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Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its antihyperglycemic action primarily through lowering hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5'-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose-2,6-bisphosphate (F-2,6-P), and knock-in (KI) of this mutant in mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The antihyperglycemic effect of metformin in high-fat diet-fed diabetic FBP1-KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action for metformin and provide further evidence that molecular targeting of FBP1 can have antihyperglycemic effects.
The Schizosaccharomyces pombe fbp1 gene, encoding fructose-1,6-bisphosphatase, is transcriptionally repressed by glucose. Mutations that confer constitutive fbp1 transcription identify git (glucose-insensitive transcription) genes that encode components of a cyclic AMP (cAMP) signaling pathway required for adenylate cyclase activation. Four of these genes encode the three subunits of a heterotrimeric G protein (gpa2, git5, and git11) and a G protein-coupled receptor (git3). Three additional genes, git1, git7, and git10, act in parallel to or downstream from the G protein genes. Here, we describe the cloning and characterization of the git7 gene. The Git7p protein is a member of the Saccharomyces cerevisiae Sgtlp protein family. In budding yeast, Sgtlp associates with Skplp and plays an essential role in kinetochore assembly, while in Arabidopsis, a pair of SGT1 proteins have been found to be involved in plant disease resistance through an interaction with RAR1. Like S. cerevisiae Sgtlp, Git7p is essential, but this requirement appears to be due to roles in septation and cell wall integrity, which are unrelated to cAMP signaling, as S. pombe cells lacking either adenylate cyclase or protein kinase A are viable. In addition, git7 mutants are sensitive to the microtubule-destabilizing drug benomyl, although they do not display a chromosome stability defect. Two alleles of git7 that are functional for cell growth and septation but defective for glucose-triggered cAMP signaling encode proteins that are altered in the highly conserved carboxy terminus. The S. cerevisiae and human SGT1 genes both suppress git7-93 but not git7-235 for glucose repression of fbp1 transcription and benomyl sensitivity. This allele-specific suppression indicates that the Git7p/Sgtlp proteins may act as multimers, such that Git7-93p but not Git7-235p can deliver the orthologous proteins to species-specific targets. Our studies suggest that members of the Git7p/Sgt1p protein family may play a conserved role in the regulation of adenylate cyclase activation in S. pombe, S. cerevisiae, and humans.
The relationship between the enhanced responses of gluconeogenesis to norepinephrine (NE) and glucagon and its zonal distribution was studied in liver lobules of cold-exposed rats by examination of preparations enriched for periportal hepatocytes (PP-H) and for perivenous hepatocytes (PV-H) by the digitonin-collagenase perfusion technique. In the control group, gluconeogenesis from lactate (10 mM) plus pyruvate (1 mM) was higher in PP-H than in PV-H. NE (100 nM) and glucagon (100 nM) increased the rate of gluconeogenesis by 80 and 70%, respectively, in both PP-H and PV-H. Gluconeogenesis in PP-H was unchanged by cold exposure. The rate in PV-H increased to the rate in PP-H at 5 days after cold exposure, and then the rate returned to the control value at 20 days. The gluconeogenic response to the alpha-adrenergic action of NE in both PP-H and PV-H doubled after 5 days. The response to glucagon tripled in PP-H and was cut in half in PV-H after 20 days. Phorbol 12-myristate 13-acetate (PMA; 1 microM), A-23187 (100 nM), and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP; 1 mM) increased the rate of gluconeogenesis by 200, 100, and 80%, respectively, in both PP-H and PV-H from the control group. The responses to PMA and A-23187 were unchanged by exposure to cold. The response to DBcAMP was doubled in PP-H and was cut in half in PV-H after 20 days of cold exposure.(ABSTRACT TRUNCATED AT 250 WORDS)