Activation of the cAMP-PKA pathway Antagonizes Metformin Suppression of Hepatic Glucose Production.

Metformin is the most commonly prescribed oral anti-diabetic agent worldwide. Surprisingly, about 35% of diabetic patients either lack or have a delayed response to metformin treatment, and many patients become less responsive to metformin over time. It remains unknown how metformin resistance or insensitivity occurs. Recently, we found that therapeutic metformin concentrations suppressed glucose production in primary hepatocytes through AMPK; activation of the cAMP-PKA pathway negatively regulates AMPK activity by phosphorylating AMPKα subunit at Ser-485, which in turn reduces AMPK activity. In this study, we find that metformin failed to suppress glucose production in primary hepatocytes with constitutively activated PKA and did not improve hyperglycemia in mice with hyperglucagonemia. Expression of the AMPKα1(S485A) mutant, which is unable to be phosphorylated by PKA, increased both AMPKα activation and the suppression of glucose production in primary hepatocytes treated with metformin. Intriguingly, salicylate/aspirin prevents the phosphorylation of AMPKα at Ser-485, blocks cAMP-PKA negative regulation of AMPK, and improves metformin resistance. We propose that aspirin/salicylate may augment metformin's hepatic action to suppress glucose production.

Metformin activates AMP-activated protein kinase by promoting formation of the αßγ heterotrimeric complex.

Metformin is the most widely prescribed oral anti-diabetic agent. Recently, we have shown that low metformin concentrations found in the portal vein suppress glucose production in hepatocytes through activation of AMPK. Moreover, low concentrations of metformin were found to activate AMPK by increasing the phosphorylation of AMPKα at Thr-172. However, the mechanism underlying the increase in AMPKα phosphorylation at Thr-172 and activation by metformin remains unknown. In the current study, we find that low concentrations of metformin promote the formation of the AMPK αßγ complex, resulting in an increase in net phosphorylation of the AMPK α catalytic subunit at Thr-172 by augmenting phosphorylation by LKB1 and antagonizing dephosphorylation by PP2C.

Low concentrations of metformin suppress glucose production in hepatocytes through AMP-activated protein kinase (AMPK).

Metformin is a first-line antidiabetic agent taken by 150 million people across the world every year, yet its mechanism remains only partially understood and controversial. It was proposed that suppression of glucose production in hepatocytes by metformin is AMPK-independent; however, unachievably high concentrations of metformin were employed in these studies. In the current study, we find that metformin, via an AMP-activated protein kinase (AMPK)-dependent mechanism, suppresses glucose production and gluconeogenic gene expression in primary hepatocytes at concentrations found in the portal vein of animals (60-80 µM). Metformin also inhibits gluconeogenic gene expression in the liver of mice administered orally with metformin. Furthermore, the cAMP-PKA pathway negatively regulates AMPK activity through phosphorylation at Ser-485/497 on the α subunit, which in turn reduces net phosphorylation at Thr-172. Because diabetic patients often have hyperglucagonemia, AMPKα phosphorylation at Ser-485/497 is a therapeutic target to improve metformin efficacy.

Control of Foxo1 gene expression by co-activator P300.

FOXO1 is an important downstream mediator of the insulin signaling pathway. In the fed state, elevated insulin phosphorylates FOXO1 via AKT, leading to its nuclear exclusion and degradation. A reduction in nuclear FOXO1 levels then leads to suppression of hepatic glucose production. However, the mechanism leading to expression of Foxo1 gene in the fasted state is less clear. We found that Foxo1 mRNA and FOXO1 protein levels of Foxo1 were increased significantly in the liver of mice after 16 h of fasting. Furthermore, dibutyrl cAMP stimulated the expression of Foxo1 at both mRNA and protein level in hepatocytes. Because cAMP-PKA regulates hepatic glucose production through cAMP-response element-binding protein co-activators, we depleted these co-activators using adenoviral shRNAs. Interestingly, only depletion of co-activator P300 resulted in the decrease of Foxo1 mRNA and FOXO1 protein levels. In addition, inhibition of histone acetyltransferase activity of P300 significantly decreased hepatic Foxo1 mRNA and FOXO1 protein levels in fasted mice, as well as fasting blood glucose levels. By characterization of Foxo1 gene promoter, P300 regulates the Foxo1 gene expression through the binding to tandem cAMP-response element sites in the proximal promoter region of Foxo1 gene.

Spatially Organized Films from Bdellovibrio bacteriovorus Prey Lysates.

Bdellovibrio bacteriovorus is a Gram-negative predator of other Gram-negative bacteria. Interestingly, Bdellovibrio bacteriovorus 109J cells grown in coculture with Escherichia coli ML-35 prey develop into a spatially organized two-dimensional film when located on a nutrient-rich surface. From deposition of 10 µl of a routine cleared coculture of B. bacteriovorus and E. coli cells, the cells multiply into a macroscopic community and segregate into an inner, yellow circular region and an outer, off-white region. Fluorescence in situ hybridization and atomic force microscopy measurements confirm that the mature film is spatially organized into two morphologically distinct Bdellovibrio populations, with primarily small, vibroid cells in the center and a complex mixture of pleomorphic cells in the outer radii. The interior region cell population exhibits the hunting phenotype while the outer region cell subpopulation does not. Crowding and high nutrient availability with limited prey appear to favor diversification of the B. bacteriovorus population into two distinct, thriving subpopulations and may be beneficial to the persistence of B. bacteriovorus in biofilms.

Studying submicrosecond protein folding kinetics using a photolabile caging strategy and time-resolved photoacoustic calorimetry.

Kinetic measurement of protein folding is limited by the method used to trigger folding. Traditional methods, such as stopped flow, have a long mixing dead time and cannot be used to monitor fast folding processes. Here, we report a compound, 4-(bromomethyl)-6,7-dimethoxycoumarin, that can be used as a "photolabile cage" to study the early stages of protein folding. The folding process of a protein, RD1, including kinetics, enthalpy, and volume change, was studied by the combined use of a phototriggered caging strategy and time-resolved photoacoustic calorimetry. The cage caused unfolding of the photolabile protein, and then a pulse UV laser (∼10(-9) s) was used to break the cage, leaving the protein free to refold and allowing the resolving of two folding events on a nanosecond time scale. This strategy is especially good for monitoring fast folding proteins that cannot be studied by traditional methods.

A new amyloid-like beta-aggregate with amyloid characteristics, except fibril morphology.

Amyloid plaques, formed from amyloid beta (Abeta) peptides (mainly Abeta40 or Abeta42), are one of the most important pathological characteristics of Alzheimer's disease. Here, a single D-form proline substitution in the 40-amino-acid Abeta40 peptide can totally change the aggregation behavior of this peptide. The residue immediately preceding each glycine in Abeta40 (S8, V24, I32, and V36) was individually replaced by D-form proline ((D)Pro). The resulting (D)P-G sequence (the (D)Pro residue and the following Gly residue) was designed as a "structural clip" to force the formation of a bend in the peptide, as this sequence has been reported to be a strong promoter of beta-hairpin formation. The mutant peptide with Val24-to-(D)Pro substitution, named V24P, formed a new amyloid-like beta-aggregate at high peptide concentration. The aggregate has most of the characteristics of amyloid fibrils, except fibril morphology. Moreover, the mutant peptide V24P, when mixed with Abeta40, can attenuate the cytotoxicity of Abeta40.