Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: a sequestration mechanism in metabolic regulation

Abstract

The importance of glucokinase (GK; EC 2.7.1.12) in glucose homeostasis has been demonstrated by the association of GK mutations with diabetes mellitus in humans and by alterations in glucose metabolism in transgenic and gene knockout mice. Liver GK activity in humans and rodents is allosterically inhibited by GK regulatory protein (GKRP). To further understand the role of GKRP in GK regulation, the mouse GKRP gene was inactivated. With the knockout of the GKRP gene, there was a parallel loss of GK protein and activity in mutant mouse liver. The loss was primarily because of posttranscriptional regulation of GK, indicating a positive regulatory role for GKRP in maintaining GK levels and activity. As in rat hepatocytes, both GK and GKRP were localized in the nuclei of mouse hepatocytes cultured in low-glucose-containing medium. In the presence of fructose or high concentrations of glucose, conditions known to relieve GK inhibition by GKRP in vitro, only GK was translocated into the cytoplasm. In the GKRP-mutant hepatocytes, GK was not found in the nucleus under any tested conditions. We propose that GKRP functions as an anchor to sequester and inhibit GK in the hepatocyte nucleus, where it is protected from degradation. This ensures that glucose phosphorylation is minimal when the liver is in the fasting, glucose-producing phase. This also enables the hepatocytes to rapidly mobilize GK into the cytoplasm to phosphorylate and store or metabolize glucose after the ingestion of dietary glucose. In GKRP-mutant mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control.

Figure 1

GKRP gene. (a) Southern hybridization analysis of BamHI-digested DNA with promoter region probe identifying the normal (4.8-kb) and the mutant (2.3-kb) alleles. (b) Northern hybridization analysis of liver RNA showing the lack of compensation from the normal allele in +/− mice and the absence of a GKRP transcript in −/− mice. (c) Western blot analysis of liver extracts showing the ≈65-kDa GKRP in +/+ and +/− mice, decreased levels of GKRP in +/− mice, and the complete absence of GKRP in −/− mice. (d) Coomassie blue-stained SDS/PAGE gel showing equivalent amounts of protein loaded.

Figure 2

GK gene expression in GKRP-mutant liver. (a) Glucose phosphorylation in liver extracts of fasted mice, showing reduced GK activity in GKRP mutants. mU, milliunits. (+/+, n = 8; +/−, n = 7; −/−, n = 7). *, P ≤ 0.05, +/+ versus +/− or −/−. (b) Representative Western blot analysis of liver extracts with GK antibodies showing the GK protein and decreased levels in +/− and in −/− livers. (c) Liver RNA analysis showing the absence of significant changes in GK mRNA levels in GKRP mutants. Data are presented as percentage of +/+ mice liver with +/+ taken as 100%. (+/+, n = 3; +/−, n = 3; −/−, n = 3). Mean ± SEM.

Figure 3

Posttranscriptional expression of GK gene is compromised in GKRP-mutant mouse liver. (A) GK expression in wild-type liver; GK mRNA (empty bars) and protein (filled bars) levels are enhanced by insulin. (B) GK expression in mutant mouse liver; GK mRNA (empty bars) is increased by insulin, whereas GK protein (filled bars) is not. Data are presented as -fold over time-0 wild-type mouse liver, with wild-type value taken as 1.0. Mean ± SEM, n = 3 for wild type and n = 3 for mutant per each time point. (C) Densitometric quantitation of the Western blot of extracts prepared from primary hepatocytes cultured in media containing 5.5 mM glucose, showing decreased levels of GK in homozygous mutant (Mut) hepatocytes. Data are presented as percentage of wild-type (WT) hepatocytes. (D) Densitometric quantitation of the Western blot of extracts from cells grown for an additional 6 hr in the presence or absence of cycloheximide showing absence of altered levels of GK degradation in Mut hepatocytes. GK values in WT and Mut cells, cultured in the absence of cycloheximide at time 0, were both considered as 100%, and data are presented as a percentage of these values.

Figure 4

Subcellular localization of GKRP and GK in mouse primary hepatocytes cultured in low-glucose-containing medium. (a–c) Immunocytochemical analysis of hepatocytes with anti-GKRP showing nuclear localization of GKRP in +/+ and +/− cells, with antibody staining decreased in +/− cells (b) and absent in −/− cells (c). (d–f) Immunocytochemical analysis of hepatocytes with anti-GK antibodies showing nuclear localization of GK in +/+ cells and +/− cells, with antibody staining reduced in +/− cells (e) and the absence of GK antibody staining in −/− cells’ nuclei, in which the staining is increased in the cytoplasm (f).

Figure 5

Subcellular localization of GKRP and GK in mouse primary hepatocytes. Immunocytochemical analysis with anti-GKRP and anti-GK antibodies. (a–c) GKRP continued to remain in the nucleus of +/+ hepatocytes cultured in low-glucose- (a), low-fructose + low-glucose- (b), or in high-glucose-containing media (c). (d–f) GK is present in the nucleus of +/+ hepatocytes cultured in low-glucose-containing medium (d), translocated into the cytoplasm of +/+ hepatocytes cultured in low-fructose + low-glucose- (e) or in high-glucose-containing media (f). (g–i) GK present in the cytoplasm of −/− hepatocytes cultured in low-glucose medium (g) continued to stay in the cytoplasm in low-fructose + glucose- (h) or in high-glucose-containing medium (i).

Figure 6

Plasma glucose, insulin, liver glycogen, and PEPCK mRNA levels. (A and B) Eight-week-old male mice were fasted for 16 hr or fed ad lib, and glucose (A) and insulin (B) levels were determined. μU, microunit. Fasted: n = 5 for wild type (WT); n = 6 for heterozygous mutant (Het); n = 6 for homozygous mutant (Mut). Fed: n = 6 for WT; n = 5 for Het; n = 5 for Mut. (C) Eight-week-old male, ad-lib fed mice were sacrificed, livers were harvested, and glycogen levels were quantitated. n = 3 for WT; n = 3 for Het; n = 3 for Mut. (D) PEPCK mRNA levels were quantitated by Northern blot analysis of total RNA prepared from fasted mouse liver. n = 3 for WT; n = 3 for Het; n = 3 for Mut. The mRNA level in WT is taken as 100%. Data are presented as mean ± SEM. *, Differences were considered statistically significant at P < 0.05.

Figure 7

Glucose homeostasis in GKRP-mutant mice. (A) Glucose tolerance test showing elevated blood glucose levels in Mut mice as compared with WT and Het mice. n = 7 for WT; n = 7 for Het; n = 13 for Mut. (B) Plasma insulin levels in mice 30 min after glucose injection, showing the absence of significant changes. n = 4 for WT; n = 4 for Het; n = 4 for Mut. (C and D) Effect of high-sucrose + high-fat feeding on body weight and plasma glucose levels. Although both groups gained body weight at similar rates (C), the plasma glucose levels are significantly elevated in Mut mice as compared with WT mice (D). n = 5 for WT; n = 5 for Mut. 1* indicates after weaning. Data represent mean ± SEM. *, Differences were considered statistically significant at P < 0.05.