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A phosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism.

ABSTRACT: Members of the protein kinase D (PKD) family (PKD1, 2, and 3) integrate hormonal and nutritional inputs to regulate complex cellular metabolism. Despite the fact that a number of functions have been annotated to particular PKDs, their molecular targets are relatively poorly explored. PKD3 promotes insulin sensitivity and suppresses lipogenesis in the liver of animals fed a high-fat diet. However, its substrates are largely unknown. Here we applied proteomic approaches to determine PKD3 targets. We identified more than 300 putative targets of PKD3. Furthermore, biochemical analysis revealed that PKD3 regulates cAMP-dependent PKA activity, a master regulator of the hepatic response to glucagon and fasting. PKA regulates glucose, lipid, and amino acid metabolism in the liver, by targeting key enzymes in the respective processes. Among them the PKA targets phenylalanine hydroxylase (PAH) catalyzes the conversion of phenylalanine to tyrosine. Consistently, we showed that PKD3 is activated by glucagon and promotes glucose and tyrosine levels in hepatocytes. Therefore, our data indicate that PKD3 might play a role in the hepatic response to glucagon.

SUBMITTER: Loza-Valdes A

PROVIDER: S-EPMC8321662 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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A phosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism.

Loza-Valdes Angel A Mayer Alexander E AE Kassouf Toufic T Trujillo-Viera Jonathan J Schmitz Werner W Dziaczkowski Filip F Leitges Michael M Schlosser Andreas A Sumara Grzegorz G

Life science alliance 20210618 8

Members of the protein kinase D (PKD) family (PKD1, 2, and 3) integrate hormonal and nutritional inputs to regulate complex cellular metabolism. Despite the fact that a number of functions have been annotated to particular PKDs, their molecular targets are relatively poorly explored. PKD3 promotes insulin sensitivity and suppresses lipogenesis in the liver of animals fed a high-fat diet. However, its substrates are largely unknown. Here we applied proteomic approaches to determine PKD3 targets. ...[more]

PMID: 34145024

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Project description:Elongation of very long-chain fatty acids protein 6 (Elovl6) has been reported to be associated with clinical treatments of a variety of metabolic diseases. However, there is no systematic and comprehensive study to reveal the regulatory role of Elovl6 in mRNA, protein and phosphorylation levels. We established the first knock-out (KO), elovl6-/-, in zebrafish. Compared with wild type (WT) zebrafish, KO presented significant higher whole-body lipid content and lower content of fasting blood glucose. We utilized RNA-Seq, tandem mass tag (TMT) labeling-based quantitative technology and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to perform the transcriptomic, proteomic and phosphoproteomic analyses of livers from WT and elovl6-/- zebrafish. There were 734 differentially expressed genes (DEG) and 559 differentially expressed proteins (DEP) between elovl6-/- and WT zebrafish, identified out of quantifiable 47251 transcripts and 5525 proteins. Meanwhile, 680 differentially expressed phosphoproteins (DEPP) with 1054 sites were found out of quantifiable 1230 proteins with 3604 sites. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis of the transcriptomic and proteomic data further suggested that the abnormal lipid metabolism and glucose metabolism in KO were mainly related to fatty acid degradation and biosynthesis, glycolysis/gluconeogenesis and PPAR signaling pathway. Based on phosphoproteomic analyses, some kinases critical for lipid metabolism and glucose metabolism, including ribosomal protein S6 kinase (Rps6kb), mitogen-activated protein kinase14 (Mapk14) and V-akt murine thymoma viral oncogene homolog 2-like (Akt2l), were identified. These results allowed us to catch on the regulatory networks of elovl6 on lipid and glucose metabolism in zebrafish. To our knowledge, this is the first multi-omic study of zebrafish lacking elovl6, which provides strong datasets to better understand many lipid/glucose metabolic risks posed to human health.

Project description:OBJECTIVE:We hypothesized that DA and L-DOPA derived from nutritional tyrosine and the resultant observed postprandial plasma excursions of L-DOPA and DA might affect glucose tolerance via their ability to be taken-up by beta cells and inhibit glucose-stimulated β-cell insulin secretion. METHODS:To investigate a possible circuit between meal-stimulated 3,4-dihydroxy-L-phenylalanine (L-DOPA) and dopamine (DA) production in the GI tract and pancreatic β-cells, we: 1) mapped GI mucosal expression of tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC); 2) measured L-DOPA and DA content of GI mucosal tissues following meal challenges with different L-tyrosine (TYR) content, 3) determined whether meal TYR content impacts plasma insulin and glucose excursions; and 4) characterized postprandial plasma excursions of L-DOPA and DA in response to meal tyrosine content in rodents and a population of bariatric surgery patients. Next, we characterized: 1) the metabolic transformation of TYR and L-DOPA into DA in vitro using purified islet tissue; 2) the metabolic transformation of orally administrated stable isotope labeled TYR into pancreatic DA, and 3) using a nuclear medicine technique, we studied endocrine beta cells in situ release and binding of DA in response to a glucose challenge. RESULTS:We demonstrate in rodents that intestinal content and circulatory concentrations L-DOPA and DA, plasma glucose and insulin are responsive to the tyrosine (TYR) content of a test meal. Intestinal expression of two enzymes, Tyrosine hydroxylase (TH) and Aromatic Amino acid Decarboxylase (AADC), essential to the transformation of TYR to DA was mapped and the metabolism of metabolism of TYR to DA was traced in human islets and a rodent beta cell line in vitro and from gut to the pancreas in vivo. Lastly, we show that β cells secrete and bind DA in situ in response to glucose stimulation. CONCLUSIONS:We provide proof-of-principle evidence for the existence of a novel postprandial circuit of glucose homeostasis dependent on nutritional tyrosine. DA and L-DOPA derived from nutritional tyrosine may serve to defend against hypoglycemia via inhibition of glucose-stimulated β-cell insulin secretion as proposed by the anti-incretin hypothesis.

Dataset Information

A phosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism.

Publications

A phosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism.

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