Development of a high-yield, high-quality purification process for adeno-associated virus vectors that can be used in vivo without ultracentrifugation: Application to a lung endothelial cell-targeted adeno-associated virus.

Recombinant adeno-associated viruses (rAAVs) are useful vectors for expressing genes of interest in vivo because of their low immunogenicity and long-term gene expression. Various mutations have been introduced in recent years and have enabled high-efficacy, stabilized, and organ-oriented transduction. Our purpose for using rAAV is to express our target gene in the mouse lung to investigate pulmonary artery hypertension. We constructed a self-complementary AAV having mutant capsids with the ESGHGYF insert, which directs the vectors to lung endothelial cells. However, when this mutant virus was purified from the producing cells by the conventional method using an ultracentrifuge, it resulted in a low yield. In addition, the purification method using an ultracentrifuge is tedious and labor-intensive. Therefore, we aimed to develop a simple, high-quality method for obtaining enough lung-targeted rAAV. First, we modified amino acids (T491V and Y730F) of the capsid to stabilize the rAAV from degradation, and we optimized culture conditions. Next, we noticed that many rAAVs were released from the cells into the culture medium. We, therefore, improved our purification method by purifying from the culture medium without the ultracentrifugation step. Purification without ultracentrifugation had the problem that impurities were mixed in, causing inflammation. However, by performing PEG precipitation and chloroform extraction twice, we were able to purify rAAV that caused only as little inflammation as that obtained by the ultracentrifuge method. Sufficient rAAV was obtained and can now be administered to a rat as well as mice from a single dish: 1.50 × 1013 ± 3.58 × 1012 vector genome from one φ150 mm dish (mean ± SEM).

PRESERVED PROINSULIN SECRETION IN LONG-STANDING TYPE 1 DIABETES.

OBJECTIVE: Recent literature has reported preserved residual beta-cell function (C-peptide "microsecretion") in many individuals with long-standing type 1 diabetes (T1D). However, the concentrations of detectable insulin/C-peptide in the serum are usually very low, and beta-cell mass is typically negligible. Proinsulin is measurable in the early years after diagnosis, consistent with the presence of residual functioning beta cells. However, individuals are not expected to secrete significant amounts of proinsulin beyond the early years after diagnosis. Our primary objective was to measure the prohormone, proinsulin, in a heterogeneous cohort of individuals with long-standing T1D. We also sought to assess whether proinsulin secretion might occur in certain individuals despite the absence of measurable C-peptide. METHODS: Random postmeal proinsulin concentrations were measured in 97 subjects with T1D (disease duration >3 years) recruited from within the T1D Exchange Clinic Network participants who took part in the Residual C-peptide Study. RESULTS: Forty-nine of these subjects had undetectable baseline and stimulated C-peptide (C-peptide [-]), and 48 of them had detectable C-peptide concentrations (C-peptide [+]). All the C-peptide (+) subjects had detectable serum proinsulin. Eight (16%) of the C-peptide (-) subjects had detectable serum proinsulin. CONCLUSION: We report the observation that proinsulin secretion persists in a proportion of individuals with long-standing T1D, even in the absence of measurable C-peptide. It is not yet clear why certain patients with T1D retain the ability to secrete proinsulin many years after diagnosis. ABBREVIATIONS: CP = C-peptide CV = coefficient of variation ELISA = enzyme-linked immunosorbent assay IQR = inter-quartile range MMTT = mixed-meal tolerance test NIBSC = National Institute for Biological Standards and Control PI = proinsulin T1D = type 1 diabetes.

Glucose and palmitate uncouple AMPK from autophagy in human aortic endothelial cells.

Dysregulated autophagy and decreased AMP-activated protein kinase (AMPK) activity are each associated with atherogenesis. Atherogenesis is preceded by high circulating concentrations of glucose and fatty acids, yet the mechanism by which these nutrients regulate autophagy in human aortic endothelial cells (HAECs) is not known. Furthermore, whereas AMPK is recognized as an activator of autophagy in cells with few nutrients, its effects on autophagy in nutrient-rich HAECs has not been investigated. We maintained and passaged primary HAECs in media containing 25 mM glucose and incubated them subsequently with 0.4 mM palmitate. These conditions impaired basal autophagy and rendered HAECs more susceptible to apoptosis and adhesion of monocytes, outcomes attenuated by the autophagy activator rapamycin. Glucose and palmitate diminished AMPK activity and phosphorylation of the uncoordinated-51-like kinase 1 (ULK1) at Ser555, an autophagy-activating site targeted by AMPK. 5-Aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR)-mediated activation of AMPK phosphorylated acetyl-CoA carboxylase, but treatment with AICAR or other AMPK activators (A769662, phenformin) did not restore ULK1 phosphorylation or autophagosome formation. To determine whether palmitate-induced ceramide accumulation contributed to this finding, we overexpressed a ceramide-metabolizing enzyme, acid ceramidase. The increase in acid ceramidase expression ameliorated the effects of excess nutrients on ULK1 phosphorylation, without altering the effects of the AMPK activators. Thus, unlike low nutrient conditions, AMPK becomes uncoupled from autophagy in HAECs in a nutrient-rich environment, such as that found in patients with increased cardiovascular risk. These findings suggest that combinations of AMPK-independent and AMPK-dependent therapies may be more effective alternatives than either therapy alone for treating nutrient-induced cellular dysfunction.

Large area fractional laser treatment of mouse skin increases energy expenditure.

Fractional laser (FL) treatment is a common dermatologic procedure that generates arrays of microscopic treatment zones separated by intact tissue, promoting fast wound healing. Using a mouse model, we introduced a large area fractional laser treatment (LAFLT) method to study metabolic effects. Using two laser modalities, ablative FL (AFL) and non-ablative FL (NAFL), and exposing different percentages of mice's total body surface area (TBSA), we followed changes in metabolic parameters in real time using metabolic cages. Additionally, body composition, markers of inflammation, neurohormonal signaling, and browning of adipocytes were investigated. LAFLT, especially in high TBSA groups, had specific metabolic effects such as significantly increased average daily energy expenditure, increased fat mass loss, systemic browning of adipocytes, and inflammatory states, without compromising other organs. The ability of LAFLT to stimulate metabolism in a controlled way could develop into a promising therapeutic treatment to induce positive metabolic changes that replace or augment systemic drugs.

A novel inverse relationship between metformin-triggered AMPK-SIRT1 signaling and p53 protein abundance in high glucose-exposed HepG2 cells.

AMP-activated protein kinase (AMPK) and the NAD(+)-dependent histone/protein deacetylase sirtuin 1 (SIRT1) are metabolic sensors that can increase each other's activity. They are also both activated by the antidiabetic drug metformin and downregulated in the liver under conditions of nutrient excess (e.g., hyperglycemia, high-fat diet, obesity). In these situations, the abundance of the tumor suppressor p53 is increased; however, the relevance of this to the changes in AMPK and SIRT1 is not known. In the present study we investigated this question in HepG2 cells under high glucose conditions. Metformin induced activation of AMPK and SIRT1 and decreased p53 protein abundance. It also decreased triglyceride accumulation and cytosolic oxidative stress (a trigger for p53 accumulation) and increased the deacetylation of p53 at a SIRT1-targeted site. The decrease in p53 abundance caused by metformin was abolished by inhibition of murine double minute 2 (MDM2), a ubiquitin ligase that mediates p53 degradation, as well as by overexpression of a dominant-negative AMPK or a shRNA-mediated knockdown of SIRT1. In addition, overexpression of p53 decreased SIRT1 gene expression and protein abundance, as well as AMPK activity in metformin-treated cells. It also diminished the triglyceride-lowering action of metformin, an effect that was rescued by incubation with the SIRT1 activator SRT2183. Collectively, these findings suggest the existence of a novel reciprocal interaction between AMPK/SIRT1 and p53 that may have implications for the pathogenesis and treatment of metabolic diseases.

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.

Acute exercise activates AMPK and eNOS in the mouse aorta.

Exercise can prevent endothelial cell (EC) dysfunction and atherosclerosis even in the absence of improvements in plasma lipids. However, the mechanisms responsible for these effects are incompletely understood. In this study we examined in mice whether an acute bout of exercise activates enzymes that could prevent EC dysfunction, such as AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). We also examined whether exercise alters known regulators of these enzymes. C57BL/6 mice underwent a single bout of exhaustive treadmill exercise after which their aortas were analyzed for activation of AMPK, AMPK regulatory proteins, eNOS, and various enzymes that, like AMPK, activate eNOS. We found that such exercise acutely activates both AMPK and eNOS in the whole aorta and that the magnitude of these effects correlated with both the distance run and activation of the AMPK regulatory proteins silent information regulator-1 (SIRT1)-LKB1 and CaMKKß. In contrast, Akt, PKA, PKG, and Src, other kinases known to activate eNOS, were unaffected. Immunohistochemical analysis revealed that AMPK and eNOS were both activated in the ECs of the aorta. This study provides the first evidence that an acute bout of exercise activates AMPK and eNOS in the endothelium of the aorta. The results also suggest that AMPK likely is the principal activator of eNOS in this setting and that its own activation may be mediated by both SIRT1-LKB1 and CaMKKß.

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.

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.

Impaired nicotinamide adenine dinucleotide (NAD+ ) metabolism in diabetes and diabetic tissues: Implications for nicotinamide-related compound treatment.

One of the biochemical abnormalities found in diabetic tissues is a decrease in the cytosolic oxidized to reduced forms of the nicotinamide adenine dinucleotide ratio (NAD+ /NADH also known as pseudohypoxia) caused by oxidation of excessive substrates (glucose through the polyol pathway, free fatty acids and lactate). Subsequently, a decline in NAD+ levels as a result of the activation of poly adenine nucleotide diphosphate-ribose polymerase (mainly in type 1 diabetes) or the inhibition of adenine nucleotide monophosphate-activated protein kinase (in type 2 diabetes). Thus, replenishment of NAD+ levels by nicotinamide-related compounds could be beneficial. However, these compounds also increase nicotinamide catabolites that cause oxidative stress. This is particularly troublesome for patients with diabetes, because they have impaired nicotinamide salvage pathway reactions at the level of nicotinamide phosphoribosyl transferase and phosphoribosyl pyrophosphate, which occurs by the following mechanisms. First, phosphoribosyl pyrophosphate synthesis from pentose phosphate pathway is compromised by a decrease in plasma thiamine and transketolase activity. Second, nicotinamide phosphoribosyl transferase expression is decreased because of reduced adenosine monophosphate-activated protein kinase activity, which occurs in type 2 diabetes. The adenosine monophosphate-activated protein kinase inhibition is caused by an activation of protein kinase C and D1 as a result of enhanced diacylglycerol synthesis caused by pseudohypoxia and increased fatty acids levels. In this regard, nicotinamide-related compounds should be given with caution to treat diabetes. To minimize the risk and maximize the benefit, nicotinamide-related compounds should be taken with insulin sensitizers (for type 2 diabetes), polyphenols, benfotiamine, acetyl-L-carnitine and aldose reductase inhibitors. The efficacy of these regimens can be monitored by measuring serum NAD+ and urinary nicotinamide catabolites.

Activation of LKB1 rescues 3T3-L1 adipocytes from senescence induced by Sirt1 knock-down: a pivotal role of LKB1 in cellular aging.

Previous reports have shown that excess calorie intake promotes p53 dependent senescence in mouse adipose tissues. The objective of the current study was to address the mechanism underlying this observation, i.e. adipocyte aging. Using cultured 3T3-L1 cells, we investigated the involvement of energy regulators Sirt1, AMPK, and LKB1 in senescence. Fifteen days post differentiation, Sirt1 knock-down increased senescence-associated beta-galactosidase (SA-ß-Gal) staining by 20-40% (p<0.05, n=12) and both cyclin kinase inhibitor p21Cip and chemokine receptor IL8Rb expression by 2-4 fold. ATP and expression of mitochondria Complex 1 were also reduced by 30% and 50%, respectively (p<0.05, n=4). Such energy depletion may have caused the observed increase in AMPK activity, despite LKB1 activity downregulation. This association between Sirt1 and LKB1 activity was confirmed in vivo in mouse adipose tissue. Upregulation of LKB1 activity by expression of the Sirt1-insensitive LKB1-K48R mutant in 3T3-L1 cells completely prevented the senescence-associated changes of Sirt1 knock-down. In addition, cellular senescence, which also occurs in cultured primary human aortic endothelial cells, was largely prevented by ectopic expression of LKB1. These results suggest that LKB1 plays a pivotal role in cellular senescence occurring in adipocytes and other cell types.

Fatty Acid Metabolites Combine with Reduced ß Oxidation to Activate Th17 Inflammation in Human Type 2 Diabetes.

Mechanisms that regulate metabolites and downstream energy generation are key determinants of T cell cytokine production, but the processes underlying the Th17 profile that predicts the metabolic status of people with obesity are untested. Th17 function requires fatty acid uptake, and our new data show that blockade of CPT1A inhibits Th17-associated cytokine production by cells from people with type 2 diabetes (T2D). A low CACT:CPT1A ratio in immune cells from T2D subjects indicates altered mitochondrial function and coincides with the preference of these cells to generate ATP through glycolysis rather than fatty acid oxidation. However, glycolysis was not critical for Th17 cytokines. Instead, ß oxidation blockade or CACT knockdown in T cells from lean subjects to mimic characteristics of T2D causes cells to utilize 16C-fatty acylcarnitine to support Th17 cytokines. These data show long-chain acylcarnitine combines with compromised ß oxidation to promote disease-predictive inflammation in human T2D.

Comparison of proinsulin and C-peptide secretion in healthy versus long-standing type 1 diabetes mellitus cohorts: A pilot study.

AIMS: Increased proinsulin (PI) compared to C-peptide (CP) concentrations have been reported, both prior to type 1 diabetes mellitus (T1D) onset, as well as early in disease. In this pilot study, we sought to define the normal PI secretion in a healthy cohort and compare this to a local T1D cohort and a separate well-defined nationally representative T1D cohort with measurable CP. METHODS: Thirteen healthy subjects and 12 T1D subjects with T1D >3 years from the local T1D cohort completed mixed meal tolerance tests (MMTT) with PI and CP measured over 90 and 240 minutes. The change in CP (maximum versus baseline, ΔCP) during MMTT in the T1D Exchange T1D cohort was stratified according to non-fasting PI concentrations, based on a fasting PI threshold, as defined by the healthy control group. RESULTS: The maximum fasting PI in the control group was 6 pmol/L. Individuals from the T1D Exchange with a non-fasting PI ≥ 6 pmol/L had a lower ΔCP during a MMTT, compared to those with a PI < 6 pmol/L. While only three individuals from the local T1D cohort had measurable CP and PI during the MMTT, those with a greater ΔCP had lower PI secretion. CONCLUSION: While all T1D subjects from the T1D Exchange secreted measurable non-fasting PI, those with a greater non-fasting PI demonstrated a decrease in ΔCP during the MMTT. PI may be preferentially secreted compared to CP in some individuals with long standing T1D.

Resveratrol-Induced AMP-Activated Protein Kinase Activation Is Cell-Type Dependent: Lessons from Basic Research for Clinical Application.

Despite the promising effects of resveratrol, its efficacy in the clinic remains controversial. We were the first group to report that the SIRT1 activator resveratrol activates AMP-activated protein kinase (AMPK) (Diabetes 2005; 54: A383), and we think that the variability of this cascade may be responsible for the inconsistency of resveratrol's effects. Our current studies suggest that the effect of SIRT1 activators such as resveratrol may not be solely through activation of SIRT1, but also through an integrated effect of SIRT1-liver kinase B1 (LKB1)-AMPK. In this context, resveratrol activates SIRT1 (1) by directly binding to SIRT1; and (2) by increasing NAD⁺ levels by upregulating the salvage pathway through Nampt activation, an effect mediated by AMPK. The first mechanism promotes deacetylation of a limited number of SIRT1 substrate proteins (e.g., PGC-1). The second mechanism (which may be more important than the first) activates other sirtuins in addition to SIRT1, which affects a broad spectrum of substrates. Despite these findings, detailed mechanisms of how resveratrol activates AMPK have not been reported. Here, we show that (1) resveratrol-induced activation of AMPK requires the presence of functional LKB1; (2) Resveratrol increases LKB1 activity, which involves translocation and phosphorylation at T336 and S428; (3) Activation of LKB1 causes proteasomal degradation of LKB1; (4) At high concentrations (50-100 µM), resveratrol also activates AMPK through increasing AMP levels; and (5) The above-mentioned activation mechanisms vary among cell types, and in some cell types, resveratrol fails to activate AMPK. These results suggest that resveratrol-induced activation of AMPK is not a ubiquitous phenomenon. In addition, AMPK-mediated increases in NAD⁺ in the second mechanism require several ATPs, which may not be available in many pathological conditions. These phenomena may explain why resveratrol is not always consistently beneficial in a clinical setting.

Tfe3 and Tfeb Transcriptionally Regulate Peroxisome Proliferator-Activated Receptor γ2 Expression in Adipocytes and Mediate Adiponectin and Glucose Levels in Mice.

Members of the MiT transcription factor family are pivotal regulators of several lineage-selective differentiation programs. We show that two of these, Tfeb and Tfe3, control the regulator of adipogenesis, peroxisome proliferator-activated receptor γ2 (Pparγ2). Knockdown of Tfeb or Tfe3 expression during in vitro adipogenesis causes dramatic downregulation of Pparγ2 expression as well as adipogenesis. Additionally, we found that these factors regulate Pparγ2 in mature adipocytes. Next, we demonstrated that Tfeb and Tfe3 act directly by binding to consensus E-boxes within the Pparγ transcriptional regulatory region. This transcriptional control also exists in vivo, as we discovered that wild-type mice in the fed state increased their expression of Tfe3, Tf3b, and Pparγ in white adipose tissue. Furthermore, Tfe3 knockout (Tfe3KO) mice in the fed state failed to upregulate Pparγ and the adiponectin gene, a Pparγ-dependent gene, confirming the in vivo role for Tfe3. Lastly, we found that blood glucose is elevated and serum adiponectin levels are suppressed in the Tfe3KO mice, indicating that the Tfe3/Tfeb/Pparγ2 axis may contribute to whole-body energy balance. Thus, we offer new insights into the upstream regulation of Pparγ by Tfe3/Tf3b and propose that targeting these transcription factors may offer opportunities to complement existing approaches for the treatment of diseases that have dysregulated energy metabolism.

Palmitate-induced apoptosis in cultured bovine retinal pericytes: roles of NAD(P)H oxidase, oxidant stress, and ceramide.

Apoptosis of pericytes (PCs) is an early event in diabetic retinopathy. It is generally thought to be a consequence of sustained hyperglycemia. In keeping with this, long-term (>7 days) incubation of cultured PCs in a high-glucose media has been shown to increase apoptosis. We examine here whether the saturated free fatty acid palmitate, the concentration of which is often elevated in diabetes, has similar effects on cultured PCs. Incubation with 0.4 mmol/l palmitate for 24 h induced both oxidant stress and apoptosis, as evidenced by a sixfold increase in DCF fluorescence and a twofold increase in caspase-3 activation, respectively. NAD(P)H oxidase appeared to be involved in these responses, since overexpression of dominant-negative subunits of NAD(P)H oxidase, such as phox47(DN), diminished oxidant stress, and phox67(DN) and N-17 RAC1(DN) prevented the increase in caspase-3 activity. Likewise, overexpression of vRAC, a constitutively active RAC1, increased caspase-3 activity to the same extent as palmitate alone. The effects of vRAC and palmitate were not additive. In parallel with the increases in oxidative stress, the redox-sensitive transcription factor nuclear factor-kappaB (NF-kappaB) was activated in cells incubated with 0.4 mmol/l palmitate. Furthermore, inhibition of NF-kappaB activation by various means inhibited caspase-3 activation. Finally, incubation with palmitate increased the cellular content of ceramide, a molecule linked to apoptosis and increases in oxidative stress and NF-kappaB activation in other cells. In keeping with such a role, in PCs both coincubation with fumonisin B1 (a ceramide synthase inhibitor) and overexpression of ceramidase I reversed the proapoptotic effect of palmitate. On the other hand, they increased rather than decreased DCF fluorescence. In conclusion, the results suggest that palmitate-induced apoptosis in PCs is associated with activation of NAD(P)H oxidase and NF-kappaB and an increase in ceramide. The precise interactions between these molecules in causing apoptosis and the importance of oxidant stress as a contributory factor remain to be determined.

Unraveling the actions of AMP-activated protein kinase in metabolic diseases: Systemic to molecular insights.

AMP-activated protein kinase (AMPK) plays a critical role both in sensing and regulating cellular energy state. In experimental animals, its activation has been shown to reduce the risk of obesity and diabetes-related co-morbidities such as insulin resistance, the metabolic syndrome and atherosclerotic cardiovascular disease. However, in humans, AMPK activation alone often does not completely resolve these conditions. Thus, an improved understanding of AMPK action and regulation in metabolic and other diseases is needed. Herein, we provide a brief description of the enzymatic regulation of AMPK and review its role in maintaining energy homeostasis. We then discuss tissue-specific actions of AMPK that become distorted during such conditions as obesity, type 2 diabetes and certain cancers. Finally, we explore recent findings regarding the interactions of AMPK with mammalian target of rapamycin complex 1 and the lysosome and discuss how changes in these relationships during overnutrition may lead to AMPK dysfunction. A more thorough understanding of AMPK's molecular interactions during diseases of overnutrition may provide key insights for the development of AMPK-based combinatorial treatments for metabolic disease.

Knockdown of GSK3ß increases basal autophagy and AMPK signalling in nutrient-laden human aortic endothelial cells.

High concentrations of glucose and palmitate increase endothelial cell inflammation and apoptosis, events that often precede atherogenesis. They may do so by decreasing basal autophagy and AMP-activated protein kinase (AMPK) activity, although the mechanisms by which this occurs are not clear. Decreased function of the lysosome, an organelle required for autophagy and AMPK, have been associated with hyperactivity of glycogen synthase kinase 3ß (GSK3ß). To determine whether GSK3ß affects nutrient-induced changes in autophagy and AMPK activity, we used a primary human aortic endothelial cell (HAEC) model of type 2 diabetes that we had previously characterized with impaired AMPK activity and autophagy [Weikel et al. (2015) Am. J. Phys. Cell Physiol. 308: , C249-C263]. Presently, we found that incubation of HAECs with excess nutrients (25 mM glucose and 0.4 mM palmitate) increased GSK3ß activity and impaired lysosome acidification. Suppression of GSK3ß in these cells by treatment with a chemical inhibitor or overexpression of kinase-dead GSK3ß attenuated these lysosomal changes. Under control and excess nutrient conditions, knockdown of GSK3ß increased autophagosome formation, forkhead box protein O1 (FOXO1) activity and AMPK signalling and decreased Akt signalling. Similar changes in autophagy, AMPK and Akt signalling were observed in aortas from mice treated with the GSK3ß inhibitor CHIR 99021. Thus, increasing basal autophagy and AMPK activity by inhibiting GSK3ß may be an effective strategy in the setting of hyperglycaemia and dyslipidaemia for restoring endothelial cell health and reducing atherogenesis.

Glucagon-like peptide-1 (GLP-1) analog liraglutide inhibits endothelial cell inflammation through a calcium and AMPK dependent mechanism.

Liraglutide is a glucagon-like peptide-1 (GLP-1) mimetic used for the treatment of Type 2 diabetes. Similar to the actions of endogenous GLP-1, liraglutide potentiates the post-prandial release of insulin, inhibits glucagon release and increases satiety. Recent epidemiological studies and clinical trials have suggested that treatment with GLP-1 mimetics may also diminish the risk of cardiovascular disease in diabetic patients. The mechanism responsible for this effect has yet to be determined; however, one possibility is that they might do so by a direct effect on vascular endothelium. Since low grade inflammation of the endothelium is an early event in the pathogenesis of atherosclerotic cardiovascular disease (ASCVD), we determined the effects of liraglutide on inflammation in cultured human aortic endothelial cells (HAECs). Liraglutide reduced the inflammatory responses to TNFα and LPS stimulation, as evidenced by both reduced protein expression of the adhesion molecules VCAM-1 and E-Selectin, and THP-1 monocyte adhesion. This was found to result from increased cell Ca2+ and several molecules sensitive to Ca2+ with known anti inflammatory actions in endothelial cells, including CaMKKß, CaMKI, AMPK, eNOS and CREB. Treatment of the cells with STO-609, a CaMKK inhibitor, diminished both the activation of AMPK, CaMKI and the inhibition of TNFα and LPS-induced monocyte adhesion by liraglutide. Likewise, expression of an shRNA against AMPK nullified the anti-inflammatory effects of liraglutide. The results indicate that liraglutide exerts a strong anti-inflammatory effect on HAECs. They also demonstrate that this is due to its ability to increase intracellular Ca2+ and activate CAMKKß, which in turn activates AMPK.