1 Gifford Laboratories, Obesity is associated with both insulin
resistance and hyperinsulinemia. Initially hyperinsulinemia compensates
for the insulin resistance and thereby maintains normal glucose
homeostasis. Obesity is also associated with increased tissue
triglyceride (TG) content. To determine whether both insulin resistance
and hyperinsulinemia might be secondary to increased tissue TG, we
studied correlations between TG content of skeletal muscle, liver, and
pancreas and plasma insulin, plasma [insulin] × [glucose], and
tissue fat; obese Zucker diabetic fatty rats; OBESITY is the most prevalent health problem in the
United States and is currently estimated to afflict ~75,000,000
Americans (28). Although the risk of diabetes is high, glucose
homeostasis remains relatively normal for long periods of time despite
insulin resistance and excessive intake of food. This ability to
maintain euglycemia despite diminished insulin effectiveness indicates that insulin production is somehow matched to insulin requirements. The
mechanism by which Tissue triglyceride (TG) content seemed to be a plausible candidate for
this dual role. Long-chain free fatty acids (FFA) have long been known
to interfere with insulin-mediated glucose metabolism (2, 8, 13, 15,
18, 24, 25, 29) and to stimulate insulin secretion acutely (5, 11, 22,
26, 31). Moreover, high FFA levels in vitro have been shown to induce in normal islets the same changes described in the compensating There being no direct way to test the possibility that the coupling of
insulin production to insulin need is mediated by tissue lipid content,
we relied on correlations between Animals.
All animals were 7-9 wk of age at the start of experiments. Obese,
prediabetic Zucker diabetic fatty (ZDF) rats
(fa/fa) were bred in our laboratory
from ZDF/Drt-fa (F10) stock purchased
from R. Peterson (University of Indiana School of Medicine,
Indianapolis, IN). Male rats exhibited the previously described
phenotype (30).
ABSTRACT
Top
Abstract
Introduction
Materials
Results
Discussion
References
-cell function in four rat models with
widely varying fat content: obese Zucker diabetic fatty rats,
free-feeding lean Wistar rats, hyperleptinemic Wistar rats with
profound tissue lipopenia, and rats pair fed to hyperleptinemics.
Correlation coefficients >0.9 (P < 0.05) were obtained among TG of skeletal muscle, liver, and pancreas and among plasma insulin, [insulin] × [glucose] product, and
-cell function as gauged by
basal, glucose-stimulated, and arginine-stimulated insulin secretion by
the isolated perfused pancreas. Although these correlations cannot
prove cause and effect, they are consistent with the hypothesis that
the TG content of tissues sets the level of both insulin resistance and
insulin production.
-cell
function
INTRODUCTION
Top
Abstract
Introduction
Materials
Results
Discussion
References
-cells recognize the level of insulin resistance
that must be overcome is unknown. Because hyperinsulinemia may be
present in obesity even when glucose tolerance is normal, subtle
glycemic elevations are not the signal for the enhanced insulin
production at that stage of the disorder. This study was undertaken to
identify a nonglycemic signal that might link insulin action to insulin
production.
-cells of obesity, such as
-cell hyperplasia and increased
insulin secretion at substimulatory glucose levels (16, 26). Third, it
has been shown that islets of hyperinsulinemic obese rats have an
extremely high TG content compared with normal littermates (20).
-cell function and insulin
effectiveness and tissue TG content of groups of rats with a widely
varying tissue fat content. At one extreme of the spectrum of fat
content were obese rats, in which tissue lipids are markedly increased
(20). At the other extreme we exploited a novel syndrome of profound
tissue lipid depletion that is the antithesis of obesity; this syndrome
was produced by inducing chronic hyperleptinemia in normal rats by
means of adenovirus-leptin gene transfer (3, 34). These animals undergo
rapid and selective loss of all grossly visible fat (3); additionally
the TG content in skeletal muscle, liver, and pancreas declines to
1/1,000 of that of obese rats and 1/10 of that of pair-fed controls
(34). Between these two extremes of severe lipopenia and obesity, we studied free-feeding normal rats and normal rats whose food intake was
restricted by pair feeding to the hyperleptinemic rats. The high
correlations between indexes of insulin resistance and insulin production and the TG content in the target tissues of insulin and in
the tissue of insulin production are consistent with the hypothesis
that tissue fat content might provide the link between insulin
resistance and hyperinsulinemia.
METHODS AND MATERIALS
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Abstract
Introduction
Materials
Results
Discussion
References
Gene transfer studies.
To clone the rat leptin cDNA and measure its mRNA, total RNA was
prepared from 1 g of epididymal adipose tissue of rats by extraction
with TRIzol as recommended by the manufacturer. Oligo(dT) was used to
prime first-strand cDNA synthesis by use of a cDNA synthesis kit
(Clontech, Palo Alto, CA). After treatment of the first-strand cDNA
with deoxyribonuclease-free ribonuclease, the leptin gene product was
amplified by polymerase chain reaction (PCR) with upstream sense primer
5'-GGAGGAATCCCTGCTCCAGC-3' and downstream antisense primer
5'-CTTCTCCTGAGGATACCTGG-3' based on the rat leptin gene
sequence (27). For both cDNA cloning and leptin mRNA measurement,
amplification was performed using 1 cycle at 94°C for 3 min,
followed by 35 cycles at 92°C for 45 s, at 53°C for 45 s, and
at 72°C for 1 min, and then final extension at 70°C for 10 min.
We also measured -actin expression with the same amplification
conditions as for leptin and a previously described oligonucleotide
pair (23).
Plasma measurements.
Beginning 1 day after adenovirus infusion, fasting blood samples
(1-3 PM) were collected from the tail vein in capillary tubes coated with EDTA. Plasma was stored at 20°C until the time
of leptin assay. Plasma leptin was assayed using the Linco leptin assay
kit (Linco Research, St. Charles, MO). Plasma insulin was assayed by
standard methods (37). Plasma glucose was measured by Glucose Analyzer
II (Beckman, Brea, CA).
Pancreas perfusion. Pancreata were isolated and perfused by the method of Grodsky and Fanska (12) as previously modified (17). The perfusate consisted of Krebs-Ringer bicarbonate buffer containing 4.5% Dextran T70, 5.6 mM glucose, 1% BSA, and 5 mM each of sodium pyruvate, sodium glutamate, and sodium fumarate. The flow rate was 3.0 ml/min. After a 20-min equilibration period, the pancreas was perfused for 10 min with 5.6 mM glucose. Then the glucose concentration was increased to 20 mM for 10 min. After a 10-min "rest," during which the glucose level was returned to baseline, arginine was perfused at a concentration of 8 mM for a total of 10 min. Samples were collected at 1-min intervals for determination of insulin concentration. They were placed in chilled tubes containing 0.3 ml of 0.15 M NaCl, 0.05 M Na2EDTA, and 0.3 M benzamide and were frozen until the time of assay.
TG content of liver, skeletal muscle, pancreas, and islets.
Tissues were dissected and placed in liquid nitrogen. About 100 mg of
tissues were placed in 4 ml of homogenizing buffer containing 18 mM of
tris(hydroxymethyl)aminomethane · HCl (pH = 7.5), 300 mM of D-mannitol, and 5 mM of
ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic
acid and were homogenized using a hand-held polytron (Kontes Glass,
Vineland, NJ) for 10 s. Lipids were extracted by the method of Folch et
al. (9). Total TG were assayed by the method of Danno et al. (6).
Statistical analysis. All data are expressed as means ± SE. Statistical difference was analyzed by unpaired t-test. P < 0.05 was considered statistically significant. Regression line was calculated using the Stat View program (Abacus Concepts, Berkeley, CA).
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RESULTS |
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Clinical and laboratory findings.
AdCMV-leptin-infused rats developed hyperleptinemia averaging
13.7 ± 2.1 ng/ml during the 14 days of the study (Fig.
1A). Compared with pair-fed normoleptinemic controls, these rats appeared hyperactive and disinterested in food but in otherwise good health. Their total feed intake over the period of 14 days averaged 58% of
that of Gal-treated controls (Fig.
1B). Body weight declined during the
1st wk and failed to increase during the 14-day period of study (Fig.
1C), thus confirming our previous
report (3). Visible body fat was absent or profoundly reduced in all
sites after 1 wk of hyperleptinemia.
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Tissue lipid content in the various groups.
Two weeks after the AdCMV-leptin infusion, the tissue content of TG was
measured in liver, skeletal muscle, whole pancreas, and islets of
age-matched hyperleptinemic rats, pair-fed and free-feeding control
rats, and obese rats. The TG content is shown in Table 1, expressed both per gram of wet weight and as a
percentage of the tissues of the three control groups. In the
hyperleptinemic rats, the TG content of liver averaged 13.3% of that
of free-feeding Gal-infused rats and was only 6.4% of that of obese
rats; the fat content of their skeletal muscle was 8% of that of
free-feeding
Gal-infused controls. In whole pancreas, TG content of
the hyperleptinemics was 5.3% of that of AdCMV-
Gal controls and
12.9% of that of pair-fed controls.
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Correlations between TG content of pancreas and the product of
plasma insulin and plasma glucose and insulin production by isolated
pancreata.
To determine the relationship between -cell function and islet fat
content, we examined the correlations between pancreatic fat content
and insulin production by isolated perfused pancreata from all groups.
The perfusate consisted of either 5.6 mM glucose, 20 mM glucose, or 8 mM arginine plus 5.6 mM glucose. We also measured the correlation
between pancreatic fat and plasma [insulin] × plasma
[glucose], which distinguished between hypoinsulinemia that
results in increased blood glucose concentrations and hypoinsulinemia associated with increased insulin effectiveness.
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DISCUSSION |
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The goal of this study was to determine whether the fat content in the
target tissues of insulin and in the pancreas is correlated with both
the effectiveness of insulin and the rate of its secretion. If so, it
would be consistent with the notion that the responses of -cells are
tailored to insulin need by a derivative of TG such as FFA. This would
facilitate maintenance of euglycemia despite the changing body
composition associated with obesity. It should, however, be stressed
that correlations cannot prove cause and effect and that alternative
interpretations of these correlations are equally possible. For
example, primary hyperinsulinemia could increase lipogenesis and cause
increased tissue TG, which could in turn give rise to insulin
resistance (24, 25).
We employed both obese ZDF rats and lean Wistar rats. The availability of a unique "fat-free" animal, the hyperleptinemic rat, a novel model of extreme lipopenia that is the antithesis of obesity, made it possible to examine these correlations over the broadest possible spectrum of TG content, ranging from extreme lipopenia to severe obesity. In acute perfusion experiments, leptin had no effect on insulin production (data not shown), but in chronic studies of cultured islets, leptin abolished the insulin responses to both glucose- and arginine-stimulated insulin secretion while depleting islet TG; FFA restored the insulin response almost immediately (19).
We observed that the production of the fasting plasma insulin and blood glucose levels, a crude index of insulin sensitivity, was significantly correlated with TG content, as was the fasting plasma insulin level by itself. Insulin sensitivity was markedly enhanced (low insulin-glucose product) in the lipopenic hyperleptinemic group. Fasting glucose levels in these groups were in a hypoglycemic range despite reduction in fasting plasma insulin levels; the insulin-glucose product of hyperleptinemic rats averaged 13% of normal controls and 4% of obese rats, evidence of exquisite insulin sensitivity. Clearly lipid content in target organs was correlated with this index of insulin sensitivity; r values ranged from 0.96 to 0.99 and were statistically significant (P < 0.05). However, because serum cortisol levels were not measured, a role of hypocortisolism in the insulin sensitivity of the hyperleptinemic rats cannot be excluded.
Pancreatic TG content was correlated with -cell function as
reflected by the fasting insulin level
(r = 0.97;
P = 0.037). All phases of insulin
secretion by the isolated perfused pancreas, basal and both glucose-
and arginine-stimulated insulin secretion, also varied directly with
the TG content of the pancreas.
The results are consistent with, but do not prove, the hypothesis that tissue TG content determines the level of insulin production and matches it to the level of insulin effectiveness, perhaps by providing an intracellular source of FFA retrieved from intracellular (TG) storage sites. Because the TG content of skeletal muscle and pancreas is also positively correlated, it is likely that it reflects generalized changes in tissue fat. Teleologically, this postulated linkage between insulin requirement and insulin secretion would serve to reduce the risk of diabetes in obesity and to minimize the possibility of hypoglycemia in undernutrition.
These observations follow more than three decades of research linking
fatty acid metabolism to carbohydrate metabolism and diabetes (2, 4, 5,
7, 8, 11, 13, 15, 16, 18, 20-22, 24-26, 29, 31-35, 38).
Most attention has been directed at the effects of plasma lipids in
reducing the insulin sensitivity of target tissues (3, 8, 13, 18, 23).
However, the initial reports of stimulatory effects of FFA on islets
(5, 11, 22) and, more recently, their inhibitory actions on -cell function (2, 7, 16, 38) have laid the groundwork for the present
studies. The main difference is the emphasis on tissue TG content
rather than perfusing levels of FFA and TG.
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ACKNOWLEDGEMENTS |
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We thank Kay McCorkle, who provided technical support, and Sharryn Harris, who provided secretarial assistance.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02700-37, the National Institutes of Health/Juvenile Diabetes Foundation Diabetes Interdisciplinary Research Program, and the Department of Veterans Affairs Institutional Research Support Grant SMI 821-109.
Address for reprint requests: R. H. Unger, Center for Diabetes Research, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8854.
Received 14 March 1997; accepted in final form 19 June 1997.
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