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Centro de Biología Molecular, Facultad de Ciencias, Universidad Autónoma (J.C.M., J.S., J.M.C.), and Instituto de Bioquímica, Centro Mixto UCM Facultad de Farmacia, Universidad Complutense (F.E., M.A., E.R., A.M.P.-L.), Madrid; and Area de Bioquímica, Facultad de Químicas, Universidad de Castilla-La Mancha (A.A.), Ciudad Real, Spain
Address all correspondence and requests for reprints to: Dr. José M. Carrascosa, Department of Molecular Biology, Molecular Biology Center, Faculty of Ciencias, Universidad Autónoma, 28049 Madrid, Spain.
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Abstract |
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Introduction |
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As skeletal muscle is the main tissue responsible for insulin-induced glucose disposal, most studies have explored possible cellular changes in muscle as a cause of the peripheral insulin resistance in the aged rat (5, 6, 9, 10, 11, 12, 13). However, the specific contribution of the different tissues that behave as targets of insulin action to the overall insulin resistance is as yet unknown. In the present work we studied the utilization of glucose by a number of tissues (brain, lung, white adipose, and several muscles) under euglycemic-hyperinsulinemic clamp conditions in young adult (3 months) and old (24 months) rats. The age of the animals was selected to analyze the effect of aging in mature animals and not the effects of growth or maturation (8, 12), as is the case in most studies in aging research that compare juvenile (1–2 month) and either adult (4–12 month) or old (>20 month) animals (6, 11, 14, 15).
Another major concern in aging research in rodents is the rat strain to be used. Thus, to analyze changes in insulin sensitivity of primary character it is important that the aged rat is able to preserve glucose homeostasis without increases in the plasma insulin concentration. On the other hand, age-associated changes in body composition, such as increased adiposity and body weight, are known to result in insulin resistance, a fact that precludes the use of rat strains with age-associated obesity. The Fisher 344 rat shows a small increase in body weight at 24 months of age and, therefore, has been used in many studies (8, 10, 11, 13, 16). However, in this strain, fat cell size increases sharply, and fasting plasma insulin concentrations are slightly higher in aged animals, a situation that does not allow the determination of whether changes in insulin sensitivity are due to the aging process or to hyperinsulinemia. This last problem is also shared by the Sprague-Dawley strain, which, in turn, shows an acute increment in body weight during aging (5, 6, 17). On the contrary, the 24-month-old Wistar rat shows fasting plasma insulin concentrations identical to those of the adult 3-month-old rat and, despite an obvious increase in body weight and fat tissue mass, the size of adipose cells remains constant (18), suggesting that the higher adipose tissue mass is a consequence of adipocyte proliferation. Consequently, studies in this report were conducted using the Wistar rat strain, which we consider more appropriate to analyze aging-associated primary alterations in peripheral insulin sensitivity. This strain has also been considered an adequate model for aging studies by other researchers (9).
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Materials and Methods |
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Euglycemic-insulin clamp
These studies were performed during the postabsorptive period,
15 h after removal of food. Rats were anesthetized with
pentobarbital (4 mg/100 g BW), and after tracheotomy (to avoid
respiratory problems), one carotid artery was catheterized for blood
sampling. Once glycemia returned to the level observed in blood
withdrawn from the tail vein of the awake animals (40 min), insulin
(Actrapid, Novo, Copenhagen, Denmark) was infused through a saphenous
vein at a constant rate to reach an insulin dose of 0.3 or 0.6
U/h·kg, respectively. A solution of 30% glucose was also infused
through the other saphenous vein beginning 5 min after the start of
hormone infusion. The infusion rate was adjusted to clamp blood glucose
at the level present in awake animals (19). To achieve this, blood
samples were taken every 5 min from the carotid artery, blood glucose
was determined within 2 min using a Reflolux II Glucose Analyzer
(Boehringer Mannheim, Mannheim, Germany) (20), and the pump dial was
adjusted according to the changes in the blood glucose level. Within 40
min of starting the clamp, plasma insulin and glucose levels remained
constant without further adjusting the pump dial. At this steady state,
insulin infusion should equal insulin clearance, and the overall
glucose utilization should reach a constant value. The rate of glucose
infusion at steady state normalized to the body weight was used to
determinate the glucose disposal rate (M) as an index of insulin
sensitivity.
Estimate of glucose utilization by individual tissues
As required by the theoretical model (21) a bolus of 80 µCi
2-deoxy-D-[1-3H]glucose (Amersham, Aylesbury,
UK) was injected iv into animals under steady state conditions. The
same bolus was administered to rats not infused with insulin to study
basal glucose utilization. Blood was sampled via arterial catheter for
determination of insulin and glucose concentrations and
2-deoxy-D-[1-3H]glucose radioactivity.
Measurements of plasma glucose and insulin levels before injecting the
radioactive bolus and at the end of the experiment (60 min) confirmed
that steady state conditions were maintained throughout the test. At
the end of the experiment, rats were killed by cervical dislocation,
and pieces of brain, lungs, white adipose tissue (mixture of epididymal
and retroperitoneal), heart (ventricular portion), diaphragm, red and
white quadriceps, and soleus muscle were rapidly removed and frozen
until processing. Tissue samples were digested at 60 C for 45 min in
plastic tubes containing 1 M NaOH and neutralized with 1
M HCl. The
2-deoxy-D-[1-3H]glucose-6-phosphate content
was determined as described previously (22). This method is based on
the fact that both 2-deoxyglucose and 2-deoxyglucose-6-phosphate remain
soluble in 6% HClO4 extracts, whereas
2-deoxyglucose-6-phosphate precipitates in the Somogyi reagent
[BaSO4/Zn(OH)2]. The rate of glucose
utilization by each tissue was calculated by dividing the
disintegrations per min of
2-deoxy-D-[1-3H]glucose-6-phosphate in the
tissue by the calculated integral of the ratio of arterial blood
2-deoxy-D-[1-3H]glucose to glucose
concentration. The data obtained are usually referred to as the glucose
metabolic index, and they can be regarded as an index of glucose
utilization at different insulin levels (23). As the protein content of
the tissues tested was not different between old and young rats, the
glucose utilization was expressed as milligrams per min/kg tissue.
Other analytical procedures
Plasma insulin was determined by RIA, using rat insulin as
standard (Incstar Corp., Stillwater, MN). Ketone bodies were determined
according to (24). Serum FFA were determined by an enzymatic kit (NEFA
C, Wako Chemicals, Neuss, Germany). Serum triglyceride were measured
with an enzymatic kit (ITC Diagnostics, Izasa, Spain).
Expression of the results
Results are expressed as the mean ± SD.
Statistical comparisons were made using Student’s t
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Young rats had plasma insulin and glucose
concentrations identical to those observed in old animals, indicating
that they were mature with regard to the preservation of glucose
homeostasis. Changes in body composition were reflected by increased
body weight and fat tissue mass (18), in agreement with data from
others (7). Old animals showed significantly elevated triglyceride
levels, a characteristic also observed in noninsulin-dependent diabetic
patients and normoglycemic/hyperinsulinemic individuals (25) as well as
in other rat strains in association with aging (26). In contrast,
although a relationship between fasting triglyceride and FFA
concentrations has been previously shown (25), FFA levels in older rats
were significantly lower. Ketone bodies were also lower in old animals
in concordance with the lower FFA concentrations.
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shows the plasma insulin and glucose
concentrations reached at steady state, 45 min after starting the
clamp. In both groups, glucose concentrations were clamped at identical
levels, which were not different from those determined in animals not
infused with insulin. Interestingly, the plasma insulin concentrations
at steady state were significantly higher in old animals at both
insulin infusion rates. This has been previously observed in elderly
humans at insulin infusion rates leading to submaximal insulin
concentrations during euglycemic clamp (3, 4), and the same can be
concluded from studies using aged Wistar rats (7). An aging-associated
decrease in the MCR of insulin seems likely to be the cause of these
findings (27, 28, 29, 30).
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) at both insulin
infusion rates, indicating the insulin-resistant condition of the aged
animals. At the highest plasma insulin levels reached during the clamp
in young and old rats, Nishimura et al. (7) reported that
hepatic glucose production was fully suppressed. Therefore, it can be
assumed that the rate of glucose infusion during the clamp is equal to
the rate of glucose utilization, and the differences observed between
young and old animals are probably due to differences in glucose
consumption by tissues and not to higher hepatic glucose production in
the older rats. However, at the lower insulin infusion rate used in
these studies, the insulin concentrations reached were reported to
elicit a lesser suppression of hepatic glucose production in old than
in adult animals (7), and it could consequently be argued that
differences in M in this case may be due to a higher hepatic glucose
production in older rats and not to the existence of insulin
resistance. However, the levels of glucose infused into old rats at
both insulin infusion rates are very similar, suggesting that the
production of glucose by the liver at the lower insulin dose represents
only a small part of the overall glucose utilization that could not
account by itself for the decrease in M found in old rats. Considering that fat tissue mass increases with aging, many investigators have proposed to express glucose disposal rates per kg lean body mass. In this work we have not determined the fat-free mass of the rats, but from the similarity in body weight between the animals in this study and those used previously (7), we can tentatively assume that the fat-free mass amounts to 62% and 55% of body weight in young and old rats, respectively. This would result in glucose disposal rates of 28 and 20 mg/min·kg fat-free mass, for young and old rats, respectively, at the highest insulin level, indicating that old animals also show insulin resistance when glucose disposal is calculated on a fat-free mass basis, in agreement to similar findings in humans (27).
Glucose utilization in various tissues
The glucose metabolic index was estimated in control animals
(i.e. without insulin infusion) and in rats under
euglycemic-hyperinsulinemic conditions. Table 3
summarizes the data obtained in eight tissues at three different
insulin levels.
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In soleus muscle and diaphragm, basal glucose uptake was also similar in both groups of rats. However, insulin-induced stimulation of glucose utilization was markedly lower in old than in young animals at both insulin infusion rates, indicating that these two tissues as well as white adipose contribute actively to the insulin-resistant state of the old rats. The increment in the glucose metabolic index at the lower insulin infusion rate over that observed under basal conditions can be compared with the maximal increment obtained at saturating insulin concentrations to obtain a raw estimation of the insulin sensitivity of the tissues. For the soleus muscle and diaphragm, the stimulatory effect elicited by this intermediate insulin concentration represents 75% and 69%, respectively, of the maximal stimulation observed in young animals, whereas in aged rats, this increment reaches only 33% and 26% of the maximal effect. These data clearly indicate that the insulin sensitivity of both tissues is diminished in old animals.
Uptake of glucose by the heart at saturating insulin concentrations was
similar in young and old animals, suggesting that the maximal capacity
of glucose utilization remains unaltered with aging. However, at
submaximal insulin concentrations, glucose uptake was significantly
higher in young rats, which indicates that at those intermediate
insulin levels the heart might also contribute to the decreased glucose
disposal observed in old animals. In contrast, under basal conditions,
glucose utilization was significantly elevated in old rats. These data
indicate that the insulin sensitivity of the heart appears to decrease
as a consequence of aging. An approximate estimation from data in Table 3 suggest that the insulin concentration necessary to induce the
half-maximal stimulation of glucose uptake in young animals should be
lower than 100 µU/ml, whereas in old animals it is around 200
µU/ml.
Concerning red and white quadriceps, the data in Table 3 indicate that
there were no significant differences in glucose uptake between young
and old animals at the insulin concentrations tested, suggesting that
these tissues are not involved in the insulin resistance associated
with aging. In young animals, the increment in glucose uptake at
intermediate insulin levels over that observed under basal conditions
represents 62% and 50% of the maximal increment for red and white
quadriceps, respectively, a percentage very similar to the 58% and
62% of the maximal increment observed in both tissues from aged
animals. Thus, from these data it can be postulated that the insulin
sensitivity of both tissues remains unchanged in old animals.
Finally, Table 3 shows that glucose utilization by brain and lung was
not stimulated by insulin in either group of rats. Interestingly,
whereas the glucose metabolic index of brain is not influenced by the
age of the animals, glucose utilization by lung was decreased in aged
rats at all three insulin concentrations tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Some researchers have claimed that peripheral insulin resistance in aged rats is simply a consequence of increases in body weight and fat mass or is due to physical inactivity. Although it is well documented that these factors affect insulin sensitivity, there is considerable experimental evidence demonstrating that insulin resistance in aged animals and humans also occurs in the absence of increased adiposity and body weight (17, 27) or after training protocols that are usually effective in improving insulin sensitivity (10, 13).
In the present work we also explored the effect of aging on the glucose metabolic index of several insulin target tissues at different plasma insulin concentrations. As the 24-month-old rats are normoinsulinemic and normoglycemic, these studies allowed us to determine which tissues contribute primarily to the development of insulin resistance. From our data it becomes evident that white adipose tissue is extremely insulin resistant in the old rats. These results are consistent with our previous observations in isolated adipocytes (18, 31) that showed a decreased insulin stimulation of several metabolic functions as well as a reduced insulin receptor kinase activity and with observations by others in human adipocytes (27). Even though adipose tissue uses only a minor part of the glucose available, it should be pointed out that a greater proportion of fat mass is present in the older animals, making its contribution to the overall insulin resistance more relevant. Reaven (25) suggested that the insulin resistance of adipose tissue in type 2 diabetics could be relevant not quantitatively, in terms of glucose utilization, but, rather, qualitatively. Thus, the lack of an antilipolytic effect of insulin or a decreased stimulation of adipocyte-associated lipoprotein lipase could lead to increases in plasma FFA and triglyceride levels that might, in turn, induce a decrease in glucose utilization by other tissues. In fact, fasting serum triglyceride levels were elevated in the old rats used in this work, in agreement with previous reports using Fischer-344 rats (26). In contrast, the serum FFA concentration appears to be decreased in old animals. More experimental work is required to elucidate whether this is due to decreased adipocyte fatty acid production or increased liver fatty acid utilization for triglyceride synthesis. Our results here indicate that the insulin insensitivity of adipose tissue could be a primary defect associated with aging, and it is of great interest to establish at which age this resistance is detectable.
We also found that insulin-stimulated glucose uptake by the diaphragm is markedly reduced in old rats at saturating and subsaturating insulin concentrations, whereas basal utilization remains unaltered. Furthermore, from our data it can be postulated that the insulin sensitivity of the diaphragm is decreased in the aged rat. A previous report using isolated diaphragm from Wistar rats claimed that insulin resistance occurs during maturation, but no further decrease in insulin action was observed with aging (32). As our data were obtained in vivo, they cannot be directly compared with the former. It is possible that in vitro studies with isolated diaphragm do not fully reproduce the conditions existing in vivo, resulting in a loss of its insulin resistance.
In contrast to the former tissues, the glucose metabolic index of heart in the presence of saturating insulin reaches similar levels in young and old animals, indicating that the metabolic capacity of heart is not influenced by aging. However, at subsaturating insulin concentrations, glucose utilization is lower in the old animals, whereas in the presence of physiological plasma insulin concentrations, glucose utilization is higher in old rats. Taken together, these data suggest that in the hearts of old animals the insulin sensitivity is decreased without changes in the maximal response to the hormone.
Finally, concerning skeletal muscles, it appears evident from our data that insulin resistance develops mainly in certain muscle types. Thus, whereas decreases in old rats in glucose utilization by the red and white portions of the quadriceps at both insulin infusion rates are rather low, the stimulation by insulin of glucose utilization in the soleus is greatly reduced in aged animals. Furthermore, the insulin sensitivity of red and white quadriceps is unaltered with aging, in contrast to the acute decrease observed in the soleus muscle of old rats. These three muscle tissues differ in fiber type composition, with the soleus being mainly constituted by type 1 fibers, and the white and red quadriceps by type 2b and 2a fibers, respectively (12). The type 1 fibers of the soleus are oxidative and can use fatty acids as fuel, whereas type 2b fibers are extremely glycolytic and cannot oxidize it (33). Thus, it can be speculated that the insulin-resistant adipose tissue of the older rats incorporates a lesser amount of fatty acids from lipoprotein triglyceride that remain available as an alternative fuel to glucose for oxidative muscles such as soleus or even heart. The greater availability of fatty acids, however, would not influence glucose utilization by glycolytic muscles such as quadriceps.
Taking into account that slow twitch fibers represent only about 10% of the rat muscle mass, it appears difficult to reconcile the observed decrement in overall glucose disposal with the development of insulin resistance in the oxidative muscles and diaphragm. In this regard there are several considerations. Firstly, the lower glucose utilization by lungs in old animals reported here could partially contribute to the decreased glucose disposal. On the other hand, it has been proposed that aging is associated with a preferential loss of fast twitch fibers and a progressive increase in the proportion of aerobic type 1 fibers that would be insulin resistant (33). Finally, it cannot be ruled out that other tissues are contributing to the decreased glucose disposal or even that, as experiments were performed with anesthetized rats, the contribution of diaphragm to the total glucose utilization might be overestimated, resulting in a higher decrement in glucose disposal.
The development of tissue-specific insulin resistance could also be due to regional changes in blood flow. However, although insulin stimulation of peripheral blood flow has been shown to be impaired in several cases of insulin resistance (34, 35, 36), considerable experimental evidence has dissociated the increase in glucose utilization from the increment in regional blood flow (37). Thus, it seems unlikely that specific changes in blood flow are the ultimate cause of the tissue-specific decrease in glucose uptake.
To summarize, our data demonstrate that normoglycemic and normoinsulinemic 24-month-old Wistar rats show insulin resistance in vivo, as manifested by the decreased glucose disposal rates under hyperinsulinemic conditions. Some insulin target tissues, such as diaphragm, soleus muscle, heart, and white adipose tissue, play a primary role in development of the insulin-resistant condition associated with aging, whereas the other mainly glycolytic muscles remain sensitive to the hormone.
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Acknowledgments |
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Footnotes |
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Received March 18, 1996.
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