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 (GL) 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 (Ppp1r3bΔhep ) 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 Ppp1r3bΔhep 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 Ppp1r3bΔhep mice. These findings indicate a major role for Ppp1r3b in regulating hepatic glycogen stores and whole-body glucose/energy homeostasis.
Highlights
Maintenance of whole-body glucose homeostasis is critical to glycemic function
Hepatic Ppp1r3b deficiency leads to depletion of hepatic glycogen stores with significant reduction in incorporation of glucose into hepatic glycogen
In Ppp1r3b⌬hep mice, transcript levels were negligible in the liver, with 97% deletion efficiency observed, and there was no compensatory increase in gene expression of other glycogen-targeting subunits: GM (Ppp1r3a) and Ppp1r3b regulates fasting energy homeostasis (PTG) (Ppp1r3c) (Fig. 1A)
Summary
To investigate the role of Ppp1r3b in the liver, we generated mice lacking this gene exclusively in the liver (see “Experimental procedures” and supplemental Fig. S1A). Hepatic glycogen content was significantly reduced in ad libitum fed state in Ppp1r3b⌬hep mice, which was rapidly depleted upon short-term, 4-h fasting as measured biochemically (Fig. 1B). We used AAV-Cre in Ppp1r3bflox/flox mice as a second approach to generate liver-specific knock-out of Ppp1r3b and found effects on hepatic glycogen and plasma glucose (supplemental Fig. S2) comparable to those in the Alb-Cre mice. Radioactivity in the glycogen pellet from tissue lysates was measured at 30 and 60 min and quantified as a percentage of glucose specific activity of total radioactivity in plasma and of total protein content In this experiment, Ppp1r3b⌬hep mice exhibited significantly reduced glycogen synthase activity in the liver, whereas its activity was not different in skeletal muscle or kidney (Fig. 1D). The loss of hepatic Ppp1r3b results in a smaller liver with reduced GS activity and glycogen content in ad libitum fed and 4-h fasted mice.
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