© 1997 American Heart Association, Inc.
Articles |
Interaction Between BTBR and C57BL/6J Genomes Produces an Insulin Resistance Syndrome in (BTBR x C57BL/6J) F1 Mice
From Departments of Biochemistry and Comparative Biosciences (T.R., K.L.S., A.D.A.) and Biodynamics Laboratory (C.D.,G.D.C.), University of Wisconsin-Madison, Madison, WI 53706.
Correspondence to Alan D. Attie, PhD, Department of Biochemistry, University of Wisconsin–Madison, 420 Henry Mall, Madison, WI 53706-1569. E-mail attie{at}biochem.wisc.edu
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Abstract Insulin resistance is a common syndrome that often precedes the development of noninsulin-dependent diabetes mellitus (NIDDM). Both diet and genetic factors are associated with insulin resistance. BTBR and C57BL/6J (B6) mice have normal insulin responsiveness and normal fasting plasma insulin levels. However, a cross between these two strains yielded male offspring with severe insulin resistance. Surprisingly, on a basal diet (6.5% fat), the insulin resistance was not associated with fasting hyperinsulinemia. However, a 15% fat diet produced significant hyperinsulinemia in the male mice (twofold at 10 weeks; P<.05). At 10 weeks of age, visceral fat contributed approximately 4.3% of the total body weight in the males versus 1.8% in females. In the males, levels of plasma triacylglycerol and total cholesterol increased 40% and 30%, respectively, compared to females. Plasma free fatty acid concentrations were unchanged. Oral glucose tolerance tests revealed significant levels of hyperglycemia and hyperinsulinemia 15 to 90 minutes after oral glucose administration in the male mice. This was particularly dramatic in males on a 15% fat diet. Glucose transport was examined in skeletal muscles in (BTBRxB6)F1 mice. In the nonhyperinsulinemic animals (females), insulin stimulated 2-deoxyglucose transport 3.5-fold in the soleus and 2.8-fold in the extensor digitorum longus muscles. By contrast, glucose transport was not stimulated in the hyperinsulinemic male mice. Hypoxia stimulates glucose transport through an insulin-independent mechanism. This is known to involve the translocation of GLUT4 from an intracellular pool to the plasma membrane. In the insulin-resistant male mice, hypoxia induced glucose transport as effectively as it did in the insulin-responsive mice. Thus, defective glucose transport in the (BTBRxB6)F1 mice is specific for insulin-stimulated glucose transport. This is similar to what has been observed in muscles taken from obese NIDDM patients. These animals represent an excellent genetic model for studying insulin resistance and investigating the transition from insulin resistance in the absence of hyperinsulinemia to insulin resistance with hyperinsulinemia.
Key Words: diabetes • genomes • insulin resistance
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Introduction |
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Insulin resistance is a syndrome in which there is a reduced response of insulin's target tissues to a given amount of insulin.1 It is an important risk factor for noninsulin-dependent diabetes mellitus (NIDDM), hypertension, and premature cardiovascular disease2,3 The syndrome affects approximately one fifth of the U.S. population. Both genetic and dietary factors have been shown to play a role in insulin resistance.4,5
People's susceptibility to insulin resistance and NIDDM appears to result from an interplay between genetic and dietary factors.6–8 The Pima Indians provide a dramatic example.9,10 The population in Mexico that consumes the traditional high-carbohydrate, low-fat diet has a very low incidence of NIDDM. In striking contrast, the population living in Arizona consumes a high-fat diet and has an incidence of NIDDM of about 65%, among the highest in the world. Thus, in this population, there appears to be a unique combination of susceptibility alleles that interact with diet to produce insulin resistance and NIDDM.
Studies in rats11–13 and mice14,15 also reveal that genetic and dietary factors play a role in insulin resistance. Certain strains are more susceptible to diet-induced insulin resistance.13,14 However, apart from the four recognized gene loci that are involved in causing obesity (and, indirectly, insulin resistance) and the agouti locus,16–20 specific insulin resistance genes have not yet been identified in rodent models. Paralleling human genetic studies, gene mapping in rodent models suggests that multiple gene loci interact to influence the susceptibility to insulin resistance.21–23
Extensive clinical genetic studies in humans have assessed the potential role of insulin signaling and glucose metabolism genes in NIDDM.5,6,24,25 In small numbers of patients, polymorphisms at candidate loci such as the GLUT2 glucose transporter,26 insulin,27,28 and the insulin receptor29 have been found.5 However, these are extremely rare alleles relative to the high incidence of NIDDM. In addition, it appears more likely that these alleles interact with particular alleles at other loci, and perhaps environmental factors, to produce the full-blown NIDDM syndrome.29 Additional genes need to be identified before a more complete understanding of these gene-gene interactions can be achieved.
Our laboratory has been using the BTBR mouse strain for chemical mutagenesis studies. This strain shows normal insulin responsiveness even when shifted to a moderately high-fat diet. In preparation for genetic mapping, we crossed BTBR mice with C57BL/6J (B6) mice, since our studies had shown that the B6 strain also exhibited no insulin resistance or hyperinsulinemia when shifted to a 15% fat diet. We were surprised to discover that the male offspring from the BTBRxB6 cross showed severe insulin resistance (without hyperinsulinemia) on the basal diet and severe hyperinsulinemia on the high-fat diet. Since this phenotype is not present in either parental strain, it results from unique interactions between alleles in the BTBR and B6 genomes.
In this report, we describe the phenotype of a new model of insulin resistance. The animals represent a valuable new model for the complex genetics of insulin resistance and for studying the relationship between dietary lipid and hyperinsulinemia.
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Methods |
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Animals and Diet
BTBR mice were obtained from Dr William F. Dove (McArdle Laboratory, University of Wisconsin-Madison). The strain is highly susceptible to ENU mutagenesis and has been used extensively for these studies in the Dove laboratory.30,31 The BTBR strain was developed by L.C. Dunn. His original stock was derived from Dobrovolskaia-Zavadskaia. He brother-sister mated the stock at the Nevis Biological Station, his emeritus laboratory from Columbia University. Around 1956, when Mary Lyon discovered tufted, he inserted it as a marker and continued to inbreed the stock. The stock was continuously maintained in the laboratory of Dunn's student, the late Dorothea Bennett, from 1962 on. In 1970, two of her graduate students, Loraine Flaherty and Karen Artzt, typed it as H-2b and began to systematically inbreed it and found it skin compatible in the ninth generation (the first time it was tested).32 Dunn had vigorously selected the stock for large litter size so that it could support the balanced lethal cross T/t x T/t, in which 50% of the embryos die. It has also been selected for 50% tail length of T/+ heterozygotes (K. Artzt, personal communication). It is thus the ideal background for genes that affect tail length. Artzt brought the mice to the laboratory of Jean-Louis Guenet, who inbred it for 45 generations before giving it to the Dove Laboratory (J.-L. Guenet, personal communication). It is still maintained today in the Artzt Laboratory but has been sent around the world. It is available from the Jackson Laboratory, Bar Harbor, Maine, USA.
C57BL/6J mice were obtained from the Jackson Laboratory. All animals were housed under controlled temperature (24°C) and lighting (12 hours light: 0600 to 1800; 12 hours dark: 1800 to 0600) with free access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Wisconsin-Madison.
Mouse diets were obtained from Harlan Teklad. The basal diet (Formulab chow, 5008) consisted of 6.5% fat and 23.5% protein (by weight), and the experimental diet (TD 94059) consisted of diet 5015 supplemented with cocoa butter to achieve a fat content of 15%. Each diet was supplemented with a vitamin and mineral mixture (Harlan Teklad).
After weaning, the animals were fed the basal diet until 6 weeks of age. Thereafter, the control group continued on the same diet, while the experimental group was switched to the 15% fat diet. Both groups were kept on the diets for 4 weeks and were immediately studied. Daily food consumption was determined for 3 weeks by weighing the food and correcting for the amount not eaten, including spillage.
Food was withdrawn from the mice at 0800 hours on the day of the experiments. The animals were weighed, and between 1200 and 1400 hours, they were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). The order of anesthetization was randomized so that the mean time of tissue sampling did not vary among the groups. Blood was sampled via the retroorbital sinus for determination of glucose, insulin, free fatty acid, triacylglycerol, and cholesterol. Right and left soleus and extensor digitorum longus (EDL) were dissected out. The following visceral fat pads were removed from the abdominal cavity and weighed: gonadal/ovarian, consisting of the adipose tissue associated with the reproductive organs; perirenal, consisting of the adipose tissue associated with the kidneys; retroperitoneal, consisting of the adipose tissue associated with the dorsal wall of the peritoneal cavity; and omental-mesenteric, consisting of the adipose tissue associated with the digestive organs.
Metabolic Analyses
Plasma glucose was measured by the glucose oxidase method (Sigma
Diagnostics). Total cholesterol and
triacylglycerol were quantified by the use of
cholesterol esterase and cholesterol oxidase
reactions and lipase, respectively, using kits from Sigma
Diagnostics. Plasma insulin was analyzed by a
radioimmunoassay from Linco. Free fatty acid levels were determined
using the acyl-CoA synthetase and acyl-CoA oxidase method (Wako
Chemicals USA).
Muscle Incubations
Muscles were incubated at 35°C in a temperature-controlled,
shaking water bath in 3 mL of pregassed Krebs-Henseleit bicarbonate
buffer (KHB)33 supplemented with 0.1% bovine
serum albumin (BSA), 8 mmol/L glucose, and 32
mmol/L mannitol with or without insulin, as specified in the
text. The gas concentrations in the 25-mL Erlenmeyer flasks were 95%
O2, 5% CO2, except in
experiments involving hypoxia, where the gas phase was 95%
N2, 5%
CO2.34 The incubation time
at 35°C was 30 minutes except in experiments involving
hypoxia. In experiments involving stimulation of glucose
transport by hypoxia, all muscles (ie, hypoxic and control
oxygenated muscles) were incubated for 45 minutes. After
the initial incubation period, all muscles were washed for 10 minutes
at 30°C in oxygenated medium to remove glucose before
assay of glucose transport activity. The medium (3 mL/flask) consisted
of KHB containing BSA and 40 mmol/L mannitol. The gas phase
in the flasks was 95% O2, 5%
CO2. If insulin was present during the
initial incubation, it was also added to the wash step.
Determination of Glucose Transport Activity
Glucose transport activity was measured with the
nonmetabolizable glucose analog, 2-deoxyglucose
(2-DG).35 Muscles were incubated for 15 minutes
at 30°C in 2 mL of oxygenated KHB supplemented with
1 mmol/L
2-deoxy-D-[1,2-3H]glucose (1.7
mCi/mmol) and 39 mmol/L
[U-14C]mannitol (8.5 µCi/mmol). If insulin
was present during the 35°C incubation, it was also included in
the transport assay. The gas phase in the flasks consisted of 95%
O2, 5% CO2. Uptake of 2-DG
into muscles under basal and stimulated conditions is linear over the
time period assayed. Furthermore, at stimulated rates of uptake, more
than 98% of the intracellular 2-DG is
phosphorylated.36 At the
conclusion of this incubation period, muscles were rapidly blotted onto
filter paper, trimmed, and quick-frozen between aluminum clamps cooled
to the temperature of liquid N2. Muscles were
stored at -70°C until use and subsequently weighed and processed by
homogenization in 0.3 mol/L perchloric acid
at 4°C. The homogenate was centrifuged, and
aliquots of the supernatant were used for determination of glucose
transport activity by quantitating the 3H and
14C radioactivity in a liquid scintillation
counter.
Oral Glucose Tolerance Tests
Food was withdrawn for 4 hours (0800 to 1200 hours), followed by
a baseline (0 minute) blood sample collected from the retroorbital
sinus, and glucose (1 g glucose/kg body weight) was administrated by
oral gavage. Blood was sampled at 15, 30, and 90 minutes for
measurements of plasma glucose and insulin concentrations.
Statistical Analysis
All values are reported as the mean±SD of indicated samples and
number of experiments. Comparison of different treatments was evaluated
by the Student's t test (two-tailed). One-way ANOVA was
used when comparisons were made among three groups. The Tukey-Kramer
Multiple Comparison post hoc test was used to locate the source of
significant variance.
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Results |
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Effect of Dietary Lipid on Fasting Insulin; Parental Strains Versus F1s
The aim of this study was to investigate genetic factors that influence the susceptibility of animals to diet-induced insulin resistance. Various mouse strains were studied by subjecting them to a baseline diet (6.5% fat by weight) until 6 weeks of age and then switching them to a moderately high-fat (15% fat by weight) diet for 4 weeks. We measured fasting serum insulin as an indicator of insulin resistance. The B6 and BTBR strains were unresponsive to the 15% fat diet (Fig 1
). However, when BTBR female
mice were bred to B6 male mice, the hybrid male offspring showed a
completely new phenotype. The male F1
animals showed a twofold increase (P<.05) in serum insulin
in response to the 15% fat diet. Female F1
animals were unresponsive to the diet (Fig 1). Insulin levels were
unchanged in male or female animals that were maintained on the basal
diet until 10 weeks (Fig 2), a finding
indicating that the hyperinsulinemia in the males
was due to the increased dietary fat and was not age related. The
hyperinsulinemia in the male
F1s was accompanied by a moderate hyperglycemia
(Fig 2). The F1 male animals from a reciprocal
cross (B6xBTBR) also were hyperinsulinemic after a
high-fat diet, but not as severely as the
(BTBRxB6)F1 males.
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The diet-induced hyperinsulinemia in the (BTBRxB6)F1 mice could be a consequence of outbreeding of the BTBR mice and the loss of homozygosity of particular BTBR alleles. Accordingly, BTBR mice were bred with A/J and with C3H/HeJ mice, strains that also do not show diet-induced hyperinsulinemia under the conditions used in these experiments. (BTBRxA/J)F1 mice and (BTBRxC3H/HeJ)F1 mice showed a phenotype that was indistinguishable from any of the parental strains, a result indicating that the diet-induced hyperinsulinemia in the (BTBRxB6)F1 mice is not simply a consequence of loss of homozygosity of BTBR alleles.
Oral Glucose Tolerance
The fasting insulin values suggested a diet-induced insulin
resistance in (BTBRxB6)F1 males. Therefore, we
performed oral glucose tolerance tests on
(BTBRxB6)F1 males that were fed either the
baseline diet or the 15% fat diet. In addition, because of the sex
difference in the F1s, we also evaluated females
on the 15% fat diet.
(BTBRxB6)F1 males on the 15% fat diet had
moderate fasting hyperglycemia compared to the other two groups (Fig 3; P<.001 versus females on
the 15% fat diet; P<.05 versus males on the baseline
diet). The blood glucose of the fat-fed males was significantly
different from that of both other groups at each time point
(P<.01 versus females; P<.05 versus males on
the baseline diet). In addition, the blood glucose levels of the males
on the baseline diet was significantly higher than that of females at
15 and 30 minutes (P<.05). In response to the oral glucose
challenge, the F1 males on the 15% fat diet
showed delayed glucose clearance and pronounced
hyperinsulinemia: Peak insulin levels were fourfold
higher in the fat-fed males than in males on the baseline diet (Fig 3).
In addition, the males on the baseline diets showed significantly
higher (P<.01 by ANOVA) insulin levels than did the fat-fed
females at 15 and 30 minutes after the glucose load (Fig 3).
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Body Weight and Fat Distribution
Obesity is strongly associated with insulin resistance. It was
therefore important to determine whether the
hyperinsulinemia in the
(BTBRxB6)F1 male mice might be a consequence of
obesity. The 15% fat diet led to a larger increase in visceral fat
mass in male than in female F1s (Table 1). In the male mice, insulin levels
correlated with visceral fat content (Fig 4). However, although the high-fat diet
produced more visceral fat in the female mice, there was not a
commensurate increase in fasting insulin levels (Fig 4).
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The 15% fat diet has a higher caloric density than the baseline diet.
The fat composition of a diet can affect feeding behavior by changing
the palatability of the food. Accordingly, we monitored food intake.
Food consumption was determined by weighing the food daily, adjusting
for spillage, over three weeks. The daily food consumption was
determined for 3 weeks by weighing the food and subtracting the amount
not eaten, including spillage. On the basal diet, the caloric intake
was 20.1 calories/day, and on the 15% fat diet, it was 23.2
calories/day. The visceral fat mass of the mice on the 15% fat diet
was increased relative to that of mice on the baseline diet in both
female and male mice (Table 1
). Nevertheless, only the males became
hyperinsulinemic on the 15% fat diet (Fig 4). In
addition, males on the baseline diet were insulin-resistant in
muscle. Therefore, the hyperinsulinemia and the
insulin resistance cannot be attributable solely to the increase in
visceral fat.
Plasma Lipids
Insulin resistance is often associated with increases in plasma
triglycerides and sometimes elevated free fatty acid
levels. Serum triglyceride was elevated by approximately
40% in the males that received either diet, compared to the females.
Free fatty acid levels were unaffected by diet and did not differ
between male and female mice. In both females and males that were fed
the high-fat diet, plasma cholesterol increased twofold and
1.7-fold, respectively, compared to animals that were given regular
chow (Table 2). Apolipoprotein B levels
were not significantly affected by the diet in either group.
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Basal and Insulin-Stimulated Glucose Transport Activity in
Skeletal Muscle
Skeletal muscle is the major site for insulin-mediated whole-body
glucose disposal; hence, this tissue is an important contributor to the
maintenance of glucose homeostasis. Accordingly, we measured
the uptake of 2-deoxyglucose in soleus and EDL muscles in the absence
(basal) and presence of 20,000 µU/ml insulin and 150 µU/ml insulin
(insert) in F1 mice on the 15% fat diet (Figs 5A and 5B).
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There was a dramatic sex difference in insulin-stimulated glucose
uptake in the (BTBRxB6)F1 mice. In female
(BTBRxB6)F1 mice on either diet, there was no
evidence of insulin resistance in muscle. In female mice eating regular
chow (Fig 5A) or a 15% fat diet (Fig 5B), maximal (20,000 µU/ml) and
physiological (150 µU/ml) insulin concentrations
stimulated 2-deoxyglucose transport approximately 3.5-fold and 2.5-fold
respectively, in the soleus muscle. The EDL muscle was similarly
responsive.
In striking contrast to the females, both muscles from male
(BTBRxB6)F1 mice were profoundly insulin
resistant, even on the basal diet. 2-Deoxyglucose transport
activity was not stimulated by insulin in the soleus (150 or 20,000
µU/ml insulin) or the EDL (20,000 µU/ml insulin), regardless of
diet (Figs 5A and 5B). Thus, in this animal model, it appears that the
degree of insulin resistance does not predict insulin levels. Placing
the animals on a moderately high-fat diet induced
hyperinsulinemia but did not exacerbate the
impaired insulin-stimulated glucose transport.
Since the (BTBRxB6)F1 animals on the baseline
diet were insulin resistant without being
hyperinsulinemic, it was conceivable that the parental
strains might also show the same phenotype. Therefore, it
became important to assess the insulin responsiveness of the parental
strains. In both strains, insulin stimulated 2-deoxyglucose transport
in soleus and EDL muscles (Figs 6A and 6B). Muscle tissue from males and females was equally responsive to
insulin. Hence, the parental strains are not insulin resistant;
therefore, the insulin resistant phenotype of the
(BTBRxB6)F1 animals is a consequence of a
synergistic combination of BTBR and B6 alleles.
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Basal and Hypoxia-Stimulated Glucose Transport Activity in
Skeletal Muscles in Mice on Fat Diet
Hypoxia stimulates glucose transport through the GLUT4
transporter by an insulin-independent pathway.34
Consequently, the measurement of hypoxia-induced glucose
transport allowed us to determine whether the impairment in
insulin-stimulated glucose transport in the
(BTBRxB6)F1 mice was specific to the
insulin-stimulated pathway. Hypoxia treatment of soleus and EDL
muscles stimulated the 2-deoxyglucose transport activity twofold to
threefold (Fig 7). In contrast to insulin
stimulation, the degree of stimulation by hypoxia was the same
in male and female mice, a finding indicating that the impaired glucose
transport was specific for an insulin-sensitive step.
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Discussion |
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This study identified an example in which the offspring of two inbred mouse strains display a phenotype that was not found in either parental strain. Both BTBR and B6 mice have a robust insulin response and do not develop insulin resistance when placed on a 15% fat diet. Unexpectedly, the (BTBRxB6)F1 mice are severely insulin resistant. This was reflected in the inability of insulin to significantly stimulate glucose transport in muscle. Since this insulin resistance phenotype is absent in the two parental strains, it results from an interaction between the BTBR and B6 genomes. It is possible that particular alleles suppress insulin resistance in the homozygous parental strains and the loss of homozygosity results in the loss of the suppressive phenotype. Since crosses of BTBR with two other mouse strains, A/J and C3H/HeJ, did not produce the phenotype of the (BTBRxB6)F1 mice, it seems unlikely that loss of homozygosity of BTBR alleles is responsible for the insulin resistance of the (BTBRxB6)F1 mice. A more likely explanation is that the phenotype of the F1 animals involves epistatic interactions between two or more gene loci with particular alleles specific for each of the BTBR and the B6 strains.
Prior studies in mice and rats have shown that some strains develop insulin resistance in response to a high-fat diet. Many of these studies used diets that are extremely high in fat for rodents even though they are only moderately high relative to normal human diets. In addition, some of the diets include cholic acid to enhance fat absorption. A potential drawback of cholic acid is that it induces inflammatory responses in mice; therefore, the induction of inflammation-related genes such as cytokines might complicate the interpretation of experiments aimed at investigating insulin responsiveness.37 In the present studies, we chose to use a moderately high-fat diet without cholate to increase the likelihood of discovering major gene effects without inducing inflammatory responses.
It is surprising to note that despite severe insulin resistance, the hybrid mice had normal insulin levels on the basal diet. In contrast, after 4 weeks on the 15% fat diet, the same animals developed significant hyperinsulinemia even though their level of insulin resistance appeared to be the same. The glucose transport studies were carried out in muscle because it is responsible for the majority of insulin-stimulated glucose clearance. Our studies cannot rule out the possibility that other tissues, such as adipose tissue, might, in the presence of muscle insulin resistance, make a larger contribution to overall glucose clearance, thus preventing hyperinsulinemia. However, the glucose tolerance tests seem to argue against such a compensatory effect because whole-animal glucose tolerance is impaired in the hybrid animals.
The effect of lipids on insulin secretion has been explained in several ways. Fatty acids have been suggested to directly influence insulin secretion.38,39 Perhaps a more important mechanism is through the well-studied effect of fatty acids on hepatic gluconeogenesis. Studies of lipolysis-induced insulin release suggest that it is an indirect consequence of fatty acid stimulation of gluconeogenesis,40,41 a result of the potent inhibitory effect of acetyl-CoA on pyruvate dehydrogenase and activation of pyruvate carboxylase.42 In the (BTBRxB6)F1 mice, the normal amount of insulin is probably just barely enough to keep pace with the rate of glucose production. A lipid load in the diet might tip the balance by inducing excess glucose production, leading to a large induction in insulin secretion.
In our studies of oral glucose tolerance, muscle glucose transport, and diet-induced hyperinsulinemia, there was a difference between male and female (BTBRxB6)F1s: Only males showed impaired glucose tolerance, insulin resistance, and diet-induced hyperinsulinemia. Sexual dimorphism in the expression of diabetic phenotypes has been well studied by Leiter and co-workers. The balance of estrogenic and androgenic hormones influences hepatic glucose output.43,44 Thus, alterations in the effective concentrations of estrogen or testosterone can interact with diabetes-susceptibility genes to exacerbate or counteract a diabetes phenotype. The sulfation of dehydroepiandrosterone (DHEA), an androgen prehormone, effectively reduces the androgen level. This sulfation reaction, catalyzed by DHEA sulfotransferase, is suppressed at puberty in lean male mice. The retention of its activity in lean female mice effectively inactivates androgens in females. In animals expressing certain diabetogenic alleles, such as db and ob, the failure of the females to induce the sulfation of DHEA makes them susceptible to hyperglycemia and insulin resistance.45 In contrast, when females retain the ability to catalyze the sulfation of DHEA, as in the fat/fat mouse, they are protected from the diabesity phenotype. It would appear that the genes contributing to the diabetes phenotypes in the (BTBRxB6)F1s do not suppress females' ability to inactivate androgens as is the case in certain strains of mice carrying the db or ob mutation.
Diabetes mellitus is currently more prevalent than at other times in recorded history. In addition, diabetes is far more common in populations that consume high-calorie, high-fat diets and have a relatively sedentary lifestyle. It has been argued that the high prevalence of diabetes is a consequence of the selection in favor of diabetes traits, which would be beneficial under other circumstances.46 For example, there might be a potential benefit of having a blunted insulin response in times of nutritional deprivation. Indeed, animal experiments suggest that diabetic animals are better able to withstand starvation than are normal animals.47 Thus, a blunted insulin response may have been selected during times of nutritional deprivation and exists as a pathological state in times of nutritional abundance.46
Given the many effects of insulin and the complexity of the insulin signaling system, one would predict that many gene loci might have alleles that influence the response to insulin. In addition, different types of nutritional adversity might interact with genes in a unique fashion to produce particular insulin-resistant syndromes.
The genetics of insulin resistance and diabetes mellitus is complex. With only a few rare exceptions, single gene loci have not yet been identified with particular diabetes alleles that act independently to produce the clinical disease with complete penetrance. Rather, the genetic data suggest that multiple genes are involved and that they interact with one another and with environmental factors.5 This inference is supported by the recent creation of an animal model of NIDDM in which one allele of the insulin receptor and of IRS-1 were disrupted by homologous recombination.48
Rodent models of insulin resistance and NIDDM also involve the interactions multiple gene loci. For example, a recent analysis of the Goto-Kakiazaki rat, a model of NIDDM, suggests that different loci contribute to different components of the diabetes phenotype.21,22 Thus, three loci influence glucose tolerance while another locus contributes to body weight. Within the three loci influencing glucose tolerance, one affects postprandial but not fasting hyperglycemia and the other two affect both.
Genetic evidence supports the notion that insulin resistance and diet-induced glucose changes are independent phenomena. Surwit et al49 showed that genetic differences between B6 and A/J strains in insulin resistance and hyperglycemia in response to a high-fat/high-carbohydrate diet did not cosegregate among recombinant inbred BxA strains, a result suggesting that different gene loci contribute to these distinct phenotypes. It is interesting to note that on the higher level of fat used in the latter studies (36%), the B6 strain developed hyperinsulinemia; there appears to be a lipid threshold in the B6 strain (somewhere between 15% and 36%) before this strain becomes hyperinsulinemic.
Genetic synergism has been described for insulin-dependent diabetes mellitus in the nonobese diabetic (NOD) mouse. In this mouse strain, several loci have been estimated to be necessary for the expression of the diabetes phenotype.50 An NOD-derived major histocompatiblity (MHC) haplotype is required; however, different combinations of non-MHC alleles can interact to produce the diabetic phenotype. Epistatic interactions have also been described for susceptibility to various kinds of cancer, indicating that certain cancer susceptibility genes can go undetected if they are not expressed in conjunction with alleles at loci that interact to influence the cancer phenotype.51–53
The insulin-resistance phenotype of the (BTBRxB6)F1 mice appears to result from the combination of alleles from these two parental strains rather than from loss of homozygosity of their alleles. This model can therefore be used for gene-mapping studies to identify gene loci that interact to produce insulin resistance.
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Acknowledgments |
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Trine Ranheim was supported by a scholarship from the Henning and Johan Throne-Holst Foundation for Scientific Sesearch. We are very grateful for the encouragement and advice from Drs William F. Dove, Alexandra Shedlovsky, and Beverly Paigen. We also thank Drs William F. Dove and Alexandra Shedlovsky for introducing us to the BTBR mouse strain. We thank Drs Jaap Twisk, Michael MacDonald, and Kimberly Dirlam for their comments on the manuscript.
Received April 4, 1997; accepted June 10, 1997.
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