Defective fatty acid uptake modulates insulin responsiveness and metabolic responses to diet in CD36-null mice (original) (raw)
Plasma glucose and insulin and glucose tolerance tests. Compared with those in WT mice, fasting glucose and insulin levels were significantly lower in CD36-null mice. Data shown (Table 1) are for both sexes combined. However, the hypoinsulinemia was more pronounced in males than in females (insulin was 54% versus 33% lower, respectively). The index 1/insulin was higher in CD36-null mice, suggesting enhanced insulin sensitivity.
Fasting plasma levels of glucose and insulin, and indices for insulin sensitivity (1/insulin) and insulin secretion (insulinogenic index) for WT and CD36-null (CD36–/–) mice fed a chow diet
CD36-null mice (males and females) had a significantly enhanced ability to clear an intraperitoneal glucose load (Figure 1a). At 20 minutes after the load, plasma glucose reached a peak concentration, which was about 30% lower in the CD36-null mice. Area under the clearance curve (inset) was 22% lower in null mice as compared with age- and sex-matched WTs (P < 0.001). To determine whether the insulin response to glucose was altered, plasma insulin was measured before and at 30 and 60 minutes after the glucose load. Insulin levels after glucose administration (Figure 1b) and the insulinogenic index (Table 1), which reflects pancreatic β cell function (35, 36), were similar for WT and CD36-null mice.
Response of blood glucose (a) and insulin (b) to a glucose load in CD36-null (CD36–/–) and WT mice fed a standard chow diet. Twelve-week-old mice fasted for 16 hours were given glucose (1.5 mg/g) intraperitoneally. (a) Blood glucose was measured before and at 10, 20, 30, 45, 60, 120, and 180 minutes after glucose administration. Two-way repeated-measures ANOVA indicated a significant effect of the genotype (P < 0.05). The change of glucose response over time in each genotype (P < 0.05) and the interaction genotype × glucose are also significant (P < 0.05). *Significant differences (t test) between CD36–/– and WT at each time point. P < 0.01 for 20–60 minutes and P < 0.05 for 120 minutes. Inset shows areas under the glucose tolerance curves (AUC) (P < 0.01). (b) Plasma insulin levels were determined before the glucose injection and at 30 and 60 minutes after injection. *Insulin levels at 0 minutes are significantly lower in CD36–/– than in WT (P < 0.05). All data are means ± SEM with n = 12 (6 males and 6 females).
Tissue uptake of glucose in vivo. To identify the tissues responsible for the increased clearance of blood glucose, we measured glucose uptake in vivo using radioactive 18F-2-FDG. As shown in Figure 2a, uptake of FDG was increased in hearts (five times), diaphragms (three times), soleus (four times), gastrocnemius (three times), and hind limb muscle (two times) from CD36-null mice as compared with WT mice. Uptake was unaltered in adipose tissue and decreased in liver (Figure 2b). During the experiment, blood glucose level was constant in both WT and CD36-null mice. However, decay of 18F-2-FDG–specific activity in blood (Figure 2c) was faster in CD36-null mice, indicating that more endogenous glucose was being released into the blood to dilute specific activity of the FDG tracer. Since the mice were fasted overnight, the major glucose source would be the liver, suggesting increased hepatic glucose output.
18F-2-FDG uptake (a and b) and FDG blood clearance (c) in CD36-null and WT mice fed the chow diet. Mice were injected with 5 μCi of 18F-2-FDG in a lateral tail vein. Blood samples were collected at 2, 10, 20, 30, 45, 60, 90, and 120 minutes after injection and were tested for radioactivity and glucose content. At the end of the experiment, tissues were removed, weighed, and counted for 18F-2-FDG radioactivity; uptake rate (a and b) is expressed per gram wet tissue. (c) Decay of FDG-specific activity (cpm/μg), calculated as percent of specific activity at 2 minutes after injection, is shown. Data are means ± SEM; n = 6 per group. *P < 0.05, **P < 0.02.
In a subsequent experiment (data not shown), we tested the response of tissue glucose uptake to exogenous administration of a high dose of insulin (0.5 IU/kg body weight). Insulin produced severalfold increases in 18F-2-FDG uptake in heart, diaphragm, soleus muscle, gastrocnemius, and adipose tissues of WT mice. In CD36-null mice, the insulin-induced increases in FGD uptake were significantly smaller as compared with those in WT, for heart, diaphragm, and soleus. This was in line with the observation that oxidative muscles of CD36-null mice had optimal rates of glucose utilization at the endogenous insulin levels present in the fasted state.
Glucose utilization by incubated muscle in vitro. To directly examine insulin sensitivity, in vitro tests were carried out using the isolated soleus, a mostly oxidative muscle; and the EDL, a mostly glycolytic muscle. As shown in Table 2, glycogenesis by the soleus in response to a maximal concentration of insulin was superior for muscles from both fed (+ 316%) and fasted (+ 495%) CD36-null mice as compared with corresponding muscles from fed (+ 111%) and fasted (+ 59%) WT mice. In contrast, there was no significant alteration of the insulin response of the EDL. Glucose oxidation by soleus and EDL muscles from WT and CD36-null mice was similar and responded equally well to insulin (data not shown). Thus, in CD36-null mice, the effect of insulin on glycogenesis in the soleus but not in the EDL was enhanced, while insulin effect on glucose oxidation was not altered.
Effect of insulin on glycogenesis in incubated soleus and EDL muscles isolated from fed or fasted WT and CD36-null (CD36–/–) mice maintained on a chow diet
Muscle glycogen and TG content. Glycogen and TG contents were measured in heart and hind limb, which are typical of oxidative and glycolytic muscles, respectively. Liver content was determined since it plays an important role in the homeostasis of both plasma glucose and lipids. As shown in Table 3, CD36-null mice had lower glycogen levels in the liver and in heart and hind limb muscles.
Glycogen and TG contents in tissues of WT and CD36-null (CD36–/–) mice fed a chow diet
TG levels were decreased in muscle and heart of CD36-null mice (by 49% and 42%, respectively) but were increased twofold in the liver (Table 3).
Effects of high-fructose and high-fat diets We next examined whether the high glucose-to-FA utilization ratio created by CD36 deficiency increases susceptibility to metabolic pathology from diets with a high glycemic load while protecting from that induced by diets high in fat.
High-fructose diet. A diet rich in fructose induces a syndrome X–like metabolic phenotype in the SHR but not in the normal control rat (16, 17). In WT and CD36-null mice (Table 4), fructose feeding did not alter blood glucose but it increased blood insulin. The increase was small in WT mice (37 versus 28), while it was more than fourfold for CD36-null mice (50 versus 12). Blood levels of FAs and TGs (Table 5) were increased by the fructose diet in both groups but were higher in null as compared with WT mice on both the chow and fructose diets.
Plasma glucose and insulin of WT and CD36-null (CD36–/–) mice fed fructose- or fat-rich diets
Fasting plasma levels of TGs and FAs for WT and CD36–/– mice maintained on chow versus fructose- or fat-rich diets
Glucose tolerance of CD36-null mice was markedly impaired by the fructose diet, while no effect was observed in WT mice (Figure 3a). Area under the clearance curve (inset) was higher in null than in WT mice fed fructose (P < 0.01). The impairment in glucose tolerance in null mice was significant after 3 weeks on the diet (data not shown), although it was less pronounced than at the time the mice were killed at 12 weeks.
Response of blood glucose (a) and insulin (b) to a glucose load in CD36-null (CD36–/–) and WT mice fed a fructose-rich diet. Mice were fed a diet containing 60% fructose for 12 weeks. After a 16-hour fast, glucose clearance (a) and plasma insulin (b) were tested in response to a glucose load as described in the legend to Figure 1 and in Methods. Data are means ± SEM (n = 7). (a) Two-way repeated-measures ANOVA indicates that change of glucose response over time in each genotype and the interaction genotype × glucose are significant (P < 0.05). *Significant differences (t test) between CD36–/– and WT at each time point. P < 0.01 for 30, 45, and 60 minutes, and P < 0.05 for 20, 90, and 120 minutes. Inset shows areas under the glucose tolerance curves (P < 0.05). (b) *Insulin levels at 0 and 30 minutes are significantly higher in CD36–/– than in WT (P < 0.05).
CD36-null mice fed fructose, secreted more insulin in response to the glucose load than did WT mice (Figure 3b). Nulls also exhibited peripheral insulin resistance, since FDG uptake by heart and gastrocnemius muscles was significantly lower than uptake by corresponding muscles from WT mice (Figure 4). This contrasted with the situation on the chow diet (Figure 2), in which FDG uptake in null muscles was severalfold higher than in WT muscles. A comparison of FDG uptake in CD36-null hearts on chow (Figure 2) versus fructose (Figure 4) yields values of 240 versus 20 μg/h/g tissue, which represents greater than a 90% drop, while FDG uptake by WT hearts averaged 50 μg/h/g for both diets.
18F-2-FDG uptake by tissues of CD36-null and WT mice fed a high-fructose diet. 18F-2-FDG (5 μCi) was injected into a lateral tail vein of mice fasted for 16 hours that were maintained on a high-fructose diet for 12 weeks. FDG uptake was determined as described in the legend to Figure 2 and in Methods. Data are means ± SEM (n = 7). *P <0.05.
High-fat diet. We examined whether CD36 deficiency, which impairs FA utilization by muscle, would protect against peripheral insulin resistance consequent to increased consumption of dietary fat. A diet rich in safflower oil (37, 38) has been shown to be the most effective in impairing glucose tolerance in the C57BL/6J mouse line and was used for these studies. This diet induces peripheral insulin resistance without increasing blood insulin and TG, since it inhibits hepatic lipogenesis and VLDL production (38).
As shown in Table 4, blood levels of glucose and insulin were similar in WT and CD36-null mice fed the high-safflower diet, while TG and FA levels (Table 5) were higher in the CD36-null mice.
Glucose tolerance was impaired by safflower feeding to an equal extent in WT and CD36-null mice (Figure 5a), and the areas under the clearance curves were similar. However, clearance curves were significantly different for each group when compared with those of the mice on the chow diet (P < 0.05). The CD36-null mice fed safflower secreted significantly more insulin than the WT mice at 30 but not at 60 minutes after the glucose load, as shown in Figure 5b.
Response of blood glucose (a) and insulin (b) to a glucose load in CD36-null (CD36–/–) and WT mice fed a high-fat diet. Mice were fed a diet high in safflower oil for 16 weeks. After a 16-hour fast, glucose clearance (a) and plasma insulin (b) were tested in response to a glucose load as described in the legend to Figure 1 and in Methods. Data are means ± SEM (n = 7). (a) Two-way repeated-measures ANOVA indicates that the interaction genotype × glucose is not significantly different between WT and CD36-null mice. Inset shows that area under the glucose response curve for safflower-fed mice (right bars) was significantly different (P < 0.05) from that for mice fed chow (left bars). Black bars, WT mice; white bars, CD36-null mice. *P <0.05. (b) Insulin response to the glucose load in WT and CD36–/– mice. *Insulin levels at 30 minutes are significantly higher in CD36–/– than in WT (P < 0.05).
Tissue TGs in mice fed fructose or high-fat diets. For the high-fructose diet (Table 6), TG levels in livers from CD36-null mice were higher than those in WT livers, while levels in heart and skeletal muscle were similar for both groups. When data are compared with those for mice on the chow diet, TG levels were increased by fructose feeding in all tissues. The relative increase was most marked in muscle (11- and 26-fold for WT and null, respectively).
TG content in tissues of WT and CD36-null (CD36–/–) mice fed either fructose or high-fat diets
For the high-safflower diet (Table 6), TG levels in heart and hind limbs were about 25% lower for CD36-null than for WT mice; however, the differences did not reach statistical significance (n = 6 per group). Levels were similar in the liver. When data are compared with those for mice on the chow diet, liver TGs for both mouse groups were lower on the safflower diet, which has been reported to inhibit hepatic lipogenesis (38).
Insulin sensitivity of glucose utilization in muscles of mice fed the safflower diet was examined in the incubated soleus in vitro. As shown in Table 7, glucose incorporation into glycogen exhibited similar responsiveness to a maximal concentration of insulin, when muscles from WT and CD36-null mice are compared. In contrast, glucose oxidation by the soleus was almost unresponsive to insulin in WT muscles, while it was still responsive in CD36-deficient muscles.
Glucose metabolism in soleus and EDL muscles from WT and CD36-null mice fed the high-fat diet