Department of Medical Biochemistry and Molecular Biology, School of Medicine, Investigation Unit of the University Hospital Virgen Macarena, Seville 41009, Spain
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ABSTRACT |
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Pancreastatin (PST), a regulatory peptide with a general inhibitory effect on secretion, is derived from chromogranin A, a glycoprotein present throughout the neuroendocrine system. We have previously demonstrated the counterregulatory role of PST on insulin action in rat hepatocytes. Here, we are reporting the PST effects on rat adipocytes. PST dose dependently inhibits basal and insulin-stimulated glucose transport, lactate production, and lipogenesis, impairing the main metabolic actions of insulin in adipocytes. These effects were observed in a wide range of insulin concentrations, leading to a shift to the right in the dose-response curve. Maximal effect was observed at 10 nM PST, and the IC50 value was ~1 nM. Moreover, PST has a lipolytic effect in rat adipocytes (ED50 0.1 nM), although it was completely inhibited by insulin. In contrast, PST dose dependently stimulated protein synthesis and enhanced insulin-stimulated protein synthesis. In summary, these data show the lipokinetic effect of PST and the inhibitory effect of PST on insulin metabolic action within a range of physiological concentrations. Therefore, these results give new pathophysiological basis for the association of PST with insulin resistance.
chromogranin A; peptide; metabolism; insulin resistance
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INTRODUCTION |
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PANCREASTATIN (PST) is a 49-amino acid peptide (33)
derived from chromogranin A (8), a glycoprotein present throughout the
neuroendocrine system (9) with a recognized role as a precursor of
biologically active peptides (4, 9, 10). PST was first described as an
inhibitor of insulin secretion (33), but many different effects were
then reported (see Ref. 23 for review). In summary, the effects of PST
on endocrine and exocrine secretion in different tissues raised the
hypothesis of PST being a general autocrine, paracrine, and endocrine
inhibitor of secretion. However, the best characterized effect of PST
has been studied in rat liver (26), where we found a counterregulatory
effect on insulin action (17). Thus we found that PST had a
calcium-dependent glycogenolytic effect in rat hepatocytes (13, 14, 20)
and an inhibitory effect on insulin-stimulated glycogen synthesis (17).
Moreover, we studied the mechanism of PST action (16, 21, 22), and we
described for the first time the characterization of PST receptors (25,
27). PST receptors seem to be coupled to a pertussis toxin-insensitive
G protein of the q /11
family, which in turn leads to the activation of phospholipase C in the
plasma membrane (30).
The PST effect on insulin secretion by the -cell and insulin action
in the hepatocyte raised the hypothesis of a role of PST in insulin
resistance (15). In fact, high PST plasma levels have been found in
type II diabetes (6), gestational diabetes (19), and essential
hypertension (28, 29). Even though we failed to find increased PST
levels in young offspring of hypertensive patients, which already had
hyperinsulinemia (24), the concurrence of high levels of PST and
catecholamines in a further step may have a deleterious effect on
insulin sensitivity (24).
To further evaluate the possible role of PST in the pathophysiology of insulin resistance, we looked for direct PST effects in another insulin target, i.e., adipose tissue. In this report, we tested the biological effect of PST on the metabolic action of insulin in rat adipocytes. We found that PST has a lipolytic effect and inhibits insulin action in a dose-dependent manner and within a physiological range of concentrations. This impairment of insulin action may contribute to the pathophysiology of insulin resistance.
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MATERIALS AND METHODS |
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Materials. Rat PST was from Peninsula Laboratories Europe (Merseyside, UK). Bovine serum albumin (BSA; fraction V) was from Sigma Chemical (St. Louis, MO). 2-deoxy-D-[3H]glucose (7 Ci/mmol) and D-[14C(U)]glucose (13 mCi/mmol) were from DuPont NEN (Bad Homburg, Germany). L-[35S]methionine (1,000 Ci/mmol) was from Amersham (Madrid, Spain). Collagenase, SDS, HEPES, and other general reagents were from Boehringer Mannheim (Barcelona, Spain).
Adipocyte isolation. Adipocytes were prepared from the epididymal fat pads of ad libitum-fed 100- to 160-g male Wistar rats, according to the method described by Rodbell (12) with minor modifications. Fat pads were minced and then digested with collagenase at 37°C for 1 h in KRB (in mM: 113 NaCl, 2 CaCl2, 5 KCl, 10 NaH2CO3, 1.18 KH2PO4, and 1.18 MgCl2), pH 7.4, supplemented with 20 mM HEPES, 6 mM glucose, and 1% BSA. Aggregated material was removed by filtration through a mesh cloth. Isolated adipocytes were washed three times, and the packed cells were subsequently suspended in the final volume of the same buffer for metabolic experiments (105 cells/ml).
Glucose transport. Glucose transport was assayed as uptake of the nonmetabolizable glucose analog 2-deoxy-D-[2,6-3H]glucose (7 Ci/mmol) as previously described (18). Adipocytes were incubated in the buffer described above without glucose at 37°C for 20 min in the presence or absence of insulin. When PST was included in the experiment, it was added 4 min before insulin stimulation. Next, 0.5 µCi 2-deoxy-D-[2,6-3H]glucose were added (0.1 mM 2-deoxy-D-glucose), and the adipocytes were incubated for a further 10 min. The assay was terminated by two rapid washes with iced PBS buffer. Cells were finally solubilized with NaOH, and radioactivity was measured by scintillation counting.
Lactate production. Lactate production was assayed to study glucose utilization by rat adipocytes. Cells were incubated in KRB buffer as described in Adipocyte isolation in the presence or the absence of different concentrations of insulin at 37°C. When PST was included in the experiment, it was added 4 min before the addition of insulin. Incubation was continued at 37°C for 2 h. Finally, cells were centrifuged at 4°C (800 g, 5 min), and the infranatant was removed for lactate measurement. Lactate was determined by enzymatic assay using a kit from Boehringer Mannheim.
Lipogenesis. Lipogenesis was measured as incorporation of D-[14C(U)]glucose into adipocyte lipids as previously described (36). Cells (1 ml, 105 adipocytes) were incubated in KRB with or without different concentrations of insulin at 37°C. When PST was included in the experiment, it was added 4 min before the addition of insulin. After 25 min, 2 µCi of D-[14C(U)]glucose were added, and incubation continued for a further 30 min. The assay was stopped by the addition of 3 ml of a mixture of chloroform-methanol (2:1 vol/vol). After centrifugation (2,000 g, 5 min), radioactivity was measured in the lipid fraction by scintillation counting.
Lipolysis. Glycerol and nonesterified fatty acids were determined to assay lipolysis rate in rat adipocytes. Cells were incubated as described in Lactate production, but the infranatant was used to measure nonesterified fatty acids and glycerol by using kits from Boehringer Mannheim.
Protein synthesis. Protein synthesis was assayed as previously described (18). Adipocytes were incubated in the buffer described in Glucose transport in the presence or absence of insulin for 20 min at 37°C. When PST was included in the experiment, it was added 4 min before insulin stimulation. Next, 0.5 µCi of L-[35S]methionine were added, and the incubation proceeded further for 30 min at 37°C. Cells were then washed two times with iced PBS and lysed with 1% SDS and 20 mM Tris · HCl, pH 7.4. After centrifugation (800 g), the infranatant was removed and supplemented with trichloroacetic acid (TCA; 10%). After incubation for 30 min on ice, precipitated proteins were collected by centrifugation (15,000 g, 15 min). The pellet was washed three times with 7% TCA, dissolved in 1 ml of 2 M NaOH, neutralized with HCl, and measured for radioactivity by scintillation counting.
Statistical analysis. Values are means ± SE. The statistical study was performed by analysis of variance to analyze the effect of PST on insulin action, and the Bonferroni posttest was used to compare the different concentration points. The degree of significance of the differences was considered at P < 0.05.
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RESULTS |
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Effect of PST on glucose transport. To study the effect of PST on basal and insulin-stimulated glucose transport in rat adipocytes, we measured the 2-deoxyglucose uptake. As shown in Fig. 1A, 10 nM PST partially inhibited the effect of insulin, producing a shift to the right of the dose-response curve. Thus EC50 of insulin was increased from 0.3 to 1 nM. Besides, the maximal effect of insulin was also inhibited, suggesting that PST affects responsiveness of the insulin receptor system. Moreover, even basal glucose transport was decreased by PST treatment compared with controls (Fig. 1A). Moreover, this effect of PST on both basal and insulin-stimulated glucose transport was dose dependent, as shown in Fig. 1B. The effect was statistically significant at 0.1 nM PST, and the IC50 was ~0.6 nM. Maximal effect was observed at 10 nM PST and inhibited the maximal insulin response ~50% (10 nM).
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Effect of PST on lactate production. To study the glucose utilization by rat adipocytes, we determined the production of lactate during 2 h of incubation. As shown in Fig. 2A, PST partially blunted the effect of insulin, increasing lactate production within a wide range of insulin concentrations. Moreover, this effect was dependent on PST concentration, as shown in Fig. 2B. This effect was significant at 10 nM PST, producing a deleterious effect on the responsiveness as well as the potency of insulin. Maximum effect of PST was observed at 10 nM, which impaired insulin action by 40%. However, PST only significantly inhibited lactate production at higher concentrations (10-100 nM), with a slight effect on basal levels (Fig. 2B) that may be due to the decrease in glucose uptake.
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Effect of PST on lipid metabolism. Next, we studied other insulin-mediated metabolic effects. Thus we studied the effect of PST on basal and insulin-stimulated lipogenesis and lipolysis. As shown in Fig. 3A, PST counteracted the effect of insulin, stimulating the lipid synthesis, shifting the dose-response to the right, and significantly blunting the responsiveness of adipocytes to insulin. This effect was dependent on PST concentration (Fig. 3B), with a maximum observed at 10 nM and an IC50 of ~1 nM. Insulin increased the rate of lipid synthesis up to threefold, and PST reduced this effect ~25%. Basal synthesis of lipids was not affected significantly by PST (Fig. 3, A and B).
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Moreover, PST also showed a lipolytic effect that was blunted by increasing concentrations of insulin. As shown in Fig. 4A, 10 nM insulin completely inhibited the lipolytic effect of PST, as determined by glycerol measurement. The effect was dependent on PST concentration (Fig. 4B), and maximal effect was observed at 10 nM PST, with an ED50 of ~0.1 nM. Similar results were obtained by determining nonesterified fatty acids (data not shown).
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Effect of PST on protein synthesis. Another metabolic action of insulin is the stimulation of protein synthesis. Therefore, we checked whether PST could also counteract this insulin effect in rat adipocytes. However, we found that PST not only did not inhibit this effect of insulin, but it potentiated the insulin-stimulated protein synthesis (~40%), shifting the dose-response curve to the left (Fig. 5A). This effect was dependent on PST concentration with a maximal effect at 10 nM and an ED50 of ~1 nM (Fig. 5B). Moreover, PST also stimulated basal protein synthesis in a dose-dependent manner, with a maximal effect at 10 nM (~30% increase) and an ED50 of ~0.6 nM.
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DISCUSSION |
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Experimental data in rat hepatocytes have supported the hypothesis of PST considered as a counterregulatory peptide of insulin action on glucose metabolism (15, 17, 23, 26). These effects of PST inhibiting insulin action, together with its inhibitory effect on insulin secretion (33), have led to the recent hypothesis that PST may have a role in the insulin resistance syndrome (15, 23, 26). Thus type II diabetes is characterized both by abnormal insulin secretion and by insulin resistance (2). Moreover, clinical data showing increased PST levels (nM range) in type II diabetes (6), gestational diabetes (19), and hypertension (24, 28, 29; correlating with catecholamine levels) pointed to a possible role of PST in the pathophysiology of insulin resistance. To further assess this hypothesis, we studied the effects of PST on insulin action in another important target of insulin: adipose tissue. Here, we report that PST produces insulin resistance in rat adipocytes in vitro. Because this is a biological effect in a target cell far from the place of synthesis (neuroendocrine system), we have found a new endocrine action of this regulatory peptide. Besides, in these insulin-sensitive cells, PST showed a lipolytic effect, suggesting a new role of PST as a lipokinetic hormone. It is worth pointing out that lipolysis was measured in the absence of adenosine deaminase; therefore, basal lipolysis was very low, as should be expected (5), due to the antilipolytic effect of adenosine (32). That is why the effect of insulin on basal lipolysis is not evident. On the other hand, the effect of PST is observed even in the presence of the antilipolytic adenosine released by adipocytes (31). Moreover, PST partially counteracts several metabolic effects of insulin: stimulation of glucose transport and lipogenesis and inhibition of lactate production. These effects were observed at physiological concentrations of both insulin and PST (nM range) and decreased the maximal response to insulin 25-50% at 10 nM. These results suggest that PST may also reduce insulin sensitivity and responsiveness in adipose tissue under in vivo conditions. However, this speculation needs confirmation by experiments in vivo. In any case, these effects of PST may reinforce the counterregulatory effect of catecholamines, whose plasma levels correlate with those of PST (19, 28). Therefore, these data further support the hypothesis of PST being considered as a stress hormone (15, 26), since the major source of circulating PST is the chromogranin A released from adrenergic and noradrenergic systems (23).
We have employed a short time for preincubation of adipocytes with PST. The rationale for this procedure is that PST signaling in the hepatocyte, which we have studied previously, is completed within minutes (16, 20-22), and a short preincubation of PST before insulin stimulation is enough to counteract insulin action in the hepatocyte. Therefore, we have assumed that a similar signaling system may account for PST effects in the adipocyte. These studies of signaling are now under progress. Thus we know that interaction of PST with insulin receptor is not responsible for the PST effect (data not shown). Therefore, PST may interact with its own receptors and interfere with insulin signaling downstream of the receptor (tyrosine phosphorylation), as we have found in hepatocytes (unpublished observations). This hypothesis remains speculative but warrants further investigation to finally elucidate the molecular mechanisms underlying PST action in the adipocyte.
The effects of PST impairing insulin action seem to be more efficient regarding glucose uptake (50% inhibition of insulin stimulation) than lipid metabolism (25% inhibition of insulin stimulation). Besides, the lipolytic effect of PST seems to be completely inhibited by high concentrations of insulin. This is consistent with a role of PST in the pathophysiology of insulin resistance, since insulin-resistant subjects have an impaired glucose metabolism, whereas lipid synthesis and adipogenesis are less affected (7). This metabolic framework is also present in obesity, which is an insulin-resistant state associated with type II diabetes and hypertension (1, 11).
Even though we observed a general counterregulatory effect of PST on insulin action, we found that PST potentiated the insulin-stimulated protein synthesis in rat adipocytes. Moreover, PST itself stimulated basal protein synthesis in the adipocyte. However, this effect of PST on protein synthesis should not be striking, since PST seems to have a parallel effect of that of other counterregulatory hormones such as growth hormone, catecholamines, and calcium-dependent hormones. Thus they have similar effects on glucose and lipid metabolism, as well as insulin secretion, whereas all of these agents have a stimulating effect on protein synthesis in adipocytes (34, 35). This effect may be mediated by protein kinase C activity, which is increased by PST in hepatocytes (21), as previously observed for vasopressin and oxytocin (3). These considerations, together with the fact that PST affects glucose metabolism stimulated by insulin, whereas the lipolytic effect is counteracted by hyperinsulinemia, support the idea of PST having a putative pathophysiological role in insulin resistance of obesity, opening an interesting line of research.
In conclusion, these data suggest a role for PST as a lipokinetic hormone and as an inhibitor of insulin action in rat adipocytes. These effects may be important in the pathophysiology of insulin resistance.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Fondo de Investigacion Sanitaria (FIS 96/1411), Ministerio de Sanidad, Spain. C. González-Yanes is a recipient of a fellowship from the Fondo de Investigacion Sanitaria (BEFI 98/9158).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: V. Sánchez-Margalet, Dept. of Medical Biochemistry and Molecular Biology, School of Medicine, Av. Sanchez Pizjuan 4, Seville 41009, Spain.
Received 2 April 1998; accepted in final form 3 September 1998.
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