Effect of glyburide on glycogen metabolism in cultured rat hepatocytes

Effect of glyburide on glycogen metabolism in cultured rat hepatocytes

Effect of Glyburide on Glycogen Metabolism in Cultured Rat Hepatocytes Mayer B. Davidson and Gwendolyn Sladen Sulfonylurea agents decrease hepatic g...

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Effect of Glyburide on Glycogen Metabolism

in Cultured Rat Hepatocytes

Mayer B. Davidson and Gwendolyn Sladen Sulfonylurea agents decrease hepatic glucose production and fasting glucose levels in type II diabetic patients without changing fasting insulin concentrations. This raises the possibility that these drugs may act directly on hepatic carbohydrate metabolism. Cultured rat hepatocytes were used to test this hypothesis. To ascertain whether this in vitro system was suitable to demonstrate an effect of sulfonylurea agents (eg, the well-documented insulin-potentiating action), we initially measured the effect of glyburide (2 pg/mL) on insulin-stimulated net glucose-‘C incorporation into glycogen. Glyburide increased sensitivity to insulin (ie, shifted the dose-response curve to the left) without affecting either responsiveness or insulin binding. Thus, the ED, was significantly lowered (8.4 v 15.2 ng/mL), whereas the percent increase (I 81% w 170%) over the basal level, specific tracer insulin binding 63% w 5.1% per mg protein), and the Scatchard plots were similar. Since an effect of sulfonylurea agents could be demonstrated in this system, and the glycogen pathways supply 75% of hepatic glucose production after an overnight fast, we next measured the direct effect of glyburide (2 bg/mL) on glycogen storage and breakdown. Glycogenolysis was assessed by measuring the breakdown of prelabeled glycogen (from galactose-‘C) and glycogen synthesis by the incorporation of glucose-C’4 into glycogen. Glyburide significantly inhibited glycogenolysis and stimulated glycogen synthesis. Furthermore, glyburide significantly stimulated glycogen synthase while glycogen phosphorylase was unaffected. In conclusion, glyburide directly inhibited glycogenolysis. stimulated glycogen synthesis and glycogen synthase, and potentiated the action of insulin on glycogen synthesis at a postbinding site in cultured rat hepatocytes. These effects, if expressed in vivo, could account for the decreased hepatic glucose production and fasting glucose concentrations in type II diabetic patients. (D 1987 by Grune & Stratton, Inc.

I

MPROVED CONTROL in diabetic patients secondary to sulfonylurea agent therapy is characterized mainly by a decreased fasting plasma glucose concentration (FPG) with much less of an effect on postprandial glycemic excursions.‘” This implies that a major response to these drugs is a reduction in the elevated rates of hepatic glucose production (HGP). At least three studies have examined changes in HGP and FPG in response to sulfonylurea agent therapy. In all three,24 there were highly statistically significant correlations between FPG and HGP both before and after treatment. Fasting insulin concentrations were unchanged by sulfonylurea agent therapy.2’4 These three studies strongly suggest that the major mechanism of improved control induced by sulfonylurea agents is a decrease in HGP. Unless hepatic insulin extraction was increased by sulfonylurea agents [there is evidence in intact dogs that it is unaffected’ but in perfused rat livers that it is enhanced6], the similar fasting peripheral insulin concentrations imply that the liver was not exposed to higher portal vein insulin levels during treatment. These observations raise the possibility of a direct effect of sulfonylurea agents on hepatic carbohydrate metabolism. Furthermore, the fact that approximately 75% of HGP is derived from glycogen stores after an overnight fast’ suggests that these drugs may affect the glycogen pathways. This possibility was tested in a system utilizing primary cultured rat hepatocytes in which glycogen synthesis and glycogenolysis could be evaluated separately. When effects on glycogen metabolism were noted, glycogen phosphorylase and glycogen synthase were measured. MATERIALS AND METHODS

Isolation of Hepatocytes

Hepatocytes were isolated on day 1 from fed Sprague-Dawley rats weighing between 250 and 350 g by a method routinely used in our laboratory,8 except that gentamycin (50 mg/L) was also added to the Swim’s 77 media. After the final wash in this medium used in the Metabolism,Vol36,

No 10 (October), 1987: pp 925-930

isolation procedure, the cells were washed twice more in the culture medium, Dulbecco’s modified Eagle’s medium (DMEM), to which was added (1 L) 3.7 g NaHCOs, 100,000 U potassium penicillin G, 10 mg streptomycin sulfate, 50 mg gentamycin and 1 g glucose (which yields a final glucose concentration of 2 mg/mL since DMEM contains 1 mg/mL). The cells were suspended in DMEM supplemented with 10% fetal calf serum and plated (30 mg cells/ mL/well) into multiwell Petri dishes (Falcon, Becton-Dickinson; Oxnard, CA) and cultured at 37OCin a CO* incubator (day 1). Insulin-Stimulated Glycogen

Net Glucose-d’ Incorporation Into

Before embarking on studies to evaluate a direct effect of glyburide on hepatic carbohydrate metabolism, it was necessary to document an effect of the sulfonylurea agent on an insulin-mediated pathway in hepatocytes, as had been reported previously,*” to give validity to our results should they be negative. The net incorporation of glucose-‘4C into glycogen was the pathway studied. On the morning of day 2, the media was removed along with the dead cells which do not attach. The cells were washed three times with DMEM without fetal calf serum. One milliter of this media containing 3.3 mg bacitracin (to inhibit insulin degradation), 0.2 &i glucose-“C, and varying amounts of insulin (0 to 400 ng) was added and the incubation continued. Triplicate wells were used for each concentration of insulin. Fifty microliters of glyburide (to yield a final concentration of 2 pg/mL) or vehicle was also added both on the morning and late afternoon of day 2. On the morning of day 3, the cells were harvested, dissolved in 1N NaOH, sonicated, and aliquots

From the Department of Medicine Cedars-Sinai Medical Center-UCLA, Los Angeles. Supported by grants from the American Diabetes Association, Southern California AJiliate, and Upjohn Pharmaceutical Company. Address reprint requests to Mayer B. Davidson, MD. Division of Endocrinology. Room 1735. CedarsSinai Medical Center, 8700 Beverly Blvd., Los Angeles. CA 90048. D 1987 by Grune & Stratton, Inc. 00%0495/87/361~0002$03.00/0

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DAVIDSON AND SLADEN

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assayed for protein” and labeled glycogen as described previously. Net incorporation of glucose-‘4C into glycogen was calculated from the specific activity of the initial medium glucose. Glucose concentrations changed very little during the 24-hour period. Insulin Binding Insulin binding to these hepatocytes was also measured to determine if glyburide was enhancing insulin action by a binding or postbinding effect. For these studies, the medium was replaced with fresh DMEM (without fetal calf serum) on day 2 and the incubation continued. On day 3, the medium was aspirated and the cells incubated with 1 mL of fresh DMEM containing 3.3 mg/mL bacitracin 0.3 to 0.6 ng/mL “‘I-insulin, and in some experiments varying amounts of unlabeled insulin (0 to 10’ ng/mL) for three hours at 1YC. The medium was aspirated, the cell monolayer washed with phosphate-buffered saline, dissolved in 1N NaOH, and aliquots taken for counting in a gamma counter and for protein determination.‘* Duplicate wells were used for each concentration of insulin. The amount of ‘2sI-insulin bound in the presence of 10’ ng/mL was subtracted from total binding to yield specific binding. Pathways of Hepatic Glycogen Metabolism After the cells were isolated and suspended in DMEM supplemented with 10% fetal calf serum, 1 ml of the cell suspension was dispensed into each of 18 multiwell Petri dishes (Falcon) and cultured at 37OC in a CO, incubator. Half of the wells contained glyburide (2 pg/mL) and 12 of them (six with and six without the sulfonylurea agent) contained galactose-“C (0.1 &i/mL). The next morning (day 2), the media was removed along with the dead cells which do not attach to the wells, the cells were washed three times with DMEM without fetal calf serum, and 1 mL of this media was added to 12 wells for a four-hour incubation. The cells in six wells were harvested as zero time controls for the pathway of glycogenolysis. The glycogen pathways were assessed as follows. Note that although only nine wells were designated, an equal number were utilized in exactly the same way except they also contained glyburide (2 pg/mL). The sulfonylurea agent was added to these 9 wells during their initial incubation (day 1) as described above and also to the media for the four-hour incubation period at 37OC in the CO2 incubator on the morning of day 2. Wells 1-3 were not incubated for four hours. Glycogenolysis. Instead, the media and cells are scraped into 12 x 75 mm tubes, centrifuged, and the media aspirated off. One milliliter of NaOH was added, the cells were sonicated briefly and digested for 15 minutes at 37°C after which a lo-NL aliquot was taken for protein determination.‘* The cell digest was transferred to 15 mL conical glass stoppered centrifuge tubes with 2 1-mL washes of 95% ethanol. Radioactive glycogen was then measured as described previously.’ Therefore, wells l-3 served as the zero time control for the amount of rqioactive glycogen left in wells 4-6 after a four-hour incubation. The amount of radioactive glycogen and protein was then measured in the cells as described above. The degree of glycogenolysis was calculated as the percent of the zero time control normalized per mg of protein as follows: CPM in Wells (l-3) - CPM in Wells (4-6) x 1oo CPM in Wells (l-3) This value represents net glycogen breakdown since during the four-hour incubation period some glucose-“C is probably reincorporated into glycogen. Glycogen synthesis. Radioactive glycogen and protein were measured in the cells of wells 7-9 after a four-hour incubation in media to which glucose-‘4C (0.2 pCi/mL) had been added. Glycogen

synthesis is calculated from the specific activity of the glucose-‘% at the beginning of the incubation and expressed as nmol glucose incorporated into glycogen per mg protein. This represents net incorporation of glucose-C’4 into glycogen since during the four-hour incubation period some breakdown of radioactive glycogen back into glucose must occur. In addition, this value represents conversion of a radioactive precursor into its product rather than true glycogen synthesis. However, absolute glycogen levels in these cells are below the threshold of an extremely sensitive method both before and after incubation, and, therefore, glycogen synthesis per se could not be evaluated. Glycogen Synthase and Glycogen Phosphorylase Cells in which enzymes were measured were isolated and incubated in the same manner as those in which the glycogen pathways were assessed. At the end of the four-hour incubation on the second day, they were washed three times with ice cold phosphate buffered saline (pH 7.0). The cells were scraped along with the third wash into centrifuge tubes and the cell pellets frozen at - 70°C until assay. Measurement of the rate-limiting enzymes of glycogen synthesis and breakdown was performed by a method” utilizing Dowex columns to isolate labeled glycogen. The procedure for phosphorylase was carried out as published while with glycogen synthase, the concentrations of glucose-6-phosphate (G6P) and uridine diphosphoglucose (UDPG) were varied as discussed below. Results of the enzyme measurements were normalized per mg of protein,‘* which was measured in the assay mixture. Statistical Analysis Comparisons between glyburide-exposed and control cells were carried out by Student’s t-test for paired observations.” Significance was accepted at the .05 level (one-tailed since direction of change could be predicted). Therefore, the inherent variability of cultured cells from different rats is not a factor in this study since only paired observations were being compared. RESULTS

The effect of insulin on net glucose-i4C

incorporation

into

glycogen is shown in Fig 1. The greatest separation between low and high concentrations of insulin occurred when the cells were exposed to glucose-‘4C and varying amounts of the 361

+ ‘,%I 1.25

1

2.5

5

10

20

40

400

INSULIN (nghl)

Fig 1. Dose-response curves of insulin-stimulated net glucow-“C incorporation into glycogen in cultured rat hepetocytes incubated in the absence W-0) and presence (O----O) of glyburide (2 PgImL).

GLYBURIDE AND HEPATIC GLYCOGEN METABOLISM

Table 1. Effect of Glyburida (2 &mLI

927

Table 2. Effect of Glyburida (2 Ccg/mL) on Hapatic Glycogan

on Insulin-Stimulated

Metabolism in 12 Experiments

Nat Glucosa-“C Incorporation Into Glycogan in 12 Experiments (+ SE) Basal lnmd glucosa/mgprotein)

Glycogan Symhssis

Maximal Response’

E&a ulg/mL)

Glyccgandysis (% glycogen hvdrolvzed)

hlmd glincwpctatedinto glvccganl

Vehicle

13.6 k 3.1

170 f 29

15.2 f 3.3

Vehicle

41 f 5.5

Glyburide

14.1 k 2.8

181 f 42

6.4 r 1.1

Glyburide

25 k 8.7

8.7 + 0.7

Difference

0.5 + 1.1

11 * 38

6.8 -?r2.8

Difference

16 k 6

2.9 * 0.7

P

NS

NS

l[Peah value

<.025

5

1: 8 I

‘tJ

0

0

a

,

of Glycogen Synthasa and,Glycogan Phorphorylese in 8

I 0.6

I 0.4 AMOUNT

two ways. One is the activity ratio (percent active) which is the activity of the enzyme in the absence of G6P divided by the maximal activity. This approach utilizes supraphysiologic concentrations of G6P and UDPG in the assay system.15 (Physiologic levels of G6P and UDPG are 0.06 and 0.25 mmol/L, respectively.)” The form of the enzyme that is active in the absence of G6P is called the independent (I) form and the activity in the presence of G6P, the dependent (D) form. (The D form is the total activity minus the I form). Sodium sulfate is often included in the assay when activity ratios are measured since it inactivates the D form and stabilizes the I form of glycogen synthase.i6 It is the I form of the enzyme that is stimulated by insulin.” The published assay for glycogen synthase13 utilizes supraphysiologic concentrations of G6P (2 mmol/L) and UDPG (1 mmol/L). Glyburide did not affect the activity ratio of glycogen synthase measured under these conditions (Table 3). The second way to evaluate glycogen synthase is to measure the activity of the enzyme in the presence of varying concentrations of G6P (with no sodium sulfate added). When this was carried out with 1 mmol/L UDPG, glyburide (2 pg/mL) did not affect the dose-response curves generated in five experiments (data not shown). Two sets of nomenclature are used for hepatic glycogen synthase.” When enzyme activity is measured with physiologic concentrations of G6P and UDPG, the I and D form are called the a and b forms, respectively. Miller” demonstrated no insulin effect on glycogen synthase activity when measured at supraphysiologic concentrations of UDPG (6.7 mmol/L) but a stimulation when assayed at physiologic levels (0.2 mmol/L) in freshly isolated hepatocytes incubated for two hours. Likewise, in our cultured hepatocytes, glycogen synthase activity when measured with physiologic concentrations of G6P and UDPG was significantly (P < .05) increased by glyburide but was unaffected by the sulfonylurea agent when supraphysiologic concentrations were used in the assay (Table 4). Furthermore, glyburide significantly stimulated glycogen synthase activity when

BOUND

Experiments

a0 0

a 0.2


Table 3. Effect of Glyburide (2pglmL) on the Activity Ratios f SE

a0

1-


P

Results are expressed per mg protein (mean f SE).

- basal value/basal value] x 100

hormone over a 24-hour period rather than eight hours or less. The dose-response relationship is analyzed in Table 1. There was no significant difference in basal incorporation (ie, in the absence of insulin) under these experimental conditions. Similarly, glyburide did not affect the maximal insulin response over basal. The dose-response curve, however, was significantly shifted to the left when the cells were exposed to glyburide signifying increased sensitivity to insulin. The sulfonylurea agent had no effect on the amount of protein in the wells (data not shown). When glyburide was added to the wells only on the morning but not in the late afternoon of day 2, the drug had no effect (data not shown). The enhancing effect of glyburide on insulin-stimulated glucose-‘4C incorporation into glycogen occurred in the absence of any changes in hormonal binding. Specific tracer binding in 23 experiments was similar in cells exposed to the vehicle (5.1% per mg protein) and those exposed to the sulfonylurea agent (5.3% per mg protein) yielding a difference k SE of only 0.2 2 0.1. Full competitive binding curves were carried out in 12 experiments. The results, depicted as Scatchard plots in Fig 2, are similar in the presence and absence of the sulfonylurea agent. Three to 5% of galactose-i4C was converted to glycogen14Con the morning of day 2. The effect of glyburide on the pathways of glycogenolysis and glycogen synthesis are shown in Table 2. The sulfonylurea agent significantly inhibited the breakdown of glycogen and increased the incorporation of glucose-“C into glycogen. Glycogen synthase activity is usually expressed in one of 6

5.8 + 0.6

(ngfmg

I

0.8

r

1.0

GlvcogenSvnthass (%I

/+I 1s

PROTEIN)

Scatcherd analysis of insulin binding in cultured rat Fig 2. hapstocytas incubated in the absence [email protected]) and presence (0) of glyburide I2 Ag/miJ.

Vehicle

34.9

f 3.3

53.3

f 2.5

Glvburide

35.0

+ 2.8

52.9

+ 2.3

Difference P

0.1 f 0.05

0.4 f 0.4

NS

NS

DAVIDSON AND SLADEN

929

Table 4. Effect of Glyburide (2 ag/mL)

on Glycogen Synthase

Activitv* N

9

5

G6P (mmol/L)

0.06

2.0

UDPG (mmol/L)

0.2

1.0

Vehicle

67.4

+ 14.6

177.5

Gwuride

60.3

f

231.0

* 61.5

Difference P

12.9

53.5

* 35.5

lpmol/min/mg

15.6

* 5.7 <.05

* 37.0

NS

protein + SE.

measured at supraphysiologic levels of G6P as long as physiologic concentrations of UDPG were used (Fig 3). Glycogen phosphorylase is usually expressed as the activity ratio (percent active), which is the ratio of phosphorylase a (measured in the presence of caffeine) to phosphorylase b (measured in the presence of adenosine monophosphate).” Glyburide did not affect glycogen phosphorylase in these cultured hepatocytes (Table 3). Since the amount of glucose14Cin glycogen is a result of the balance between glycogen synthesis and breakdown, it is likely that the inhibition of glycogenolysis by the drug simply reflects an enhanced incorporation of g1ucose-‘4C into glycogen during the period of incubation. DISCUSSION

Glyburide enhanced the insulin effect on net glucose-‘4C incorporation into glycogen by significantly increasing the sensitivity (ie, shift of the insulin dose-response curve to the left) without affecting responsiveness (ie, maximal increase over basal) (Table 1, Fig 1). It should be pointed out that this enhanced sensitivity occurred with concentrations of insulin normally present in the portal vein attesting to the physiologic significance of these results. This action of insulin was noted in the absence of any effect of the sulfonylurea agent on insulin binding (Fig 2). These results indicate that the site of action of the sulfonylurea agent lies beyond the binding step, ie, the drug exerts a postbinding effect. There have been a few examples of a postreceptor insulin antagonistic defect 500

.g 400 aa ij b cn 300 E 2 E ” >0

200

1 1

2

(n = 9 Experiments)

3

4

GLUCOSE-6-PHOSPHATE

6

5

10

(mM)

Fig 3. Dose-response curves of glycogen synthase activity in cultured hepatocytes incubated in the absence (G--G) and presence (O----O) of glyburide (2 pg/mL). The enzyme was assayed with 0.2 mmol/L UDPG and varying concentrations of G6P. For vehicle vglyburide et 0.6, 1,2, 4. 6. end 10 mmol/L G6P. P =z .05, .06. .026. .05. .Ol, .025. respectively.

signified by decreased sensitivity (ie, a rightward shift of the insulin dose-response curve) without a change in maximal responsiveness.” Conversely, these data indicate that a postreceptor potentiation of insulin action can be manifested by increased sensitivity only. The enhancement by glyburide of insulin-stimulated net glucose-‘4C incorporation into glycogen in these cultured hepatocytes confirms reports in which sulfonylurea agents potentiated the actions of insulin in vivo.‘-4*‘9-23 The effect of sulfonylurea agents on insulin binding is more controversial. When these drugs were administered in vivo and tissue was subsequently harvested for the measurement of insulin bindof studies reported that it was ing, a number increased.2*2’~22~32~33 However, a substantial number of studies reported no change in insulin binding in this situation.3*23.3’3* Furthermore, when sulfonylurea agents were added to tissues in vitro, they did not affect insulin binding in this (Fig 2) and in most studies.‘0V29933*39~3 In the few in which insulin binding did increase?30v”“5 changes were small (12% to 44%). It should be emphasized that in many instances in which insulin binding was unaffected by sulfonylurea agents, either diabetic control was significantly improved2*3*37or insulinmediated metabolism was enhanced9-“*@ (Fig 1). We interpret all of these data to show that sulfonylurea agents work at a postbinding site. The increased insulin binding noted in some studies after in vivo administration of sulfonylurea agents must be an epiphenomenon not related to the primary mode of action of these drugs. The conclusion that these agents potentiate the action of insulin at a postbinding step is supported by the direct effect (in the absence of insulin) of glyburide on the glycogen pathways in these cultured hepatocytes (Table 2). That is, these drugs may affect an intracellular site or sites that enhance carbohydrate metabolism unrelated to the specific actions of insulin. There have been other attempts to determine if sulfonylurea agents can affect hepatic carbohydrate metabolism directly with mixed results. Some studies failed to demonstrate a direct effect in liver,‘0~“~M~48 while other data showed that sulfonylurea agents do affect hepatic carbohydrate metabolism in the absence of insulin.b54 Studies in which a direct effect of sulfonylurea agents on hepatic carbohydrate metabolism could be demonstratedG54 were all carried out for several hours only. Experiments performed over many hours in cultured hepatocytes could show no direct effect.“” This is consistent with our inability to demonstrate a direct effect of glyburide over a 24-hour period (Table 1) whereas we could over a four-hour period (Table 2). Since hepatocytes can autoregulate glucose production by changes in the glycogen pathwayq5’ perhaps this process occurring over many hours reversed the direct effect of glyburide noted after four hours. The glyburide stimulation of glycogen synthase in these cultured hepatocytes (Table 4, Fig 3) suggests that dephosphorylation reactions may be involved in the mechanism of sulfonylurea agent action since the enzyme is activated by glycogen synthase phosphatase. Activation of heart pyruvate dehydrogenases6 and adipocyte glycogen synthase” by sulfonylurea agents also points toward a dephosphorylation mechanism. Although the activation of adipocyte glycogen syn-

GLYBURIDE

AND HEPATIC GLYCOGEN

METABOLISM

929

thase was postulated to be secondary to enhanced glucose transport,” sulfonylurea agents did not affect glucose transport in adipocytes.40*58,59 Thus, glycogen synthase is probably stimulated directly by sulfonylurea agents in both adipose and hepatic tissue since in the latter glucose transport is not a rate-limiting step in carbohydrate metabolism. The activation by glyburide of hepatic glycogen synthase could be demonstrated when the enzyme was measured in the presence of physiologic concentrations (0.2 mmol/L) (Table 4, Fig 3) but not supraphysiologic levels (1.0 mmol/L) of UDPG. The stimulation of adipocyte glycogen synthase also

occurred when the enzyme was assayed with 0.2 mmol/L UDPG.” Taken all together, these data suggest that the sulfonyiurea agents may act at a subcellular ievel by affecting one or several of the complex phosphorylation-dephosphorylation reactions although their exact mechanism of action remains far from clear. ACKNOWLEDGMENT

We gratefully acknowledge the excellent technical assistance of Robert G. Karjala and the secretarial support of Margo E. Black-

burn.

REFERENCES

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