Reduced beta -cell function contributes to impaired glucose tolerance in dogs made obese by high-fat feeding

Karl J. Kaiyala1, Ronald L. Prigeon2, Steven E. Kahn2, Stephen C. Woods3, Daniel Porte Jr.2, and Michael W. Schwartz2

1 Department of Psychology and 2 Department of Medicine, University of Washington School of Medicine, Seattle 98195; and Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108, and 3 Department of Psychiatry, University of Cincinnati, Cincinnati, Ohio 45267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The ability to increase beta -cell function in the face of reduced insulin sensitivity is essential for normal glucose tolerance. Because high-fat feeding reduces both insulin sensitivity and glucose tolerance, we hypothesized that it also reduces beta -cell compensation. To test this hypothesis, we used intravenous glucose tolerance testing with minimal model analysis to measure glucose tolerance (Kg), insulin sensitivity (SI), and the acute insulin response to glucose (AIRg) in nine dogs fed a chow diet and again after 7 wk of high-fat feeding. Additionally, we measured the effect of consuming each diet on 24-h profiles of insulin and glucose. After high-fat feeding, SI decreased by 57% (P = 0.003) but AIRg was unchanged. This absence of beta -cell compensation to insulin resistance contributed to a 41% reduction of Kg (P = 0.003) and abolished the normal hyperbolic relationship between AIRg and SI observed at baseline. High-fat feeding also elicited a 44% lower 24-h insulin level (P = 0.004) in association with an 8% reduction of glucose (P = 0.0003). We conclude that high-fat feeding causes insulin resistance that is not compensated for by increased insulin secretion and that this contributes to the development of glucose intolerance. These effects of high-fat feeding may be especially deleterious to individuals predisposed to type 2 diabetes mellitus.

glucose tolerance; glucose effectiveness; insulin secretion; diabetes; obesity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

the regulation of blood glucose levels by insulin depends on insulin sensitivity and the amount of insulin delivered to target tissues. Because a variety of acute and chronic factors can substantially alter insulin sensitivity, the ability of the beta -cell to respond with compensatory changes of insulin secretion should play an important role in the maintenance of normal glucose tolerance. Thus it was hypothesized almost two decades ago that insulin sensitivity and pancreatic beta -cell function are coupled so that a change of insulin sensitivity engenders a proportionate reciprocal change in glucose-stimulated insulin secretion in healthy individuals (5, 8). Accordingly, in the face of reduced insulin sensitivity, proportionate beta -cell compensation acts to maintain normal glucose homeostasis (29, 30), whereas impaired beta -cell compensation contributes to the pathophysiology of glucose intolerance and type 2 diabetes mellitus (5, 24, 32, 42).

Empirical confirmation of reciprocity between levels of insulin sensitivity and beta -cell function derive in large part from cross-sectional studies of subjects with apparently normal glucose tolerance but wide variation in adiposity and insulin sensitivity. As predicted by Bergman et al. in 1981 (8), the mathematical model best describing the relationship between insulin sensitivity, SI, quantified by the minimal model method (7), and beta -cell function, measured as the acute insulin response to glucose (AIRg), was hyperbolic such that their product, SI · AIRg [the disposition index (8)], equals a constant (29). Consequently, normal glucose tolerance was preserved even among very obese insulin-resistant individuals as long as beta -cell function remained intact (29).

Although the beta -cell response to altered insulin sensitivity has been examined in only a handful of longitudinal investigations involving a limited number of subjects, their findings are largely consistent with a dynamic reciprocal relationship between insulin sensitivity and beta -cell function. In a study of older adults who responded to prolonged exercise training with decreased adiposity and improved insulin sensitivity, the disposition index and intravenous glucose tolerance both remained unchanged due to a reciprocal decrease of AIRg (28). Conversely, reduced SI associated with late pregnancy is accompanied by a compensatory increase of AIRg (17), a response that is required if gestational diabetes mellitus is to be avoided. Similarly, experimental insulin resistance induced by administration of nicotinic acid to human subjects for 2 wk was accompanied by an increase of AIRg. Because the magnitude of this response was insufficient to fully offset the reduction of insulin sensitivity, however, glucose tolerance decreased (27). This outcome underscores the importance of the beta -cell response to insulin resistance in preserving normal glucose tolerance.

Existing data suggest that high-fat feeding alters the relationship between insulin sensitivity and beta -cell function. High levels of dietary fat intake are associated with impaired glucose tolerance (14, 16, 34, 35, 45, 53, 54) and insulin resistance (16, 33, 36, 44, 51, 52). Collectively, these data suggest that high-fat feeding may induce insulin resistance without proportionate beta -cell compensation, a possibility of particular concern to those at risk for the development of type 2 diabetes. To test this hypothesis, we used tolbutamide-modified frequently sampled intravenous glucose tolerance tests (FSIGT) analyzed by the minimal model (7) to quantify insulin sensitivity and to measure beta -cell function as AIRg in nine dogs before and after 7 wk of ad libitum high-fat feeding. Additionally, 24-h measurements of glucose and insulin levels were obtained to determine the effect of the high-fat diet on circulating insulin and glucose levels under free living conditions in animals with ad libitum access to food.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Studies were performed in nine adult male mongrel dogs weighing 31-40 kg at baseline. Each dog served as its own control in a repeated-measures design. All studies were approved by the Animal Care Committee at the Puget Sound Veterans Affair Health Care System.

Study protocol. After catheter implantation (see Surgical methods), animals were provided ad libitum access to standard laboratory chow for at least 5 wk and body weight was recorded at 3-day intervals to document weight stability at the time of the baseline study. During this time, dogs were accustomed to the Pavlov sling used during the FSIGT protocol (see FSIGT procedure). Animals underwent body composition testing, 24-h studies for insulin and glucose levels, and the FSIGT protocol at baseline while on the standard chow diet and again after a 7-wk period on a high-fat diet consumed ad libitum.

Surgical methods. Chronic indwelling arterial catheters were placed under general anesthesia. Tygon (Akron, OH) tubing (0.07 in. OD by 0.04 in. ID) was inserted via the omocervical artery and advanced under guidance of fluoroscopy until the catheter tip resided within the aorta at the level of the diaphragm. The proximal end of the catheter was externalized at the back of the neck via an arterial valve connector (Harvard Apparatus, Holliston, MA) to enable blood sampling. Between studies, patency was maintained with heparin. Dogs were allowed to recover for at least 5 wk before being studied.

Diets. The baseline diet consisted of standard dog chow (Harlan Teklad, Madison, WI) providing 17% of calories as fat. The caloric density of this diet is 3.47 kcal/g. The high-fat diet provides 80% of calories as fat and was developed in this laboratory. This diet entailed two feedings per day, morning and afternoon. The afternoon component consisted of a homogeneous mixture of 454 g of lard (Armour Foods, Omaha, NE) and 748 g of canned dog food. Variety was increased by use of two brands of dog food in three flavors (chicken, beef, and turkey; Blue Mountain Special Menu, Lehigh Valley, PA, and Friskies Alpo Prime Cuts, Glendale, CA). Additionally, 71 g of chicken or beef baby food (Heinz, Pittsburgh, PA) were added to each 1,202 g serving of the lard-dog food mixture. This mixture was estimated to contain 4.13 kcal/g. The morning feeding component consisted of peanut butter coated (57 g)-portions of lard (57 g) with oil-based tuna fish centers (19 g). Each portion was estimated to represent 1,150 kcal. Sufficient amounts of both diets were provided to ensure ad libitum access to food at all times. All animals remained healthy througout the study period. Estimates of caloric intake were based on the weight of food consumed.

Quantification of body composition. Body composition was estimated using the isotope dilution technique (23). A stock solution of sterile isotonic saline containing 2 µCi/ml of 3H2O (24 ml) was administered intravenously as a bolus. The syringe was weighed before and after injection for precise determination of the volume administered. Blood samples were collected before and 3 h after the injection for determination of plasma radioactivity (dpm). For each body composition determination, a single assay utilizing a liquid-scintillation counter (Packwood Tri-Carb 1,600 TR, Meriden, CT) quantified dpm in each of three 0.3-ml samples of baseline and equilibrium plasma and in three 0.3-ml samples of scintillation fluid containing 1 µl of radioactive stock. The difference in mean dpm/ml between pre- and postinjection plasma samples was taken as the equilibrium concentration of the 3H2O, from which lean body mass was calculated (23). Based on the study by Widdowson and Dickerson (56), we assumed that body water represents 74% of the lean body mass, that total body water equals 95.2% of the distribution volume of 3H2O, and that plasma equals 94% water. Percent body fat was calculated as 100 · fat mass-1 · total body mass-1.

Measurement of 24-h insulin and glucose levels. Around-the-clock sampling of arterial plasma was performed in the animal's home cage during ad libitum provision of food and water. Dogs were not fasted overnight for these studies, which began at 9:30 AM. A total of 44 blood samples were obtained. Samples were drawn every 30 min for the first 15 h, every hour for the next 4 h, and at 30-min intervals for the final 5 h. Plasma glucose was quantified as the mean of the 44 values obtained. Plasma insulin was quantified as the time-weighted mean of the 44 values by using the trapezoidal rule to compute the area under the 24-h (1,440 min) insulin curve and dividing by 1,440.

FSIGT procedure. Tolbutamide-modified FSIGT studies were conducted at 10:30 AM following an overnight fast. At t = 0 min, a glucose bolus (0.3 g/kg of the baseline body weight) was infused over 40 s into a forelimb vein. At t = 20 min, tolbutamide was administered (3 mg/kg iv of the baseline body weight) to improve the precision of parameter estimates derived from minimal model analysis (4). Arterial blood was collected at t = -20, -10, -1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 19, 22, 23, 24, 25, 27, 30, 35, 40, 52, 60, 70, 80, 90, 100, 120, 140, 160, and 180 min.

Determination of glucose tolerance. The glucose disappearance constant (Kg) was used as a measure of intravenous glucose tolerance. Kg is an estimate of the disappearance rate (%/min) of plasma glucose based on the slope of the line derived from least-squares regression of the natural logarithm of plasma glucose on time from min 10 through 19 during the FSIGT.

Quantification of insulin sensitivity and glucose effectiveness. The minimal model method (5-7), developed originally in dogs, was used to analyze FSIGT for quantification of the insulin sensitivity index (SI) and glucose effectiveness at basal insulin (Sg). SI measures the ability of a given amount of insulin to enhance glucose disappearance and Sg measures the ability of glucose to enhance its own disposal at basal insulin. Sg is comprised of an insulin-dependent component (SI · basal insulin) and a noninsulin-dependent component (glucose effectiveness at zero insulin, GEZI) (30). Thus GEZI is derived from Sg as GEZI = Sg - (SI · basal insulin). Studies were analyzed by a co-author (R. L. Prigeon) blind to the condition (baseline or high-fat diet) under which the study was done.

Measurement of pancreatic beta -cell function and disposition index. The AIRg was used as a measure of beta -cell function in response to a glucose challenge. AIRg was determined as the mean incremental plasma insulin level in samples collected at t = 2, 3, 4, 5, 6, 8, and 10 min following intravenous glucose administration during the FSIGT. The product of SI · AIRg, the disposition index (8, 9), was computed as an index of insulin-mediated glucose disposal during a FSIGT.

Quantification of relationship between insulin sensitivity and beta -cell function. The relationship between insulin sensitivity measured as SI and beta -cell function measured as AIRg was analyzed via the approach previously used in human subjects for this purpose (29). The concept underlying this analysis is that AIRg · SI = a constant, which is a rectangular hyperbolic function. After expressing AIRg as a function of SI and taking the natural logarithm (ln) of both sides of the equation, ln(AIRg) = -1.0 · ln(SI) + a constant (29). The logarithmic model is favorable in terms of the homoscedasticity of variance assumption of linear regression (2). A slope of -1.0 implies that AIRg · SI = a constant, as predicted for a hyperbolic relationship between insulin sensitivity and beta -cell function.

Insulin and glucose assays. Blood samples were immediately placed on ice in EDTA and later centrifuged for storage of plasma at -20°C. Plasma insulin-like immunoreactivity was assayed using a modification of the double-antibody method (37). Plasma glucose was analyzed by the Trinder method (3).

Statistical analyses. Paired t-tests were used to analyze the change (Delta ) of body composition, plasma glucose levels, insulin levels, Kg, SI, Sg, GEZI, and AIRg (computed as value after high-fat feeding minus value at baseline). Data are summarized as the means ± SE. If not normally distributed, data were log transformed before statistical analysis. Classical least-squares linear regression was used to evaluate the associations between Kg and the disposition index, Kg, and GEZI, Delta Kg and Delta GEZI, and Delta Kg and Delta disposition index. In accordance with the approach taken by Kahn et al. (29), we regressed both ln(AIRg) on ln(SI), and ln(fasting insulin) on ln(SI), using a method [orthogonal distance regression (12)] that takes into account error in both the x and y variables. This method provides an unbiased estimate of the slope of the relationship between two variables that are each measured with error, since classical linear regression (which assumes that x is measured without error) tends to underestimate the slope of the relationship between x and y variables in this setting (2). Significance was established at a two-sided alpha -level of 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Body composition. As shown in Table 1, high-fat feeding for 7 wk significantly increased mean values of body weight (Delta  = 6.6 ± 1.9 kg, P = 0.009), percent body fat (Delta  = 12.4 ± 3.3%fat units, P = 0.005), and fat mass (Delta  = 6.8 ± 1.9 kg, P = 0.006). Two of the nine dogs, however, did not increase in adiposity and a third had only a slight increase. Lean body mass was unchanged by the high-fat diet (Delta  = -0.83 ± 1.60 kg, P = 0.60).

                              
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Table 1.   Body composition and fasting plasma glucose and insulin values at baseline and after 7 wk of high-fat feeding

Fasting plasma insulin and glucose levels. The mean fasting insulin concentration (Fig. 1A) increased by 50% from 56.00 ± 6.40 to 84.00 ± 7.87 pM (P = 0.008) after 7 wk on the high-fat diet. This increase occurred in association with a 6.2% increase in the mean fasting plasma glucose level (Fig. 1B) from 4.68 ± 0.14 to 4.97 ± 0.16 mM (P = 0.05).


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Fig. 1.   Effects of high-fat feeding on fasting plasma insulin levels (A), fasting plasma glucose levels (B), mean 24-h insulin levels (C), and mean 24-h glucose levels (D). black-diamond , Mean values. Consumption of high-fat diet for 7 wk significantly increased fasting levels of plasma insulin and glucose but significantly reduced the mean 24-h insulin and glucose levels.

Insulin and glucose levels during 24-h studies. When measured after 7-wk of high-fat feeding, there was a significant 44% decrease in the mean time-weighted average 24-h insulin levels generated during ad libitum food intake. During the chow and high-fat feeding studies, the mean time-weighted average insulin levels (Fig. 1C) were 253.47 ± 32.64 and 140.28 ± 22.22 pM (P = 0.004), respectively. This decrease in circulating insulin levels occurred in association with a significant (7.8%) decrease in the 24-h mean glucose level (Fig. 1D) from 4.86 ± 0.05 to 4.48 ± 0.08 mM (P = 0.0003).

Caloric intake during 24-h studies. Mean caloric intakes during the 24-h chow and high-fat feeding studies were not significantly different (control 3,798 ± 481 kcal, high-fat 4,260 ± 636 kcal, P = 0.59).

Glucose tolerance. Mean glucose tolerance, quantified as Kg, decreased markedly from 3.61 ± 0.38 to 2.12 ± 0.16%/min (-41.3%, P = 0.003, Fig. 2A) after 7 wk of high-fat feeding. Decreases in glucose tolerance were not significantly associated with Delta body weight (r = +0.40, P = 0.28), Delta fat mass (r = +0.50, P = 0.17), or Delta % fat units (r = +0.46, P = 0.21).


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Fig. 2.   Effects of high-fat feeding on glucose disappearance constant (Kg; A), acute insulin response to glucose (AIRg; B), insulin sensitivity index (SI; C), and glucose effectiveness at basal insulin (Sg; D). black-diamond , Mean values. Consumption of high-fat diet for 7 wk significantly reduced Kg, SI, and Sg, whereas AIRg was unchanged.

Acute insulin response to glucose. The mean AIRg decreased nonsignificantly from 393.2 ± 62.3 to 336.0 ± 27.5 pM (P = 0.37, Fig. 2B).

Insulin sensitivity and glucose effectiveness. Insulin sensitivity was markedly decreased after high-fat feeding in all dogs. The mean value of SI decreased from 13.55 ± 1.74 to 5.87 ± 1.21 · 10-5 · min-1 · pM-1, a reduction of 56.6% (P = 0.003, Fig. 2C). Changes in SI were not significantly correlated with Delta fat mass (r = -0.40, P = 0.29), Delta body weight (r = -0.44, P = 0.22), or Delta % fat units (r = -0.48, P = 0.19). Similarly, percent changes in SI were not significantly associated with percent changes in fat mass (r = -0.44, P = 0.23) or percent changes in body weight (r = -0.46, P = 0.22). The effect of high-fat feeding to reduce insulin sensitivity therefore was not accounted for by changes in adiposity.

High-fat feeding also significantly reduced Sg (Fig. 2D) from 3.88 ± 0.43 to 2.49 ± 0.22 · 10-2 · min-1, a 35.5% decline (P = 0.03). Similarly, glucose effectiveness independent of basal insulin (GEZI) was reduced by 35.2% from 3.08 ± 0.40 to 1.99 ± 0.26 · 10-2 · min-1, although this change did not achieve statistical significance (P = 0.06).

Relationship between insulin levels and insulin sensitivity. To determine if AIRg and SI are reciprocally related in dogs consuming the chow diet, we modeled the relationship at baseline between ln(AIRg) and ln(SI) using orthogonal regression (see MATERIALS AND METHODS). The resulting model was described by the equation ln(AIRg) = -1.05 · ln(SI- 3.53 (P = 0.011). Consistent with the hypothesis that AIRg · SI = a constant, the slope estimate of this equation is very close to -1.0 (P = 0.88 for comparison with -1.0). Consequently, the model was refitted with a slope of -1.0 to compute an intercept term corresponding to a rectangular hyperbolic relationship between AIRg and SI. This resulted in the equation ln(AIRg) = -1.0 · ln(SI- 3.10, which after exponentiation translates to SI · AIRg = 4.50 · 10-2 · min-1 (Fig. 3A). Supporting the validity of this regression-based model, the actual geometric and arithmetic means for the SI · AIRg product (the disposition index) were 4.50 and 4.72 · 10-2 · min-1, respectively. During chow feeding therefore a significant inverse association existed between a measure of first-phase insulin secretion and insulin sensitivity such that the product of AIRg · SI (the disposition index) tended to remain constant across a broad range of insulin sensitivity.


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Fig. 3.   Relationship between AIRg and SI at baseline (A) and after high-fat feeding (B). Curved line in A represents hyperbola fit to baseline data (black-triangle) in accordance with procedure used by Kahn et al. (29) in humans. Hyperbola is described by the equation SI · AIRg = 4.5 · 10-2 · min-1 (r = 0.79, P = 0.011). In B this baseline hyperbola is recapitulated to demonstrate effect of high-fat feeding to cause relative impairment of the insulin response ().

In contrast, the association between ln(AIRg) and ln(SI) during high-fat feeding was not significant (P = 0.90), and AIRg clearly failed to increase in proportion to the decline in SI (Fig. 3B). Thus, after high-fat feeding, a hyperbolic relationship between AIRg and SI was not observed, and AIRg was low for the prevailing level of insulin sensitivity. Consistent with this interpretation, the mean value of the disposition index was reduced from 4.72 ± 0.47 to 1.93 ± 0.39 · 10-2 · min-1 in response to high-fat feeding, a 59.1% decrease (P = 0.001). High-fat feeding therefore resulted in a state of reduced insulin sensitivity that was not compensated for by increased beta -cell function measured as AIRg.

Similar results were obtained from the relationships between fasting insulin and SI. The relationship at baseline between ln(fasting insulin) and ln(SI) during chow feeding was significant and was modeled by the equation ln(fasting insulin) = -0.62 · ln(SI- 1.62 (P = 0.03). The slope estimate of -0.62 is not significantly different from a value of -1.0 (P = 0.13). After high-fat feeding, however, the relationship between ln(fasting insulin) and ln(SI) was not significant (P = 0.45). At baseline therefore the relationship between fasting insulin and SI was suggestive of a rectangular hyperbolic function as has been found in human subjects (see materials and methods), but this relationship was no longer detected after 7 wk of high-fat feeding.

Interaction of insulin sensitivity and insulin secretion as determinants of glucose tolerance. Figure 4A depicts the associations between Kg and the disposition index both at baseline and after high-fat feeding. At baseline, Kg was strongly associated with the disposition index (r = 0.89, P = 0.001), which accounted for 80% of the variance in Kg. After high-fat feeding, the association between Kg and the disposition index was similar but did not reach statistical significance (r = 0.63, P = 0.07). Based on a linear regression analysis of the combined data obtained from the same animals on both diets, the disposition index accounted for 79.4% of the variance in Kg (P = 0.001). Similarly, the association between Delta Kg and Delta disposition index was significant (Fig. 4B, r = 0.68, P = 0.04), indicating that a significant proportion of the change in Kg induced by high-fat feeding could be accounted for by changes in the disposition index.


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Fig. 4.   A: relationship between Kg and disposition index at baseline () and after high-fat feeding (). Disposition index accounts for 79% of variance in Kg (P = 0.001 based on 7 df) based on a regression model using the data set comprised of both chow and high-fat fed values (represented by line shown). B: linear regression of association between high-fat diet-induced changes of Kg and changes of disposition index (r = 0.68, P = 0.04).

However, unlike previous work in humans (30), we found no evidence of an association between Kg and GEZI, either at baseline (r = 0.19, P = 0.63) or at followup (r = -0.26, P = 0.51). Similarly, the association between Delta Kg and Delta GEZI was not statistically significant (r = 0.12, P = 0.75). Hence, differences in glucose tolerance, both at baseline across dogs and after high-fat feeding within dogs, were largely accounted for by differences in the disposition index but not in glucose effectiveness.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several studies (28, 29) support the hypothesis (5, 8) that altered insulin sensitivity induces a proportionate reciprocal change in beta -cell function that serves to minimize changes of glucose tolerance. In the case of high-fat feeding, however, reduced glucose tolerance develops (14, 16, 34, 35, 45, 53, 54) in association with insulin resistance (16, 36, 44, 51, 52) and weight gain (31, 44, 48, 49). We therefore hypothesized that high-fat diets impede the increase of beta -cell function that normally accompanies reduced insulin sensitivity. In direct support of this hypothesis, we found that dogs consuming a high-fat diet for 7 wk had a 57% decrease of SI that clearly was not accompanied by an increase of mean AIRg and consequently, glucose tolerance was significantly reduced. Moreover, we demonstrated that the reciprocal relationship between beta -cell function and insulin sensitivity that exists during chow feeding is lost after animals are switched to a high-fat diet.

The 57% reduction of mean SI observed in our study is consistent with a recent report that 6 wk of high-fat feeding in dogs was associated with a 60% reduction in whole body insulin-mediated glucose uptake as measured by the hyperinsulinemic euglycemic clamp technique (44). Although the results of human studies are more variable regarding the effect of high-fat feeding on insulin sensitivity (reviewed in Ref. 50), they are generally consistent with data reported here. Using the minimal model approach, Chen et al. (16) found that SI was significantly reduced after only 3-5 days of high-fat feeding (55% of kcal) in young, nonobese subjects, whereas Swinburn et al. (53) reported that 14 days of high-fat feeding (50% of kcal) did not significantly reduce SI in human subjects that were already obese and insulin resistant. This discrepancy likely reflects the difficulty inherent in detecting a decrease in SI among the insulin-resistant subjects in the latter study (53). In rodents, increased dietary fat intake is clearly associated with reduced insulin sensitivity (reviewed in Ref. 50), even when the caloric content of the high-fat diet is matched to that of the control diet (51). Collectively, these findings indicate that high-fat diets may induce insulin resistance even in the absence of increased body adiposity. Consistent with this possibility is our finding that SI was reduced in each of the three dogs that exhibited little or no change of adiposity in response to high-fat feeding. Additionally, the observation that changes of SI were not strongly associated with changes of body weight (r = -0.44) or fat mass (r = -0.40) suggests that increased body adiposity was not the dominant mechanism by which the high-fat diet reduced insulin sensitivity. It is possible, however, that increases of central adiposity, a measure strongly linked to insulin resistance (18, 19) but not evaluated here, contributed to the decrease of insulin sensitivity induced by the high-fat diet in our study, even among animals that did not exhibit a detectable increase of overall fat mass.

Although we did not assess the mechanism by which high-fat feeding reduces insulin sensitivity, a large portion of literature (reviewed in Ref. 10) suggests that this effect may involve a glucose-fatty acid cycle in which increased lipid oxidation in insulin-sensitive tissues inhibits the action of insulin to stimulate cellular glucose uptake, a mechanism first proposed by Randle in 1963 (reviewed in Ref. 43). Support for the concept that high-fat diets increase lipid oxidation is provided by the finding that high-fat feeding significantly reduces the 24-h respiratory quotient (1, 22, 55), such that daily fat oxidation is increased as much as twofold (22). This shift in fuel metabolism elevates mitochondrial acetyl-CoA and increases citrate formation, which inhibits key glycolytic enzymes (43) and increases the flux of fructose 6-phosphate into the hexosamine pathway, which is hypothesized to generate a signal that induces insulin resistance (20). Several additional mechanisms by which dietary fat may impair insulin sensitivity have also been proposed, including reduced expression of insulin-sensitive glucose transporters (26), a decrease in the active form of glycogen synthase in skeletal muscle (21), reduced insulin binding to its receptor (38), and impaired activation of receptor tyrosine kinase (38).

Consistent with the results of Swinburn et al. (53), we observed that high-fat feeding was also associated with a significant 36% reduction in Sg. This finding reflects a reduction in the insulin-independent component of glucose effectiveness (GEZI), because GEZI was diminished by 35% (P = 0.06) after high-fat feeding. However, our data do not support the conclusion of Swinburn et al. that the predominant effect of increased fat intake on glucose disposal is to impair the insulin-independent component of glucose uptake. To the contrary, our results suggest that glucose intolerance induced by high-fat feeding is the result of defects in insulin sensitivity and beta -cell function, as well as reduced insulin-independent glucose uptake.

In contrast to the reliable decreases observed in insulin sensitivity and glucose effectiveness, high-fat feeding had mixed effects on the three different insulin responses measured in this study. Specifically, the fasting plasma insulin level was increased by 50%, whereas the 24-h plasma insulin profile was reduced by 44% and the measure of first-phase insulin secretion (AIRg) was essentially unchanged. Increased fasting insulin is therefore unreliable as a surrogate measure of glucose-stimulated and feeding-induced insulin secretion. Whereas the increase of fasting insulin could reflect a compensatory response to the decrease of insulin sensitivity, other factors may also have contributed to this response. Specifically, the rate of insulin secretion rises sharply with increases of circulating glucose within the physiological range (15). Elevated fasting glucose levels during high-fat feeding therefore may have contributed to the increase of fasting insulin. Decreased insulin clearance (25) is a second mechanism that could have raised fasting insulin levels independent of beta -cell compensation. Hence, the extent to which the increase of fasting insulin observed after high-fat feeding reflects a compensatory response of the beta -cell to insulin resistance is uncertain. In contrast, AIRg provides a more direct measure of beta -cell function, because the glucose stimulus is constant across conditions and because the insulin secretion rate greatly predominates over insulin clearance as a determinant of the acute-phase insulin level achieved during a glucose challenge.

The substantial (44%) reduction in the 24-h circulating insulin level during ad libitum intake of the high-fat diet in comparison to chow feeding was unexpected, given the concomitant reduction of SI. This observation raises the possibility that the insulin resistance detected under fasting conditions was not present during ad libitium consumption of the high-fat diet. However, we favor the alternative explanation that beta -cell stimulation was reduced during consumption of the high-fat diet, because dietary lipids are weak stimulants of insulin secretion (47). The significant reduction in 24-h plasma glucose levels reflects the reduced carbohydrate stimulus to beta -cells associated with consumption of the high-fat diet.

Consistent with data obtained in humans (29), we detected a hyperbolic relationship between beta -cell function and insulin sensitivity, described by the equation AIRg · SI = a constant, during consumption of the chow diet. This relationship is a quantitative reflection of the hypothesized mechanism by which glucose tolerance is preserved under conditions associated with a decline in insulin sensitivity (5, 27, 29, 42). After high-fat feeding, however, the relationship between insulin response and insulin sensitivity was markedly changed (Fig. 3). Specifically, the observed 57% decrease of SI was actually accompanied by a small, albeit nonsignificant, decrease of AIRg. This failure of AIRg to increase in the face of reduced SI provides direct evidence that high-fat feeding interferes with beta -cell adaptations that would normally compensate for insulin resistance, as proposed previously by Swinburn et al. (53). Chen et al. (16) also provided data in humans concordant with the possibility of adverse effects of elevated fat intake on beta -cell function. Our data therefore provide further support for the concept that high-fat feeding reduces the ability of the beta -cell to compensate for insulin resistance.

The mechanisms underlying these effects on insulin responses may involve chronic exposure of beta -cells to elevated free fatty acid (FFA) levels, although the effect of FFAs on insulin secretion is complex. Thus long-term, but not short-term (39), elevations of FFAs can impair glucose-stimulated insulin secretion, as demonstrated by both in vitro rodent studies (46, 57, 58) and an in vivo human study in which a two- to threefold increase of FFAs maintained for 24 h induced a 50% decrease in AIRg (39). Mechanisms that may explain this effect include glucose-FFA substrate competition within beta -cells (58) and depletion of insulin stores caused by an effect of FFAs to stimulate basal insulin secretion without a matching increase of insulin synthesis (13).

On the other hand, acute exposure of beta -cells to FFAs increases insulin release from cultured islets, and a 48-h experimental increase of FFAs potentiated the insulin secretion rate in a human study conducted by Boden et al. (11). Because plasma glucose was clamped at a high level (~9 mM) in this study, however, both insulin secretion and insulin synthesis may have been augmented. Thus the effect of elevated FFAs on glucose-stimulated insulin responses may depend on the ambient glucose level (40) such that prolonged elevation of FFAs without a concomitant elevation of glucose favors reduced beta -cell function. The absence of beta -cell compensation measured in terms of AIRg in our study may therefore reflect an effect of the high-fat diet to cause insulin resistance without concomitant hyperglycemia, as suggested by the reduction in 24-h glucose levels observed during consumption of the high-fat diet.

If the lack of increased beta -cell function observed in our study stemmed from low carbohydrate ingestion, it could represent an adaptive, carbohydrate-sparing response analogous to that seen during starvation rather than a maladaptive impairment of beta -cell function. Low glucose levels in starvation, analogous to our high-fat diet, occur in conjunction with increased lipid oxidation and insulin resistance and reduced beta -cell sensitivity to glucose (15). However, in previous work AIRg was halved by a long-term fatty acid infusion despite a progressive increase of glucose to almost 7 mM (39). Similarly, reducing circulating fatty acids potentiated AIRg in normoglycemic first-degree relatives of people with type 2 diabetes (41). Thus chronically elevated fatty acid concentrations can suppress beta -cell function under conditions that do not involve decreased circulating glucose levels. Our finding that beta -cell function did not increase in the face of reduced insulin sensitivity caused by high-fat feeding may therefore reflect the combined effects of low plasma glucose and high fatty acid levels, and additional studies are warranted to assess this interaction and the extent to which it pertains to the consumption of more typical high-fat diets.

Bergman et al. (8) predicted that development of reduced insulin sensitivity without a quantitatively equivalent compensatory beta -cell response would reduce glucose tolerance. Consistent with this proposal, mean Kg in our study was reduced by 41% after high-fat feeding, and regression analysis indicated that 46% of the variability in the change in Kg was explained by the decrease of the disposition index. Whether adaptive or maladaptive, therefore, this observation attaches pathogenic significance to the failure to increase AIRg during high-fat feeding and suggests that impaired insulin-stimulated glucose disposal is a major determinant of the decline of glucose tolerance

We conclude that a high-fat diet reduces the ability of the beta -cell to compensate for insulin resistance and that this leads to glucose intolerance. The concept that high-fat diets cause insulin resistance and oppose the compensatory response of the beta -cell has potentially important implications for the pathogenesis of impaired glucose tolerance and type 2 diabetes.


    ACKNOWLEDGEMENTS

We are grateful for the expert technical assistance provided by Rix Keuster, Hong Nguyen, Ruth Hollingworth, and Vicki Hoagland.


    FOOTNOTES

This work was supported by the National Institutes of Health Grants DK-17047, DK-35816, DK-12829, DK-52989, NS-32273, and DE-07132.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. W. Schwartz, Metabolism (151), Veterans Affairs Puget Sound Health Care System, 1660 S. Columbian Way, Seattle, WA 98108 (E-mail: mschwart{at}u.washington.edu).

Received 18 December 1998; accepted in final form 26 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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