Control of Expression of Insulin

Resistance and Hyperglycemia

by Different Genetic Factors
in Diabetic C57BL/6J Mice
RICHARD S. SURWIT, MICHAEL F. SELDIN, CYNTHIA M. KUHN,
CHRISTINA COCHRANE, AND MARK N. FEINGLOS
The inheritance of the tendency to develop diet-
induced non-insulin-dependent (type II) diabetes was
analyzed in crosses between diabetes-prone C57BL/6J
(BL/6) mice and diabetes-resistant A/J mice. The
effects of a diabetogenic diet on blood glucose and
insulin levels, insulin sensitivity, and weight were
evaluated in F, and both (BL/6 x A/J) F, x BL/6 and
(BL/6 x A/J) F, x A/J backcross mice. These results
suggest that diet-induced hyperglycemia is largely
determined by a recessive gene and diet-induced
insulin resistance by a dominant gene. Analyses of
both backcrosses indicated that insulin sensitivity
and blood glucose levels were unrelated, suggesting
that they are controlled by different genetic factors.
This conclusion was supported by data from nine
recombinant inbred BXA strains in which no
correlation was observed between these variables.
Furthermore, insulin sensitivity and body weight
correlated differently in the two backcross groups,
suggesting that insulin resistance is not simply
a function of obesity. The number of genes that
predominantly influence diabetic traits was estimated
by comparing the variance observed in (BL/6 x A/J)
F
t
x BL/6 backcross mice with that observed in
parental mice. The data suggest that relatively few
genes predominantly affect the diabetic phenotype
in this murine model. Diabetes 40:82-87,1991
U
nderlying genetic factors are probably critical in
the pathogenesis of non-insulin-dependent (type
II) diabetes mellitus. Although studies of identical
twins have shown 95% concordance of disease
(1) and abnormal glucose tolerance in as many as 30% of
relatives of patients with type II diabetes (2), little is known
From the Departments of Psychiatry, Medicine, and Pharmacology, Duke
University Medical Center, Durham, North Carolina.
Address correspondence and reprint requests to Dr. Richard S. Surwit, Box
3842, Duke University Medical Center, Durham, NC 27710.
Received for publication 19 February 1990 and accepted in revised form
8 August 1990.
about the specific inherited defects. In certain populations,
a form of type II diabetes appears to be inherited in a com-
plex fashion, with insulin resistance and impaired pancreatic
activity segregating independently (3,4). Linkage studies
have failed to demonstrate a clear association of these traits
with any genetic locus, including those of the gene encoding
either insulin or insulin receptors (5).
Animal models of type II diabetes are amenable to genetic
analysis. Inbred mouse strains are of particular interest be-
cause several forms of type II diabetes exist in inbred strains,
which provide large numbers of genetically identical indi-
viduals (6). Finally, the mouse genome has been extensively
mapped, and much of it is conserved in humans, which
suggests that identification of a genetic locus or region in
the mouse genome may facilitate human genetic analyses
(7).
Several mutations in C57BL/6J (BL/6) mice have resulted
in type II diabetes-like syndromes (8), suggesting to us that
this strain may carry many of the genes necessary for this
disease (9). Consistent with this hypothesis, studies in our
laboratory demonstrated that the BL/6 mouse but not the
A/J mouse will develop an analogue of type II diabetes if
weaned onto a high-fat high-simple-carbohydrate diet and
allowed to become obese (9).
In this article, we describe the metabolic characteristics
of an FT cross of diabetes-prone BL/6 mice and diabetes-
resistant A/J mice raised on the diabetogenic diet and those
of the backcross of the F, onto the BL/6 and A/J strains and
a few recombinant inbred (Rl) strains. The distribution of
these values supports the hypothesis that different genetic
factors influence the expression of diet-induced insulin re-
sistance and hyperglycemia.
RESEARCH DESIGN AND METHODS
Twenty BL/6 mice, 20 A/J mice, 20 F, crosses (BXA), and
55 backcrosses on the BL/6 parent strain (BL/6 x A/J fe-
male) F, x BL/6 were obtained at 1 mo of age from Jackson
(Bar Harbor, ME). A second group of 44 (BL/6 x A/J)
F, x A/J backcross mice were bred at Duke University from
parental stock obtained from Jackson. Finally, 5 animals from
82 DIABETES, VOL. 40, JANUARY 1991

R.S. SURWIT AND ASSOCIATES
each of Rl BXA strains 8, 10, 11, 13, 19, 24, and 25 and 4
each from strains 7 and 17 were obtained from the laboratory
of M. Nesbitt (University of California, San Diego). All animals
studied were male. Note that Rl strains are derived from
crosses between two inbred progenitor strains in which the
mice from the F
2
generation and each subsequent genera-
tion are mated in accordance with a strict inbreeding pro-
gram. After 20 generations, each of the resultant Rl strains
has a unique contribution from each original progenitor in
which the alleles from either progenitor strain have become
fixed at each locus. Thus, observations may be made on
different individuals, each of which has an identical geno-
type.
On their arrival at the laboratory, half of the animals of
each strain were placed on a high-fat high-simple-carbo-
hydrate diet previously shown to induce type II diabetes in
BL/6 mice (9). The diet contained 20.5% protein, 35.8% fat,
0.4% fiber, 3.6% ash, 3.1% moisture, and 36.8% carbohy-
drate (primarily disaccharides; diet 1850, Bio-Serve, French-
town, NJ). Typically, BL/6 animals fed this diet for 3 mo show
fasting plasma glucose >11.11 mM and plasma insulin
>898.2 pM. The rest of the animals were maintained on
standard Purina Rodent Chow (Ralston-Purina, Richmond,
IN). This diet contained 23% protein, 4.5% fat, 6% fiber, 8%
ash, and 56% complex carbohydrate.
All animals were housed in the vivarium at Duke University
in group cages, with five animals to each cage. They were
given food and water ad libitum and maintained on a 12-h
light-dark diurnal cycle. All animals were weighed weekly.
Once per month, beginning with mo 2, all animals were
fasted for 8 h, after which blood was sampled for serum
glucose (assayed by Beckman autoanalyzer) and serum in-
sulin (assayed by a Cambridge Medical Diagnostics ra-
dioimmunoassay).
After 4.5 mo, five animals of each parental strain and all
backcrosses were given an insulin-sensitivity test. Animals
were fasted for 8 h before testing and then given an injection
of regular pork insulin (0.5 lU/kg i.p.; Lilly, Indianapolis, IN).
Blood was sampled at 15 and 30 min. Blood samples were
analyzed for serum glucose as described above. Insulin sen-
sitivity (K|) was measured as the slope of the fall in glucose
over time after the injection of insulin. This technique, when
applied to humans, has been shown to give results com-
parable to those obtained with glucose-insulin-clamp tech-
niques (10). All blood samples were collected by retro-orbital
sinus puncture.
Data from the 5th mo were used to calculate estimates of
genetic dominance for fasting glucose, insulin, insulin sen-
sitivity (mo 4.5), and body weight. These data were also used
to calculate the degree to which these traits segregated
together and the number of genetic loci that determined each
trait. Month five was selected because previous studies have
shown that diabetes is fully developed in these mice at this
time (9).
To analyze the genetics of diet-induced diabetes, diabe-
tes-prone BL/6 mice were crossed with diabetes-resistant
A/J mice. The mice were given either a control or diabeto-
genic diet as previously described (9). We then compared
the parental strains to the F, on both control and experimental
diets to determine the dominance of each of the above meta-
bolic characteristics. Dominance {h) of the BL/6 genotype
was estimated by the formula (11)
mean BL/6 x A/J F, - mean A/J
h =
mean BL/6 - mean A/J
Variance (var) of h was estimated with the following formula
(kindly provided by B. Weir, Dept. of Statistics, North Carolina
State Univ., Raleigh). For the quantity
h =
X] X2
AQ AD
where X, is the mean BL/6 x A/J F,, X
2
is the mean A/J,
and X
3
is the mean BL/6, an approximate expression for the
variance is
var(h) = var(X,)+ var(X2)+ ^ -
- x-
var(X,)
X, - X
3
Y
{A3 A2) /
var(X2)
x, - x
2
Y
\A$ A2J /
var(X3)
where d represents the derivative of the function. Note that
there are no covariances, because the three Xs are found
from different samples. To further characterize the relation-
ship of genetic factors that result in the diabetic phenotype,
the F, mice were backcrossed with the diabetes-prone
BL/6 parent and diabetes-resistant A/J parent. The analysis
of correlation in genetically heterogeneous populations
(backcross mice) allows us to assess the degree to which
the same genetic factors control phenotypic expression of
the various traits, provided that environmental factors are not
as important as genetic factors in determining disease.
The method of Wright (11), after modifications suggested
by Lande (12) and Cockerham (13), was used to estimate
the number of genes responsible for each biological property
measured between mo 4 and 5. The following formulas were
applied to data obtained from each parent, F,, and (BL/6 x
A/J) F, x BL/6 backcross to determine the mean SD
number of effective loci (S,) in which the F, differs from the
BL/6 parent. Note that these methods are imprecise and
serve as estimates of the number of loci that predominantly
affect each trait, because many additional genes may make
minor contributions that are quantitatively important. For as-
sumptions and limitations of these procedures, see refs. 11-
13.
S, =
(mean BL/6 - mean F,)
2
2as
oy
2
is the additive genetic variance stemming from differences
among gene frequencies of the two parental populations
us
2
= 2a
2
BC, -
1
/6(7aBL/6
2
+ crA/J
2
4<rF,
2
)
where BC, = (BL/ 6 x A/ J) F, x BL/ 6 backcross mice. The
SD of S, was cal cul ated as
, , /4(a
2
BL/6/n
BL
,
6
+ q
Fl
2
/n
Fl
) var q
s
CTs
^' ' \ (mean BL/6 - mean F,)
2
a
s
4
DIABETES, VOL. 40, JANUARY 1991 83

GENETIC FACTORS IN DIABETIC C57BL/6J MICE
where
var a
s
2
=
( 3a
BL/ 6
2
qA/ J
2
2a
s
8n
B
and n is the number of mice in each group.
Similar analysis with data from the reciprocal backcross
(BL/6 x A/J) F, x A/J to determine S
2
was not performed
because parental strains were not run simultaneously.
RESULTS
Effect of diabetogenic diet on cross between diabetes-
prone and diabetes-resistant strains. The mo-5 values for
glucose, insulin, and weight and mo-4.5 values for /<, are
shown in Fig. 1. BL/6 mice fed the control diet had slightly
higher glucose values than the A/J or F, mice given this diet,
but insulin levels, K
u
and body weight were the same as
those of A/J and F, animals. The experimental diet raised
glucose and insulin levels and body weight of all animals,
but this effect was much more pronounced for the diabetes-
prone BL/6 mice. Although the effect of the diabetogenic
diet on insulin levels and body weight became more pro-
nounced during the 5 mo of analyses, hyperglycemia was
evident after 1 mo (data not shown).
The FT mice on the diabetogenic diet showed glucose and
insulin levels and body weights that were intermediate be-
tween the parental strains; fasting insulin and body weights
were significantly lower than those of BL/6 mice and signif-
icantly higher than those of A/J mice (Fig. 1). The diabeto-
genic diet greatly reduced the K, of BL/6 mice but did not
affect ft, of A/J mice. In contrast, the K
x
values for F, animals
on the experimental diet were less than those of A/J mice
but not significantly greater than those of BL/6 mice.
At mo 5, the mean SD h of the BL/6 genotype for blood
glucose was estimated to be 0.19 0.09, suggesting that
this characteristic is recessive. Mean SD h for insulin-
sensitivity was measured at 0.71 0.21, suggesting that
this trait is controlled mostly by dominant genes. Thus, hy-
perglycemia and insulin sensitivity appear to be inherited
differently.
Correlation analysis of genetic and nongenetic factors
influencing different diabetic characteristics. The regres-
sion values for four dependent variables are listed for both
backcross groups in Table 1. In only one case was the cor-
relation in one backcross significantly different from that in
the other backcross. Although K, was significantly correlated
with body weight in the (BL/6 x A/J) F, x A/J backcross
(r = 0.44, P < 0.01), it was not correlated at all in the
1000-
BL/ 6
FIG. 1. Metabolic parameters of C57BL/6J (BL/6), A/J, and (BL/6 x A/J) F,, mice on diabetogenic and control diets. Fasting serum glucose (F,)
fasting serum insulin, and body weight after 5 mo and insulin sensitivity (K,) after 4.5 mo in BL/6, A/J, and F, mice on control (open bars) or
diabetogenic (hatched bars) diet. Results are expressed as means SE (n = 8-10/group for fasting serum glucose, fasting serum insulin, and
body weight; n = 4-5/group for insulin sensitivity). There was significant effect of diet, strain, and diet x strain interaction by analysis of
variance allowing following pairwise comparisons. Diet had significant effect on glucose for BL/6 and A/J (P < 0.02 or better), on insulin and
body weight for all strains (P < 0.001), and on insulin sensitivity for BL/6 and F, mice only (P < 0.05 or better). On control diet, F, animals
differed from BL/6 mice for body weight only and from A/J mice for glucose and body weight. On diabetogenic diet, F, animals differed from
BL/6 mice in glucose, insulin, and body weight and from A/J mice in all except glucose. P < 0.05 or better for latter probabilities.
84 DIABETES, VOL. 40, JANUARY 1991

R.S. SURWIT AND ASSOCIATES
TABLE 1
Correlation coefficients of diabetic parameters at mo 5 in backcross C57BL/6J (BL/6) mice on diabetogenic diet
BG and BW IRI and BW BG and IRI K, and BW K, and BG K, and IRI
(BL/6 x A/J) F, x BL/6 55
(BL/6 x A/J) F, x A/J 44
(BL/6 x A/J) F, x BL/6 55
(BL/6 x A/J) F, x A/J 41
0.10
0.28
0.26
0.45
0.43
0.14
0.00
0.44*
0.17
0.10
0.26
0.47
BG, blood glucose; BW, body weight; IRI, immunoreactive insulin; K
u
insulin sensitivity.
*P < 0.01 such that r, ^ r
2
for crosses (BL/6 x A/J) F, x BL/6 vs. (BL/6 x A/J) F, x A/J.
(BL/6 x A/J) F, x BL/6 backcross (r = 0). These corre-
lations were significantly different (P < 0.01) from each other
(14). This suggests that the presence of animals homozy-
gous for the BL/6 genotype in the (BL/6 x A/J) F, x BL/6
backcross adds significant variability to the relationship be-
tween insulin resistance and body weight above that attrib-
utable to the environment. Thus, insulin resistance is not
simply a result of obesity; the degree of insulin resistance
depends on genetic background.
Another notable finding is the lack of correlation between
K, values and blood glucose. In both backcross groups, the
relationship between these variables appears to be random.
This is in contrast to a strong correlation observed in the
BL/6 parental group (r = 0.96). The probability that the
correlation observed in the BL/6 parental group is different
from the observed correlations in either backcross group is
P< 0.05 (14).
Analyses of hyperglycemia and insulin resistance in Rl
strains. To further investigate the inheritance of genes pre-
disposing to the diabetic phenotype, nine BXA Rl strains
were examined. Because of the few animals per strain, glu-
cose values were averaged over mo 3, 4, and 5. K, values
were determined at mo 4.5. Mice from each of these strains
are homozygous at each locus, but each strain contains a
unique contribution of the two progenitor strains. There is no
relationship between serum glucose values and K, values
(Fig. 2). Rank-sum correlations between K, values and serum
11 8 17 25 7 13 10 19
(BXA) Rl Strains
24
FIG. 2. Serum glucose and insulin sensitivity (K,) of recombinant
inbred (Rl) strains on diabetogenic diet. Mean SE serum glucose
(open bars) and K, {hatched bars) values in 9 C57BL/6J x A/J (BXA)
Rl strains. Blood glucose values are averaged over mo 3, 4, and 5.
Glucose data are based on 15 observations for strains 8, 10, 11, 13, 24,
and 25, with 14 observations for strain 19,12 for strain 7, and 11 for
strain 17. Insulin-sensitivity determinations were made at mo 4.5. n =
5 mice/group except for strains 11 and 17 (n = 4).
glucose or insulin levels were not significant (data not
shown). These data support the conclusion that insulin re-
sistance and hyperglycemia are determined by different ge-
netic factors in this mouse model.
Analyses of genetic factors influencing diabetic pheno-
type with backcross mice. Glucose, insulin, and weight
data of all strains measured at mo 5 and K, values at mo 4.5
are presented in Table 2, and the individual glucose values
are depicted in Fig. 3.
On the diabetogenic diet, diabetes-prone BL/6 and dia-
betes-resistant A/J animals were >3SD apart for each pa-
rameter except AC,. Furthermore, differences in variance
between parental and F, mice were small, indicating the
genetic homogeneity of parental populations and the rela-
tively small environmental variance. Therefore, the large vari-
ance of backcross values relative to the parental and F, mice
indicates genetic heterogeneity of the backcross mice. A
clear bimodal or trimodal distribution of glucose (Fig. 3),
insulin (data not shown), and K, (data not shown) was not
observed. However, with the method of Wright (11) as
adapted and modified by Lande (12) and Cockerham (13),
the number of major genetic factors (S,) for which the dia-
betes-prone BL/6 mouse differed from the relatively dia-
betes-resistant F, mouse was calculated. S, was <1 for
glucose, log glucose, and insulin; 4.1 for weight; and 1.10
for log insulin. Similarly, S, for /<, was 0.06 (calculated at mo
4.5). These data suggest that relatively few genes are prob-
ably responsible for differences in glucose, insulin, insulin
sensitivity, and weight observed between F, mice and
(BL/6 x A/J) F, x BL/6 backcross mice maintained on the
diabetogenic diet.
DISCUSSION
These data allow us to draw several conclusions about the
inheritance of the tendency to develop type II diabetes in
the BL/6 mouse. Of primary importance is that diet-induced
insulin resistance and hyperglycemia are controlled by dif-
ferent genetic factors. Three lines of evidence allow us to
reach this conclusion. First, in both backcross groups, fast-
ing glucose and insulin sensitivity do not correlate, indicating
that insulin resistance and hyperglycemia segregate inde-
pendently. Second, the differential dominance of blood glu-
cose levels and insulin sensitivity in the F, crosses show that,
although insulin resistance is inherited in a largely dominant
fashion, hyperglycemia appears to be a recessive trait. Third,
and most conclusively, hyperglycemia and insulin sensitivity
segregated independently in Rl strains. Thus, at least two
distinct genetic factors are necessary for the appearance of
type II diabetes characteristic of the BL/6 mouse. Unfortu-
nately, the physiological basis for hyperglycemia or insulin
DIABETES, VOL. 40, JANUARY 1991 85

GENETIC FACTORS IN DIABETIC C57BL/6J MICE
TABLE 2
Serum glucose, log glucose, insulin, log insulin, insulin sensitivity, and body weight for experimental animals at mo 5
Glucose (mM)
BL/6
A/J
(BL/6 x A/J) F,
(BL/6 x A/J) F, x BL/6
Log glucose
BL/6
A/J
(BL/6 x A/J) F,
(BL/6 x A/J) F, x BL/6
Insulin (pM)
BL/6
A/J
(BL/6 x A/J) F,
(BL/6 x A/J) F, x BL/6
Log insulin
BL/6
A/J
(BL/6 x A/J) F,
(BL/6 x A/J) F, x BL/6
Insulin sensitivity (/<,)
BL/6
A/J
(BL/6 x A/J) F,
(BL/6 x A/J) F, x BL/6
Body weight (g)
BL/6
A/J
(BL/6 x A/J) F,
(BL/6 x A/J) F, x BL/6
Mean SD
12.5 1.3
8.3 1.1
9.1 0.8
12.3 2.8
0.130 0.130
0.120 0.120
0.122 0.002
0.128 0.005
893.6 128.3
187.9 59.6
401.4 188.3
985.7 377.1
14.286 0.487
9.989 0.924
12.164 1.417
15.000 1.900
-2. 10 1.09
-5. 17 1.17
-3. 04 1.15
-0. 60 2.18
49.5 4.0
35.2 4.3
41.1 3.8
50.5 4.5
Variance
1.7
1.3
0.7
6.1
6.98 x 10"
6
1.0 x 10"
5
3.4 x 10"
6
2.3 x 10"
5
16556.8
3520.3
34789.8
150844.2
0.227
0.869
2.045
2.759
1.18
1.38
1.33
4.79
16.1
18.2
14.6
20.0
n
10
9
10
55
10
9
10
55
10
8
10
55
10
8
10
55
5
4
5
55
10
10
10
55
S, SD
0.65 0. 18
0.95 0.36
0.49 0. 10
1.10 0.37
0.06 0.06
4.10 2.49
BL/6 = C57BL/6J. S, represents an estimate of the number of major genetic factors for which the diabetes-prone BL/6 mouse differs
from the relatively diabetes-resistant F, mouse. The formula for calculation of S, is given in METHODS.
resistance in the BL/6 mouse remains uncertain. As in hu-
man type II diabetes, hyperglycemia is accompanied by
hyperinsulinemia in our mouse model (15). However, subtle
defects in pancreatic function, such as impaired first-phase
insulin release, are characteristic of BL/6 mice (16).
Although we cannot immediately generalize these findings
to the phenomenon of human diabetes, several studies have
suggested that insulin sensitivity and impaired pancreatic
activity may be separate phenomena, which do not have to
occur together in human type II diabetes (17-20). Lillioja et
al. (17) found that impaired glucose tolerance in Pima In-
1 0 '
20 30
animal number
50
FIG. 3. Individual serum glucose values for (BL/6 x A/J) F, x BL/6
backcross mice after 5 mo on diabetogenic diet. Note, BL/6 is our
abbreviation for C57BL/6J.
dians was due to insulin resistance, whereas the develop-
ment of frank hyperglycemia was associated with a defect
in pancreatic function. Furthermore, in the Pima population,
insulin resistance appears to be determined by one codom-
inant gene, whereas an additional recessive gene is thought
to be responsible for hyperglycemia (18). Eriksson et al. (19)
showed that even insulin sensitivity alone could not explain
impaired glucose tolerance in a Scandanavian population;
a defect in first-phase insulin release had to be present as
well. Finally, Banerji and Lebovitz (20) described two forms
of type II diabetes, one in which insulin sensitivity is normal
and a significant pancreatic defect exists and another in
which there is a mild impairment of glucose-stimulated in-
sulin secretion accompanied by significant insulin resist-
ance. Reaven (21) has suggested that insulin resistance is
a very common problem, and therefore cannot be by itself
the cause of type II diabetes. Our data support the conclu-
sion that impaired insulin sensitivity does not cause hyper-
glycemia in the BL/6 mouse and that the development of
diabetes in this animal model is primarily dependent on some
other genetic factor.
Another conclusion that can be reached from our data is
that insulin sensitivity is not simply a function of body weight.
The different correlations between insulin sensitivity and
weight observed in the two backcross groups suggest that
insulin sensitivity is dependent in part on genetic back-
ground. Thus, insulin resistance may be inherited indepen-
dent of obesity. Studies in several human populations have
suggested similar conclusions. Lillioja et al. (17) reported
that the familial component of insulin action and hyperin-
86 DIABETES, VOL. 40, JANUARY 1991

R.S. SURWIT AND ASSOCIATES
sulinemia occurs in addition to the effects of obesity in Pima
Indians, and Haffner et al. (22) found a similar independence
of genetic and environmental effects in Mexican Americans.
Finally, estimation of the number of genes that differentiate
the F, from the BL/6 strain suggests that relatively few genes
are primarily responsible for the manifestation of diet-in-
duced insulin resistance and diet-induced hyperglycemia.
These estimates suggest that the diabetes of the BL/6
mouse is amenable to genetic analysis. Future studies of Fa-
generation crosses of BL/6 and A/J mice will provide further
precision in estimating the number of genes responsible for
hyperglycemia and insulin resistance. Furthermore, gene-
mapping studies with available Rl lines may allow identifi-
cation of one or more genetic loci that are important in the
pathogenesis of this murine model of type II diabetes and
perhaps eventual identification of the human homologues.
ACKNOWLEDGMENTS
This research was supported by a grant from the American
Diabetes Association, the John D. and Catherine T. Mac-
Arthur Foundation Research Network on the Consequences
and Determinants of Health Promoting and Health Damaging
Behavior, and by Research Scientist Development Award
K01-MH-00303 from the National Institute of Mental Health.
M.F.S. is a Charles E. Culpeper Foundation Scholar; the work
was supported in part by this foundation.
We are indebted to Dr. Muriel Nesbitt for providing BXA
strains, Dr. Bruce Weir for valuable discussions on estimating
dominance and the number of genes for a quantitative char-
acter, and Dr. Marc K. Drezner for critical review of the manu-
script.
Some of the research contained in this article was pre-
sented at the scientific sessions of the 50th annual meeting
of the American Diabetes Association, Atlanta, Georgia, 16-
19 June 1990.
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