Domenico Accili

Insulin-regulated hepatic gluconeogenesis through FOXO1–PGC-1α interaction

Hepatic gluconeogenesis is absolutely required for survival during prolonged fasting or starvation, but is inappropriately activated in diabetes mellitus. Glucocorticoids and glucagon have strong gluconeogenic actions on the liver. In contrast, insulin suppresses hepatic gluconeogenesis. Two components known to have important physiological roles in this process are the forkhead transcription factor FOXO1 (also known as FKHR) and peroxisome proliferative activated receptor-γ co-activator 1 (PGC-1α; also known as PPARGC1), a transcriptional co-activator; whether and how these factors collaborate has not been clear. Using wild-type and mutant alleles of FOXO1, here we show that PGC-1α binds and co-activates FOXO1 in a manner inhibited by Akt-mediated phosphorylation. Furthermore, FOXO1 function is required for the robust activation of gluconeogenic gene expression in hepatic cells and in mouse liver by PGC-1α. Insulin suppresses gluconeogenesis stimulated by PGC-1α but co-expression of a mutant allele of FOXO1 insensitive to insulin completely reverses this suppression in hepatocytes or transgenic mice. We conclude that FOXO1 and PGC-1α interact in the execution of a programme of powerful, insulin-regulated gluconeogenesis.

FoxOs at the crossroads of cellular metabolism, differentiation, and transformation

Forkhead transcription factors of the FoxO subfamily are emerging as a shared component among pathways regulating diverse cellular functions, such as differentiation, metabolism, proliferation, and survival. Their transcriptional output is controlled via a two-tiered mechanism of phosphorylation and acetylation. Modest alterations of this balance can result in profound effects. The gamut of phenotypes runs from protection against diabetes and predisposition to neoplasia, conferred by FoxO loss of function, to increased cellular survival and a marked catabolic response associated with gain of function.

The Forkhead Transcription Factor Foxo1 Regulates Adipocyte Differentiation

An outstanding question in adipocyte biology is how hormonal cues are relayed to the nucleus to activate the transcriptional program that promotes adipogenesis. The forkhead transcription factor Foxo1 is regulated by insulin via Akt-dependent phosphorylation and nuclear exclusion. We show that Foxo1 is induced in the early stages of adipocyte differentiation but that its activation is delayed until the end of the clonal expansion phase. Constitutively active Foxo1 prevents the differentiation of preadipocytes, while dominant-negative Foxo1 restores adipocyte differentiation of fibroblasts from insulin receptor-deficient mice. Further, Foxo1 haploinsufficiency protects from diet-induced diabetes in mice. We propose that Foxo1 plays an important role in the integration of hormone-activated signaling pathways with the complex transcriptional cascade that promotes adipocyte differentiation.

Regulation of insulin action and pancreatic β-cell function by mutated alleles of the gene encoding forkhead transcription factor Foxo1

Type 2 diabetes results from impaired action and secretion of insulin. It is not known whether the two defects share a common pathogenesis. We show that haploinsufficiency of the Foxo1 gene, encoding a forkhead transcription factor (forkhead box transcription factor O1), restores insulin sensitivity and rescues the diabetic phenotype in insulin-resistant mice by reducing hepatic expression of glucogenetic genes and increasing adipocyte expression of insulin-sensitizing genes. Conversely, a gain-of-function Foxo1 mutation targeted to liver and pancreatic β-cells results in diabetes arising from a combination of increased hepatic glucose production and impaired β-cell compensation due to decreased Pdx1 expression. These data indicate that Foxo1 is a negative regulator of insulin sensitivity in liver, adipocytes and pancreatic β-cells. Impaired insulin signaling to Foxo1 provides a unifying mechanism for the common metabolic abnormalities of type 2 diabetes.NOTE: In the AOP version of this article, the name of the fourth author was misspelled as W K Cavanee rather than the correct spelling: W K Cavenee. This has been corrected in the full-text online version of the article. The name will appear correctly in the print version.

Pancreatic β Cell Dedifferentiation as a Mechanism of Diabetic β Cell Failure

Diabetes is associated with β cell failure. But it remains unclear whether the latter results from reduced β cell number or function. FoxO1 integrates β cell proliferation with adaptive β cell function. We interrogated the contribution of these two processes to β cell dysfunction, using mice lacking FoxO1 in β cells. FoxO1 ablation caused hyperglycemia with reduced β cell mass following physiologic stress, such as multiparity and aging. Surprisingly, lineage-tracing experiments demonstrated that loss of β cell mass was due to β cell dedifferentiation, not death. Dedifferentiated β cells reverted to progenitor-like cells expressing Neurogenin3, Oct4, Nanog, and L-Myc. A subset of FoxO1-deficient β cells adopted the α cell fate, resulting in hyperglucagonemia. Strikingly, we identify the same sequence of events as a feature of different models of murine diabetes. We propose that dedifferentiation trumps endocrine cell death in the natural history of β cell failure and suggest that treatment of β cell dysfunction should restore differentiation, rather than promoting β cell replication.

SirT1 Gain of Function Increases Energy Efficiency and Prevents Diabetes in Mice

In yeast, worms, and flies, an extra copy of the gene encoding the Sirtuin Sir2 increases metabolic efficiency, as does administration of polyphenols like resveratrol, thought to act through Sirtuins. But evidence that Sirtuin gain of function results in increased metabolic efficiency in mammals is limited. We generated transgenic mice with moderate overexpression of SirT1, designed to mimic the Sirtuin gain of function that improves metabolism in C. elegans. These mice exhibit normal insulin sensitivity but decreased food intake and locomotor activity, resulting in decreased energy expenditure. However, in various models of insulin resistance and diabetes, SirT1 transgenics display improved glucose tolerance due to decreased hepatic glucose production and increased adiponectin levels, without changes in body weight or composition. We conclude that SirT1 gain of function primes the organism for metabolic adaptation to insulin resistance, increasing hepatic insulin sensitivity and decreasing whole-body energy requirements. These findings have important implications for Sirtuin-based therapies in humans.

The forkhead transcription factor Foxo1 links insulin signaling to Pdx1 regulation of pancreatic β cell growth

Diabetes is caused by an absolute (type 1) or relative (type 2) deficiency of insulin-producing beta cells. The mechanisms governing replication of terminally differentiated beta cells and neogenesis from progenitor cells are unclear. Mice lacking insulin receptor substrate-2 (Irs2) develop beta cell failure, suggesting that insulin signaling is required to maintain an adequate beta cell mass. We report that haploinsufficiency for the forkhead transcription factor Foxo1 reverses beta cell failure in Irs2(-/-) mice through partial restoration of beta cell proliferation and increased expression of the pancreatic transcription factor pancreas/duodenum homeobox gene-1 (Pdx1). Foxo1 and Pdx1 exhibit mutually exclusive patterns of nuclear localization in beta cells, and constitutive nuclear expression of a mutant Foxo1 is associated with lack of Pdx1 expression. We show that Foxo1 acts as a repressor of Foxa2-dependent (Hnf-3beta-dependent) expression from the Pdx1 promoter. We propose that insulin/IGFs regulate beta cell proliferation by relieving Foxo1 inhibition of Pdx1 expression in a subset of cells embedded within pancreatic ducts.

The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression

Type 2 diabetes is characterized by the inability of insulin to suppress glucose production in the liver and kidney. Insulin inhibits glucose production by indirect and direct mechanisms. The latter result in transcriptional suppression of key gluconeogenetic and glycogenolytic enzymes, phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6p). The transcription factors required for this effect are incompletely characterized. We report that in glucogenetic kidney epithelial cells, Pepck and G6p expression are induced by dexamethasone (dex) and cAMP, but fail to be inhibited by insulin. The inability to respond to insulin is associated with reduced expression of the forkhead transcription factor Foxo1, a substrate of the Akt kinase that is inhibited by insulin through phosphorylation. Transduction of kidney cells with recombinant adenovirus encoding Foxo1 results in insulin inhibition of dex/cAMP-induced G6p expression. Moreover, expression of dominant negative Foxo1 mutant results in partial inhibition of dex/cAMP-induced G6p and Pepck expression in primary cultures of mouse hepatocyes and kidney LLC-PK1-FBPase(+) cells. These findings are consistent with the possibility that Foxo1 is involved in insulin regulation of glucose production by mediating the ability of insulin to decrease the glucocorticoid/cAMP response of G6p.

Structure-based prediction of protein–protein interactions on a genome-wide scale

The genome-wide identification of pairs of interacting proteins is an important step in the elucidation of cell regulatory mechanisms. Much of our present knowledge derives from high-throughput techniques such as the yeast two-hybrid assay and affinity purification, as well as from manual curation of experiments on individual systems. A variety of computational approaches based, for example, on sequence homology, gene co-expression and phylogenetic profiles, have also been developed for the genome-wide inference of protein–protein interactions (PPIs). Yet comparative studies suggest that the development of accurate and complete repertoires of PPIs is still in its early stages. Here we show that three-dimensional structural information can be used to predict PPIs with an accuracy and coverage that are superior to predictions based on non-structural evidence. Moreover, an algorithm, termed PrePPI, which combines structural information with other functional clues, is comparable in accuracy to high-throughput experiments, yielding over 30,000 high-confidence interactions for yeast and over 300,000 for human. Experimental tests of a number of predictions demonstrate the ability of the PrePPI algorithm to identify unexpected PPIs of considerable biological interest. The surprising effectiveness of three-dimensional structural information can be attributed to the use of homology models combined with the exploitation of both close and remote geometric relationships between proteins.

Dual role of transcription factor FoxO1 in controlling hepatic insulin sensitivity and lipid metabolism.

Hepatic insulin resistance affects both carbohydrate and lipid metabolism. It has been proposed that insulin controls these 2 metabolic branches through distinct signaling pathways. FoxO transcription factors are considered effectors of the pathway regulating hepatic glucose production. Here we show that adenoviral delivery of constitutively nuclear forkhead box O1 (FoxO1) to mouse liver results in steatosis arising from increased triglyceride accumulation and decreased fatty acid oxidation. FoxO1 gain of function paradoxically increased insulin sensitivity by promoting Akt phosphorylation, while FoxO1 inhibition via siRNA decreased it. We show that FoxO1 regulation of Akt phosphorylation does not require DNA binding and is associated with repression of the pseudokinase tribble 3 (Trb3), a modulator of Akt activity. This unexpected dual role of FoxO1 in promoting insulin sensitivity and lipid synthesis in addition to glucose production has the potential to explain the peculiar admixture of insulin resistance and sensitivity that is commonly observed in the metabolic syndrome.