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OBJECTIVE Atherosclerotic cardiovascular disease is the leading cause of death among people with diabetes. Generation of oxidized LDLs and reduced nitric oxide (NO) availability because of endothelial NO synthase (eNOS) dysfunction are critical events in atherosclerotic plaque formation. Biochemical mechanism leading from hyperglycemia to oxLDL formation and eNOS dysfunction is unknown. RESEARCH DESIGN AND METHODS We show that glucose, acting through oxidative stress, activates the transcription factor Foxo1 in vascular endothelial cells. RESULTS Foxo1 promotes inducible NOS (iNOS)-dependent NO-peroxynitrite generation, which leads in turn to LDL oxidation and eNOS dysfunction. We demonstrate that Foxo1 gain-of-function mimics the effects of hyperglycemia on this process, whereas conditional Foxo1 knockout in vascular endothelial cells prevents it. CONCLUSIONS The findings reveal a hitherto unsuspected role of the endothelial iNOS-NO-peroxynitrite pathway in lipid peroxidation and eNOS dysfunction and suggest that Foxo1 activation in response to hyperglycemia brings about proatherogenic changes in vascular endothelial cell function.

Type 2 diabetes is associated with accelerated atherogenesis, which may result from a combination of factors, including dyslipidemia characterized by increased VLDL secretion, and insulin resistance. To assess the hypothesis that both hepatic and peripheral insulin resistance contribute to atherogenesis, we crossed mice deficient for the LDL receptor (Ldlr-/- mice) with mice that express low levels of IR in the liver and lack IR in peripheral tissues (the L1B6 mouse strain). Unexpectedly, compared with Ldlr-/- controls, L1B6Ldlr-/- mice fed a Western diet showed reduced VLDL and LDL levels, reduced atherosclerosis, decreased hepatic AKT signaling, decreased expression of genes associated with lipogenesis, and diminished VLDL apoB and lipid secretion. Adenovirus-mediated hepatic expression of either constitutively active AKT or dominant negative glycogen synthase kinase (GSK) markedly increased VLDL and LDL levels such that they were similar in both Ldlr-/- and L1B6Ldlr-/- mice. Knocking down expression of hepatic IR by adenovirus-mediated shRNA decreased VLDL triglyceride and apoB secretion in Ldlr-/- mice. Furthermore, knocking down hepatic IR expression in either WT or ob/ob mice reduced VLDL secretion but also resulted in decreased hepatic Ldlr protein. These findings suggest a dual action of hepatic IR on lipoprotein levels, in which the ability to increase VLDL apoB and lipid secretion via AKT/GSK is offset by upregulation of Ldlr.

OBJECTIVE—Endoplasmic reticulum stress increases macrophage apoptosis, contributing to the complications of atherosclerosis. Insulin-resistant macrophages are more susceptible to endoplasmic reticulum stress–associated apoptosis probably contributing to macrophage death and necrotic core formation in atherosclerotic plaques in type 2 diabetes. However, the molecular mechanisms of increased apoptosis in insulin-resistant macrophages remain unclear. RESEARCH DESIGN AND METHODS—The studies were performed in insulin-resistant macrophages isolated from insulin receptor knockout or ob/ob mice. Gain- or loss-of-function approaches were used to evaluate the roles of forkhead transcription factors (FoxOs) in endoplasmic reticulum stress–associated macrophage apoptosis. RESULTS—Insulin-resistant macrophages showed attenuated Akt activation and increased nuclear localization of FoxO1 during endoplasmic reticulum stress induced by free cholesterol loading. Overexpression of active FoxO1 or FoxO3 failed to induce apoptosis in unchallenged macrophages but exacerbated apoptosis in macrophages with an active endoplasmic reticulum stress response. Conversely, macrophages with genetic knockouts of FoxO1, -3, and -4 were resistant to apoptosis in response to endoplasmic reticulum stress. FoxO1 was shown by chromatin immunoprecipitation and promoter expression analysis to induce inhibitor of κBε gene expression and thereby to attenuate the increase of nuclear p65 and nuclear factor-κB activity during endoplasmic reticulum stress, with proapoptotic and anti-inflammatory consequences. CONCLUSIONS—Decreased Akt and increased FoxO transcription factor activity during the endoplasmic reticulum stress response leads to increased apoptosis of insulin-resistant macrophages. FoxOs may have a dual cellular function, resulting in either proapoptotic or anti-inflammatory effects in an endoplasmic reticulum stress–modulated manner. In the complex plaque milieu, the ultimate effect is likely to be an increase in macrophage apoptosis, plaque inflammation, and destabilization.

Forkhead box O (Foxo) transcription factors govern metabolism and cellular differentiation. Unlike Foxo-dependent metabolic pathways and target genes, the mechanisms by which these proteins regulate differentiation have not been explored. Activation of Notch signaling mimics the effects of Foxo gain of function on cellular differentiation. Using muscle differentiation as a model system, we show that Foxo physically and functionally interacts with Notch by promoting corepressor clearance from the Notch effector Csl, leading to activation of Notch target genes. Inhibition of myoblast differentiation by constitutively active Foxo1 is partly rescued by inhibition of Notch signaling while Foxo1 loss of function precludes Notch inhibition of myogenesis and increases myogenic determination gene (MyoD) expression. Accordingly, conditional Foxo1 ablation in skeletal muscle results in increased formation of MyoD-containing (fast-twitch) muscle fibers and altered fiber type distribution at the expense of myogenin-containing (slow-twitch) fibers. Notch/Foxo1 cooperation may integrate environmental cues through Notch with metabolic cues through Foxo1 to regulate progenitor cell maintenance and differentiation.

SummaryThe hallmark of type 2 diabetes is excessive hepatic glucose production. Several transcription factors and coactivators regulate this process in cultured cells. But gene ablation experiments have yielded few clues as to the physiologic mediators of this process in vivo. We show that inactivation of the gene encoding forkhead protein Foxo1 in mouse liver results in 40% reduction of glucose levels at birth and 30% reduction in adult mice after a 48 hr fast. Gene expression and glucose clamp studies demonstrate that Foxo1 ablation impairs fasting- and cAMP-induced glycogenolysis and gluconeogenesis. Pgc1α is unable to induce gluconeogenesis in Foxo1-deficient hepatocytes, while the cAMP response is significantly blunted. Conversely, Foxo1 deletion in liver curtails excessive glucose production caused by generalized ablation of insulin receptors and prevents neonatal diabetes and hepatosteatosis in insulin receptor knockout mice. The data provide a unifying mechanism for regulation of hepatic glucose production by cAMP and insulin.

OBJECTIVE— Adiponectin increases insulin sensitivity and contributes to insulin's indirect effects on hepatic glucose production.RESEARCH DESIGN AND METHODS— To examine adiponectin's contribution to insulin action, we analyzed adiponectin levels and activation of AMP-activated protein kinase (AMPK) in insulin receptor transgenic/knockout mice (L1), a genetic model of resistance to insulin's indirect effects on hepatic glucose production.RESULTS— In euglycemic, insulin-resistant L1 mice, we detected hyperadiponectinemia with normal levels of adiponectin receptor-1 and -2. Moreover, adiponectin administration is unable to lower glucose levels or induce activation of AMPK, consistent with a state of adiponectin resistance. In a subset of hyperglycemic L1 mice, we observed decreased mRNA expression of AdipoR2 in liver and muscle, as well as decreased peroxisome proliferator–activated receptor (PPAR)α target gene expression in liver, raising the possibility that deterioration of adiponectin/AdipoR2 signaling via PPARα activation contributes to the progression from compensated insulin resistance to diabetes. In contrast, we failed to detect changes in other markers of the systemic or local inflammatory response.CONCLUSIONS— These data provide evidence for a mechanism of adiponectin resistance and corroborate the notion that adiponectin potentiates hepatic insulin sensitivity.

Type 2 diabetes results from impaired insulin action and beta-cell dysfunction. There are at least two components to beta-cell dysfunction: impaired insulin secretion and decreased beta-cell mass. To analyze how these two variables contribute to the progressive deterioration of metabolic control seen in diabetes, we asked whether mice with impaired beta-cell growth due to Irs2 ablation would be able to mount a compensatory response in the background of insulin resistance caused by Insr haploinsufficiency. As previously reported, approximately 70% of mice with combined Insr and Irs2 mutations developed diabetes as a consequence of markedly decreased beta-cell mass. In the initial phases of the disease, we observed a robust increase in circulating insulin levels, even as beta-cell mass gradually declined, indicating that replication-defective beta-cells compensate for insulin resistance by increasing insulin secretion. These data provide further evidence for a heterogeneous beta-cell response to insulin resistance, in which compensation can be temporarily achieved by increasing function when mass is limited. The eventual failure of compensatory insulin secretion suggests that a comprehensive treatment of beta-cell dysfunction in type 2 diabetes should positively affect both aspects of beta-cell physiology.

Insulin, proinsulin, and C-peptide levels increase with sulfonylurea exposure but the acuity of increase has not been described in dialysis patients. We present a case of a dialysis patient who presented with hypoglycemia and was found to have accidental sulfonylurea ingestion. This is a 73-year-old man with ESRD on peritoneal dialysis, without history of diabetes, who presented with hypoglycemia. Past medical history includes multiple myeloma, congestive heart failure, and hypertension. At initial presentation, his blood glucose was 47 mg/dL, with concomitant elevations in the following: C-peptide 30.5 (nl: 0.8–3.5 ng/mL), insulin 76 (nl: 3–19 μIU/mL), and proinsulin 83.3 (nl: ≤8.0 pmol/L). During the 72-hour fast, which he completed without hypoglycemia, insulin declined to be within normal limits (to 12 μIU/mL); proinsulin (to 12.1 pmol/L) and C-peptide (to 7.2 ng/mL) levels decreased but remained elevated. The sulfonylurea screen ultimately returned positive for glipizide, clinching the diagnosis. This is the first reported case which characterizes the chronic elevation of proinsulin in a patient with ESRD, as well as its dramatic increase after a presumed solitary exposure to sulfonylurea. The 72-hour fast conducted gives insight into the clearance of insulin, proinsulin, and C-peptide after sulfonylurea ingestion in ESRD.

The development of insulin resistance (IR) in the liver is a key pathophysiologic event in the development of type 2 diabetes. Although insulin loses its ability to suppress glucose production, it largely retains its capacity to drive lipogenesis. This selective IR results in the characteristic hyperglycemia and dyslipidemia of type 2 diabetes. The delineation of two branched pathways of insulin receptor (InsR) signaling to glucose versus triglyceride production, one through FoxO and the other through SREBP-1c, provides a mechanism to account for this pathophysiological abnormality. We tested the complementary hypothesis that selective IR arises due to different intrinsic sensitivities of glucose production versus de novo lipogenesis to insulin as a result of cell-autonomous down-regulation of InsR number in response to chronic hyperinsulinemia. We demonstrate in mouse primary hepatocytes that chronic hyperinsulinemia abrogates insulin's inhibition of glucose production, but not its stimulation of de novo lipogenesis. Using a competitive inhibitor of InsR, we show that there is a 4-fold difference between levels of InsR inhibition required to cause resistance of glucose production versus lipogenesis to the actions of insulin. Our data support a parsimonious model in which differential InsR activation underlies the selective IR of glucose production relative to lipogenesis, but both processes require signaling through Akt1/2.

Leptin controls food intake by regulating the transcription of key neuropeptides in the hypothalamus. The mechanism by which leptin regulates gene expression is unclear, however. Here we show that delivery of adenovirus encoding a constitutively nuclear mutant FoxO1, a transcription factor known to control liver metabolism and pancreatic beta-cell function, to the hypothalamic arcuate nucleus of rodents results in a loss of the ability of leptin to curtail food intake and suppress expression of Agrp. Conversely, a transactivation-deficient FoxO1 mutant prevents induction of Agrp by fasting. We also find that FoxO1 and the transcription factor Stat3 exert opposing actions on the expression of Agrp and Pomc through transcriptional squelching. FoxO1 promotes opposite patterns of coactivator-corepressor exchange at the Pomc and Agrp promoters, resulting in activation of Agrp and inhibition of Pomc. Thus, FoxO1 represents a shared component of pathways integrating food intake and peripheral metabolism.

The ectoenzyme PC-1 is an insulin receptor inhibitor that is elevated in cells and tissues of humans with type 2 diabetes (T2D). We have recently shown that acute PC-1 overexpression in liver causes insulin resistance and glucose intolerance in mice ( 3 ), but the chronic effects of PC-1 overexpression on these functions are unknown. Herein we produced transgenic mice overexpressing the potent q allele of human PC-1 in muscle and liver. Compared with controls, these mice had 2- to 3-fold elevations of PC-1 content in liver and 5- to 10-fold elevations in muscle. In the fed state, the PC-1 animals had 100 mg/dl higher glucose levels and sixfold higher insulin levels compared with controls. During glucose tolerance tests, these PC-1 animals had peak glucose levels that were >150 mg/dl higher than controls. In vivo uptake of 2-deoxy-d-glucose in muscle during insulin infusion was decreased in the PC-1 animals. These in vivo data support the concept, therefore, that PC-1 plays a role in insulin resistance and hyperglycemia and suggest that animals with overexpression of human PC-1 in insulin-sensitive tissues may be important models to investigate insulin resistance.

Activation of inflammatory pathways may contribute to the beginning and the progression of both atherosclerosis and type 2 diabetes. Here we report a novel interaction between insulin action and control of inflammation, resulting in glucose intolerance and vascular inflammation and amenable to therapeutic modulation. In insulin receptor heterozygous (Insr+/-) mice, we identified the deficiency of tissue inhibitor of metalloproteinase 3 (Timp3, an inhibitor of both TNF-alpha-converting enzyme [TACE] and MMPs) as a common bond between glucose intolerance and vascular inflammation. Among Insr+/- mice, those that develop diabetes have reduced Timp3 and increased TACE activity. Unchecked TACE activity causes an increase in levels of soluble TNF-alpha, which subsequently promotes diabetes and vascular inflammation. Double heterozygous Insr+/-Timp3+/- mice develop mild hyperglycemia and hyperinsulinemia at 3 months and overt glucose intolerance and hyperinsulinemia at 6 months. A therapeutic role for Timp3/TACE modulation is supported by the observation that pharmacological inhibition of TACE led to marked reduction of hyperglycemia and vascular inflammation in Insr+/- diabetic mice, as well as by the observation of increased insulin sensitivity in Tace+/- mice compared with WT mice. Our results suggest that an interplay between reduced insulin action and unchecked TACE activity promotes diabetes and vascular inflammation.

Type 2 diabetes arises from a combination of impaired insulin action and defective pancreatic β-cell function. Classically, the two abnormalities have been viewed as distinct yet mutually detrimental processes. The combination of impaired insulin-dependent glucose metabolism in skeletal muscle and impaired β-cell function causes an increase of hepatic glucose production, leading to a constellation of tissue abnormalities that has been referred to as the diabetes “ruling triumvirate.” Targeted mutagenesis in mice has led to a critical reappraisal of the integrated physiology of insulin action. These studies indicate that insulin resistance in skeletal muscle and adipose tissue does not necessarily lead to hyperglycemia, so long as insulin sensitivity in other tissues is preserved. Additional data suggest a direct role of insulin signaling in β-cell function and regulation of β-cell mass, thus raising the possibility that insulin resistance may be the overarching feature of diabetes in all target tissues. I propose that we replace the original picture of a ruling triumvirate with that of a squabbling republic in which every tissue contributes to the onset of the disease.

Accelerated atherosclerosis is a major cause of morbidity and death in insulin-resistant states such as obesity and the metabolic syndrome, but the underlying mechanisms are poorly understood. We show that macrophages from obese (ob/ob) mice have increased binding and uptake of oxidized LDL, in part due to a post-transcriptional increase in CD36 protein. Macrophages from ob/ob mice are also insulin resistant, as shown by reduced expression and signaling of insulin receptors. Three lines of evidence indicate that the increase in CD36 is caused by defective insulin signaling: (a) Treatment of wild-type macrophages with LY294002, an inhibitor of insulin signaling via PI3K, results in an increase in CD36; (b) insulin receptor knockout macrophages show a post-transcriptional increase in CD36 protein; and (c) administration of thiazolidinediones to intact ob/ob mice and ob/ob, LDL receptor-deficient mice results in a reversal of macrophage insulin receptor defects and decreases CD36 protein. The last finding contrasts with the increase in CD36 that results from treatment of macrophages with these drugs ex vivo. The results suggest that defective macrophage insulin signaling predisposes to foam cell formation and atherosclerosis in insulin-resistant states and that this is reversed in vivo by treatment with PPAR-gamma activators.

Insulin promotes both metabolism and growth. However, it is unclear whether insulin-dependent growth is merely a result of its metabolic actions. Targeted ablation of insulin receptor (Insr) has not clarified this issue, because of early postnatal lethality. To examine this question, we generated mice with variable cellular mosaicism for null Insr alleles. Insr ablation in approximately 80% of cells caused extreme growth retardation, lipoatrophy, and hypoglycemia, a clinical constellation that resembles the human syndrome of leprechaunism. Insr ablation in 98% of cells, while resulting in similar growth retardation and lipoatrophy, caused diabetes without beta-cell hyperplasia. The growth retardation was associated with a greater than 60-fold increase in the expression of hepatic insulin-like growth factor binding protein-1. These findings indicate that insulin regulates growth independently of metabolism and that the number of insulin receptors is an important determinant of the specificity of insulin action.

In mice, gonads are formed shortly before embryonic day 10.5 by the thickening of the mesonephros and consist of somatic cells and migratory primordial germ cells. The male sex-determining process is set in motion by the sex-determining region of the Y chromosome (Sry), which triggers differentiation of the Sertoli cell lineage. In turn, Sertoli cells function as organizing centres and direct differentiation of the testis. In the absence of Sry expression, neither XX nor XY gonads develop testes, and alterations in Sry expression are often associated with abnormal sexual differentiation. The molecular signalling mechanisms by which Sry specifies the male pathway and models the undifferentiated gonad are unknown. Here we show that the insulin receptor tyrosine kinase family, comprising Ir, Igf1r and Irr, is required for the appearance of male gonads and thus for male sexual differentiation. XY mice that are mutant for all three receptors develop ovaries and show a completely female phenotype. Reduced expression of both Sry and the early testis-specific marker Sox9 indicates that the insulin signalling pathway is required for male sex determination.

Insulin-like growth factors promote myoblast differentiation through phosphoinositol 3-kinase and Akt signaling. Akt substrates required for myogenic differentiation are unknown. Forkhead transcription factors of the forkhead box gene, group O (Foxo) subfamily are phosphorylated in an insulin-responsive manner by phosphatidylinositol 3-kinase–dependent kinases. Phosphorylation leads to nuclear exclusion and inactivation. We show that a constitutively active Foxo1 mutant inhibits differentiation of C2C12 cells and prevents myotube differentiation induced by constitutively active Akt. In contrast, a transcriptionally inactive mutant Foxo1 partially rescues inhibition of C2C12 differentiation mediated by wortmannin, but not by rapamycin, and is able to induce aggregation-independent myogenic conversion of teratocarcinoma cells. Inhibition of Foxo expression by siRNA resulted in more efficient differentiation, associated with increased myosin expression. These observations indicate that Foxo proteins are key effectors of Akt-dependent myogenesis.

Mice bearing targeted gene mutations that affect insulin receptor (Insr) function have contributed important new information on the pathogenesis of type 2 diabetes. Whereas complete Insr ablation is lethal, conditional mutagenesis in selected tissues has more limited consequences on metabolism. Studies of mice with tissue-specific ablation of Insr have indicated that both canonical (e.g. muscle and adipose tissue) and noncanonical (e.g. liver, pancreatic beta-cells, and brain) insulin target tissues can contribute to insulin resistance, albeit in a pathogenically distinct fashion. Furthermore, experimental crosses of Insr mutants with mice carrying mutations that affect insulin action at more distal steps of the insulin signaling cascade have begun to unravel the genetics of type 2 diabetes. These studies are consistent with an oligogenic inheritance, in which synergistic interactions among few alleles may account for the genetic susceptibility to diabetes. In addition to mutant alleles conferring an increased risk of diabetes, these studies have uncovered mutations that protect against insulin resistance, thus providing proof-of-principle for the notion that certain alleles may confer resistance to diabetes.

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.

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 beta-cells results in diabetes arising from a combination of increased hepatic glucose production and impaired beta-cell compensation due to decreased Pdx1 expression. These data indicate that Foxo1 is a negative regulator of insulin sensitivity in liver, adipocytes and pancreatic beta-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.

Insulin and insulin-like growth factors (IGF) play overlapping and complementary roles in pancreatic beta-cell function and peripheral metabolism. In this study, we have analyzed mice bearing loss-of-function mutations of the insulin/IGF signaling systems. Combined inactivation of insulin receptor (Insr) and Igf1 receptor (Igf1r), but not of either receptor alone, resulted in a 90% decrease in the size of the exocrine pancreas, because of decreased cellular proliferation. In contrast to the findings in the exocrine compartment, endocrine alpha- and beta-cell development was unperturbed. Combined ablation of Igf1 and Igf2, the ligands for these two receptors, resulted in an identical phenotype. We also examined the effect of heterozygous null Igf1r mutations on glucose homeostasis in adult mice. Igf1r haploinsufficiency did not affect insulin action and compensatory beta-cell growth in insulin-resistant mice with combined Insr and Igf1r heterozygous null mutations, resulting in a considerably milder phenotype than combined haploinsufficiency for Insr and its main signaling substrates, Irs1 and Irs2. We conclude that Igf1r and Insr are required for embryonic development of the exocrine but not of the endocrine pancreas and that defects of Igf1r do not alter glucose homeostasis as long as the insulin receptor system remains intact.

Receptor tyrosine kinases of the insulin-insulin-like growth factor (IGF) family promote growth and mediate metabolic signals. Despite their extensive structural homology, genetic evidence indicates that their physiological functions are distinct. Nevertheless, there is limited evidence from cell culture systems suggesting that their signalling capabilities differ. Thus, it remains unclear whether the different physiological roles of insulin and IGF-I receptors result from intrinsic differences in their abilities to activate distinct signalling pathways, or arise from extrinsic differences, such as tissue distribution, relative abundance and developmental regulation.

On the basis of the recommendation of the American Diabetes Association’s Panel on Ethical Scientific Programs (ESP), the American Diabetes Association, the publisher of Diabetes, is issuing this expression of concern to alert readers to questions about the reliability of the data in the above-cited article. After readers of the journal contacted Diabetes about potentially duplicated images in the article, the ESP reviewed the following issues

Insulin resistance is a hallmark of diabetes and an unmet clinical need. Insulin inhibits hepatic glucose production and promotes lipogenesis by suppressing FOXO1-dependent activation of G6pase and inhibition of glucokinase, respectively. The tight coupling of these events poses a dual conundrum: mechanistically, as the FOXO1 corepressor of glucokinase is unknown, and clinically, as inhibition of glucose production is predicted to increase lipogenesis. Here, we report that SIN3A is the insulin-sensitive FOXO1 corepressor of glucokinase. Genetic ablation of SIN3A abolishes nutrient regulation of glucokinase without affecting other FOXO1 target genes and lowers glycemia without concurrent steatosis. To extend this work, we executed a small-molecule screen and discovered selective inhibitors of FOXO-dependent glucose production devoid of lipogenic activity in hepatocytes. In addition to identifying a novel mode of insulin action, these data raise the possibility of developing selective modulators of unliganded transcription factors to dial out adverse effects of insulin sensitizers.

Type 2 diabetes mellitus and its complications are, with cardiovascular diseases, leading threats to public health in the 21st century. In the United States, type 2 diabetes care accounts for a third of federal health insurance (Medicare) expenditures—nearly half of it to treat associated macrovascular problems ( 1 ). The cornerstone of type 2 diabetes is insulin resistance—a decreased sensitivity of tissues and organs, such as the liver, to the metabolic effects of the hormone insulin (the other major cause is failure of the pancreas to produce insulin). Yet, except for thiazolidinediones—whose checkered safety history, troublesome side effects, and regulatory setbacks stifled widespread adoption by clinicians—treatment options for insulin resistance have generally remained unchanged since the 1940s. Although insulin signaling pathways in cells have been largely deciphered ( 2 ), the key mediators of insulin signaling are poor drug targets because they either lack a suitable ligand-binding domain, or are shared with other cellular pathways that regulate cell growth and proliferation. This realization spawned research into “alternative pathways” that control insulin resistance. On page 1621 of this issue, Kir et al. ( 3 ) find that human fibroblast growth factor 19 (FGF19) can boost certain effects of insulin on the mammalian liver, raising interest and questions about possible therapies involving this molecule.

β-Cell dysfunction in diabetes results from abnormalities of insulin production, secretion, and cell number. These abnormalities may partly arise from altered developmental programming of β-cells. Foxo1 is important to maintain adult β-cells, but little is known about its role in pancreatic progenitor cells as determinants of future β-cell function. We addressed this question by generating an allelic series of somatic Foxo1 knockouts at different stages of pancreatic development in mice. Surprisingly, ablation of Foxo1 in pancreatic progenitors resulted in delayed appearance of Neurogenin3+ progenitors and their persistence into adulthood as a self-replicating pool, causing a fourfold increase of β-cell mass. Similarly, Foxo1 ablation in endocrine progenitors increased their numbers, extended their survival, and expanded β-cell mass. In contrast, ablation of Foxo1 in terminally differentiated β-cells did not increase β-cell mass nor did it affect Neurogenin3 expression. Despite the increased β-cell mass, islets from mice lacking Foxo1 in pancreatic or endocrine progenitors responded poorly to glucose, resulting in glucose intolerance. We conclude that Foxo1 integrates cues that determine developmental timing, pool size, and functional features of endocrine progenitor cells, resulting in a legacy effect on adult β-cell mass and function. Our results illustrate how developmental programming predisposes to β-cell dysfunction in adults and raise questions on the desirability of increasing β-cell mass for therapeutic purposes in type 2 diabetes.

Insulin integrates hepatic glucose and lipid metabolism, directing nutrients to storage as glycogen and triglyceride. In type 2 diabetes, levels of the former are low and the latter are exaggerated, posing a pathophysiologic and therapeutic conundrum. A branching model of insulin signalling, with FoxO1 presiding over glucose production and Srebp-1c regulating lipogenesis, provides a potential explanation. Here we illustrate an alternative mechanism that integrates glucose production and lipogenesis under the unifying control of FoxO. Liver-specific ablation of three FoxOs (L-FoxO1,3,4) prevents the induction of glucose-6-phosphatase and the repression of glucokinase during fasting, thus increasing lipogenesis at the expense of glucose production. We document a similar pattern in the early phases of diet-induced insulin resistance, and propose that FoxOs are required to enable the liver to direct nutritionally derived carbons to glucose versus lipid metabolism. Our data underscore the heterogeneity of hepatic insulin resistance during progression from the metabolic syndrome to overt diabetes, and the conceptual challenge of designing therapies that curtail glucose production without promoting hepatic lipid accumulation.

Generation of surrogate sources of insulin-producing β-cells remains a goal of diabetes therapy. While most efforts have been directed at differentiating embryonic or induced pluripotent stem (iPS) cells into β-like-cells through endodermal progenitors, we have shown that gut endocrine progenitor cells of mice can be differentiated into glucose-responsive, insulin-producing cells by ablation of transcription factor Foxo1. Here we show that FOXO1 is present in human gut endocrine progenitor and serotonin-producing cells. Using gut organoids derived from human iPS cells, we show that FOXO1 inhibition using a dominant-negative mutant or lentivirus-encoded small hairpin RNA promotes generation of insulin-positive cells that express all markers of mature pancreatic β-cells, release C-peptide in response to secretagogues and survive in vivo following transplantation into mice. The findings raise the possibility of using gut-targeted FOXO1 inhibition or gut organoids as a source of insulin-producing cells to treat human diabetes.

Insulin signaling in the CNS modulates satiety and glucose metabolism, but insulin target neurons are poorly defined. We have previously shown that ablation of insulin receptors (InsR) in Glut4-expressing tissues results in systemic abnormalities of insulin action. We propose that Glut4 neurons constitute an insulin-sensitive neuronal subset. We determined their gene expression profiles using flow-sorted hypothalamic Glut4 neurons. Gene ontology analyses demonstrated that Glut4 neurons are enriched in olfacto-sensory receptors, M2 acetylcholine receptors, and pathways required for the acquisition of insulin sensitivity. Following genetic ablation of InsR, transcriptome profiling of Glut4 neurons demonstrated impairment of the insulin, peptide hormone, and cAMP signaling pathways, with a striking upregulation of anion homeostasis pathway. Accordingly, hypothalamic InsR-deficient Glut4 neurons showed reduced firing activity. The molecular signature of Glut4 neurons is consistent with a role for this neural population in the integration of olfacto-sensory cues with hormone signaling to regulate peripheral metabolism.

Background and aim: We sought to identify mechanisms of beta cell failure in genet- ically obese mice. Little is known about the role of pancreatic innervation in the progression of beta cell failure. In this work we studied adrenergic innervation, in view of its potent inhibitory effect on insulin secretion. We analyzed genetically obese ob/ob and db/db mice at different ages (6- and 15-week-old), corresponding to different compensatory stages in the course of beta cell dysfunction. 15 week-old HFD mice were also studied. Methods and results: All mice were characterized by measures of plasma glucose, insulin, and HOMA. After perfusion, pancreata were dissected and studied by light microscopy, electron mi- croscopy, and morphometry. Insulin, Tyrosine Hydroxylase-positive fibers and cells and Neuro- peptide Y-positive cells were scored by immunohistochemistry. Islets of obese mice showed increased noradrenergic fiber innervation, with significant in- creases of synaptoid structures contacting beta cells compared to controls. Noradrenergic inner- vation of the endocrine area in obese db/db mice tended to increase with age, as diabetes progressed. In ob/ob mice, we also detected an age-dependent trend toward increased noradren- ergic innervation that, unlike in db/db mice, was unrelated to glucose levels. We also observed a progressive increase in Neuropeptide Y-immunoreactive elements localized to the islet core. Conclusions: Our data show increased numbers of sympathetic nerve fibers with a potential to convey inhibitory signals on insulin secretion in pancreatic islets of genetically obese animals, regardless of their diabetic state. The findings suggest an alternative interpretation of the path- ogenesis of beta cell failure, as well as novel strategies to reverse abnormalities in insulin secre- tion.

Targeted gene mutations have established distinct, yet overlapping, developmental roles for receptors of the insulin/IGF family. IGF-I receptor mediates IGF-I and IGF-II action on prenatal growth and IGF-I action on postnatal growth. Insulin receptor mediates prenatal growth in response to IGF-II and postnatal metabolism in response to insulin. In rodents, unlike humans, insulin does not participate in embryonic growth until late gestation. The ability of the insulin receptor to act as a bona fide IGF-II-dependent growth promoter is underscored by its rescue of double knockout Igf1r/Igf2r mice. Thus, IGF-II is a true bifunctional ligand that is able to stimulate both insulin and IGF-I receptor signaling, although with different potencies. In contrast, the IGF-II/cation-independent mannose-6-phosphate receptor regulates IGF-II clearance. The growth retardation of mice lacking IGF-I and/or insulin receptors is due to reduced cell number, resulting from decreased proliferation. Evidence from genetically engineered mice does not support the view that insulin and IGF receptors promote cellular differentiation in vivo or that they are required for early embryonic development. The phenotypes of insulin receptor gene mutations in humans and in mice indicate important differences between the developmental roles of insulin and its receptor in the two species.

We have previously shown that hepatocytes lacking insulin receptors (Ir−/−) fail to mediate metabolic responses, such as stimulation of glycogen synthesis, while retaining the ability to proliferate in response to IGFs. In this study we have asked whether overexpression of type I IGF receptors would rescue the metabolic response of Ir−/− hepatocytes. After IGF-I stimulation, insulin receptor substrate-1 and -2 phosphorylation and PI3K activity were restored to levels similar to or greater than those seen in wild-type cells. Rates of cell proliferation in response to IGF-I increased approximately 2-fold, whereas glycogen synthesis was restored to wild-type levels, but was comparatively smaller than that elicited by overexpression of insulin receptors. In summary, overexpression of IGF-I receptors in Ir−/− hepatocytes normalized insulin receptor substrate-2 phosphorylation and glycogen synthesis to wild-type levels, whereas it increased cell proliferation above wild-type levels. Moreover, stimulation of glycogen synthesis was submaximal compared with the effect of insulin receptor overexpression. We conclude that IGF-I receptors are more efficiently coupled to cell proliferation than insulin receptors, but are less potent than insulin receptors in stimulating glycogen synthesis. The data are consistent with the possibility that there exist intrinsic signaling differences between insulin and IGF-I receptors.

Receptors of the insulin/insulinlike growth factor (IGF) family have been implicated in the regulation of pancreatic beta-cell growth and insulin secretion. The insulin receptor-related receptor (IRR) is an orphan receptor of the insulin receptor gene (Ir) subfamily. It is expressed at considerably higher levels in beta cells than either insulin or IGF-1 receptors, and it has been shown to engage in heterodimer formation with insulin or IGF-1 receptors. To address whether IRR plays a physiologic role in beta-cell development and regulation of insulin secretion, we have characterized mice lacking IRR and generated a combined knockout of Ir and Irr. We report that islet morphology, beta-cell mass, and secretory function are not affected in IRR-deficient mice. Moreover, lack of IRR does not impair compensatory beta-cell hyperplasia in insulin-resistant Ir(+/-) mice, nor does it affect beta-cell development and function in Ir(-/-) mice. We conclude that glucose-stimulated insulin secretion and embryonic beta-cell development occur normally in mice lacking Irr.

Altered skin wound healing is a common cause of morbidity and mortality among diabetic patients. However, the molecular mechanisms whereby diabetes alters skin physiology have not been elucidated. In this study, we investigated the relative roles of hyperglycemia, insulin, and IGF-I, all of which are abnormal in diabetes, in primary murine skin keratinocytes. These cells proliferate and differentiate in vitro in a manner similar to skin in vivo. It was found that in the presence of high glucose (20 mmol/l), the glucose transport rate of primary proliferating or differentiating keratinocytes was downregulated, whereas at 2 mmol/l glucose, the transport rate was increased. These changes were associated with changes in the GLUT1 expression and with changes in the affinity constant (Km) of the transport. Exposure to high glucose was associated with changes in cellular morphology, as well as with decreased proliferation and enhancement of Ca2+-induced differentiation of keratinocytes. Furthermore, in the presence of high glucose, ligand-induced IGF-I receptor but not insulin receptor (IR) autophosphorylation was decreased. Consequently, in high glucose, the effects of IGF-I on glucose uptake and keratinocyte proliferation were inhibited. Interestingly, lack of IR expression in IR-null keratinocytes abolished insulin-induced glucose uptake and partially decreased insulin- and IGF-I–induced proliferation, demonstrating the direct involvement of the IR in these processes. Our results demonstrate that hyperglycemia and impaired insulin signaling might be directly involved in the development of chronic complications of diabetes by impairing glucose utilization of skin keratinocytes as well as skin proliferation and differentiation.

Impaired wound healing of skin is one of the most serious complications of diabetes. However, the pathogenesis of this process is not known, and it is unclear whether impaired insulin signaling could directly affect skin physiology. To elucidate the role of insulin in skin, we studied skin insulin receptor (IR) null mice. The morphology of the skin of newborn IR null mice was normal; however, these mice exhibited decreased proliferation of skin keratinocytes and changes in expression of skin differentiation markers. Due to the short life span of the IR null mice, further characterization was performed in cultured skin keratinocytes that can be induced to differentiate in vitro, closely following the maturation pattern of epidermis in vivo. It was found that despite a compensatory increase in the insulin-like growth factor I receptor autophosphorylation, differentiation of cultured IR null keratinocytes was markedly impaired. In vitro proliferation was not affected as much. Furthermore, although the basal glucose transport system of the null mice was not defective, the insulin-induced increase in glucose transport was abrogated. These results suggest that insulin regulates, via the IR, the differentiation and glucose transport of skin keratinocytes, whereas proliferation is affected by the diabetes milieu of IR knockout mice.

Diabetes affects millions of people worldwide, and its chronic complications are a leading cause of death in many industrialized countries. In a minority of patients, diabetes is brought about by the auto-immune destruction of insulin-producing pancreatic beta cells (Type 1 diabetes). In the vast majority of patients, diabetes is brought about by a combination of genetic and environmental factors that affect the organism's ability to respond to insulin (Type 2 diabetes). This impairment is due to a complex abnormality involving insulin action at the periphery and insulin production in the beta cell. Genetic factors play a key role in the development of type 2 diabetes. However, the inheritance of diabetes is non-Mendelian in nature, due to genetic heterogeneity, polygenic pathogenesis and incomplete penetrance. For these reasons, many laboratories have developed "designer" mice bearing targeted mutations in genes of the insulin action and insulin secretion pathways in order to develop a better model for the inheritance and pathogenesis of type 2 diabetes. These mutant mice are beginning to challenge established paradigms in the pathogenesis of type 2 diabetes and to shed light onto the genetic interactions underlying its complex inheritance. Here we review recent progress in the field and assess its impact on human studies of the genetics, prevention and treatment of type 2 diabetes.

The pleiotropic actions of insulin are mediated by a single receptor tyrosine kinase. Structure/function relationships of the insulin receptor have been conclusively established, and the early steps of insulin signaling are known in some detail. A generally accepted paradigm is that insulin receptors, acting through insulin receptor substrates, stimulate the lipid kinase activity of phosphatidylinositol 3-kinase. The rapid rise in Tris-phosphorylated inositol (PIP(3)) that ensues triggers a cascade of PIP(3)-dependent serine/threonine kinases. Among the latter, Akt (a product of the akt protooncogene) and atypical protein kinase C isoforms are thought to be involved in insulin regulation of glucose transport and oxidation; glycogen, lipid, and protein synthesis; and modulation of gene expression. The presence of multiple insulin-regulated, PIP(3)-dependent kinases is consistent with the possibility that different pathways are required to regulate different biological actions of insulin. Additional work remains to be performed to understand the distal components of insulin signaling. Moreover, there exists substantial evidence for insulin receptor substrate- and/or phosphatidylinositol 3-kinase-independent pathways of insulin action. The ultimate goal of these investigations is to provide clues to the pathogenesis and treatment of the insulin resistant state that is characteristic of type 2 diabetes.

International audience; Exposure to psychostimulants and antipsychotics increases neurotensin (NT) gene expression in the striatum and nucleus accumbens. To investigate the contribution of D(3) receptors to these effects we used mice with targeted disruption of the D(3) receptor gene. Basal NT mRNA expression was similar in D(3) receptor mutant mice and wild-type animals. Acute administration of haloperidol increased NT gene expression in the striatum in D(3)+/+, D(3)+/- and D(3)-/- mice. Similarly, acute cocaine and amphetamine induced NT mRNA expression in the nucleus accumbens shell and olfactory tubercle to a comparable extent in D(3) mutants and wild-type mice. Daily injection of cocaine for seven days increased NT mRNA in a restricted population of neurons in the dorsomedial caudal striatum of D(3)+/+ mice, but not in D(3)-/- and D(3)+/- animals. No differences were observed between D(3) receptor mutant mice and wild-type littermates in the locomotor activity and stereotyped behaviors induced by repeated cocaine administration. These findings demonstrate that dopamine D(3) receptors are not necessary for the acute NT mRNA response to drugs of abuse and antipsychotics but appear to play a role in the regulation of NT gene induction in striatal neurons after repeated cocaine. In addition, our results indicate that the acute locomotor response to cocaine and development of psychostimulant-induced behavioral sensitization do not require functional D(3) receptors.

In patients harboring the IR1152 mutant insulin receptor, hepatic glucose production was normally suppressed by insulin. Hepatocytes without the insulin receptor gene and expressing IR1152 (Hep(MUT)) also showed normal insulin suppression of glucose production and full insulin response of glycogen synthase. In contrast, expression of the IR1152 mutant in skeletal muscle maximally increased glucose uptake and storage, preventing further insulin stimulation. IRS-1 phosphorylation was normally stimulated by insulin in both intact Hep(MUT) and L6 skeletal muscle cells expressing the IR1152 mutant (L6(MUT)). At variance, IRS-2 phosphorylation exhibited high basal levels with no further insulin-dependent increase in L6(MUT) but almost normal phosphorylation, both basal and insulin-stimulated, in the Hep(MUT) cells. In vitro, IR1152 mutant preparations from both the L6(MUT) and the Hep(MUT) cells exhibited increased basal and no insulin-stimulated phosphorylation of IRS-2 immobilized from either muscle or liver cells. IR1152 internalization in liver and muscle cells closely paralleled the ability of this mutant to phosphorylate IRS-2 in vivo in these cells. Block of receptor internalization (wild-type and mutant) in the liver and muscle cells also inhibited IRS-2, but not IRS-1, phosphorylation. Thus, the mechanisms controlling insulin receptor internalization differ in liver and skeletal muscle cells and may enable IR1152 to control glucose metabolism selectively in liver. In both cell types, receptor internalization seems necessary for IRS-2 but not IRS-1 phosphorylation.

Observations in targeted mouse mutants and patients with genetic abnormalities grant insight into the various and distinct roles of insulin-like growth factor receptors (IGF-Rs) and insulin receptors (IRs) during early development. While IGF-1Rs (mediating both IGF-1 and IGF-2 actions) are important for embryonic and fetal growth, IRs (mediating IGF-2 rather than insulin action) play a minor role. However, it is an oversimplification to conclude that IGF-1Rs mediate growth and IRs mediate metabolic responses. Mice lacking both IRs and IGF-1Rs are more severely growth retarded than mice lacking either receptor alone. The phenotype of combined deficiency of IRs and IGF-1Rs is similar to the phenotype caused by the absence of IGF-1 and IGF-2. This provides genetic proof that these two receptors account for the entirety of the growth promoting effects of IGF-1 and IGF-2. There is little evidence that hybrid insulin/IGF-1 receptors promote embryonic growth to a significant degree. The clinical presentation regarding severity of growth retardation and metabolic disturbances observed in animal models versus in humans may differ greatly and the reasons will be reviewed in detail.

Molecular scanning of insulin receptor substrate-1 (IRS-1) revealed several amino acid substitutions. The most common IRS-1 variant, a Gly to Arg972 change, is more prevalent among type 2 diabetic patients. In this study we overexpressed wild-type and Arg972IRS-1 variant in L6 skeletal muscle cells and examined the functional consequences of this polymorphism on insulin metabolic signaling. L6 cells expressing Arg972-IRS-1 (L6-Arg972) showed a decrease in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity compared with L6 cells expressing wild-type IRS-1 (L6-WT) as a consequence of decreased binding of p85 subunit of PI 3-kinase to IRS-1. L6-Arg972 exhibited a decrease in both basal and insulin-stimulated glucose transport due to a reduction in the amount of both GLUT1 and GLUT4 translocated to the plasma membrane. Both basal and insulin-stimulated Akt phosphorylations were decreased in L6-Arg972 compared with L6-WT. Basal glycogen synthase kinase-3 (GSK-3) activity was increased in L6-Arg972 compared with L6-WT, and insulin-induced inactivation of GSK-3 was also reduced in L6-Arg972. This change was associated with a significant decrease in insulin-stimulated glucose incorporation into glycogen and glycogen synthase activity in L6-Arg972 compared with L6-WT. These results indicate that the Arg972-IRS-1 polymorphism impairs the ability of insulin to stimulate glucose transport, glucose transporter translocation, and glycogen synthesis by affecting the PI 3-kinase/Akt/GSK-3 signaling pathway. The present data indicate that the polymorphism at codon 972 of IRS-1 may contribute to the in vivo insulin resistance observed in carriers of this variant.

Insulin resistance can result from genetic interactions among susceptibility alleles. To identify genetic loci predisposing to insulin resistance, we used crosses between different strains of mice with a targeted null allele of the insulin receptor gene. On the genetic background of B6 mice, the insulin receptor gene mutation causes mild hyperinsulinemia. In contrast, on the genetic background of 129/Sv mice, the same mutation causes severe hyperinsulinemia, suggesting that the 129/Sv strain harbors alleles that interact with the insulin receptor mutation and predispose to insulin resistance. As a first step to identify these alleles, we generated an F2 intercross between insulin receptor heterozygous mutant mice on B6 and 129/Sv backgrounds (B6IR x 129IR) and performed a genome-wide scan with polymorphic markers at a 20-cM resolution. We report the identification of loci on chromosomes 2 (logarithm of odds [LOD] 5.58) and 10 (LOD 5.58) that show significant evidence for linkage to plasma insulin levels as a quantitative trait. These findings indicate that targeted mutations in knockout mice can be used to unravel the complex genetic interactions underlying insulin resistance.

The transcription factor FKHR is inhibited by phosphorylation in response to insulin and IGF-1 through Akt kinase. Here we show that FKHR phosphorylation in hepatocytes conforms to a hierarchical pattern in which phosphorylation of the Akt site at S(253), in the forkhead DNA binding domain, is a prerequisite for the phosphorylation of two additional potential Akt sites, T(24) and S(316). Using insulin receptor-deficient hepatocytes, we show that T(24) fails to be phosphorylated by IGF-1 receptors, suggesting that this residue is targeted by a kinase specifically activated by insulin receptors. Lack of T(24) phosphorylation is associated with the failure of IGF-1 to induce nuclear export of FKHR, and to inhibit expression of a reporter gene under the transcriptional control of the IGF binding protein-1 insulin response element. We propose that site-specific phosphorylation of FKHR is one of the mechanisms by which insulin and IGF-1 receptors exert different effects on gene expression.

The forkhead rhabdomyosarcoma transcription factor (FKHR) is a promising candidate to be the transcription factor that binds to the insulin response element of the insulin-like growth factor-binding protein-1 (IGFBP-1) promoter and mediates insulin inhibition of IGFBP-1 promoter activity. Cotransfection of mouse FKHR increased IGFBP-1 promoter activity 2–3-fold in H4IIE rat hepatoma cells; insulin inhibited FKHR-stimulated promoter activity ∼70%. A C-terminal fragment of mouse FKHR (residues 208–652) that contains the transcription activation domain fused to a Gal4 DNA binding domain potently stimulated Gal4 promoter activity. Insulin inhibited FKHR fragment-stimulated promoter activity by ∼70%. Inhibition was abolished by coincubation with the phosphatidylinositol-3 kinase inhibitor, LY294002. The FKHR 208–652 fragment contains two consensus sites for phosphorylation by protein kinase B (PKB)/Akt, Ser-253 and Ser-316. Neither site is required for insulin inhibition of promoter activity stimulated by the FKHR fragment, and overexpression of Akt does not inhibit FKHR fragment-stimulated Gal4 promoter activity. These results suggest that insulin- and phosphatidylinositol-3 kinase-dependent phosphorylation of another site in the fragment by a kinase different from PKB/Akt inhibits transcription activation by the fragment. Phosphorylation of this site also may be involved in insulin inhibition of transcription activation by full-length FKHR, but only after phosphorylation of Ser-253 by PKB/Akt.

Biomedical research is not immune to economic imperatives. But as the realities of profit encroach ever closer on what was once regarded as an idealistic and selfless endeavor, the author reflects on four trends that are hollowing out the research enterprise.

Skeletal muscle insulin resistance is among the earliest detectable defects in humans with type 2 diabetes mellitus. To determine the contribution of muscle insulin resistance to the metabolic phenotype of diabetes, we used the Cre-loxP system to disrupt the insulin receptor gene in mouse skeletal muscle. The muscle-specific insulin receptor knockout mice exhibit a muscle-specific > 95% reduction in receptor content and early signaling events. These mice display elevated fat mass, serum triglycerides, and free fatty acids, but blood glucose, serum insulin, and glucose tolerance are normal. Thus, insulin resistance in muscle contributes to the altered fat metabolism associated with type 2 diabetes, but tissues other than muscle appear to be more involved in insulin-regulated glucose disposal than previously recognized.