Effects of insulin and transgenic overexpression of UDP-glucose pyrophosphorylase on UDP-glucose and glycogen accumulation in skeletal muscle fibers

Abstract

UDP-glucose (UDP-Glc) and glycogen levels in skeletal muscle fibers of defined fiber type were measured using microanalytical methods. Infusing rats with insulin increased glycogen in both Type I and Type II fibers. Insulin was without effect on UDP-Glc in Type I fibers but decreased UDP-Glc by 35-40% in Type IIA/D and Type IIB fibers. The reduction in UDP-Glc suggested that UDP-Glc pyrophosphorylase (PPL) activity might limit glycogen synthesis in response to insulin. To explore this possibility, we generated mice overexpressing a UDP-Glc PPL transgene in skeletal muscle. The transgene increased both UDP-Glc PPL activity and levels of UDP-Glc in skeletal muscles by approximately 3-fold. However, overexpression of UDP-Glc PPL was without effect on either the levels of skeletal muscle glycogen or glucose tolerance in vivo. The transgene was also without effect on either control or insulin-stimulated rates of (14)C-glucose incorporation into glycogen in muscles incubated in vitro. The results indicate that UDP-Glc PPL activity is not limiting for glycogen synthesis.

Fig. 1. Fiber type assignments of rectus abdominis fibers from control and insulin-treated rats

Rats were infused with saline (open symbols) or insulin (10 milliunits/min/kg) (filled symbols), while clamping blood glucose at 5 mM. This required infusing glucose at 2.9 ± 0.75 and 142 ± 4 μmol/min/kg in the control and insulin-treated animals, respectively. After 2 h, the rectus abdominus muscle was freeze-clamped in situ. Hind limb glucose uptake measured over this time was increased from 0.20 ± 0.4 to 0.52 ± 0.13 μmol/min by insulin. Single fibers were dissected from three control and three insulin-stimulated muscles. LDH and MDH activities were measured in individual fibers. Based on these activities the fibers were divided into the following three categories: Type I, Type IIA/D, and Type IIB fibers, identified as described by Hintz et al. (12). This strategy does not distinguish between IIA and IID fibers, so these fibers are grouped together. The mean (± S.E.) LDH activities (in mmol/hr/kg, dry weight) in the control and insulin-treatment groups, respectively, were as follows: Type I, 18.7 ± 2.2 and 20.2 ± 1.1; Type IIA/D, 44.1 ± 2.9 and 47.2 ± 3.0; Type IIB, 81.6 ± 1.6 and 80.0 ± 1.8. MDH activities were as follows: Type I, 12.0 ± 0.9 and 12.8 ± 0.4; Type IIA/D, 18.4 ± 0.5 and 19.0 ± 0.5; and Type IIB, 3.7 ± 0.2 and 3.5 ± 0.2.

Fig. 2. Effects of insulin on levels of UDP-Glc and glycogen in single fibers

Levels of glycogen in equivalents of glucose (A) and UDP-Glc (B) were measured in single muscle fibers, which were divided into the fiber type categories described in the legend to Fig. 1. Mean values ± S.E. (where n = number of fibers) are presented. The significance of differences between insulin and control values were evaluated by a two-tailed heteroscedastic t test (36). *, not significantly different; **, p <0.01; ***, p <0.001.

Fig. 3. Transgenic overexpression of UDP-Glc PPL

Tibialis anterior (TA), EDL, gastrocnemius (Gastroc.), and soleus muscles were dissected from two lines of mice, PPL-1 and PPL-2, expressing the UDP-Glc PPL transgene and from wild type littermates. UDP-Glc PPL activity was measured in extracts of these muscles and expressed relative to extract protein.

Fig. 4. Relative levels of UDP-Glc PPL achieved by transgenic overexpression in different skeletal muscles

Quadricep, EDL, and diaphragm muscles were dissected from two PPL-1 mice and from two nontransgenic littermates. Samples of extracts were subjected to SDS-PAGE and immunoblotted with antibodies to UDP-Glc PPL.

Fig. 5. Effects of transgenic overexpression of UDP-Glc PPL on the UDP-Glc and glycogen contents in skeletal muscle

Glycogen and UDP-Glc were measured in samples of quadricep muscles from PPL-1 mice and nontransgenic littermates. The results are expressed relative to wet weight of the tissues and are the mean values ± S.E. of measurements obtained from muscles of three wild type mice and four transgenic mice.

Fig. 6. Glucose tolerance is unaffected by transgenic overexpression of UDP-Glc PPL

PPL-1 mice (•) and nontransgenic littermates (○) were fasted overnight. A, blood glucose was measured at increasing times after injecting glucose (1 mg/g of body weight) intraperitoneally. Mean values ± S.E. from 27 wild type mice and 33 trans-genic mice are presented. B, blood glucose was measured at increasing times after injecting glucose (0.5 mg/g of body weight) intravenously. Mean values ± S.E. from 28 wild type and 34 transgenic mice are presented.

Fig. 7. Failure of UDP-Glc PPL overexpression to increase [U-¹⁴C]glucose incorporation into glycogen in vitro

Muscles from PPL-1 mice and nontransgenic littermates were incubated for 30 min at 37 °C without or with either 100 microunits/ml insulin (A) or 250 milliunits/ml insulin (B and C) in Krebs-Ringer bicarbonate buffer containing 5 mM [U-¹⁴C]glucose (500 cpm/nmol). [U-¹⁴C]Glucose incorporation into glycogen was determined and expressed relative to the wet weight of the tissues. Mean values ± S.E. from eight wild type and eight transgenic EDL muscles (A), four wild type and four transgenic EDL muscles (B), and three wild type and three transgenic hemidiaphragms (C) are presented.