HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Holness, M. J. Articles by Sugden, M. C. Search for Related Content PubMed PubMed Citation Articles by Holness, M. J. Articles by Sugden, M. C. Social Bookmarking                What's this?

Peroxisome Proliferator–Activated Receptor- and Glucocorticoids Interactively Regulate Insulin Secretion During Pregnancy

From the Centre for Diabetes and Metabolic Medicine, Institute of Cell and Molecular Science, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, Queen Mary, University of London, London, U.K

Address correspondence and reprint requests to Professor Mary C. Sugden, Centre for Diabetes and Metabolic Medicine, Institute of Cell and Molecular Science, 4 Newark St., Whitechapel, London E1 2AT, U.K. E-mail: m.c.sugden{at}qmul.ac.uk

Abbreviations: GSIS, glucose-stimulated insulin secretion; I, incremental plasma insulin values integrated over the period of increased glucose; PDK, pyruvate dehydrogenase kinase; PPAR, peroxisome proliferator-activated receptor

	ABSTRACT

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
We evaluated the impact of peroxisome proliferator–activated receptor (PPAR) activation and dexamethasone treatment on islet adaptations to the distinct metabolic challenges of fasting and pregnancy, situations where lipid handling is modified to conserve glucose. PPAR activation (24 h) in vivo did not affect glucose-stimulated insulin secretion (GSIS) in nonpregnant female rats in the fasted state, although fasting suppressed GSIS. Dexamethasone treatment (5 days) of nonpregnant rats lowered the glucose threshold and augmented GSIS at high glucose; the former effect was selectively opposed by PPAR activation. Pregnancy-induced changes in GSIS were opposed by PPAR activation at day 19 of pregnancy. Dexamethasone treatment from day 14 to 19 of pregnancy did not modify the GSIS profile of perifused islets from 19-day pregnant rats but rendered the islet GSIS profile refractory to PPAR activation. During sustained hyperglycemia in vivo, dexamethasone treatment augmented GSIS in nonpregnant rats but limited further modification of GSIS by pregnancy. We propose that the effect of PPAR activation to oppose lowering of the glucose threshold for GSIS by glucocorticoids is important as part of the fasting adaptation, and modulation of the islet GSIS profile by glucocorticoids toward term facilitates the transition of maternal islet function from the metabolic demands of pregnancy to those imposed after parturition.

Peroxisome proliferator–activated receptor (PPAR) is a transcription factor upregulating expression of genes involved in lipid catabolism (1), is critical to adaptations of glucose handling to fasting (2), and is a major determinant of fatty acid oxidation in rodent islets and INS cells (3–6). Since despite hypoglycemia, PPAR-null mice exhibit hyperinsulinemia on fasting (5,7), islet PPAR expression is increased on fasting (7), and islets from PPAR-null mice show enhanced glucose-stimulated insulin secretion (GSIS) after starvation (5), it is proposed that PPAR signaling is required to suppress GSIS during fasting (5,7). Suppression of GSIS occurs in conjunction with suppression of irreversible glucose disposal via oxidation, an event linked to increased expression of the PPAR target gene pyruvate dehydrogenase kinase (PDK)4 (8–12, rev. in 13) and enhanced expression of genes related to lipid catabolism (2) in a range of tissues. Enhanced PDK4 expression and activity suppress glucose oxidation, allowing increased anaplerotic entry of pyruvate into the tricarboxylic acid cycle (rev. in 13). As mitochondrial anaplerotic products are important for GSIS (rev. in 14,15), potentiated GSIS following culture with the PPAR agonist WY14,643 by adenoviral transduction with PPAR plus its heterodimerization partner, the retinoid X receptor, may be explained by PPAR-stimulated PDK4 expression and augmented anaplerosis (6,11). However, PPAR activation could, under some conditions, promote exhaustion of a lipid-derived molecule important for optimal GSIS, thereby blunting GSIS (5). PPAR activation reverses insulin resistance (and thus the requirement for islet compensation) induced by high–saturated fat diets (16,17). The increase in lipid delivery associated with high–saturated fat diets leads to the accumulation of excess lipids ectopically in muscle, liver, and the pancreatic islet that affects metabolic signaling (rev. in 18). Such accumulated lipid-derived compounds can be cleared through PPAR activation (18).

Excess endogenous (19,20) and exogenous (21,22) glucocorticoids impair insulin sensitivity. Signaling via PPAR is essential for the induction of hepatic insulin resistance by the synthetic glucocorticoid dexamethasone (23). Glucocorticoids induce PDK4 expression in a rat hepatoma cell line, an effect opposed by insulin (24). In vivo, dexamethasone treatment of rats during the last third of pregnancy modifies regulation of maternal glucose metabolism by insulin (25). Dexamethasone also counteracts the effect of placental lactogens and prolactin to enhance insulin secretion by pancreatic islets in culture (26). These hormones have been proposed to mediate some of the islet adaptations to pregnancy (27–29).

Since effects of PPAR ablation are observed after fasting, where glycemia is in the low physiological range, and because islets from PPAR-null mice show increased insulin secretion at low glucose when previously exposed to high glucose in culture compared with islets from wild-type mice (30), we examined whether PPAR activation may regulate the threshold of GSIS during fasting (where GSIS is suppressed) or in pregnancy (where islet compensation for maternal insulin resistance involves a lowered threshold for stimulation of insulin secretion by glucose, as well as enhanced insulin secretion at high glucose) (rev. in 31). In addition, since maternal glucocorticoids rise during starvation and during pregnancy toward term (rev. in 32), we evaluated whether there was interactive regulation of GSIS by PPAR and glucocorticoids. Our data suggest that PPAR is important for the islet response to the distinct metabolic challenges imposed by both fasting and pregnancy, situations that are associated with modified lipid handling to conserve glucose.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Kits for measurement of insulin by enzyme-linked immunosorbent assay were purchased from Mercodia (Uppsala, Sweden). Kits for determination of glucose (glucose oxidase) were purchased from Roche Diagnostics (Lewes, East Sussex, U.K.). Wako kits for the spectrophotometric determination of triglyceride levels were purchased from Alpha Labs (Eastleigh, U.K.). Dexamethasone (sodium phosphate) was obtained from David Bull Laboratories (Warwick, U.K.). The PPAR agonist WY14,643 (pirinixic acid) was purchased from Sigma (Poole, Dorset, U.K.). Human actrapid insulin was purchased from Novo Nordisk (Bagsvaerd, Denmark). Female Wistar rats (200–250 g) and mini-osmotic pumps were purchased from Charles River (Margate, Kent, U.K.). Standard pelleted rodent diet was purchased from Special Diet Services (Witham, Essex, U.K.). General laboratory reagents were from Roche Diagnostics or from Sigma.

Studies were conducted in adherence with the regulations of the U.K. Animal Scientific Procedures Act (1986). Age-matched female rats maintained on a standard light (0800–2000)-dark (2000–0800) cycle at 21 ± 2°C were individually housed. Rats were fed ad libitum or fasted for 48 h, as specified, with free access to water. Pregnant rats were time mated by the appearance of sperm plugs (day 0 of pregnancy). Dexamethasone was administered (100 µg/kg body wt per day) via a chronically implanted osmotic pump to nonpregnant female rats or pregnant rats from day 14 to day 19 of pregnancy (term = 22–23 days) (25). This procedure leads to almost total suppression of endogenous corticosterone levels (33). Sham operations were undertaken on the control groups. At the same time, rats were fitted with chronic indwelling jugular cannulae for infusion and sampling (34,35). WY14,643 was administered to nonpregnant or 18-day pregnant rats as a single injection (50 mg/kg body wt i.p.), and rats were sampled after a further 24 h (36,37). This period of exposure to the PPAR agonist is adequate to elicit markedly enhanced protein expression of the PPAR-linked gene PDK4 in islets of nonpregnant rats (11). We selected this period of exposure to WY14,643 since previous studies show that as the period of PPAR activation increases beyond 2 days, pregnant (but not unmated) rats become refractory to its action (38). In addition, more prolonged PPAR activation suppresses fetal growth and increases circulating triglycerides, most likely as a consequence of increased adipocyte lipolysis (38). Control nonpregnant and 19-day pregnant rats were injected with vehicle.

Intravenous glucose tolerance tests.
Glucose was administered as an intravenous bolus (0.5 g glucose/kg; 150 µl/100 g body wt) to conscious, unrestrained rats via the indwelling jugular cannula (34,35). Blood samples (100 µl) were withdrawn from the indwelling cannula, which was flushed with saline after the injection of glucose to remove residual glucose. Samples of whole blood (50 µl) were deproteinized [ZnSO₄/Ba(OH)₂], centrifuged (10,000g at 4°C), and the supernatant assayed for glucose. The remaining sample was immediately centrifuged (10,000g at 4°C) and plasma stored at –20°C until assayed for insulin. Insulin and glucose responses were used for calculation of the incremental plasma insulin values integrated over the 30-min period after the injection of glucose (I_0–30).

Islet isolation and stepwise glucose perifusions.
Rats were anesthetized (sodium pentobarbital; 60 mg/ml in 0.9% NaCl, 1 ml/kg body wt i.p.). Once locomotor activity had ceased, pancreases were excised and islets isolated by collagenase digestion. Insulin release from freshly isolated islets was measured in a perifusion system as described previously (36). Islets were perifused in basal medium (Krebs-Ringer buffer containing 20 mmol/l HEPES, pH 7.4, 5 mg/ml BSA, and 2 mmol/l glucose) for 60 min at a flow rate of 1 ml/min at 37°C before collection of fractions. Perifused islets were then subjected to square-wave stimulation, where the perifusate glucose concentration was raised to 8 mmol/l for 16 min and subsequently to 16 mmol/l for 16 min. After exposure to 16 mmol/l glucose, perifusate glucose was then lowered by switching back to basal medium. Fractions (2 ml) were collected at 2-min intervals and stored at –20°C before assay for glucose and insulin. To compare insulin secretory responses during islet perifusions, areas under the insulin curves were calculated for the entire period of the perifusion study (I_60–140).

Hyperglycemic clamps.
An intravenous glucose bolus was administered to conscious, unrestrained postabsorptive rats via an indwelling cannula, and 25% glucose solution was subsequently infused to maintain blood glucose concentrations at a target concentration of 10 mmol/l. Blood samples were obtained before the glucose bolus and at 5-min intervals thereafter for 40 min. Samples of whole blood (50 µl) were treated as described for the intravenous glucose tolerance tests. Insulin responses were used for calculation of the incremental plasma insulin values integrated over the period of glucose infusion (I_0–40).

Insulin clearance.
A constant infusion of human actrapid insulin (4 mU · kg^–1 · min^–1) was given for 2 h. Exogenous glucose infusion was initiated at 1 min after the start of insulin infusion. Blood was sampled from the right jugular vein at 5-min intervals. Blood glucose concentrations during the clamp were determined using a glucose analyzer (YSI, Yellow Springs, OH). The exogenous glucose infusion rate was adjusted to maintain glycemia. Plasma insulin concentrations reached steady state by 90 min after initiating insulin infusion; thereafter, three blood samples (0.1 ml) were obtained at 15-min intervals for measurements of plasma insulin concentrations. Insulin clearance rates were calculated by dividing the rate of insulin infusion by the steady-state insulin concentration during euglycemic hyperinsulinemia.

Statistical analyses.
Results are means ± SE. Numbers of litters, rats, or islet preparations are in parentheses. Statistical analysis was by ANOVA, followed by Fisher’s post hoc tests for individual comparisons or Student’s t test as appropriate (Statview; Abacus Concepts, Berkeley, CA). A P value <0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
PPAR activation has little effect on GSIS in the fasted state with nonpregnant female rats.
We have previously demonstrated that PPAR activation in postabsorptive rats (6 h after food withdrawal) does not affect GSIS following intravenous glucose challenge in vivo or during stepwise glucose islet perifusions in vitro (36). We therefore examined GSIS after fasting (48 h), which causes increased islet PPAR signaling, indicated by enhanced islet expression of PDK4 (11) and other PPAR target genes (5). GSIS was substantially suppressed by 48 h fasting (fast group), demonstrated by a 60% decline (P < 0.001) in total suprabasal 30-min area under the insulin curve (I_0–30), following an intravenous glucose tolerance test in vivo (control 265 ± 33 vs. fast 105 ± 12 µU · ml^–1 · min^–1), and a 73% decline (P < 0.01) in the areas under the insulin curve for GSIS with perifused islets ex vivo (I_60–140) compared with the fed group (control 922 ± 127 vs. fast 248 ± 90 µU · ml^–1 · min^–1) (Fig. 1A). Administration of the PPAR agonist WY14,643 after 24 h fasting, at a dose and over the time scale previously shown to increase PDK4 expression in pancreatic islets and liver (8,11), with sampling after 24 h (fast+WY group) had no further significant effect on GSIS in vivo (a I_0–30 value of 114 ± 19 µU · ml^–1 · min^–1). Areas under the insulin curves (I_60–140 values) for ex vivo islet perifusions were similarly not significantly affected by WY14,643 treatment during fasting (fast+WY 180 ± 18 µU · ml^–1 · min^–1) (Fig. 1A).

View larger version (39K): [in this window] [in a new window]   FIG. 1. Starvation and dexamethasone (Dex) treatment of nonpregnant rats modify GSIS in islet perifusions. Islets were harvested from nonpregnant ad libitum–fed rats (Con), 48-h fasted rats (Fast), 48-h fasted rats treated with WY14,643 (50 mg/kg body wt; 24 h exposure) (Fast+WY), nonpregnant ad libitum–fed rats treated with dexamethasone (100 µg/kg body wt per day; 5 days) (Dex), or nonpregnant ad libitum–fed rats treated with dexamethasone plus WY14,643 (24 h exposure, day 4–5 of dexamethasone treatment) (Dex+WY group). Islets were perifused (2 mmol/l glucose for 60 min; flow rate 1 ml/min) before being subjected to square-wave stimulation by raising the perifusate glucose concentration to 8 mmol/l (16 min) and subsequently to 16 mmol/l (16 min). After exposure to 16 mmol/l glucose, perifusate glucose was then lowered by switching back to basal medium. Fractions (2 ml) collected at 2-min intervals were assayed for insulin and glucose. A: Patterns of GSIS by perifused islets isolated from nonpregnant control rats (), 48-h starved rats (•), and 48-h starved rats treated with WY14,643 (). B: Patterns of GSIS by perifused islets isolated from nonpregnant control rats (), Dex rats (•), and Dex+WY rats (). C: Data for the control, Dex, and Dex+WY groups are expressed relative to perfusate insulin concentrations at 100 min (time = 100). D–G: Areas under the insulin curves for the control, Dex, and Dex+WY groups were calculated for discrete 16-min periods during perifusion in which the perifusate glucose concentration varied between 2 and 8 mmol/l and between 8 and 16 mmol/l (1 and 2, respectively), the immediate poststimulation transition (3), and the longer-term poststimulation period (4). Data are means ± SE for 10 control rats, 5 fast rats, 5 fast+WY rats, 5 Dex rats, and 7 Dex+WY rats. Statistically significant effects of dexamethasone treatment are indicated by ***P < 0.001 and **P < 0.01. Statistically significant effects of WY14,643 treatment are indicated by P < 0.01.

PPAR activation specifically opposes lowering of the glucose threshold by dexamethasone with islets from nonpregnant rats.
We examined effects of 24 h in vivo exposure to the PPAR agonist WY14,643 in perifused islets from dexamethasone-treated (nonpregnant) female rats (Dex+WY group; Fig. 1B). PPAR activation failed to modify glucose responsiveness of GSIS but modulated glucose sensitivity. To quantify differences in the glucose threshold for GSIS between perifused islets from control, Dex, and Dex+WY groups, areas under the insulin curves were calculated for 16-min periods, during which perifusate glucose was rising: 1, during which perifusate glucose varied between 2 and 8 mmol/l, and 2, during which the perifusate glucose concentration varied between 8 and 16 mmol/l. Similarly, areas under the insulin curves were calculated for equivalent 16-min periods of perifusion after switching to basal glucose: 3, the immediate poststimulation transition after removal of the 16 mmol/l glucose stimulus and restoration of basal glucose, and 4, the longer-term poststimulation period. Data are shown in Fig. 1D–G. Dexamethasone treatment of nonpregnant rats increased GSIS over the entire range of glucose concentrations, as shown by significantly higher values of 1, 2, 3, and 4. Contrasting with its failure to significantly affect GSIS at low glucose concentrations with perifused islets from nonpregnant rats (36), antecedent PPAR activation lowered 1 and 2 values for GSIS in the dexamethasone-treated nonpregnant rats (Fig. 1D and E). Antecedent PPAR activation did not affect 3 values in the dexamethasone-treated group (Fig. 1F). The overall pattern of the responses of 4 values to antecedent PPAR activation (Fig. 1G) qualitatively resemble that of 2, indicating that GSIS in dexamethasone-treated rats regains susceptibility to suppression by PPAR activation as extracellular glucose concentrations fall. PPAR activation therefore selectively opposes the leftward shift in the glucose threshold induced by dexamethasone treatment, while exhibiting a negligible effect on GSIS at high glucose concentrations.

Differential responsiveness of GSIS to antecedent PPAR activation is observed as the duration of pregnancy proceeds.
Pregnant rats were treated with WY14,643 at day 18 of pregnancy, and islets were prepared at day 19 for perifusion (19dP group). The efficacy of PPAR activation during late (18–19 days) pregnancy was demonstrated by the action of WY14,643 to significantly decrease circulating triglyceride concentrations by 41% (P < 0.05) (19dP 1.40 ± 0.31 vs. 19dP+WY 0.82 ± 0.17 mmol/l). GSIS by perifused islets from 15-day pregnant rats is unaffected by antecedent PPAR activation in vivo (36). In marked contrast, GSIS by perifused islets from 19-day pregnant rats (Fig. 2A) was markedly suppressed by antecedent PPAR activation in vivo (19P+WY group). Both the glucose threshold and glucose responsiveness of GSIS were affected by PPAR activation at this stage of pregnancy, as shown by lower values for 1, 2, 3, and 4 (Fig. 2D–G). It can therefore be concluded that the progression of pregnancy from day 15 (mid pregnancy) to day 19 (late pregnancy) is associated with increased susceptibility of GSIS to modulation by PPAR activation.

View larger version (35K): [in this window] [in a new window]   FIG. 2. The pattern of response of GSIS with perifused islets from 19-day pregnant rats, with or without dexamethasone (Dex) treatment, to antecedent PPAR activation in vivo. Dexamethasone was administered subcutaneously (100 µg/kg body wt per day) for 5 days from day 14 of pregnancy (19dP+Dex group). WY14,643 (50 mg/kg body wt i.p.) was administered to pregnant rats with (19dP+Dex+WY group) or without (19dP+WY) dexamethasone treatment on day 18 of pregnancy and rats sampled at day 19 of pregnancy. A: Data for perifused islets from 19-day pregnant rats (19dP; open symbols) and 19dP+WY rats (closed symbols). B: Data for perifused islets from 19dP+Dex and 19dP+Dex+WY rats (). C: Areas under the insulin curves calculated for the period over which perifusate glucose exceeded basal (I60–140). D–G: 1, 2, 3, and 4 values. Data are means ± SE for six 19dP, five 19dP+WY, five 19dP+Dex, and five 19dP+Dex+WY rats. Statistically significant differences from the untreated 19-day pregnant group are indicated by *P < 0.05 and **P < 0.01. Statistically significant differences from the dexamethasone-treated 19-day pregnant group are indicated by P < 0.05. Statistically significant effects of dexamethasone treatment are indicated by P < 0.05.

Effects of dexamethasone on GSIS are dominant to those of pregnancy.
To summarize, PPAR activation selectively opposes the leftward shift in the glucose threshold induced by dexamethasone treatment in nonpregnant rats, while exhibiting a negligible effect on GSIS at high glucose concentrations (Fig. 1). In contrast, PPAR activation opposes both the leftward shift in the glucose threshold and the increase in glucose responsiveness observed at day 19 of pregnancy (Fig. 2). The similarities between the GSIS profiles for 19dP and 19dP+Dex islets (Fig. 2) suggest that the stable modifications of islet function that result in altered GSIS, elicited by either dexamethasone treatment or late pregnancy, might be exclusive. We examined the pattern of response to PPAR activation in pregnant rats treated with dexamethasone from day 14 to 19 of pregnancy to examine whether the pattern of response more closely resembled that seen after dexamethasone treatment in nonpregnant rats or that observed at late (19 day) pregnancy (without dexamethasone treatment). Data are shown in Fig. 2. As was observed for the untreated 19-day pregnant rats, WY14,643 treatment of dexamethasone-treated pregnant rats elicited a significant 53% (P < 0.05) decline in circulating triglyceride concentrations, indicating the efficacy of WY14,643 treatment. WY14,643 treatment of dexamethasone-treated pregnant rats had a very limited effect on glucose responsiveness, but there was modest suppression of 1 and 2 (Fig. 2D and E). Thus, the effect of dexamethasone on glucose responsiveness appears dominant to that of pregnancy. Furthermore, the pattern of response of GSIS to PPAR activation at day 19 of pregnancy, following dexamethasone administration from day 14 to day 19 of pregnancy, is reminiscent of that observed at day 15 of pregnancy (without dexamethasone treatment [36]).

Dexamethasone treatment augments insulin secretion during hyperglycemic clamps in nonpregnant rats but not late pregnant rats.
To test whether the effect of dexamethasone treatment from day 14 to day 19 of pregnancy limits islet adaptations occurring from day 15 to day 19 of pregnancy, we analyzed insulin responses during hyperglycemic clamp studies in vivo, in which glucose was infused to maintain glycemia at 10 mmol/l for 40 min. Data for nonpregnant rats (with or without dexamethasone treatment) are shown in Fig. 3A–C. At similar steady-state glycemia, steady-state plasma insulin levels were 2.6-fold higher (P < 0.05) in the Dex group versus the control group (Fig. 3B), and the Dex group exhibited a 2-fold greater incremental area under the insulin curve (I_0–40) (Fig. 3C). Data for 19-day pregnant rats (with or without dexamethasone treatment) are shown in Fig. 3D–F. Steady-state plasma insulin levels were 4.1-fold higher (P < 0.001) in the 19dP group compared with unmated control group (compare open symbols in Fig. 3B and E). I_0–40 was 4.7-fold higher (P < 0.001) in the 19dP group compared with the control group (compare open bars in Fig. 3C and F). Steady-state plasma insulin levels did not differ significantly between Dex and 19dP+Dex groups (compare closed bars in Fig. 3C and F). Comparison of the 19dP and 19dP+Dex group shows that dexamethasone treatment during pregnancy decreases I_0–40 by 51% (P < 0.05) and steady-state plasma insulin levels by 57% (P < 0.05) to values not different from those of the Dex group. Thus, dexamethasone treatment and pregnancy do not exert additive effects on GSIS in vivo. We infused insulin at a fixed rate (4 mU · kg^–1 · min^–1), while maintaining glycemia, to determine whether decreased insulin levels in the 19dP+Dex group, compared with the 19dP group, reflected increased insulin clearance. The coefficients of variance for glycemia were <15%, and steady-state glycemia attained during euglycemic hyperinsulinemia were similar in the control and dexamethasone-treated groups of pregnant rats (data not shown). At similar steady-state glycemia, steady-state plasma insulin concentrations during insulin infusion did not differ significantly between control and dexamethasone-treated dams (19dP 69 ± 7 µU/ml [n = 10] vs. 19dP+Dex 67 ± 5 µU/ml [n = 6]). Hence, insulin clearance is unaffected by dexamethasone treatment during pregnancy (19dP 22.3 ± 2.2 ml/min [n = 10] vs. 19dP+Dex 18.6 ± 1.1 ml/min [n = 6]).

View larger version (35K): [in this window] [in a new window]   FIG. 3. Dexamethasone (Dex) treatment augments insulin secretion during hyperglycemic clamp in vivo in nonpregnant but not in late (19 day)-pregnant rats. Blood samples were withdrawn from nonpregnant control rats (open symbols/bars) or dexamethasone-treated nonpregnant rats (Dex group; closed symbols/bars) (A–C) or untreated 19-day pregnant rats (19dP; open symbols/bars) or dexamethasone-treated 19-day pregnant rats (19dP+Dex; closed symbols/bars) (D–F) at intervals during sustained intravenous glucose infusion for measurement of blood glucose (A and D) and plasma insulin concentrations (B and E). Mean steady-state blood glucose concentrations over the period of 20–40 min after initiating glucose infusion were: control 10.7 ± 0.8 mmol/l (5), Dex 10.8 ± 0.4 mmol/l (5), 19P 9.2 ± 0.3 mol/l (6), and 19P+Dex 10.5 ± 0.2 mmol/l (5). Corresponding steady-state plasma insulin concentrations were: control 101 ± 9 µU/ml (5), Dex 258 ± 41 µU/ml (5), 19P 416 ± 77 µU/ml (6), and 19P+Dex 179 ± 33 µU/ml (4). Incremental plasma insulin values integrated over the period of 0–40 min after the initiating hyperglycemia (I0–40) are also shown (C and F). Data are means ± SE for five control, five Dex, six 19P, or five 19P+Dex rats. Statistically significant effects of dexamethasone treatment are indicated by *P < 0.05. Statistically significant effects of pregnancy are reported in the text.

	DISCUSSION

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

Pregnancy is a lifestyle factor that leads to adaptive changes in pancreatic ß-cell function, including increased GSIS and a reduction in the glucose-stimulated threshold for insulin secretion. The latter is thought to be the primary mechanism by which the pancreatic ß-cells can release significantly more insulin at low blood glucose concentrations, resulting in an altered set point for insulin secretion and action during late pregnancy. We have previously observed that PPAR activation fails to impact on the characteristics of GSIS at day 15 of pregnancy (36). In contrast, in the present study during late pregnancy (day 19; term = 22–23 days), PPAR activation both increased the glucose threshold for GSIS and lowered glucose responsiveness. There is therefore a shift in the functional characteristics of GSIS, with respect to PPAR activation, as pregnancy proceeds toward term. Importantly, PPAR activation later in pregnancy leads to a pattern of GSIS at day 19 of pregnancy (i.e., late gestation, seen in the present study) reminiscent of that seen earlier at day 15 of pregnancy (36). This suggests that the enhancement of GSIS observed at day 19 of pregnancy compared with day 15 of pregnancy is opposed by increased PPAR signaling. It is likely that the effects of PPAR activation on GSIS in late pregnancy observed in the present studies reflect changes in the acute stimulation of the release of stored insulin from docked granules by the ATP-sensitive K⁺ channel–dependent mechanism; however, we were unable to detect a clear first- and second-phase secretion under either the conditions of the rat islet perifusions in vitro or in vivo during long-term hyperglycemic clamp studies.

Glucocorticoid concentrations increase during late pregnancy to assist maturation of fetal tissue function (32). We aimed to establish whether increased exposure to glucocorticoids might contribute to the mechanism of this phenotypic shift in the characteristics of GSIS. Glucocorticoid treatment from day 14 to day 19 of pregnancy in the absence of PPAR activation appeared to exert only a minimal effect on the characteristic pattern of the GSIS profile ex vivo with perifused islets from late pregnant rats; a lowered glucose threshold and increased glucose responsiveness continued to be observed. However, following antecedent treatment with exogenous glucocorticoids, the glucose responsiveness of perifused islets of late-pregnant rats became refractory to suppression by PPAR activation, as is the increased glucose responsiveness observed with perifused islets from nonpregnant rats treated with dexamethasone or with islets from pregnant rats at day 15 of pregnancy (36). Dexamethasone has been shown by others to exert a significant inhibitory effect on insulin secretion by islets from neonatal rats previously cultured with lactogens (26). Our experiments also demonstrated that glucocorticoid treatment suppresses GSIS in vivo during hyperglycemic clamps during late pregnancy to levels of GSIS elicited by glucocorticoid treatment of nonpregnant rats. The use of islet perifusions eliminates neuro, hormonal, and metabolic influences on GSIS. The disparity between the data obtained in vivo (suppression of GSIS) and in vitro with perifused islets (unimpaired GSIS) in the pregnant rats may reflect that dexamethasone-induced insulin resistance causes islet adaptations to augment GSIS, which are stable to islet isolation and perifusion, but that GSIS is suppressed in vivo by one or more neuro/hormonal influences that prevents the full function of these adaptations. Based on these data, we propose that increased exposure to glucocorticoids, as the duration of pregnancy proceeds beyond 19 days toward term, could have a physiological function in that islet glucose responsiveness is increasingly and selectively governed by the maternal glucocorticoid level rather than "pregnancy adaptations" introduced by placental lactogens and prolactin, which occur earlier during gestation. This may allow a smoother transition of maternal islet function from the metabolic demands imposed by pregnancy to those imposed after parturition.

	ACKNOWLEDGMENTS

 
We are grateful to the Wellcome Trust (060965/Z/00) and Diabetes U.K. (BDA:RD01/0002345) for financial support. G.K.G. was a recipient of a Diabetes U.K. studentship.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication May 15, 2006 and accepted in revised form September 6, 2006

	REFERENCES

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Aoyama T, Peters JM, Iritani N, Nakajima T, Furihata K, Hashimoto T, Gonzalez FJ: Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem 273:5678–5684, 1998[Abstract/Free Full Text]
2. Kersten S, Seydoux J, Peters JM, Gonzalez FJ, Desvergne B, Wahli W: Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting. J Clin Invest 103:1489–1498, 1999[Medline]
3. Zhou YT, Shimabukuro M, Wang MY, Lee Y, Higa M, Milburn JL, Newgard CB, Unger RH: Role of peroxisome proliferator-activated receptor alpha in disease of pancreatic beta cells. Proc Natl Acad Sci U S A 95:8898–8903, 1998[Abstract/Free Full Text]
4. Tordjman K, Standley KN, Bernal-Mizrachi C, Leone TC, Coleman T, Kelly DP, Semenkovich CF: PPARalpha suppresses insulin secretion and induces UCP2 in insulinoma cells. J Lipid Res 43:936–943, 2002[Abstract/Free Full Text]
5. Gremlich S, Nolan C, Roduit R, Burcelin R, Peyot ML, Delghingaro-Augusto V, Desvergne B, Michalik L, Prentki M, Wahli W: Pancreatic islet adaptation to fasting is dependent on peroxisome proliferator-activated receptor alpha transcriptional up-regulation of fatty acid oxidation. Endocrinology 146:375–382, 2005[Abstract/Free Full Text]
6. Ravnskjaer K, Boergesen M, Rubi B, Larsen JK, Nielsen T, Fridriksson J, Maechler P, Mandrup S: Peroxisome proliferator-activated receptor alpha (PPARalpha) potentiates, whereas PPARgamma attenuates, glucose-stimulated insulin secretion in pancreatic beta-cells. Endocrinology 146:3266–3276, 2005[Abstract/Free Full Text]
7. Sugden MC, Bulmer K, Gibbons GF, Knight BL, Holness MJ: Peroxisome-proliferator-activated receptor-alpha (PPARalpha) deficiency leads to dysregulation of hepatic lipid and carbohydrate metabolism by fatty acids and insulin. Biochem J 364:361–368, 2002[Medline]
8. Holness MJ, Bulmer K, Smith ND, Sugden MC: Investigation of potential mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase isoforms 2 and 4 by fatty acids and thyroid hormone. Biochem J 369:687–695, 2003[Medline]
9. Holness MJ, Bulmer K, Gibbons GF, Sugden MC: Up-regulation of pyruvate dehydrogenase kinase isoform 4 (PDK4) protein expression in oxidative skeletal muscle does not require the obligatory participation of peroxisome-proliferator-activated receptor alpha (PPARalpha). Biochem J 366:839–846, 2002[Medline]
10. Holness MJ, Smith ND, Bulmer K, Hopkins T, Gibbons GF, Sugden MC: Evaluation of the role of peroxisome-proliferator-activated receptor alpha in the regulation of cardiac pyruvate dehydrogenase kinase 4 protein expression in response to starvation, high-fat feeding and hyperthyroidism. Biochem J 364:687–694, 2002[Medline]
11. Sugden MC, Bulmer K, Augustine D, Holness MJ: Selective modification of pyruvate dehydrogenase kinase isoform expression in rat pancreatic islets elicited by starvation and activation of peroxisome proliferator–activated receptor-: implications for glucose-stimulated insulin secretion. Diabetes 50:2729–2736, 2001[Abstract/Free Full Text]
12. Sugden MC, Bulmer K, Gibbons GF, Holness MJ: Role of peroxisome proliferator-activated receptor-alpha in the mechanism underlying changes in renal pyruvate dehydrogenase kinase isoform 4 protein expression in starvation and after refeeding. Arch Biochem Biophys 395:246–252, 2001[Medline]
13. Sugden MC, Holness MJ: Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs. Am J Physiol Endocrinol Metab 284:E855–E862, 2003[Abstract/Free Full Text]
14. MacDonald MJ, Fahien LA, Brown LJ, Hasan NM, Buss JD, Kendrick MA: Perspective: emerging evidence for signaling roles of mitochondrial anaplerotic products in insulin secretion. Am J Physiol Endocrinol Metab 288:E1–E15, 2005[Abstract/Free Full Text]
15. Lu D, Mulder H, Zhao P, Burgess SC, Jensen MV, Kamzolova S, Newgard CB, Sherry AD: 13C NMR isotopomer analysis reveals a connection between pyruvate cycling and glucose-stimulated insulin secretion (GSIS). Proc Natl Acad Sci U S A 99:2708–2713, 2002[Abstract/Free Full Text]
16. Holness MJ, Smith ND, Greenwood GK, Sugden MC: Acute (24 h) activation of peroxisome-proliferator-activated receptor (PPAR) reverses high-fat feeding induced insulin hypersecretion in vivo and in perifused pancreatic islets. J Endocrinol 177:197–205, 2003[Abstract]
17. Holness MJ, Smith ND, Greenwood GK, Sugden MC: Interactive influences of peroxisome proliferator-activated receptor alpha activation and glucocorticoids on pancreatic beta cell compensation in insulin resistance induced by dietary saturated fat in the rat. Diabetologia 48:2062–2068, 2005[Medline]
18. Muoio DM, Newgard CB: Obesity-related derangements in metabolic regulation. Annu Rev Biochem 75:367–401, 2006[Medline]
19. Phillips DI, Barker DJ, Fall CH, Seckl JR, Whorwood CB, Wood PJ, Walker BR: Elevated plasma cortisol concentrations: a link between low birth weight and the insulin resistance syndrome? J Clin Endocrinol Metab 83:757–760, 1998[Abstract/Free Full Text]
20. Rosmond R, Dallman MF, Bjorntorp P: Stress-related cortisol secretion in men: relationships with abdominal obesity and endocrine, metabolic and hemodynamic abnormalities. J Clin Endocrinol Metab 83:1853–1859, 1998[Abstract/Free Full Text]
21. Stojanovska L, Rosella G, Proietto J: Evolution of dexamethasone-induced insulin resistance in rats. Am J Physiol Endocrinol Metab 258:E748–E756, 1990[Abstract/Free Full Text]
22. Severino C, Brizzi P, Solinas A, Secchi G, Maioli M, Tonolo G: Low-dose dexamethasone in the rat: a model to study insulin resistance. Am J Physiol Endocrinol Metab 283:E367–E373, 2002[Abstract/Free Full Text]
23. Bernal-Mizrachi C, Weng S, Feng C, Finck BN, Knutsen RH, Leone TC, Coleman T, Mecham RP, Kelly DP, Semenkovich CF: Dexamethasone induction of hypertension and diabetes is PPAR-alpha dependent in LDL receptor-null mice. Nat Med 9:1069–1075, 2003[Medline]
24. Huang B, Wu P, Bowker-Kinley MM, Harris RA: Regulation of pyruvate dehydrogenase kinase expression by peroxisome proliferator–activated receptor- ligands, glucocorticoids, and insulin. Diabetes 51:276–283, 2002[Abstract/Free Full Text]
25. Holness MJ, Sugden MC: Dexamethasone during late gestation exacerbates peripheral insulin resistance and selectively targets glucose-sensitive functions in beta cell and liver. Endocrinology 142:3742–3748, 2001[Abstract/Free Full Text]
26. Weinhaus AJ, Bhagroo NV, Brelje TC, Sorenson RL: Dexamethasone counteracts the effect of prolactin on islet function: implications for islet regulation in late pregnancy. Endocrinology 141:1384–1393, 2000[Abstract/Free Full Text]
27. Weinhaus AJ, Stout LE, Sorenson RL: Glucokinase, hexokinase, glucose transporter 2, and glucose metabolism in islets during pregnancy and prolactin-treated islets in vitro: mechanisms for long term up-regulation of islets. Endocrinology 137:1640–1649, 1996[Abstract]
28. Weinhaus AJ, Bhagroo NV, Brelje TC, Sorenson RL: Role of cAMP in upregulation of insulin secretion during the adaptation of islets of Langerhans to pregnancy. Diabetes 47:1426–1435, 1998[Abstract/Free Full Text]
29. Vasavada RC, Garcia-Ocana A, Zawalich WS, Sorenson RL, Dann P, Syed M, Ogren L, Talamantes F, Stewart AF: Targeted expression of placental lactogen in the beta cells of transgenic mice results in beta cell proliferation, islet mass augmentation, and hypoglycemia. J Biol Chem 275:15399–15406, 2000[Abstract/Free Full Text]
30. Bihan H, Rouault C, Reach G, Poitout V, Staels B, Guerre-Millo M: Pancreatic islet response to hyperglycemia is dependent on peroxisome proliferator-activated receptor alpha (PPARalpha). FEBS Lett 579:2284–2288, 2005[Medline]
31. Sorenson RL, Brelje TC: Adaptation of islets of Langerhans to pregnancy: beta-cell growth, enhanced insulin secretion and the role of lactogenic hormones. Horm Metab Res 29:301–307, 1997[Medline]
32. Fowden AL, Li J, Forhead AJ: Glucocorticoids and the preparation for life after birth: are there long-term consequences of the life insurance? Proc Nutr Soc 57:113–122, 1998[Medline]
33. Langdown ML, Smith ND, Sugden MC, Holness MJ: Excessive glucocorticoid exposure during late intrauterine development modulates the expression of cardiac uncoupling proteins in adult hypertensive male offspring. Pflugers Arch 442:248–255, 2001[Medline]
34. Holness MJ: Impact of early growth retardation on glucoregulatory control and insulin action in mature rats. Am J Physiol Endocrinol Metab 270:E946–E954, 1996[Abstract/Free Full Text]
35. Holness MJ, Sugden MC: Antecedent protein restriction exacerbates development of impaired insulin action after high-fat feeding. Am J Physiol Endocrinol Metab 276:E85–E93, 1999[Abstract/Free Full Text]
36. Sugden MC, Greenwood GK, Smith ND, Holness MJ: Peroxisome proliferator-activated receptor-alpha activation during pregnancy attenuates glucose-stimulated insulin hypersecretion in vivo by increasing insulin sensitivity, without impairing pregnancy-induced increases in beta-cell glucose sensing and responsiveness. Endocrinology 144:146–153, 2003[Abstract/Free Full Text]
37. Brun S, Carmona MC, Mampel T, Vinas O, Giralt M, Iglesias R, Villarroya F: Activators of peroxisome proliferator–activated receptor- induce the expression of the uncoupling protein-3 gene in skeletal muscle: a potential mechanism for the lipid intake-dependent activation of uncoupling protein-3 gene expression at birth. Diabetes 48:1217–1222, 1999[Abstract]
38. Soria A, Bocos C, Herrera E: Opposite metabolic response to fenofibrate treatment in pregnant and virgin rats. J Lipid Res 43:74–81, 2002[Abstract/Free Full Text]
39. Kwon HS, Harris RA: Mechanisms responsible for regulation of pyruvate dehydrogenase kinase 4 gene expression. Adv Enzyme Regul 44:109–121, 2004[Medline]
40. Karlsson S, Ostlund B, Myrsen-Axcrona U, Sundler F, Ahren B: Beta cell adaptation to dexamethasone-induced insulin resistance in rats involves increased glucose responsiveness but not glucose effectiveness. Pancreas 22:148–156, 2001[Medline]
41. Langdown ML, Holness MJ, Sugden MC: Early growth retardation induced by excessive exposure to glucocorticoids in utero selectively increases cardiac GLUT1 protein expression and Akt/protein kinase B activity in adulthood. J Endocrinol 169:11–22, 2001[Abstract]
42. Dobbins RL, Chester MW, Stevenson BE, Daniels MB, Stein DT, McGarry JD: A fatty acid-dependent step is critically important for both glucose- and non-glucose-stimulated insulin secretion. J Clin Invest 101:2370–2376, 1998[Medline]
43. Dobbins RL, Chester MW, Daniels MB, McGarry JD, Stein DT: Circulating fatty acids are essential for efficient glucose-stimulated insulin secretion after prolonged fasting in humans. Diabetes 47:1613–1618, 1998[Abstract]

CiteULike

Del.icio.us

Digg

Reddit

Technorati

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Holness, M. J. Articles by Sugden, M. C. Search for Related Content PubMed PubMed Citation Articles by Holness, M. J. Articles by Sugden, M. C. Social Bookmarking                What's this?