Peroxisome Proliferator-Activated Receptor- Activation during Pregnancy Attenuates Glucose-Stimulated Insulin Hypersecretion in Vivo by Increasing Ins
Abstractd!p, http://www.100md.com
We investigated the effects of acute (24-h) peroxisome proliferator-activated receptor (PPAR) activation by WY14,643 (pirinixic acid) treatment on glucose-stimulated insulin secretion (GSIS) during pregnancy, in the rat, in relation to insulin sensitivity. GSIS after iv glucose challenge (500 mg/kg) was increased at d 15 of pregnancy but was attenuated by WY14,643 treatment in vivo, with decreases in acute insulin response (51%; P < 0.001) and total suprabasal 30-min area under the insulin curve (I) (55%; P < 0.001). GSIS was unaffected by WY14,643 treatment in unmated rats. Islet perifusions were employed to identify persistent effects of PPAR{alpha} activation. GSIS was enhanced, and the glucose threshold was reduced in perifused islets from pregnant rats, but WY14,643 treatment failed to reverse these effects. WY14,643 treatment of 15-d-pregnant rats significantly lowered (by 63%; P < 0.01) the insulin resistance index [total suprabasal 30-min area under insulin curve x suprabasal 30-min area under glucose curve (IxG)]. A strong positive linear relationship (r = 0.92) between acute insulin response and IxG was evident between groups. Our studies show that acute PPAR activation reverses the augmented GSIS evoked by pregnancy in vivo, whereas the isolated islets retain pregnancy-induced enhancement of ß-cell glucose sensing and responsiveness. Normalization of maternal GSIS to that found in the nonpregnant state is observed in association with alleviation of maternal insulin resistance.
Introductionnm7:n, 百拇医药
ABNORMAL LIPID PARTITIONING, resulting in either depletion of islet lipid or accumulation of excessive islet lipid, is important for the development of pancreatic ß-cell failure (1). Long-term lipid sensing by the pancreatic ß-cell is coordinated through peroxisome proliferator-activated receptors (PPARs) (2, 3). PPAR{alpha} is expressed in islets and islet cell lines, and it controls genes involved in lipid metabolism, in particular those implicated in fatty acid (FA) transport and ß-oxidation (2, 3). Islet expression of PPAR{alpha} and enzymes of FA oxidation are induced by high FA concentrations (3) and exposure to PPAR{alpha} receptor ligands (3, 4). Signaling via PPAR{alpha} also influences whole-body insulin sensitivity, particularly under conditions where peripheral insulin action is impaired because of increased lipid delivery. Thus, chronic (15-d) administration of the PPAR{alpha} activator cipofibrate to 5-wk-old obese Zucker rats reduces plasma insulin concentrations without causing glucose intolerance during an iv glucose tolerance test, suggesting improved insulin action (5). Similarly, administration of fenofibrate, as part of the diet, reverses basal hyperinsulinemia and hyperglycemia in high-fat-fed C57BL/6 mice (5) and WY14,643 [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid] increases insulin sensitivity in high-fat-fed rats (6). It was suggested that PPAR{alpha} activation opposes the development of peripheral insulin resistance by relieving lipid-mediated inhibition of insulin-stimulated glucose disposal (6).
Pregnancy leads to adaptations of maternal carbohydrate metabolism, including a progressive state of maternal insulin resistance, to confer a competitive advantage to the developing fetus (7, 8, 9, 10, 11, 12, 13, 14). As in other insulin-resistant states, mid-late pregnancy is associated with elevated maternal circulating triacylglycerol (TAG) levels (15). Maternal hypertriglyceridemia is caused by enhanced maternal adipose tissue lipolysis (16, 17, 18), enhanced maternal hepatic very low density lipoprotein production (19, 20), and decreased maternal extrahepatic lipoprotein lipase activity (21, 22). Maternal insulin resistance is observed in conjunction with enhanced pancreatic ß-cell insulin secretion in response to glucose and a lowered threshold for stimulation of insulin secretion by glucose (reviewed in Ref. 23). Treatment of pregnant rats with fenofibrate, for 1–2 d from d 16 of pregnancy, decreases maternal hypertriglyceridemia (24). Possible effects of PPAR{alpha} activation on maternal glucose-insulin homeostasis have not been delineated.
In the present study, we investigated the effects of PPAR{alpha} activation by WY14,643 in vivo on the modulation of insulin secretion and sensitivity that is observed during mid-late pregnancy in the rat. Because maternal triglyceridemia becomes refractory to fenofibrate if treatment is continued beyond 48 h (24), and because 24 h of WY14,643 treatment is adequate to enhance the expression of islet PPAR{alpha} and PPAR{alpha} -linked enzymes in vitro (25) and in vivo (4), WY14,643 was administered for 24 h only. Studies were conducted in the intact animal at d 15 of pregnancy (term is 23 d). In addition, studies of glucose-stimulated insulin secretion (GSIS) were conducted ex vivo using islet perifusions.kdy, 百拇医药
Materials and Methodskdy, 百拇医药
Materialskdy, 百拇医药
Analytical-grade organic solvents were obtained from BDH (Poole, Dorset, UK). General laboratory reagents were purchased from Roche Diagnostics Ltd. (Lewes, East Sussex, UK) or from Sigma (Poole, Dorset, UK). WY14,643 was purchased from Sigma. Kits for determination of insulin and glucose concentrations were purchased from Mercodia (Uppsala, Sweden) and Sigma, respectively. Kits for determination of plasma nonesterified FA (NEFA) and TAG concentrations were purchased from Alpha Labs (Eastleigh, Hants, UK).
Animals\}?, http://www.100md.com
All studies were conducted in adherence to the regulations of the United Kingdom Animal Scientific Procedures Act (1986). Female albino Wistar rats (200–250 g) were purchased from Charles River Laboratories, Inc. (Margate, Kent, UK). Rats were maintained at a temperature of 22 ± 2 C and subjected to a 12-h light, 12-h dark cycle. Rats were given free access to standard, pelleted rodent diet purchased from Special Diet Services (Witham, Essex, UK) [52% carbohydrate, 15% protein, 3% lipid, and 30% nondigestible residue (by wt); 2.61 kcal metabolizable energy/g]. Rats were time-mated by the appearance of sperm plugs (d 0 of pregnancy). Pregnant rats with less than 8 fetuses were not included in the study. WY14,643 was administered to unmated and 14-d-pregnant rats with free access to diet, as a single ip injection (50 mg/kg body wt.); and rats were sampled after a further 24 h (26). Control, unmated, and 14-d-pregnant rats were injected with vehicle. In all experiments, rats were allowed access ad libitum to water.
The iv glucose challenge;?4g, http://www.100md.com
Glucose was administered as an iv bolus (0.5-g glucose/kg body weight; 150 µl per 100 g body weight) to conscious, unrestrained rats (see Ref. 27). Glucose was injected via a chronic indwelling jugular cannula, and blood samples (100 µl) were withdrawn at intervals 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 with ZnSO4/Ba(OH)2, centrifuged (10,000 x g) at 4 C, and the supernatant was retained for subsequent assay of blood glucose. The remaining blood sample was immediately centrifuged (10,000 x g) at 4 C, and plasma was stored at -20 C until assayed for insulin. The calculated acute insulin response (AIR) was calculated as the mean of suprabasal 2- and 5-min plasma insulin values. Insulin and glucose responses during the glucose tolerance test were used for calculation of the incremental plasma insulin values integrated over the 30-min period after the injection of glucose (I) and the corresponding incremental integrated plasma glucose values (). The insulin resistance index (IR index) was calculated as the product of IxG after glucose challenge. The rate of glucose disappearance (k) was calculated from the slope of the regression line obtained with log-transformed glucose values from 2–15 min after glucose administration.
Islet isolation and perifusiono, 百拇医药
Rats were anesthetized by injection of sodium pentobarbital (60 mg/ml in 0.9% NaCl; 1 ml/kg body wt, ip); and, once locomotor activity had ceased, pancreases were excised and islets were isolated by collagenase digestion (28). Free islets were collected, under a dissecting microscope, with a 20 µl pipette, into HEPES-buffered Hanks’ balanced salts solution containing 5% BSA. Insulin release from freshly isolated islets was measured in a perifusion system as described by Hughes et al., 1992 (29). In this system, 50 islets were housed in small chambers on Millicell culture inserts. Islets were perifused in basal medium (Krebs-Ringer containing 20 mM HEPES, pH 7.4; 5 mg/ml BSA; and 2 mM glucose), for 60 min at a flow rate of 1 ml/min at 37 C, before collection of fractions. Glucose concentrations were then modified as indicated. Fractions (2 ml) were collected at 2-min intervals and stored at -20 C before assay for insulin.o, 百拇医药
Analytical methods\, 百拇医药
Plasma glucose concentrations were determined by a glucose oxidase method (Sigma). Plasma immunoreactive insulin concentrations were measured by ELISA, using rat insulin as a standard (Mercodia). Plasma NEFA and TAG levels were determined with commercial kits (Alpha Labs).\, 百拇医药
Statistical analysis\, 百拇医药
Results are presented as the mean ± SEM, with the numbers of rats or islet preparations in parentheses. Statistical analysis was performed by ANOVA followed by Fisher’s post hoc tests for individual comparisons or Student’s t test, as appropriate (Statview; Abacus Concepts, Inc., Berkeley, CA). A P value < 0.05 was considered to be statistically significant.\, 百拇医药
Results\, 百拇医药
Effect of acute PPAR activation, for 24 h, in vivo, on food intake and body weight gain in unmated and pregnant rats\, 百拇医药
Food intakes and body weight gain of untreated and WY14,643-treated unmated and 15-d-pregnant rats over the period of WY14,643 treatment are shown in Table 1. WY14,643 treatment did not significantly affect either food intake or body weight gain in unmated rats (Table 1). Pregnancy was associated with hyperphagia and a progressive increase in body weight from d 0-d 15 of pregnancy (results not shown; see also Ref. 30). Body weights of untreated dams increased by 3% from d 14–15 of pregnancy, and this was accompanied by significantly higher (40%; P < 0.05) food intake, compared with unmated rats. Acute (24-h) PPAR{alpha} activation in vivo did not influence maternal food intake or body weight gain (Table 1), and fetal numbers per dam were unchanged [control, 14 ± 1 (n = 6 litters); WY14,643-treated, 14 ± 1 (n = 10 litters); not significant (NS)].
fig.ommitteed!^ogp, 百拇医药
Table 1. Effects of 24-h WY14,643 treatment on food intake and body weight gain and on postabsorptive circulating insulin and glucose concentrations and the insulin-to-glucose ratio in unmated and 15-d-pregnant rats!^ogp, 百拇医药
Acute PPAR{alpha} activation, for 24 h, in vivo, leads to modest hypoinsulinemia in 15-d-pregnant rats in the postabsorptive state!^ogp, 百拇医药
Plasma insulin concentrations were lower (by 32%) in WY14,643-treated dams, compared with the untreated pregnant group in the postabsorptive state, reversing the effect of pregnancy to increase basal insulin concentrations by 58% (Table 1). This relative hypoinsulinemia was not accompanied by postabsorptive hyperglycemia in the WY14,643-treated pregnant group (Table 1). The insulin-to-glucose ratio, which is an index of the relative response of insulin to fasting glycemia, was therefore lower (by 27%) in the WY14,643-treated pregnant group, compared with the untreated pregnant group, such that the effect of pregnancy to increase the basal insulin-to-glucose ratio was reversed by 60% (Table 1). WY14,643 treatment of nonpregnant rats also modestly lowered postabsorptive plasma insulin concentrations, compared with those found in nonpregnant control rats; and again, postabsorptive glycemia was unchanged (Table 1).
Effect of acute PPAR{alpha} activation, for 24 h, in vivo, on maternal plasma NEFA and TAG concentrations at d 15 of pregnancyw!, 百拇医药
We analyzed the effects of acute (24-h) PPAR{alpha} activation in vivo on circulating lipid-fuel concentrations in postabsorptive unmated and 15-d-pregnant rats, in relation to plasma insulin levels (Fig. 1). As expected, plasma NEFA (Fig. 1A) concentrations in the postabsorptive state were increased in 15-d-pregnant rats, compared with unmated controls (by 3.1-fold; P < 0.01). Furthermore, plasma NEFA levels in pregnant rats were higher than would be predicted from the relationship between NEFA and insulin levels in unmated rats (Fig. 1A) confirming the existence of maternal insulin resistance, with respect to adipose-tissue antilipolysis, in late pregnancy. Treatment with WY14,643 failed to modify plasma NEFA concentrations in unmated rats but partially reversed the pregnancy-induced increase in NEFA concentrations (by 34%). Nevertheless, plasma NEFA levels in postabsorptive WY14,643-treated pregnant rats remained significantly higher than plasma NEFA levels in either postabsorptive untreated controls (2.4-fold; P < 0.001) or postabsorptive WY14,643-treated unmated rats (by 2.2-fold: P < 0.01) (Fig. 1A). Plasma TAG concentrations in the postabsorptive state were increased in 15-d-pregnant rats, compared with unmated controls (by 1.4-fold; P < 0.05) (Fig. 1B). Treatment with WY14,643 led to a small and nonsignificant (17%) decline in plasma TAG concentrations in postabsorptive untreated controls. As expected for rats at this stage of pregnancy (24), plasma TAG levels were lowered (by 23%) by PPAR{alpha} activation, in vivo, for 24 h, in 15-d-pregnant rats (Fig. 1B), reversing the pregnancy-induced elevation in plasma TAG levels by 81%. As a result, plasma TAG concentrations in unmated and 15-d-pregnant rats were no longer statistically significantly different after 24-h treatment with WY14,643 (Fig. 1B). WY14,643 treatment did not modify plasma 3-hydroxybutyrate levels in either pregnant or unmated rats (results not shown).
fig.ommitteedh7/tv's, 百拇医药
Figure 1. Acute (24-h) PPAR{alpha} activation in vivo lowers plasma TAG but does not affect plasma NEFA concentrations in 15-d-pregnant rats. Blood samples were withdrawn from untreated (C, open circles) or WY14,643-treated (C+WY, closed circles) unmated, or untreated (P, open triangles) or WY14,643-treated (P+WY, closed triangles) 15-d-pregnant conscious, unrestrained rats in the postabsorptive state, for measurement of plasma NEFA (A) and TAG (B) concentrations, using commercial kits. Results are means ± SEM for 11 untreated unmated rats, 5 untreated 15-d-pregnant rats, 10 WY14,643-treated unmated rats, or 8 WY14,643-treated 15-d-pregnant rats. Statistically significant effects of pregnancy are indicated by: *, P < 0.05; ***, P < 0.001. There were no statistically significant effects of WY14,643 treatment.h7/tv's, 百拇医药
GSIS in vivo is enhanced in conjunction with the existence of insulin resistance at d 15 of pregnancyh7/tv's, 百拇医药
The calculated AIR (i.e. the means of suprabasal 2- and 5-min plasma insulin) and I after iv administration of glucose (500 mg/kg) in 15-d-pregnant and unmated rats are shown in Fig. 2, A and B, respectively. AIR and I after glucose administration were significantly increased at d 15 of pregnancy, compared with unmated female rats [by 2.3-fold (P < 0.001) and 2.5-fold (P < 0.001), respectively].
fig.ommitteedq, 百拇医药
Figure 2. Acute (24-h) PPAR activation in vivo markedly lowers glucose-stimulated hypersecretion of insulin after iv glucose challenge in 15-d-pregnant rats. Glucose was administered as an iv bolus (0.5-g glucose/kg body weight) to untreated or WY14,643-treated unmated (C, open bars) or 15-d-pregnant (P, closed bars) conscious, unrestrained rats in the postabsorptive state. Blood samples were withdrawn, at intervals, for measurement of plasma insulin, using a commercial kit. AIR values, calculated as the mean of suprabasal 2- and 5-min plasma insulin, are shown in A. Insulin responses during the glucose tolerance test were used for calculation of the incremental plasma insulin values integrated over 30 min, I, shown in B. Results are means ± SEM for 9 untreated unmated rats, 5 untreated 15-d-pregnant rats, 8 WY14,643-treated unmated rats, or 7 WY14,643-treated 15-d-pregnant rats. Statistically significant effects of pregnancy are indicated by: ***, P < 0.001. Statistically significant effects of WY14,643 treatment are indicated by: , P < 0.001.q, 百拇医药
Acute PPAR{alpha} activation, for 24 h, in vivo, markedly and selectively lowers GSIS after iv glucose challenge in 15-d-pregnant rats
We analyzed the effects of acute (24-h) PPAR{alpha} activation in vivo on insulin secretion in unmated and 15-d-pregnant rats, in response to an acute glucose challenge. In unmated rats, 24-h treatment with WY14,643 did not significantly affect AIR or I (Fig. 2, A and B). By contrast, the relative response of insulin to a rapid increase in glycemia in the 15-d-pregnant group was greatly lowered by 24-h treatment with WY14,643, as reflected by substantial decreases in both AIR (51%; P < 0.001) and I (55%; P < 0.001). Thus, WY14,643 treatment reversed the effect of pregnancy to elevate AIR and I by 92% and 91%, respectively. It is concluded that acute (24-h) PPAR{alpha} activation in vivo greatly attenuates differences in insulin secretion in vivo in response to iv glucose challenge induced by pregnancy. Furthermore, activation of PPAR{alpha} by WY14,643 administration only affected hypersecretion of insulin, because GSIS was unaffected by WY14,643 treatment in control (unmated) rats (Fig. 2, A and B).
Relationship between changes in insulin secretion and action after PPAR{alpha} activation in pregnancy?@&c, 百拇医药
Failure of the pancreatic ß-cells to compensate for insulin resistance is critical to the pathology of type 2 diabetes (reviewed in Ref. 31). In healthy individuals, a hyperbolic relationship has been observed between insulin sensitivity and secretion (32, 33). Pregnancy-induced hyperinsulinemia is also considered as a compensatory response to the development of insulin resistance. The development of insulin resistance in healthy pregnant women is accompanied by a reciprocal increase in insulin secretion (34). The IR index IxG was significantly increased by 3.8-fold at d 15 of pregnancy (P < 0.001; Fig. 3A). Acute (24-h) treatment of 15-d-pregnant rats with WY14,643 did not significantly affect G (untreated, 12.8 ± 0.5 mmol/min·liter; WY14,643-treated, 12.3 ± 1.1 mmol/min·liter; NS) and only slightly decreased (by 13%) the rate of glucose disappearance calculated over the first 15 min after iv glucose injection (untreated, 3.7 ± 0.3%/min; WY14,643-treated, 3.2 ± 0.5%/min; NS). By contrast, IxG was significantly lowered (by 63%; P < 0.001) by WY14,643 treatment in 15-d-pregnant rats (Fig. 3A), reversing the pregnancy-induced increase in the IxG by 86%. Hence, acute (24-h) PPAR activation significantly improves insulin sensitivity in the pregnant group. Importantly, the positive linear relationship (r = 0.92) between AIR and IR index, shown in Fig. 3B, demonstrates that insulin secretion in vivo in pregnancy can be acutely modulated in concert with the requirement for insulin-stimulated glucose disposal.
fig.ommitteed85w}qh, http://www.100md.com
Figure 3. Acute (24-h) PPAR activation in vivo markedly lowers IxG in 15-d-pregnant rats. Glucose was administered as an iv bolus (0.5-g glucose/kg body weight) to untreated or WY14,643-treated unmated (C, open bars) or 15-d-pregnant (P, closed bars) conscious, unrestrained rats in the postabsorptive state. Blood samples were withdrawn, at intervals, for measurement of plasma insulin and blood glucose, using commercial kits. Insulin and glucose responses during the glucose tolerance test were used for calculation of the incremental plasma insulin values integrated over 30 min, I and the corresponding incremental integrated plasma glucose values (G). The IR index IxG, calculated as the product of the areas under the glucose and insulin curves after glucose challenge, are shown in A. The relationship between AIR and IxG in untreated or WY14,643-treated unmated rats (circles) or 15-d-pregnant rats (triangles) in the postabsorptive state is shown in panel B. Results are means ± SEM for 8 untreated control rats, 4 untreated 15-d-pregnant rats, 8 WY14,643-treated control rats, or 6 WY14,643-treated 15-d-pregnant rats. Statistically significant effects of pregnancy are indicated by: ***, P < 0.001. Statistically significant effects of WY14,643 treatment are indicated by: , P < 0.001.
Islets from 15-d-pregnant rats exhibit enhanced glucose sensitivity and responsiveness of insulin secretion during perifusion ex vivo2rdo\(, http://www.100md.com
The preceding findings raise the question of whether the reversal of insulin hypersecretion in pregnant rats in vivo, achieved by activation of PPAR by WY14,643 treatment, represents a stable modification of islet function or is an indirect response of the islets to the improvement in whole-body insulin sensitivity. We therefore tested whether the effect of pregnancy to alter pancreatic ß-cell sensitivity and responsiveness to glucose is modified by acute (24-h) activation of PPAR, by measuring insulin release by perifused islets. Overall patterns of insulin release during stepwise glucose perifusion, designed to generate a steady rise in perifusate glucose concentrations to a target concentration of 6.8 mM, then a rise in perifusate glucose concentrations to a target concentration of 13.5 mM, followed by a decline in perifusate glucose concentrations to basal levels, over a total 2-h perifusion period, are shown for islets from untreated and WY14,643-treated unmated and 15-d-pregnant groups in Fig. 4. Rates of insulin release by islets isolated from untreated unmated rats were not significantly increased by raising the perifusate glucose concentration to a mean concentration of 6.9 ± 0.2 mM. However, raising the perifusate glucose concentration to a mean concentration of 13.8 ± 0.3 mM led to a significant increase in insulin release to peak rates of 97 µU/min, which were 13.5-fold above basal (P < 0.01) (Fig. 4A). Insulin release steadily declined back to basal once perifusate glucose concentrations returned to basal levels of 1.9 ± 0.1 mM (Fig. 4A). Basal rates of insulin release by perifused islets isolated from 15-d-pregnant rats were significantly higher (by 24%) than rates of insulin release by perifused islets from unmated rats (Fig. 4A). Raising the perifusate glucose concentration to a mean concentration of 7.1 ± 0.8 mM significantly increased rates of insulin release by perifused islets from 15-d-pregnant rats by up to 5.7-fold (P < 0.05), such that rates were progressively higher than corresponding rates from perifused islets from unmated rats. Consequently, as demonstrated by the leftward shift in the profile of insulin secretion, significant stimulation of insulin secretion was achieved at a lower perifusate glucose concentration with islets from pregnant rats, compared with islets from unmated rats (Fig. 4A). aising the perifusate glucose concentration to a mean concentration of 14.1 ± 0.7 mM increased insulin secretion by islets from pregnant rats, to rates that were initially 6.2-fold higher (P < 0.05) than those by islets from unmated rats, declining to 2.0-fold higher (P < 0.05) with time (Fig. 4A). Although insulin release rates by perifused islets from 15-d-pregnant rats steadily declined toward basal values once perifusate glucose concentrations returned to basal levels (2.2 ± 0.2 mM), they remained 1.3- to 2.9-fold higher than corresponding values for unmated rats. The increased insulin release by perifused islets from 15-d-pregnant rats, compared with perifused islets from control rats, resulted in a 2.6-fold higher (P < 0.05) {Delta} I for insulin during the 2-h perifusion period [unmated, 890 ± 128 µU/min·liter (n = 10); 15-d-pregnant, 2335 ± 318 µU/min·liter (n = 4); P < 0.05]. Our data therefore demonstrate that the overall effect of 15 d of pregnancy, to enhance glucose sensitivity and responsiveness, is retained ex vivo and can be clearly demonstrated during islet perifusions. This result seems to eliminate any acute influence of circulating factors, including acute changes in systemic lipid delivery to the islet, and implicates a stable change in islet function.
fig.ommitteedms@p1cd, http://www.100md.com
Figure 4. Effects of glucose on insulin release by perifused islets isolated from untreated or WY14,643-treated unmated or 15-d-pregnant rats. Islets isolated from unmated (C, open circles) or 15-d-pregnant (P, closed circles) untreated rats (A) or WY14,643-treated rats (B) were perifused in basal medium containing 2 mM glucose, for 60 min, at a flow rate of 1 ml/min at 37 C, before collection of fractions. Perifusate glucose concentrations were then modified to 8 and 16 mM, as indicated. Perifusate fractions (2 ml) were collected at 2-min intervals and stored at -20 C, before assay for insulin and glucose, using commercial kits. Assayed perifusate glucose concentrations are provided in the text. Results are means ± SEM for 10 untreated unmated rats, 4 untreated 15-d-pregnant rats, 5 WY14,643-treated unmated rats, or 5 WY14,643-treated 15-d-pregnant rats. Statistically significant effects of pregnancy are indicated by *, P < 0.05.ms@p1cd, http://www.100md.com
Enhanced insulin secretion by perifused islets from 15-d-pregnant rats is not reversed by acute (24-h) antecedent PPAR{alpha} activation in vivo
Acute (24-h) WY14,643 treatment of unmated rats did not significantly affect insulin release at basal (2 mM), mid- (6.7 ± 0.6 mM), or high (12.6 ± 0.8 mM) perifusate glucose (compare Fig. 4, A and B). Consequently, I during the 2-h perifusion period was unchanged by acute activation of PPAR{alpha} by WY14,643 treatment [unmated, 890 ± 128 µU/min·liter (n = 10); unmated + WY14,643, 930 ± 237 µU/min·liter (n = 5)]. Acute (24-h) WY14,643 treatment of 15-d-pregnant rats increased insulin release rates by perifused islets at basal (2 mM) glucose (by 54%). Although insulin release rates by perifused islets from WY14,643-treated pregnant rats were not significantly affected at mid- (7.1 ± 0.8 mM) or high (14.1 ± 0.7 mM) glucose concentrations, compared with perifused islets from untreated pregnant rats (compare Fig. 4, A and B), rates of insulin release tended to be higher with islets from WY14,643-treated pregnant rats (by 1.3- to 1.9-fold at mid- and by up to 1.4-fold at high perifusate glucose). As a consequence, I, during the 2-h perifusion period, was increased by 19% by acute WY14,643 treatment during pregnancy [15-d-pregnant, 2335 ± 318 µU/min·liter (n = 4); 15-d-pregnant + WY14,643, 2718 ± 480 µU/min·liter (n = 5)]. Thus, contrasting with effects to suppress GSIS in vivo, acute PPAR{alpha} activation in vivo for 24 h fails to reverse the effects of 15 d of pregnancy to enhance insulin secretion by perifused islets ex vivo (Fig. 4). These findings demonstrate that effects of pregnancy to increase the glucose sensitivity and responsiveness of insulin secretion are not modified by acute PPAR{alpha} activation in vivo. It is also implied that suppression of GSIS by acute PPAR{alpha} activation in vivo reflects changes in circulating factors, which may include acute changes in systemic lipid delivery to the islet, which suppress insulin hypersecretion but do not involve reversal of the stable changes in islet function evoked by pregnancy.
Discussiony, 百拇医药
Pregnancy is a nongenetic factor that leads to adaptive changes in ß-cell function, including increased GSIS and a reduction in the glucose-stimulation threshold. The latter is thought to be the primary mechanism by which the ß-cells can release significantly more insulin under normal blood glucose concentrations. Mid-to-late pregnancy is also associated with insulin resistance, hypertriglyceridemia, and altered maternal TAG handling and production. PPARs exert important lipid-lowering effects in vivo and, in models of hypertriglyceridemia, can oppose the development of peripheral insulin resistance by relieving inhibition of insulin-stimulated glucose disposal by muscle. Long-term lipid sensing by the pancreatic ß-cell is coordinated via the PPARs. Thus, an investigation of the effects of PPAR{alpha} activation in vivo in pregnancy was considered to be potentially useful for analysis of the relationship between maternal insulin resistance and augmented insulin secretion. We demonstrate that acute (24-h) activation of PPAR{alpha} by WY14,643 reverses the augmented GSIS evoked by pregnancy in vivo. In addition, studies using perifused islets show that this reversal is not a consequence of modification of pregnancy-induced changes in ß-cell glucose sensing and responsiveness, which are retained ex vivo after PPAR{alpha} activation in vivo. However, normalization of maternal GSIS to that found in the nonpregnant state is observed in association with alleviation of maternal insulin resistance. The data provide insight into the relationships between maternal insulin resistance, changes in islet glucose sensing, and responsiveness and enhanced insulin secretion during pregnancy.
First, we confirmed previous findings of augmented insulin secretion in vivo in response to hyperglycemia in mid-late pregnant rats (35). Studies using perifused islets demonstrated that the effect of pregnancy to lower the threshold for GSIS and enhance GSIS was retained ex vivo, supporting previous studies (36, 37). Increased sensitivity and responsiveness of insulin secretion to glucose was observed in association with pregnancy-induced insulin resistance, as assessed by a 3.8-fold increase in IxG. Whereas insulin secretion after iv glucose challenge was greatly increased at d 15 of pregnancy, glucose tolerance was only modestly affected, compatible with the concept that insulin hypersecretion is largely compensatory for insulin resistance during pregnancy. In vivo, the relative response of insulin to a rapid increase in glycemia, in the 15-d-pregnant group, elicited by iv glucose challenge was greatly attenuated by 24-h treatment with WY14,643. Activation of PPAR{alpha} by WY14,643 administration only affected pregnancy-induced hypersecretion of insulin, given that GSIS was unaffected by WY14,643 treatment in unmated rats. The present data therefore clearly demonstrate an acute (24-h) effect of PPAR{alpha} activation to attenuate differences in GSIS between unmated and 15-d-pregnant rats in vivo.
A lowering of the threshold for GSIS, thought to be mediated by the placental lactogens-I and -II and PRL (see, e.g., Refs. 37 and 38), is primary to the mechanism by which ß-cells release significantly more insulin under normal blood glucose concentrations during pregnancy. The lowered insulin-to-glucose concentration ratio observed in the pregnant group in the postabsorptive state as a consequence of WY14,643 administration, taken in conjunction with the marked lowering in GSIS in vivo, suggested to us that insulin secretion in response to glucose during pregnancy might require a higher level of glycemia than normal, as a consequence of PPAR{alpha} activation. However, studies of the characteristics of the insulin secretory response to a rise in glucose, during islet perifusion, eliminated the possibility that PPAR{alpha} activation might reverse these stable modifications of islet function induced by pregnancy. Our data clearly show that, whereas reversal of insulin hypersecretion elicited by pregnancy is one outcome of acute activation of PPAR{alpha} elicited by WY14,643 administration in vivo, effects are not mediated via stable changes in ß-cell function that are detectable ex vivo: pregnancy-induced increases in GSIS and a decreased glucose-stimulation threshold are evident with isolated perifused islets, but these parameters were unaffected by acute PPAR{alpha} activation in vivo. Hence, antecedent PPAR{alpha} activation does not result in attenuation of the intrinsic adaptations of the ß-cell to pregnancy, and the enhanced capacity for glucose sensing is not associated with augmented GSIS in vivo under conditions of PPAR{alpha} activation.
Altered GSIS in vivo could result from nonpersistent direct effects of PPAR{alpha} activation on the islet, or from effects of PPAR{alpha} activation to modify circulating factors (metabolites, hormones) that influence insulin secretion. FAs are important nutrients for normal ß-cell function. In addition, they play an auxiliary role to heighten the responsiveness of the ß-cell to a variety of insulin secretogogues (see Ref. 39 for review). However, chronically elevated FA are linked to the development of insulin resistance and impaired insulin secretion, although the nature of this relationship remains to be clarified (reviewed in Ref. 40). Input of FA into the islet can occur directly by uptake of circulating NEFA, which is critically dependent on the circulating NEFA concentration, or from circulating TAG via islet lipoprotein lipase (41). In the present study, postabsorptive FA concentrations were increased in pregnancy, which is consistent with the accelerated entry into starvation that is characteristic of pregnancy (Ref. 12 ; reviewed in Ref. 42). However, under conditions of PPAR{alpha} activation, plasma NEFA levels remained significantly higher in pregnant than in unmated rats, suggesting that reversal of enhanced GSIS by WY14,643 is not a consequence of lowered FA delivery to the islet. By contrast, PPAR{alpha} activation in vivo led to its characteristic lowering of plasma TAG and, importantly, plasma TAG concentrations after WY14,643 treatment did not differ between unmated and pregnant rats.
Normalization of circulating TAG between unmated and pregnant rats, in conjunction with normalization of GSIS in vivo, raises the possibility that raised concentrations of lipoproteins, with associated increases in local delivery of FA to the ß-cell via islet lipoprotein lipase, may contribute to enhanced GSIS in vivo during pregnancy. An equally attractive possibility is that altered maternal insulin secretory responses to glucose in vivo (but not with perifused islets) represent a decreased requirement for insulin hypersecretion because of alleviation of maternal insulin resistance by PPAR{alpha} activation, i.e. the reversal of insulin hypersecretion during pregnancy is a secondary consequence of WY14,643 treatment specifically targeting the decreased insulin sensitivity of pregnancy, rather than directly affecting islet function. In human subjects, insulin-resistant subjects have higher insulin levels and AIR to glucose than insulin-sensitive subjects (33, 43). In obesity, there is an inverse relationship between insulin sensitivity and insulin secretion, which is indicative of ß-cell compensation for insulin resistance (44). During healthy pregnancies, the development of insulin resistance is accompanied by a reciprocal increase in insulin secretion (34). The effect of PPAR{alpha} activation, to lower maternal GSIS in vivo, was observed in association with enhanced maternal insulin sensitivity, as demonstrated by a lowered IxG and, despite markedly lowered insulin secretion, almost normal glucose tolerance. As shown in Fig. 3B, the overall sensitivity of the ß-cell to glucose during pregnancy, as reflected by AIR, is strongly positively correlated with resistance to the action of insulin. Our studies are important because they show that changes in maternal insulin secretion and action induced during pregnancy can be modified in the absence of any modification of plasma NEFA delivery, indicating that insulin resistance at the level of the adipocyte lipolysis is insufficient alone to induce whole-body insulin resistance. In addition, they suggest that PPAR{alpha} could participate in altered glucose homeostasis in pregnancy via modulation of maternal insulin sensitivity, extending the established requirement for PPAR{alpha} for normal islet lipid homeostasis.
In conclusion, our studies show that acute activation of PPAR{alpha} reverses the augmented GSIS evoked by pregnancy in vivo, while the islets retain pregnancy-induced changes in ß-cell glucose sensing and responsiveness. A key finding is that a stable enhancement in the capacity and sensitivity for GSIS, as observed in pregnancy, does not, of necessity, lead to insulin secretion inappropriate for the requirement for glucose disposal to maintain normoglycemia. Hence, our studies indicate that manipulations designed to enhance the ß-cell insulin secretory capacity and responsiveness to glucose in type 2 diabetes are of potential therapeutic value.6s, http://www.100md.com
Acknowledgments6s, http://www.100md.com
We extend our thanks to Harjinder S. Lall for expert technical assistance.6s, http://www.100md.com
Received August 5, 2002.6s, http://www.100md.com
Accepted for publication September 20, 2002.6s, http://www.100md.com
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We investigated the effects of acute (24-h) peroxisome proliferator-activated receptor (PPAR) activation by WY14,643 (pirinixic acid) treatment on glucose-stimulated insulin secretion (GSIS) during pregnancy, in the rat, in relation to insulin sensitivity. GSIS after iv glucose challenge (500 mg/kg) was increased at d 15 of pregnancy but was attenuated by WY14,643 treatment in vivo, with decreases in acute insulin response (51%; P < 0.001) and total suprabasal 30-min area under the insulin curve (I) (55%; P < 0.001). GSIS was unaffected by WY14,643 treatment in unmated rats. Islet perifusions were employed to identify persistent effects of PPAR{alpha} activation. GSIS was enhanced, and the glucose threshold was reduced in perifused islets from pregnant rats, but WY14,643 treatment failed to reverse these effects. WY14,643 treatment of 15-d-pregnant rats significantly lowered (by 63%; P < 0.01) the insulin resistance index [total suprabasal 30-min area under insulin curve x suprabasal 30-min area under glucose curve (IxG)]. A strong positive linear relationship (r = 0.92) between acute insulin response and IxG was evident between groups. Our studies show that acute PPAR activation reverses the augmented GSIS evoked by pregnancy in vivo, whereas the isolated islets retain pregnancy-induced enhancement of ß-cell glucose sensing and responsiveness. Normalization of maternal GSIS to that found in the nonpregnant state is observed in association with alleviation of maternal insulin resistance.
Introductionnm7:n, 百拇医药
ABNORMAL LIPID PARTITIONING, resulting in either depletion of islet lipid or accumulation of excessive islet lipid, is important for the development of pancreatic ß-cell failure (1). Long-term lipid sensing by the pancreatic ß-cell is coordinated through peroxisome proliferator-activated receptors (PPARs) (2, 3). PPAR{alpha} is expressed in islets and islet cell lines, and it controls genes involved in lipid metabolism, in particular those implicated in fatty acid (FA) transport and ß-oxidation (2, 3). Islet expression of PPAR{alpha} and enzymes of FA oxidation are induced by high FA concentrations (3) and exposure to PPAR{alpha} receptor ligands (3, 4). Signaling via PPAR{alpha} also influences whole-body insulin sensitivity, particularly under conditions where peripheral insulin action is impaired because of increased lipid delivery. Thus, chronic (15-d) administration of the PPAR{alpha} activator cipofibrate to 5-wk-old obese Zucker rats reduces plasma insulin concentrations without causing glucose intolerance during an iv glucose tolerance test, suggesting improved insulin action (5). Similarly, administration of fenofibrate, as part of the diet, reverses basal hyperinsulinemia and hyperglycemia in high-fat-fed C57BL/6 mice (5) and WY14,643 [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid] increases insulin sensitivity in high-fat-fed rats (6). It was suggested that PPAR{alpha} activation opposes the development of peripheral insulin resistance by relieving lipid-mediated inhibition of insulin-stimulated glucose disposal (6).
Pregnancy leads to adaptations of maternal carbohydrate metabolism, including a progressive state of maternal insulin resistance, to confer a competitive advantage to the developing fetus (7, 8, 9, 10, 11, 12, 13, 14). As in other insulin-resistant states, mid-late pregnancy is associated with elevated maternal circulating triacylglycerol (TAG) levels (15). Maternal hypertriglyceridemia is caused by enhanced maternal adipose tissue lipolysis (16, 17, 18), enhanced maternal hepatic very low density lipoprotein production (19, 20), and decreased maternal extrahepatic lipoprotein lipase activity (21, 22). Maternal insulin resistance is observed in conjunction with enhanced pancreatic ß-cell insulin secretion in response to glucose and a lowered threshold for stimulation of insulin secretion by glucose (reviewed in Ref. 23). Treatment of pregnant rats with fenofibrate, for 1–2 d from d 16 of pregnancy, decreases maternal hypertriglyceridemia (24). Possible effects of PPAR{alpha} activation on maternal glucose-insulin homeostasis have not been delineated.
In the present study, we investigated the effects of PPAR{alpha} activation by WY14,643 in vivo on the modulation of insulin secretion and sensitivity that is observed during mid-late pregnancy in the rat. Because maternal triglyceridemia becomes refractory to fenofibrate if treatment is continued beyond 48 h (24), and because 24 h of WY14,643 treatment is adequate to enhance the expression of islet PPAR{alpha} and PPAR{alpha} -linked enzymes in vitro (25) and in vivo (4), WY14,643 was administered for 24 h only. Studies were conducted in the intact animal at d 15 of pregnancy (term is 23 d). In addition, studies of glucose-stimulated insulin secretion (GSIS) were conducted ex vivo using islet perifusions.kdy, 百拇医药
Materials and Methodskdy, 百拇医药
Materialskdy, 百拇医药
Analytical-grade organic solvents were obtained from BDH (Poole, Dorset, UK). General laboratory reagents were purchased from Roche Diagnostics Ltd. (Lewes, East Sussex, UK) or from Sigma (Poole, Dorset, UK). WY14,643 was purchased from Sigma. Kits for determination of insulin and glucose concentrations were purchased from Mercodia (Uppsala, Sweden) and Sigma, respectively. Kits for determination of plasma nonesterified FA (NEFA) and TAG concentrations were purchased from Alpha Labs (Eastleigh, Hants, UK).
Animals\}?, http://www.100md.com
All studies were conducted in adherence to the regulations of the United Kingdom Animal Scientific Procedures Act (1986). Female albino Wistar rats (200–250 g) were purchased from Charles River Laboratories, Inc. (Margate, Kent, UK). Rats were maintained at a temperature of 22 ± 2 C and subjected to a 12-h light, 12-h dark cycle. Rats were given free access to standard, pelleted rodent diet purchased from Special Diet Services (Witham, Essex, UK) [52% carbohydrate, 15% protein, 3% lipid, and 30% nondigestible residue (by wt); 2.61 kcal metabolizable energy/g]. Rats were time-mated by the appearance of sperm plugs (d 0 of pregnancy). Pregnant rats with less than 8 fetuses were not included in the study. WY14,643 was administered to unmated and 14-d-pregnant rats with free access to diet, as a single ip injection (50 mg/kg body wt.); and rats were sampled after a further 24 h (26). Control, unmated, and 14-d-pregnant rats were injected with vehicle. In all experiments, rats were allowed access ad libitum to water.
The iv glucose challenge;?4g, http://www.100md.com
Glucose was administered as an iv bolus (0.5-g glucose/kg body weight; 150 µl per 100 g body weight) to conscious, unrestrained rats (see Ref. 27). Glucose was injected via a chronic indwelling jugular cannula, and blood samples (100 µl) were withdrawn at intervals 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 with ZnSO4/Ba(OH)2, centrifuged (10,000 x g) at 4 C, and the supernatant was retained for subsequent assay of blood glucose. The remaining blood sample was immediately centrifuged (10,000 x g) at 4 C, and plasma was stored at -20 C until assayed for insulin. The calculated acute insulin response (AIR) was calculated as the mean of suprabasal 2- and 5-min plasma insulin values. Insulin and glucose responses during the glucose tolerance test were used for calculation of the incremental plasma insulin values integrated over the 30-min period after the injection of glucose (I) and the corresponding incremental integrated plasma glucose values (). The insulin resistance index (IR index) was calculated as the product of IxG after glucose challenge. The rate of glucose disappearance (k) was calculated from the slope of the regression line obtained with log-transformed glucose values from 2–15 min after glucose administration.
Islet isolation and perifusiono, 百拇医药
Rats were anesthetized by injection of sodium pentobarbital (60 mg/ml in 0.9% NaCl; 1 ml/kg body wt, ip); and, once locomotor activity had ceased, pancreases were excised and islets were isolated by collagenase digestion (28). Free islets were collected, under a dissecting microscope, with a 20 µl pipette, into HEPES-buffered Hanks’ balanced salts solution containing 5% BSA. Insulin release from freshly isolated islets was measured in a perifusion system as described by Hughes et al., 1992 (29). In this system, 50 islets were housed in small chambers on Millicell culture inserts. Islets were perifused in basal medium (Krebs-Ringer containing 20 mM HEPES, pH 7.4; 5 mg/ml BSA; and 2 mM glucose), for 60 min at a flow rate of 1 ml/min at 37 C, before collection of fractions. Glucose concentrations were then modified as indicated. Fractions (2 ml) were collected at 2-min intervals and stored at -20 C before assay for insulin.o, 百拇医药
Analytical methods\, 百拇医药
Plasma glucose concentrations were determined by a glucose oxidase method (Sigma). Plasma immunoreactive insulin concentrations were measured by ELISA, using rat insulin as a standard (Mercodia). Plasma NEFA and TAG levels were determined with commercial kits (Alpha Labs).\, 百拇医药
Statistical analysis\, 百拇医药
Results are presented as the mean ± SEM, with the numbers of rats or islet preparations in parentheses. Statistical analysis was performed by ANOVA followed by Fisher’s post hoc tests for individual comparisons or Student’s t test, as appropriate (Statview; Abacus Concepts, Inc., Berkeley, CA). A P value < 0.05 was considered to be statistically significant.\, 百拇医药
Results\, 百拇医药
Effect of acute PPAR activation, for 24 h, in vivo, on food intake and body weight gain in unmated and pregnant rats\, 百拇医药
Food intakes and body weight gain of untreated and WY14,643-treated unmated and 15-d-pregnant rats over the period of WY14,643 treatment are shown in Table 1. WY14,643 treatment did not significantly affect either food intake or body weight gain in unmated rats (Table 1). Pregnancy was associated with hyperphagia and a progressive increase in body weight from d 0-d 15 of pregnancy (results not shown; see also Ref. 30). Body weights of untreated dams increased by 3% from d 14–15 of pregnancy, and this was accompanied by significantly higher (40%; P < 0.05) food intake, compared with unmated rats. Acute (24-h) PPAR{alpha} activation in vivo did not influence maternal food intake or body weight gain (Table 1), and fetal numbers per dam were unchanged [control, 14 ± 1 (n = 6 litters); WY14,643-treated, 14 ± 1 (n = 10 litters); not significant (NS)].
fig.ommitteed!^ogp, 百拇医药
Table 1. Effects of 24-h WY14,643 treatment on food intake and body weight gain and on postabsorptive circulating insulin and glucose concentrations and the insulin-to-glucose ratio in unmated and 15-d-pregnant rats!^ogp, 百拇医药
Acute PPAR{alpha} activation, for 24 h, in vivo, leads to modest hypoinsulinemia in 15-d-pregnant rats in the postabsorptive state!^ogp, 百拇医药
Plasma insulin concentrations were lower (by 32%) in WY14,643-treated dams, compared with the untreated pregnant group in the postabsorptive state, reversing the effect of pregnancy to increase basal insulin concentrations by 58% (Table 1). This relative hypoinsulinemia was not accompanied by postabsorptive hyperglycemia in the WY14,643-treated pregnant group (Table 1). The insulin-to-glucose ratio, which is an index of the relative response of insulin to fasting glycemia, was therefore lower (by 27%) in the WY14,643-treated pregnant group, compared with the untreated pregnant group, such that the effect of pregnancy to increase the basal insulin-to-glucose ratio was reversed by 60% (Table 1). WY14,643 treatment of nonpregnant rats also modestly lowered postabsorptive plasma insulin concentrations, compared with those found in nonpregnant control rats; and again, postabsorptive glycemia was unchanged (Table 1).
Effect of acute PPAR{alpha} activation, for 24 h, in vivo, on maternal plasma NEFA and TAG concentrations at d 15 of pregnancyw!, 百拇医药
We analyzed the effects of acute (24-h) PPAR{alpha} activation in vivo on circulating lipid-fuel concentrations in postabsorptive unmated and 15-d-pregnant rats, in relation to plasma insulin levels (Fig. 1). As expected, plasma NEFA (Fig. 1A) concentrations in the postabsorptive state were increased in 15-d-pregnant rats, compared with unmated controls (by 3.1-fold; P < 0.01). Furthermore, plasma NEFA levels in pregnant rats were higher than would be predicted from the relationship between NEFA and insulin levels in unmated rats (Fig. 1A) confirming the existence of maternal insulin resistance, with respect to adipose-tissue antilipolysis, in late pregnancy. Treatment with WY14,643 failed to modify plasma NEFA concentrations in unmated rats but partially reversed the pregnancy-induced increase in NEFA concentrations (by 34%). Nevertheless, plasma NEFA levels in postabsorptive WY14,643-treated pregnant rats remained significantly higher than plasma NEFA levels in either postabsorptive untreated controls (2.4-fold; P < 0.001) or postabsorptive WY14,643-treated unmated rats (by 2.2-fold: P < 0.01) (Fig. 1A). Plasma TAG concentrations in the postabsorptive state were increased in 15-d-pregnant rats, compared with unmated controls (by 1.4-fold; P < 0.05) (Fig. 1B). Treatment with WY14,643 led to a small and nonsignificant (17%) decline in plasma TAG concentrations in postabsorptive untreated controls. As expected for rats at this stage of pregnancy (24), plasma TAG levels were lowered (by 23%) by PPAR{alpha} activation, in vivo, for 24 h, in 15-d-pregnant rats (Fig. 1B), reversing the pregnancy-induced elevation in plasma TAG levels by 81%. As a result, plasma TAG concentrations in unmated and 15-d-pregnant rats were no longer statistically significantly different after 24-h treatment with WY14,643 (Fig. 1B). WY14,643 treatment did not modify plasma 3-hydroxybutyrate levels in either pregnant or unmated rats (results not shown).
fig.ommitteedh7/tv's, 百拇医药
Figure 1. Acute (24-h) PPAR{alpha} activation in vivo lowers plasma TAG but does not affect plasma NEFA concentrations in 15-d-pregnant rats. Blood samples were withdrawn from untreated (C, open circles) or WY14,643-treated (C+WY, closed circles) unmated, or untreated (P, open triangles) or WY14,643-treated (P+WY, closed triangles) 15-d-pregnant conscious, unrestrained rats in the postabsorptive state, for measurement of plasma NEFA (A) and TAG (B) concentrations, using commercial kits. Results are means ± SEM for 11 untreated unmated rats, 5 untreated 15-d-pregnant rats, 10 WY14,643-treated unmated rats, or 8 WY14,643-treated 15-d-pregnant rats. Statistically significant effects of pregnancy are indicated by: *, P < 0.05; ***, P < 0.001. There were no statistically significant effects of WY14,643 treatment.h7/tv's, 百拇医药
GSIS in vivo is enhanced in conjunction with the existence of insulin resistance at d 15 of pregnancyh7/tv's, 百拇医药
The calculated AIR (i.e. the means of suprabasal 2- and 5-min plasma insulin) and I after iv administration of glucose (500 mg/kg) in 15-d-pregnant and unmated rats are shown in Fig. 2, A and B, respectively. AIR and I after glucose administration were significantly increased at d 15 of pregnancy, compared with unmated female rats [by 2.3-fold (P < 0.001) and 2.5-fold (P < 0.001), respectively].
fig.ommitteedq, 百拇医药
Figure 2. Acute (24-h) PPAR activation in vivo markedly lowers glucose-stimulated hypersecretion of insulin after iv glucose challenge in 15-d-pregnant rats. Glucose was administered as an iv bolus (0.5-g glucose/kg body weight) to untreated or WY14,643-treated unmated (C, open bars) or 15-d-pregnant (P, closed bars) conscious, unrestrained rats in the postabsorptive state. Blood samples were withdrawn, at intervals, for measurement of plasma insulin, using a commercial kit. AIR values, calculated as the mean of suprabasal 2- and 5-min plasma insulin, are shown in A. Insulin responses during the glucose tolerance test were used for calculation of the incremental plasma insulin values integrated over 30 min, I, shown in B. Results are means ± SEM for 9 untreated unmated rats, 5 untreated 15-d-pregnant rats, 8 WY14,643-treated unmated rats, or 7 WY14,643-treated 15-d-pregnant rats. Statistically significant effects of pregnancy are indicated by: ***, P < 0.001. Statistically significant effects of WY14,643 treatment are indicated by: , P < 0.001.q, 百拇医药
Acute PPAR{alpha} activation, for 24 h, in vivo, markedly and selectively lowers GSIS after iv glucose challenge in 15-d-pregnant rats
We analyzed the effects of acute (24-h) PPAR{alpha} activation in vivo on insulin secretion in unmated and 15-d-pregnant rats, in response to an acute glucose challenge. In unmated rats, 24-h treatment with WY14,643 did not significantly affect AIR or I (Fig. 2, A and B). By contrast, the relative response of insulin to a rapid increase in glycemia in the 15-d-pregnant group was greatly lowered by 24-h treatment with WY14,643, as reflected by substantial decreases in both AIR (51%; P < 0.001) and I (55%; P < 0.001). Thus, WY14,643 treatment reversed the effect of pregnancy to elevate AIR and I by 92% and 91%, respectively. It is concluded that acute (24-h) PPAR{alpha} activation in vivo greatly attenuates differences in insulin secretion in vivo in response to iv glucose challenge induced by pregnancy. Furthermore, activation of PPAR{alpha} by WY14,643 administration only affected hypersecretion of insulin, because GSIS was unaffected by WY14,643 treatment in control (unmated) rats (Fig. 2, A and B).
Relationship between changes in insulin secretion and action after PPAR{alpha} activation in pregnancy?@&c, 百拇医药
Failure of the pancreatic ß-cells to compensate for insulin resistance is critical to the pathology of type 2 diabetes (reviewed in Ref. 31). In healthy individuals, a hyperbolic relationship has been observed between insulin sensitivity and secretion (32, 33). Pregnancy-induced hyperinsulinemia is also considered as a compensatory response to the development of insulin resistance. The development of insulin resistance in healthy pregnant women is accompanied by a reciprocal increase in insulin secretion (34). The IR index IxG was significantly increased by 3.8-fold at d 15 of pregnancy (P < 0.001; Fig. 3A). Acute (24-h) treatment of 15-d-pregnant rats with WY14,643 did not significantly affect G (untreated, 12.8 ± 0.5 mmol/min·liter; WY14,643-treated, 12.3 ± 1.1 mmol/min·liter; NS) and only slightly decreased (by 13%) the rate of glucose disappearance calculated over the first 15 min after iv glucose injection (untreated, 3.7 ± 0.3%/min; WY14,643-treated, 3.2 ± 0.5%/min; NS). By contrast, IxG was significantly lowered (by 63%; P < 0.001) by WY14,643 treatment in 15-d-pregnant rats (Fig. 3A), reversing the pregnancy-induced increase in the IxG by 86%. Hence, acute (24-h) PPAR activation significantly improves insulin sensitivity in the pregnant group. Importantly, the positive linear relationship (r = 0.92) between AIR and IR index, shown in Fig. 3B, demonstrates that insulin secretion in vivo in pregnancy can be acutely modulated in concert with the requirement for insulin-stimulated glucose disposal.
fig.ommitteed85w}qh, http://www.100md.com
Figure 3. Acute (24-h) PPAR activation in vivo markedly lowers IxG in 15-d-pregnant rats. Glucose was administered as an iv bolus (0.5-g glucose/kg body weight) to untreated or WY14,643-treated unmated (C, open bars) or 15-d-pregnant (P, closed bars) conscious, unrestrained rats in the postabsorptive state. Blood samples were withdrawn, at intervals, for measurement of plasma insulin and blood glucose, using commercial kits. Insulin and glucose responses during the glucose tolerance test were used for calculation of the incremental plasma insulin values integrated over 30 min, I and the corresponding incremental integrated plasma glucose values (G). The IR index IxG, calculated as the product of the areas under the glucose and insulin curves after glucose challenge, are shown in A. The relationship between AIR and IxG in untreated or WY14,643-treated unmated rats (circles) or 15-d-pregnant rats (triangles) in the postabsorptive state is shown in panel B. Results are means ± SEM for 8 untreated control rats, 4 untreated 15-d-pregnant rats, 8 WY14,643-treated control rats, or 6 WY14,643-treated 15-d-pregnant rats. Statistically significant effects of pregnancy are indicated by: ***, P < 0.001. Statistically significant effects of WY14,643 treatment are indicated by: , P < 0.001.
Islets from 15-d-pregnant rats exhibit enhanced glucose sensitivity and responsiveness of insulin secretion during perifusion ex vivo2rdo\(, http://www.100md.com
The preceding findings raise the question of whether the reversal of insulin hypersecretion in pregnant rats in vivo, achieved by activation of PPAR by WY14,643 treatment, represents a stable modification of islet function or is an indirect response of the islets to the improvement in whole-body insulin sensitivity. We therefore tested whether the effect of pregnancy to alter pancreatic ß-cell sensitivity and responsiveness to glucose is modified by acute (24-h) activation of PPAR, by measuring insulin release by perifused islets. Overall patterns of insulin release during stepwise glucose perifusion, designed to generate a steady rise in perifusate glucose concentrations to a target concentration of 6.8 mM, then a rise in perifusate glucose concentrations to a target concentration of 13.5 mM, followed by a decline in perifusate glucose concentrations to basal levels, over a total 2-h perifusion period, are shown for islets from untreated and WY14,643-treated unmated and 15-d-pregnant groups in Fig. 4. Rates of insulin release by islets isolated from untreated unmated rats were not significantly increased by raising the perifusate glucose concentration to a mean concentration of 6.9 ± 0.2 mM. However, raising the perifusate glucose concentration to a mean concentration of 13.8 ± 0.3 mM led to a significant increase in insulin release to peak rates of 97 µU/min, which were 13.5-fold above basal (P < 0.01) (Fig. 4A). Insulin release steadily declined back to basal once perifusate glucose concentrations returned to basal levels of 1.9 ± 0.1 mM (Fig. 4A). Basal rates of insulin release by perifused islets isolated from 15-d-pregnant rats were significantly higher (by 24%) than rates of insulin release by perifused islets from unmated rats (Fig. 4A). Raising the perifusate glucose concentration to a mean concentration of 7.1 ± 0.8 mM significantly increased rates of insulin release by perifused islets from 15-d-pregnant rats by up to 5.7-fold (P < 0.05), such that rates were progressively higher than corresponding rates from perifused islets from unmated rats. Consequently, as demonstrated by the leftward shift in the profile of insulin secretion, significant stimulation of insulin secretion was achieved at a lower perifusate glucose concentration with islets from pregnant rats, compared with islets from unmated rats (Fig. 4A). aising the perifusate glucose concentration to a mean concentration of 14.1 ± 0.7 mM increased insulin secretion by islets from pregnant rats, to rates that were initially 6.2-fold higher (P < 0.05) than those by islets from unmated rats, declining to 2.0-fold higher (P < 0.05) with time (Fig. 4A). Although insulin release rates by perifused islets from 15-d-pregnant rats steadily declined toward basal values once perifusate glucose concentrations returned to basal levels (2.2 ± 0.2 mM), they remained 1.3- to 2.9-fold higher than corresponding values for unmated rats. The increased insulin release by perifused islets from 15-d-pregnant rats, compared with perifused islets from control rats, resulted in a 2.6-fold higher (P < 0.05) {Delta} I for insulin during the 2-h perifusion period [unmated, 890 ± 128 µU/min·liter (n = 10); 15-d-pregnant, 2335 ± 318 µU/min·liter (n = 4); P < 0.05]. Our data therefore demonstrate that the overall effect of 15 d of pregnancy, to enhance glucose sensitivity and responsiveness, is retained ex vivo and can be clearly demonstrated during islet perifusions. This result seems to eliminate any acute influence of circulating factors, including acute changes in systemic lipid delivery to the islet, and implicates a stable change in islet function.
fig.ommitteedms@p1cd, http://www.100md.com
Figure 4. Effects of glucose on insulin release by perifused islets isolated from untreated or WY14,643-treated unmated or 15-d-pregnant rats. Islets isolated from unmated (C, open circles) or 15-d-pregnant (P, closed circles) untreated rats (A) or WY14,643-treated rats (B) were perifused in basal medium containing 2 mM glucose, for 60 min, at a flow rate of 1 ml/min at 37 C, before collection of fractions. Perifusate glucose concentrations were then modified to 8 and 16 mM, as indicated. Perifusate fractions (2 ml) were collected at 2-min intervals and stored at -20 C, before assay for insulin and glucose, using commercial kits. Assayed perifusate glucose concentrations are provided in the text. Results are means ± SEM for 10 untreated unmated rats, 4 untreated 15-d-pregnant rats, 5 WY14,643-treated unmated rats, or 5 WY14,643-treated 15-d-pregnant rats. Statistically significant effects of pregnancy are indicated by *, P < 0.05.ms@p1cd, http://www.100md.com
Enhanced insulin secretion by perifused islets from 15-d-pregnant rats is not reversed by acute (24-h) antecedent PPAR{alpha} activation in vivo
Acute (24-h) WY14,643 treatment of unmated rats did not significantly affect insulin release at basal (2 mM), mid- (6.7 ± 0.6 mM), or high (12.6 ± 0.8 mM) perifusate glucose (compare Fig. 4, A and B). Consequently, I during the 2-h perifusion period was unchanged by acute activation of PPAR{alpha} by WY14,643 treatment [unmated, 890 ± 128 µU/min·liter (n = 10); unmated + WY14,643, 930 ± 237 µU/min·liter (n = 5)]. Acute (24-h) WY14,643 treatment of 15-d-pregnant rats increased insulin release rates by perifused islets at basal (2 mM) glucose (by 54%). Although insulin release rates by perifused islets from WY14,643-treated pregnant rats were not significantly affected at mid- (7.1 ± 0.8 mM) or high (14.1 ± 0.7 mM) glucose concentrations, compared with perifused islets from untreated pregnant rats (compare Fig. 4, A and B), rates of insulin release tended to be higher with islets from WY14,643-treated pregnant rats (by 1.3- to 1.9-fold at mid- and by up to 1.4-fold at high perifusate glucose). As a consequence, I, during the 2-h perifusion period, was increased by 19% by acute WY14,643 treatment during pregnancy [15-d-pregnant, 2335 ± 318 µU/min·liter (n = 4); 15-d-pregnant + WY14,643, 2718 ± 480 µU/min·liter (n = 5)]. Thus, contrasting with effects to suppress GSIS in vivo, acute PPAR{alpha} activation in vivo for 24 h fails to reverse the effects of 15 d of pregnancy to enhance insulin secretion by perifused islets ex vivo (Fig. 4). These findings demonstrate that effects of pregnancy to increase the glucose sensitivity and responsiveness of insulin secretion are not modified by acute PPAR{alpha} activation in vivo. It is also implied that suppression of GSIS by acute PPAR{alpha} activation in vivo reflects changes in circulating factors, which may include acute changes in systemic lipid delivery to the islet, which suppress insulin hypersecretion but do not involve reversal of the stable changes in islet function evoked by pregnancy.
Discussiony, 百拇医药
Pregnancy is a nongenetic factor that leads to adaptive changes in ß-cell function, including increased GSIS and a reduction in the glucose-stimulation threshold. The latter is thought to be the primary mechanism by which the ß-cells can release significantly more insulin under normal blood glucose concentrations. Mid-to-late pregnancy is also associated with insulin resistance, hypertriglyceridemia, and altered maternal TAG handling and production. PPARs exert important lipid-lowering effects in vivo and, in models of hypertriglyceridemia, can oppose the development of peripheral insulin resistance by relieving inhibition of insulin-stimulated glucose disposal by muscle. Long-term lipid sensing by the pancreatic ß-cell is coordinated via the PPARs. Thus, an investigation of the effects of PPAR{alpha} activation in vivo in pregnancy was considered to be potentially useful for analysis of the relationship between maternal insulin resistance and augmented insulin secretion. We demonstrate that acute (24-h) activation of PPAR{alpha} by WY14,643 reverses the augmented GSIS evoked by pregnancy in vivo. In addition, studies using perifused islets show that this reversal is not a consequence of modification of pregnancy-induced changes in ß-cell glucose sensing and responsiveness, which are retained ex vivo after PPAR{alpha} activation in vivo. However, normalization of maternal GSIS to that found in the nonpregnant state is observed in association with alleviation of maternal insulin resistance. The data provide insight into the relationships between maternal insulin resistance, changes in islet glucose sensing, and responsiveness and enhanced insulin secretion during pregnancy.
First, we confirmed previous findings of augmented insulin secretion in vivo in response to hyperglycemia in mid-late pregnant rats (35). Studies using perifused islets demonstrated that the effect of pregnancy to lower the threshold for GSIS and enhance GSIS was retained ex vivo, supporting previous studies (36, 37). Increased sensitivity and responsiveness of insulin secretion to glucose was observed in association with pregnancy-induced insulin resistance, as assessed by a 3.8-fold increase in IxG. Whereas insulin secretion after iv glucose challenge was greatly increased at d 15 of pregnancy, glucose tolerance was only modestly affected, compatible with the concept that insulin hypersecretion is largely compensatory for insulin resistance during pregnancy. In vivo, the relative response of insulin to a rapid increase in glycemia, in the 15-d-pregnant group, elicited by iv glucose challenge was greatly attenuated by 24-h treatment with WY14,643. Activation of PPAR{alpha} by WY14,643 administration only affected pregnancy-induced hypersecretion of insulin, given that GSIS was unaffected by WY14,643 treatment in unmated rats. The present data therefore clearly demonstrate an acute (24-h) effect of PPAR{alpha} activation to attenuate differences in GSIS between unmated and 15-d-pregnant rats in vivo.
A lowering of the threshold for GSIS, thought to be mediated by the placental lactogens-I and -II and PRL (see, e.g., Refs. 37 and 38), is primary to the mechanism by which ß-cells release significantly more insulin under normal blood glucose concentrations during pregnancy. The lowered insulin-to-glucose concentration ratio observed in the pregnant group in the postabsorptive state as a consequence of WY14,643 administration, taken in conjunction with the marked lowering in GSIS in vivo, suggested to us that insulin secretion in response to glucose during pregnancy might require a higher level of glycemia than normal, as a consequence of PPAR{alpha} activation. However, studies of the characteristics of the insulin secretory response to a rise in glucose, during islet perifusion, eliminated the possibility that PPAR{alpha} activation might reverse these stable modifications of islet function induced by pregnancy. Our data clearly show that, whereas reversal of insulin hypersecretion elicited by pregnancy is one outcome of acute activation of PPAR{alpha} elicited by WY14,643 administration in vivo, effects are not mediated via stable changes in ß-cell function that are detectable ex vivo: pregnancy-induced increases in GSIS and a decreased glucose-stimulation threshold are evident with isolated perifused islets, but these parameters were unaffected by acute PPAR{alpha} activation in vivo. Hence, antecedent PPAR{alpha} activation does not result in attenuation of the intrinsic adaptations of the ß-cell to pregnancy, and the enhanced capacity for glucose sensing is not associated with augmented GSIS in vivo under conditions of PPAR{alpha} activation.
Altered GSIS in vivo could result from nonpersistent direct effects of PPAR{alpha} activation on the islet, or from effects of PPAR{alpha} activation to modify circulating factors (metabolites, hormones) that influence insulin secretion. FAs are important nutrients for normal ß-cell function. In addition, they play an auxiliary role to heighten the responsiveness of the ß-cell to a variety of insulin secretogogues (see Ref. 39 for review). However, chronically elevated FA are linked to the development of insulin resistance and impaired insulin secretion, although the nature of this relationship remains to be clarified (reviewed in Ref. 40). Input of FA into the islet can occur directly by uptake of circulating NEFA, which is critically dependent on the circulating NEFA concentration, or from circulating TAG via islet lipoprotein lipase (41). In the present study, postabsorptive FA concentrations were increased in pregnancy, which is consistent with the accelerated entry into starvation that is characteristic of pregnancy (Ref. 12 ; reviewed in Ref. 42). However, under conditions of PPAR{alpha} activation, plasma NEFA levels remained significantly higher in pregnant than in unmated rats, suggesting that reversal of enhanced GSIS by WY14,643 is not a consequence of lowered FA delivery to the islet. By contrast, PPAR{alpha} activation in vivo led to its characteristic lowering of plasma TAG and, importantly, plasma TAG concentrations after WY14,643 treatment did not differ between unmated and pregnant rats.
Normalization of circulating TAG between unmated and pregnant rats, in conjunction with normalization of GSIS in vivo, raises the possibility that raised concentrations of lipoproteins, with associated increases in local delivery of FA to the ß-cell via islet lipoprotein lipase, may contribute to enhanced GSIS in vivo during pregnancy. An equally attractive possibility is that altered maternal insulin secretory responses to glucose in vivo (but not with perifused islets) represent a decreased requirement for insulin hypersecretion because of alleviation of maternal insulin resistance by PPAR{alpha} activation, i.e. the reversal of insulin hypersecretion during pregnancy is a secondary consequence of WY14,643 treatment specifically targeting the decreased insulin sensitivity of pregnancy, rather than directly affecting islet function. In human subjects, insulin-resistant subjects have higher insulin levels and AIR to glucose than insulin-sensitive subjects (33, 43). In obesity, there is an inverse relationship between insulin sensitivity and insulin secretion, which is indicative of ß-cell compensation for insulin resistance (44). During healthy pregnancies, the development of insulin resistance is accompanied by a reciprocal increase in insulin secretion (34). The effect of PPAR{alpha} activation, to lower maternal GSIS in vivo, was observed in association with enhanced maternal insulin sensitivity, as demonstrated by a lowered IxG and, despite markedly lowered insulin secretion, almost normal glucose tolerance. As shown in Fig. 3B, the overall sensitivity of the ß-cell to glucose during pregnancy, as reflected by AIR, is strongly positively correlated with resistance to the action of insulin. Our studies are important because they show that changes in maternal insulin secretion and action induced during pregnancy can be modified in the absence of any modification of plasma NEFA delivery, indicating that insulin resistance at the level of the adipocyte lipolysis is insufficient alone to induce whole-body insulin resistance. In addition, they suggest that PPAR{alpha} could participate in altered glucose homeostasis in pregnancy via modulation of maternal insulin sensitivity, extending the established requirement for PPAR{alpha} for normal islet lipid homeostasis.
In conclusion, our studies show that acute activation of PPAR{alpha} reverses the augmented GSIS evoked by pregnancy in vivo, while the islets retain pregnancy-induced changes in ß-cell glucose sensing and responsiveness. A key finding is that a stable enhancement in the capacity and sensitivity for GSIS, as observed in pregnancy, does not, of necessity, lead to insulin secretion inappropriate for the requirement for glucose disposal to maintain normoglycemia. Hence, our studies indicate that manipulations designed to enhance the ß-cell insulin secretory capacity and responsiveness to glucose in type 2 diabetes are of potential therapeutic value.6s, http://www.100md.com
Acknowledgments6s, http://www.100md.com
We extend our thanks to Harjinder S. Lall for expert technical assistance.6s, http://www.100md.com
Received August 5, 2002.6s, http://www.100md.com
Accepted for publication September 20, 2002.6s, http://www.100md.com
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