Fasting induces ketoacidosis and hypothermia in PDHK2/PDHK4-double-knockout mice

Abstract

The importance of PDHK (pyruvate dehydrogenase kinase) 2 and 4 in regulation of the PDH complex (pyruvate dehydrogenase complex) was assessed in single- and double-knockout mice. PDHK2 deficiency caused higher PDH complex activity and lower blood glucose levels in the fed, but not the fasted, state. PDHK4 deficiency caused similar effects, but only after fasting. Double deficiency intensified these effects in both the fed and fasted states. PDHK2 deficiency had no effect on glucose tolerance, PDHK4 deficiency produced only a modest effect, but double deficiency caused a marked improvement and also induced lower insulin levels and increased insulin sensitivity. In spite of these beneficial effects, the double-knockout mice were more sensitive than wild-type and single-knockout mice to long-term fasting, succumbing to hypoglycaemia, ketoacidosis and hypothermia. Stable isotope flux analysis indicated that hypoglycaemia was due to a reduced rate of gluconeogenesis and that slightly more glucose was converted into ketone bodies in the double-knockout mice. The findings establish that PDHK2 is more important in the fed state, PDHK4 is more important in the fasted state, and survival during long-term fasting depends upon regulation of the PDH complex by both PDHK2 and PDHK4.

Figure 1. Improved glucose-tolerance and increased insulin-sensitivity in PDHK2/PDHK4-DKO mice, but not PDHK2-KO mice

(A) Glucose-tolerance tests were performed on wild-type (WT) and PDHK2-KO mice; n = 4 for both. (B) Glucose-tolerance tests were conducted with wild-type (WT) and PDHK2/PDHK4-DKO mice; n = 4 for both. (C) Food was removed from mice of the indicated genotypes for 24 h. Blood glucose levels were determined 0, 12 or 24 h after removal of food; n = 6 for wild-type (WT), PDHK2-KO and PDHK4-KO mice; n = 5 for DKO mice. (D) Insulin-tolerance tests were performed on wild-type (WT) and PDHK2/PDHK4-DKO mice; n = 6 for both. Results are means ± S.E.M. *P < 0.05 relative to wild-type mice determined by Student's t test.

Figure 2. Decreased phosphorylation of the PDH complex E1α subunit in the skeletal muscle of PDHK-KO mice

(A) Representative immunoblots of the Ser²⁹³-phosphorylated form of the PDH complex E1α subunit of skeletal muscle from wild-type (WT), PDHK2-KO, PDHK4-KO and PDHK2/PDHK4-DKO mice. Molecular masses are indicated in kDa (KD). (B) Histograms constructed from data obtained by Western blot analysis. Results are means ± S.D. with n = 3 mice per group. **P < 0.01; ***P < 0.001 relative to wild-type mice determined by one-way ANOVA.

Figure 3. Glycogen levels are reduced, pyruvate clearance is increased and the rate of glucose production is reduced in PDHK2/PDHK4-DKO mice

Liver glycogen levels in wild-type (WT), PDHK2-KO, PDHK4-KO and DKO mice in the fed state (A) and 24 h fasted state (B); n = 5 mice per group. (C) Blood glucose levels during pyruvate-tolerance test with wild-type (WT) and PDHK2/PDHK4-DKO mice; n = 4 mice for both. (D) Glucose production rate in wild-type (WT) and DKO after 18 h of fasting was determined using constant infusion of [U-¹³C₆]glucose; n = 5 for wild-type; n = 6 for DKO. Results are means ± S.E.M. *P < 0.05; **P < 0.01 relative to wild-type mice determined by Student's t test.

Figure 4. Conversion of glucose into ketone bodies and blood ketone bodies is increased in PDHK2/PDHK4-DKO mice

(A) Plasma ketone bodies (KB) (β-hydroxybutyrate + acetoacetate) produced from glucose in wild-type (WT) and DKO mice after 18 h of fasting was determined using constant infusion of [U-¹³C₆]glucose; n = 5 for wild-type; n = 6 for DKO. (B) Plasma NEFA levels were determined in male wild-type (WT) and PDHK2/PDHK4-DKO mice after overnight fasting; n = 4 for both. (C and D) Plasma acetoacetate (C) and β-hydroxybutyrate (D) levels were determined in male wild-type (WT), PDHK2-KO, PDHK4-KO and PDHK2/PDHK4-DKO mice 0, 12, 24 or 36 h after removal of food. Results are means ± S.E.M. with n = 5 in each group for each time point. *P < 0.05; **P < 0.01; ***P < 0.001 relative to wild-type mice determined by Student's t test.

Figure 5. Fasting induces acidosis in PDHK2/PDHK4-DKO mice

(A) Blood pH was determined in wild-type (WT) and PDHK2/PDHK4-DKO mice during fasting; n = 5 for both. (B and C) Blood pH (B) and bicarbonate (C) were determined in wild-type (WT), PDHK2-KO, PDHK4-KO and PDHK2/PDHK4-DKO mice during fasting. Results are means ± S.E.M. with n = 4 per group. *P < 0.05; **P < 0.01; ***P < 0.001 relative to wild-type mice determined by Student's t test.

Figure 6. Deficiency of PDHK2, PDHK4, and both PDHK2 and PDHK4 does not increase expression of other PDHK isoforms in the heart, liver and skeletal muscle

Protein levels of PDHK2, PDHK4 and PDHK1 were determined by Western blot analysis in the heart (A), liver (B) and skeletal muscle (C) of wild-type (WT), PDHK4-KO mice, PDHK2-KO mice and PDHK2/PDHK4-DKO mice.