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Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3A 1R9
Submitted 18 July 2003 ; accepted in final form 2 September 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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ethanol; fetal growth restriction
Whereas the association of adverse events during pregnancy with glucose intolerance has been well documented, little is known regarding the effects of events occurring only during the postnatal period. It is particularly important to know the effects of EtOH exposure through lactation, because alcohol consumption among nursing women is quite common, and there is a popular belief that EtOH promotes lactation (23). Ingested EtOH is secreted in the milk, with the potential for exposing the developing offspring to toxic effects of EtOH (15). EtOH treatment of lactating rats has been reported to cause growth restriction in suckling pups (38), but it is not known whether this results in alterations of glucose homeostasis. A few studies have suggested that the postnatal period may be metabolically vulnerable because key metabolic processes are still developing (25). The present study was performed to determine the effects of maternal EtOH consumption during gestation or lactation on insulin sensitivity. Differences in birth weight between pups allowed the examination of the effects of EtOH-induced IUGR or EtOH exposure per se on insulin resistance.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Intravenous glucose tolerance test. At 16 wk of age, offspring from each group underwent a frequently sampled intravenous glucose tolerance test (IVGTT). The rats were fasted overnight, and by 9:00 AM the next morning they were anesthetized with an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). The rats lay on a surgical table with temperature control during the duration of the experiments. Catheters were quickly inserted into the right femoral vein and the carotid artery. Baseline blood samples (0.2 ml) were collected from the arterial catheter into heparinized tubes 30 and 1 min before dextrose (40% wt/vol, 0.4 g/kg body wt) was administered through the femoral vein catheter. Blood (0.1 ml) was subsequently drawn 1, 2, 3, 5, and 8 min after glucose injection. Regular insulin (Humulin, 0.1 U/kg; Eli Lilly, Indianapolis, IN) was then injected through the femoral vein, and blood collection continued at 10, 12, 14, 16, 18, 20, 22, 25, 30, 40, 60, and 90 min. The withdrawn volume was replaced with normal saline. Blood glucose was determined immediately, but plasma was stored at -20°C for insulin determination. The rats were killed by exsanguinations, and liver and gastrocnemius muscle were rapidly trimmed of any visible fat, snap frozen in liquid nitrogen, and stored at -70°C until used.
Incremental areas under the glucose and insulin curves were calculated by using the trapezoidal rule after subtracting basal values. Acute insulin response to glucose (AIR) was calculated as the area under the insulin curve for the first 8 min after the glucose challenge. A glucose tolerance index (KG), representing the net glucose elimination rate in response to both endogenous and exogenous insulin, was calculated as the negative slope of a regression line between glucose concentrations expressed in natural logarithms and time, in minutes, between 1 and 25 min. The latter time point was the time at which the glucose curve in the control rats stopped decreasing. The units of KG are percent per minute (1). Insulin sensitivity index (SI) was determined by minimal modeling (27, 28, 31, 40) by using Pacini and Bergman's MINMOD program (30). Products of SI with AIR were used as the disposition index (DI), which has been proposed in dogs (7), humans (18), and mice (31) as representing insulin effect.
Tissue triglycerides. Tissue triglycerides were extracted as described by Atkinson et al. (4). Frozen gastrocnemius muscle and liver tissues (30 mg) were extracted for 90 min in 2 ml of 2:1 chloroformmethanol. Methanol (0.4 ml) was then added, and the extract was vortexed for 30 s. After centrifugation at 1,100 g for 10 min, the supernatant was collected, mixed with 0.5 ml of 0.04% CaCl2, and centrifuged at 550 g for 20 min. The upper phase was removed, and the interface was washed three times with a mixture of chloroform (1.5 ml), methanol (24 ml), and water (23.5 ml). The final wash was removed, and methanol (0.05 ml) was added to obtain one phase. The samples were dried at 60°C under nitrogen and redissolved in 0.05 ml of 3:2 tert-butyl alcohol-Triton X-100. Triglycerides were determined spectrophotometrically by using the TRIG GPO kit from Roche Diagnostics (Indianapolis, IN).
Other assays. Plasma glucose was measured by the glucose oxidase method by using a YSI2300 glucose analyzer (YSI, Yellow Springs, OH). Plasma insulin was measured with a sensitive rat radioimmunoassay kit (Linco Research, St. Charles, MO), which has a 100% cross-reactivity with human insulin. Plasma free fatty acids (FFA) were determined by using the NEFA C kit from Wako (Richmond, VA).
Statistics. Statistical analyses were conducted with SPSS software (version 11.0 for Windows, SPSS, Chicago, IL). Differences between groups were evaluated by ANOVA with Dunnett's multiple-comparison test. Insulin values were log transformed before analysis. Values are expressed as means ± SE. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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10.5 kcal/day), caloric intake of EtOH-exposed dams amounted to 91.7 kcal/day. Weight gain during pregnancy was not statistically different between EtOHexposed dams (142.8 ± 25.1 g) and the other two groups (160.0 ± 10.2 g).
The frequency distribution of birth weight among all of the pups taken together was bimodal (not shown), and the demarcation of the two weight subpopulations was 6.0 g. All male offspring in the control group and in the group exposed to EtOH during lactation had a birth weight >6.0 g. Among male offspring exposed to EtOH during pregnancy, 12 had a birth weight <6.0 g, whereas 9 were within the control range. The offspring exposed to EtOH during pregnancy were, therefore, further separated into two subgroups: small and normal weight at birth.
Offspring weights and food intake are shown in Table 1. In the prenatal EtOH group, offspring that were small at birth had a catch-up growth after 4 wk of age, at which time their growth rate became indistinguishable from that of controls. This was also the time that their food intake, which was slightly reduced during the first 2 wk postweaning, increased to control levels. Surprisingly, however, offspring in the prenatal EtOH group that had a normal birth weight had a slower growth rate by 11 wk of age, despite normal food intake. Offspring exposed to EtOH during lactation had a growth curve that was comparable to that of controls, with a slightly increased food intake.
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Glucose tolerance and insulin sensitivity. Glucose and insulin curves during the IVGTT are shown in Fig. 1. The glucose curves were similar among the three EtOH groups, but showed higher glycemia compared with control rats, especially during the last 70 min. The areas under the glucose curves (mmol·l-1·min-1) were also similar among the EtOH groups (postnatal EtOH: 1,026 ± 57 mmol·l-1·min-1, n = 8; prenatal EtOH-normal birth weight: 994 ± 40 mmol·l-1·min-1, n = 8; prenatal EtOH-small birth weight: 1,030 ± 80 mmol·l-1· min-1, n = 5) and were significantly greater in all three groups compared with controls (798 ± 32 mmol·l-1·min-1, n = 5, P < 0.05). Both groups of offspring that were exposed to EtOH before birth had elevated AIR (1,168 ± 225 and 2,020 ± 881 pmol/l), with the greater value in the lighter group, compared with controls (406 ± 37 pmol/l, P < 0.05–0.01). AIR in offspring exposed to EtOH during lactation (521 ± 61 pmol/l) was similar to that of controls. As shown in Table 2, KG, SI, and DI were significantly decreased in the three EtOH groups compared with controls. Glucose effectiveness (SG) was not different between groups. Pooled data showed a hyperbolic relationship between AIR and SI (Fig. 2).
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FFA and triglycerides. Because hypertriglyceridemia and accumulation of triglycerides in nonadipose tissues have been associated with insulin resistance, we measured plasma FFA and triglyceride as well as muscle and liver triglyceride. Plasma FFA levels were similar between EtOH groups and controls. However, plasma, muscle, and liver triglyceride levels were about twofold higher in EtOH rats that were born small compared with controls. Surprisingly, plasma and tissue triglyceride levels in the other two EtOH groups were comparable to that in controls (Table 3).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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All groups of rats exposed to EtOH were insulin resistant, because they remained hyperglycemic after insulin injection during the IVGTT, an indication that their glucose disposal was lower than in control rats to which the same amount of insulin had been administered. In addition, when the data were analyzed by Pacini and Bergman's minimal model (30), which has been validated in rodents (27, 28, 31, 40), the three EtOH groups of rats had a decreased SI. There was a hyperbolic relationship between AIR and SI, indicating that insulin resistance was associated with a compensatory increase in insulin secretion (7, 18). We also found that the DI, an index of insulin action calculated as the product of insulin area and SI, was decreased in the three EtOH groups. A normal DI is usually associated with normal glucose tolerance, and it is believed to represent an adequate compensation of the decreasing insulin sensitivity by increasing insulin secretion (7, 18, 31).
It has been proposed that insulin action is not the sole factor accounting for glucose disposal and that other factors, such as the effect of glucose per se, should be considered (7, 18, 31). SG has been defined as the whole body effect of hyperglycemia to enhance glucose disposal and to suppress endogenous glucose production (8). It has been suggested that glucose is the major determinant of glucose disposal in rats (21). Elevated SG has been reported in normoglycemic, insulin-resistant relatives of patients with Type 2 diabetes, and it was proposed that this represents an upregulation of glucose-mediated glucose uptake and may be a compensatory mechanism against the developing insulin resistance (17). Glucose intolerance in mice fed a high-fat diet is initially dependent on a reduction in SG, whereas, after obesity has progressed, insufficient 
-cell compensation to insulin resistance contributes to a higher extent (3, 31). In the present study, however, SG was normal in all groups of rats exposed to EtOH, an indication that it is an unlikely explanation for the observed glucose intolerance in these animals. We have previously reported that rats exposed to EtOH in utero have a normal -cell mass (10, 11). In the present study, the rats exposed to EtOH in utero had an increased insulin secretion compared with controls. Glucose intolerance in the EtOH-exposed rats, therefore, appeared to have been determined primarily by insulin resistance rather than impaired insulin secretion.
There have been several reports of altered glucose homeostasis in adult rat offspring as a consequence of maternal EtOH ingestion during pregnancy. Most studies reported normal glucose concentrations with increased insulin levels as a manifestation of insulin resistance (reviewed in Refs. 10, 11, 14). Only one previous study formally assessed insulin sensitivity in these rats. Elton et al. (14) reported that rats exposed to EtOH in utero had a reduced glucose uptake in soleus muscle, but whole body insulin sensitivity measured during euglycemic clamp was not affected. The discrepancy between in vivo and vitro insulin sensitivity was attributed to a predominance of white muscle over red muscle in rats (14). Of note, this study assessed insulin responsiveness expressed as glucose clearance per gram body weight, as opposed to insulin sensitivity, which takes into account prevailing insulin levels (19, 33). It is also possible that the findings were confounded by the fact that the clamp does not differentiate between insulin action and glucose effect on glucose disposal, especially if glucose plays a major role in glucose disposal in rats (21). We show here a reduction of insulin sensitivity and glucose intolerance in rats exposed to EtOH in utero using the minimal model approach. We have previously reported that these rats have a reduced GLUT4 content in gastrocnemius muscle (10, 11), which is in agreement with findings of reduced glucose uptake in isolated soleus muscle of rats with EtOH-induced IUGR (14). In addition, EtOH-exposed rats had increased resistin expression, which may have contributed to insulin resistance (10, 11). We now show that the rats were insulin resistant, regardless of their birth weight, an indication that insulin sensitivity was reduced not solely because of IUGR, but as a result of EtOH exposure early in life.
As reported before (11), insulin resistance in this model was not associated with changes in circulating FFA. We found increased plasma and tissue triglyceride concentrations only in EtOH rats that had a small birth weight. Hypertriglyceridemia was recently reported in rat offspring exposed to EtOH in utero (32). Our findings indicate that EtOH-induced IUGR is associated with a proatherogenic phenotype, in addition to insulin resistance of glucose metabolism. The association of elevated triglycerides with decreased SI in these animals might represent a more severe form of insulin resistance. Indeed, accumulation of triglycerides in nonadipose tissues has been reported in insulin-resistant states and may play a role in the pathogenesis of obesity and diabetes (35). It is unclear why prenatal EtOH exposure caused IUGR in some offspring only. Because the mothers were given a similar amount of EtOH, gained similar weight during pregnancy, and had a similar litter size, the differential effect of EtOH on fetal growth probably represents differences in sensitivity to EtOH.
This study is an extension of reports showing an association between adverse perinatal events and the development of insulin resistance in later life, as reported in humans (5, 9) and animals (10, 11, 14, 16, 36, 41). In contrast to these numerous studies on the effects of the intrauterine milieu, the influence of events occurring during the early postnatal period on glucose homeostasis is relatively unknown. Experiments on protein malnutrition during lactation in rats have only been reported by a few investigators. One group first reported reduced insulin secretion with increased insulin sensitivity at 1 yr of age in undernourished rats (13) but subsequently followed up with a report of insulin resistance during a clamp in undernourished female rats (24). Rao (34) reported hepatic insulin resistance in weanling rats after chronic food restriction. In a different approach, one dose of testosterone given only to female newborn rats caused insulin resistance at adult age (29). Some studies of EtOH exposure during lactation in rats resulted in decreased growth rate and blood glucose and elevated circulating triglycerides, FFA, and glycerol at 12 days of age (38). In our study, insulin sensitivity was reduced, but no changes in FFA or triglycerides were found, in adult rats born with a normal weight that were exposed to EtOH during lactation only. This observation indicates that the period of lactation is vulnerable to EtOH exposure in terms of glucose homeostasis. To our knowledge, this has not been reported before.
Because early exposure to EtOH was a common denominator in the rat offspring in this study, it is possible that EtOH exposure per se was the cause of insulin resistance. EtOH causes insulin resistance, and potential mechanisms include an interference with membrane trafficking of glucose transporters (26), oxidative injury to insulin target tissues (22), and induction of tumor necrosis factor-
expression (2). EtOH also increases glucocorticoid secretion (42), and hypercortisolism has been reported in adult offspring after prenatal EtOH exposure (39). It is also possible that the three groups of offspring developed insulin resistance through separate mechanisms. The two groups exposed to EtOH in utero might have acquired insulin resistance via maternal transmittal. Proving such transmission, however, would require studying offspring of the present generation of rats without exposing the second generation of offspring to EtOH (20). Factors related to IUGR may provide alternative mechanisms of insulin resistance in the group with low birth weight (5, 16, 41). Adverse events during pregnancy are associated with elevated glucocorticoid levels in the adult offspring, and this has been proposed to be the defect programming the fetus with IUGR to metabolic diseases in later life (6). Lipotoxicity may also have been contributory in offspring with IUGR, as they had elevated tissue triglycerides (35).
In summary, exposure to EtOH during early development, albeit not necessarily during pregnancy, may program the offspring to abnormal glucose homeostasis later in life. The underlying mechanisms are still unknown and will require further investigation.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This study was supported by grants from the Canadian Institutes of Health Research and the Manitoba Health Research Council.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, Control (n = 5); 
, postnatal ethanol (EtOH; n = 8); 
, prenatal EtOH with normal birth weight (n = 8); 
, prenatal EtOH group with small birth weight (n = 5).