolphospholipids, is extremely rapidly reequilibrated into eachinositol phospholipid.

Incorporation of D-[5-3H]Glucose into Water. A possiblemechanism for the increase of de novo synthesis of DAG byelevated glucose levels could be an enhanced glucose fluxthrough the glycolytic pathway. Thus, we examined the ratesof glucose metabolism in retinal capillary endothelial cells bymeasuring the rate of conversion of [3H]glucose metabolitesinto water. Upon exposure to 22.2 mM (400 mg/dl) glucose,a 110% increase was observed within 3 hr. It returned to

HYPERGLYCEMIA

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DIHYDROXYACETONE PHOSPHATE MEMBRANE

GLYCEROL-3- PHOSPHATE 1

LYSOPHOSPHATIDIC ACID t

PHOSPHATIDIC ACID t

DIACYLGLYCEROL 1

C-KINASE 1

GROWTH FACTOROR

INSULIN RECEPTORPROCESSI NG

DNA SYNTHESIS

cAMP LEVELS BASEMENTMEMBRANESYNTHESIS

NEOVASCULARIZATION

FIG. 2. Schematic diagram of the de novo synthesis of DAG and the activation of PKC. Those arrows with question marks are theoreticalpossibilities.

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control levels after 2 days and increased again by 30% on day3 and 90% on day 5 (Fig. 4). This interesting biphasic patternof glucose metabolism suggests that an elevated glucose levelcan enhance the amount of glucose metabolized by multiplemechanisms, with the second phase being correlated intochanges in DAG and PKC activities.

2 3 4 5 6DURATION OF EXPOSURE (DAYS)

FIG. 4. Effect of elevated glucose level on the rate of glucosemetabolism in bovine retinal capillary endothelial cells. After reach-ing confluence, medium was changed to DMEM containing 22.2 mMglucose or 5.6mM glucose (as the control). After the indicated periodof time, cells were radiolabeled with D-[5-3H]glucose for 3 hr, andradioactivity incorporated into water was measured.

Total Cellular DAG Mass. After elevating the mediumglucose concentration from 5.6 to 22.2 mM for 5 days, therewas a 19% increase of total cellular DAG content in retinalcapillary endothelial cells, from 128.4 + 10.5 to 153.4 ± 18.8pmol per 106 cells (n = 5, P < 0.05).

DISCUSSIONWe have demonstrated in retinal capillary and aortic vascularcells and vascular tissues from diabetic animals that theelevation ofthe glucose level can increase PKC activity in themembranous pool. Elevation ofPKC activities in the kidneyhas also been observed, confirming a recent report of in-creased PKC activity in renal glomeruli from diabetic animals(34). Together, these results suggest that hyperglycemia cancause an elevation of membranous PKC activity both incultured vascular cells and in vascular tissues in vivo such asretina, aorta, and kidney. The increase of membranous PKCactivity with a decrease in the cytosolic pool stimulated byglucose can be explained by a translocation of the enzymemolecules (35). Kinetic studies showing that the increasedmembranous activity is due to a Vm,, rather than a Km changealso favor the existence of physical translocation of theenzyme from the cytosol to the membrane.Recent models envision the activated PKC as a component

of a quaternary complex consisting of the enzyme, Ca2+, acidphospholipids, and DAG, presumably associated with acellular membrane structure (36). Thus, the observed in-crease in membranous PKC may be due to increased mem-branous DAG and/or acidic phospholipid levels. DAG, inaddition to being derived from the breakdown of inositolphospholipids, can also be synthesized de novo from theglycolytic intermediates dihydroxyacetone phosphate andglycerol 3-phosphate (37-39) by means of a stepwise acyla-tion (Fig. 2). Our data of increased incorporation of 14C fromglucose into DAG (Fig. 3) suggest that the rate of de novosynthesis of DAG from glucose is enhanced by elevatedglucose levels because stimulation of phospholipid break-down does not favor glucose carbon incorporation into DAG(37).The findings that the incorporation of 14C into PA and

inositol phospholipids is also enhanced by elevated glucoselevels suggest that a common underlying pathway for allthese lipids is stimulated. Since PA, a precursor for bothDAG and inositol phospholipids, can also be synthesized denovo from the glycolytic intermediates (Fig. 2), one reason-able explanation is that the de novo synthesis of PA isenhanced by elevated glucose levels first and then the in-creased PA levels flux into DAG and inositol phospholipids.Indeed, a sustained increase in PA elicited by glucose hasbeen reported in pancreatic islets by using steady-state32P-labeling (38) and [3H]glycerol labeling (39).The finding that glucose metabolism is increased by ele-

vation of extracellular glucose levels suggested that themechanisms for the increase of de novo synthesis of PA andDAG could be an enhanced glucose flux through the glyco-lytic pathway. This is an unexpected finding since glucosemetabolism in most tissues other than pancreas and liver isregulated by the phosphorylation of glucose involving hex-okinases having Km values less than 0.4 mM (40). Thus,elevating glucose levels from 5 to 20 mM as in the presentstudy should not have altered the glucose utilization unlessthere are unusual glucokinases induced or the intracellularglucose levels are much lower than those measured in theextracellular pool. These possibilities will have to be differ-entiated. A biphasic pattern of glucose utilization was found,which perhaps reflects this complex nature in the regulationof glucose metabolism. Since changes in de novo synthesis ofDAG and PKC activity changes correlated only with thesecond phase, the data would suggest that the initial increase

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of glucose metabolism was not translated into DAG synthe-sis, denoting other regulating sites.

Chronologically, the increase in membranous PKC activityand the second phase of increase in the rate of glucosemetabolism correlated well with each other, supporting thepossibility that increased membranous PKC activity wasmediated through an enhanced glycolytic pathway. The ex-planation for a delay of 3-5 days is unclear. However, it ispossible that elevated glucose levels result in induction oftranscription or translation ofglycolytic enzyme(s) or that thekinetic properties of the glycolytic enzymes may be alteredthrough nonenzymatic glycosylation (5). The temporal se-quence of the changes of the rate of glucose metabolism,DAG de novo synthesis, and total DAG content is complex.The decrease of cytosolic PKC activity after a 24-hr

exposure to 22.2 mM glucose without any change in themembranous PKC activity is of interest. It suggests thatglucose probably has other effects on PKC besides stimulat-ing the translocation of PKC by enhancing DAG synthesis.We have shown that elevated glucose levels can cause an

elevation of membranous PKC activities, both in culturedvascular cells and in vascular tissues in vivo. This elevationis due to a translocation of PKC from cytosol to membraneby means of an increase of de novo synthesis of DAG as aresult ofa hyperglycemia-induced increase in the metabolismof glucose (Fig. 2). Since an increase in DAG levels in thehearts of diabetic animals has recently been reported (41), wesuggest that this activation ofPKC by glucose by means ofdenovo synthesis of DAG could be generalized to all vasculartissues involved in diabetic vascular complications. Theelevation of PKC activities has been shown to alter manycellular functions that are common in diabetic vascularcomplications (1-6, 22), such as stimulating neovasculariza-tion (20), altering collagen synthesis (42), and affecting hor-mone and growth factor receptor recycling (12, 43). Inconclusion, we suggest that the alterations in the levels ofmetabolites encountered in pathophysiological state such asdiabetes mellitus can affect PKC activities. The consequenceof the activation of PKC by hyperglycemia by means of thebiochemical mechanisms described in this report may beresponsible for some of the vascular changes observed indiabetic patients.

We wish to express our appreciation to Ms. Leslie Balmat forexcellent secretarial assistance. We thank Dr. Jeffrey S. Flier and Dr.Mary J. Spiro for reviewing the manuscript. These studies weresupported by National Institutes of Health Grant EY05110 (G.L.K.),Diabetes Endocrinology Research Center Grant DK36836, JuvenileDiabetes Foundation Grant 187408, and institutional funds from theJoslin Diabetes Center.

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