Increased insulin action in cultured hepatocytes from rats with diabetes induced by neonatal streptozotocin.

0013-7227/91/1284-1693$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 128, No. 4 Printed in U.S.A.

Increased Insulin Action in Cultured Hepatocytes from Rats with Diabetes Induced by Neonatal Streptozotocin* BRIGITTE MELIN, MARTINE CARON, GISELE CHERQUI, MARIE-JOSE BLIVET, DANIELLE BAILBE, JACQUES PICARD, JACQUELINE CAPEAU, AND BERNARD PORTHA Laboratoire de Biologie Cellulaire (B.M., M.C., G.C., M.-J.B., J.P., J.C.), INSERM U. 181, Faculte de Medecine Saint-Antoine, 75571 Paris Cedex 12, France; and Laboratoire de Physiologie du Deueloppement (D.B., B.P.), CNRS URA 307, Universite Paris 7, Tour 33, 75251 Paris Cedex 05, France

larly, insulin action on glucose incorporation into glycogen, lipogenesis, and amino acid transport were enhanced in diabetic hepatocytes. The hormone effect was manifested by an increase in the sensitivity and/or in the responsiveness, reflecting the multiplicity of the pathways whereby the insulin signal is transduced through the insulin receptor to multiple postreceptor sites. To gain insight into the possible mechanism of these disturbances, we evaluated the initial insulin receptor interaction and the kinase activity of the receptor /3-subunit. In accordance with our previous study on intact livers, we found no alteration in either of these parameters in nO-STZ rat hepatocytes. Thus, the present study clearly demonstrates that these diabetic rats exhibit a postreceptor hyperresponsiveness to insulin at the cellular level. It strengthens the notion that a /?-cell deficiency with glucose intolerance does not necessarily lead to a hepatic insulin resistance. (Endocrinology 128: 1693-1701,1991)

ABSTRACT. Previous studies have shown that Wistar rats injected at birth (nO) with STZ (nO-STZ) develop as adults a noninsulin-dependent diabetic state characterized by a lack of insulin response to glucose in vivo, a mild basal hyperglycemia, and an impaired glucose tolerance. Our former in vivo studies using the insulin-glucose clamp technique revealed an increased insulin action upon hepatic glucose production in these animals. We have now cultured hepatocytes from these mildly diabetic rats in parallel with hepatocytes from control rats, to examine more closely basal and insulin-regulated glucose production and glucose incorporation into glycogen. In addition, we extended our investigation to other hepatic functions such as lipid synthesis and amino acid transport, which could not be studied in vivo. Although glucose production from glycogenolysis or gluconeogenesis in absence or presence of glucagon was identical in the two cell populations, glucagon-stimulated glycogenolysis was more sensitive to insulin action in diabetic hepatocytes. Simi-

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WO DEFECTS, insulin deficiency and insulin resistance, usually coexist in overt non-insulin-dependent diabetes mellitus (NIDDM), because they are interrelated at multiple levels (1). To help gain information concerning the pathogenesis of these defects, animal models of NIDDM have been developed (for review see Refs. 2, 3). Among them, the experimental rat model of NIDDM resulting from the spontaneous evolution of streptozotocin-induced neonatal diabetes (nOSTZ model) was first developed by Portha et al. (4). This model and some of its variations developed by other groups (5, 6) have received considerable attention during the last 10 yr, because their physiopathology evokes the early stages of the human disease with an insulin secretory abnormality in response to glucose, a moderate Received August 16,1990. Address all correspondence and requests for reprints to: Dr. Jacqueline Capeau, Laboratoire de Biologie Cellulaire, INSERM U. 181, Faculte de Medecine Saint-Antoine, 27, rue Chaligny, 75571 Paris Cedex 12, France. * This work was supported by grants from INSERM (CRE no. 874013) and from the Universite Pierre et Marie Curie (Paris, France).

elevation of basal plasma glucose value, and an impaired glucose tolerance (7, 8). Thus, such experimental models represented valuable tools to study the alterations in /3cell function and the relationship between insulin deficiency and insulin action. Recent investigations attempted to address the question of whether a primary pancreatic failure invariably led to insulin resistance. Using exogenous insulin to measure glucose kinetics or insulin glucose clamp techniques to evaluate insulin action, studies conducted in vivo in the nO-STZ rat model (injected with STZ on the day of birth) have shown that basal hepatic glucose production was abnormally high, an observation that evoked a hepatic insulin resistance, whereas insulin action on the pathways of glucose production was enhanced, an observation which rather suggested a hypersensitivity to insulin (9, 10). This raised the question of whether this dysregulation of glucose metabolism manifested in vivo in these diabetic rats reflects an intrinsic defect of their liver cells, which would also affect other major hepatic functions. To address this issue, we used hepatocytes cultured on a fibronectin

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INCREASED INSULIN ACTION IN DIABETIC RATS

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matrix, a model that proved valuable to accurately examine, in the absence of serum, the influence of physiological or pharmacological stimuli on liver metabolism (11, 12). Thus, we compared basal and insulin-regulated glucose production as well as glucose incorporation into glycogen in parallel cultures from control and diabetic rats. We extended our investigation to lipid synthesis and amino acid transport, two metabolic pathways that could not be approached in in vivo studies. Our results show that hepatocytes derived from nO-STZ diabetic rats exhibit in vitro not only an increased sensitivity to insulin for glucose production, in accordance with the in vivo observations, but also an increased response to the hormone for other metabolic pathways. Their hyperresponsive state was independent of alterations in the binding of insulin or in the kinase activity of its receptors as compared to control hepatocytes. Materials and Methods The nO-STZ rat model On the day of birth (nO), Wistar pups were injected ip with 100 mg/kg STZ diluted in citrate buffer (0.05 M, pH 4.5), whereas control pups received the solvent only. Control and nO-STZ pups were weaned 28 days after birth, and were then fed standard laboratory chow ad libitum. Spontaneous evolution of this treatment led to a noninsulin-dependent diabetic (NIDD) state in the adult, which was stable and chronic, as previously described (7). Isolation and incubation of hepatocytes Hepatocytes were isolated from control and nO-STZ adult female rats between 3-5 months of age by the collagenase perfusion method, as described previously (11). Microscopic examination of these hepatocytes before plating revealed a high index (>95%) of cell viability in both control and diabetic rats, as assessed by Trypan blue exclusion. There was no obvious difference in the size of hepatocytes from diabetic rats as compared to those from control rats, and the cellular DNA content was the same in both cell populations (4.7 ± 0.4 and 4.5 ± 0.5 ng DNA/106 hepatocytes in control and diabetic, respectively). However, the yield of the cells was consistently greater for the diabetic livers (129 ± 8%) as compared to control rat livers. Hepatocytes from both animals were suspended at a final concentration of 106 cells/ml in Dulbecco's Modified Eagle's (DME) medium containing penicillin (50 Mg/nil), streptomycin (50 U/ml), NaHCO3 (12 mM), and 5.5 mM glucose unless otherwise indicated. They were then plated onto fibronectinprecoated dishes (10 ^g/dish) at a concentration averaging 2 x 106 cells/35 mm dishes. The advantage of this method is that neither insulin nor serum is required for adhesion and spreading of the cells. Three hours after plating, the initial medium was changed at 37 C to remove cellular debris and loosely adherent cell aggregates. The adherent cells were subsequently cultured in the same medium for 16 h, unless otherwise specified. The

Endo • 1991 Voll28«No4

hepatocytes were then washed again at room temperature and treated according to each procedure. Glucose production To study glucose production from glycogenolysis, hepatocytes from 30-h fasted rats were preincubated with [U-14C} glucose (3 /tCi/ml, 10 /XM) in insulin-free DME medium containing 25 mM glucose for 20 h. The cells were then washed 3 times with DME medium at 37 C. Some dishes were stopped at that step by washing the cells with ice-cold PBS and adding 0.5 ml 20% KOH for cell solubilization. To measure radioactive glucose incorporation into glycogen, glycogen was extracted and ethanol precipitated as described previously (12, 13). The precipitate was solubilized in 200 fil water, and its radioactivity was counted in 10 ml of a toluene scintillant (ACS, Amersham). This value was used as the zero time control for the amount of radioactive glycogen accumulated during this preincubation period. The other prelabeled dishes were incubated for 30 min in the absence or in the presence of 0.5 nM glucagon with or without increasing concentrations of insulin (0-100 ng/ml) in 5.5 mM glucose DME medium. They were then treated as above to measure the residual amount of radioactive glycogen. The degree of net glycogenolysis was calculated from the difference between radioactive glycogen measured before and after 30 min incubation. Values were expressed as nanomoles of glucose from glycogen per mg cell protein/h. Glucose production through gluconeogenesis was assessed in 30-h fasted rats by incubating hepatocytes for 1 h at 37 C in the absence and in the presence of 10 mM pyruvate as a gluconeogenic precursor in HEPES-bicarbonate buffer (4.2 mM HEPES, 12 mM NaHCO3, 5.4 mM KC1, 1.3 mM CaCl2, 0.4 mM KH2PO4, 1 mM MgSO4, 0.3 mM Na2HPO4, 140 mM NaCl, 5.5 mM glucose, and 0.1% BSA, pH 7.4). The amount of glucose in the buffer was assayed, after deproteinization with perchloric acid, by a glucose oxidase method (Kit 716251, Boehringer Mannheim, Meylan, France). The difference in glucose production in the presence and in the absence of pyruvate represented net pyruvate-mediated gluconeogenesis in these cells. It was expressed as nanomoles of glucose formed per mg cell protein/ h. Glucose incorporation into glycogen For this determination, the cells from fed animals were preincubated overnight in 5.5 mM glucose. Glucose incorporation into glycogen was then estimated by measuring the level of [U-14C]glucose incorporated into cellular glycogen after 1 h at 37 C. Hepatocyte monolayers from fed animals were first incubated for 15 min at 37 C in 1 ml DME medium containing 25 mM glucose with or without increasing concentrations of insulin (1-100 ng/ml). Then, [U-14C]glucose (1.3 MCi/ml) was added for 1 h. The incorporation was stopped by rapidly washing the cells with ice-cold PBS. The cells were solubilized in 0.5 ml 20% KOH. Aliquots were removed for protein determination. Glycogen was extracted, ethanol precipitated, solubilized, and counted as described above. Data are presented as nanomoles of glucose incorporated into glycogen per mg cell protein/h.


INCREASED INSULIN ACTION IN DIABETIC RATS Lipid synthesis To assess lipogenesis, the hepatocyte monolayers from fed animals were incubated in 25 mM glucose DME medium throughout the study. Increasing concentrations of insulin (0100 ng/dish in 1 ml medium) were added for 30 min at 37 C, before the addition of [1-14C]acetate (0.4 mM, 2 /iCi/ml). The incubation continued for an additional 2 h. The plates were then washed three times with ice-cold PBS, the cells scraped in 1 ml water and transferred into ice-cold scintillation vials, following the procedure described by Moody et al. (14), to be counted as described previously (12). Data are given as nanomoles of acetate incorporated into total lipids per mg cell protein/2 h. Amino acid transport The effect of insulin on amino acid transport was measured by using a-aminoisobutyric acid (AIB), a nonmetabolizable analog of alanine (15). Hepatocytes isolated from fed animals were preincubated for 3 h at 37 C in the HEPES-bicarbonate buffer used above in the absence or the presence of increasing insulin concentrations (1-100 ng/ml). The rate of transport was measured after 10 min at 37 C in the presence of [14C]AIB (0.22 AtCi/ml) at a final concentration of 75 nM AIB. The reaction was stopped by washing the cells three times with icecold PBS. The cells were solubilized in 20% KOH, and aliquots were removed for protein determination. The radioactivity was then determined in 10 ml of a toluene scintillant (ACS, Amersham). Extracellular trapping and nonspecific transport of AIB were estimated by measuring AIB uptake at 4 C. This value was subtracted from each sample. The data are presented as picomoles of AIB transported per mg cell protein/min. Insulin binding Suspensions of control and diabetic hepatocytes (2 x 105 cells/0.25 ml) were incubated (15 h at 4 C) with A14-[125I] insulin (100 pM) in the absence or the presence of increasing concentrations (0, 1, 5, 10, 100, 500 ng/0.25 ml) of native insulin. The binding buffer was bicarbonate-HEPES buffer containing 10 mM glucose and 1% albumine (vol/vol) at pH 7.65. All data were corrected for nonspecific [125I]insulin binding measured in the presence of an excess (2 jtg/0.25 ml) of unlabeled insulin. After three extensive washings in Krebs Ringer phosphate buffer 0.1% BSA, the cell pellet was counted. Specific binding is expressed as picograms of insulin bound per mg cell protein. Tyrosine kinase activity The insulin receptor-mediated tyrosine kinase activity of control and diabetic hepatocytes was assayed on an exogenous substrate, as described by Kasuga et al. (16). Briefly, wheat germ-purified receptors (2 ng protein) were first incubated without or with insulin for 16 h at 4 C, and then phosphorylated in the presence of 2 mM MnCl2, 15 mM MgCl2, 48 /xM cold ATP, 2 MM [7-32P]ATP for 10 min at 4 C (in a final vol of 100 /A). The synthetic substrate [poly(GluNa, Tyr), 10 mg/ml] was then added for 20 min at 4 C. After addition of 20 ^1 of a stopping solution, aliquots of 50 /x\ were spotted on phosphocellulose paper squares (1 X 1 cm), which were then extensively

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washed for 24 h with 75 mM phosphoric acid. Their radioactivity was then determined by the Cerenkov radiation method. [7-32P]ATP trapping into control squares was subtracted from each value. Other analyses Glycogen concentration was determined after precipitation by ethanol as described above and enzymatic hydrolysis by the method of Roehrig and Allred (17). Glucose was assayed by a glucose oxidase method (Kit 716251, Boehringer Mannheim France SA). The data are expressed as nanomoles of glycogen glucose per mg cellular protein. The total amount of cellular protein per dish was measured by the method of Lowry et al. (18), and the DNA content by a fluorometric procedure, as described by Labarca and Paigen (19). Hepatocyte triglycerides were measured by an enzymatic method (Kit 125032, Boehringer Mannheim France SA). Results are given as the means ± SEM for the indicated numbers of independently performed experiments. Differences between the mean values were evaluated by Student's t test.

Results Characteristics of the rats and cellular parameters The mean values for basal plasma glucose and insulin levels in 3-5-month-old control and nO-STZ female rats in the nonfasted state were 136 ± 2 and 214 ± 13 mg/dl for glucose (P < 0.01), and 95 ± 9 and 85 ± 13 /iU/ml for insulin (NS), respectively (n = 8). Thus, diabetic animals exhibited moderate nonfasting hyperglycemia, and their insulin level was slightly lower than that of control animals. Some characteristics of the hepatocytes isolated from the experimental animals are presented in Table 1. DNA, protein, and triglyceride contents of the hepatocytes were not significantly affected by the diabetic state of the animals. However, in the hepatocytes of the nO-STZ animals, a significant decrease in glycogen content could be observed. Glucose production Increased glucose output is a major metabolic disturbance that accounts for fasting hyperglycemia in NIDDM. TABLE 1. Some biochemical characteristics of hepatocytes from control and nO-STZ diabetic rats Content Protein (mg/106 hepatocytes) DNA (/Ltg/mg protein) Triglycerides (Mg/mg protein) Glycogen (jig/mg protein)

Control

Diabetic

0.72 ± 0.05

0.76 ± 0.05

6.5 ± 0.1 138.6 ± 26.6

5.9 ± 0.1 113.1 ± 6.0

103.2 ± 8.2

70.0 ± 8.6°

Results are expressed as mean ± SEM of hepatocytes from five to eight separate preparations. 0 Significantly different from control: P < 0.05.


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This enhanced hepatic glucose production could result from glycogenolysis, gluconeogenesis, or a combination of both processes. We have carried out experiments in hepatocytes from both control and diabetic rats in a fasting state, to evaluate basal glucose output through both metabolic pathways. The measurement of glycogenolysis was assessed in hepatocytes from 30-h fasted control and diabetic rats, after repletion of their glycogen stores in vitro by an overnight incubation with [14C]glucose in 25 mM glucose medium. The cells were then washed and incubated for 30 min in fresh medium containing 5.5 mM glucose. The difference in the amount of labeled glycogen measured before and after this last incubation represented the extent of net basal glycogen breakdown. From the data presented in Table 2, it appears that basal

glycogenolysis was similar in control and diabetic hepatocytes. Basal glucose output resulting from gluconeogenesis was assessed in glycogen-depleted hepatocytes from 30h fasted rats, by measuring glucose production in the presence of 10 mM pyruvate for 1 h in the absence of other precursors interfering in the pathway. As demonstrated by Katz (20), no valid measure of gluconeogenesis can be obtained by measuring the incorporation of labeled three-carbon precursors into glucose, because of obvious carbon exchanges in the Krebs cycle. The difference in glucose production in the absence and in the presence of pyruvate as a gluconeogenic substrate could thus provide a good estimation of this pathway. Similarly, it was not altered in diabetic cells (Table 2). Thus, the data for glucose production in the basal state by hepatocytes from fasted animals through either glycogenolysis or gluconeogenesis did not reveal any significant difference in cells from diabetic rats as compared to cells from control rats. We next examined these two processes after an acute treatment with insulin. Preliminary experiments indicated that insulin could not bring about any modification in the basal amount of glucose produced through either glycogenolysis and gluconeogenesis (data not shown). Thus, to enhance the sensitivity of the cells to insulin TABLE 2. Basal glucose production through glycogenolysis and gluconeogenesis by control and diabetic hepatocytes

Control Diabetic

Glycogenolysis (nmol glucose/mg cell protein • h)

Glucose from 10 mM pyruvate (nmol glucose/mg cell protein • h)

118 ± 4 119 ± 9

96 ± 8 104 ± 8

Control and diabetic rats were fasted for 30 h, and hepatocytes were then isolated. Basal glycogenolysis and glucose production from pyruvate were measured as described in Materials and Methods. Data are given as the mean ± SEM of six experiments.

Endo•1991 Voll28«No4

action, the pathways of glucose production were stimulated by glucagon. Indeed, in vivo, glucagon is a major controller of hepatic glucose output, and its action is primarily antagonized by insulin. Glucagon (0.5 nM) stimulated basal hepatic glucose production through either glycogenolysis or gluconeogenesis by about 2-fold in control hepatocytes (213 ± 21% and 213 ± 14% of the basal values for glycogenolysis and gluconeogenesis, respectively). The action of glucagon was similar in diabetic hepatocytes (191 ± 14% on glycogenolysis, 205 ± 19% on gluconeogenesis). In our experimental conditions, the cellular glycogen stores were repleted with labeled glucose. Since the stimulation of glucose production from glycogen by glucagon is known to account for 75% of hepatic glucose production after an overnight fast (21), we thus focused on the ability of insulin to antagonize the pathway of glycogenolysis. Figure 1A shows that in both cell populations insulin partially inhibited, in a dose-dependent fashion, glycogen breakdown triggered by 0.5 nM glucagon. The ED50 in control cells was 0.7 nM insulin. In diabetic cells, the maximal inhibition elicited by insulin was similar to that measured in control cells, i.e. about 50% of the maximal glucagon stimulation. However, the curve was significantly shifted to the left, thus indicating an increased sensitivity to insulin in these diabetic hepatocytes (ED50 = 0.1 nM). Glucose incorporation into glycogen

To examine whether the above observations reflected a general effect of insulin on glucose metabolism, we next studied insulin action on [14C] glucose incorporation into glycogen. Basal glucose incorporation was essentially similar in both groups of cells (25.6 ± 6.4 and 24.7 ± 4.2 nmol glucose incorporated into glycogen/mg cell protein • h in control and diabetic cells, respectively). However, as depicted in Fig. IB, insulin-stimulated [14C]glucose incorporation into glycogen was significantly increased in diabetic cells at concentrations ranging from 5-100 ng/ml (30% and 50% over control cells at 5 ng and 100 ng, respectively), this latter concentration giving consistently the maximal response. Thus, whereas diabetic cells showed a greater responsiveness to insulin, the sensitivity to the hormone was the same (0.8 nM) in both cells. Lipid synthesis and AIB uptake To further investigate whether other metabolic pathways of insulin action are also modified in diabetic hepatocytes, we next evaluated the effect of insulin on lipid and protein metabolism. Lipid synthesis was studied over a 2-h period by the incorporation of [l-14C]acetate into lipids accumulating in hepatocytes exposed to various insulin concentrations



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incorporated/mg cell protein-2 h in diabetics). Insulin stimulated lipogenesis in a dose-dependent manner with a maximal effect at 50 ng/ml. As shown in Fig. 2, there was a tendency for nO-STZ hepatocytes to be more responsive to insulin than control hepatocytes, but this effect was not statistically significant. The sensitivity to insulin was similar in both cell populations (0.7 nM). We also assessed insulin action on protein metabolism in hepatocytes by studying AIB uptake, a function requiring a longer incubation to allow the transcriptional activation of new amino acid transporters. Insulin is well known to stimulate this process (22). This assay was performed in parallel dishes of control and diabetic cells after a 3-h incubation with increasing insulin concentrations. Figure 3 shows that, in both cell populations, insulin stimulated AIB uptake in a dose-dependent manner with the maximal effect occurring at 50 ng/ml. However, whereas the basal uptake of the amino acid was not significantly altered, diabetic hepatocytes were clearly more responsive to insulin than control hepatocytes (mean increment in diabetic cells averaging 90% over control cells for insulin concentrations ranging between 1-10 ng/ml) and also more sensitive to the hormone (ED50 was 0.3 nM in diabetic vs. 0.8 nM in control cells).

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Insulin binding and kinase activity of the insulin receptor in control and nO-STZ rat hepatocytes cultured in vitro

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insulin (ng/ml) FlG. 1. Insulin action on glucose metabolism in control and nO-STZdiabetic rat cultured hepatocytes. A, Inhibition of glucagon-induced glycogenolysis by insulin in control (•) and nO-STZ-diabetic (•) rat cultured hepatocytes. [14C]Glucose prelabeled hepatocytes were washed and incubated in the presence of glucagon (0.5 nM) with or without increasing insulin concentrations (0-100 ng/ml) in DME medium for 30 min, as indicated in Materials and Methods. Each point represents the mean ± SEM of four experiments performed in triplicate. Significant differences between diabetic and control cells are indicated; **, P < 0.02, ***, P < 0.01 B, Stimulation of glycogenesis by insulin in control (•) and nO-STZ-diabetic (O) rat cultured hepatocytes. Cultured hepatocytes were preincubated for 15 min at 37 C in DME medium in the absence (basal) or the presence of increasing insulin concentrations (0-100 ng/ml). [14C]Glucose was then added for 1 h. The assay for glycogenesis was conducted as indicated in Materials and Methods. The basal values of glycogenesis were 25.6 ± 6.4 and 24.7 ± 4.2 nmol glucose incorporated into glycogen/mg protein • h for control and diabetic hepatocytes, respectively. Each point represents the mean ± SEM of four to six experiments performed in duplicate. Values significantly different from the respective control values are indicated: **, P < 0.02; ***, P < 0.01.

(1-100 ng/ml). Basal lipogenesis was slightly but not significantly decreased in diabetic hepatocytes (16.7 ± 1.6 nmol in controls vs. 13.7 ± 1.7 nmol of acetate

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FlG. 2. Insulin stimulation of lipid synthesis in hepatocyte cultures from control (•) and nO-STZ-diabetic (O) rats. Hepatocytes were preincubated for 30 min at 37 C without (basal) and with increasing insulin concentrations before the addition of [14C]acetate for a final incubation of 2 h, as indicated in Materials and Methods. The basal values (100%) were 16.7 ± 1.6 and 13.7 ± 1.7 nmol of [14C]acetate incorporated into lipids/2 h in control and diabetic hepatocytes, respectively. Each point represents the mean ± SEM of four to six experiments performed in duplicate. Values significantly different from the respective control values are indicated: ***, P < 0.01.



1698

Endo• 1991 Voll28»No4

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insulin (ng/ml) FIG. 3. Insulin stimulation of amino acid transport in control (•) and nO-STZ-diabetic (O) rat cultured hepatocytes. Monolayers were incubated in the absence or the presence of increasing concentrations of insulin for 3 h at 37 C. Amino acid transport was then evaluated in the presence of [NC]AIB at a final concentration of 75 MM for 10 min at 37 C. Basal transport was 48 ± 3 and 41 ± 3 pmol of AIB/mg proteinmin in control and diabetic hepatocytes, respectively. Each point represents the mean ± SEM of four to six experiments performed in duplicate. Values significantly different from the respective control values are indicated: *,P< 0.05; ***, P < 0.01; ****, P < 0.001.

the insulin binding and kinase activity of the insulin receptors in diabetic rats as compared to control rats (23). Because of possible influences due to the environment to which the cells were exposed in vivo as compared with their in vitro culture conditions, we reinvestigated these two metabolic parameters on hepatocytes cultured for 20 h. The specific binding of insulin was measured on suspensions from control and diabetic hepatocytes, as described in Materials and Methods. Neither the binding of a tracer amount (100 pM) of [125I]insulin (16.3 ± 2.4 pg/mg and 16.2 ±1.9 pg/mg cell protein in control and diabetic hepatocytes, respectively) nor the concentration of insulin that displaced 50% of the bound hormone, an index of receptor affinity (3.0 ± 1.1 nM and 3.0 ± 0.8 nM in control and diabetic cells, respectively) was altered by the diabetic state (Fig. 4). We also checked the insulin receptor kinase activity by assaying the ability of purified receptor preparations to phosphorylate an exogenous protein substrate, the synthetic polymer poly(GluNa,Tyr) on tyrosine residues. Insulin receptors purified from control and diabetic cultured hepatocytes phosphorylated this substrate; basal phosphorylation was 0.42 ±0.1 pmol/mg and 0.30 ± 0.05 pmol/mg substrate in control and diabetic cells, respectively. Insulin (5 and 50 nM) induced a dose-dependent increase in this process, which was similar in both preparations (172% and 213% of the basal level in control us. 177% and 225% in diabetic, n = 2). Thus, these results

1000

native insulin (ng/incubation) FIG. 4. Competition-inhibition curve of specific [125I]insulin binding to control and nOSTZ-diabetic rat hepatocytes. Control ( • •) and diabetic (O O) hepatocytes were incubated overnight at 4 C with a tracer concentration (100 pM) of the radioligand in the absence (100%) or the presence of increasing concentrations of native hormone, as described in Materials and Methods. Each point represents the mean ± SEM of four separate experiments.

obtained in cultured hepatocytes are in accordance with the studies previously carried out on intact livers of these animals (23) and confirm that neither insulin binding nor insulin receptor kinase activity were altered by the diabetic state in the nO-STZ rat model.

Discussion Our previous in vivo studies showed that nO-STZ diabetic rats exhibit an elevated basal glucose production, an observation that may reflect a hepatic insulin resistance, while displaying an increased response to insulin on the pathways of glucose production, an observation that suggests a hypersensitivity to insulin. These apparent discrepancies led us to investigate: 1) the degree of alteration of the hepatic response to insulin for glucose metabolism; and 2) the response to insulin of other major hepatic functions, so as to evaluate the extent of the hepatic disturbances. The present study was carried out on cultured hepatocytes from nO-STZ diabetic and control rats handled in parallel. All diabetic rats were given STZ on the day of birth and were used as adults between 3-5 months of age. They all displayed glucose intolerance and moderate hyperglycemia. The diabetic state did not alter their growth rate nor their average weight. Moreover, the ability of the hepatocytes to sustain the culture conditions was not different for the diabetic animals as compared to control animals. Our present results provide evidence that cultured hepatocytes from these mildly diabetic rats display an enhanced response to insulin in vitro not only for glucose metabolism, but also for other metabolic pathways. This enhanced response is unlikely to result from decreased



insulin degradation in hepatocytes from nO-STZ rats, as the degradation rate of the hormone was previously shown to be similar to that measured in cells from control rats (24). It rather reflects a frank intrinsic defect of hepatic functions at the cellular level. To optimize the physiological conditions for the study of glycogenolysis and gluconeogenesis in vitro, rats were kept in a fasted state before hepatocyte isolation. In the absence of hormonal and neuroendocrine factors, glucose level per se is known to be a regulator of its own production by the liver; i.e. glucose production is an inverse function of the ambient plasma glucose concentration, a phenomenon that has been referred to as hepatic autoregulation (25, 26). Increasing glucose concentration was shown to inhibit glucose release from hepatocyte cultures and perfused liver (27). The cells were therefore maintained at a low glucose concentration (5.5 mM) at which no significant net glucose uptake could occur, whereas glucose production was not restrained by extracellular glucose (28). Basal glucose production through either pathway did not differ between the two hepatocyte populations under the above conditions. Such an observation might suggest that a normalization of this parameter occurs in vitro, which could result from the incubation of control and diabetic cells in the same extracellular medium in the absence of serum. This means, for diabetic hepatocytes, a protection from an impaired hormonal regulation, and an exposure to the same nutritional environment than control hepatocytes. Such identical conditions tend eventually to equalize the availability of intracellular substrates in either cell type. Since all of these factors participate in the regulation of basal glucose output, it may not be surprising to find a similar level of basal hepatic glucose production in controls and diabetics in vitro, in contrast to what has been found in vivo (10). Preliminary assays showed that basal glycogenolysis and gluconeogenesis were not significantly modified after incubation of control and diabetic cells with insulin (data not shown). The sensitivity of the cells to insulin in these pathways was improved by taking advantage of the ability of the hormone to antagonize the action of glucagon. Basal amounts of glucagon trigger hepatic glucose output primarily by stimulating glycogenolysis (29), and the suppression of glucose production by physiological increments of insulin after an overnight fast is attributable almost exclusively to an inhibition of glycogenolysis (21, 30). Indeed, the sensitivity of hepatocytes to insulin was such that a concentration as low as 1 ng/ml of the hormone inhibited glucagon-induced glycogenolysis by 15% in control cells. Diabetic hepatocytes appeared much more sensitive to insulin than control hepatocytes, since, at the same hormone concentration, the inhibition of glycogenolysis reached 30%. Thus, the increased

1699

suppression of glycogenolysis found in vitro reflected an increased sensitivity of diabetic hepatocytes to insulin. In the course of our investigations, we observed that, although insulin efficiently restrained glycogenolysis, it had a rather limited ability to suppress gluconeogenesis in vitro (data not shown). Such a lack of effect of insulin on gluconeogenesis has already been reported in vivo (30, 31) and may be explained by the fact that the substrates (pyruvate or lactate) used to estimate the gluconeogenic pathway are more glycogenic than glucose and serve to supply glycogen synthesis and repletion after a fast, as demonstrated by numerous studies (32-34). Glucose incorporation into glycogen was evaluated in hepatocytes from animals in the postabsorptive state. At first, it appeared that the glycogen content of hepatocytes from the fed diabetic animals was significantly decreased as compared to controls. A similar glycogen depletion has been described in diabetic animals (35, 36) and was not attributable to a deficiency of glycogen synthase or to an increased kinase-mediated phosphorylation, but to a failure of the diabetic liver to activate the synthase in response to the appropriate substrate stimulus (37, 38). The stimulation of glycogen synthesis by glucose could be restored after insulin injection (39). As suggested above, the insulin deficiency and particularly the defective surge of insulin in response to a carbohydrate ingestion that characterize nO-STZ rats might well account for the slight decrease in their hepatocyte glycogen content, since insulin is required not only to build up but also to maintain maximal glycogen stores (40). The acute effect of insulin on glucose incorporation into glycogen was enhanced in diabetic hepatocytes and resulted in a marked increase in their maximal response to the hormone, although their sensitivity was unchanged. This was in contrast with the insulin effect on glucagoninduced glucose production, which resulted in an increased sensitivity to the hormone, although the maximal response remained unaltered. The complexity of the insulin response in diabetic hepatocytes was further evidenced when studying insulin action on other hepatic functions; regarding lipid synthesis, diabetic hepatocytes appeared more responsive to insulin than controls, while exhibiting a similar sensitivity to the hormone. However, diabetic hepatocytes exhibited an increase in both the sensitivity and the maximal response to the hormone with respect to amino acid transport. These alterations are probably located at a postreceptor step, since neither insulin binding nor insulin receptor kinase activity were altered in nO-STZ rat hepatocytes, in accordance with previous studies performed on intact livers (23). Thus, cultured hepatocytes from nO-STZ rats do not exhibit an insulin-resistant state, but rather express an enhanced response to insulin on several metabolic path-


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ways. This latter was manifested, depending on the pathway considered, as an increase in either the responsiveness, or the sensitivity, or the two parameters, reflecting the heterogeneity in the effector systems by which the various insulin-activated pathways are mediated. Our experimental approach enabled us to show that the impairment in insulin action reflects intrinsic cellular abnormalities located at a step distal to the insulin-receptor interaction. Our results of enhanced hepatic insulin action in this model appear at variance with those currently observed in human hypoinsulinemic type 2 diabetes. Indeed, in this human disease, the insulin secretory disturbances and the insulin deficiency proved impossible to separate from an overall insulin resistance (for review see Ref. 1). In attempts to understand the relationship between specific /3-cell damage and emergence of insulin resistance, various chronic and stable NIDD rat models of graded severity were elaborated (2,4,6,41). Comparative studies on these models suggested the existence of a relationship between the degree of pancreatic destruction, the level of hyperglycemia and the development of peripheral insulin resistance, the higher the reduction in /3-cell mass and the degree of hyperglycemia, and the greater the occurrence of insulin resistance. In the nO-STZ model, the reduction in /3-cell mass led to only mild basal hypoinsulinemia and hyperglycemia; normal or enhanced insulin response was observed in adipocytes (Kergoat, M., M. Guerre-Millo, M. Lavau, and B. Portha, submitted) and hepatocytes (present study). Other experimental models (n2-STZ, n5-STZ, adult-STZ) in which a drastic reduction in /3-cell mass (more than 50%) was associated with an overt basal hypoinsulinemia and a more severe hyperglycemia were shown to develop frank insulin resistance, as assessed in vivo by glucose-insulin tolerance tests (42) and glucose-clamp studies (35, 41, 43), or in vitro on glucose transport by adipocytes (44, 45), muscle (35, 43) and isolated hearts (36). Only one study by Maloff (46) reported no alteration of insulin action in adipocytes. These discrepancies between the nO-STZ model and the other models of neonatal or adult STZ injection might be accounted for by differences in the severity and the chronicity of hyperglycemia. In fact, all these STZ rat models including ours are only partially representative of human NIDDM, a disorder in which complex interrelations between various defects (insulin deficiency, insulin resistance, and obesity) usually coexist. In conclusion, although our in vitro model was shown to have some limitations for the estimation of basal hepatic glucose output, it constituted a simplified system that revealed the insulin hyperresponsiveness of diabetic animals as being an intrinsic defect of their hepatic cells. From our overall in vivo and in vitro observations on this

Endo • 1991 Voll28»No4

model, it may be assumed that /3-cell deficiency and glucose intolerance do not necessarily lead to insulin resistance.

Acknowledgments The authors thank Betty Jacquin for her expert secretarial assistance and her help in preparing the manuscript.

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Spontaneous recovery from non-insulin-dependent diabetes mellitus induced by neonatal streptozotocin treatment in spontaneously hypertensive rats.

Impairment of glycosyl-phosphatidylinositol-dependent insulin signaling system in isolated rat hepatocytes by streptozotocin-induced diabetes.

Insulin responsiveness of isolated perfused livers from rats with streptozotocin induced diabetes.

Reversal of streptozotocin-induced diabetes in rats by intraperitoneal implantation of encapsulated neonatal rabbit pancreatic tissue.

Adipocyte dysfunction in rats with streptozotocin-nicotinamide-induced diabetes.

Lycopene ameliorates renal function in rats with streptozotocin-induced diabetes.

Serum lipoproteins and apolipoproteins in rats with streptozotocin-induced diabetes.

Dual effect of metformin in cultured rat hepatocytes: potentiation of insulin action and prevention of insulin-induced resistance.

Increased intrinsic mitochondrial respiratory capacity in skeletal muscle from rats with streptozotocin-induced hyperglycemia.

Production of corticosteroid hormones in vitro by adrenals in rats with streptozotocin-induced diabetes.

Increased ketogenesis related to insulin deficiency in isolated hepatocytes from NIDDM model rats.

Chronic hypertension induced by streptozotocin in rats.

Interactions of okadaic acid with insulin action in hepatocytes: role of protein phosphatases in insulin action.

Sesame effects on testicular damage in streptozotocin-induced diabetes rats.

Experimental Nonalcoholic Steatohepatitis Induced by Neonatal Streptozotocin Injection and a High-Fat Diet in Rats.

Insulin reverses anxiety-like behavior evoked by streptozotocin-induced diabetes in mice.

Hypothalamic 5-hydroxytryptamine metabolism is not influenced by streptozotocin-diabetes induced at neonatal age.

Effect of probucol on recovery from streptozotocin diabetes in rats.

Dexamethasone stimulates vitamin K-dependent carboxylase activity in neonatal rats and cultured fetal hepatocytes.

Effects of aldose reductase inhibitors on prostacyclin (PGI2) synthesis by aortic rings from rats with streptozotocin-induced diabetes.

Improvement in glucose-induced insulin secretion in diabetic rats after long-term gliclazide treatment: a comparative study using different models of non-insulin-dependent diabetes mellitus induced by neonatal streptozotocin.

Insulin Restores an Altered Corneal Epithelium Circadian Rhythm in Mice with Streptozotocin-induced Type 1 Diabetes.

Amino acid uptake regulation by cell growth in cultured hepatocytes isolated from fetal and adult rats.

Antioxidant icariside II combined with insulin restores erectile function in streptozotocin-induced type 1 diabetic rats.