Simulated Hyperglycemic Hyperosmolar Syndrome

IMPAIRED INSULIN ANDEPINEPHRINE EFFECTSUPONLIPOLYSIS

IN THE ISOLATED RAT FAT CELL

B. PAULTURPIN, WILLIAM C. DUCKWORTH,and SOLOMONS. SOLOMON,Departmentsof Research and Medicine, Veterans Administration Medical Center andUniversity of Tennessee Center for the Health Sciences,Memphis, Tennessee 38104

A B S T RA C T These investigations were designed toevaluate the effect of excess glucose and sodiumchloride on lipolysis in the isolated adipocyte undernormal and modelled pathological conditions simulat-ing the hyperglycemic hyperosmolar syndrome.

Isolated rat fat cells were incubated in the presenceof various combinations of sodium chloride, glucose,epinephrine, and insulin. Lipolysis was measured asglycerol and free fatty acid release, and total mediumosmolarity as milliosmoles per liter by freezing pointdepression.

Basal lipolysis was unaffected by changes in os-molarity with sodium chloride, but glucose and glucoseplus sodium chloride increased basal glycerol release.Increasing osmolarity with sodium chloride diminishedthe lipolytic response to epinephrine. Increasingosmolarity with glucose augmented the lipolytic re-sponse to epinephrine up to a total medium osmolarityof 550 mosmol. Higher osmolarities produced withglucose suppressed the .epinephrine-induced lipolyticresponse.

When the hyperglycemic hyperosmolar syndromewas simulated with 100 mMglucose and 50 mMsodiumchloride (total osmolarity = 460 mosmol) the epi-nephrine-stimulated lipolysis dose-response curve inthe isolated fat cell was shifted to the right. Further-more, in the presence of 100 mMglucose + 50 mMsodium chloride, physiological concentrations of insulinwere less effective in opposing epinephrine-stimulated

This work was presented, in part, at the Midwest SectionAmerican Federation for Clinical Research, 1977.

Dr. Turpin is an Associate Investigator of the VeteransAdministration. Dr. Duckworth is the recipient of ResearchCareer Development Award AM 00187-01 from the U. S.Public Health Service.

Received for publication 23 February 1978 and in revisedform 27 September 1978.

lipolysis. In the presence of 50 mMglucose and 25mMsodium chloride (total osmolarity = 370 mosmol)epinephrine-stimulated lipolysis measured as freefatty acid release was decreased by 50%.

Under conditions simulating the hyperglycemic hy-perosmolar syndrome in the isolated rat adipocyte,altered lipolysis reflects impaired effectiveness of bothinsulin and epinephrine as antilipolytic and lipolytichormones, respectively. Furthermore, the attenuatedresponse to both hormones appears to be primarily afunction of extracellular solute composition. The lack ofketosis is the result of diminished release of free fattyacids from peripheral adipose cells.

INTRODUCTION

The syndrome of hyperglycemia in the absence ofketonemia, hyperosmolarity of the serum, and lowlevels of peripheral blood immunoreactive insulin iswell-established clinically as the hyperglycemichyperosmolar syndrome (HHS)' (1-3). Several hy-potheses have been proposed to explain the absenceof ketoacidosis in this diabetic condition. The develop-ment of ketoacidosis in the diabetic patient is a con-sequence of the relative insulin deficiency and result-ant lack of antilipolysis. Excessive quantities of freefatty acids are mobilized from adipose tissue as a resultof the unrestrained lipolysis. These free fatty acidsare subsequently converted to acetate via acetyl-CoA.The elevated levels of free fatty acids also inhibit themetabolism of acetate through all pathways exceptthose involved in ketone body formation (4). In HHS,plasma free fatty acid levels are low or normal (5) sug-gesting an effect on ketogenesis attributable to lack ofsubstrate. Thus, absence of ketosis might reflect the

'Abbreviation used in this paper: HHS, hyperglycemichyperosmolar syndrome.

The Journal of Clinical Investigation Volume 63 March 1979 - 403 -409 403

influence of multiple factors which regulate lipolysis andfree fatty acid release from adipose tissue. It has beenshown that insulin in low concentrations is more effec-tive in inhibiting lipolysis in adipose tissue than inenhancing glucose use by peripheral tissues (6). Thissuggests that the low levels of insulin present in theHHSmight be sufficient to suppress lipolysis. How-ever, the similarities in insulin levels in hyperosmolarvs. ketoacidotic states militate against insulin beingthe sole antilipolytic factor in the HHS (5, 7).

METHODSAnimals. Male Holtzman rats which weighed 150-180 g

were used. The animals were fed Purina Laboratory Chow(Ralston Purina Co., St. Louis, Mo.) ad libitum until time ofsacrifice by decapitation.

Chemicals. Fatty acid-poor bovine serum albumin, frac-tion V, lot 27, was obtained from Miles Laboratories, Inc.,Miles Research Products, Elkhart, Ind. Fatty acid-free bovineserum albumin fraction V, lot 21, was obtained from the samesource. Crude bacterial collagenase, lot 43N111, was pur-chased from Worthington Biochemical Corp., Freehold, N. J.Sodium Hepes and Hepes were purchased from Calbiochem,San Diego, Calif. Porcine insulin, lot 615-D63, was obtainedfrom Eli Lilly and Company, Indianapolis, Ind. All otherchemicals were of analytical grade and obtained fromstandard commercial sources.

Preparation of adipose cells. Isolated fat cells were pre-pared by the methods of Rodbell (8) and Gliemann (9) employ-ing collagenase digestion as modified by Solomon et al. (10).Approximately 1.2 g of rat epididymal adipose tissue was usedfor each experiment. After collagenase digestion, cells werewashed and then suspended in Krebs-Ringer Hepes bufferwhich contained 4% bovine serum albumin at pH 7.4 and37°C. Cells were used both for incubation (8) and perifusionas described previously (11-13).

Incubation of isolated adipose cells. Cells were incubatedin plastic vials in a total volume of 2.0 ml. Cells suspendedin 1.0 ml of isosmolar Krebs-Ringer Hepes buffer were addedto 1.0 ml of buffer containing various substances, dependingupon the experiment. Vials were incubated in a metabolicshaker for 120 min. The incubation was terminated by decant-ing the contents into chilled glass conical tubes and placingthem in ice. The tubes were centrifuged at 1,800 rpm for 20min at 4°C. The fat cake was removed and the supernatant fluidwas assayed for glycerol and free fatty acid content. An aliquotof cells was taken for a cell count by the hanging-droptechnique (10).

Perifusion of isolated fat cells. Isolated fat cells werestudied with the perifusion system as previously described(11) with the following exceptions. During perifusion, glycerolrelease was continually monitored by the introduction into thesystem of an automated assay system (14) with the fluorometricmethod (15). The perifusion system was combined with agradient maker to provide a perifusion medium with os-molarity decreasing from 1,000 mosmol to 280 mosmol.

Measurement of lipolysis. Glycerol content of the peri-fusate and the incubation medium was determined by thefluorometric method of Chernick (15). Glycerol release ex-pressed as nanomoles glycerol per 105 cells per hour wasused as an index of lipolytic rate. Because of interexperimen-tal variation in adipose cell responsiveness to hormone stimu-lation, results of some experiments are expressed on a per-centage basis (11).

Free fatty acid concentration in the incubation medium wasmeasured by chloroform extraction and colorimetric de-termination (16).

Measurement of osmolality. Hyperosmolar solutions ofvarious agents were added to the incubation vials as 0.2-mlvol. The final osmolarity of the 2.0-ml vol was measuredwith a Fiske osmometer (Fiske Associates, Inc., Uxbridge,Mass.) to determine the freezing point depression.

Analysis of data. The data was evaluated for significanceby the Student's t test for non-paired data (17).

RESULTS

Effect of sodium chloride on basal lipolysis. TableI shows basal lipolysis under isosmolar (280 mosmol)and hyperosmolar conditions (350, 450, and 1,000mosmol). It is evident that basal lipolysis wasunaffected through this spectrum of extracellularosmolarity.

Effect of sodiu as chloride on epinephrine-stinmulatedlipolysis. Table I summarizes the effects of variationin extracellular sodium chloride osmoles on epineph-rine-stimulated lipolysis in the incubation system. Thisgroup of experiments shows response to a submaximalstimulatory dose of epinephrine, 0.3 ,M. Under hyper-osmolar conditions, although lipolysis was augmentedsomewhat, this was not a statistically significant in-crease (data not shown). Epinephrine-stimulatedlipolysis at 280 mosmol was 157+15 (SEM) nmol glycerol/105 cells per h. With increasing osmolarity, the lipolyticresponse to epinephrine was progressively sup-pressed as compared with 280 mosmol. The effect ofsodium chloride on epinephrine-stimulated lipolysiswas also evaluated with perifused isolated fat cells(11-13). This system was used to determine if the ef-fects of hyperosniolarity were reversible. 0.5 ,uM epi-nephrine was present in the perifusion medium. Fig. 1is a representative experiment showing the effect ofhyperosmolarity in suppressing lipolysis in the

TABLE IThe Effect of Changiing Extracellular Osmolarity with Sodiumn

Chloride on Basal and Epinephrine-StimulatedLipolysis in the Incubated Isolated Fat Cell

Glycerol release

Epinephrine stimulationOsmolarity Basal, n = 3 (0.3 aM) n = 9

mosmollliter nneol/105 cellslh

280 11+1* 157+15350 12+14 111+12§450 13± 11 38±6§

1,000 9+1j 6±1§

* Values shown are SEM.4 NS compared with basal at 280 mosmol.§ P < 0.001 compared with epinephrine response at 280mosmol.

404 B. P. Turpin, W. C. Duckworth, and S. S. Solomon

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FIGURE 1 The effect of hyperosmolarity as a result ofsodium chloride on lipolysis in the perifused isolated ratadipocyte. The perifusion buffer contained 0.5 uM epi-nephrine. As the osmolarity (0 *) decreases, lipolysis(0 --- 0) increases. For comparison, the effect of a constantinfusion of epinephrine in isosmolar buffer is shown (O 0).

presence of a submaximal stimulatory dose of epi-nephrine. As the osmolarity declined, the lipolyticeffect of the epinephrine became apparent at -650mosmol. Lipolysis increased in a smooth fashion untilthe isosmolar state was reached. The magnitude of the

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lipolytic response was similar to that seen when theisolated fat cells were perifused with 0.5 ,uM epinephl-rine in isosmolar buffer. These results demonstrate thathypertonicity produces no irreversible alteration inlipolytic response. The contour of the graph showiinglipolysis suggests also that there may be more than onecomponent to the sodium chloride effect. The incre-ment in effectiveness of epinephrine between 750 and550 mosmol was less than between 550 and 350mosmol. Similarly, the effectiveness of epinephrinemarkedly increased between 350 and 280 mosimol.

Effect of glucose on basal atnd epinelArine-stiimu-lated lipolysis. Fig. 2 shows the effect of glucose onlipolysis in the incubated isolated fat cell. Basal lipoly-sis was progressively augmented by increases in extra-cellular osmolarity with glucose. In the absence of glu-cose (280 mosmol) basal lipolysis was 4 nmol glycerol/105 cells per h; addition of 100 mMglucose increasedthe lipolytic rate to 13±+1. With 300 mMglucose,lipolysis was 20± 1; and with 1,000 mMglutcose, 31 ± 1.Adjacent values are significantly different from eachother with P < 0.001.

Lipolytic response to epinephrine was augmeinted byaddition of glucose. Increasing the concentration ofglucose from 0 to 300 mM(-550 mosmol total) wasassociated with an increase in response. Higher con-centrations of glucose produced less augmenitation ofthe response to epinephrine, and at 500 mMglucosethe response to epinephrine was less than under isos-molar conditions. No response to epinephrine was seenat 1,000 mMglucose.

Total Osmolarity (mosmol / liter)FIGURE 2 A representative experiment showing the effect of increasing osmolarity produicedby glucose on lipolysis in the isolated rat adipocyte. Each point represents the mean-SEMI(n = 3). There is no glucose present at 280 mosmol. Response to 0.5 yM epinephrine(0 *) is augmented by addition of glucose to produce total osmolarity of 580 mosmol (300 mnMglucose). Basal lipolysis (---- 0) is progressively increased by addition of glucose. *P < 0.02;**P < 0.01; ***P < 0.001.

Effects of Hyperosmolarity otn Lipolysis 405

---I I I I I I I I I

200 300 400 500 600 700 800 900 1,000 J,100 1,200 1,300

I

These results indicate that in the isolated fat cell,hyperosmolarity as a result of glucose at concentrations<300 mMaugments both basal and epinephrine-stimu-lated lipolysis. This contrasts with the effect of sodiumchloride on lipolysis. Basal lipolysis was unaffected bychanges in extracellular sodium chloride concentra-tion, but epinephrine-sensitive lipolysis could be com-pletely inhibited by sodium chloride. Because glucoseand sodium chloride are the major determinants ofhyperosmolarity in the HHS, an in vitro simulation ofthe syndrome was formulated with 100 mMglucose and50 mMsodium chloride added to isosmolar buffer.

Effect of glucose and sodium chloride on insulininhibition of stimulated lipolysis. In the series ofincubation experiments represented in Table II, 0.5,tM epinephrine, a submaximal stimulatory dose, in-creased lipolysis 300+25 nmol glycerol/105 cells perh above basal (20±4). A maximal response was at-tained with 1.0 ,uM epinephrine, 625±80. Insulin, 5,uU/ml, inhibited the response to 0.5 ,uM epinephrineby 71% (P < 0.001). It is also seen in these experimentsthat 50 mMsodium chloride and 100 mMglucose in-hibited and augmented, respectively, the lipolytic re-sponse to 0.5 AM epinephrine. Insulin, 5 ,U/ml, in-hibited lipolysis in the presence of 0.5 ,M epinephrineand 50 mMsodium chloride by 39%. This compareswith 55% inhibition by insulin in the presence of 100 mMglucose. When0.5 ,uM epinephrine was combined with50 mMsodium chloride and 100 mMglucose, the lipolyticresponse was attenuated when compared with epi-nephrine alone (P < 0.01). The effectiveness of insulinas an antilipolytic hormone in the presence of thesesolutes was likewise attenuated, 47% inhibition. Thiseffect was examined more closely in the followingseries of experiments.

Effectiveness of insulin and epinephrine in simu-

TABLE IIThe Effect of Insulin on Epinephrine-Stimulated Lipolysis

under Hyperosmolar Conditions

Lipolysis

0.5 M0.5 MAM EPI + 5

Addition Osmolarity EPI* MUIn/mlt

mosmol/liter nmol glycerollO'5 cellslhabove basal

None 280 300+255 86±+1650 mMNaCl 380 160+23 99+8100 mMGlucose 380 700+36 316±3150 mMNaCl and

100 mMGlucose 460 240+27 125±16

* EPI = Epinephrine.t In= Insulin.5 Values shown are SEM.

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=60-

80-

100-7f/- , , ,2 5 20 50 100

Log [Insulin](j.U/mI)

FIGURE 3 Inhibition by insulin of epinephrine-stimulatedlipolysis. In hyperosmolar medium (460 mosmol, *- *)produced with 100 mMglucose and 50 mMsodium chloride,all concentrations of insulin examined are less effective inopposing epinephrine-stimulated lipolysis than in isosmolarmedium (O-O). n = 6; *P < 0.05; **P < 0.001.

lated HHS. The dose-response curves for insulin in-hibition of epinephrine-stimulated lipolysis in the in-cubation system under isosmolar and hyperosmolarconditions are represented in Fig. 3. In isosmolarmedium (280 mosmol), 0.5 ,uM epinephrine-stimu-lated lipolysis above basal 485±30 nmol glycerol/105cells per h. In hyperosmolar medium (100 mMglucoseand 50 mMsodium chloride, -460 mosmol), lipolyticresponse to 0.5 uM epinephrine was 342±24 nmolglycerol/105 cells per h above basal. All concentrationsof insulin were less effective under hyperosmolar con-ditions in opposing epinephrine-stimulated lipolysisthan in isosmolar medium. 5 ,uU insulin/ml in hyper-osmolar medium inhibited lipolysis by 31%. This com-pares with 62% inhibition in isosmolar medium(P < 0.05). With 20 ,U insulin/ml, inhibition was 63%vs. 85%(P < 0.001). Even at 100 ,uU insulin/ml, lipoly-sis was not completely suppressed in hyperosmolarmedium.

Impaired effectiveness of insulin under conditionssimulating the HHS does not explain the restrainedlipolysis characteristic of the syndrome. Therefore, thelipolytic potency of epinephrine was examined undersimilar experimental conditions. Fig. 4 is a representa-tive experiment showing the dose-response curves forepinephrine-stimulated lipolysis under both isosmolarand hyperosmolar conditions.. It is seen that hyper-osmolar medium is associated with a decrease in re-sponsiveness of the adipocyte to epinephrine.

Effect of glucose and sodium chloride on free fattyacid release. Although glycerol release is an accuratemeasurement of total hydrolysis of triglyceride, releaseof free fatty acid more accurately reflects the net con-

406 B. P. Turpin, W. C. Duckworth, and S. S. Solomon

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FIGURE 4 Responsiveness of adipocytes to epinephrine(EPI) in hyperosmolar medium. Hyperosmolar medium(O - - - 0) produced with 100 mMglucose and 50 mMsodiumchloride is associated with a decreased responsiveness of theadipocytes to epinephrine. n = 3; *P < 0.01; **P < 0.001.

version of triglyceride to free fatty acids. The nextseries of incubation experiments was performed with50 mMglucose (a concentration well within the rangeseen in HHS) and excess sodium chloride 12.5, 25, and50 mMin the presence of 1.0 mMepinephrine (a sub-maximal stimulatory dose in this series of experi-ments). Fig. 5 shows the change from basal in freefatty acid release under various conditions. 50 mMglu-cose inhibits both stimulated (P < 0.05) and basal (P< 0.05) fatty acid release. Glucose in the presence of12.5 mMNaCl is not more effective than glucose alonein suppressing fatty acid release; but 25 mMNaCl (totalmedium sodium osmoles = 170) in the presence of glu-cose markedly diminishes fatty acid release (P < 0.01).This effect is even more pronounced with 50 mMsodium chloride (P < 0.001). Epinephrine-inducedfree fatty acid release was also inhibited by hyperos-molarity as a result of sodium chloride excess alone.Sodium chloride excess (25,50, and 100 mM) inhibitedfatty acid release by 12, 19, and 65%, respectively. Thisdegree of inhibition of free fatty acid release wasvery similar to that of inhibition of glycerol release(Table I) which shows that glycerol release was in-hibited by sodium chloride excess (70 and 170 mM)by 28 and 76%, respectively.

DISCUSSION

Investigations of adipose tissue and isolated adiposecells with hyperosmolar solutions of sucrose, mannitol,sodium chloride, and urea have shown increased trans-port of glucose across the plasma membrane (18-21);increased incorporation of "4C-labeled glucose intoCO2, total lipid, free fatty acids, and glycerol (18); at-tenuation of lipolysis and release of free fatty acidsin response to lipolytic agents (18, 22); and augmenta-tion of increases in intracellular concentrations of

cyclic AMPin response to lipolytic hormones (22). Withthe exception of the last mentioned effect, thesechanges in metabolic parameters are similar to, andparallel those, produced by insulin.

In 1963, Jungas and Ball showed that epinephrine,in the presence of glucose, was more effective in stimu-lating lipolysis in epididymal adipose tissue than epi-nephrine alone (23). These studies were confirmed bylater work which emphasized the role of nonesterifiedfatty acids as regulators of net lipolysis (24, 25). Physio-logic or superphysiologic concentrations of glucose areassociated with reesterification of intracellular freefatty acids with a subsequent lowering of the concentra-tion of these organic acids. This is associated withincreased net lipolysis, both basal and stimulated, asreflected by glycerol which is the most accurate meas-urement of triglyceride hydrolysis (25-28). Because ofthe reesterification of the fatty acids, however, fattyacid release is usually decreased when glucose ispresent in the incubation media (18, 29).

The distinguishing feature of the HHS, when com-

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FIGURE 5 Inhibition of epinephrine (EPI)-stimulated freefatty acid release by hyperosmolar medium containing 50 mMglucose alone and combined with sodium chloride (mean±SEM; n = 4); *P < 0.05; **P < 0.01; ***P < 0.001. Statisticalcomparison with 1.0 ,uM epinephrine alone or with basal forglucose alone.

Effects of Hyperosmolarity on Lipolysis 407

pared with other insulinopenic states, is the absenceof ketogenesis of sufficient magnitude to produce keto-acidosis. It'has been proposed that ketone body produc-tion is low because of deficient substrate in the form offree fatty acids. It would appear that in HHSthe livermay not be exposed to sufficient insulin to prevent keto-genesis (5) as plasma insulin levels are similar to thoseseen in diabetic ketoacidosis. However, the low levelsof plasma free fatty acids seen in the majority of pa-tients with the HHS do indicate that free fatty acidrelease by peripheral adipose tissue is inhibited. Onemajor unanswered question relates to the factorsresponsible for restrained lipolysis in the adiposecell under these conditions. Dehydration and volumedepletion associated with decreased food intake havebeen shown to have an effect in lowering circulatingfree fatty acids (30-32). Similarly, a nonsuppressibleinsulin-like agent (a somatomedin) has been suggestedas an antilipolytic factor in this syndrome (33). How-ever, the effects of these and other hormones withantilipolytic properties have not been evaluated in thissyndrome.

The results of our studies and others have shown thatglucose enhances both basal and stimulated lipolysis,as reflected in glycerol release, in adipose tissue, andin isolated adipocytes (24-26). The maximal effect onepinephrine-stimulated glycerol output was seen at100 mMglucose with higher levels of glucose thatproduced less of a stimulatory effect as well as veryhigh glucose levels, actually blocking epinephrine-induced lipolysis. In contrast, basal lipolysis was in-creased at all levels of glucose.

Although most concentrations of glucose augmentedepinephrine-induced glycerol output, sodium chloridewas able to abolish this effect of glucose. Indeed, theeffect of sodium chloride as an inhibitor of lipolysiswas more striking against the combination of epi-nephrine and glucose than against epinephrine alone.Sodium chloride negated the enhancement producedby glucose of epinephrine-stimulated lipolysis. Theseobservations demonstrate that the net effect of sodiumchloride in the presence of glucose is antilipolytic.

Hyperosmolarity induced by excess sodium chloridealone inhibited epinephrine-induced lipolysis. Bothglycerol and fatty acid release were decreased in thepresence of as little as 25 mMsodium chloride ex-cess. Sodium chloride is apparently capable of restrain-ing lipolysis measured as glycerol or free fatty acidrelease under conditions simulating the HHS. It isthought that hyperosmolarity increases glucose uptakethrough a nonspecific effect of tonicity rather thanthrough the effects of a particular solute (20). The anti-lipolytic effect of sodium chloride might be producedthrough inhibition of free fatty acid reesterification orinhibition of the passage of products of lipolysis

through the cell membrane. Either mechanism wouldresult in levels of intracellular free fatty acids suf-ficiently high to inhibit lipolysis. Extracellular sodiumchloride in higher than physiologic concentrations mayinterfere with the Na+, K+-ATPase mechanism, whichhas been linked with active transport of free fattyacids (34, 35).

When insulin was present in physiologic concentra-tions, its effect in opposing lipolysis was less underhyperosmolar than isosmolar conditions. This mightrepresent a combined enhancement of hyperosmolarityand insulin on glucose uptake. However, the ability ofinsulin to stimulate glucose uptake in muscle prepara-tions under hyperosmolar conditions has been shownto be diminished (4). Furthermore, the antilipolyticeffect of hyperosmolarity is not additive to insulin. Incontrast, insulin was less effective under these condi-tions. These results are most interesting when com-pared with a recent report of diminished lipolysis as-sociated with increased intracellular cyclic AMPaf-ter lipolytic hormone stimulation under hyperosmolarconditions. Although hyperosmolarity in those experi-ments augmented increases in intracellular cyclic AMPin response to a lipolytic hormone, lipolysis did notproceed at a commensurate rate (7). This strongly sug-gests inhibition of the system beyond the step of cyclicAMP accumulation. When hyperosmolar conditionswere produced with glucose and sodium chloride, ourresults showed a decreased responsiveness of the adi-pocyte to epinephrine, but no change in maximallipolysis.

Wehave shown that physiological concentrations ofinsulin are less effective in opposing lipolysis underconditions simulating the HHS. Hence, the dimin-ished lipolysis seen in this insulinopenic state cannotbe attributed to increased effectiveness of insulin.Rather, it appears that the absence of ketosis in thissyndrome might represent decreased responsivenessto catecholamines as reflected in the release of freefatty acids from peripheral adipose tissue.

Our results suggest that specific solute effects aremore likely responsible for the clinical presentation inthis syndrome, perhaps through perturbations in levelsof free fatty acids.

ACKNOWLEDGMENTSThe authors wish to thank Dr. C. T. Huber for technical ad-vice on the perifusion of isolated fat cells, and also Ms.Margaret Goessling and Ms. Janice New for expert technicalassistance.

This study was conducted under Veterans AdministrationResearch Projects 8036-01, 8036-04, 1942-04, and 8569-01; andwas supported, in part, by National Institutes of Health grantAM 18022, and by a grant from the Juvenile DiabetesFoundation.

408 B. P. Turpin, W. C. Duckworth, and S. S. Solomon

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