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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 18, 2003 as doi:10.1096/fj.03-0081fje. |
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Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, and Center of Animal Biotechnology and Gene Therapy, Universitat Autònoma de Barcelona, E-08193-Bellaterra, Spain
2Correspondence: E-mail: fatima.bosch{at}uab.es
SPECIFIC AIMS
In type 2 diabetes, glucose phosphorylation, a regulatory step in glucose utilization by skeletal muscle, is impaired. Since glucokinase (GK) expression in skeletal muscle of transgenic mice increases glucose phosphorylation, the aim of this study was to examine whether such mice counteract the obesity and insulin resistance induced by a high-fat diet.
PRINCIPAL FINDINGS
1. Transgenic mice expressing GK in skeletal muscle counteracted the obesity induced by a high-fat diet
After 12 wk on a high-fat diet, control mice had gained 
60% of body weight and were obese (from 28.0±0.6 g to 45.3±0.9 g) (Fig. 1
) while transgenic mice showed only a 35% increase (from 26.0±0.8 g to 35.1±0.9 g for Tg1 and from 26.2±0.7 g to 35.5±0.8 g for Tg2) (Fig. 1)
. Nevertheless, control and transgenic mice fed a standard diet showed a similar increase in body weight (25% and 22% respectively) (from 28.0±0.3 g to 35.2±0.8 g for controls, from 26.0±0.8 g to 31.6±0.6 g for Tg1, and from 25.7±0.4 g to 31.7±0.9 g for Tg2). Both control and transgenic mice had similar food intake (4.5±0.4 g/day of standard diet and 4.0±0.4 g/day of high-fat diet).
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2. Prevention of glucose intolerance and insulin resistance by glucokinase expression in skeletal muscle
After ingesting a high-fat diet, control mice were hyperglycemic when fed (187±7 mg/dL vs. 152±4 mg/dL standard diet-fed control mice) and after an overnight fast (136±15 mg/dL vs. 88±3 mg/dL) and hyperinsulinemic (about threefold increase) (9.2±0.5 ng/mL vs. 3±0.2 ng/mL standard diet-fed control mice). In contrast, transgenic mice fed a high-fat diet remained normoglycemic (162±7 mg/dL Tg1 and 158±8 mg/dL Tg2) and showed only a mild increase in insulinemia (1.5-fold) (5.2±0.5 ng/mL Tg1 and 4.6±0.3 ng/mL Tg2). When an intraperitoneal glucose tolerance test was performed after 12 wk on a high-fat diet, blood glucose levels in the transgenic group were lower than in controls (Fig. 2
A). This indicated that the transgenic mice maintained whole-body glucose tolerance. Moreover, during an intraperitoneal insulin tolerance test performed after 12 wk on a high-fat diet, a significantly increased hypoglycemic response was observed in transgenic mice compared with control mice (Fig. 2B
). Thus, by 60 min after insulin injection, transgenic mice fed a high-fat diet showed a 55% decrease in blood glucose levels whereas control mice fed a high-fat diet had only a 25% reduction in glycemia. This indicated that transgenic mice were more sensitive to the hypoglycemic effects of insulin. All these results show that after a high-fat diet control mice developed insulin resistance, whereas transgenic mice expressing GK in the skeletal muscle showed improved glucose tolerance and were normoglycemic and insulin sensitive.
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3. Increased intramuscular concentrations of glucose 6-phosphate and glycogen in GK-expressing skeletal muscle of transgenic mice
After 12 wk on a high-fat diet, skeletal muscle of Tg1 and Tg2 mice had levels of GK protein similar to those fed a standard diet. No expression of the transgene was detected in the white or brown adipose tissue, heart, spleen, or brain of the transgenic mice (data not shown). The presence of GK increased (about twofold) the intracellular concentration of glucose 6-phosphate in skeletal muscle of Tg1 and Tg2 (Con 0.379±0.04, Tg1 0.708±0.06, and Tg2 0.810±0.06). This increase in glucose phosphorylation was parallel to a rise in glycogen con10t (50%) in both transgenic lines (Con 3.1±0.1, Tg1 4.4±0.2 and Tg2 5.1±0.05).
4. UCP-3 overexpression was induced in skeletal muscle of transgenic mice expressing glucokinase
The lower body weight increase together with the same food intake as the controls observed in the transgenic mice suggested an increase in energy expenditure by the skeletal muscle of these animals. Expression of the uncoupling protein-3 (UCP3) was determined in skeletal muscle of control and transgenic mice fed a high-fat diet for 12 wk. An increase (about twofold) in the UCP3 mRNA levels was detected in high-fat fed transgenic mice. This finding suggests that GK expression in skeletal muscle induced UCP3 overexpression in this tissue.
CONCLUSIONS AND SIGNIFICANCE
Our results indicate that the increase in glucose phosphorylation by GK gene expression in skeletal muscle led to increased energy expenditure that counteracted weight gain and mimicked a situation of insulin sensitivity (Fig. 3
). After 12 wk on this diet, MLC/GK transgenic mice were normoglycemic whereas control mice were hyperglycemic in both fed and fasted conditions, and highly hyperinsulinemic. Moreover, the normal blood glucose levels of transgenic mice attenuated the secretory response of pancreatic ß cells, which might also have contributed to the reduction of hyperinsulinemia observed in these mice. This prevented insulin resistance in skeletal muscle and maintained glucose tolerance and insulin sensitivity in the whole animal. The reduction in body weight gain observed in the high-fat fed MLC/GK transgenic mice, although attributable to the improved insulin sensitivity itself, was accompanied by a similar food intake in controls and transgenic mice, which suggests increased energy expenditure. Furthermore, similar to transgenic mice expressing GK, transgenic mice overexpressing UCP1 or UCP3 in skeletal muscle show increased glucose uptake by skeletal muscle, improved insulin sensitivity, and resistance to the obesity induced by a high-fat diet. Thus, we suggest that the increased expression of UCP3 in skeletal muscle of high-fat fed transgenic mice expressing GK reflects an increase in energy expenditure that prevents the development of obesity and insulin resistance. Therefore, our data suggest that modulation of skeletal muscle phosphorylation through the expression of the GK gene in skeletal muscle may be a therapeutic approach to improve insulin sensitivity in type 2 diabetes, obesity, and other insulin-resistant conditions.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0081fje; doi: 10.1096/fj.03-0081fje 
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), Tg1 (n=15) (
), and Tg2 (n=23) (•) transgenic mice fed a high-fat diet for 3 months are shown. Data are the mean ± SE of the mice in each group.
) were given an i.p. injection of 0.5 mg of glucose/g body weight. At the times indicated, blood samples were taken from the tail vein and glucose was determined. Results are means ± SE of 10 mice in each group (*P<0.05 vs. control mice fed a high-fat diet). B) Intraperitoneal insulin tolerance test. Awake high-fat fed transgenic Tg1 (