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1.  Postreceptor insulin resistance contributes to human dyslipidemia and hepatic steatosis 
Metabolic dyslipidemia is characterized by high circulating triglyceride (TG) and low HDL cholesterol levels and is frequently accompanied by hepatic steatosis. Increased hepatic lipogenesis contributes to both of these problems. Because insulin fails to suppress gluconeogenesis but continues to stimulate lipogenesis in both obese and lipodystrophic insulin-resistant mice, it has been proposed that a selective postreceptor defect in hepatic insulin action is central to the pathogenesis of fatty liver and hypertriglyceridemia in these mice. Here we show that humans with generalized insulin resistance caused by either mutations in the insulin receptor gene or inhibitory antibodies specific for the insulin receptor uniformly exhibited low serum TG and normal HDL cholesterol levels. This was due at least in part to surprisingly low rates of de novo lipogenesis and was associated with low liver fat content and the production of TG-depleted VLDL cholesterol particles. In contrast, humans with a selective postreceptor defect in AKT2 manifest increased lipogenesis, elevated liver fat content, TG-enriched VLDL, hypertriglyceridemia, and low HDL cholesterol levels. People with lipodystrophy, a disorder characterized by particularly severe insulin resistance and dyslipidemia, demonstrated similar abnormalities. Collectively these data from humans with molecularly characterized forms of insulin resistance suggest that partial postreceptor hepatic insulin resistance is a key element in the development of metabolic dyslipidemia and hepatic steatosis.
PMCID: PMC2631303  PMID: 19164855
2.  Activated AKT/PKB signaling in C. elegans uncouples temporally distinct outputs of DAF-2/insulin-like signaling 
In the nematode, Caenorhabditis elegans, a conserved insulin-like signaling pathway controls larval development, stress resistance and adult lifespan. AGE-1, a homolog of the p110 catalytic subunit of phosphoinositide 3-kinases (PI3K) comprises the major known effector pathway downstream of the insulin receptor, DAF-2. Phospholipid products of AGE-1/PI3K activate AKT/PKB kinase signaling via PDK-1. AKT/PKB signaling antagonizes nuclear translocation of the DAF-16/FOXO transcription factor. Reduced AGE-1/PI3K signaling permits DAF-16 to direct dauer larval arrest and promote long lifespan in adult animals. In order to study the downstream effectors of AGE-1/PI3K signaling in C. elegans, we conducted a genetic screen for mutations that suppress the constitutive dauer arrest phenotype of age-1(mg109) animals.
This report describes mutations recovered in a screen for suppressors of the constitutive dauer arrest (daf-C) phenotype of age-1(mg109). Two mutations corresponded to alleles of daf-16. Two mutations were gain-of-function alleles in the genes, akt-1 and pdk-1, encoding phosphoinositide-dependent serine/threonine kinases. A fifth mutation, mg227, located on chromosome X, did not correspond to any known dauer genes, suggesting that mg227 may represent a new component of the insulin pathway. Genetic epistasis analysis by RNAi showed that reproductive development in age-1(mg109);akt-1(mg247) animals was dependent on the presence of pdk-1. Similarly, reproductive development in age-1(mg109);pdk-1(mg261) animals was dependent on akt-1. However, reproductive development in age-1(mg109); mg227 animals required only akt-1, and pdk-1 activity was dispensable in this background. Interestingly, while mg227 suppressed dauer arrest in age-1(mg109) animals, it enhanced the long lifespan phenotype. In contrast, akt-1(mg247) and pdk-1(mg261) did not affect lifespan or stress resistance, while both daf-16 alleles fully suppressed these phenotypes.
A screen for suppressors of PI3K mutant phenotypes identified activating mutations in two known pathway components, providing insights into their regulation. In particular, the interdependence of akt-1 and pdk-1, even in activated forms, supports the existence of AGE-1-independent pathways for these phospholipid-dependent kinases. Phenotypic analysis of these alleles shows that the larval and adult outputs of AGE-1/PI3K are fully separable in these mutants.
PMCID: PMC1609106  PMID: 17020605
3.  Tuberous Sclerosis Complex-1 Deficiency Attenuates Diet-Induced Hepatic Lipid Accumulation 
PLoS ONE  2011;6(3):e18075.
Non-alcoholic fatty liver disease (NAFLD) is causally linked to type 2 diabetes, insulin resistance and dyslipidemia. In a normal liver, insulin suppresses gluconeogenesis and promotes lipogenesis. In type 2 diabetes, the liver exhibits selective insulin resistance by failing to inhibit hepatic glucose production while maintaining triglyceride synthesis. Evidence suggests that the insulin pathway bifurcates downstream of Akt to regulate these two processes. Specifically, mTORC1 has been implicated in lipogenesis, but its role on hepatic steatosis has not been examined. Here, we generated mice with hepatocyte-specific deletion of Tsc1 to study the effects of constitutive mTORC1 activation in the liver. These mice developed normally but displayed mild hepatomegaly and insulin resistance without obesity. Unexpectedly, the Tsc1-null livers showed minimal signs of steatosis even under high-fat diet condition. This ‘resistant’ phenotype was reversed by rapamycin and could be overcome by the expression of Myr-Akt. Moreover, rapamycin failed to reduce hepatic triglyceride levels in models of steatosis secondary to Pten ablation in hepatocytes or high-fat diet in wild-type mice. These observations suggest that mTORC1 is neither necessary nor sufficient for steatosis. Instead, Akt and mTORC1 have opposing effects on hepatic lipid accumulation such that mTORC1 protects against diet-induced steatosis. Specifically, mTORC1 activity induces a metabolic shift towards fat utilization and glucose production in the liver. These findings provide novel insights into the role of mTORC1 in hepatic lipid metabolism.
PMCID: PMC3066210  PMID: 21479224
4.  Chronic Antidiabetic Sulfonylureas In Vivo: Reversible Effects on Mouse Pancreatic β-Cells 
PLoS Medicine  2008;5(10):e206.
Pancreatic β-cell ATP-sensitive potassium (KATP) channels are critical links between nutrient metabolism and insulin secretion. In humans, reduced or absent β-cell KATP channel activity resulting from loss-of-function KATP mutations induces insulin hypersecretion. Mice with reduced KATP channel activity also demonstrate hyperinsulinism, but mice with complete loss of KATP channels (KATP knockout mice) show an unexpected insulin undersecretory phenotype. Therefore we have proposed an “inverse U” hypothesis to explain the response to enhanced excitability, in which excessive hyperexcitability drives β-cells to insulin secretory failure without cell death. Many patients with type 2 diabetes treated with antidiabetic sulfonylureas (which inhibit KATP activity and thereby enhance insulin secretion) show long-term insulin secretory failure, which we further suggest might reflect a similar progression.
Methods and Findings
To test the above hypotheses, and to mechanistically investigate the consequences of prolonged hyperexcitability in vivo, we used a novel approach of implanting mice with slow-release sulfonylurea (glibenclamide) pellets, to chronically inhibit β-cell KATP channels. Glibenclamide-implanted wild-type mice became progressively and consistently diabetic, with significantly (p < 0.05) reduced insulin secretion in response to glucose. After 1 wk of treatment, these mice were as glucose intolerant as adult KATP knockout mice, and reduction of secretory capacity in freshly isolated islets from implanted animals was as significant (p < 0.05) as those from KATP knockout animals. However, secretory capacity was fully restored in islets from sulfonylurea-treated mice within hours of drug washout and in vivo within 1 mo after glibenclamide treatment was terminated. Pancreatic immunostaining showed normal islet size and α-/β-cell distribution within the islet, and TUNEL staining showed no evidence of apoptosis.
These results demonstrate that chronic glibenclamide treatment in vivo causes loss of insulin secretory capacity due to β-cell hyperexcitability, but also reveal rapid reversibility of this secretory failure, arguing against β-cell apoptosis or other cell death induced by sulfonylureas. These in vivo studies may help to explain why patients with type 2 diabetes can show long-term secondary failure to secrete insulin in response to sulfonylureas, but experience restoration of insulin secretion after a drug resting period, without permanent damage to β-cells. This finding suggests that novel treatment regimens may succeed in prolonging pharmacological therapies in susceptible individuals.
In a mouse study aiming to understand why long-term treatment for type 2 diabetes with sulfonylureas eventually fails, Colin Nichols and Maria Remedi suggest that slow restoration of insulin secretion may be possible after a drug-resting period.
Editors' Summary
Diabetes is an increasingly common chronic disease characterized by high blood sugar (glucose) levels. In normal people, blood sugar levels are controlled by the hormone insulin. Insulin is released by β-cells in the pancreas when blood glucose levels rise after eating (glucose is produced by the digestion of food). In fasting people, membrane proteins called ATP-sensitive potassium (KATP) channels keep the β-cell in a “hyperpolarized” state in which they do not secrete insulin. After a meal, glucose enters the β-cell where its chemical breakdown converts ADP into ATP (the molecule that provides the energy that drives cellular processes). The increased ratio of ATP to ADP closes the KATP channels, “depolarizes” the β-cells, and allows the entry of calcium ions, which trigger insulin release. The released insulin then “instructs” insulin-responsive muscle and fat cells to take up glucose from the bloodstream. In type 2 diabetes, the commonest type of diabetes, the muscle and fat cells gradually become nonresponsive to insulin and consequently blood glucose levels rise. Over time, this hyperglycemia increases the risk of heart attacks, kidney failure, and other life-threatening complications. On average, people with diabetes die 5–10 y younger than people without diabetes.
Why Was This Study Done?
People with type 2 diabetes are often initially treated with drugs called sulfonylureas (for example, glibenclamide). Sulfonylureas help to reduce blood glucose levels by inhibiting (in effect, closing) the KATP channels, which enhances insulin secretion. Unfortunately, after patients have been treated for several years with sulfonylureas, their β-cells often stop secreting insulin and the patients then have to inject insulin to control their blood sugar levels. The mechanism by which chronic sulfonylurea treatment affects β-cell behavior is poorly understood, which means that it is hard to improve this antidiabetes treatment. Mice that have been genetically altered so that they have no KATP channels (KATP knockout mice) also rapidly lose their ability to secrete insulin, although they secrete unusually large amounts at birth. This suggests that permanent membrane depolarization (β-cell hyperexcitability) may cause insulin secretory failure. In this study, the researchers investigate whether this mechanism might be responsible for sulfonylurea-induced loss of insulin secretion.
What Did the Researchers Do and Find?
The researchers implanted slowly releasing pellets of glibenclamide into wild-type mice and then monitored their blood glucose levels and glucose tolerance (the speed of glucose removal from the blood after a glucose “meal”) for up to 128 d; the pellets released drug for 90 d. The glibenclamide-implanted mice progressively developed diabetes, lost the ability to secrete insulin in response to glucose and, after 1 wk of treatment, were as glucose intolerant as adult KATP knockout mice. Compared to freshly isolated β-cells from untreated wild-type mice, glucose-stimulated insulin secretion by β-cells isolated from glibenclamide-treated wild-type mice and from KATP knockout mice was reduced to a similar degree. However, the secretory capacity of β-cells isolated from the glibenclamide-treated wild-type mice was restored to normal within hours of drug washout and was normal in β-cells isolated from treated mice 1 mo after exhaustion of the slow-release pellets. Consistent with this result, there was no obvious β-cell death in the glibenclamide-treated mice.
What Do These Findings Mean?
Although findings from animal studies do not always reflect what happens in people, these findings suggest that insulin secretion might sometimes fail in people who take sulfonylureas for a long time, because these drugs cause β-cell hyperexcitability. The finding that the secretory failure caused by sulfonylurea treatment is reversible is important because it suggests that short-acting sulfonylureas might be re-evaluated to see whether they could delay sulfonylurea-induced failure of the insulin secretory response by providing the pancreatic β-cells with periods when they are not depolarized. This finding (and the absence of β-cells death in the glibenclamide-treated mice) also suggests that there may be a way to reverse the loss of the insulin secretory response in patients who have taken sulfonylureas for a long time. Both approaches could help patients with diabetes delay or even avoid the need for insulin injections.
Additional Information.
Please access these Web sites via the online version of this summary at
This study is further discussed in a PLoS Medicine Perspective by Renstrom and colleagues
The MedlinePlus encyclopedia provides information for patients about diabetes (in English and Spanish)
The US National Diabetes Information Clearinghouse provides information on all aspects of diabetes (in English and Spanish)
The International Diabetes Federation also provides comprehensive information about diabetes
Wikipedia has pages on KATP channels and on sulfonylurea drugs (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2573909  PMID: 18959471
5.  A signaling pathway AKTing up in schizophrenia 
The Journal of Clinical Investigation  2008;118(6):2018-2021.
The serine/threonine protein kinase AKT (also known as PKB) signaling pathway has been associated with several human diseases, including schizophrenia. Studies in preclinical models have demonstrated that impaired AKT signaling affects neuronal connectivity and neuromodulation and have identified AKT as a key signaling intermediary downstream of dopamine (DA) receptor 2 (DRD2), the best-established target of antipsychotic drugs. A study by Tan et al. in this issue of the JCI strengthens links among AKT signaling, DA transmission, and cognition in healthy individuals and offers potential avenues to explore in an effort to find more effective pharmacotherapies for schizophrenia and related disorders (see the related article beginning on page 2200).
PMCID: PMC2391280  PMID: 18497888
6.  Postprandial Hepatic Lipid Metabolism Requires Signaling though Akt2 Independent of the Transcription Factors FoxA2, FoxO1 and SREBP1c 
Cell metabolism  2011;14(4):516-527.
Under conditions of obesity and insulin resistance, the serine/threonine protein kinase Akt/PKB is required for lipid accumulation in liver. Two forkhead transcription factors, FoxA2 and FoxO1 have been suggested to function downstream of and to be negatively regulated by Akt and proposed as key determinants of hepatic triglyceride content. In this study, we utilize genetic loss of function experiments to show that constitutive activation of neither FoxA2 nor FoxO1 can account for the protection from steatosis afforded by deletion of Akt2 in liver. Rather, another downstream target positively regulated by Akt, the mTORC1 complex, is required in vivo for de novo lipogenesis and Srebp1c expression. Nonetheless, activation of mTORC1 and SREBP1c are not sufficient to drive postprandial lipogenesis in the absence of Akt2. These data show that insulin signaling through Akt2 promotes anabolic lipid metabolism independent of Foxa2 or FoxO1 and through pathways additional to the mTORC1-dependent activation of SREBP1c.
PMCID: PMC3190164  PMID: 21982711
7.  EAK proteins: novel conserved regulators of C. elegans lifespan 
Aging (Albany NY)  2010;2(10):742-747.
FoxO transcription factors (TFs) extend lifespan in invertebrates and may participate in the control of human longevity. The role of FoxO TFs in lifespan regulation has been studied most extensively in C. elegans, where a conserved insulin/insulin-like growth factor signaling (IIS) pathway and the germline both control lifespan by regulating the subcellular localization of the FoxO transcription factor DAF-16. Although the control of FoxO activity through modulation of its subcellular localization is well established, nuclear translocation of FoxO is not sufficient for full FoxO activation, suggesting that undiscovered inputs regulate FoxO activity after its translocation to the nucleus. We have recently discovered a new conserved pathway, the EAK (enhancer-of-akt-1) pathway, which acts in parallel to the Akt/PKB family of serine-threonine kinases to regulate DAF-16/FoxO activity. Whereas mutation of Akt/PKB promotes the nuclear accumulation of DAF-16/FoxO, mutation of eak genes increases nuclear DAF-16/FoxO activity without influencing DAF-16/FoxO subcellular localization. Thus, EAK proteins regulate the activity of nuclear DAF-16/FoxO. Two EAK proteins, EAK-2/HSD-1 and EAK-7, influence C. elegans lifespan and are conserved in mammals. The discovery of the EAK pathway defines a new conserved FoxO regulatory input and may have implications relevant to aging and the pathogenesis of aging-associated diseases.
PMCID: PMC2993804  PMID: 20975207
C. elegans; aging; lifespan; longevity; insulin signaling; Akt; FoxO; eak; diabetes; cancer; osteoporosis
8.  Requirement of Atypical Protein Kinase Cλ for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes 
Molecular and Cellular Biology  1998;18(12):6971-6982.
Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCζ and PKCλ) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKCλ in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKCλ (λKD or λΔNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKCλ, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by λKD or λΔNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKCλ was ∼50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKCλ mutant that lacks the pseudosubstrate domain (λΔPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of λΔPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKCλ. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKCλ pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.
PMCID: PMC109280  PMID: 9819385
9.  Akt1 and insulin-like growth factor 2 (Igf2) regulate placentation and fetal/postnatal development 
Phenotypic characterization of Akt1 and Igf2 null mice has revealed roles for each in the regulation of placentation, and fetal and postnatal growth. Insulin-like growth factor 2 (IGF2) is encoded by the Igf2 gene and influences cellular function, at least in part, through activation of an intracellular serine/threonine kinase called AKT1. Akt1 and Igf2 null mice were originally characterized on inbred and mixed genetic backgrounds, prohibiting direct comparisons of their phenotypes. The impact of loss of AKT1 or IGF2 on placental, fetal, and postnatal function were examined following transfer of Akt1 and Igf2 null mutations to an outbred CD1 genetic background. Disruption of IGF2 did not affect AKT expression or activation. Both Akt1−/− and Igf2−/− mice exhibited decreased placental weight, fetal weight and viability. Deregulation of placental growth was similar in Akt1 and Igf2 nulls; however, disruption of Igf2 had a more severe impact on prenatal survival and postnatal growth. Placental structure, including organization of junctional and labyrinth zones and development of the interstitial, invasive, trophoblast lineage, were similar in mutant and wild-type mice. Akt1 and Igf2 null mutations affected postnatal growth. The relative impact of each gene differed during pre-weaning versus post-weaning growth phases. AKT1 had a more significant role during pre-weaning growth, whereas IGF2 was a bigger contributor to post-weaning growth. Akt1 and Igf2 null mutations impact placental, fetal and postnatal growth. Placental phenotypes are similar; however, fetal and postnatal growth patterns are unique to each mutation.
PMCID: PMC3894249  PMID: 22562201
AKT1; IGF2; trophoblast; placentation
10.  PTEN Mutations as a Cause of Constitutive Insulin Sensitivity and Obesity 
The New England journal of medicine  2012;367(11):1002-1011.
Epidemiologic and genetic evidence links type 2 diabetes, obesity, and cancer. The tumor-suppressor phosphatase and tensin homologue (PTEN) has roles in both cellular growth and metabolic signaling. Germline PTEN mutations cause a cancer-predisposition syndrome, providing an opportunity to study the effect of PTEN haploinsufficiency in humans.
We measured insulin sensitivity and beta-cell function in 15 PTEN mutation carriers and 15 matched controls. Insulin signaling was measured in muscle and adipose-tissue biopsy specimens from 5 mutation carriers and 5 well-matched controls. We also assessed the effect of PTEN haploinsufficiency on obesity by comparing anthropometric indexes between the 15 patients and 2097 controls from a population-based study of healthy adults. Body composition was evaluated by means of dual-emission x-ray absorptiometry and skinfold thickness.
Measures of insulin resistance were lower in the patients with a PTEN mutation than in controls (e.g., mean fasting plasma insulin level, 29 pmol per liter [range, 9 to 99] vs. 74 pmol per liter [range, 22 to 185]; P = 0.001). This finding was confirmed with the use of hyperinsulinemic euglycemic clamping, showing a glucose infusion rate among carriers 2 times that among controls (P = 0.009). The patients’ insulin sensitivity could be explained by the presence of enhanced insulin signaling through the PI3K-AKT pathway, as evidenced by increased AKT phosphorylation. The PTEN mutation carriers were obese as compared with population-based controls (mean body-mass index [the weight in kilograms divided by the square of the height in meters], 32 [range, 23 to 42] vs. 26 [range, 15 to 48]; P<0.001). This increased body mass in the patients was due to augmented adiposity without corresponding changes in fat distribution.
PTEN haploinsufficiency is a monogenic cause of profound constitutive insulin sensitization that is apparently obesogenic. We demonstrate an apparently divergent effect of PTEN mutations: increased risks of obesity and cancer but a decreased risk of type 2 diabetes owing to enhanced insulin sensitivity. (Funded by the Wellcome Trust and others.)
PMCID: PMC4072504  PMID: 22970944
11.  Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region 
Cell Division  2006;1:11.
p27kip1 (p27) is a multifunctional protein implicated in regulation of cell cycling, signal transduction, and adhesion. Its activity is controlled in part by Phosphatylinositol-3-Kinase (PI3K)/Akt1 signaling, and disruption of this regulatory connection has been identified in human breast cancers. The serine/threonine protein kinase Akt1 directly phosphorylates p27, so identifying the modified residue(s) is essential for understanding how it regulates p27 function. Various amino acids have been suggested as potential targets, but recent attention has focused on threonine 157 (T157) because it is located in a putative Akt1 consensus site. However, T157 is not evolutionarily conserved between mouse and human. We therefore re-evaluated Akt1 phosphorylation of p27 using purified proteins and in cells.
Here we show purified Akt1 phosphorylates human and mouse p27 equally well. Phospho-peptide mapping indicates Akt1 targets multiple sites conserved in both species, while phospho-amino acid analysis identifies the targeted residues as serine rather than threonine. P27 deletion mutants localized these sites to the N-terminus, which contains the major p27 phosphorylation site in cells (serine 10). P27 phosphorylated by Akt1 was detected by a phospho-S10 specific antibody, confirming this serine was targeted. Akt1 failed to phosphorylate p27S10A despite evidence of a second site from mapping experiments. This surprising result suggested S10 phosphorylation might be required for targeting the second site. We tested this idea by replacing S10 with threonine, which as expected led to the appearance of phospho-threonine. Phospho-serine was still present, however, confirming Akt1 sequentially targets multiple serines in this region. We took two approaches in an attempt to explain why different residues were previously implicated. A kinetic analysis revealed a putative Akt1 binding site in the C-terminus, which may explain why mutations in this region affect p27 phosphorylation. Furthermore, commercially available recombinant Akt1 preparations exhibit striking differences in substrate specificity and site selectivity. To confirm S10 is a relevant site, we first showed that full-length wild type Akt1 purified from mammalian cells phosphorylates both human and mouse p27 on S10. Finally, we found that in cultured cells under physiologically relevant conditions such as oxidative stress or growth factor deprivation, endogenous Akt1 causes p27 accumulation by phosphorylating S10.
Identifying where Akt1 phosphorylates p27 is essential for understanding its functional implications. We found that full-length wild type Akt1 – whether purified, transiently overexpressed in cells, or activated in response to cellular stress – phosphorylates p27 at S10, a noncanonical but evolutionarily conserved site known to regulate p27 activity and stability. Using recombinant Akt1 recapitulating this specificity, we showed modification of p27S10 also leads to phosphorylation of an adjacent serine. These results integrate PI3K/Akt1 signaling in response to stress with p27 regulation through its major phosphorylation site in cells, and thus identify new avenues for understanding p27 deregulation in human cancers.
PMCID: PMC1524731  PMID: 16780593
12.  β Cell transplantation and immunosuppression: can’t live with it, can’t live without it 
The Journal of Clinical Investigation  2007;117(9):2380-2382.
Since the first diabetic was treated with insulin in 1922, millions of patients have relied on frequent insulin injections and glucose monitoring to combat the disease and its complications. Improved immunosuppressive regimens in islet transplantation developed in the Edmonton protocol raised the hopes of diabetics worldwide for a complete cure and insulin independence. However, transplant success has proven to be short-lived and accompanied by significant side effects. Using a clever genetic model for conditional ablation of pancreatic β cells in vivo, Nir and colleagues show in this issue of the JCI that the immunosuppressant drugs clinically inhibit β cell proliferation in the diabetic setting (see the related article beginning on page 2553). They also demonstrate that β cells have a remarkable regenerative capacity and that normal β cell mass can recover even in the setting of hyperglycemia. Their new mouse model should aid in the development of improved immunoregulatory strategies and in the elucidation of the molecular pathways that govern β cell regeneration.
PMCID: PMC1952647  PMID: 17786232
13.  Phosphorylation of Human Insulin Receptor Substrate-1 at Serine 629 Plays a Positive Role in Insulin Signaling 
Endocrinology  2007;148(10):4895-4905.
The function of insulin receptor substrate-1 (IRS-1) is regulated by both tyrosine and serine/threonine phosphorylation. Phosphorylation of some serine/threonine residues in IRS-1 dampens insulin signaling, whereas phosphorylation of other serine/threonine residues enhances insulin signaling. Phosphorylation of human IRS-1 at Ser629 was increased by insulin in Chinese hamster ovary cells expressing the insulin receptor (1.26 ± 0.09-fold; P < 0.05) and L6 cells (1.35 ± 0.29-fold; P < 0.05) expressing human IRS-1. Sequence analysis surrounding Ser629 revealed conformity to the consensus phosphorylation sequence recognized by Akt. Phosphorylation of IRS-1 at Ser629 in cells was decreased upon treatment with either an Akt inhibitor or by coexpression with kinase dead Akt, whereas Ser629 phosphorylation was increased by coexpression with constitutively active Akt. In addition, Ser629 of IRS-1 is directly phosphorylated by Akt in vitro. In cells, preventing phosphorylation of Ser629 by a Ser629Ala mutation resulted in increased phosphorylation of Ser636, a known negative regulator of IRS-1, without affecting phosphorylation of Tyr632 or Ser616. Cells expressing the Ser629Ala mutation, along with increased Ser636 phosphorylation, had decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol 3′-kinase with IRS-1 and decreased phosphorylation of Akt at Ser473. Finally, in vitro phosphorylation of a Ser629-containing IRS-1 fragment with Akt reduces the subsequent ability of ERK to phosphorylate Ser636/639. These results suggest that a feed-forward mechanism may exist whereby insulin activation of Akt leads to phosphorylation of IRS-1 at Ser629, resulting in decreased phosphorylation of IRS-1 at Ser636 and enhanced downstream signaling. Understanding the complex phosphorylation patterns of IRS-1 is crucial to elucidating the factors contributing to insulin resistance and, ultimately, the pathogenesis of type 2 diabetes.
PMCID: PMC3581341  PMID: 17640984
14.  Chronic Rapamycin Treatment Causes Glucose Intolerance and Hyperlipidemia by Upregulating Hepatic Gluconeogenesis and Impairing Lipid Deposition in Adipose Tissue 
Diabetes  2010;59(6):1338-1348.
The mammalian target of rapamycin (mTOR)/p70 S6 kinase 1 (S6K1) pathway is a critical signaling component in the development of obesity-linked insulin resistance and operates a nutrient-sensing negative feedback loop toward the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Whereas acute treatment of insulin target cells with the mTOR complex 1 (mTORC1) inhibitor rapamycin prevents nutrient-induced insulin resistance, the chronic effect of rapamycin on insulin sensitivity and glucose metabolism in vivo remains elusive.
To assess the metabolic effects of chronic inhibition of the mTORC1/S6K1 pathway, rats were treated with rapamycin (2 mg/kg/day) or vehicle for 15 days before metabolic phenotyping.
Chronic rapamycin treatment reduced adiposity and fat cell number, which was associated with a coordinated downregulation of genes involved in both lipid uptake and output. Rapamycin treatment also promoted insulin resistance, severe glucose intolerance, and increased gluconeogenesis. The latter was associated with elevated expression of hepatic gluconeogenic master genes, PEPCK and G6Pase, and increased expression of the transcriptional coactivator peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α) as well as enhanced nuclear recruitment of FoxO1, CRTC2, and CREB. These changes were observed despite normal activation of the insulin receptor substrate/PI 3-kinase/Akt axis in liver of rapamycin-treated rats, as expected from the blockade of the mTORC1/S6K1 negative feedback loop.
These findings unravel a novel mechanism by which mTORC1/S6K1 controls gluconeogenesis through modulation of several key transcriptional factors. The robust induction of the gluconeogenic program in liver of rapamycin-treated rats underlies the development of severe glucose intolerance even in the face of preserved hepatic insulin signaling to Akt and despite a modest reduction in adiposity.
PMCID: PMC2874694  PMID: 20299475
15.  Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans 
PLoS Medicine  2014;11(11):e1001755.
Theo Rein and colleagues examine the role of FKBP51 in the actions of antidepressants, with a particular focus on pathways of autophagy.
Please see later in the article for the Editors' Summary
FK506 binding protein 51 (FKBP51) is an Hsp90 co-chaperone and regulator of the glucocorticoid receptor, and consequently of stress physiology. Clinical studies suggest a genetic link between FKBP51 and antidepressant response in mood disorders; however, the underlying mechanisms remain elusive. The objective of this study was to elucidate the role of FKBP51 in the actions of antidepressants, with a particular focus on pathways of autophagy.
Methods and Findings
Established cell lines, primary neural cells, human blood cells of healthy individuals and patients with depression, and mice were treated with antidepressants. Mice were tested for several neuroendocrine and behavioral parameters. Protein interactions and autophagic pathway activity were mainly evaluated by co-immunoprecipitation and Western blots. We first show that the effects of acute antidepressant treatment on behavior are abolished in FKBP51 knockout (51KO) mice. Autophagic markers, such as the autophagy initiator Beclin1, were increased following acute antidepressant treatment in brains from wild-type, but not 51KO, animals. FKBP51 binds to Beclin1, changes decisive protein interactions and phosphorylation of Beclin1, and triggers autophagic pathways. Antidepressants and FKBP51 exhibited synergistic effects on these pathways. Using chronic social defeat as a depression-relevant stress model in combination with chronic paroxetine (PAR) treatment revealed that the stress response, as well as the effects of antidepressants on behavior and autophagic markers, depends on FKBP51. In human blood cells of healthy individuals, FKBP51 levels correlated with the potential of antidepressants to induce autophagic pathways.
Importantly, the clinical antidepressant response of patients with depression (n = 51) could be predicted by the antidepressant response of autophagic markers in patient-derived peripheral blood lymphocytes cultivated and treated ex vivo (Beclin1/amitriptyline: r = 0.572, p = 0.003; Beclin1/PAR: r = 0.569, p = 0.004; Beclin1/fluoxetine: r = 0.454, p = 0.026; pAkt/amitriptyline: r = −0.416, p = 0.006; pAkt/PAR: r = −0.355, p = 0.021; LC3B-II/PAR: r = 0.453, p = 0.02), as well as by the lymphocytic expression levels of FKBP51 (r = 0.631, p<0.0001), pAkt (r = −0.515, p = 0.003), and Beclin1 (r = 0.521, p = 0.002) at admission. Limitations of the study include the use of male mice only and the relatively low number of patients for protein analyses.
To our knowledge, these findings provide the first evidence for the molecular mechanism of FKBP51 in priming autophagic pathways; this process is linked to the potency of at least some antidepressants. These newly discovered functions of FKBP51 also provide novel predictive markers for treatment outcome, consistent with physiological and potential clinical relevance.
Please see later in the article for the Editors' Summary
Editors' Summary
Everyone feels miserable sometimes, but about one in six people will have an episode of clinical depression during their lifetime. For people who are clinically depressed, overwhelming feelings of sadness, anxiety, and hopelessness can last for months or years. Affected individuals lose interest in activities they used to enjoy, they sometimes have physical symptoms such as disturbed sleep, and they may contemplate suicide. Clinicians diagnose depression and determine its severity using questionnaires (“depression rating scales”) that explore the patient's feelings and symptoms. Mild depression is often treated with talking therapies (psychotherapy) such as cognitive behavioral therapy, which helps people change negative ways of thinking. For more severe depression, patients are also usually prescribed an antidepressant, most commonly a “selective serotonin reuptake inhibitor” such as paroxetine or a tricyclic antidepressant such as amitriptyline.
Why Was This Study Done?
Unfortunately, antidepressants don't work for more than half of patients. Moreover, because it is unclear how antidepressants work, it is not possible to predict which patients will respond to which antidepressants. Thus, matching patient to drug can be a lengthy, sometimes unsuccessful, process. Here, the researchers use several approaches to test the hypothesis that a protein called FK506 binding protein 51 (FKBP51) is involved in the actions of antidepressants and to investigate whether the ability of both FKBP51 and antidepressants to regulate a process called autophagy underlies the impact of FKBP51 on antidepressant responses. FKBP51 is a regulator of stress physiology, which is connected to the development and treatment of depression; genetic studies have suggested a link between FKBP51 expression and the antidepressant response rate. Some antidepressants are known to alter the initial steps in the autophagy pathway, a multistep process that maintains the integrity of cells through regulated degradation and recycling of cellular components; however, the potential synergistic role of FKBP51 and antidepressants in regulating pathways of autophagy are unknown.
What Did the Researchers Do and Find?
The researchers first treated wild-type mice and FKBP51 knockout mice (genetically altered animals that make no FKBP51) with an acute dose of antidepressant and compared their behavior in a forced swim test, an assay that measures the action of antidepressants in mice by determining how long the mice struggle or float inertly when placed in deep water. As expected, acute antidepressant treatment increased the time that wild-type mice spent struggling. However, this effect of antidepressant treatment was greatly attenuated in the FKBP51 knockout mice. Moreover, the levels of several autophagy markers increased in the brains of wild-type mice following antidepressant treatment but not in the brains of FKBP51 knockout mice. Next, using “chronic social defeat stress” to model the “endophenotype” of depression (a combination of physiological, hormonal, and behavioral traits seen in people with depression) in mice, the researchers showed that the stress response and the effect of chronic antidepressants on behavior and on autophagic markers all depend on FKBP51. Using cell-based assays, the researchers showed that antidepressants and FKBP51 had synergistic (interactive) effects on the autophagic pathway and that, in human blood cells, FKBP51 levels correlated with the potential of antidepressants to induce autophagic pathways. Finally, the researchers report that the clinical response to antidepressant treatment in 51 patients with depression was associated with the response of autophagic markers in their peripheral blood lymphocytes to antidepressant treatment in test tubes, and that the expression levels of FKBP51 and autophagy markers in patient lymphocytes at admission were associated with subsequent clinical responses to antidepressants.
What Do These Findings Mean?
These findings suggest that the protein FKBP51 is required for the effects of both acute and chronic treatment with some antidepressants on behavior and on autophagic pathways in mice. These findings also reveal an association between antidepressant treatment responses in patients and both the expression levels of FKBP51 and autophagy markers in lymphocytes at admission and the response of autophagic markers to antidepressant treatment in patient lymphocytes. The accuracy of these findings is limited by the small number of clinical samples available for analysis, by the use of only male mice in the animal experiments, and by the inability of animal models of depression to fully replicate the human condition. Nevertheless, these findings identify the early stages of autophagy as potential targets for the development of new antidepressants and identify several potential biomarkers that might, after further clinical validation, help clinicians predict antidepressant efficacy in patients with depression.
Additional Information
Please access these websites via the online version of this summary at
The US National Institute of Mental Health provides information on all aspects of depression (in English and Spanish), including information on antidepressants
The UK National Health Service Choices website provides detailed information about depression and about antidepressants; it also provides personal stories about depression
The UK charity Mind provides information on depression, including some personal stories about depression
More personal stories about depression are available from
MedlinePlus provides links to other resources about depression
Wikipedia has a page on autophagy (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The patients included in this study were all enrolled in the Munich Antidepressant Response Signature project, which aims to identify gene variants and biomarkers that predict treatment outcomes with antidepressants
PMCID: PMC4227651  PMID: 25386878
16.  Activation of skeletal muscle casein kinase II by insulin is not diminished in subjects with insulin resistance. 
Journal of Clinical Investigation  1991;87(3):1017-1022.
Insulin resistance, which may precede the development of non-insulin-dependent diabetes mellitus in Pima Indians, appears to result from a postreceptor defect in signal transduction in skeletal muscle. To identify the putative postreceptor lesion responsible for insulin resistance in Pima Indians, we investigated the influence of insulin on the activity of casein kinase II (CKII) in skeletal muscle of seven insulin-sensitive, four insulin-resistant, nondiabetic, and five insulin-resistant diabetic Pima Indians during a 2 h hyperinsulinemic, euglycemic clamp. In sensitive subjects, CKII was transiently activated reaching a maximum over basal activity (42%) at 45 min before declining. CKII was also stimulated in resistant (19%) and diabetic (34%) subjects. Basal CKII activity in resistant subjects was 40% higher than in either sensitive or diabetic subjects, although the concentration of CKII protein, as determined by Western blotting, was equal among the three groups. Basal CKII activity was correlated with fasting plasma insulin concentrations, suggesting that the higher activity in resistant subjects resulted from insulin action. Extracts of muscle obtained from all three groups either before or after insulin administration were treated with immobilized alkaline phosphatase, which reduced and equalized CKII activity. These results suggest that insulin stimulates CKII activity in human skeletal muscle by a mechanism involving phosphorylation of either CKII or of an effector molecule, and support the idea that elevated basal activity in resistant subjects results from insulin action. It appears that the ability of insulin to activate CKII in skeletal muscle is not impaired in insulin-resistant Pima Indians, and that the biochemical lesion responsible for insulin resistance occurs either downstream from CKII or in a different pathway of insulin action.
PMCID: PMC329895  PMID: 1999482
17.  Targeting the Phosphatidylinositol 3-Kinase Signaling Pathway in Breast Cancer 
The Oncologist  2011;16(4):404-414.
The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) network plays a key regulatory function in cell survival, proliferation, migration, metabolism, angiogenesis, and apoptosis. Genetic aberrations found at different levels make this pathway one of the most commonly disrupted in human breast cancer. Because the PI3K pathway has divergent downstream effects, the identification of the key effectors of the pathway and their presence in the different subtypes of breast tumors will allow the development of ideal targeted therapies with meaningful clinical efficacy.
Learning Objectives
After completing this course, the reader will be able to: Describe how PTEN loss, PIK3CA mutations, and AKT dysregulation affect the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling network in human breast cancer.Review the current state of AKT and mTOR inhibitor development, and describe its potential for clinical applications.
This article is available for continuing medical education credit at
The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) network plays a key regulatory function in cell survival, proliferation, migration, metabolism, angiogenesis, and apoptosis. Genetic aberrations found at different levels, either with activation of oncogenes or inactivation of tumor suppressors, make this pathway one of the most commonly disrupted in human breast cancer. The PI3K-dependent phosphorylation and activation of the serine/threonine kinase AKT is a key activator of cell survival mechanisms. The activation of the oncogene PIK3CA and the loss of regulators of AKT including the tumor suppressor gene PTEN are mutations commonly found in breast tumors. AKT relieves the negative regulation of mTOR to activate protein synthesis and cell proliferation through S6K and 4EBP1. The common activation of the PI3K pathway in breast cancer has led to the development of compounds targeting the effector mechanisms of the pathway including selective and pan-PI3K/pan-AKT inhibitors, rapamycin analogs for mTOR inhibition, and TOR-catalytic subunit inhibitors. The influences of other oncogenic pathways such as Ras-Raf-Mek on the PI3K pathway and the known feedback mechanisms of activation have prompted the use of compounds with broader effect at multiple levels and rational combination strategies to obtain a more potent antitumor activity and possibly a meaningful clinical effect. Here, we review the biology of the network, its role in the development and progression of breast cancer, and the evaluation of targeted therapies in clinical trials.
PMCID: PMC3228119  PMID: 21406469
18.  Requirement for Activation of the Serine-Threonine Kinase Akt (Protein Kinase B) in Insulin Stimulation of Protein Synthesis but Not of Glucose Transport 
Molecular and Cellular Biology  1998;18(7):3708-3717.
A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ∼75% in CHO cells and ∼30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.
PMCID: PMC108953  PMID: 9632753
19.  Palmitate Impairs and Eicosapentaenoate Restores Insulin Secretion Through Regulation of SREBP-1c in Pancreatic Islets 
Diabetes  2008;57(9):2382-2392.
OBJECTIVE—Chronic exposure to fatty acids causes β-cell failure, often referred to as lipotoxicity. We investigated its mechanisms, focusing on contribution of SREBP-1c, a key transcription factor for lipogenesis.
RESEARCH DESIGN AND METHODS—We studied in vitro and in vivo effects of saturated and polyunsaturated acids on insulin secretion, insulin signaling, and expression of genes involved in β-cell functions. Pancreatic islets isolated from C57BL/6 control and SREBP-1–null mice and adenoviral gene delivery or knockdown systems of related genes were used.
RESULTS—Incubation of C57BL/6 islets with palmitate caused inhibition of both glucose- and potassium-stimulated insulin secretion, but addition of eicosapentaenoate (EPA) restored both inhibitions. Concomitantly, palmitate activated and EPA abolished both mRNA and nuclear protein of SREBP-1c, accompanied by reciprocal changes of SREBP-1c target genes such as insulin receptor substrate-2 (IRS-2) and granuphilin. These palmitate-EPA effects on insulin secretion were abolished in SREBP-1–null islets. Suppression of IRS-2/Akt pathway could be a part of the downstream mechanism for the SREBP-1c–mediated insulin secretion defect because adenoviral constitutively active Akt compensated it. Uncoupling protein-2 (UCP-2) also plays a crucial role in the palmitate inhibition of insulin secretion, as confirmed by knockdown experiments, but SREBP-1c contribution to UCP-2 regulation was partial. The palmitate-EPA regulation of insulin secretion was similarly observed in islets from C57BL/6 mice pretreated with dietary manipulations. Furthermore, administration of EPA to diabetic KK-Ay mice ameliorated impairment of insulin secretion in their islets.
CONCLUSIONS—SREBP-1c plays a dominant role in palmitate-mediated insulin secretion defect, and EPA prevents it through SREBP-1c inhibition, implicating a therapeutic potential for treating diabetes related to lipotoxicity.
PMCID: PMC2518489  PMID: 18458149
20.  Inflammation, Insulin Resistance, and Diabetes—Mendelian Randomization Using CRP Haplotypes Points Upstream 
PLoS Medicine  2008;5(8):e155.
Raised C-reactive protein (CRP) is a risk factor for type 2 diabetes. According to the Mendelian randomization method, the association is likely to be causal if genetic variants that affect CRP level are associated with markers of diabetes development and diabetes. Our objective was to examine the nature of the association between CRP phenotype and diabetes development using CRP haplotypes as instrumental variables.
Methods and Findings
We genotyped three tagging SNPs (CRP + 2302G > A; CRP + 1444T > C; CRP + 4899T > G) in the CRP gene and measured serum CRP in 5,274 men and women at mean ages 49 and 61 y (Whitehall II Study). Homeostasis model assessment-insulin resistance (HOMA-IR) and hemoglobin A1c (HbA1c) were measured at age 61 y. Diabetes was ascertained by glucose tolerance test and self-report. Common major haplotypes were strongly associated with serum CRP levels, but unrelated to obesity, blood pressure, and socioeconomic position, which may confound the association between CRP and diabetes risk. Serum CRP was associated with these potential confounding factors. After adjustment for age and sex, baseline serum CRP was associated with incident diabetes (hazard ratio = 1.39 [95% confidence interval 1.29–1.51], HOMA-IR, and HbA1c, but the associations were considerably attenuated on adjustment for potential confounding factors. In contrast, CRP haplotypes were not associated with HOMA-IR or HbA1c (p = 0.52–0.92). The associations of CRP with HOMA-IR and HbA1c were all null when examined using instrumental variables analysis, with genetic variants as the instrument for serum CRP. Instrumental variables estimates differed from the directly observed associations (p = 0.007–0.11). Pooled analysis of CRP haplotypes and diabetes in Whitehall II and Northwick Park Heart Study II produced null findings (p = 0.25–0.88). Analyses based on the Wellcome Trust Case Control Consortium (1,923 diabetes cases, 2,932 controls) using three SNPs in tight linkage disequilibrium with our tagging SNPs also demonstrated null associations.
Observed associations between serum CRP and insulin resistance, glycemia, and diabetes are likely to be noncausal. Inflammation may play a causal role via upstream effectors rather than the downstream marker CRP.
Using a Mendelian randomization approach, Eric Brunner and colleagues show that the associations between serum C-reactive protein and insulin resistance, glycemia, and diabetes are likely to be noncausal.
Editors' Summary
Diabetes—a common, long-term (chronic) disease that causes heart, kidney, nerve, and eye problems and shortens life expectancy—is characterized by high levels of sugar (glucose) in the blood. In people without diabetes, blood sugar levels are controlled by the hormone insulin. Insulin is released by the pancreas after eating and “instructs” insulin-responsive muscle and fat cells to take up the glucose from the bloodstream that is produced by the digestion of food. In the early stages of type 2 diabetes (the commonest type of diabetes), the muscle and fat cells become nonresponsive to insulin (a condition called insulin resistance), and blood sugar levels increase. The pancreas responds by making more insulin—people with insulin resistance have high blood levels of both insulin and glucose. Eventually, however, the insulin-producing cells in the pancreas start to malfunction, insulin secretion decreases, and frank diabetes develops.
Why Was This Study Done?
Globally, about 200 million people have diabetes, but experts believe this number will double by 2030. Ways to prevent or delay the onset of diabetes are, therefore, urgently needed. One major risk factor for insulin resistance and diabetes is being overweight. According to one theory, increased body fat causes mild, chronic tissue inflammation, which leads to insulin resistance. Consistent with this idea, people with higher than normal amounts of the inflammatory protein C-reactive protein (CRP) in their blood have a high risk of developing diabetes. If inflammation does cause diabetes, then drugs that inhibit CRP might prevent diabetes. However, simply measuring CRP and determining whether the people with high levels develop diabetes cannot prove that CRP causes diabetes. Those people with high blood levels of CRP might have other unknown factors in common (confounding factors) that are the real causes of diabetes. In this study, the researchers use “Mendelian randomization” to examine whether increased blood CRP causes diabetes. Some variants of CRP (the gene that encodes CRP) increase the amount of CRP in the blood. Because these variants are inherited randomly, there is no likelihood of confounding factors, and an association between these variants and the development of insulin resistance and diabetes indicates, therefore, that increased CRP levels cause diabetes.
What Did the Researchers Do and Find?
The researchers measured blood CRP levels in more than 5,000 people enrolled in the Whitehall II study, which is investigating factors that affect disease development. They also used the “homeostasis model assessment-insulin resistance” (HOMA-IR) method to estimate insulin sensitivity from blood glucose and insulin measurements, and measured levels of hemoglobin A1c (HbA1c, hemoglobin with sugar attached—a measure of long-term blood sugar control) in these people. Finally, they looked at three “single polynucleotide polymorphisms” (SNPs, single nucleotide changes in a gene's DNA sequence; combinations of SNPs that are inherited as a block are called haplotypes) in CRP in each study participant. Common haplotypes of CRP were related to blood serum CRP levels and, as previously reported, increased blood CRP levels were associated with diabetes and with HOMA-IR and HbA1c values indicative of insulin resistance and poor blood sugar control, respectively. By contrast, CRP haplotypes were not related to HOMA-IR or HbA1c values. Similarly, pooled analysis of CRP haplotypes and diabetes in Whitehall II and another large study on health determinants (the Northwick Park Heart Study II) showed no association between CRP variants and diabetes risk. Finally, data from the Wellcome Trust Case Control Consortium also showed no association between CRP haplotypes and diabetes risk.
What Do These Findings Mean?
Together, these findings suggest that increased blood CRP levels are not responsible for the development of insulin resistance or diabetes, at least in European populations. It may be that there is a causal relationship between CRP levels and diabetes risk in other ethnic populations—further Mendelian randomization studies are needed to discover whether this is the case. For now, though, these findings suggest that drugs targeted against CRP are unlikely to prevent or delay the onset of diabetes. However, they do not discount the possibility that proteins involved earlier in the inflammatory process might cause diabetes and might thus represent good drug targets for diabetes prevention.
Additional Information.
Please access these Web sites via the online version of this summary at
This study is further discussed in a PLoS Medicine Perspective by Bernard Keavney
The MedlinePlus encyclopedia provides information about diabetes and about C-reactive protein (in English and Spanish)
US National Institute of Diabetes and Digestive and Kidney Diseases provides patient information on all aspects of diabetes, including information on insulin resistance (in English and Spanish)
The International Diabetes Federation provides information about diabetes, including information on the global diabetes epidemic
The US Centers for Disease Control and Prevention provides information for the public and professionals on all aspects of diabetes (in English and Spanish)
Wikipedia has a page on Mendelian randomization (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2504484  PMID: 18700811
21.  A PP2A regulatory subunit PPTR-1 regulates the C. elegans Insulin/IGF-1 signaling pathway by modulating AKT-1 phosphorylation 
Cell  2009;136(5):939-951.
The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in the regulation of lifespan, dauer diapause, metabolism and stress response. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. In a RNAi screen for serine/threonine protein phosphatases that counter-balance the effect of the kinases in the IIS pathway, we identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects phenotypes associated with the IIS pathway including lifespan, dauer, stress resistance and fat storage. We show that PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56β regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this modulation ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in modulating insulin signaling through AKT dephosphorylation and thereby has widespread implications in cancer and diabetes research.
PMCID: PMC2707143  PMID: 19249087
22.  Genome-Wide Analysis of Germline Signaling Genes Regulating Longevity and Innate Immunity in the Nematode Pristionchus pacificus 
PLoS Pathogens  2012;8(8):e1002864.
Removal of the reproductive system of many animals including fish, flies, nematodes, mice and humans can increase lifespan through mechanisms largely unknown. The abrogation of the germline in Caenorhabditis elegans increases longevity by 60% due to a signal emitted from the somatic gonad. Apart from increased longevity, germline-less C. elegans is also resistant to other environmental stressors such as feeding on bacterial pathogens. However, the evolutionary conservation of this pathogen resistance, its genetic basis and an understanding of genes involved in producing this extraordinary survival phenotype are currently unknown. To study these evolutionary aspects we used the necromenic nematode Pristionchus pacificus, which is a genetic model system used in comparison to C. elegans. By ablation of germline precursor cells and subsequent feeding on the pathogen Serratia marcescens we discovered that P. pacificus shows remarkable resistance to bacterial pathogens and that this response is evolutionarily conserved across the Genus Pristionchus. To gain a mechanistic understanding of the increased resistance to bacterial pathogens and longevity in germline-ablated P. pacificus we used whole genome microarrays to profile the transcriptional response comparing germline ablated versus un-ablated animals when fed S. marcescens. We show that lipid metabolism, maintenance of the proteasome, insulin signaling and nuclear pore complexes are essential for germline deficient phenotypes with more than 3,300 genes being differentially expressed. In contrast, gene expression of germline-less P. pacificus on E. coli (longevity) and S. marcescens (immunity) is very similar with only 244 genes differentially expressed indicating that longevity is due to abundant gene expression also involved in immunity. By testing existing mutants of Ppa-DAF-16/FOXO and the nuclear hormone receptor Ppa-DAF-12 we show a conserved function of both genes in resistance to bacterial pathogens and longevity. This is the first study to show that the influence of the reproductive system on extending lifespan and innate immunity is conserved in evolution.
Author Summary
Removal of the germline in the nematode Caenorhabditis elegans can increase lifespan and resistance to bacterial pathogens. Currently there is no information on what genes are regulated to produce this resistance phenotype in other nematodes and whether they are the same as genes involved in lifespan regulation. We used the necromenic nematode, Pristionchus pacificus, a species that diverged from C. elegans 250–400 MYA, ablated its germline and found increased resistance to the pathogens Serratia marcescens and Xenorhabdus nematophila. In a novel manner we performed cell ablation of the germline, exposure to bacterial pathogens and used whole genome microarrays of the same animals to find that this resistance is due to expression of genes involved in insulin signaling, nuclear pore complexes, ribosomal translation and lipid production. Furthermore, we see little difference between germline ablated lifespan and immunity leading us to believe that living longer is due to an abundance of genes also being involved with immunity. We could also show that, similar to C. elegans, the transcription factor DAF-16/FOXO and nuclear hormone receptor DAF-12, are integral for this response. Our study is the first to understand how the reproductive system regulates both lifespan and innate immunity transcriptionally and offers insights into the signaling cascades involved with resisting pathogen attack.
PMCID: PMC3415453  PMID: 22912581
23.  Genetic variation in a metabolic signaling pathway and colon and rectal cancer risk: mTOR, PTEN, STK11, RPKAA1, PRKAG2, TSC1, TSC2, PI3K and Akt1 
Carcinogenesis  2010;31(9):1604-1611.
Serine/threonine protein kinase 11 (STK11) and phosphatase tensin homolog deleted on chromosome 10 (PTEN) link insulin sensitivity and metabolic signaling to inflammation and other hormonal factors and colorectal cancer. We evaluate genetic variation in nine genes in a candidate pathway as follows: STK11 (3 tagSNPs), PTEN (9 tagSNPs), FRAP1 (mTOR) (4 tagSNPs), TSC1 (14 tagSNPs), TSC2 (8 tagSNPs), Akt1 (2 tagSNPs), PIK3CA (7 tagSNPs), PRKAA1 (13 tagSNPs) and PRKAG2 (68 tagSNPs) in two population-based case–control studies of colon (n = 1574 cases, 1940 controls) and rectal (n = 91 cases, 999 controls) cancer. FRAP1, PRKAA1, PRKAG2 and TSC2 genes were significantly associated with colon cancer; risk estimates ranged from 1.21 [95% confidence interval (CI) 1.05–1.38] for FRAP1rs1057079 for the AG/GG genotype to 1.51 (95% CI 1.09–2.09) for PRKAG2rs9648723 CC genotype. PIK3CA, PRKAG2, PTEN, STK11 and TSC1 were significantly associated with rectal cancer overall. The strongest association was observed for PIK3CA rs7651265 GG genotype (odds ratio 2.32 95% CI 1.02–5.30). FRAP1 was associated with microsatellite instability (MSI)+ colon tumors; PRKAA1, CpG island methylator phenotype (CIMP)+ and MSI+ colon tumors; PRKAG2 and KRAS2 colon tumors; TSC1 and CIMP+ and MSI+ colon tumors; TSC2 with MSI+ colon tumors; PIK3CA with KRAS2-mutated rectal tumors; PRKAG2 (rs6964824) with KRAS2- and TP53-mutated rectal tumors and with PRKAG2 (rs412396 and rs4725431) with CIMP+ rectal tumors. These data suggest that genetic variation in a predefined candidate pathway for colorectal cancer contributes to both colon and rectal cancer risk. Associations appear to be strongest for CIMP+ and MSI+ tumors.
PMCID: PMC2930805  PMID: 20622004
24.  Grape seed proanthocyanidins and metformin act by different mechanisms to promote insulin signaling in rats fed high calorie diet 
Key pathways like insulin signaling, AMP activated kinase (AMPK) activation and inflammatory signaling are involved in the complex pathological network of hepatic insulin resistance. Our aim is to investigate whether grape seed proanthocyanidins (GSP) and metformin (MET) target any of these pathways in insulin resistant rat liver. Albino Wistar rats were rendered insulin resistant by feeding a high fat-fructose diet (HFFD). Either GSP (100 mg/kg b.w), MET(50 mg/kg b.w) or both were administered to insulin resistant rats as therapeutic options. HFFD-feeding caused hyperglycemia, hyperinsulinemia, increased gluconeogenesis, decreased tyrosine phosphorylation of insulin receptor-β(IR-β) and insulin receptor substrate-1 (IRS-1) and increased serine phosphorylation of IRS-1. The association of p85α subunit of phosphotidyl inositol 3 kinase(PI3K) with IRS-1 and subsequent Akt phosphorylation were reduced while the expression of mitogen activated protein kinases (MAPK) were increased in HFFD rats. Both MET and GSP reduced hyperglycemia and hyperinsulinemia and improved glycolysis, tyrosine phosphorylation of IR-β and IRS-1, IRS-1-PI3K association and Akt activation. However, activation of tumor necrosis factor-α, interleukin-6, leptin and suppressor of cytokine signaling-3 and reduction in adiponectin caused by chronic HFFD feeding were reversed by GSP better than by MET. Activation of AMPK by GSP was much less compared to that by MET. These findings suggest that GSP might activate PI3K pathway and promote insulin action by reducing serine kinase activation and cytokine signaling and MET by targeting AMPK. The beneficial effects were enhanced during combination therapy. Thus, combination therapy with MET and GSP may be considered for the management of metabolic syndrome.
PMCID: PMC3972396  PMID: 24026800
High fat-fructose diet; Grape seed proanthocyanidins; Metformin; Insulin signaling; Inflammation
25.  Prime suspect: the TCF7L2 gene and type 2 diabetes risk  
Journal of Clinical Investigation  2007;117(8):2077-2079.
Transcription factor-7–like 2 (TCF7L2) is the most important type 2 diabetes susceptibility gene identified to date, with common intronic variants strongly associated with diabetes in all major racial groups. This ubiquitous transcription factor in the Wnt signaling pathway was not previously known to be involved in glucose homeostasis, so defining the underlying mechanism(s) will provide new insights into diabetes. In this issue of the JCI, Lyssenko and colleagues report on their human and isolated islet studies and suggest that the risk allele increases TCF7L2 expression in the pancreatic β cell, reducing insulin secretion and hence predisposing the individual to diabetes (see the related article beginning on page 2155).
PMCID: PMC1934573  PMID: 17671643

Results 1-25 (389904)