Glucose Intolerance and Resistin Expression in Rat Offspring Exposed to Ethanol in Utero: Modulation by Postnatal High-Fat Diet
Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3A1R9$3;^}9/, 百拇医药
Abstract$3;^}9/, 百拇医药
High-fat diet and intrauterine growth retardation may predispose to obesity, insulin resistance, and type 2 diabetes. Because prenatal ethanol (ETOH) exposure causes intrauterine growth retardation, we investigated its interactions with postnatal high-fat diet on glucose tolerance and adipocyte-derived hormones in the rat offspring.$3;^}9/, 百拇医药
High-fat-fed offspring had increased adiposity, serum leptin, and muscle uncoupling protein-3, but decreased adiponectin mRNA, compared with corresponding chow-fed groups. ETOH-exposed offspring had normal adiponectin, but increased resistin mRNA and protein, compared with controls, regardless of postnatal diet. Skeletal muscle glucose transporter-4 content was decreased after both ETOH exposure and high-fat feeding. Glycemic and insulin responses to an ip glucose challenge were equally increased in non-ETOH-exposed high-fat-fed offspring and in ETOH-exposed chow-fed offspring, with additive effects of ETOH and high-fat diet. Pancreatic insulin content was elevated only in non-ETOH-exposed high-fat-fed offspring.
The data suggest that high-fat diet worsens glucose intolerance in offspring of rats exposed to ETOH. Prenatal ETOH exposure and postnatal high-fat diet might cause insulin resistance through separate mechanisms, involving resistin and adiponectin, respectively.\gu, 百拇医药
Introduction\gu, 百拇医药
THE NATURAL HISTORY of type 2 diabetes is characterized by a period of insulin resistance, with euglycemia and compensatory hyperinsulinemia, followed by a period of impaired glucose tolerance during which insulin secretion becomes altered, leading to overt diabetes when insulin secretion is inadequate (1). Genetic, congenital, and environmental factors are thought to determine the development of insulin resistance and its worsening, which results in glucose intolerance and diabetes. Though several genes encoding enzymes of insulin action have been identified, they do not seem to play a major role in the pathogenesis of diabetes in the majority of patients (2). Among environmental determinants of insulin resistance, the role of diets high in saturated fat has been extensively studied. High-fat diets, which characteristically are consumed in modern Western societies, adversely affect glucose metabolism by decreasing the sensitivity of glucose transport to insulin. These diets induce insulin resistance by increasing body fat accretion, promoting skeletal muscle triglycerides synthesis, and augmenting circulating free fatty acids (FFAs; Ref. 3). High-fat diets also cause liver insulin resistance, evident by an impaired ability of insulin to suppress hepatic glucose production (4).
The importance of congenital or gestational factors was first recognized by epidemiological studies describing associations between intrauterine growth retardation (IUGR) and insulin resistance, impaired glucose tolerance, type 2 diabetes, and cardiovascular diseases later in life (5). Other studies have found associations between intrauterine exposure to the diabetic milieu and obesity, insulin resistance, and glucose intolerance in the offspring (6). Similar results have been reported in regards to prenatal exposure to famine, which causes IUGR (7). Animal models of IUGR [including placental ischemia (8), glucocorticoid exposure (9), and global or protein malnutrition (10, 11, 12) during pregnancy] agree with these epidemiological studies; and it is now generally believed that adverse events during pregnancy may interfere with fetal physiology, metabolism, and development and may program the fetus to later develop metabolic diseases in adulthood (13).ty3\+%, http://www.100md.com
Ethanol (ETOH) ingestion during pregnancy can lead to abnormal fetal development, sometimes manifested as the fetal alcohol syndrome (FAS) (14). IUGR is a hallmark of FAS, and the prevalence of FAS is elevated in populations with lower socioeconomic status (15), where type 2 diabetes is also common (16). A few studies in humans (17) and rodents (18, 19) have reported the presence of glucose intolerance in offspring exposed to ETOH in utero. It has been suggested that ETOH-induced IUGR is associated with insulin resistance, but its pathogenesis remains unclear. In a recent study, glucose uptake was decreased in red muscle of offspring of rats exposed to ETOH in vitro, but insulin receptor tyrosine kinase activity was not altered (20).
Recent reports suggest that adipocytokines may be involved in the control of energy homeostasis and insulin action. Congenital or acquired lipoatrophy is associated with insulin resistance, which can be reduced by adipose tissue transplantation or by leptin, a product of the ob gene secreted by the adipocyte (21). Leptin also inhibits food intake, reduces body weight, stimulates energy expenditure, and decreases hyperglycemia and hyperinsulinemia in leptin-deficient, ob/ob mice (21, 22). Adiponectin, another adipocyte protein also known as ACRP30 or adipoQ, has been proposed to be an important link between obesity and type 2 diabetes. The expression and circulating levels of adiponectin are reduced in obese rodents, monkeys, and humans and increased by weight loss and thiazolidinediones (21, 23, 24, 25). Circulating adiponectin levels are inversely related with fasting insulin concentrations and positively correlated with insulin sensitivity, determined by euglycemic clamps in humans and monkeys (26, 27). In addition, elevation in circulating adiponectin levels inhibited endogenous glucose production and reduced glycemia in mice (28, 29, 30).
Resistin, a more recently identified adipocyte hormone, has also been proposed to link obesity to type 2 diabetes (31). Resistin was elevated in obese diabetic mice, and its administration or neutralization resulted in impaired or improved insulin action, respectively. In a recent report, we have shown that prenatal ETOH exposure results in IUGR, with impaired glucose homeostasis and increased resistin expression, in newborn and adult offspring (32). The adult offspring had glucose intolerance, hyperinsulinemia, and reduced skeletal muscle glucose transporter (GLUT)4 expression, but there were no alterations of adipose tissue mass, leptin, or leptin receptor expression. The present study was undertaken to determine the interaction of prenatal ETOH exposure with postnatal high-fat diet on glucose tolerance and the expression of adipocytokines in rats..jlw*, 百拇医药
Materials and Methods.jlw*, 百拇医药
Materials.jlw*, 百拇医药
Mouse adiponectin and rat insulin and leptin RIA kits were from Linco Research, Inc. (St. Charles, MO). Resistin enzyme immunoassay kit was from Phoenix Pharmaceuticals, Inc. (Belmont, CA). FFA assay kit (NEFA C) was from Wako Pure Chemical Industries Ltd. (Richmond, VA). Anti-GLUT4 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-uncoupling protein (UCP)-3 antibody was from Chemicon International (Temecula, CA). Electrophoresis reagents were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). ECL enhanced chemiluminescence kit was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Trizol, SuperScript reverse transcriptase, Taq DNA polymerase, and oligo(deoxythymidine) primers were obtained from Life Technologies, Inc. (Rockville, MD). cDNA primers were synthesized by Life Technologies, Inc. Insulin immunohistochemistry kit was from DAKO Corp. Diagnostics (Mississauga, Ontario, Canada). The high-fat diet (D12451) was purchased from Research Diets (New Brunswick, NJ). ETOH to be administered to rats was obtained from pharmaceutical services of the Health Sciences Centre (Winnipeg, Manitoba, Canada). Isopropyl alcohol and methanol were from Fisher Scientific (Nepean, Ontario, Canada). All other chemicals were purchased from Sigma-Aldrich Corp. (Oakville, Ontario, Canada).
Animals and experimental design\e!u, http://www.100md.com
Virgin Sprague Dawley rats were purchased from Charles River Laboratories, Inc. (Saint Constant, Quebec, Canada) and housed in individual cages under controlled temperature, humidity, and light cycle. They were allowed free access to tap water and commercial rat chow (Agway Prolab, Syracuse, NY), providing a balanced amount of minerals and vitamins and containing 20% proteins, 11% fat, and 69% carbohydrates. The rats were randomly divided into two weight-matched groups, and pregnancy was timed using the vaginal sperm plug. Throughout gestation, one group was given ETOH, 2 g/kg (36%) by gavage twice daily at 0900 and 1600 h, and the second group (control) was given the same volume of water instead of ETOH. Body weight and food intake were recorded from d 14 of gestation to parturition. Rat offspring were weaned onto either normal chow or high-fat diet providing 45% calories as fat, 35% carbohydrates, and 20% proteins. For body weight and food studies, the rats were housed 1 d per week in individual plastic cages with metal wire basket tops. Food was weighed and placed on the basket top. After 24 h, remaining food was weighed, and food intake was calculated as the difference between the two weights, corrected for any food spill, assessed by scanning the cage bedding. Body weights were recorded at the same time. At 13 wk of age, the rats were fasted overnight and underwent an ip glucose tolerance test (IPGTT) by 0900 h the next morning. Glucose (30% wt/vol), 2 g/kg body weight, was injected ip; and tail blood (40 µl) was collected at 0, 30, 60, and 120 min for glucose determination. The rats were killed by exsanguination, through cardiac puncture under light ether anesthesia, and gastrocnemius muscle was stored at -70 C until used. Another set of rats were killed without prior overnight fast; and gastrocnemius muscle, pancreas, and epididymal adipose tissues were obtained. Aliquots of serum were stored at -20 C until assayed. The protocol was approved by the Committee for Animal Use in Research and Teaching of the University of Manitoba.
Adiponectin and resistin expression4, http://www.100md.com
Total RNA was extracted from approximately 100 mg adipose tissue, by the Trizol method (Life Technologies, Inc.). The first-strand cDNAs for resistin and adiponectin were synthesized from 5 µg total RNA, using SuperScript reverse transcriptase and oligo(deoxythymidine) primers. The reverse transcription products (5 µl) were amplified by PCR, using Taq DNA polymerase and specific primers for adiponectin (sense: 5'-GTTCTCTTCACCTACGACCAG-3'; antisense: 5'-GGAAGAGAAGAAAGCCAGTAA-3) and resistin (sense: 5'-TTTTCCTTTTCTTCCTTGTCC-3'; antisense: 5'-TGCTGTCCAGTCTATCCTTGC-3'). Another 5 µl of the reverse transcription product was amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers as an internal control. The amplification conditions for both PCRs were 94 C for 45 sec, 55 C for 45 sec, and 72 C for 90 sec (30 cycles). The expected RT-PCR products of resistin and adiponectin are 267 and 178 bp in length, respectively. RT-PCR products (10 µl) were electrophoresed in a 1.5% agarose gel, stained with ethidium bromide, and densitometrically analyzed using NIH Image software.
GLUT-4 content*:r&^]3, http://www.100md.com
Gastrocnemius muscle tissue (500 mg) was homogenized for 5 sec using a Brinkmann Instruments, Inc. (Westbury, NY) homogenizer in ice-cold TES buffer [20 mM Tris-HCl (pH 7.4), containing 250 mM sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 0.01 mM leupeptin, and 5 µg/ml aprotinin]. The homogenate was spun twice at 3,000 x g for 10 min at 4 C, the supernatant spun again at 100,000 x g for 90 min at 4 C, and the resultant precipitate suspended in ice-cold TES by shearing using 22-, 25-, and 30-gauge needles. Protein (50 µg per lane) was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto nitrocellulose membranes. Blots were blocked with 5% dry milk for 1 h and incubated with rabbit anti-GLUT4 antiserum at 1:1,000 dilution for 1 h at room temperature. Blots were then washed in Tris-buffered saline (TBS)-Tween for 15 min, incubated with goat antirabbit horseradish peroxidase-conjugated secondary antibody at 1:3,000 for 1 h at room temperature, and washed in TBS-Tween for 15 min. Immune complexes were detected using the ECL chemiluminescent detection kit, after exposing the blots to an Eastman Kodak Co. (Rochester, NY) Biomax Light Film. GLUT4 protein was quantified by densitometry using NIH Image software.
Uncoupling protein-3 content(zh', 百拇医药
Gastrocnemius muscle tissue was homogenized using a Brinkmann Instruments, Inc. homogenizer in a solution of 10 mM Tris-HCl buffer (pH 7.4), containing 250 mM sucrose and 1 mM EDTA. Mitochondrial fractions were then prepared as described by Cannon and Lindberg (33). Briefly, the homogenate was centrifuged at 12,000 x g for 30 min. The pellet was resuspended in the same buffer and was centrifuged at 800 x g for 10 min. After saving the supernatant, the nuclear pellet was resuspended and centrifuged as above. Both supernatants were combined and centrifuged at 10,000 x g for 30 min. The mitochondrial pellet was resuspended in the same buffer. Protein (50 µg per lane) was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto nitrocellulose membranes. Blots were blocked with 5% dry milk for 1 h and incubated overnight at 4 C with anti-UCP-3 antibody at 1:800 dilution. Blots were then washed in TBS-Tween for 15 min, incubated with goat antirabbit horseradish peroxidase-conjugated secondary antibody at 1:20,000 for 1 h at room temperature, and washed in TBS-Tween for 15 min. Immune complexes were detected using the ECL chemiluminescent detection kit as described above.
Pancreatic insulin content0ht;8?#, http://www.100md.com
Pancreases were dissected and immediately frozen at -70 C. Proteins were extracted using the acid-ETOH method (34). Briefly, pancreases weighing 350–450 mg were homogenized in 5 ml acid-ETOH buffer (1.5 ml HCl, 12 M in 100 ml 70% ETOH) and incubated overnight at 4 C for further extraction. On the next day, samples were centrifuged at 3000 x g for 15 min at 4 C, and supernatants were stored at -70 C for assay. Insulin content was measured with a rat insulin RIA kit.0ht;8?#, http://www.100md.com
Pancreatic histomorphometry0ht;8?#, http://www.100md.com
The whole pancreas was dissected out, weighed, and fixed in 10% buffered formaldehyde solution. The tissue was dehydrated and embedded in paraffin, and 6-µm sections were stained with hematoxylin and eosin by standard procedures. Paraffin-embedded sections were also immunostained using the DAKO Corp. Envision+ system according to the manufacturer’s recommendations. Sections were first deparaffinized with xylene and rehydrated with ETOH and distilled water. Sections were next incubated with a rabbit antiinsulin Igs (1:100), followed by peroxidase-labeled polymer conjugated to goat antirabbit Igs. Sections were then stained with 3,3'-diaminobenzidine substrate-chromogen, counterstained with hematoxylin and eosin, and examined by light microscopy for islet counting. Morphometric analysis was performed using a light microscope connected to a Microprojection System and Northern Exposure software (Nikon, Hollywood, CA). The relative cross-sectional area of ß-cells was determined by marking the threshold of the captured image for brown tissue (ß-cells) and for blue tissue (exocrine pancreas). These analyses were performed by three individuals, two of whom were blinded to the study. Total tissue area was corrected for the unstained area. ß-Cell density was calculated as the cross-sectional area of ß-cells per tissue section area. ß-Cell mass per pancreatic piece was estimated as the product of ß-cell density and the weight of the fresh pancreatic piece, and total ß-cell mass per animal was estimated from the fresh weight of the pancreas.
Other assays5(4|, 百拇医药
Serum insulin and leptin were measured with rat-specific RIA kits (Linco Research, Inc.). Serum adiponectin was measured with a mouse RIA kit (Linco Research, Inc.), which (according to the manufacturer) has a high cross-reactivity with rat adiponectin. Serial dilutions of rat serum in our laboratory produced values that paralleled the standard curve. Serum resistin was determined by enzyme immunoassay (Phoenix Pharmaceuticals, Inc.), with an antibody raised against the human resistin (51–108)-NH2, which (according to the manufacturer) recognizes rat resistin. Values we obtained with serial dilutions of rat serum paralleled the standard curve in this assay. Glucose was measured using a 2300 glucose analyzer (YSI, Inc., Yellow Springs, OH). Tissue protein was determined by the Bradford method, using BSA as standard.5(4|, 百拇医药
Statistics5(4|, 百拇医药
All analyses were conducted with SPSS, Inc. software (version 10.1 for Windows; SPSS, Inc., Chicago, IL). Differences between two groups were evaluated by unpaired t test. Individual glucose and insulin measurements during IPGTT were compared by repeated-measures ANOVA. Effects of prenatal ETOH exposure and postnatal high-fat diet were analyzed by two-way ANOVA using ETOH and fat diet as fixed factors. Insulin values were log-transformed before analysis. Values are expressed as the mean ± SEM. P < 0.05 was considered significant.
Results6]lh*8@, 百拇医药
Animal weight and food intake6]lh*8@, 百拇医药
The amount of ETOH ingested (1.5 g/d) provided approximately 7 kcal/g body weight or approximately 10.5 kcal/d; and ETOH dams consumed 19.6 ± 0.9 g chow per day, providing 68.6 ± 3.2 kcal daily. The total daily caloric intake (79.0 kcal) was not significantly different than that of control dams, whose daily chow consumption was 24.9 ± 2.2 g, providing 87.2 ± 7.8 kcal, and weight gain during pregnancy was similar between the ETOH and control groups (116.0 ± 14.4 vs. 118.0 ± 15.5 g). Litter size per dam was 15.4 ± 1.0 in ETOH and 16.0 ± 1.7 in controls [P = not significant (NS)].6]lh*8@, 百拇医药
At birth, ETOH pups weighed significantly less than controls (5.1 ± 0.1 vs. 6.3 ± 0.1 g, P < 0.001), and weight difference persisted until 6 wk of age. Weight of ETOH rats then caught up to control weight, and rats from both groups weaned onto normal chow subsequently had comparable weights (Fig. 1A). Weaning onto a high-fat diet resulted in more weight gain in both groups, with, however, a slower weight gain in offspring of ETOH dams. Postnatal diet (P < 0.001), but not prenatal ETOH (P = 0.139), was a significant determinant of weight at 13 wk of age, and there was a significant interaction between diet and prenatal ETOH exposure (P < 0.025). The weight of epididymal fat pads was higher in rats fed a high-fat diet, compared with those on normal chow, but ETOH offspring on a high-fat diet tended to have larger epididymal fat than controls (Table 1). Food intake was significantly lower in ETOH offspring, compared with controls, at 4 wk of age; but it reached control level by 5 wk and subsequently increased, with age, similarly in both groups until 8 wk and then plateaued (Fig. 1B). Daily food ingestion was quantitatively greater in rats weaned onto normal chow than in those on high-fat diet, regardless of prenatal conditions, but caloric intakes were similar except for being reduced in high-fat-fed ETOH offspring after 8 wk of age (Fig. 1C).
fig.ommitteed{e#oz, http://www.100md.com
Figure 1. Body weight (A), food consumption (B), and energy intake (C) of rat offspring. CONT, No ETOH exposure during pregnancy; ETOH, ETOH exposure during pregnancy; CHOW, postnatal chow feeding; HIFA, postnatal high-fat feeding. Data are shown as the mean ± SEM (n = 12 rats/group).{e#oz, http://www.100md.com
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Table 1. Animal characteristics{e#oz, http://www.100md.com
Adipose hormones, FFA, and UCP-3{e#oz, http://www.100md.com
Because of difference in adiposity and food intake between dietary groups, we measured serum leptin and resistin and determined adipose tissue resistin mRNA and skeletal muscle UCP-3. Serum leptin concentration was higher in high-fat-fed rats than in those on normal chow; and among the high-fat diet groups, there was a tendency for elevated leptin in ETOH offspring, compared with controls (Fig. 2A). Similar to leptin, skeletal muscle UCP-3 was elevated in rats fed a high-fat diet, compared with those on normal chow (Fig. 2B). Adipose tissue resistin mRNA and serum resistin concentration were significantly elevated in ETOH offspring, compared with controls, but were not affected by high-fat diet (Fig. 3. We also determined serum adiponectin, adipose tissue adiponectin mRNA, and serum FFA. Serum adiponectin levels were normal in all groups (Fig. 4A). A significant reduction in adipose tissue adiponectin mRNA was found only in non-ETOH-exposed high-fat-fed rats (Fig. 4B). ETOH had no effect on serum FFA level, but there was a surprisingly significant effect of high-fat diet to decrease FFA (Table 1).
fig.ommitteedw9x$, 百拇医药
Figure 2. Serum leptin and gastrocnemius muscle UCP-3 protein in rat offspring at 13 wk of age. ETOH effect on leptin (P = 0.09) and UCP-3 (P = NS). HIFA effect on both leptin and UCP-3 (P < 0.001). Data are shown as the mean ± SEM (n = 6 rats/group). O.D, Optical density (arbitrary units).w9x$, 百拇医药
fig.ommitteedw9x$, 百拇医药
Figure 3. Serum resistin (n = 6 rats/group) (A) and epididymal fat resistin mRNA expression (n = 3 rats/group) (B) in rat offspring at 13 wk of age. Resistin mRNA was measured by RT-PCR (representative blot shown) and expressed in arbitrary units relative to GAPDH mRNA levels (graph shown). ETOH effect, P < 0.005; HIFA effect, P = NS. Data are shown as the mean ± SEM.w9x$, 百拇医药
fig.ommitteedw9x$, 百拇医药
Figure 4. Serum adiponectin (A) and epididymal fat adiponectin mRNA (B) in rat offspring at 13 wk of age. Adiponectin mRNA (representative blot shown) was measured by RT-PCR and expressed in arbitrary units relative to GAPDH mRNA levels (graph shown). ETOH and HIFA effects on serum adiponectin, P = NS; ETOH effect on mRNA, P = NS; HIFA effect on mRNA, P = 0.014. Data are shown as the mean ± SEM (n = 6 rats/group).
Glucose tolerance, insulin, and GLUT4no, 百拇医药
To study the interaction between prenatal ETOH exposure and postnatal diet on glucose homeostasis, we measured serum glucose and insulin levels in the nonfasting state and during an IPGTT after an overnight fast. The fasting and nonfasting glucose levels were similar among all four groups. Both nonfasting (Table 1) and fasting insulin levels were elevated in the ETOH offspring, compared with controls, and increased further on high-fat diet. During IPGTT, glucose levels increased most in ETOH offspring on high-fat diet, and intermediate increases were found in chow-fed ETOH rats and in high-fat-fed controls, which showed comparable glucose values (Fig. 5A). The area under the glucose curve correlated with glycemic increases. Insulin response peak to ip glucse challenge and the area under the insulin curve were greatest in high-fat-fed ETOH offspring and smallest in controls, with intermediate values in high-fat-fed controls and chow-fed ETOH rats (Fig. 5B).
fig.ommitteed2a-0]jn, 百拇医药
Figure 5. Serum glucose (A) and insulin (B) levels during an IPGTT in 13-wk-old rat offspring. Data are shown as the mean ± SEM (n = 6) of each time point. The areas under the glucose curves (mM/min) of the four groups were 1247 ± 57 (CONT-CHOW), 1469 ± 87 (CONT-HIFA), 1516 ± 107 (ETOH-CHOW), and 1789 ± 101 (ETOH-HIFA), with significant effects of ETOH (P < 0.005) and HIFA (P < 0.025). Their respective areas under the insulin curves (ng/ml·min) were 78.5 ± 6.8, 159.5 ± 20.4, 110.5 ± 14.2, and 200.0 ± 18.5, with significant effects of ETOH (P < 0.05) and HIFA (P < 0.0001).2a-0]jn, 百拇医药
Because glucose intolerance in association with hyperinsulinemia suggests the presence of insulin resistance, we measured GLUT4 in skeletal muscle membranes (Fig. 6). In the random-fed state, GLUT4 level was not different among groups, despite differences in circulating insulin levels. After glucose challenge, GLUT4 was significantly lower in high-fat-fed normal rats and in chow-fed ETOH rats, compared with controls, and tended to further decrease in high-fat-fed ETOH animals.
fig.ommitteed2.!fi^, http://www.100md.com
Figure 6. Gastrocnemius muscle GLUT4 in rat offspring at 13 wk of age. This figure shows representative GLUT4 immunoblots and densitometric analyses of the blots carried out after an IPGTT (n = 6 rats/group). ETOH effect, P = 0.03; HIFA effect, P < 0.001. Data are shown as the mean ± SEM.2.!fi^, http://www.100md.com
Pancreas morphometry and insulin content2.!fi^, http://www.100md.com
Because changes in insulin levels could be caused by ß-cell changes, we measured pancreatic insulin content and performed histomorphometric analyses of pancreatic islets (Table 2). The pancreas was significantly larger in the two groups of rats fed high-fat diet, compared with those on normal chow, and the islet density was higher in the three study groups, compared with controls. Pancreatic insulin content, ß-cell density, and ß-cell mass significantly increased in high-fat-fed normal rats, but this increase was offset in high-fat-fed ETOH rats.2.!fi^, http://www.100md.com
fig.ommitteed
Table 2. Pancreatic insulin content and morphometrywcr{{, 百拇医药
Discussionwcr{{, 百拇医药
We have recently shown that alcohol ingestion by the mother during pregnancy results in IUGR with impaired glucose homeostasis and increased resistin expression in newborn and adult offspring. The adult offspring had glucose intolerance, hyperinsulinemia, and reduced skeletal muscle GLUT4 expression. These manifestations of insulinresistance were not associated with alterations of adipose tissue mass, leptin, or leptin receptor expression (32). In the current study, we have extended these investigations and now report on the effects of a postnatal high-fat diet on weight and glucose regulation in the offspring of dams ingesting ETOH during pregnancy. We show that high-fat diet worsens glucose intolerance and decreases adiponectin mRNA, but has no effect on resistin expression, above the level achieved with in utero ETOH exposure. In addition, prenatal ETOH exposure did not alter adiponectin expression in these animals.
High-fat diet caused less weight gain in offspring of ETOH dams, which also had less caloric intake, compared with controls. However, even though consuming less calories, these ETOH offspring had the largest epididymal fat pads and gained more weight than controls and chow fed ETOH rats, indicating that the high-fat diet increased energy storage in ETOH offspring through appetite-independent mechanisms. Leptin and UCP-3 increased equally with high-fat feeding in both ETOH and control offspring, probably reflecting protective mechanisms against further weight gain (35, 36).p+o:c&d, 百拇医药
High-fat diet reduced GLUT4 expression, increased insulin secretion, and worsened glucose tolerance (37, 38). Glucose transport across the plasma membrane is the ratelimiting step for glucose metabolism in skeletal muscle, and the insulin-dependent glucose transporter GLUT4 is a primary determinant of insulin-stimulated glucose uptake and metabolism in this tissue. Increasing muscle GLUT4 content by transgenic overexpression or by increased contractile activity enhances insulin-stimulated muscle glucose uptake, whereas reducing the content of GLUT4 by gene knockout, denervation, or aging impairs insulin-mediated muscle glucose uptake (39). The decreased skeletal muscle GLUT4 content in ETOH offspring on chow or high-fat diet, therefore, provides a mechanism for insulin resistance in these animals. Compared with the high-fat diet alone, the combination of ETOH and high-fat diet decreased pancreatic insulin content while increasing circulating insulin levels. This is a sign of early ß-cell failure, with insufficient insulin synthesis or storage, but exaggerated insulin secretion to maintain normoglycemia in the presence of insulin resistance (40, 41).
Because adipocyte factors have been implicated in the regulation of insulin action, we examined FFA, adiponectin, and resistin levels in rat offspring. FFAs are well known to cause insulin resistance of both skeletal muscle and liver, where they interfere with insulin signaling (4, 42). However, high-fat diet, in this study, was not associated with increases in circulating FFA. This paradoxical observation has been reported before (43) and attributed to an increased efficiency of FFA clearance by skeletal muscle (44). We found no elevation of FFA in rat offspring exposed to ETOH in utero. Of note, FFA levels in this study were measured after an overnight fast; and it has been suggested that, because of their variability, fasting FFA levels often fail to correlate with manifestations of insulin resistance (45).k-s%-, 百拇医药
Adiponectin has been implicated as a factor that can mediate FFA lowering, and recent evidence suggests a major role for this protein in the regulation of insulin action (21). Adiponectin expression is reduced in insulin-resistant states, increased with caloric restriction and thiazolidinedione treatment (21, 24, 25), and correlates with insulin sensitivity determined by euglycemic clamps (26, 27). In addition, adiponectin administration increases insulin sensitivity and reduces glycemia in mice (28, 29, 30). In the current study, we found decreased adiponectin expression by high-fat diet in rats, in agreement with the above reports. However, circulating adiponectin levels were normal. A lack of correlation between adiponectin protein and adiponectin mRNA or insulin sensitivity has been reported by others (26). In addition, we found no effect of prenatal ETOH exposure on adiponectin expression, despite glucose intolerance and hyperinsulinemia, suggesting that these abnormalities are not explained by changes in adiponectin levels.
We found elevated resistin mRNA and protein in rats after prenatal ETOH exposure but not solely after high-fat diet. Steppan et al. (31) found elevated resistin levels in both genetic (ob/ob and db/db) and diet-induced obese diabetic mice. These authors proposed that resistin is a link between obesity and type 2 diabetes. In their study, antiresistin antibody improved blood glucose and insulin sensitivity in mice with diet-induced obesity, whereas administration of recombinant resistin impaired insulin action in vivo in mice and ex vivo in adipocytes. In addition, circulating resistin levels and adipocyte resistin gene expression were markedly decreased by treatment with the insulin sensitizer, antidiabetic drug rosiglitazone. Subsequent reports have confirmed (46, 47) or questioned (48, 49, 50) the conclusion that resistin levels are elevated in obese insulin-resistant states. Recent genomic studies have shown that noncoding single nucleotide polymorphisms in the resistin gene are associated with obesity (51) or may influence insulin sensitivity in interaction with obesity (52). In line with these reports, our current results and those of a recent study (32) support a role for resistin in the glucose intolerance associated with ETOH-induced IUGR.
The regulation of adiponectin and resistin levels is not yet well understood. Both hormones are inhibited by ß-adrenergic agonists (53, 54), cAMP activators (21, 54), and TNF- (21, 53, 54), which can cause insulin resistance. In addition, an increase in the expression of both adipocytokines has been observed during high-fat feeding in rats (55). However, these hormones are usually regulated in opposite directions by diet-induced obesity and glucocorticoids, which down-regulate adiponectin (21, 25, 54) and up-regulate resistin (31, 48, 53, 56). Conversely, thiazolidinediones stimulate adiponectin (21, 24) but inhibit resistin expression (31, 53, 56). An elevated cortisone level in ETOH-exposed offspring (57) could explain increased resistin but not normal adiponectin expression in these animals. Insulin has been reported to stimulate adiponectin secretion (58), but its effects on resistin expression have been variable (53, 56, 59). We have recently shown increased resistin expression in hypoinsulinemic newborn rats exposed to ETOH during pregnancy, suggesting that hyperinsulinemia is an unlikely factor in the increased resistin expression in ETOH offspring (32). Elevated serum glucose concentrations in these pups may have contributed to an increase in resistin expression at this early age (53, 59). It is uncertain, however, whether this explains the persistently elevated resistin levels during adulthood. Thus, the regulation of both adiponectin and resistin involves multiple hormonal and nutritional factors, and the reason why prenatal ETOH exposure and postnatal high-fat diet have differential effects on these hormones is presently unclear.
In summary, prenatal ETOH exposure elevated resistin expression, whereas postnatal high-fat diet reduced adiponectin expression in rats. In the presence of both conditions, adiponectin was normal, whereas resistin remained elevated. Both conditions resulted in glucose intolerance and hyperinsulinemia with additive effects.7'wqrpv, http://www.100md.com
Received June 14, 2002.7'wqrpv, http://www.100md.com
Accepted for publication October 16, 2002.7'wqrpv, http://www.100md.com
References7'wqrpv, http://www.100md.com
Lillioja S, Mott DM, Howard BV, Bennett PH, Yki-Jarvinen H, Freymond D, Nyomba BL, Zurlo F, Swinburn B, Bogardus C 1988 Impaired glucose tolerance as a disorder of insulin action: cross-sectional and longitudinal studies in Pima Indians. N Engl J Med 318:1217–12257'wqrpv, http://www.100md.com
Groop L 2000 Genetics of the metabolic syndrome. Br J Nutr 83(Suppl 1):S39–S487'wqrpv, http://www.100md.com
Lichtenstein AH, Schwab US 2000 Relationship of dietary fat to glucose metabolism. Atherosclerosis 150:227–2437'wqrpv, http://www.100md.com
Oakes ND, Cooney GJ, Camilleri S, Chisholm DJ, Kraegen EW 1997 Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding. Diabetes 46:1768–1774
Barker DJ, Hales CN, Fall CH, Osmond C, Phillips K, Clark PM 1993 Type 2 (non-insulin-dependent) diabetes mellitus, hypertension and hyperlipidemia (syndrome X): relation to reduced fetal growth. Diabetologia 36:62–67/kj@, 百拇医药
Pettitt DJ, Bennett PH, Saad MF, Charles MA, Nelson RG, Knowler WC 1991 Abnormal glucose tolerance during pregnancy in Pima Indian women: long-term effects on offspring. Diabetes 40(Suppl 2):126–130/kj@, 百拇医药
Phillips DIW 1998 Birth weight and the future development of diabetes: a review of the evidence. Diabetes Care 21(Suppl 2):B150–B155/kj@, 百拇医药
Simmons RA, Templeton LJ, Gertz SJ 2001 Intrauterine growth retardation leads to the development of type 2 diabetes in the rat. Diabetes 50:2279–2286/kj@, 百拇医药
Benediktsson R, Lindsay RS, Noble J, Seckl JR, Edwards CR 1993 Glucocorticoid exposure in utero: a new model for adult hypertension. Lancet 341:339–341/kj@, 百拇医药
Ozanne SE, Wang CL, Coleman N, Smith GD 1996 Altered muscle insulin sensitivity in the male offspring of protein-malnourished rats. Am J Physiol 271:E1128–E1134
Martin MA, Alvarez C, Goya L, Portha B, Pascual-Leone AM 1997 Insulin secretion in adult rats that had experienced different underfeeding patterns during their development. Am J Physiol 272:E634–E6404#da/, 百拇医药
Vickers MH, Breier BH, Cutfield WS, Hofman PL, Gluckman PD 2000 Fetal origins of hyperphagia, obesity, and hypertension and postnatal amplification by hypercaloric nutrition. Am J Physiol 279:E83–E874#da/, 百拇医药
Nilsson C, Larsson BM, Jennische E, Eriksson E, Bjorntorp P, York DA, Holmang A 2001 Maternal endotoxemia results in obesity and insulin resistance in adult male offspring. Endocrinology 142:2622–26304#da/, 百拇医药
Clarren SK, Smith DW 1978 The fetal alcohol syndrome. N Engl J Med 298:1063–10674#da/, 百拇医药
Bingol N, Schuster C, Fuchs M, Iosub S, Turner G, Stone RK, Gromisch DS 1987 The influence of socioeconomic factors on the occurrence of fetal alcohol syndrome. Adv Alcohol Subst Abuse 6:105–1184#da/, 百拇医药
Marshall JA, Hamman RF, Baxter J, Mayer EJ, Fulton DL, Orleans M, Rewers M, Jones RH 1993 Ethnic differences in risk factors associated with the prevalence of non-insulin dependent diabetes mellitus. The San Luis Valley Diabetes Study. Am J Epidemiol 137:706–718
Castells S, Mark E, Abaci F, Schwartz E 1981 Growth retardation in fetal alcohol syndrome. Unresponsiveness to growth-promoting hormones. Dev Pharmacol Ther 3:232–241#.6--v, 百拇医药
Villaroya F, Mampel T 1985 Glucose tolerance and insulin response in offspring of ethanol-treated pregnant rats. Gen Pharmacol 16:415–418#.6--v, 百拇医药
Lopez-Tejero D, Llobera M, Herrera E 1989 Permanent abnormal response to glucose load after prenatal ethanol exposure in rats. Alcohol 6:469–473#.6--v, 百拇医药
Elton CW, Pennington JS, Lynch SA, Carver FM, Pennington SN 2002 Insulin resistance in adult rat offspring associated with maternal dietary fat and alcohol consumption. J Endocrinol 173:63–71#.6--v, 百拇医药
Havel PJ 2002 Control of energy homeostasis and insulin action by adipocyte hormones: leptin, acylation stimulating protein, and adiponectin. Curr Opin Lipidol 13:51–59#.6--v, 百拇医药
Friedman JM, Halaas JL 1998 Leptin and the regulation of body weight in mammals. Nature 395:763–770#.6--v, 百拇医药
Hu E, Liang P, Spiegelman BM 1996 AdipoQ is a novel adipose-specific gene dysregulted in obesity. J Biol Chem 271:10697–10703
Combs TP, Wagner JA, Berger J, Doebber T, Wang WJ, Zhang BB, Tanen M, Berg AH, O’Rahilly S, Savage DB, Chatterjee K, Weiss S, Larson PJ, Gottesdiener KM, Gertz BJ, Charron MJ, Scherer PE, Moller DE 2002 Induction of adipocyte complement-related protein of 30 kilodaltons by PPAR{gamma} agonists: a potential mechanism of insulin sensitization. Endocrinology 143:998–1007r], 百拇医药
Stanhope KL, Graham JL, Sinha M, Havel PJ 2002 Low circulating adiponectin levels and reduced adipocyte adiponectin production in obese, insulin-resistant Sprague-Dawley rats. Diabetes 51(Suppl 2):A404 (Abstract)r], 百拇医药
Hotta K, Funahashi T, Bodkin NL, Ortmeyer HK, Arita Y, Hansen BC, Matsuzawa Y 2001 Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys. Diabetes 50:1126–1133r], 百拇医药
Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, Pratley RE, Tataranni PA 2001 Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab 86:1930–1935
Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson MR, Yen FT, Bihain BE, Lodish HF 2001 Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci USA 98:2005–2010+0+(, 百拇医药
Berg AH, Combs TP, Du X, Brownlee M, Scherer PE 2001 The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat Med 7:947–953+0+(, 百拇医药
Combs TP, Berg AH, Obici S, Scherer PE, Rossetti L 2001 Endogenous glucose production is inhibited by the adipose-derived protein Acrp30. J Clin Invest 108:1875–1881+0+(, 百拇医药
Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS, Lazar MA 2001 The hormone resistin links obesity to diabetes. Nature 409:307–312+0+(, 百拇医药
Chen L, Nyomba G Prenatal alcohol exposure impairs glucose tolerance in adult rat offspring. Canadian Diabetes Association meeting, Edmonton, Canada, 2001, p 33 (Abstract 121)+0+(, 百拇医药
Cannon B, Lindberg O 1979 Mitochondria from brown adipose tissue: isolation and properties. Methods Enzymol 55F:65–78
Best HC, Haist RE, Ridout JE 1939 Diet and the insulin content of the pancreas. J Physiol 97:107–119l2fmhv\, 百拇医药
Matsuda J, Hosoda K, Itoh H, Son C, Doi K, Tanaka T, Fukunaga Y, Inoue G 1997 Cloning of rat uncoupling protein-3 and uncoupling protein-2 cDNAs: their gene expression in rats fed high-fat diet. FEBS Lett 418:200–204l2fmhv\, 百拇医药
Samec S, Seydoux J, Dulloo AG 1999 Post-starvation gene expression of skeletal muscle uncoupling protein 2 and uncoupling protein 3 in response to dietary fat levels and fatty acid composition. A link with insulin resistance. Diabetes 48:436–441l2fmhv\, 百拇医药
Kolterman OG, Greenfield M, Reaven GM, Saekow M, Olefsky JM 1979 Effect of a high carbohydrate diet on insulin binding to adipocytes and on insulin action in vivo in man. Diabetes 28:731–736l2fmhv\, 百拇医药
Kahn BB, Pedersen O 1993 Suppression of GLUT4 expression in skeletal muscle of rats that are obese from high fat feeding but not from high carbohydrate feeding or genetic obesity. Endocrinology 132:13–22l2fmhv\, 百拇医药
Charron MJ, Katz EB, Olson AL 1999 GLUT4 gene regulation and manipulation. J Biol Chem 274:3253–3256
Larsson LI, Boder GB, Shaw WN 1977 Changes in the islets of Langerhans in the obese Zucker rat. Lab Invest 36:593–598t{y, http://www.100md.com
Frankel BJ, Cajander S, Boquist L 1987 Islet morphology in young, genetically diabetic Chinese hamsters during the hyperinsulinemic phase. Pancreas 2:625–631t{y, http://www.100md.com
Dresner A, Laurent D, Marcucci M, Griffin ME, Dufour S, Cline GW, Slezak LA, Andersen DK, Hundal RS, Rothman DL, Petersen KF, Shulman GI 1999 Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidylinositol 3-kinase activity. J Clin Invest 103:253–259t{y, http://www.100md.com
Helge JW, Ayre K, Chaunchaiyakul S, Hulbert AJ, Kiens B, Storlien LH 1998 Endurance in high-fat-fed rats: effects of carbohydrate content and fatty acid profile. J Appl Physiol 85:1342–1348t{y, http://www.100md.com
Hegarty BD, Cooney GJ, Kraegen EW, Furler SM 2002 Increased efficiency of fatty acid uptake contributes to lipid accumulation in skeletal muscle of high fat-fed insulin-resistant rats. Diabetes 51:1477–1484t{y, http://www.100md.com
Steinberg HO, Tarshoby M, Monestel R, Hook G, Cronin J, Johnson A, Bayazeed B, Baron A 1997 Elevated circulating free fatty acid levels impair endothelium-dependent vasodilation. J Clin Invest 100:1230–1239
Degawa-Yamauchi M, Bovenkerk JE, Zhu Q, Considine RV 2002 Serum resistin is significantly elevated in obese humans. Diabetes 51(Suppl 2):A405 (Abstract)a, 百拇医药
Moore GB, Chapman H, Holder JC, Lister CA, Piercy V, Smith SA, Clapham JC 2001 Differential regulation of adipocytokine mRNAs by rosiglitazone in db/db mice. Biochem Biophys Res Commun 286:735–741a, 百拇医药
Levy JR, Davenport B, Clore JN, Stevens W 2002 Lipid metabolism and resistin gene expression in insulin-resistant Fischer 344 rats. Am J Physiol 282:E626–E633a, 百拇医药
Way JM, Gorgun CZ, Tong Q, Uysal KT, Brown KK, Harrington WW, Oliver Jr WR, Willson TM, Kliewer SA, Hotamisligil GS 2001 Adipose tissue resistin expression is severely suppressed in obesity and stimulated by peroxisome proliferator-activated receptor gamma agonists. J Biol Chem 276:25651–25653a, 百拇医药
Le Lay S, Boucher J, Rey A, Castan-Laurell I, Krief S, Ferre P, Valet P, Dugail I 2001 Decreased resistin expression in mice with different sensitivities to a high-fat diet. Biochem Biophys Res Commun 289:564–567
Engert JC, Vohl MC, Williams SM, Lepage P, Loredo-Osti JC, Faith J, Dore C, Renaud Y, Burtt NP, Villeneuve A, Hirschhorn JN, Altshuler D, Groop LC, Despres JP, Gaudet D, Hudson TJ 2002 5' flanking variants of resistin are associated with obesity. Diabetes 51:1629–16346110, http://www.100md.com
Wang H, Chu WS, Hemphill C, Elbein SC 2002 Human resistin gene: molecular scanning and evaluation of association with insulin sensitivity and type 2 diabetes in Caucasians. J Clin Endocrinol Metab 87:2520–25246110, http://www.100md.com
Shojima N, Sakoda H, Ogihara T, Fujishiro M, Katagiri H, Anai M, Onishi Y, Ono H, Inukai K, Abe M, Fukushima Y, Kikuchi M, Oka Y, Asano T 2002 Humoral regulation of resistin expression in 3T3-L1 and mouse adipose cells. Diabetes 51:1737–17446110, http://www.100md.com
Fasshauer M, Klein J, Neumann S, Eszlinger M, Paschke R 2002 Hormonal regulation of adiponectin gene expression in 3T3-L1 adipocytes. Biochem Biophys Res Commun 290:1084–10896110, http://www.100md.com
Li J, Yu X, Pan W, Unger RH 2002 Gene expression profile of rat adipose tissue at the onset of high-fat-diet obesity. Am J Physiol 282:E1334–E13416110, http://www.100md.com
Haugen F, Jorgensen A, Drevon CA, Trayhurn P 2001 Inhibition by insulin of resistin gene expression in 3T3-L1 adipocytes. FEBS Lett 507:105–1086110, http://www.100md.com
Ogilvie KM, Rivier C 1997 Prenatal alcohol exposure results in hyperactivity of the hypothalamic-pituitary-adrenal axis of the offspring: modulation by fostering at birth and postnatal handling. Alcohol Clin Exp Res 21:424–4296110, http://www.100md.com
Bogan JS, Lodish HF 1999 Two compartments for insulin-stimulated exocytosis in 3T3-L1 adipocytes defined by endogenous ACRP30 and GLUT4. J Cell Biol 146:609–6206110, http://www.100md.com
Kim KH, Lee K, Moon YS, Sul HS 2001 A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation. J Biol Chem 276:11252–11256(Li Chen and B. L. G. Nyomba)
Abstract$3;^}9/, 百拇医药
High-fat diet and intrauterine growth retardation may predispose to obesity, insulin resistance, and type 2 diabetes. Because prenatal ethanol (ETOH) exposure causes intrauterine growth retardation, we investigated its interactions with postnatal high-fat diet on glucose tolerance and adipocyte-derived hormones in the rat offspring.$3;^}9/, 百拇医药
High-fat-fed offspring had increased adiposity, serum leptin, and muscle uncoupling protein-3, but decreased adiponectin mRNA, compared with corresponding chow-fed groups. ETOH-exposed offspring had normal adiponectin, but increased resistin mRNA and protein, compared with controls, regardless of postnatal diet. Skeletal muscle glucose transporter-4 content was decreased after both ETOH exposure and high-fat feeding. Glycemic and insulin responses to an ip glucose challenge were equally increased in non-ETOH-exposed high-fat-fed offspring and in ETOH-exposed chow-fed offspring, with additive effects of ETOH and high-fat diet. Pancreatic insulin content was elevated only in non-ETOH-exposed high-fat-fed offspring.
The data suggest that high-fat diet worsens glucose intolerance in offspring of rats exposed to ETOH. Prenatal ETOH exposure and postnatal high-fat diet might cause insulin resistance through separate mechanisms, involving resistin and adiponectin, respectively.\gu, 百拇医药
Introduction\gu, 百拇医药
THE NATURAL HISTORY of type 2 diabetes is characterized by a period of insulin resistance, with euglycemia and compensatory hyperinsulinemia, followed by a period of impaired glucose tolerance during which insulin secretion becomes altered, leading to overt diabetes when insulin secretion is inadequate (1). Genetic, congenital, and environmental factors are thought to determine the development of insulin resistance and its worsening, which results in glucose intolerance and diabetes. Though several genes encoding enzymes of insulin action have been identified, they do not seem to play a major role in the pathogenesis of diabetes in the majority of patients (2). Among environmental determinants of insulin resistance, the role of diets high in saturated fat has been extensively studied. High-fat diets, which characteristically are consumed in modern Western societies, adversely affect glucose metabolism by decreasing the sensitivity of glucose transport to insulin. These diets induce insulin resistance by increasing body fat accretion, promoting skeletal muscle triglycerides synthesis, and augmenting circulating free fatty acids (FFAs; Ref. 3). High-fat diets also cause liver insulin resistance, evident by an impaired ability of insulin to suppress hepatic glucose production (4).
The importance of congenital or gestational factors was first recognized by epidemiological studies describing associations between intrauterine growth retardation (IUGR) and insulin resistance, impaired glucose tolerance, type 2 diabetes, and cardiovascular diseases later in life (5). Other studies have found associations between intrauterine exposure to the diabetic milieu and obesity, insulin resistance, and glucose intolerance in the offspring (6). Similar results have been reported in regards to prenatal exposure to famine, which causes IUGR (7). Animal models of IUGR [including placental ischemia (8), glucocorticoid exposure (9), and global or protein malnutrition (10, 11, 12) during pregnancy] agree with these epidemiological studies; and it is now generally believed that adverse events during pregnancy may interfere with fetal physiology, metabolism, and development and may program the fetus to later develop metabolic diseases in adulthood (13).ty3\+%, http://www.100md.com
Ethanol (ETOH) ingestion during pregnancy can lead to abnormal fetal development, sometimes manifested as the fetal alcohol syndrome (FAS) (14). IUGR is a hallmark of FAS, and the prevalence of FAS is elevated in populations with lower socioeconomic status (15), where type 2 diabetes is also common (16). A few studies in humans (17) and rodents (18, 19) have reported the presence of glucose intolerance in offspring exposed to ETOH in utero. It has been suggested that ETOH-induced IUGR is associated with insulin resistance, but its pathogenesis remains unclear. In a recent study, glucose uptake was decreased in red muscle of offspring of rats exposed to ETOH in vitro, but insulin receptor tyrosine kinase activity was not altered (20).
Recent reports suggest that adipocytokines may be involved in the control of energy homeostasis and insulin action. Congenital or acquired lipoatrophy is associated with insulin resistance, which can be reduced by adipose tissue transplantation or by leptin, a product of the ob gene secreted by the adipocyte (21). Leptin also inhibits food intake, reduces body weight, stimulates energy expenditure, and decreases hyperglycemia and hyperinsulinemia in leptin-deficient, ob/ob mice (21, 22). Adiponectin, another adipocyte protein also known as ACRP30 or adipoQ, has been proposed to be an important link between obesity and type 2 diabetes. The expression and circulating levels of adiponectin are reduced in obese rodents, monkeys, and humans and increased by weight loss and thiazolidinediones (21, 23, 24, 25). Circulating adiponectin levels are inversely related with fasting insulin concentrations and positively correlated with insulin sensitivity, determined by euglycemic clamps in humans and monkeys (26, 27). In addition, elevation in circulating adiponectin levels inhibited endogenous glucose production and reduced glycemia in mice (28, 29, 30).
Resistin, a more recently identified adipocyte hormone, has also been proposed to link obesity to type 2 diabetes (31). Resistin was elevated in obese diabetic mice, and its administration or neutralization resulted in impaired or improved insulin action, respectively. In a recent report, we have shown that prenatal ETOH exposure results in IUGR, with impaired glucose homeostasis and increased resistin expression, in newborn and adult offspring (32). The adult offspring had glucose intolerance, hyperinsulinemia, and reduced skeletal muscle glucose transporter (GLUT)4 expression, but there were no alterations of adipose tissue mass, leptin, or leptin receptor expression. The present study was undertaken to determine the interaction of prenatal ETOH exposure with postnatal high-fat diet on glucose tolerance and the expression of adipocytokines in rats..jlw*, 百拇医药
Materials and Methods.jlw*, 百拇医药
Materials.jlw*, 百拇医药
Mouse adiponectin and rat insulin and leptin RIA kits were from Linco Research, Inc. (St. Charles, MO). Resistin enzyme immunoassay kit was from Phoenix Pharmaceuticals, Inc. (Belmont, CA). FFA assay kit (NEFA C) was from Wako Pure Chemical Industries Ltd. (Richmond, VA). Anti-GLUT4 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-uncoupling protein (UCP)-3 antibody was from Chemicon International (Temecula, CA). Electrophoresis reagents were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). ECL enhanced chemiluminescence kit was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Trizol, SuperScript reverse transcriptase, Taq DNA polymerase, and oligo(deoxythymidine) primers were obtained from Life Technologies, Inc. (Rockville, MD). cDNA primers were synthesized by Life Technologies, Inc. Insulin immunohistochemistry kit was from DAKO Corp. Diagnostics (Mississauga, Ontario, Canada). The high-fat diet (D12451) was purchased from Research Diets (New Brunswick, NJ). ETOH to be administered to rats was obtained from pharmaceutical services of the Health Sciences Centre (Winnipeg, Manitoba, Canada). Isopropyl alcohol and methanol were from Fisher Scientific (Nepean, Ontario, Canada). All other chemicals were purchased from Sigma-Aldrich Corp. (Oakville, Ontario, Canada).
Animals and experimental design\e!u, http://www.100md.com
Virgin Sprague Dawley rats were purchased from Charles River Laboratories, Inc. (Saint Constant, Quebec, Canada) and housed in individual cages under controlled temperature, humidity, and light cycle. They were allowed free access to tap water and commercial rat chow (Agway Prolab, Syracuse, NY), providing a balanced amount of minerals and vitamins and containing 20% proteins, 11% fat, and 69% carbohydrates. The rats were randomly divided into two weight-matched groups, and pregnancy was timed using the vaginal sperm plug. Throughout gestation, one group was given ETOH, 2 g/kg (36%) by gavage twice daily at 0900 and 1600 h, and the second group (control) was given the same volume of water instead of ETOH. Body weight and food intake were recorded from d 14 of gestation to parturition. Rat offspring were weaned onto either normal chow or high-fat diet providing 45% calories as fat, 35% carbohydrates, and 20% proteins. For body weight and food studies, the rats were housed 1 d per week in individual plastic cages with metal wire basket tops. Food was weighed and placed on the basket top. After 24 h, remaining food was weighed, and food intake was calculated as the difference between the two weights, corrected for any food spill, assessed by scanning the cage bedding. Body weights were recorded at the same time. At 13 wk of age, the rats were fasted overnight and underwent an ip glucose tolerance test (IPGTT) by 0900 h the next morning. Glucose (30% wt/vol), 2 g/kg body weight, was injected ip; and tail blood (40 µl) was collected at 0, 30, 60, and 120 min for glucose determination. The rats were killed by exsanguination, through cardiac puncture under light ether anesthesia, and gastrocnemius muscle was stored at -70 C until used. Another set of rats were killed without prior overnight fast; and gastrocnemius muscle, pancreas, and epididymal adipose tissues were obtained. Aliquots of serum were stored at -20 C until assayed. The protocol was approved by the Committee for Animal Use in Research and Teaching of the University of Manitoba.
Adiponectin and resistin expression4, http://www.100md.com
Total RNA was extracted from approximately 100 mg adipose tissue, by the Trizol method (Life Technologies, Inc.). The first-strand cDNAs for resistin and adiponectin were synthesized from 5 µg total RNA, using SuperScript reverse transcriptase and oligo(deoxythymidine) primers. The reverse transcription products (5 µl) were amplified by PCR, using Taq DNA polymerase and specific primers for adiponectin (sense: 5'-GTTCTCTTCACCTACGACCAG-3'; antisense: 5'-GGAAGAGAAGAAAGCCAGTAA-3) and resistin (sense: 5'-TTTTCCTTTTCTTCCTTGTCC-3'; antisense: 5'-TGCTGTCCAGTCTATCCTTGC-3'). Another 5 µl of the reverse transcription product was amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers as an internal control. The amplification conditions for both PCRs were 94 C for 45 sec, 55 C for 45 sec, and 72 C for 90 sec (30 cycles). The expected RT-PCR products of resistin and adiponectin are 267 and 178 bp in length, respectively. RT-PCR products (10 µl) were electrophoresed in a 1.5% agarose gel, stained with ethidium bromide, and densitometrically analyzed using NIH Image software.
GLUT-4 content*:r&^]3, http://www.100md.com
Gastrocnemius muscle tissue (500 mg) was homogenized for 5 sec using a Brinkmann Instruments, Inc. (Westbury, NY) homogenizer in ice-cold TES buffer [20 mM Tris-HCl (pH 7.4), containing 250 mM sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 0.01 mM leupeptin, and 5 µg/ml aprotinin]. The homogenate was spun twice at 3,000 x g for 10 min at 4 C, the supernatant spun again at 100,000 x g for 90 min at 4 C, and the resultant precipitate suspended in ice-cold TES by shearing using 22-, 25-, and 30-gauge needles. Protein (50 µg per lane) was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto nitrocellulose membranes. Blots were blocked with 5% dry milk for 1 h and incubated with rabbit anti-GLUT4 antiserum at 1:1,000 dilution for 1 h at room temperature. Blots were then washed in Tris-buffered saline (TBS)-Tween for 15 min, incubated with goat antirabbit horseradish peroxidase-conjugated secondary antibody at 1:3,000 for 1 h at room temperature, and washed in TBS-Tween for 15 min. Immune complexes were detected using the ECL chemiluminescent detection kit, after exposing the blots to an Eastman Kodak Co. (Rochester, NY) Biomax Light Film. GLUT4 protein was quantified by densitometry using NIH Image software.
Uncoupling protein-3 content(zh', 百拇医药
Gastrocnemius muscle tissue was homogenized using a Brinkmann Instruments, Inc. homogenizer in a solution of 10 mM Tris-HCl buffer (pH 7.4), containing 250 mM sucrose and 1 mM EDTA. Mitochondrial fractions were then prepared as described by Cannon and Lindberg (33). Briefly, the homogenate was centrifuged at 12,000 x g for 30 min. The pellet was resuspended in the same buffer and was centrifuged at 800 x g for 10 min. After saving the supernatant, the nuclear pellet was resuspended and centrifuged as above. Both supernatants were combined and centrifuged at 10,000 x g for 30 min. The mitochondrial pellet was resuspended in the same buffer. Protein (50 µg per lane) was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto nitrocellulose membranes. Blots were blocked with 5% dry milk for 1 h and incubated overnight at 4 C with anti-UCP-3 antibody at 1:800 dilution. Blots were then washed in TBS-Tween for 15 min, incubated with goat antirabbit horseradish peroxidase-conjugated secondary antibody at 1:20,000 for 1 h at room temperature, and washed in TBS-Tween for 15 min. Immune complexes were detected using the ECL chemiluminescent detection kit as described above.
Pancreatic insulin content0ht;8?#, http://www.100md.com
Pancreases were dissected and immediately frozen at -70 C. Proteins were extracted using the acid-ETOH method (34). Briefly, pancreases weighing 350–450 mg were homogenized in 5 ml acid-ETOH buffer (1.5 ml HCl, 12 M in 100 ml 70% ETOH) and incubated overnight at 4 C for further extraction. On the next day, samples were centrifuged at 3000 x g for 15 min at 4 C, and supernatants were stored at -70 C for assay. Insulin content was measured with a rat insulin RIA kit.0ht;8?#, http://www.100md.com
Pancreatic histomorphometry0ht;8?#, http://www.100md.com
The whole pancreas was dissected out, weighed, and fixed in 10% buffered formaldehyde solution. The tissue was dehydrated and embedded in paraffin, and 6-µm sections were stained with hematoxylin and eosin by standard procedures. Paraffin-embedded sections were also immunostained using the DAKO Corp. Envision+ system according to the manufacturer’s recommendations. Sections were first deparaffinized with xylene and rehydrated with ETOH and distilled water. Sections were next incubated with a rabbit antiinsulin Igs (1:100), followed by peroxidase-labeled polymer conjugated to goat antirabbit Igs. Sections were then stained with 3,3'-diaminobenzidine substrate-chromogen, counterstained with hematoxylin and eosin, and examined by light microscopy for islet counting. Morphometric analysis was performed using a light microscope connected to a Microprojection System and Northern Exposure software (Nikon, Hollywood, CA). The relative cross-sectional area of ß-cells was determined by marking the threshold of the captured image for brown tissue (ß-cells) and for blue tissue (exocrine pancreas). These analyses were performed by three individuals, two of whom were blinded to the study. Total tissue area was corrected for the unstained area. ß-Cell density was calculated as the cross-sectional area of ß-cells per tissue section area. ß-Cell mass per pancreatic piece was estimated as the product of ß-cell density and the weight of the fresh pancreatic piece, and total ß-cell mass per animal was estimated from the fresh weight of the pancreas.
Other assays5(4|, 百拇医药
Serum insulin and leptin were measured with rat-specific RIA kits (Linco Research, Inc.). Serum adiponectin was measured with a mouse RIA kit (Linco Research, Inc.), which (according to the manufacturer) has a high cross-reactivity with rat adiponectin. Serial dilutions of rat serum in our laboratory produced values that paralleled the standard curve. Serum resistin was determined by enzyme immunoassay (Phoenix Pharmaceuticals, Inc.), with an antibody raised against the human resistin (51–108)-NH2, which (according to the manufacturer) recognizes rat resistin. Values we obtained with serial dilutions of rat serum paralleled the standard curve in this assay. Glucose was measured using a 2300 glucose analyzer (YSI, Inc., Yellow Springs, OH). Tissue protein was determined by the Bradford method, using BSA as standard.5(4|, 百拇医药
Statistics5(4|, 百拇医药
All analyses were conducted with SPSS, Inc. software (version 10.1 for Windows; SPSS, Inc., Chicago, IL). Differences between two groups were evaluated by unpaired t test. Individual glucose and insulin measurements during IPGTT were compared by repeated-measures ANOVA. Effects of prenatal ETOH exposure and postnatal high-fat diet were analyzed by two-way ANOVA using ETOH and fat diet as fixed factors. Insulin values were log-transformed before analysis. Values are expressed as the mean ± SEM. P < 0.05 was considered significant.
Results6]lh*8@, 百拇医药
Animal weight and food intake6]lh*8@, 百拇医药
The amount of ETOH ingested (1.5 g/d) provided approximately 7 kcal/g body weight or approximately 10.5 kcal/d; and ETOH dams consumed 19.6 ± 0.9 g chow per day, providing 68.6 ± 3.2 kcal daily. The total daily caloric intake (79.0 kcal) was not significantly different than that of control dams, whose daily chow consumption was 24.9 ± 2.2 g, providing 87.2 ± 7.8 kcal, and weight gain during pregnancy was similar between the ETOH and control groups (116.0 ± 14.4 vs. 118.0 ± 15.5 g). Litter size per dam was 15.4 ± 1.0 in ETOH and 16.0 ± 1.7 in controls [P = not significant (NS)].6]lh*8@, 百拇医药
At birth, ETOH pups weighed significantly less than controls (5.1 ± 0.1 vs. 6.3 ± 0.1 g, P < 0.001), and weight difference persisted until 6 wk of age. Weight of ETOH rats then caught up to control weight, and rats from both groups weaned onto normal chow subsequently had comparable weights (Fig. 1A). Weaning onto a high-fat diet resulted in more weight gain in both groups, with, however, a slower weight gain in offspring of ETOH dams. Postnatal diet (P < 0.001), but not prenatal ETOH (P = 0.139), was a significant determinant of weight at 13 wk of age, and there was a significant interaction between diet and prenatal ETOH exposure (P < 0.025). The weight of epididymal fat pads was higher in rats fed a high-fat diet, compared with those on normal chow, but ETOH offspring on a high-fat diet tended to have larger epididymal fat than controls (Table 1). Food intake was significantly lower in ETOH offspring, compared with controls, at 4 wk of age; but it reached control level by 5 wk and subsequently increased, with age, similarly in both groups until 8 wk and then plateaued (Fig. 1B). Daily food ingestion was quantitatively greater in rats weaned onto normal chow than in those on high-fat diet, regardless of prenatal conditions, but caloric intakes were similar except for being reduced in high-fat-fed ETOH offspring after 8 wk of age (Fig. 1C).
fig.ommitteed{e#oz, http://www.100md.com
Figure 1. Body weight (A), food consumption (B), and energy intake (C) of rat offspring. CONT, No ETOH exposure during pregnancy; ETOH, ETOH exposure during pregnancy; CHOW, postnatal chow feeding; HIFA, postnatal high-fat feeding. Data are shown as the mean ± SEM (n = 12 rats/group).{e#oz, http://www.100md.com
fig.ommitteed{e#oz, http://www.100md.com
Table 1. Animal characteristics{e#oz, http://www.100md.com
Adipose hormones, FFA, and UCP-3{e#oz, http://www.100md.com
Because of difference in adiposity and food intake between dietary groups, we measured serum leptin and resistin and determined adipose tissue resistin mRNA and skeletal muscle UCP-3. Serum leptin concentration was higher in high-fat-fed rats than in those on normal chow; and among the high-fat diet groups, there was a tendency for elevated leptin in ETOH offspring, compared with controls (Fig. 2A). Similar to leptin, skeletal muscle UCP-3 was elevated in rats fed a high-fat diet, compared with those on normal chow (Fig. 2B). Adipose tissue resistin mRNA and serum resistin concentration were significantly elevated in ETOH offspring, compared with controls, but were not affected by high-fat diet (Fig. 3. We also determined serum adiponectin, adipose tissue adiponectin mRNA, and serum FFA. Serum adiponectin levels were normal in all groups (Fig. 4A). A significant reduction in adipose tissue adiponectin mRNA was found only in non-ETOH-exposed high-fat-fed rats (Fig. 4B). ETOH had no effect on serum FFA level, but there was a surprisingly significant effect of high-fat diet to decrease FFA (Table 1).
fig.ommitteedw9x$, 百拇医药
Figure 2. Serum leptin and gastrocnemius muscle UCP-3 protein in rat offspring at 13 wk of age. ETOH effect on leptin (P = 0.09) and UCP-3 (P = NS). HIFA effect on both leptin and UCP-3 (P < 0.001). Data are shown as the mean ± SEM (n = 6 rats/group). O.D, Optical density (arbitrary units).w9x$, 百拇医药
fig.ommitteedw9x$, 百拇医药
Figure 3. Serum resistin (n = 6 rats/group) (A) and epididymal fat resistin mRNA expression (n = 3 rats/group) (B) in rat offspring at 13 wk of age. Resistin mRNA was measured by RT-PCR (representative blot shown) and expressed in arbitrary units relative to GAPDH mRNA levels (graph shown). ETOH effect, P < 0.005; HIFA effect, P = NS. Data are shown as the mean ± SEM.w9x$, 百拇医药
fig.ommitteedw9x$, 百拇医药
Figure 4. Serum adiponectin (A) and epididymal fat adiponectin mRNA (B) in rat offspring at 13 wk of age. Adiponectin mRNA (representative blot shown) was measured by RT-PCR and expressed in arbitrary units relative to GAPDH mRNA levels (graph shown). ETOH and HIFA effects on serum adiponectin, P = NS; ETOH effect on mRNA, P = NS; HIFA effect on mRNA, P = 0.014. Data are shown as the mean ± SEM (n = 6 rats/group).
Glucose tolerance, insulin, and GLUT4no, 百拇医药
To study the interaction between prenatal ETOH exposure and postnatal diet on glucose homeostasis, we measured serum glucose and insulin levels in the nonfasting state and during an IPGTT after an overnight fast. The fasting and nonfasting glucose levels were similar among all four groups. Both nonfasting (Table 1) and fasting insulin levels were elevated in the ETOH offspring, compared with controls, and increased further on high-fat diet. During IPGTT, glucose levels increased most in ETOH offspring on high-fat diet, and intermediate increases were found in chow-fed ETOH rats and in high-fat-fed controls, which showed comparable glucose values (Fig. 5A). The area under the glucose curve correlated with glycemic increases. Insulin response peak to ip glucse challenge and the area under the insulin curve were greatest in high-fat-fed ETOH offspring and smallest in controls, with intermediate values in high-fat-fed controls and chow-fed ETOH rats (Fig. 5B).
fig.ommitteed2a-0]jn, 百拇医药
Figure 5. Serum glucose (A) and insulin (B) levels during an IPGTT in 13-wk-old rat offspring. Data are shown as the mean ± SEM (n = 6) of each time point. The areas under the glucose curves (mM/min) of the four groups were 1247 ± 57 (CONT-CHOW), 1469 ± 87 (CONT-HIFA), 1516 ± 107 (ETOH-CHOW), and 1789 ± 101 (ETOH-HIFA), with significant effects of ETOH (P < 0.005) and HIFA (P < 0.025). Their respective areas under the insulin curves (ng/ml·min) were 78.5 ± 6.8, 159.5 ± 20.4, 110.5 ± 14.2, and 200.0 ± 18.5, with significant effects of ETOH (P < 0.05) and HIFA (P < 0.0001).2a-0]jn, 百拇医药
Because glucose intolerance in association with hyperinsulinemia suggests the presence of insulin resistance, we measured GLUT4 in skeletal muscle membranes (Fig. 6). In the random-fed state, GLUT4 level was not different among groups, despite differences in circulating insulin levels. After glucose challenge, GLUT4 was significantly lower in high-fat-fed normal rats and in chow-fed ETOH rats, compared with controls, and tended to further decrease in high-fat-fed ETOH animals.
fig.ommitteed2.!fi^, http://www.100md.com
Figure 6. Gastrocnemius muscle GLUT4 in rat offspring at 13 wk of age. This figure shows representative GLUT4 immunoblots and densitometric analyses of the blots carried out after an IPGTT (n = 6 rats/group). ETOH effect, P = 0.03; HIFA effect, P < 0.001. Data are shown as the mean ± SEM.2.!fi^, http://www.100md.com
Pancreas morphometry and insulin content2.!fi^, http://www.100md.com
Because changes in insulin levels could be caused by ß-cell changes, we measured pancreatic insulin content and performed histomorphometric analyses of pancreatic islets (Table 2). The pancreas was significantly larger in the two groups of rats fed high-fat diet, compared with those on normal chow, and the islet density was higher in the three study groups, compared with controls. Pancreatic insulin content, ß-cell density, and ß-cell mass significantly increased in high-fat-fed normal rats, but this increase was offset in high-fat-fed ETOH rats.2.!fi^, http://www.100md.com
fig.ommitteed
Table 2. Pancreatic insulin content and morphometrywcr{{, 百拇医药
Discussionwcr{{, 百拇医药
We have recently shown that alcohol ingestion by the mother during pregnancy results in IUGR with impaired glucose homeostasis and increased resistin expression in newborn and adult offspring. The adult offspring had glucose intolerance, hyperinsulinemia, and reduced skeletal muscle GLUT4 expression. These manifestations of insulinresistance were not associated with alterations of adipose tissue mass, leptin, or leptin receptor expression (32). In the current study, we have extended these investigations and now report on the effects of a postnatal high-fat diet on weight and glucose regulation in the offspring of dams ingesting ETOH during pregnancy. We show that high-fat diet worsens glucose intolerance and decreases adiponectin mRNA, but has no effect on resistin expression, above the level achieved with in utero ETOH exposure. In addition, prenatal ETOH exposure did not alter adiponectin expression in these animals.
High-fat diet caused less weight gain in offspring of ETOH dams, which also had less caloric intake, compared with controls. However, even though consuming less calories, these ETOH offspring had the largest epididymal fat pads and gained more weight than controls and chow fed ETOH rats, indicating that the high-fat diet increased energy storage in ETOH offspring through appetite-independent mechanisms. Leptin and UCP-3 increased equally with high-fat feeding in both ETOH and control offspring, probably reflecting protective mechanisms against further weight gain (35, 36).p+o:c&d, 百拇医药
High-fat diet reduced GLUT4 expression, increased insulin secretion, and worsened glucose tolerance (37, 38). Glucose transport across the plasma membrane is the ratelimiting step for glucose metabolism in skeletal muscle, and the insulin-dependent glucose transporter GLUT4 is a primary determinant of insulin-stimulated glucose uptake and metabolism in this tissue. Increasing muscle GLUT4 content by transgenic overexpression or by increased contractile activity enhances insulin-stimulated muscle glucose uptake, whereas reducing the content of GLUT4 by gene knockout, denervation, or aging impairs insulin-mediated muscle glucose uptake (39). The decreased skeletal muscle GLUT4 content in ETOH offspring on chow or high-fat diet, therefore, provides a mechanism for insulin resistance in these animals. Compared with the high-fat diet alone, the combination of ETOH and high-fat diet decreased pancreatic insulin content while increasing circulating insulin levels. This is a sign of early ß-cell failure, with insufficient insulin synthesis or storage, but exaggerated insulin secretion to maintain normoglycemia in the presence of insulin resistance (40, 41).
Because adipocyte factors have been implicated in the regulation of insulin action, we examined FFA, adiponectin, and resistin levels in rat offspring. FFAs are well known to cause insulin resistance of both skeletal muscle and liver, where they interfere with insulin signaling (4, 42). However, high-fat diet, in this study, was not associated with increases in circulating FFA. This paradoxical observation has been reported before (43) and attributed to an increased efficiency of FFA clearance by skeletal muscle (44). We found no elevation of FFA in rat offspring exposed to ETOH in utero. Of note, FFA levels in this study were measured after an overnight fast; and it has been suggested that, because of their variability, fasting FFA levels often fail to correlate with manifestations of insulin resistance (45).k-s%-, 百拇医药
Adiponectin has been implicated as a factor that can mediate FFA lowering, and recent evidence suggests a major role for this protein in the regulation of insulin action (21). Adiponectin expression is reduced in insulin-resistant states, increased with caloric restriction and thiazolidinedione treatment (21, 24, 25), and correlates with insulin sensitivity determined by euglycemic clamps (26, 27). In addition, adiponectin administration increases insulin sensitivity and reduces glycemia in mice (28, 29, 30). In the current study, we found decreased adiponectin expression by high-fat diet in rats, in agreement with the above reports. However, circulating adiponectin levels were normal. A lack of correlation between adiponectin protein and adiponectin mRNA or insulin sensitivity has been reported by others (26). In addition, we found no effect of prenatal ETOH exposure on adiponectin expression, despite glucose intolerance and hyperinsulinemia, suggesting that these abnormalities are not explained by changes in adiponectin levels.
We found elevated resistin mRNA and protein in rats after prenatal ETOH exposure but not solely after high-fat diet. Steppan et al. (31) found elevated resistin levels in both genetic (ob/ob and db/db) and diet-induced obese diabetic mice. These authors proposed that resistin is a link between obesity and type 2 diabetes. In their study, antiresistin antibody improved blood glucose and insulin sensitivity in mice with diet-induced obesity, whereas administration of recombinant resistin impaired insulin action in vivo in mice and ex vivo in adipocytes. In addition, circulating resistin levels and adipocyte resistin gene expression were markedly decreased by treatment with the insulin sensitizer, antidiabetic drug rosiglitazone. Subsequent reports have confirmed (46, 47) or questioned (48, 49, 50) the conclusion that resistin levels are elevated in obese insulin-resistant states. Recent genomic studies have shown that noncoding single nucleotide polymorphisms in the resistin gene are associated with obesity (51) or may influence insulin sensitivity in interaction with obesity (52). In line with these reports, our current results and those of a recent study (32) support a role for resistin in the glucose intolerance associated with ETOH-induced IUGR.
The regulation of adiponectin and resistin levels is not yet well understood. Both hormones are inhibited by ß-adrenergic agonists (53, 54), cAMP activators (21, 54), and TNF- (21, 53, 54), which can cause insulin resistance. In addition, an increase in the expression of both adipocytokines has been observed during high-fat feeding in rats (55). However, these hormones are usually regulated in opposite directions by diet-induced obesity and glucocorticoids, which down-regulate adiponectin (21, 25, 54) and up-regulate resistin (31, 48, 53, 56). Conversely, thiazolidinediones stimulate adiponectin (21, 24) but inhibit resistin expression (31, 53, 56). An elevated cortisone level in ETOH-exposed offspring (57) could explain increased resistin but not normal adiponectin expression in these animals. Insulin has been reported to stimulate adiponectin secretion (58), but its effects on resistin expression have been variable (53, 56, 59). We have recently shown increased resistin expression in hypoinsulinemic newborn rats exposed to ETOH during pregnancy, suggesting that hyperinsulinemia is an unlikely factor in the increased resistin expression in ETOH offspring (32). Elevated serum glucose concentrations in these pups may have contributed to an increase in resistin expression at this early age (53, 59). It is uncertain, however, whether this explains the persistently elevated resistin levels during adulthood. Thus, the regulation of both adiponectin and resistin involves multiple hormonal and nutritional factors, and the reason why prenatal ETOH exposure and postnatal high-fat diet have differential effects on these hormones is presently unclear.
In summary, prenatal ETOH exposure elevated resistin expression, whereas postnatal high-fat diet reduced adiponectin expression in rats. In the presence of both conditions, adiponectin was normal, whereas resistin remained elevated. Both conditions resulted in glucose intolerance and hyperinsulinemia with additive effects.7'wqrpv, http://www.100md.com
Received June 14, 2002.7'wqrpv, http://www.100md.com
Accepted for publication October 16, 2002.7'wqrpv, http://www.100md.com
References7'wqrpv, http://www.100md.com
Lillioja S, Mott DM, Howard BV, Bennett PH, Yki-Jarvinen H, Freymond D, Nyomba BL, Zurlo F, Swinburn B, Bogardus C 1988 Impaired glucose tolerance as a disorder of insulin action: cross-sectional and longitudinal studies in Pima Indians. N Engl J Med 318:1217–12257'wqrpv, http://www.100md.com
Groop L 2000 Genetics of the metabolic syndrome. Br J Nutr 83(Suppl 1):S39–S487'wqrpv, http://www.100md.com
Lichtenstein AH, Schwab US 2000 Relationship of dietary fat to glucose metabolism. Atherosclerosis 150:227–2437'wqrpv, http://www.100md.com
Oakes ND, Cooney GJ, Camilleri S, Chisholm DJ, Kraegen EW 1997 Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding. Diabetes 46:1768–1774
Barker DJ, Hales CN, Fall CH, Osmond C, Phillips K, Clark PM 1993 Type 2 (non-insulin-dependent) diabetes mellitus, hypertension and hyperlipidemia (syndrome X): relation to reduced fetal growth. Diabetologia 36:62–67/kj@, 百拇医药
Pettitt DJ, Bennett PH, Saad MF, Charles MA, Nelson RG, Knowler WC 1991 Abnormal glucose tolerance during pregnancy in Pima Indian women: long-term effects on offspring. Diabetes 40(Suppl 2):126–130/kj@, 百拇医药
Phillips DIW 1998 Birth weight and the future development of diabetes: a review of the evidence. Diabetes Care 21(Suppl 2):B150–B155/kj@, 百拇医药
Simmons RA, Templeton LJ, Gertz SJ 2001 Intrauterine growth retardation leads to the development of type 2 diabetes in the rat. Diabetes 50:2279–2286/kj@, 百拇医药
Benediktsson R, Lindsay RS, Noble J, Seckl JR, Edwards CR 1993 Glucocorticoid exposure in utero: a new model for adult hypertension. Lancet 341:339–341/kj@, 百拇医药
Ozanne SE, Wang CL, Coleman N, Smith GD 1996 Altered muscle insulin sensitivity in the male offspring of protein-malnourished rats. Am J Physiol 271:E1128–E1134
Martin MA, Alvarez C, Goya L, Portha B, Pascual-Leone AM 1997 Insulin secretion in adult rats that had experienced different underfeeding patterns during their development. Am J Physiol 272:E634–E6404#da/, 百拇医药
Vickers MH, Breier BH, Cutfield WS, Hofman PL, Gluckman PD 2000 Fetal origins of hyperphagia, obesity, and hypertension and postnatal amplification by hypercaloric nutrition. Am J Physiol 279:E83–E874#da/, 百拇医药
Nilsson C, Larsson BM, Jennische E, Eriksson E, Bjorntorp P, York DA, Holmang A 2001 Maternal endotoxemia results in obesity and insulin resistance in adult male offspring. Endocrinology 142:2622–26304#da/, 百拇医药
Clarren SK, Smith DW 1978 The fetal alcohol syndrome. N Engl J Med 298:1063–10674#da/, 百拇医药
Bingol N, Schuster C, Fuchs M, Iosub S, Turner G, Stone RK, Gromisch DS 1987 The influence of socioeconomic factors on the occurrence of fetal alcohol syndrome. Adv Alcohol Subst Abuse 6:105–1184#da/, 百拇医药
Marshall JA, Hamman RF, Baxter J, Mayer EJ, Fulton DL, Orleans M, Rewers M, Jones RH 1993 Ethnic differences in risk factors associated with the prevalence of non-insulin dependent diabetes mellitus. The San Luis Valley Diabetes Study. Am J Epidemiol 137:706–718
Castells S, Mark E, Abaci F, Schwartz E 1981 Growth retardation in fetal alcohol syndrome. Unresponsiveness to growth-promoting hormones. Dev Pharmacol Ther 3:232–241#.6--v, 百拇医药
Villaroya F, Mampel T 1985 Glucose tolerance and insulin response in offspring of ethanol-treated pregnant rats. Gen Pharmacol 16:415–418#.6--v, 百拇医药
Lopez-Tejero D, Llobera M, Herrera E 1989 Permanent abnormal response to glucose load after prenatal ethanol exposure in rats. Alcohol 6:469–473#.6--v, 百拇医药
Elton CW, Pennington JS, Lynch SA, Carver FM, Pennington SN 2002 Insulin resistance in adult rat offspring associated with maternal dietary fat and alcohol consumption. J Endocrinol 173:63–71#.6--v, 百拇医药
Havel PJ 2002 Control of energy homeostasis and insulin action by adipocyte hormones: leptin, acylation stimulating protein, and adiponectin. Curr Opin Lipidol 13:51–59#.6--v, 百拇医药
Friedman JM, Halaas JL 1998 Leptin and the regulation of body weight in mammals. Nature 395:763–770#.6--v, 百拇医药
Hu E, Liang P, Spiegelman BM 1996 AdipoQ is a novel adipose-specific gene dysregulted in obesity. J Biol Chem 271:10697–10703
Combs TP, Wagner JA, Berger J, Doebber T, Wang WJ, Zhang BB, Tanen M, Berg AH, O’Rahilly S, Savage DB, Chatterjee K, Weiss S, Larson PJ, Gottesdiener KM, Gertz BJ, Charron MJ, Scherer PE, Moller DE 2002 Induction of adipocyte complement-related protein of 30 kilodaltons by PPAR{gamma} agonists: a potential mechanism of insulin sensitization. Endocrinology 143:998–1007r], 百拇医药
Stanhope KL, Graham JL, Sinha M, Havel PJ 2002 Low circulating adiponectin levels and reduced adipocyte adiponectin production in obese, insulin-resistant Sprague-Dawley rats. Diabetes 51(Suppl 2):A404 (Abstract)r], 百拇医药
Hotta K, Funahashi T, Bodkin NL, Ortmeyer HK, Arita Y, Hansen BC, Matsuzawa Y 2001 Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys. Diabetes 50:1126–1133r], 百拇医药
Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, Pratley RE, Tataranni PA 2001 Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab 86:1930–1935
Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson MR, Yen FT, Bihain BE, Lodish HF 2001 Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci USA 98:2005–2010+0+(, 百拇医药
Berg AH, Combs TP, Du X, Brownlee M, Scherer PE 2001 The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat Med 7:947–953+0+(, 百拇医药
Combs TP, Berg AH, Obici S, Scherer PE, Rossetti L 2001 Endogenous glucose production is inhibited by the adipose-derived protein Acrp30. J Clin Invest 108:1875–1881+0+(, 百拇医药
Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS, Lazar MA 2001 The hormone resistin links obesity to diabetes. Nature 409:307–312+0+(, 百拇医药
Chen L, Nyomba G Prenatal alcohol exposure impairs glucose tolerance in adult rat offspring. Canadian Diabetes Association meeting, Edmonton, Canada, 2001, p 33 (Abstract 121)+0+(, 百拇医药
Cannon B, Lindberg O 1979 Mitochondria from brown adipose tissue: isolation and properties. Methods Enzymol 55F:65–78
Best HC, Haist RE, Ridout JE 1939 Diet and the insulin content of the pancreas. J Physiol 97:107–119l2fmhv\, 百拇医药
Matsuda J, Hosoda K, Itoh H, Son C, Doi K, Tanaka T, Fukunaga Y, Inoue G 1997 Cloning of rat uncoupling protein-3 and uncoupling protein-2 cDNAs: their gene expression in rats fed high-fat diet. FEBS Lett 418:200–204l2fmhv\, 百拇医药
Samec S, Seydoux J, Dulloo AG 1999 Post-starvation gene expression of skeletal muscle uncoupling protein 2 and uncoupling protein 3 in response to dietary fat levels and fatty acid composition. A link with insulin resistance. Diabetes 48:436–441l2fmhv\, 百拇医药
Kolterman OG, Greenfield M, Reaven GM, Saekow M, Olefsky JM 1979 Effect of a high carbohydrate diet on insulin binding to adipocytes and on insulin action in vivo in man. Diabetes 28:731–736l2fmhv\, 百拇医药
Kahn BB, Pedersen O 1993 Suppression of GLUT4 expression in skeletal muscle of rats that are obese from high fat feeding but not from high carbohydrate feeding or genetic obesity. Endocrinology 132:13–22l2fmhv\, 百拇医药
Charron MJ, Katz EB, Olson AL 1999 GLUT4 gene regulation and manipulation. J Biol Chem 274:3253–3256
Larsson LI, Boder GB, Shaw WN 1977 Changes in the islets of Langerhans in the obese Zucker rat. Lab Invest 36:593–598t{y, http://www.100md.com
Frankel BJ, Cajander S, Boquist L 1987 Islet morphology in young, genetically diabetic Chinese hamsters during the hyperinsulinemic phase. Pancreas 2:625–631t{y, http://www.100md.com
Dresner A, Laurent D, Marcucci M, Griffin ME, Dufour S, Cline GW, Slezak LA, Andersen DK, Hundal RS, Rothman DL, Petersen KF, Shulman GI 1999 Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidylinositol 3-kinase activity. J Clin Invest 103:253–259t{y, http://www.100md.com
Helge JW, Ayre K, Chaunchaiyakul S, Hulbert AJ, Kiens B, Storlien LH 1998 Endurance in high-fat-fed rats: effects of carbohydrate content and fatty acid profile. J Appl Physiol 85:1342–1348t{y, http://www.100md.com
Hegarty BD, Cooney GJ, Kraegen EW, Furler SM 2002 Increased efficiency of fatty acid uptake contributes to lipid accumulation in skeletal muscle of high fat-fed insulin-resistant rats. Diabetes 51:1477–1484t{y, http://www.100md.com
Steinberg HO, Tarshoby M, Monestel R, Hook G, Cronin J, Johnson A, Bayazeed B, Baron A 1997 Elevated circulating free fatty acid levels impair endothelium-dependent vasodilation. J Clin Invest 100:1230–1239
Degawa-Yamauchi M, Bovenkerk JE, Zhu Q, Considine RV 2002 Serum resistin is significantly elevated in obese humans. Diabetes 51(Suppl 2):A405 (Abstract)a, 百拇医药
Moore GB, Chapman H, Holder JC, Lister CA, Piercy V, Smith SA, Clapham JC 2001 Differential regulation of adipocytokine mRNAs by rosiglitazone in db/db mice. Biochem Biophys Res Commun 286:735–741a, 百拇医药
Levy JR, Davenport B, Clore JN, Stevens W 2002 Lipid metabolism and resistin gene expression in insulin-resistant Fischer 344 rats. Am J Physiol 282:E626–E633a, 百拇医药
Way JM, Gorgun CZ, Tong Q, Uysal KT, Brown KK, Harrington WW, Oliver Jr WR, Willson TM, Kliewer SA, Hotamisligil GS 2001 Adipose tissue resistin expression is severely suppressed in obesity and stimulated by peroxisome proliferator-activated receptor gamma agonists. J Biol Chem 276:25651–25653a, 百拇医药
Le Lay S, Boucher J, Rey A, Castan-Laurell I, Krief S, Ferre P, Valet P, Dugail I 2001 Decreased resistin expression in mice with different sensitivities to a high-fat diet. Biochem Biophys Res Commun 289:564–567
Engert JC, Vohl MC, Williams SM, Lepage P, Loredo-Osti JC, Faith J, Dore C, Renaud Y, Burtt NP, Villeneuve A, Hirschhorn JN, Altshuler D, Groop LC, Despres JP, Gaudet D, Hudson TJ 2002 5' flanking variants of resistin are associated with obesity. Diabetes 51:1629–16346110, http://www.100md.com
Wang H, Chu WS, Hemphill C, Elbein SC 2002 Human resistin gene: molecular scanning and evaluation of association with insulin sensitivity and type 2 diabetes in Caucasians. J Clin Endocrinol Metab 87:2520–25246110, http://www.100md.com
Shojima N, Sakoda H, Ogihara T, Fujishiro M, Katagiri H, Anai M, Onishi Y, Ono H, Inukai K, Abe M, Fukushima Y, Kikuchi M, Oka Y, Asano T 2002 Humoral regulation of resistin expression in 3T3-L1 and mouse adipose cells. Diabetes 51:1737–17446110, http://www.100md.com
Fasshauer M, Klein J, Neumann S, Eszlinger M, Paschke R 2002 Hormonal regulation of adiponectin gene expression in 3T3-L1 adipocytes. Biochem Biophys Res Commun 290:1084–10896110, http://www.100md.com
Li J, Yu X, Pan W, Unger RH 2002 Gene expression profile of rat adipose tissue at the onset of high-fat-diet obesity. Am J Physiol 282:E1334–E13416110, http://www.100md.com
Haugen F, Jorgensen A, Drevon CA, Trayhurn P 2001 Inhibition by insulin of resistin gene expression in 3T3-L1 adipocytes. FEBS Lett 507:105–1086110, http://www.100md.com
Ogilvie KM, Rivier C 1997 Prenatal alcohol exposure results in hyperactivity of the hypothalamic-pituitary-adrenal axis of the offspring: modulation by fostering at birth and postnatal handling. Alcohol Clin Exp Res 21:424–4296110, http://www.100md.com
Bogan JS, Lodish HF 1999 Two compartments for insulin-stimulated exocytosis in 3T3-L1 adipocytes defined by endogenous ACRP30 and GLUT4. J Cell Biol 146:609–6206110, http://www.100md.com
Kim KH, Lee K, Moon YS, Sul HS 2001 A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation. J Biol Chem 276:11252–11256(Li Chen and B. L. G. Nyomba)