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Endocrinology, doi:10.1210/en.2003-1543
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Endocrinology Vol. 145, No. 10 4667-4676
Copyright © 2004 by The Endocrine Society

Muscle-Specific Overexpression of CD36 Reverses the Insulin Resistance and Diabetes of MKR Mice

Lisa Héron-Milhavet, Martin Haluzik, Shoshana Yakar, Oksana Gavrilova, Stephanie Pack, William C. Jou, Azeddine Ibrahimi, Hyunsook Kim, Desmond Hunt, Daphne Yau, Zeenat Asghar, Jamie Joseph, Michael B. Wheeler, Nada A. Abumrad and Derek LeRoith

Diabetes Branch (L.H.-M., M.H., S.Y., O.G., S.P., W.C.J., H.K., D.H., D.L.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; Mouse Metabolic Core Facility (O.G., S.P., W.C.J.), Université Sidi Mohammed Ben Abdellah (A.I.), Centre d’Etudes Universitaires de Taza, Taza 1223, Morocco; Endocrine and Diabetes Research Group (D.Y., Z.A., J.J., M.B.W.), Department of Physiology, University of Toronto, Toronto, Canada M5S 1AB; and Department of Physiology and Biophysics (N.A.A.), State University of New York, Stony Brook, New York 11794

Address all correspondence and requests for reprints to: Derek LeRoith, M.D., Ph.D., Diabetes Branch, NIDDK, Room 8D12, Building 10, National Institutes of Health, Bethesda, Maryland 20892-1758. E-mail: Derek{at}helix.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin resistance is one of the primary characteristics of type 2 diabetes. Mice overexpressing a dominant-negative IGF-I receptor specifically in muscle (MKR mice) demonstrate severe insulin resistance with high levels of serum and tissue lipids and eventually develop type 2 diabetes at 5–6 wk of age. To determine whether lipotoxicity plays a role in the progression of the disease, we crossed MKR mice with mice overexpressing a fatty acid translocase, CD36, in skeletal muscle. The double-transgenic MKR/CD36 mice showed normalization of the hyperglycemia and the hyperinsulinemia as well as a marked improvement in liver insulin sensitivity. The MKR/CD36 mice also exhibited normal rates of fatty acid oxidation in skeletal muscle when compared with the decreased rate of fatty acid oxidation in MKR. With the reduction in insulin resistance, ß-cell function returned to normal. These and other results suggest that the insulin resistance in the MKR mice is associated with increased muscle triglycerides levels and that whole-body insulin resistance can be, at least partially, reversed in association with a reduction in muscle triglycerides levels, although the mechanisms are yet to be determined.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PATHOPHYSIOLOGY OF type 2 diabetes mellitus involves defects in tissue sensitivity to insulin and perturbations in insulin secretion. Under normal conditions, insulin binds to insulin receptors (IRs) on target cells, resulting in cellular cascades that promote intracellular glucose transport and metabolism (1). Insulin resistance in target tissues (muscle and liver) is observed early in the disease process and is rapidly followed by decreased insulin secretion as a result of progressive pancreatic ß-cell dysfunction. This combination leads to overt diabetes with fasting and postprandial hyperglycemia. Insulin resistance is a common defect in type 2 diabetes, with the liver continuing to produce glucose and the uptake of glucose into muscle being impaired. A number of animal models have been developed using gene-targeting technology to study the molecular basis of diabetes. IR gene disruption in mice leads to severe diabetes and death from ketoacidosis in the immediate postnatal period (2, 3). Moreover, deletion of the IR gene in pancreatic ß-cells resulted in mice with only mild diabetes (4, 5). Muscle-specific IR knockout mice (MIRKO) show moderate insulin resistance and a mild metabolic disorder but do not develop type 2 diabetes or show any major metabolic changes (6). When GLUT4 is disrupted selectively in muscle, the mice show severe insulin resistance and glucose intolerance from an early age (7). Taken together, these studies suggest that type 2 diabetes has a heterogeneous etiology. Recently, the MKR transgenic mouse, overexpressing a dominant-negative IGF-I receptor (IGF-IR) specifically in muscle was characterized as a model of severe insulin resistance and type 2 diabetes (8). MKR mice exhibit insulin resistance initially in muscle followed by liver and fat. These changes are associated with increases in serum fatty acids (FAs) and triglyceride levels as well as in triglyceride deposits in liver and muscle tissues. The accumulation of triglycerides in muscle and in liver may alter the response of these tissues to insulin, and this effect has been invoked as an additional factor in the pathogenesis of type 2 diabetes.

FAs are the predominant fuel used by skeletal muscle. It has been shown that increased FA availability alters glucose metabolism by affecting glucose transport and glucose metabolism in insulin-sensitive cells. This would result in increased fat metabolism and inhibition of carbohydrate metabolism (reviewed in Refs.9 and 10). Increased muscle triglycerides have been correlated with reduced insulin action (11). Indeed, several clinical investigations have demonstrated a potential link between insulin-resistant glucose metabolism and muscle triglyceride levels (reviewed in Ref.12). This link appears to reflect the tissue’s metabolic balance between FA uptake and oxidation. Resistance would be associated with conditions where FA uptake exceeds oxidative capacity (13). Impaired muscle FA oxidation with type 2 diabetes (14) would lead to accumulation of intermediates of fatty acid metabolism, such as fatty acyl-CoA, which impair insulin signaling. In line with this, exercise, which increases muscle FA uptake and oxidation improves insulin sensitivity of diabetic subjects (13).

An important site for regulation of muscle lipid metabolism is at the level of FA uptake at the sarcolemmal membrane. Recent work documented a major role of the membrane protein CD36 (14) (also called FAT for FA translocase) in facilitating FA uptake and use by muscle (15, 16). Mice with muscle-specific CD36 overexpression (15) have enhanced FA oxidation in response to stimulation/contraction, whereas CD36 deficiency is associated with defective FA uptake and oxidation in muscle and adipose tissues (13, 15). The importance of CD36 and of optimal muscle FA use for insulin action was illustrated by the studies of Hevener et al. (16). Treatment of rats with the insulin-sensitizing drug pioglitazone, prevented the drop in muscle CD36 and the induction of insulin resistance that are induced by lipid administration. More directly, the presence of a functional CD36 was shown to be essential for the actions of pioglitazone (17) and roziglitazone (18) to improve insulin sensitivity in general and that of muscle in particular. These observations support the interpretation that CD36-facilitated FA uptake could be an important determinant of insulin sensitivity.

To explore whether abnormalities of fatty acid metabolism are important contributors to the appearance of type 2 diabetes after insulin resistance in the MKR mice, we have crossed MKR and muscle-specific CD36-overexpressing mice to generate double-transgenic MKR/CD36 animals. We hypothesized that impaired FA oxidation may be responsible for the increased triglyceride content in the muscle of MKR mice. In addition, increased FA flux to the liver as a result of poor peripheral FA use would compromise hepatic insulin sensitivity. We reasoned that muscle overexpression of CD36 should enhance peripheral FA uptake and oxidation, decrease hepatic FA flux, and possibly improve insulin sensitivity in the MKR mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and genotyping
Mice were kept under 12-h light, 12-h dark cycles and fed standard diets. To generate the double-transgenic MKR/CD36 line, homozygous MKR mice were crossed with hemizygous mice possessing CD36 overexpression in muscle. Both lines are derived from identical genetic backgrounds (pure FVB/N) and have both been previously characterized (8, 15). For genotyping, we established a method by PCR [as opposed to the Southern blotting used before (8)]. MKR mice were genotyped by PCR using oligos specifically spanning the human IGF-IR (direct primer, 5'-gagtggccattaaaacagtgaacgag-3', and reverse primer, 5'-tcttgctcaggcttggaggtgctagg-3'). CD36 mice were genotyped using specific oligos amplifying the rat CD36 transgene (direct primer, 5'-cgatcggaactgtgggctcattac-3', and reverse primer, 5'-ctctgtgccaacagacag-3'). Double-transgenic MKR/CD36 mice were genotyped using both PCR settings. All studies were conducted on mice hemizygous for each transgene. Control males consisted of age-matched wild-type (WT) mice with the same genetic background. The Animal Care and Use Committee of the National Institute of Diabetes and Digestive and Kidney Diseases approved all procedures and studies were conducted in accordance with National Institutes of Health guidelines.

Body composition
Body composition (fat distribution) was measured in awake mice using an NMR analyzer (Bruker Minispec mq10, Bruker Optics Inc., Billerica, MA).

Analytical assays and metabolic rate
Blood glucose levels were determined from the tail vein using a glucometer (One Touch, Lifescan, Milpitas, CA). Blood was collected from the retroorbital sinus of male mice under nonfasting conditions for insulin determination and under fasting conditions for FA and triglyceride levels determination. Serum insulin levels were measured by RIA (Linco Research Inc., St. Charles, MO). Serum FA and triglyceride levels were analyzed in fasted mice using a commercial FA kit (Roche, Indianapolis, IN) and the GPO-Trinder kit (Sigma Chemical Co., St. Louis, MO), respectively. Liver and muscle triglyceride levels were measured in fasted mice as previously described (19). Briefly, after tissue extraction with chloroform/methanol, measurement of triglyceride levels was performed by ethanolic KOH hydrolysis and radiometric assays to assess glycerol levels. Metabolic rate was measured in fed male mice 11–12 wk of age by indirect calorimetry using the Oxymax system (Columbus Instruments, Columbus, OH) as described by Gavrilova et al. (20). Data were collected for 24 h at room temperature (24 C) and for 24 h at thermoneutral temperature (30 C). Data are expressed as the average of 24 h and normalized to total body weight. Serum leptin, adiponectin, and resistin were measured using RIA kits from Linco. TNF{alpha} and IL-6 were measured using immunoassays from ALPCO Diagnostics (Windham, NH).

Hyperinsulinemic-euglycemic clamp
The clamp protocols are based on those of Kim and Shulman and were performed as described (21, 22). Briefly, the mice were fasted for approximately 16 h, and the insulin infusion was as a bolus of 300 mU/kg over 3 min followed by an infusion of 2.5 mU/kg·min (Humulin R, Eli Lilly, Indianapolis, IN). The period of tracer equilibration was 90 min, and the steady-state specific activity was calculated as an average of four samples between 90 and 120 min.

Pancreatic perfusion studies
Nonfasted mice were anesthetized ip with a combination of 20 mg/kg xylazine and 100 mg/kg ketamine. Pancreata were isolated and perfused as described previously (23), a protocol originally based upon that of Grodsky et al. (24). Briefly, the pancreas was isolated, via ligature, from the stomach, spleen, and duodenum in vivo. After a 20-min preperfusion with 1.4 mM glucose to allow equilibration of insulin secretion, the pancreas was perfused with 1.4 mM glucose for 4 min, 16.7 mM glucose for 20 min, 1.4 mM glucose for 15 min, and 20 mM L-arginine plus 16.7 mM glucose for 10 min at a rate of 1ml/min. Samples were collected from the portal vein and assayed for insulin by RIA.

Islet morphometric analysis
Six hours before removal of the pancreas, mice were injected with 5-bromo-2'-deoxyuridine (BrdU) (100 mg/kg ip; Sigma). Upon removal, pancreata were fixed in 4% paraformaldehyde and paraffin embedded 12 h after fixation. Sections approximately 4 µm thick were double immunostained, first for insulin using a rabbit anti-guinea pig primary antibody (Dako, Mississauga, Canada) and a biotinylated goat antirabbit secondary antibody (Vector Laboratories Inc., Burlington, Canada) and subsequently for BrdU using a mouse anti-BrdU primary antibody (Clone IU4, Caltag Laboratories, Burlingame, CA) and a biotinylated horse antimouse secondary antibody (Vector). The same USA Level 2 Detection System (Signet Laboratories, Dedham, MA) was used for both insulin and BrdU. Hematoxylin was used for counterstaining. Images of each section were acquired using an Olympus BX60 microscope connected to a Photometrics CoolSNAP color camera (Roper Scientifc, Trenton, NJ). Each slide was covered systematically, and 12 fields at a final magnification of x10 were analyzed for each pancreas. ß-Cell area (insulin-positive area) was measured, and the numbers of insulin-positive and BrdU-positive nuclei were counted. ß-Cell mass was determined by calculating the ratio of ß-cell area to total pancreatic area and multiplying this value by the pancreatic weight (25). The percentage of proliferating ß-cells was calculated as the percentage of insulin-positive cells staining positive for BrdU.

FA oxidation on isolated soleus muscle
All experiments were performed in Krebs-Henseleit buffer (KHB) (118.5 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 0.5 mM EDTA, and 25 mM NaHCO3, supplemented with 5 mM D-glucose) (15). All BSA and palmitate/BSA complexes used were dialyzed extensively against KHB, and all buffers were continuously gassed with 95% O2/5% CO2. Soleus muscles, including tendons, were rapidly and carefully excised from 12-wk-old animals and placed in capped 10-ml vials containing 5 ml KHB supplemented with 2% BSA. After a 20-min incubation in a shaking water bath (150 strokes/min), muscles were incubated with 0.2 mM palmitate and 0.45 µCi/ml (1-14C) palmitate (ICN, Irvine, CA) complexed to 2% BSA. Antibiotic solution (Sigma) was added to prevent bacterial growth. Incubations were carried out at 30 C for 30 min in vials that were sealed with rubber stoppers (Kontes, Vineland, NJ) to maintain the interior of the vial at 95% O2/5% CO2. The incubation medium was quickly transferred to new capped vials equipped with a center well containing 200 µl of ethanolamine/ethylene glycol (1:2, vol/vol). Perchloric acid was added through the cap to a final concentration of 0.6 M and vials incubated overnight with gentle shaking. The amount of CO2 produced was determined by liquid scintillation counting of the ethanolamine/ethylene glycol mixture.

Glucose uptake in isolated muscle
Mice were anesthetized with an ip injection of pentobarbital (40 mg/kg body weight ip) after an overnight fast. Soleus and extensor digitorum longus muscles were isolated and removed intact. After a 1-h preequilibration in KHB buffer, muscles were incubated for 10 min in the absence or presence of insulin (14 nM/2000 µU/ml). Thereafter, muscles were incubated for 20 min in the presence of [C14]2-deoxyglucose (2 µCi/ml) and [H3]mannitol (0.3 µCi/ml). After this 20-min incubation, muscles were quickly blotted on filter paper and frozen in liquid nitrogen. Muscles were digested for 1 h at 50 C in KOH 1 N and then counted for radioactive tracer incorporation (8).

Statistical analysis
The data are expressed as average ± SEM. Statistically significant differences between genotypes were determined using a one-factor ANOVA followed by a t test, except for perfusion and islet morphology data for which the Bonferroni multiple comparisons test was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Muscle-specific CD36 overexpression in MKR mice improves hyperglycemia and hyperinsulinemia without altering serum free FA and triglyceride levels
MKR mice were crossed with mice overexpressing the CD36 FA translocase specifically in skeletal muscle. When followed from 3–15 wk, the double-transgenic MKR/CD36 mice did not show any significant differences in body weight when compared with their MKR or CD36 littermates (data not shown). However, MKR, CD36, and double-transgenic mice all showed a 10–20% reduction in body weight (P < 0.05) up to 4 wk compared with the WT mice. After 4 wk of age, the body weight of MKR/CD36 was normal compared with the WT mice, whereas the MKR mice showed less than 10% reduction. Total adipose mass (percentage of body weight) as well as fat distribution were measured by NMR and are shown in Table 1Go. Adipose tissue mass of MKR mice was significantly lower than that of WT and MKR/CD36 mice. Adipose mass in MKR/CD36 mice was restored almost back to WT levels. Metabolic rate was measured in all groups of mice (Table 1Go). MKR mice had slightly higher metabolic rate than the WT mice, whereas the rate for CD36 and MKR/CD36 mice was comparable to the WT mice. MKR/CD36 mice had significantly lower respiratory exchange ratio, suggesting that they oxidized relatively more FA. Finally, we did not observe any major differences in the activity between these various groups of mice. To explore the consequence of the muscle-specific overexpression of CD36 in MKR mice, we measured metabolic responses in WT, MKR, CD36, and MKR/CD36 mice. At 5 wk of age, fed blood glucose levels (Fig. 1AGo) as well as fed serum insulin levels (Fig. 1BGo) were elevated in MKR mice, which are both insulin resistant and diabetic at this age. In contrast, when CD36 was overexpressed in MKR muscle, the resulting double-transgenic mice showed normal fed blood glucose levels (Fig. 1AGo) and fed serum insulin levels (Fig. 1BGo). Weekly measurements revealed that these normal levels observed in MKR/CD36 mice were maintained from 5–15 wk of age (data not shown). Serum FA and triglycerides were significantly higher in MKR mice and CD36 mice (Fig. 1Go, C and D). This difference in the findings of serum FA and triglycerides in CD36 mice in two separate studies (Ref.15 vs. Fig. 1AGo) may be explained by the fact that younger animals (males only) were analyzed in this study, as opposed to the 16- to 20-wk-old mice (males and females) studied previously (15). Another factor potentially contributing to this difference is the significant gender differences in the metabolic effects of abnormal CD36 expression in transgenic mice, and CD36 expression levels may differ between males and females (Ibrahimi, A., and N. A. Abumrad, unpublished observations). Interestingly, we did not observe any reduction in serum FA or serum triglycerides when CD36 was overexpressed in MKR mice (Fig. 1Go, C and D). When the mice were followed to 12 wk of age, similar observations were made for the various genotypes (data not shown). A similar profile was obtained for insulin, glucose, and lipid levels in both fed and fasted animals (data not shown). Thus, these results indicate that serum FA and triglyceride levels may not be the only factors implicated in the restoration of normal levels in blood glucose and serum insulin in the double-transgenic MKR/CD36 mice. We then examined whether changes in adipocytokines could be involved. Leptin, TNF-{alpha}, resistin, and IL-6 levels did not show major differences between all four groups (data not shown). Adiponectin levels rose significantly in MKR/CD36 mice compared with MKR mice (11.61 ± 0.7 vs. 7.84 ± 0.4; P < 0.01).


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  		TABLE 1. Metabolic rate and total body composition

 


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  		FIG. 1. Metabolic parameters in serum. Glucose (A) and insulin (B) levels were measured in the serum of 5-wk-old fed mice. FAs (C) and triglycerides (TG) (D) were measured in the serum of 5-wk-old 12-h-fasted animals. Blood was collected from the tail vein directly into non-EDTA-coated capillary tubes and centrifuged to separate the serum, which was used to assay triglycerides, FAs, and insulin. Glucose values were obtained with one drop of blood from the tail vein applied to a glucometer strip. All serum parameters were similar for 5- to 20-wk-old mice. Values shown are the mean ± SEM; *, significant differences (P < 0.05) from WT mice (n = 5–8 in each group).

 
Hyperinsulinemic-euglycemic clamp shows that muscle-specific CD36 overexpression improves insulin sensitivity in MKR mice
To determine whether the improvements in blood glucose levels in MKR/CD36 mice compared with MKR mice were mediated by improved insulin sensitivity, we performed hyperinsulinemic-euglycemic clamp studies in 6- to 8-wk-old mice. After an overnight fast, MKR mice were markedly hyperglycemic, whereas MKR/CD36 mice had only mildly increased blood glucose compared with CD36 and WT mice. Fasting insulin levels were 2- to 3-fold higher in MKR mice compared with other groups (Table 2Go). Glucose infusion rate, a measure of whole-body insulin sensitivity, was decreased in MKR relative to WT and CD36 mice and improved in MKR/CD36 mice (Fig. 2AGo). Clamp endogenous glucose production, a measurement of liver insulin sensitivity, was significantly (~4.5-fold) increased in MKR mice, indicating severe liver insulin resistance (Fig. 2BGo). Clamp endogenous glucose production in MKR/CD36 and CD36 mice was comparable with that of the control group (Fig. 2BGo). Whole-body glucose uptake under hyperinsulinemic-euglycemic clamp conditions is chiefly a measure of insulin-induced muscle glucose uptake, at least in humans and rats (26). This was dramatically (~2.5-fold) reduced in MKR mice relative to WT and CD36 groups, respectively (Fig. 2CGo). Thus, muscle insulin sensitivity is markedly impaired in MKR mice. The MKR/CD36 mice showed some improvements in whole-body insulin sensitivity with whole-body glucose uptake being significantly higher than in MKR mice but still significantly lower than in WT and CD36 mice, respectively. It is important to note that the same results were obtained with 15- to 16-wk-old mice (data not shown). Muscle glucose uptake measured at the end of the hyperinsulinemic-euglycemic clamp tended to be lower in MKR mice and showed a very minimal trend toward improvement in MKR/CD36 mice (Fig. 2DGo). Therefore, in vitro studies were performed (see below). In summary, hyperinsulinemic-euglycemic data suggest that muscle CD36 overexpression improves whole-body to some extent and strongly improves liver insulin sensitivity in MKR mice.


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  		TABLE 2. Metabolic parameters during basal and hyperinsulinemic-euglycemic clamp periods

 


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  		FIG. 2. Metabolic parameters during hyperinsulinemic-euglycemic clamp periods in mice. Six- to 8-wk-old male WT, MKR, CD36, and MKR/CD36 mice were tested during hyperinsulinemic-euglycemic clamp analysis. Various parameters were determined and are shown in Table 2Go. Glucose infusion rate (A), clamp endogenous glucose production (EGP) (B), whole-body glucose uptake (C), and muscle glucose uptake (D) are shown. *, Significant differences from WT mice (P < 0.05); **, statistically significant differences from MKR mice (P < 0.05). Data are expressed as mean ± SEM (n = 6 in each group).

 
Glucose uptake in isolated muscle
Soleus and extensor digitorum longus muscles were isolated and removed intact and basal and insulin-stimulated glucose uptake studied. In both muscle types, insulin significantly stimulated glucose uptake in WT and CD36 mice (P < 0.05; Fig. 3Go). The effect of insulin in MKR and MKR/CD36 mice was markedly reduced or absent as we have previously shown for MKR mice (8).



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  		FIG. 3. Insulin-stimulated glucose uptake in incubated (ex vivo) skeletal muscle. Soleus (A) and extensor digitorum longus (EDL) (B) muscles were studied in vitro as described in Materials and Methods. Basal and insulin-stimulated glucose uptake was measured in six to 10 mice per group and the results expressed as mean ± SEM; *, significantly different from basal (P < 0.05).

 
Muscle, but not liver, triglyceride contents are reduced in MKR mice overexpressing muscle-specific CD36
Muscle and liver triglyceride content was measured to determine whether reduced tissue triglyceride levels are associated with liver insulin sensitivity improvement. Muscle triglyceride levels were significantly reduced by 30% in MKR/CD36 mice compared with MKR mice (Fig. 4AGo). Unexpectedly, liver triglyceride levels were not elevated in the MKR mice as previously described in adult mice (8), and no significant differences were observed between MKR/CD36 and MKR mice, although there was a tendency toward elevated triglyceride content (Fig. 4BGo). In the original study of the MKR mice, we used a colorimetric detection technique to determine tissue triglycerides, whereas in the present study, we used a radioactive detection technique, which might explain the difference in the results obtained. To confirm our findings, liver cross-sections from all four genotypes were stained with O-Red-Oil, and no differences in fat content were detected (data not shown).



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  		FIG. 4. Muscle and liver triglyceride (TG) content. Triglyceride content was assessed in muscle (A) and liver (B). Tissue triglyceride content was determined for all mice genotypes and is shown for 5-wk-old mice after an overnight fast. Similar results were obtained in mice from 5–20 wk of age. *, Significant differences between MKR and WT mice (P < 0.05); **, significant differences between MKR/CD36 and MKR (P < 0.05). Data are expressed as an average ± SEM (n = 5 in each group).

 
Liver glycogen content is decreased in MKR mice overexpressing muscle-specific CD36
Morphologically, no notable differences were observed between the livers of adult (8 wk old) MKR/CD36 and MKR mice (as well as CD36 and WT), as determined by hematoxylin and eosin staining (Fig. 5Go, top). However, staining for periodic acid Schiff (PAS), revealed a marked decrease in glycogen deposits in MKR/CD36 mice compared with MKR mice, which demonstrated increased glycogen stores compared with WT and CD36 mice (Fig. 5Go, bottom).



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  		FIG. 5. Staining of liver sections. Hematoxylin and eosin (H&E) staining (top) and glycogen (PAS) staining (bottom) of liver sections from WT (A), MKR (B), CD36 (C), and MKR/CD36 (D) 8-wk-old mice. Magnification, x10.

 
Muscle FA oxidation rate is restored to WT levels in MKR mice overexpressing muscle-specific CD36
Previous studies showed that CD36 mice display an enhanced capacity for FA oxidation in response to contraction (15). We show in this study that FA (palmitate) oxidation, measured (using palmitate oxidation) in isolated soleus muscle, was decreased in the MKR mice compared with WT mice and CD36 mice (Fig. 6Go). On the other hand, the MKR/CD36 mice showed an increase in FA oxidation when compared with MKR mice with levels similar to WT mice (Fig. 6Go). Thus, CD36 expression in MKR mice muscle restored muscle FA oxidation rates to those of WT mice.



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  		FIG. 6. Palmitate oxidation by soleus muscle. Palmitate oxidation rates by isolated soleus muscle from fed WT, MKR, CD36, and MKR/CD36 mice were determined at a FA/BSA ratio of 0.67 (six in each group). Data are the mean ± SEM; *, significantly different from WT, from CD36, and from MKR/CD36 (P < 0.05).

 
Insulin secretion and islet morphological analysis
The insulin response of the in situ-perfused pancreas was measured to assess the dynamics of ß-cell glucose responsiveness. A substimulatory concentration of 1.4 mM glucose was first introduced to the pancreas to establish a basal rate of secretion, followed by stimulation with 16.7 mM glucose to assess first and second phases of secretion. MKR mice exhibited an elevated rate of basal secretion (Fig. 7AGo) with the integrated response approximately 6.5-fold greater than CD36 mice (Table 3Go). The MKR/CD36 double-transgenic mice exhibited a significantly reduced rate of basal secretion compared with MKR mice (Fig. 7AGo), demonstrated by an integrated response only marginally higher than in CD36 mice (Table 3Go). Upon stimulation with glucose, first and second phases were seen in CD36 mice, whereas MKR mice exhibited extremely high rates of secretion and a response profile in which separate phases were difficult to distinguish (Fig. 7AGo). MKR/CD36 mice, however, displayed a distinct first phase of secretion with a greater peak response than CD36 mice, followed by a second-phase response similar to CD36 mice and significantly less than MKR mice (Fig. 7AGo). The integrated response of the double-transgenic mice under stimulatory conditions was comparable to that of CD36 mice, whereas that of MKR mice was approximately 6.5-fold greater than CD36 and MKR/CD36 mice (Table 3Go). After allowing secretion rates to return to basal levels, a combined 16.7 mM glucose and 20 mM arginine pulse was introduced to assess functional ß-cell capacity. The arginine-glucose integrated response of MKR mice was more than 4-fold greater than CD36 mice. The integrated response of MKR/CD36 mice was half that of MKR mice but more than twice the response of CD36 mice as shown in Table 3Go, although this latter difference was not statistically significant.



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  		FIG. 7. A, Profiles of insulin secretion under basal (1.4 mM glucose) and stimulatory (16.7 mM glucose) conditions. Glucose concentrations are indicated for MKR/CD36 ({blacktriangleup}), MKR ({cjs2108}), and CD36 ({circ}). B, ß-Cell mass data are expressed as the mean ± SEM (n = 4 for WT and MKR, and n = 3 for CD36 and MKR/CD36). C, ß-Cell proliferation, or percentage of BrdU-positive ß-cells, is shown. ***, P < 0.001 vs. WT; #, P < 0.01 vs. MKR.

 

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  		TABLE 3. Integrated perfusion response for basal, glucose-stimulated, and arginine + glucose-stimulated insulin secretion

 
To gain insight into the results obtained from the perfused pancreas model, ß-cell mass and percent proliferation were assessed in pancreatic sections. In MKR mice, ß-cell mass was increased approximately 5-fold over WT and CD36 mice. In contrast, MKR/CD36 mice displayed an almost 50% reduction in ß-cell mass when compared with MKR mice (Fig. 7BGo), although this difference was not statistically significant. However, this was still more than 2-fold greater than WT and CD36 mice, which showed comparable ß-cell mass. Similar results were seen upon examination of ß-cell proliferation. Proliferation was quantified as the percentage of insulin-positive cells also staining positive for BrdU. MKR mice exhibited an approximately 3.5-fold increase in proliferation compared with WT mice and an approximately 3-fold increase compared with CD36 mice, whereas MKR/CD36 mice displayed an almost 50% reduction in the amount of proliferation relative to MKR mice, although, again, this reduction did not achieve statistical significance (Fig. 7CGo). These results suggest that the expanded ß-cell mass in MKR mice is permissive for the exaggerated insulin secretion seen during pancreas perfusion and that the expansion is mediated, at least in part, by an increased number of proliferating ß-cells. Consistent with this, the normalized secretion in MKR/CD36 mice is associated with only a moderate expansion in mass and an attenuated incidence of proliferation, thus demonstrating the relationship between size of the ß-cell mass and the amount of insulin secretion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The MKR mouse model was developed using a dominant-negative IGF-IR (KR-IGF-IR) specifically targeted to skeletal muscle. This resulted in the formation of hybrid receptors between the mutant and the endogenous IGF-I and insulin receptors, thereby markedly affecting their normal function leading to severe muscle insulin resistance (8). The MKR mice lacked IGF-I-induced glucose uptake in muscle and exhibited a major reduction in insulin-induced glucose uptake into skeletal muscle (8). This led to insulin resistance in liver and fat, as demonstrated by the hyperinsulinemic-euglycemic clamp assay (8). Pancreatic ß-cell dysfunction developed relatively quickly and resulted in the development of type 2 diabetes. The development of diabetes in MKR mice was associated with elevated serum FAs and triglycerides as well as with accumulation of triglycerides in skeletal muscle. This indicated that FA use by muscle may be impaired leading to conditions that promote insulin resistance. Depressed FA oxidation has been reported in the muscle of patients with type 2 diabetes (14).

Because our objective was to determine whether improving muscle FA use by constitutive expression of CD36 would be associated with an improvement of insulin sensitivity in MKR mice, we crossed them with mice overexpressing CD36 specifically in muscle (15) to generate double-transgenic MKR/CD36 mice. CD36 is a FA translocase that facilitates FA uptake by muscle and other tissues, and its translocation from intracellular compartments to the plasma membrane (27) is involved in acute regulation of FA uptake during muscle contraction. Insulin was also shown to induce membrane recruitment of CD36 (28). These effects are consistent with the demonstrated roles of CD36 in promoting both esterification and oxidation (Fig. 6Go) of exogenous FA by muscle tissues. Other observations with cultured muscle cells overexpressing CD36 support the interpretation that CD36 may target the FA to a metabolically active TAG pool and that high turnover of this pool limits lipid accumulation (Bastie, C., T. Hajri, P. A. Grimaldi, V. Drover, and N. A. Abumrad, unpublished observations).

The double-transgenic MKR/CD36 exhibited normal levels of blood glucose and serum insulin compared with the high levels of the MKR mice. However, this was not accompanied by decreased serum lipid levels, because serum FA and triglycerides remained at the same high levels observed in the MKR mice. These results support the interpretation that high circulating FA levels do not directly cause insulin resistance. Using the hyperinsulinemic-euglycemic clamp technique, we demonstrated that normalization of glucose homeostasis was associated with improvements in whole-body insulin sensitivity and in liver insulin responsiveness as determined by suppression of hepatic glucose production. However, the marked improvement in whole-body glucose disposal observed in MKR/CD36 mice could not be related to an amelioration of muscle insulin sensitivity because this did not seem to change, as determined both in vivo and in vitro. This could reflect the fact that whole-body glucose disposal measured during the clamp detects changes in insulin-stimulated glucose uptake by organs such as heart, fat, and liver (8) and not only skeletal muscle. Thus, the whole body glucose uptake measured during the hyperinsulinemic clamp may not always be a reflection of muscle glucose uptake only, such as is seen in humans. Interestingly, of all the adipokines measured, only adiponectin showed a marked improvement in the NKR/CD36 mice when compared with MKR. It is conceivable that some of the improvement in insulin action on hepatic glucose production may have been the result of this change in adiponectin.

Plasma FAs and insulin sensitivity are usually negatively correlated (29). Indeed, it is widely accepted that elevated FA levels can exert a deleterious effect on the overall action of insulin, and this has been demonstrated both in animals and humans (30, 31, 32). It has been recently shown that fenofibrates induce insulin sensitivity by lowering both serum and im lipid levels (33). The negative correlation is especially strong for im triglycerides (34, 35), which are likely to reflect the state of FA use by muscle. Specifically, it has been noted that insulin resistance in rodents and humans is associated with increased triglyceride deposition within skeletal muscle cells (36, 37). In the present study, we show that the enhanced FA oxidation by muscles of MKR/CD36 mice was reflected in decreased muscle triglyceride levels when compared with the levels in the MKR mice. This improvement in muscle FA use was not associated with enhanced insulin-stimulated glucose uptake in muscle as would be expected because the IGF-I and insulin receptors are genetically blocked. However, it was associated with a marked improvement in whole-body insulin responsiveness. To our knowledge, these data are the first to suggest that impaired muscle FA use can contribute to inhibition of insulin signaling in other tissues by some as yet undetermined signaling molecule(s). One possibility how this may occur is through modulation of the glycerol/glucose cycle. For example, using 13C-mass isotopomer distribution analysis, it has been shown that in the peroxisome proliferator-activated receptor-{alpha} null mouse, which exhibits defective FA use in muscle, the contribution of plasma glycerol to hepatic gluconeogenesis is increased to approximately 50% from the 10% measured in the control mouse. Also, hepatic glucose production from glycerol was not suppressed by feeding, implying hepatic insulin resistance (38). Thus, by analogy, it is conceivable that CD36 expression in muscles of MKR/CD36 mice by enhancing FA turnover (both esterification and oxidation of FAs) by this tissue may contribute to decreased glycerol release and to a diminished operation of the glycerol/glucose cycle. This would be reflected in a larger proportion of hepatic glucose production being susceptible to suppression by insulin.

Our findings also suggest the possibility that the IGF signaling pathway may play a role in regulating lipid metabolism in muscle. Previous studies have examined the effects of IGF-I on blood free FAs and reported decreases at high IGF-I concentrations (39) with resistance of diabetic subjects to these effects. Similarly, transgenic mice overexpressing GH and having high levels of IGF-I exhibited low circulating free FAs despite the increased lipolysis indicating an enhanced ability for FA oxidation. The fact that triglyceride secretion by the liver was not increased suggested that the increased FA use was by the muscle (40). Our data suggest that it would be important to examine the role of the IGF-I pathway in regulating muscle FA uptake and oxidation and possibly CD36 expression levels and localization. Thus, our results suggest that the overexpression of CD36 in the muscle of MKR mice improves insulin sensitivity, possibly by reducing im lipid content via stimulation of ß-oxidation in muscles despite the lack of reducing serum lipids.

This study also shows that overexpressing CD36 in the skeletal muscle of the MKR mice led to a normalization of the insulin secretion that was shown to be exaggerated in the MKR mice. We suspect this is because of the improved insulin sensitivity. This would alleviate the demand for insulin, leading to a decrease in its secretion as well as a decrease in ß-cell mass because mass is a major factor determining the amount of insulin that can be secreted.

Interestingly, no increase in liver triglycerides was observed in MKR mice maintained on a regular chow diet, and we did not observe changes in liver histology between the different genotypes. On the contrary, when the livers were stained with PAS to detect glycogen storage, MKR mice showed a significant increase in glycogen storage as opposed to MKR/CD36 where glycogen levels were restored to normal (WT) levels. This may be related to the restoration of normoglycemia in MKR/CD36 mice. Studies in vitro (41) and in vivo (42) have shown that hyperglycemia per se induces a translocation of hepatic glucokinase to the cytosol, thereby increasing its activity. An increased glucokinase activity regulates hepatic glucose production and glycogen synthase activity (43) and increases glycogen synthesis.

In conclusion, this study demonstrates that increased muscle triglyceride content is related to insulin resistance because by lowering muscle triglyceride levels without affecting serum levels, we were able to improve whole-body insulin sensitivity. Although we have not measured the effect of CD36 expression on MKR phenotype before 5 wk of age, it is possible that the effects are induced at an earlier developmental stage. By overexpressing CD36, a FA translocase in muscle of MKR mice, we could reverse their type 2 diabetes phenotype as early as 5 wk old and at least till the age of 5 months.


   Acknowledgments
 
We are thankful to Bethel Stannard for technical support as well as Dr. P. Pennisi and Dr. H. Zhao for helpful discussions.


   Footnotes
 
This study was supported by a 2003 Diabetes Young Investigator grant (to L.H.-M.) from the Association Française des Diabétiques, Paris, France, and operating grants from the Canadian Diabetes Association and the Canadian Institutes of Health (CIHR MOP-12898) to M.B.W. D.Y. and Z.A. are supported by studentships from the Banting and Best Diabetes Center. The study was also supported in part by a grant from the American Diabetes Association to D.L.

Present address for L.H.-M.: Cell Biology Unit, IGH, CNRS UPR1142, Montpellier, France.

Present address for M.H.: Faculty of Medicine, Charles University, Prague, Czech Republic.

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; FA, fatty acid; IGF-IR; IGF-I receptor; IR, insulin receptor; KHB, Krebs-Henseleit buffer; WT, wild-type.

Received November 12, 2003.

Accepted for publication June 15, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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