Short-term caloric restriction in db/db mice improves myocardial function and increases high molecular weight (HMW) adiponectin.

BACKGROUND: Obesity and metabolic syndrome lead to the development of metabolic heart disease (MHD) that is characterized by left ventricular hypertrophy (LVH), diastolic dysfunction, and increased mitochondrial ROS. Caloric restriction (CR) is a nutritional intervention that protects against obesity, diabetes, and cardiovascular disease. Healthy adipose tissue is cardioprotective via releasing adipokines such as adiponectin. We tested the hypothesis that CR can ameliorate MHD and it is associated with improved adipose tissue function as reflected by increased circulating levels of high molecular weight (HMW) adiponectin and AMP-activated protein kinase (AMPK) in db/db mice. METHODS: Genetically obese db/db and lean db/+ male mice were fed either ad libitum or subjected to 30% CR for 5 weeks. At the end of the study period, echocardiography was carried out to assess diastolic function. Blood, heart, and epididymal fat pads were harvested for mitochondrial study, ELISA, and Western blot analyses. RESULTS: CR reversed the development of LVH, prevented diastolic dysfunction, and decreased cardiac mitochondrial H2O2 in db/db (vs. ad lib) mice. These beneficial effects on the heart were associated with increased circulating level of HMW adiponectin. Furthermore, CR increased AMPK and eNOS activation in white adipose tissue of db/db mice, but not in the heart. CONCLUSIONS: These findings indicate that even short-term CR protects the heart from MHD. Whether the beneficial effects of CR on the heart could be related to the improved adipose tissue function warrants future investigation.

AMPK Activation by Metformin Suppresses Abnormal Extracellular Matrix Remodeling in Adipose Tissue and Ameliorates Insulin Resistance in Obesity.

Fibrosis is emerging as a hallmark of metabolically dysregulated white adipose tissue (WAT) in obesity. Although adipose tissue fibrosis impairs adipocyte plasticity, little is known about how aberrant extracellular matrix (ECM) remodeling of WAT is initiated during the development of obesity. Here we show that treatment with the antidiabetic drug metformin inhibits excessive ECM deposition in WAT of ob/ob mice and mice with diet-induced obesity, as evidenced by decreased collagen deposition surrounding adipocytes and expression of fibrotic genes including the collagen cross-linking regulator LOX Inhibition of interstitial fibrosis by metformin is likely attributable to the activation of AMPK and the suppression of transforming growth factor-ß1 (TGF-ß1)/Smad3 signaling, leading to enhanced systemic insulin sensitivity. The ability of metformin to repress TGF-ß1-induced fibrogenesis is abolished by the dominant negative AMPK in primary cells from the stromal vascular fraction. TGF-ß1-induced insulin resistance is suppressed by AMPK agonists and the constitutively active AMPK in 3T3L1 adipocytes. In omental fat depots of obese humans, interstitial fibrosis is also associated with AMPK inactivation, TGF-ß1/Smad3 induction, aberrant ECM production, myofibroblast activation, and adipocyte apoptosis. Collectively, integrated AMPK activation and TGF-ß1/Smad3 inhibition may provide a potential therapeutic approach to maintain ECM flexibility and combat chronically uncontrolled adipose tissue expansion in obesity.

Improved Insulin Sensitivity 3 Months After RYGB Surgery Is Associated With Increased Subcutaneous Adipose Tissue AMPK Activity and Decreased Oxidative Stress.

Morbidly obese individuals are predisposed to a wide range of disorders, including type 2 diabetes, atherosclerotic cardiovascular disease, fatty liver disease, and certain cancers. Remarkably, all of these disorders can be improved or prevented by Roux-en-Y gastric bypass (RYGB) surgery. We have reported that decreased AMPK activity, together with increased oxidative stress and inflammation in adipose tissue, is associated with insulin resistance in morbidly obese bariatric surgery patients. In the current study, we assessed how these parameters are affected by RYGB surgery. Eleven patients (average age of 46 ± 4 years) were studied immediately prior to surgery and 3 months postoperatively. We measured subcutaneous adipose tissue AMPK phosphorylation (threonine 172, an index of its activation), malonyl-CoA content, protein carbonylation (a marker of oxidative stress), plasma adiponectin, and mRNA expression of several inflammatory cytokines. After surgery, AMPK activity increased 3.5-fold and oxidative stress decreased by 50% in subcutaneous adipose tissue. In addition, malonyl-CoA levels were reduced by 80%. Furthermore, patients had improvements in their BMI and insulin sensitivity (HOMA) and had increased circulating high-molecular weight adiponectin and decreased fasting plasma insulin levels. In contrast, the expression of inflammatory markers in subcutaneous adipose tissue was unchanged postoperatively, although plasma CRP was diminished by 50%.

AMP-activated Protein Kinase (AMPK): Does This Master Regulator of Cellular Energy State Distinguish Insulin Sensitive from Insulin Resistant Obesity?

Although a correlation exists between obesity and insulin resistance, roughly 25 % of obese individuals are insulin sensitive. AMP-activated protein kinase (AMPK) is a cellular energy sensor that among its many actions, integrates diverse physiological signals to restore energy balance. In addition, in many situations it also increases insulin sensitivity. In this context, AMPK activity is decreased in very obese individuals undergoing bariatric surgery who are insulin resistant compared to equally obese patients who are insulin sensitive. In this review, we will both explore what distinguishes these individuals, and evaluate the evidence that diminished AMPK is associated with insulin resistance and metabolic syndrome-associated disorders in other circumstances.

What distinguishes adipose tissue of severely obese humans who are insulin sensitive and resistant?

PURPOSE OF REVIEW: Despite a strong correlation between obesity and insulin resistance, 25% of severely obese (BMI >40) individuals are insulin sensitive. In this review, we will examine the factors in adipose tissue that distinguish the two groups, as well as reasons for believing the insulin-sensitive group will be less disease prone. RECENT FINDINGS: Obesity has been linked to the metabolic syndrome with an increase in visceral (intra-abdominal) compared to subcutaneous fat. Recent studies in which adipose tissue of insulin-sensitive and insulin-resistant patients with severe obesity were compared indicate that the insulin-resistant group is also distinguished by increases in oxidative stress and decreases in AMP-activated protein kinase (AMPK) activity. In contrast, changes in the expression of genes for SIRT1, inflammatory cytokines, mitochondrial biogenesis and function, and the two α-isoforms of AMPK showed more depot variation. Studies of how these and other changes in adipose tissue respond to bariatric surgery are still in their infancy. SUMMARY: Available data suggest that increases in oxidative stress, decreases in AMPK activity and SIRT1 gene expression, depot-specific changes in inflammatory, mitochondrial and other genes distinguish adipose tissue of insulin resistant from insulin-sensitive individuals with severe obesity.

Insulin sensitive and resistant obesity in humans: AMPK activity, oxidative stress, and depot-specific changes in gene expression in adipose tissue.

We previously reported that adenosine monophosphate-activated protein kinase (AMPK) activity is lower in adipose tissue of morbidly obese individuals who are insulin resistant than in comparably obese people who are insulin sensitive. However, the number of patients and parameters studied were small. Here, we compared abdominal subcutaneous, epiploic, and omental fat from 16 morbidly obese individuals classified as insulin sensitive or insulin resistant based on the homeostatic model assessment of insulin resistance. We confirmed that AMPK activity is diminished in the insulin resistant group. A custom PCR array revealed increases in mRNA levels of a wide variety of genes associated with inflammation and decreases in PGC-1α and Nampt in omental fat of the insulin resistant group. In contrast, subcutaneous abdominal fat of the same patients showed increases in PTP-1b, VEGFa, IFNγ, PAI-1, and NOS-2 not observed in omental fat. Only angiotensinogen and CD4(+) mRNA levels were increased in both depots. Surprisingly, TNFα was only increased in epiploic fat, which otherwise showed very few changes. Protein carbonyl levels, a measure of oxidative stress, were increased in all depots. Thus, adipose tissues of markedly obese insulin resistant individuals uniformly show decreased AMPK activity and increased oxidative stress compared with insulin sensitive patients. However, most changes in gene expression appear to be depot-specific.

Insulin resistance due to nutrient excess: is it a consequence of AMPK downregulation?

It has long been known that excesses of glucose and branched chain amino acids, such as leucine, lead to insulin resistance in skeletal muscle. A recent study in incubated rat muscle suggests that both molecules may do so by virtue of their ability to downregulate the fuel sensing and signaling enzyme AMP-activated protein kinase (AMPK) and activate mTOR/p70S6 kinase (p70S6K) signaling. The results also demonstrated that inhibition of mTOR/p70S6K with rapamycin prevented the development of insulin resistance but had no effect on AMPK activity (Thr172 phosphorylation of its catalytic subunit). In contrast, activation of AMPK by both AICAR and α-lipoic acid led to the phosphorylation of specific molecules that diminished both mTOR/p70S6K signaling and insulin resistance. These findings suggest that downregulation of AMPK precedes mTOR/p70S6K activation in mediating glucose and leucine-induced insulin resistance, although the mechanism by which it does so remains to be determined. Also requiring study is how an excess of the two nutrients leads to AMPK downregulation.

Mitochondrial transporter ATP binding cassette mitochondrial erythroid is a novel gene required for cardiac recovery after ischemia/reperfusion.

BACKGROUND: Oxidative stress and mitochondrial dysfunction are central mediators of cardiac dysfunction after ischemia/reperfusion. ATP binding cassette mitochondrial erythroid (ABC-me; ABCB10; mABC2) is a mitochondrial transporter highly induced during erythroid differentiation and predominantly expressed in bone marrow, liver, and heart. Until now, ABC-me function in heart was unknown. Several lines of evidence demonstrate that the yeast ortholog of ABC-me protects against increased oxidative stress. Therefore, ABC-me is a potential modulator of the outcome of ischemia/reperfusion in the heart. METHODS AND RESULTS: Mice harboring 1 functional allele of ABC-me (ABC-me(+/-)) were generated by replacing ABC-me exons 2 and 3 with a neomycin resistance cassette. Cardiac function was assessed with Langendorff perfusion and echocardiography. Under basal conditions, ABC-me(+/-) mice had normal heart structure, hemodynamic function, mitochondrial respiration, and oxidative status. However, after ischemia/reperfusion, the recovery of hemodynamic function was reduced by 50% in ABC-me(+/-) hearts as a result of impairments in both systolic and diastolic function. This reduction was associated with impaired mitochondrial bioenergetic function and with oxidative damage to both mitochondrial lipids and sarcoplasmic reticulum calcium ATPase after reperfusion. Treatment of ABC-me(+/-) hearts with the superoxide dismutase/catalase mimetic EUK-207 prevented oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and restored mitochondrial and cardiac function to wild-type levels after reperfusion. CONCLUSIONS: Inactivation of 1 allele of ABC-me increases the susceptibility to oxidative stress induced by ischemia/reperfusion, leading to increased oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and to impaired functional recovery. Thus, ABC-me is a novel gene that determines the ability to tolerate cardiac ischemia/reperfusion.

Decreased AMP-activated protein kinase activity is associated with increased inflammation in visceral adipose tissue and with whole-body insulin resistance in morbidly obese humans.

Inflammation and infiltration of immune cells in white adipose tissue have been implicated in the development of obesity-associated insulin resistance. Likewise, dysregulation of the fuel-sensing enzyme AMP-activated protein kinase (AMPK) has been proposed as a pathogenetic factor for these abnormalities based on both its links to insulin action and its anti-inflammatory effects. In this study, we examined the relationships between AMPK activity, the expression of multiple inflammatory markers in visceral (mesenteric and omental) and abdominal subcutaneous adipose tissue, and whole-body insulin sensitivity in morbidly obese patients (BMI 48±1.9 kg/m(2)) undergoing gastric bypass surgery. AMPK activity was assessed by Western-blots (P-AMPK/T-AMPK) and mRNA levels of various markers of inflammation by qRT-PCR. Patients were stratified as insulin sensitive obese or insulin-resistant obese according to their HOMA-IR values. The results indicate that AMPK activity is lower in visceral than in subcutaneous abdominal adipose tissue of these patients and that this is associated with an increased expression of multiple inflammatory genes. They also revealed that AMPK activity is lower in adipose tissue of obese patients who are insulin resistant (HOMA-IR>2.3) than in BMI-matched insulin sensitive subjects. Furthermore, this difference was evident in all three fat depots. In conclusion, the data suggest that there are close links between reduced AMPK activity and inflammation in white adipose tissue, and whole-body insulin resistance in obese humans. Whether adipose tissue AMPK dysregulation is a causal factor for the development of the inflammation and insulin resistance remains to be determined.

Downregulation of AMPK accompanies leucine- and glucose-induced increases in protein synthesis and insulin resistance in rat skeletal muscle.

OBJECTIVE: Branched-chain amino acids, such as leucine and glucose, stimulate protein synthesis and increase the phosphorylation and activity of the mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase (p70S6K). We examined in skeletal muscle whether the effects of leucine and glucose on these parameters and on insulin resistance are mediated by the fuel-sensing enzyme AMP-activated protein kinase (AMPK). RESEARCH DESIGN AND METHODS: Rat extensor digitorum longus (EDL) muscle was incubated with different concentrations of leucine and glucose with or without AMPK activators. Muscle obtained from glucose-infused rats was also used as a model. RESULTS: In the EDL, incubation with 100 or 200 µmol/l leucine versus no added leucine suppressed the activity of the α2 isoform of AMPK by 50 and 70%, respectively, and caused concentration-dependent increases in protein synthesis and mTOR and p70S6K phosphorylation. Very similar changes were observed in EDL incubated with 5.5 or 25 mmol/l versus no added glucose and in muscle of rats infused with glucose in vivo. Incubation of the EDL with the higher concentrations of both leucine and glucose also caused insulin resistance, reflected by a decrease in insulin-stimulated Akt phosphorylation. Coincubation with the AMPK activators AICAR and α-lipoic acid substantially prevented all of those changes and increased the phosphorylation of specific sites of mTOR inhibitors raptor and tuberous sclerosis complex 2 (TSC2). In contrast, decreases in AMPK activity induced by leucine and glucose were not associated with a decrease in raptor or TSC2 phosphorylation. CONCLUSIONS: The results indicate that both leucine and glucose modulate protein synthesis and mTOR/p70S6 and insulin signaling in skeletal muscle by a common mechanism. They also suggest that the effects of both molecules are associated with a decrease in AMPK activity and that AMPK activation prevents them.

AMPK and SIRT1: a long-standing partnership?

AMP-activated protein kinase (AMPK) and the histone/protein deacetylase SIRT1 are fuel-sensing molecules that have coexisted in cells throughout evolution. When a cell's energy state is diminished, AMPK activation restores energy balance by stimulating catabolic processes that generate ATP and downregulating anabolic processes that consume ATP but are not acutely needed for survival. SIRT1 in turn is best known historically for producing genetic changes that mediate the increase in longevity caused by calorie restriction. Although the two molecules have been studied intensively for many years, only recently has it become apparent that they have similar effects on diverse processes such as cellular fuel metabolism, inflammation, and mitochondrial function. In this review we will examine the evidence that these similarities occur because AMPK and SIRT1 both regulate each other and share many common target molecules. In addition, we will discuss the clinical relevance of these interactions and in particular the possibility that their dysregulation predisposes to disorders such as type 2 diabetes and atherosclerotic cardiovascular disease and is a target for their therapy.

Prostaglandin E2 suppresses lipopolysaccharide-stimulated IFN-beta production.

Macrophages activate the production of cytokines and chemokines in response to LPS through signaling cascades downstream from TLR4. Lipid mediators such as PGE(2), which are produced during inflammatory responses, have been shown to suppress MyD88-dependent gene expression upon TLR4 activation in macrophages. The study reported here investigated the effect of PGE(2) on TLR3- and TLR4-dependent, MyD88-independent gene expression in murine J774A.1 macrophages, as well as the molecular mechanism underlying such an effect. We demonstrate that PGE(2) strongly suppresses LPS-induced IFN-beta production at the mRNA and protein levels. Poly (I:C)-induced IFN-beta and LPS-induced CCL5 production were also suppressed by PGE(2). The inhibitory effect of PGE(2) on LPS-induced IFN-beta expression is mediated through PGE(2) receptor subtypes EP(2) and EP(4), and mimicked by the cAMP analog 8-Br-cAMP as well as by the adenylyl cyclase activator forskolin. The downstream effector molecule responsible for the cAMP-induced suppressive effect is exchange protein directly activated by cAMP (Epac) but not protein kinase A. Moreover, data demonstrate that Epac-mediated signaling proceeds through PI3K, Akt, and GSK3beta. In contrast, PGE(2) inhibits LPS-induced TNF-alpha production in these cells through a distinct pathway requiring protein kinase A activity and independent of Epac/PI3K/Akt. In vivo, administration of a cyclooxygenase inhibitor before LPS injection resulted in enhanced serum IFN-beta concentration in mice. Collectively, data demonstrate that PGE(2) is a negative regulator for IFN-beta production in activated macrophages and during endotoxemia.

Impaired insulin and insulin-like growth factor expression and signaling mechanisms in Alzheimer's disease--is this type 3 diabetes?

The neurodegeneration that occurs in sporadic Alzheimer's disease (AD) is consistently associated with a number of characteristic histopathological, molecular, and biochemical abnormalities, including cell loss, abundant neurofibrillary tangles and dystrophic neurites, amyloid-beta deposits, increased activation of pro-death genes and signaling pathways, impaired energy metabolism/mitochondrial function, and evidence of chronic oxidative stress. The general inability to convincingly link these phenomena has resulted in the emergence and propagation of various heavily debated theories that focus on the role of one particular element in the pathogenesis of all other abnormalities. However, the accumulating evidence that reduced glucose utilization and deficient energy metabolism occur early in the course of disease, suggests a role for impaired insulin signaling in the pathogenesis of AD. The present work demonstrates extensive abnormalities in insulin and insulin-like growth factor type I and II (IGF-I and IGF-II) signaling mechanisms in brains with AD, and shows that while each of the corresponding growth factors is normally made in central nervous system (CNS) neurons, the expression levels are markedly reduced in AD. These abnormalities were associated with reduced levels of insulin receptor substrate (IRS) mRNA, tau mRNA, IRS-associated phosphotidylinositol 3-kinase, and phospho-Akt (activated), and increased glycogen synthase kinase-3beta activity and amyloid precursor protein mRNA expression. The strikingly reduced CNS expression of genes encoding insulin, IGF-I, and IGF-II, as well as the insulin and IGF-I receptors, suggests that AD may represent a neuro-endocrine disorder that resembles, yet is distinct from diabetes mellitus. Therefore, we propose the term, "Type 3 Diabetes" to reflect this newly identified pathogenic mechanism of neurodegeneration.