PPP1R3B Human

What is the function of PPP1R3B in human metabolism?

PPP1R3B (also known as GL) serves as a regulatory subunit of protein phosphatase 1 (PP1), a serine/threonine phosphatase that plays a crucial role in modulating glycogen synthesis in the liver and skeletal muscle. The PPP1R3B protein specifically regulates the activity of glycogen synthase, effectively controlling the rate at which glucose is stored as glycogen, particularly in hepatic tissue . This regulatory function makes PPP1R3B essential for maintaining glucose homeostasis in the body.

Research methodologically, this function has been elucidated through a combination of biochemical assays, knockout studies, and the analysis of human genetic variants associated with metabolic phenotypes. Most significantly, hepatocyte-specific gene deletion and overexpression studies in mouse models have demonstrated that PPP1R3B acts as a metabolic switch determining whether the liver stores energy as glycogen or diverts it to lipid synthesis pathways .

Where is the PPP1R3B gene located and what is its structure?

The PPP1R3B gene is located on chromosome 8p23 in the human genome, a region that has been linked with type 2 diabetes and maturity-onset diabetes of the young (MODY) in multiple genetic studies . Structurally, RT-PCR analysis has revealed that the gene consists of two exons and possesses two alternative promoters . This genomic location is of particular interest because the PPP1R3B locus has been associated with various cardiometabolic traits through genome-wide association studies (GWAS) .

The gene is situated approximately 175 kilobases from a long non-coding RNA gene, LOC157273, which has been shown to regulate PPP1R3B expression . This close proximity and regulatory relationship highlight the complex genomic architecture surrounding PPP1R3B.

How does PPP1R3B expression vary across human tissues?

PPP1R3B demonstrates a tissue-specific expression pattern, with predominant expression in the liver, which aligns with its primary role in hepatic glycogen metabolism and glucose homeostasis. While the gene is also expressed to a lesser extent in skeletal muscle and other tissues, its highest functional activity appears to be in hepatocytes.

This tissue-specific expression profile corresponds to the physiological role of different tissues in glycogen metabolism. The liver, as the primary site of postprandial glycogen storage and subsequent glucose release during fasting, shows robust PPP1R3B expression. In contrast, skeletal muscle, which primarily stores glycogen for local energy utilization, expresses other PP1 glycogen-targeting subunits more prominently.

How is PPP1R3B regulated at the transcriptional level?

PPP1R3B transcription involves a complex regulatory network including the presence of two alternative promoters as identified through RT-PCR analysis . A particularly interesting aspect of PPP1R3B regulation involves the long non-coding RNA LOC157273, which acts as a negative regulator of PPP1R3B expression in human hepatocytes .

Research has demonstrated that siRNA-mediated knockdown of LOC157273, decreasing its transcript levels by approximately 80%, results in a 1.7-fold increase in PPP1R3B mRNA levels in primary human hepatocytes . This finding establishes LOC157273 as an effector transcript at the PPP1R3B locus, providing insight into the molecular mechanisms controlling PPP1R3B expression.

The variant rs4841132, associated with an insulin-resistant diabetes risk phenotype, is located in the second exon of LOC157273. Carriers with the A/G heterozygous genotype demonstrate reduced LOC157273 abundance due to decreased transcription of the A allele, which correspondingly increases PPP1R3B expression and glycogen deposition compared to G/G carriers .

What signaling pathways interact with PPP1R3B in glycogen metabolism?

PPP1R3B functions within an intricate network of metabolic signaling pathways:

• Insulin signaling pathway: Insulin activates Akt, which inhibits GSK3β, reducing inhibitory phosphorylation of glycogen synthase. Concurrently, the PPP1R3B-PP1 complex further activates glycogen synthase through dephosphorylation, creating a coordinated response involving both kinase and phosphatase regulation.

• Glucagon signaling pathway: During fasting, glucagon activates protein kinase A (PKA), which can phosphorylate and regulate PPP1R3B, modifying its ability to target PP1 to glycogen. This helps coordinate the switch between glycogen synthesis and breakdown.

• AMPK pathway: AMP-activated protein kinase senses cellular energy status and can regulate glycogen metabolism, potentially through direct or indirect effects on PPP1R3B function.

Research methodologically, these pathway interactions have been studied using phosphoproteomic analysis, metabolic flux studies, and the examination of metabolic phenotypes in genetically modified mouse models with altered Ppp1r3b expression .

What PPP1R3B variants have been associated with metabolic disorders?

Multiple genomic studies have identified PPP1R3B variants associated with metabolic disorders:

In a targeted resequencing study of 8,710 samples, researchers identified 23 PPP1R3B missense mutations. The burden of likely deleterious PPP1R3B variants was significantly increased in patients with type 2 diabetes (0.58%, 95% CI: 0.36-0.93) compared to non-diabetic individuals (0.31%, 95% CI: 0.20-0.49) .

Interestingly, carriers with diabetes had distinct phenotypic characteristics, including less abdominal fat and higher serum LDL-cholesterol compared to T2D patients without rare missense PPP1R3B variants. Additionally, non-diabetic carriers had higher birth weights compared to non-carriers .

How do PPP1R3B variants contribute to metabolic disorder pathophysiology?

PPP1R3B variants contribute to metabolic disorder pathophysiology through multiple mechanisms:

• Altered glycogen metabolism: Loss-of-function variants reduce hepatic glycogen synthesis capacity, impairing the liver's ability to clear glucose from the bloodstream after meals, resulting in postprandial hyperglycemia.

• Metabolic substrate switching: When glycogen synthesis is impaired, glucose is redirected toward lipogenesis. This metabolic shift is evident in PPP1R3B-deficient mouse models (Ppp1r3bΔhep), which show negligible liver glycogen but increased hepatic triglycerides compared to wild-type mice .

• Impaired glucose homeostasis: Ppp1r3bΔhep mice develop dramatically impaired glucose tolerance and insulin insensitivity when challenged with a high sucrose diet, demonstrating the critical role of PPP1R3B in maintaining glucose homeostasis under metabolic stress .

• Lipid metabolism alterations: Human carriers of rare PPP1R3B variants show altered lipid profiles, including higher LDL-cholesterol levels, indicating cross-talk between glycogen metabolism and lipid regulatory pathways .

This pathophysiological understanding comes from integration of human genetic studies with functional characterization of variants in cellular and animal models.

What are optimal protocols for studying PPP1R3B-mediated glycogen synthesis?

Researchers investigating PPP1R3B function can employ the following optimized protocols:

• Cell models:

  • Primary human hepatocytes cultured on collagen-coated plates

  • HepaRG or HepG2 cells as alternatives with appropriate validation

  • siRNA knockdown for reduced expression (typically achieving 70-80% reduction)

  • Adenoviral vectors for overexpression studies

• Insulin-stimulated glycogen synthesis assay:

  • Serum starvation (6-12 hours) to reduce basal glycogen levels

  • Treatment with insulin (10-100 nM) to stimulate glycogen synthesis

  • Cell harvesting at multiple timepoints (0-4 hours)

  • Biochemical measurement of glycogen content using amyloglucosidase digestion

• Analysis of PPP1R3B function:

  • Western blotting to assess phosphorylation of key proteins in the pathway

  • Glycogen synthase activity assays (±glucose-6-phosphate)

  • Co-immunoprecipitation to assess PPP1R3B-PP1 complex formation

The study by Manning et al. demonstrated the effectiveness of siRNA knockdown of LOC157273 followed by measurement of PPP1R3B expression and glycogen deposition in primary human hepatocytes, providing a methodological framework for investigating the regulatory relationships in this pathway .

How can researchers effectively model PPP1R3B function in vivo?

Several experimental models have proven effective for studying PPP1R3B function in vivo:

• Genetically modified mouse models:

  • Hepatocyte-specific deletion of Ppp1r3b (Ppp1r3bΔhep) - Results in dramatic reduction in glycogen synthase activity, depletion of liver glycogen stores, and rapid fasting hypoglycemia

  • Hepatocyte overexpression of murine Ppp1r3b (Ppp1r3bhepOE) - Increases liver glycogen content and preserves blood glucose levels even after prolonged fasting

• Metabolic challenge experiments:

  • High sucrose diet (66% sucrose) challenges reveal the importance of PPP1R3B in maintaining glucose homeostasis under metabolic stress

  • Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) demonstrate the metabolic consequences of altered PPP1R3B expression

  • Fasting/refeeding protocols to assess dynamic glycogen metabolism

• Analytical techniques:

  • Liver glycogen content measurement (Ppp1r3bΔhep mice show negligible liver glycogen)

  • Hepatic triglyceride quantification (Ppp1r3bΔhep mice show increased hepatic TG compared to wild-type)

  • Histological assessment for lipid droplet accumulation, particularly in pericentral regions

These in vivo modeling approaches have revealed that PPP1R3B acts as a metabolic switch that effectively causes a shift between lipid and glucose utilization as energetic substrates in the liver.

What is the relationship between PPP1R3B and non-alcoholic fatty liver disease (NAFLD)?

The relationship between PPP1R3B and NAFLD presents an intriguing paradox that requires nuanced interpretation:

The research suggests that optimal glycogen metabolism, facilitated by proper PPP1R3B function, protects against the development of fatty liver by channeling dietary carbohydrates into glycogen rather than lipid synthesis pathways.

How does the long non-coding RNA LOC157273 regulate PPP1R3B expression?

LOC157273 has been identified as a key regulatory factor for PPP1R3B expression:

• Genomic context:

  • LOC157273 is a long non-coding RNA gene located near PPP1R3B on chromosome 8p23.1

  • The variant rs4841132, associated with an insulin-resistant diabetes risk phenotype, is located in the second exon of LOC157273

• Regulatory relationship:

  • LOC157273 acts as a negative regulator of PPP1R3B expression in human hepatocytes

  • siRNA knockdown reducing LOC157273 transcript levels by ~80% resulted in:

    • 1.7-fold increase in PPP1R3B mRNA levels

    • 50% increase in glycogen deposition in primary human hepatocytes

• Allele-specific effects:

  • A/G heterozygous carriers of rs4841132 show reduced LOC157273 abundance

  • This reduction is due to decreased transcription of the A allele

  • The resulting increased PPP1R3B expression leads to enhanced glycogen deposition compared to G/G carriers

• Research methodology:

  • The relationship was characterized using:

    • Fluorescent in situ hybridization to determine LOC157273 localization

    • siRNA knockdown with RT-PCR quantification

    • RNA-seq to measure transcriptome-wide responses

    • Insulin-stimulated glycogen deposition assays

This regulatory mechanism provides insight into how non-coding elements in the genome can influence metabolic processes through modulation of key regulatory genes like PPP1R3B.

What is the potential of PPP1R3B as a therapeutic target for metabolic disorders?

PPP1R3B presents several promising characteristics as a potential therapeutic target:

• Type 2 diabetes applications:

  • Enhancing PPP1R3B function could improve postprandial glucose clearance

  • Mouse models overexpressing Ppp1r3b show improved glucose tolerance and insulin sensitivity

  • This approach could address a fundamental aspect of T2D pathophysiology by enhancing the liver's capacity for glucose disposal

• NAFLD applications:

  • Increasing PPP1R3B function could redirect energy storage from lipids to glycogen

  • This metabolic rewiring might reduce hepatic steatosis in early NAFLD

  • Ppp1r3bhepOE mice demonstrate reduced hepatic triglyceride content

• Potential therapeutic strategies:

  • Small molecule enhancers of PPP1R3B-PP1 interaction

  • Antisense oligonucleotides targeting LOC157273 to increase PPP1R3B expression

  • Gene therapy approaches to increase hepatic PPP1R3B expression

• Considerations and challenges:

  • Tissue-specific targeting would be essential to avoid unwanted effects

  • Careful dosing required to prevent hypoglycemia

  • Individual variation in response based on genetic background

  • Long-term effects on liver physiology require thorough investigation

The metabolic switch function of PPP1R3B makes it particularly interesting as a therapeutic target that could address multiple aspects of metabolic syndrome simultaneously.

What are critical outstanding questions in PPP1R3B research?

Despite significant advances, several critical questions remain in PPP1R3B research:

• Population-specific effects:

  • While PPP1R3B genetic variability does not appear to contribute significantly to diabetes in Caucasian populations, its role cannot be excluded in other populations such as the Japanese, among whom linkage to diabetes is observed at 8p23 and a non-synonymous mutation has been detected

  • More research is needed to understand these population-specific differences

• Tissue-specific regulation:

  • How does PPP1R3B regulation differ between hepatocytes and other tissues expressing the gene?

  • What factors determine tissue-specific responses to PPP1R3B variants?

• Developmental aspects:

  • Non-diabetic carriers of PPP1R3B variants have higher birth weights

  • The developmental roles of PPP1R3B and implications for metabolic programming remain largely unexplored

• Epigenetic regulation:

  • How do epigenetic mechanisms regulate PPP1R3B expression in different metabolic states?

  • What role might epigenetic dysregulation play in altered PPP1R3B function in metabolic disease?

• Therapeutic translation:

  • Can the insights from mouse models be effectively translated to human therapeutics?

  • What biomarkers might predict responsiveness to PPP1R3B-targeted therapies?

Addressing these questions will require integrative approaches combining human genetic studies, functional genomics, metabolic phenotyping, and translational research methodologies.