Impaired insulin-stimulated glycogen synthesis of peripheral tissues is a characteristic feature of many patients with non-insulin-dependent diabetes mellitus (NIDDM) and their first-degree relatives with normal glucose tolerance, suggesting putative inherited defects in this metabolic pathway. In previous studies, we have failed to reveal mutations in the coding regions of the muscle-specific glycogen synthase gene and the three genes that encode the catalytic subunits of protein phosphatase 1 (PP1) as frequent causes of insulin resistance. Because the glycogen-associated regulatory subunit of protein phosphatase 1 (PP1 G-subunit) plays a key role in the insulin stimulation of glycogen synthesis and the activity of PP1 is decreased in insulin-resistant subjects, we have now cloned the human G-subunit cDNA to search for abnormalities in the corresponding gene (designated PPP1R3 in the human genome nomenclature) in patients with NIDDM. The human cDNA was isolated from a skeletal muscle cDNA library and was found to encode a 126-kDa protein, which shows 73% amino acid identity to the rabbit PP1 G-subunit. The human G-subunit cDNA from 30 insulin-resistant NIDDM patients was analyzed for genetic variations in the G-subunit by using single-stranded conformation polymorphism (SSCP) scanning of reversely transcribed mRNA. One variant SSCP profile was detected in the region encoding the COOH-terminal part of the PP1 G-subunit in only one NIDDM patient, and subsequent nucleotide sequencing showed a C to A transversion on one allele at base position 2792. This change predicts an amino acid substitution from alanine to glutamic acid. The carrier of this mutation was characterized by reduced insulin-stimulated nonoxidative glucose metabolism when examined with the euglycemic hyperinsulinemic clamp. SSCP scanning of the 2584–2844 nucleotide fragment of PP1 G-subunit cDNA from an additional 22 NIDDM patients and 29 control subjects did not reveal additional genetic variants. To indirectly screen for abnormalities in PP1 G-subunit gene regulation, we measured the mRNA level of the G-subunit in skeletal muscle. However, no difference in the abundance of mRNA of PP1 G-subunit was found between patients with diabetes and control subjects.
Sequence of the Human Glycogen-Associated Regulatory Subunit of type 1 Protein Phosphatase and Analysis of Its Coding Region and mRNA Level in Muscle From Patients With NIDDM
NIDDM, non-insulin-dependent diabetes mellitus; GS, glycogen synthase; PP1, protein phosphatase type 1; C-subunit, catalytic subunit; G-subunit, glycogenassociated regulatory subunit; PKA, protein kinase A; ISPK-1, insulin-stimulated protein kinase 1; PCR, polymerase chain reaction; RIA, radioimmunoassay; NEFA, nonesterified fatty acid; nt, nucleotide; kb, kilobase; dNTP, 2′deoxynucleoside 5′ triphosphate; bp, base pair; SSCP, single-stranded conformation polymorphism; BMI, body mass index.
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Yu Hua Chen, Lars Hansen, Mao Xiang Chen, Christian Bjørbæk, Henrik Vestergaard, Torben Hansen, Paticia T W Cohen, Oluf Pedersen; Sequence of the Human Glycogen-Associated Regulatory Subunit of type 1 Protein Phosphatase and Analysis of Its Coding Region and mRNA Level in Muscle From Patients With NIDDM. Diabetes 1 October 1994; 43 (10): 1234–1241. https://doi.org/10.2337/diab.43.10.1234
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