In Vitro Studies on the Action of CS-045,A New Antidiabetic Agent
Theodore P. Ciaraldi,Allison Gilmore, Jerrold M. Olefsky,Michelle Goldberg, and Kim A. Heidenreich
The mechanism of action of CS-045, a new orally active antidiabetic agent, was studied in vitro using cultured hepatoma cells (Hep G2) and muscle cells (BC3H-1). Treatment of both types of cultured cells with varying doses of CS-045 did not significantly alter insulin receptor binding. Basal and insulin-stimulated glucose transport in BC3H-1 cells was also unaltered by the drug. In contrast,CS-045 increased glycogen synthase I activity in both cell types. This effect was maximal after 24 hours and in Hep G2 cells was associated with a threefold increase in the apparent affinity of the enzyme for glucose-6-phosphate.Gluconeogenesis from lactate in Hep G2 cells was greatly reduced by CS-045 treatment. We conclude that CS-045 may act directly on muscle and liver cells to increase glucose utilization. It is also effective in reducing glucose production.These multiple effects may account in part for the ability of CS-045 to reduce blood sugar levels in vivo. 1990 by W.B. Saunders Company.
INSULIN RESISTANCE is an important pathophysio-logic feature of non-insulin-dependent diabetes mellitus (NIDDM)’ and is characterized by decreased glucose utiliza-tion by peripheral tissues such as muscle, and a failure of insulin to fully suppress glucose production by the liver.2 Increased hepatic glucose output in the basal postabsorptive state is also a major contributor to the hyperglycemia of NIDDM.? A wide variety of orally active hypoglycemic agents (including several generations of sulfonylurea drugs and the biguanides) have been developed to treat these problems. There is a large body of evidence indicating that these compounds have extrapancreatic actions.3-6 For exam-ple,sulfonylureas and biguanides have been shown in vivo7 and in vitro’2-18 to potentiate insulin’s actions and/or to have direct effects on carbohydrate and lipid metabolism in target tissues. The mechanisms of the extrapancreatic effects are still unknown and controversy exists as to whether they act to increase insulin receptors in target tissues, or exert their effects solely at the postreceptor level.7-19
Recently,a new oral antidiabetic drug has been reported.20 The agent, CS-045, is a hindered phenolic compound contain-ing a thiazolidene ring, and thus differs markedly in structure from sulfonylureas and biguanides.The agent was effective in a number of insulin-resistant, diabetic animal models in lowering hyperglycemia and hypertriglyceridemia to near normal values.20 It also corrected much of the hyperinsuline-mia in these animals. The major results of CS-045 treatment were improved glucose tolerance with decreased insulin secretion.The means by which CS-045 exerts these effects is unknown,but could potentially involve both receptor and postreceptor sites of action. In this study,we examined the ability of CS-045 to influence multiple events in carbohy-drate metabolism in cultured human hepatoma cells(Hep G2) and BC3H-1 muscle cells.
From the Division of Endocrinology/Metabolism,Department of Medicine.University of California.San Diego.La Jolla,CA;and The Veterans Administration Medical Center,Medical Research Service,San Diego,CA92161.
Supported by a grant from Sankyo Company,Ltd.
Address reprint requests to Theodore P. Ciaraldi, PhD, Veterans Administration Medical Center,Medical Research Service (V-11IG).3350 La Jolla Village Dr,San Diego, CA 92161.
1990 by W.B.Saunders Company.
0026-0495/90/3910-0010$3.00/0
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MATERIALS AND METHODS
Materials
CS-04520 and metabolites were kindly provided by Dr Hiroyoshi Horikoshi of Sankyo (Tokyo,Japan).Procine insulin was a gift from Dr R.E. Chance of Eli Lilly (Indianapolis,IN).[('1)Tyr4Jinsulin was from Dr Bruce Frank,also of Eli Lilly. Bovine serum albumin (BSA)was purchased from Boehringer-Mannheim(Indianapolis. IN). The AG 1-X8 anion exchange resin was from Bio-Rad (Richmond, CA).Tissue culture media, pyruvate, glutamine, and all antibiotics were purchased from Grand Island Biological (Grand Island, NY). Fetal calf serum (FCS) was from Irvine Scientific (Irvine,CA).2-Deoxyglucose,L-glucose,glucose-6-phosphate,and uridine-diphosphoglucose(UDPG)were obtained from Sigma Chem-ical (St Louis, MO). 'H-2-deoxyglucose, 'H-L-glucose, '4C-lactate, and 4C-UDP-glucose were all purchased from New England Nu-clear(Boston,MA).
Cell Culture
The Hep G2 cell line is a minimal deviation human hepatoma that maintains liver cell morphology and function." Hep G2 celIls express insulin receptors, display a number of metabolic responses to the hormone,21-23 and have been routinely used as a model cell for studying insulin action in the liver.21-27 Cells were maintained in continuous culture in plastic T7s flasks. The culture media was Minimal Essential Media, Earls salts, supplemented with L-glu-tamine (2 mmol/L), gentamycin sulfate (50μg/mL),penicillin(100 U/mL),streptomycin (100 μg/mL), and FCS (10%). Cells were suspended by treatment with trypsin/EDTA, seeded on 60-mm dishes (7.5 x 10° cells/dish), and cultured in a humidified atmo-sphere containing 5% CO,. After 4 days in culture,the media was replaced with fresh media. At this time, CS-045 or vehicle (DMSO at a final concentration of 0.05%) was added to the cells.
The murine BC3H-1 muscle cell line spontaneously differentiates into nonfusing myocytes as determined by a number of criteria.28.29 While the tissue of origin of these cells,skeletal or smooth muscle,is uncertain,2 they have been widelyused as a model system for studying insulin action in muscle.30-33 BC3H-1 cells were cultured in Dulbecco's modified Eagle medium (DMEM) (25 mmol/L glucose) supplemented with FCS (10%),sodium pyruvate(1 mmol/L), L-glutamine (4 mmol/L), penicillin G (100 U/mL), and streptomy-cin(100μg/mL) in a humidified atmosphere of10% CO2:90% air. Stock cultures were maintained in a logarithmic growth phase by subculture cvcry 4 days.Experimental cells were plated at an initial density of 4,000 cells/35-mm dish and cultured 10 to 12 days.The medium was changed every 4 days and fresh CS-045 added at this time.There was no detectable metabolism of the drug in muscle cells as determined by high-performance liquid chromatography.Previ-ous studies from our laboratory (K.Heidenreich, unpublished data)
Metabolism,Vol 39,No 10 (October), 1990: pp 1056-1062 
IN VITRO STUDIES ON THE ACTION OF CS-045
and others,3 indicate that insulin receptors and the glucose transport system are fully developed after 10 days in culture. Experiments were routinely performed on day 11 cultures. CS-045, or vehicle (0.05% DMSO),was either added to the medium throughout the culture period or 2 hours before assay.
Insulin Binding
Control and CS-045-treated cells were rinsed four times with 2 mL minimal essential media (MEM), pH 7.9 containing 1% BSA. The cells were then incubated with 2 mL of the same media containing 2I-insulin (34.7 pmol/L) and varying concentrations of unlabeled insulin at 16°C for 3 hours. The binding reaction was terminated by 5 washes with 2 mL chilled phosphate-buffered saline (PBS).One milliliter of 1N NaOH was added to each dish to solubilize the cells and this volume, together with a second 1-mL rinse of the dish was placed in 12 x 75 mm tubes for determination of radioactivity.Nonspecific binding was determined in the presence of 17.4 μmol/L unlabeled insulin.
Glucose Transport in BC3H-1 Cells
The initial rates of hexose transport in BC3H-1 cells(1x10° cells/dish) was measured using 2-deoxyglucose as the substrate. Cells were washed twice and then incubated in Krebs-Ringer-phosphate buffer (pH 7.6) containing 1% BSA with and without insulin (17.4 nmol/L) for 60 minutes at 37°C. The transport reaction was initiated by the addition of 'H-2-deoxyglucose(0.4 μCi) and unlabeled 2-deoxyglucose (0.1 mmol/L).After 3 minutes, transport was terminated by removal of the reaction medium and washing each monolayer four times with ice-cold PBS containing 0.3 mmol/L phloretin.Nonspecific uptake and trapping was determined using the same procedure with 'H-L-glucose.Cells were solubilized in 1 mL IN NaOH and counted in 15 mL ACS II (Amersham, Arlington Heights,IL).
Glycogen Synthase Assay
After treatment with or without CS-045,the culture media was removed and the cells were rinsed four times with 2 mL of MEM containing 2% BSA.pH 7.6.Cells were incubated with varying concentrations of insulin in the same media for 2 hours at 37°C.The cells were rapidly rinsed three times with 2 mL of chilled PBS,then rinsed two times with 0.5 mL of 5 mmol/L Tris, 2 mmol/L EDTA, 2% glycerol(TEG),as described by Miller et al. Another 0.5 mL of TEG was placed on the dish and the cells frozen by floating on liquid N2.Dishes were thawed,scrapped with a rubber policeman into flat-bottom vials, and homogenized by two pulses (15 seconds)with a Polytron(Brinkman,Westbury,NY) tissue grinder (setting 7).The total cell homogenate was used for the assay of glycogen synthase by the method of Thomas et al."Enzyme activity was measured in the presence of varying concentrationsof glucose-6-phosphate.In the standard assay system two glucose-6-phosphate levels were used:low (0.02 mmol/L)and high (6.1 mmol/L),while the UDPG concentra-tion was kept constant at 0.3 mmol/L. The reaction was for 30 minutes at 30°C where activity was linear with time in both cell types and all groups tested.Tissue blank reactions contained cell homoge-nates boiled for 10 minutes before use. This value was subtracted from all results.For the purpose of these studies,the activity determined in the presence of the low glucose-6-phosphate level (0.02 mmol/L)was termed glycogen synthase I activity.
Gluconeogenesis in Hep G2 Cells
Gluconeogenesis was measured as the incorporation of C-lactate into glucose by the method of Probst et al.3After drug treatment, dishes were washed three times with 2 mL of MEM containing 2%

1057
BSA and varying concentrations of lactate. After a 20-minute incubation with or without insulin, "C-lactate (0.5 μCi) was added. A 100-μL aliquot of the media was removed after 0,15,and 120 minutes.The media samples were loaded onto columns containing AG 1-X8 anion exchange resin.200 to 400 mesh. in the formate form.The columns were first washed with 1.5 mL H,O and the initial flow through containing glucose retained.Lactate was eluted from the column with 0.4 mol/L Na formate.Radioactivity in the different fractions was determined by liquid scintillation counting.
Statistical Analysis
Statistical significance was evaluated using Student's t test for paired observations and two-tailed P values were calculated.
RESULTS
Cell Growth and Viability
CS-045 was added to Hep G2 cells 2 to 4 days after subculture or at the time of subculture for BC3H-1 cells.The cells continued to divide during the treatment period.Concen-trations of CS-045 up to 20 μg/mL had no effect on either cell number,protein content, or cell viability (as measured by trypan blue exclusion) for either cell type. Higher concentra-tions were deleterious,causing a loss of cells.
Insulin Binding
An initial step at which CS-045 might exert an extrapan-creatic effect on carbohydrate metabolism could be an alteration in insulin binding to target cells.71 To determine if CS-045 alters insulin binding, we performed “in vitro”insulin receptor binding assays in control and CS-045-treated Hep G2 and BC3H-1 cells. Binding was performed for 3 hours at 16°C since these conditions permit measure-ment of cell surface binding. Direct addition of CS-045(20 ug/mL)to the binding assay did not alter insulin binding to either cell type (data not shown).Treatment of Hep G2 cells for 48 hours with CS-045 also had no effect (Table 1). Complete competition curves generated by measuring the inhibition of radiolabeled insulin binding by varying concen-trations of unlabeled insulin were also similar in control and CS-045-treated Hep G2 cells. Insulin degradation was low (~2%/10°cells) in both control and drug-treated cells.
Treatment of BC3H-1 cells for up to 11 days with CS-045 (20μg/mL)resulted in a small but not statistically signifi-cant (P>.05) increase in insulin binding (Table 1). The affinity of the insulin receptor and insulin degradation by BC3H-I cells were not altered by the drug.
Table 1. Effect of CS-045 on Insulin Binding to Cultured Muscle
and Liver Cells
-Bound (% of Total Bound/10° Cells)
Cell Type Control +CS-045
Hep G2 9.27±2.00 8.65±1.80
BC3H-1 1.90±0.23 2.38±0.22
NOTE.Cll d ih ith
NOTE.Cells were treated with or without CS-045 (20 μg/mL)for either 48 hours (Hep G2) or 11 days (BC3H-1).Binding was measured as described in Methods and represents specific binding of at tracer concentration of 1251.Results are the average ± SE of three experiments in Hep G2 cells and four experiments in BC3H-1 cells. 
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Glucose Transport
An early event in insulin action is the transport of glucose into cells. In BC3H-1 cells this process can be stimulated nearly threefold by acute exposure to insulin(Fig 1).Chronic treatment (11 days) of BC3H-1 cells with CS-045 had no effect on either basal or maximal insulin-stimulated glucose transport (Fig 1). Acute(2-hour) exposure of BC3H-1 cells to CS-045 also had no direct effect on basal or insulin-stimulated glucose transport(data not shown).
Glycogen Synthase Activity
A major event in glucose utilization after transport into the cell is storage as glycogen.The activity of the major glycogen synthetic enzyme,glycogen synthase, was studied in cell homogenates from both Hep G2 and BC3H-1 cells. The initial assay was performed at concentrations of substrate ([UDPG]=.3 mmol/L) and allosteric regulator ([G-6-P]=.02 mmol/L) that measured primarily the glucose-6-phosphate independent form of the enzyme.3 In liver,and presumably Hep G2 cells,glucose is the major agent respon-sible for stimulation of this form of the enzyme.38 Treatment of Hep G2 cells with CS-045 (20 μg/mL) caused a 3.5-fold increase in GS I activity (Fig 2). In muscle, a different isoenzyme is present.3839 However,drug treatment also increased GS I activity, although not to the same extent as in liver cells (Fig 2). The magnitude of the stimulation of glycogen synthase was dependent on the dose of CS-045(Fig 3) and was maximal at 20 μg/mL in both cell types. Direct addition of CS-045 did not influence glycogen synthase activity. Insulin (8.4 nmol/L) acutely(2 hour) stimulated glycogen synthase activity by 25% ± 12% in Hep G2 and 61%±13% in BC3H-1 cells.The combination of chronic CS-045 and acute insulin treatment did not cause any response beyond that seen with CS-045 alone.
The time course of stimulation of GS I activity by CS-045

CIARALDI ET AL
(uleoJd Bu/ulu/aloud)
Fig 2. Effect of CS-045 on glycogen synthase I activity in BC3H-1 and Hep G2 cells. Cells were treated with (■)or without (□) CS-045 (20 μg/mL) as indicated in the Methods. Cells were harvested, and glycogen synthase I activity, as defined in the text, was measured in cellular homogenates. In the assay, the concen-trations of glucose-6-phosphate and UDP-glucose were 0.02 mmol/L and 0.3 mmol/L,respectively.Results are the average ± SEM of six experiments in Hep G2 cells and three experiments in BC3H-1 cells.
in Hep G2 cells is shown in Fig 4. With a maximal dose of the drug (20 μg/mL), a large increase in GS I activity was seen by 4 hours.However,the full effect was not obtained until 24 hours.Glycogen synthase activity in control cells increased

(6ua/ulu/aloud)

[CS-045] (ug/ml)
Fig1. Effect of CS-045 on basal and insulin-stimulated glucose transport in BC3H-1 cells. Cells were grown in the presence (■)or absence(□)of CS-045(20μg/mL) for 11 days.Insulin(8.7 nmol/L) was added 1 hour before the 2-deoxyglucose transport assay. Results are the average ± SE of three experiments.

Fig 3. Concentration dependence of CS-045 on glycogen synthase I activity in Hep G2 cells. Cells were treated with varying concentrations of CS-045 for 48 hours. Cells were then harvested and assayed for glycogen synthase I activity as described in the Methods.Results are average ± SE of four experiments. 
IN VITRO STUDIES ON THE ACTION OF CS-045
Time(hours)
Fig4. Time course of CS-045 stimulation of glycogen synthase I activity in Hep G2 cells.After 2 days in culture,the culture medium was replaced with fresh medium and cells cultured with (O) or without (·) CS-045 (20 μg/mL). At the indicated times,the cells were harvested and assayed for glycogen synthase I activity as described in the Methods.Results are average ± SE of four experiments.

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Table 2.Effect of CS-045 on Glycogen Synthase Activity
CS-045 Total Activity
Cell Type (ug/mL) Activity Ratio (nmol/min/mg)
0
Mo02 20 28:00/0≤.00 043±231
012+00/0<.00 2241000 NS
NOTE.Cells were treated with or without CS-045 for either 48 hours (Hep G2)or 11 days(BC3H-1).Glycogen synthase activity waB measured as described in the Methods.Activity Ratio= (Activity measured at [G-6-P]=0.02 mmol/Ll/Activity measured at[G-6-G-6-P]=6.16.1 mmol/ L)x 100,where[UDP-glucose]=0.3mmol/L.
*Total activity was measured at maximal concentrations of G-6-P (6.1 mmol/L)and UDP-glucosa(3.2 mmol/L).Results are average ±SE for Hep G2 cells (n = 8) and BC3H-1 cells (n = 3).
and regulator in control and CS-045-treated Hep G2 cells. The data in Table 3 show that CS-045 treatment resulted in a threefold increase in the apparent affinity for glucose-6-phosphate.The predicted increase in affinity for UDP-glucose after CS-045 exposure was also seen in Hep G2 cells (Table 3). The K., for UDP-glucose was approximately 40% lower than the Km in control cells. At high[G-6-P]the K.for UDP-glucose was decreased to 0.14 mmol/L in control cells and was not lowered further in CS-045-treated cells(0.12 mmol/L.).
only slightly over the 3-day treatment period.One possible cause for the slow stimulation of the enzyme could be that CS-045 must be metabolized to be active. The major circulating metabolites of CS-045 are the glycuronide and sulfonate derivatives.2° When these compounds were studied directly,neither was able to influence GS I activity “in vitro”(not shown). A prolonged time course of action could also indicate that new protein synthesis is involved.To investigate this possibility,we cultured Hep G2 cells in the presence of cycloheximide(5 μg/mL) for 48 hours in the absence or presence of CS-045.This dose of cycloheximide inhibited protein synthesis by 95%. Cycloheximide depressed control and drug-stimulated GS I activity equally (to 42% to 46% of control),but CS-045 still caused a threefold increase in GS I activity.
The increase in glycogen synthase activity seen after CS-045 treatment could theoretically be due to either an increase in the amount of enzyme or to a conversion of the enzyme from the less active D form to the I form.38.39 The results of experiments to discriminate between these two possibilities are shown in Table 2. Total enzyme activity was measured at optimal concentrations of substrate and allo-steric regulator.Under these conditions, CS-045 had no effect on total enzyme activity in BC3H-1 muscle cells,and paridoxically decreased activity in Hep G2 cells.Thus,it appears that the effect of CS-045 is to increase the portion of the enzyme that is in the I form, independent of glucose-6-phosphate (Table 2). A possible mechanism by which CS-045 may stimulate GS I is to alter the affinity of the enzyme for glucose-6-phosphate. To examine this possibility,we measured the affinities of the synthase for both its substrate

Gluconeogenesis
A major contributor to the hypoglycemia of NIDDM is elevated hepatic glucose output.2 This glucose can arise from glycogcnolysis and from gluconcogenesis-utilizing sub-strates such as lactate, the levels of which are also elevated in NIDDM.2Glucose production from lactate was measured in control and CS-045-treated Hep G2 cells (Fig 5). CS-045 treatment (48 hours) resulted in a greater than 50% reduc-tion in gluconeogenesis measured in the presence of 1 mmol/L lactate over a 2-hour period.The results also show that the reduction of gluconcogenesis by CS-045 was even greater at higher substrate levels. Insulin had only a small effect on gluconeogenesis (10% reduction). The effects of insulin and CS-045 treatment were not additive.
Table 3. Effect of CS-045 on Affinity Constants of Glycogen
Synthase in Hep G2 Cells
G-6-P
CS-045 Apparent UDP-Glucose
(μg/mL) Affinity(mmol/L) Km(mmol/L)
0 .30±.02 .95±.11
P<.0005 P<.05
20 .09±.02 .52±.07
NOTE. Cells were treated with or without CS-045 for 48 hours and then harvested for the glycogen synthase assay.The apparent affinity of G-6-P was determined by varying [G-6-P] from 0 to 6.1 mmol/L at a constant [UDP-glucose]-0.3 mmol/L.Results are average ±SE of values calculatod after Edie-Hofatee transformation of the data from six individual experiments. The K for UDP-glucose was determined at [G-6-P]=0.02 mmol/L,while [UDP-glucose] varied from 0.05 to 10 mmol/L.Results are average +SE of individual determinations of four experiments. 
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[Lactate](mM)
Fig 5. Effect of CS-045 on gluconeogenesis in Hep G2 cells. Cells were treated with (O) or without (·) CS-045(20μg/mL)for 48 hours.Dishes were then rinsed and incubated with varying concentrations of lactate for 2 hours at 37℃. '*C-lactate incorpora-tion into glucose was then measured on samples of the media. Results are average ± SE of three experiments. Standard error bars,if not visible,are contained within the symbols, P<.05 at all points.
DISCUSSION
A wide variety of agents have been used to treat the hyperglycemia of diabetes.A new potential candidate for treatment is the recently described drug,CS-045.2 This agent differs in structure from the other major classes of hypoglycemic drugs,the sulfonylureas and biguanides,and appears to have a different mechanism of action.Given orally or as a food admixture, CS-045 lowers hyperglycemia and hyperinsulinemia,decreases lactate, free fatty acid and ketone body levels,and reduces hypertriglyceridemia.20 All of these effects are seen in insulin-resistant animal models where hyperinsulinemia is present. The ability of CS-045 to reduce glucose levels while also lowering insulin levels suggests an extrapancreatic site of action. The current studies were undertaken to elucidate the means by which CS-045 can exert its extrapancreatic action to lower blood glucose levels.Since liver and muscle are the major insulin-scnsitive tissues controlling carbohydrate utilization and production,we have employed cell lines maintained in continuous culture that have been widely used as models of liver(Hep G2 cells) and muscle (BC3H-1).
This study shows that CS-045 treatment stimulates glyco-gen synthase activity in both cultured muscle and liver cells. The ability of CS-045 treatment to directly increase glyco-gen synthase activity could possibly account for an increase in glucose utilization by both liver and muscle.Several findings suggest that CS-045 does not increase glycogen synthase activity by increasing enzyme synthesis.One is the failure of the drug to increase the total enzyme activity (Table 2), while the other is the normal (near threefold) stimulation of activity that occurs in the presence of cyclohex-imide.Previous studies have shown that glycogen synthase activity can be stimulated by increasing the fraction of the enzyme that is in the I form,independent of glucose-6-phosphate. This conversion is the result of a dephosphoryla-tion,which in muscle is primarily under the control of insulin,39 while in liver, glucose is the major regulator.3 The

CIARALDI ET AL
prolonged time course of CS-045 action (Fig 4) suggests that accumulation of some unknown material may be involved, although metabolites of CS-045 do not appear to be involved. Whatever the exact mechanism, CS-045 appears to act by converting synthase from the D to I form (Table 2), increasing the apparent affinity of the liver enzyme for the allosteric regulator and, as well,for the substrate (Table 3). Sulfonylureas also stimulate glycogen synthase I activity in cultured fat cells and primary cultured rat hepatocytes.14.18 However,the effects of sulfonylureas are due to a potentia-tion of insulin action, with little direct effect on the enzyme. Insulin action on glycogen synthase is not influenced by CS-045 treatment.This represents yet another difference between CS-045 and the sulfonylureas.
Another interesting finding is the large reduction in gluconeogenesis after CS-045 treatment of Hep G2 cells(Fig 5).Since a major cause of fasting hyperglycemia in NIDDM is elevated basal hepatic glucose production secondary to cnhanced gluconcogenesis,23.40 an agent that can spccifically decrease hepatic glucose output would be advantageous in the treatment of NIDDM.The major determinate of gluco-neogenesis under these conditions is the Vmax, which ap-pears to be decreased fivefold after CS-045 treatment(Fig 5).The breakdown of glycogen also contributes to hepatic glucose output,and the effect of CS-045 on this process is unknown.Glyburidchas been shown to reduce glycogenoly-sis in primary cultured hepatocytes,4 so this is a direction for further study.Another agent,ciglitazone,has been reported to decrease gluconeogenesis from alanine in the perfused liver,yet to a far smaller extent than that seen with CS-045." The biguanide metformin can also reduce gluconeogenesis, but the major effect is to act synergistically with insulin.2 CS-045 differs from these agents in that its effect on gluconeogenesis is independent of insulin and drug treatment does not potentiate insulin action.
In addition to direct effects of the drug,it is possible that CS-045 could also influence insulin action. The first step of insulin action is interaction with specific receptors located on the surface of target cells.2 Treatment of cultured BC3H-1 or Hep G2 cells with CS-045 had no significant effect on tracer insulin receptor binding (Table 1).This is in contrast to results from in vivo studies,where adipocyte binding was elevated.20 The discrepancy between effects observed “in vivo” and “in vitro” after drug treatment has also been a common observation with the sulfonylureas.711.13-19 In most instances,in vivo treatment with sulfonylureas results in an increase in insulin binding to many cell types,711 while direct exposure of cells usually is without effect.12-18 Variations in cell type and treatment protocol have shown in vitro effects in some cases.'°Elevated insulin levels, either in vivo or in vitro, have been shown to downregulate receptors leading to decreased insulin receptor binding.4.4 It is possible that the lowering of insulin levels with CS-045 in vivo may permit receptors to up-regulate. Such an effect would not be observed in tissue culture and may explain the discrepancy between in vivo and in vitro results. Another possible explanation is that the drug inhibits insulin-induced receptor down-regulation,blunting the receptor loss seen during hyperinsulinemia but having no effect in the absence of 
IN VITRO STUDIES ON THE ACTION OF CS-045
insulin (as in the culture environment).Evidence for the latter possibility has been reported for sulfonylureas acting on human skin fibroblasts or monocytes.7.19
An early action after insulin binding is stimulation of glucose transport.In BC3H-1 cells,insulin causes a threefold increase in transport.3° Treatment of the muscle cells with CS-045 had no influence on basal glucose transport or the maximal responsiveness of the transport system to insulin (Fig 1).yet these parameters were improved in adipocytes from treated Zucker fatty rats.20 Since it has been shown, both in vivo and in vitro, that high glucose and insulin levels can lead to decreased glucose transport,145the ability of CS-045 to increase transport in vivo may again be a result of lowering biood sugar and insulin levels, without any direct action on the transport system. Treatment with sulfonylureas also increases glucose transport into perfused rat hind-quarters4 and in isolated adipocytes from diabetic subjects. Sulfonylureas also have direct effects on glucose transport in vitro,elevating glucose transport into BC3H-1 myocytes,L6 muscle cells, and rat adipocytes.41.48-50 The in vivo and in vitro effects of sulfonylureas are greatest on insulin-stimulated transport.50 The lack of any direct effect on CS-045 on

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glucose transport studied in vitro is one difference from the sulfonylureas.
In summary,CS-045 has been shown by the current studies to have multiple effects on cultured liver and muscle cells.It has effects on a number of pathways controlling the balance between glucose production and utilization. The sum of these effects is to increase glucose utilization and reduce hepatic glucose production, effects that might explain its hypoglycemic activity in vivo.The drug does not appear to influence insulin action to any great extent in vitro,as neither insulin binding nor insulin effects on glucose transport, glycogen synthase,or gluconeogenesis were altered by CS-045 treatment. CS-045 differs in many ways from sulfo-nylurcas and represents a new and different type of hypogly-cemic drug. Its large effects on hepatic glucose production in vitro suggest that it may have major value in treatment of the component of hyperglycemia due to increased hepatic glu-cose production in NIDDM.
ACKNOWLEDGMENT
We wish to thank Elizabeth Martinez and Cleon Tate for expert assistance in the preparation of the manuscript.
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