Hepatic protein phosphatase 1 regulatory subunit 3B (Ppp1r3b) promotes hepatic glycogen synthesis and thereby regulates fasting energy homeostasis

Abstract

Maintenance of whole-body glucose homeostasis is critical to glycemic function. Genetic variants mapping to chromosome 8p23.1 in genome-wide association studies have been linked to glycemic traits in humans. The gene of known function closest to the mapped region, PPP1R3B (protein phosphatase 1 regulatory subunit 3B), encodes a protein (G_L) that regulates glycogen metabolism in the liver. We therefore sought to test the hypothesis that hepatic PPP1R3B is associated with glycemic traits. We generated mice with either liver-specific deletion (Ppp1r3b^Δhep ) or liver-specific overexpression of Ppp1r3b The Ppp1r3b deletion significantly reduced glycogen synthase protein abundance, and the remaining protein was predominantly phosphorylated and inactive. As a consequence, glucose incorporation into hepatic glycogen was significantly impaired, total hepatic glycogen content was substantially decreased, and mice lacking hepatic Ppp1r3b had lower fasting plasma glucose than controls. The concomitant loss of liver glycogen impaired whole-body glucose homeostasis and increased hepatic expression of glycolytic enzymes in Ppp1r3b^Δhep mice relative to controls in the postprandial state. Eight hours of fasting significantly increased the expression of two critical gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, above the levels in control livers. Conversely, the liver-specific overexpression of Ppp1r3b enhanced hepatic glycogen storage above that of controls and, as a result, delayed the onset of fasting-induced hypoglycemia. Moreover, mice overexpressing hepatic Ppp1r3b upon long-term fasting (12-36 h) were protected from blood ketone-body accumulation, unlike control and Ppp1r3b^Δhep mice. These findings indicate a major role for Ppp1r3b in regulating hepatic glycogen stores and whole-body glucose/energy homeostasis.

Keywords: Alb-Cre; Ppp1r3b; gluconeogenesis; glycogen; glycogen storage disease; glycogen synthase; glycolysis; liver.

Figure 1.

Liver-specific deletion of Ppp1r3b results in depletion of hepatic glycogen content. A, hepatic mRNA levels of Ppp1r3a, Ppp1r3b, and Ppp1r3c measured by SYBR-Green RT quantitative PCR in control and Ppp1r3b^Δhep [Alb-Cre] mice (n = 10–14/genotype). B, hepatic glycogen content in ad libitum chow-fed and 4-h fasted control and Ppp1r3b^Δhep mice (n = 6/genotype). C, PAS staining of paraffin-embedded liver sections from ad lib fed and 4-h fasted Ppp1r3b^Δhep and control mice shows depletion of glycogen (purple stain). Note that the 4-h fasted sections were not counterstained with hemotoxylin. Scale bar, 1 mm. D, 2-deoxy-d-[³H] glucose (2-DOG) incorporation into glycogen was measured after 6 h of fasting in control and Ppp1r3b^Δhep mice (n = 3–4/genotype). Label incorporation was measured as cpm from precipitated glycogen from tissue lysates and normalized to plasma cpm/mg protein. The results were replicated in at least two independent experiments. All mice were adults (2–8 months) and were age-matched within experiments. The data are expressed as the means ± S.E. Significance was determined in all panels by unpaired Student's t test. *, p < 0.05; **, p < 0.005; ***, p < 0.0001. ns, not significant.

Figure 2.

Ppp1r3b-deficient livers have reduced total GS protein but higher relative phospho-GS. A, 4-h fasted liver lysates were used for measurement of transcript levels of Gys2 and Pygl (n = 6–9/genotype). B, protein expression of total and p-GS GS in primary hepatocytes isolated from ad libitum chow-fed control and Ppp1r3b^Δhep mice. C, protein expression of total GS, P-GS (Ser-641), total GP, and PP1 in liver lysates from 4-h fasted control and Ppp1r3b^Δhep mice. The ratio of total GS to β-actin is decreased, and P-GS to total GS is increased Ppp1r3b^Δhep compared with control mice (n = 6/genotype). D, liver GS protein expression and PAS staining in ad libitum chow-fed and overnight fasted Ppp1r3b^Δhep mice compared with control mice (n = 4–6/genotype). Scale bar, 1 mm. All mice were adults (2–8 months) and were age-matched within experiments. The results were replicated in at least two independent experiments. The data are expressed as the means ± S.E. Significance was determined in all panels by unpaired Student's t test. *, p < 0.05; **, p < 0.005; ***, p < 0.0001. n.s, not significant.

Figure 3.

Plasma glucose homeostasis is impaired in Ppp1r3b^Δhep mice. A, blood glucose levels of ad libitum chow-fed and 4-h fasted mice (n = 10–14/genotype). B, blood glucose levels during a 36-h fasting period in Ppp1r3b^Δhep mice compared with control mice (n = 6/genotype). C, blood lactate levels in ad libitum chow-fed and in 4-h fasted control and Ppp1r3b^Δhep mice (n = 10–14/genotype). D, total ketone bodies in blood measured during ad libitum ad fed and 12-, 24-, and 36-h fasting conditions in Ppp1r3b^Δhep mice compared with control mice (n = 6–8/genotype). E, PTT in control and Ppp1r3b^Δhep mice (n = 6/genotype). PTT was performed in mice by administering 2 g/kg body weight sodium pyruvate by intraperitoneal injection after overnight (14–16 h) fasting. The values are reported as percentages of basal glucose levels; the area under the curve (AUC) is expressed as arbitrary units. All mice were adults (2–8 months) and were age-matched within experiments). The results were replicated in at least two independent experiments. The data are expressed as the means ± S.E. Significance was determined in all panels by unpaired Student's t test. *, p < 0.05; **, p < 0.005; ***, p < 0.0001. n.s, not significant.

Figure 4.

Ppp1r3b-deficient livers undergo rapid switching from glycolysis to gluconeogenesis. A, ad libitum chow-fed liver lysates were used for measurement of transcript levels of glycolytic and gluconeogenic genes (Gck, Gpi1, Pfk1, Aldob, Tpi1, Gapdh, Pgk1, Pgam1, Eno1, Pklr, Pepck, and G6pc (n = 6–9/genotype). B, 8-h fasted liver lysates were used for measurement of transcript levels of the above glycolytic and gluconeogenic genes (n = 5/genotype). The results were replicated in at least two independent experiments. The data are expressed as the means ± S.E. Significance was determined in all panels by unpaired Student's t test. *, p < 0.05; **, p < 0.005; ***, p < 0.0001. n.s, not significant.

Figure 5.

Overexpression of Ppp1r3b in liver enhances hepatic glycogen storage and delayed responses to fasting. A, 4-h fasted liver lysates were used for measurement of transcript levels of Ppp1r3b, Ppp1r3c (PTG), Ppp1r3a (G_M), Gys2, and Pygl from C57BL/6J mice injected with AAV-null and AAV-Ppp1r3b (n = 4/group). B, hepatic glycogen content in 4- and 8-h fasted liver lysates from C57BL/6J mice injected with AAV-null and AAV-Ppp1r3b (n = 3–4/group). C, PAS staining of paraffin-embedded liver sections from 4- and 8-h fasted C57BL/6J mice injected with AAV-null and AAV-Ppp1r3b, showing increased amount of glycogen (purple stain) in AAV-Ppp1r3b-overexpressing mice. D, protein expression of total GS, P-GS (Ser-641), total GP, and PP1 in liver lysates from 4-h fasted from C57BL/6J mice injected with AAV-null and AAV-Ppp1r3b (n = 4/group). The ratio of total GS to β-actin is increased and P-GS to total GS is decreased in AAV-Ppp1r3b-overexpressing mice compared with the Null group (n = 4/genotype). E, blood glucose levels during a 36-h fasting period in C57BL/6J mice injected with AAV-null and AAV-Ppp1r3b (n = 7–8/group). F, total ketone bodies in blood measured during ad libitum fed and 12-, 24-, and 36-h fasting conditions in C57BL/6J mice injected with AAV-null and AAV-Ppp1r3b (n = 7–8/group). The mice were injected at 9 weeks of age, and liver lysates were analyzed at 11–12 weeks post injection. The results were replicated in at least two independent experiments. The data are expressed as the means ± S.E. Significance was determined in all panels by unpaired Student's t test. *, p < 0.05; **, p < 0.005; ***, p < 0.0001. n.s, not significant.