Yasuo Ido

Hyperglycemic Pseudohypoxia and Diabetic Complications

Vasodilation and increased blood flow are characteristic early vascular responses to acute hyperglycemia and tissue hypoxia. In hypoxic tissues these vascular changes are linked to metabolic imbalances associated with impaired oxidation of NADH to NAD+ and the resulting increased ratio of NADH/NAD+. In hyperglycemic tissues these vascular changes also are linked to an increased ratio of NADH/NAD+, in this case because of an increased rate of reduction of NAD+ to NADH. Several lines of evidence support the likelihood that the increased cytosolic ratio of free NADH/NAD+ caused by hyperglycemia, referred to as pseudohypoxia because tissue partial pressure oxygen is normal, is a characteristic feature of poorly controlled diabetes that mimics the effects of true hypoxia on vascular and neural function and plays an important role in the pathogenesis of diabetic complications. These effects of hypoxia and hyperglycemia-induced pseudohypoxia on vascular and neural function are mediated by a branching cascade of imbalances in lipid metabolism, increased production of superoxide anion, and possibly increased nitric oxide formation.

Aminoguanidine, a Novel Inhibitor of Nitric Oxide Formation, Prevents Diabetic Vascular Dysfunction

Increased blood flow and vascular leakage of proteins preferentially affect tissues that are sites of diabetic complications in humans and animals. These vascular changes in diabetic rats are largely prevented by aminoguanidine. Glucose-induced vascular changes in nondiabetic rats are also prevented by aminoguanidine and by NG-monomethyl-L-arginine (NMMA), an established inhibitor of nitric oxide (NO·) formation from L-arginine. Aminoguanidine and NMMA are equipotent inhibitors of interleukin-1 β-induced 1) nitrite formation (an oxidation product of NO·) and cGMP accumulation by the rat β-cell insulinoma cell line RINm5F, and 2) inhibition of glucose-stimulated insulin secretion and formation of iron-nitrosyl complexes by islets of Langerhans. In contrast, NMMA is ∼ 40 times more potent than aminoquanidine in elevating blood pressure in nondiabetic rats. These results demonstrate that aminoguanidine inhibits NO. production and suggest a role for NO· in the pathogenesis of diabetic vascular complications.

SIRT1 Regulates Hepatocyte Lipid Metabolism through Activating AMP-activated Protein Kinase

Resveratrol may protect against metabolic disease through activating SIRT1 deacetylase. Because we have recently defined AMPK activation as a key mechanism for the beneficial effects of polyphenols on hepatic lipid accumulation, hyperlipidemia, and atherosclerosis in type 1 diabetic mice, we hypothesize that polyphenol-activated SIRT1 acts upstream of AMPK signaling and hepatocellular lipid metabolism. Here we show that polyphenols, including resveratrol and the synthetic polyphenol S17834, increase SIRT1 deacetylase activity, LKB1 phosphorylation at Ser428, and AMPK activity. Polyphenols substantially prevent the impairment in phosphorylation of AMPK and its downstream target, ACC (acetyl-CoA carboxylase), elevation in expression of FAS (fatty acid synthase), and lipid accumulation in human HepG2 hepatocytes exposed to high glucose. These effects of polyphenols are largely abolished by pharmacological and genetic inhibition of SIRT1, suggesting that the stimulation of AMPK and lipid-lowering effect of polyphenols depend on SIRT1 activity. Furthermore, adenoviral overexpression of SIRT1 stimulates the basal AMPK signaling in HepG2 cells and in the mouse liver. AMPK activation by SIRT1 also protects against FAS induction and lipid accumulation caused by high glucose. Moreover, LKB1, but not CaMKKβ, is required for activation of AMPK by polyphenols and SIRT1. These findings suggest that SIRT1 functions as a novel upstream regulator for LKB1/AMPK signaling and plays an essential role in the regulation of hepatocyte lipid metabolism. Targeting SIRT1/LKB1/AMPK signaling by polyphenols may have potential therapeutic implications for dyslipidemia and accelerated atherosclerosis in diabetes and age-related diseases.

SIRT1 Modulation of the Acetylation Status, Cytosolic Localization, and Activity of LKB1 POSSIBLE ROLE IN AMP-ACTIVATED PROTEIN KINASE ACTIVATION

SIRT1, a histone/protein deacetylase, and AMP-activated protein kinase (AMPK) are key enzymes responsible for longevity and energy homeostasis. We examined whether a mechanistic connection exists between these molecules that involves the major AMPK kinase LKB1. Initial studies demonstrated that LKB1 is acetylated in cultured (HEK293T) cells, mouse white adipose tissue, and rat liver. In the 293T cells, SIRT1 overexpression diminished lysine acetylation of LKB1 and concurrently increased its activity, cytoplasmic/nuclear ratio, and association with the LKB1 activator STRAD. In contrast, short hairpin RNA for SIRT1, where studied, had opposite effects on these parameters. Mass spectrometric analysis established that acetylation of LKB1 occurs on multiple, but specific, lysine residues; however, only mutation of lysine 48 to arginine, which mimics deacetylation, reproduced all of the effects of activated SIRT1. SIRT1 also affected downstream targets of LKB1. Thus its overexpression increased AMPK and acetyl-CoA carboxylase phosphorylation, and conversely, RNA interference-mediated SIRT1 knockdown reduced AMPK phosphorylation and that of another LKB1 target MARK1. Consistent with the results in cultured cells, total LKB1 lysine acetylation was decreased by 60% in the liver of 48-h starved rats compared with starved-refed rats, and this was associated with modest but significant increases in both LKB1 and AMPK activities. These results suggest that LKB1 deacetylation is regulated by SIRT1 and that this in turn influences its intracellular localization, association with STRAD, kinase activity, and ability to activate AMPK.

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 cells 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.

Concurrent regulation of AMP-activated protein kinase and SIRT1 in mammalian cells

We examined in HepG2 cells whether glucose-induced changes in AMP-activated protein kinase (AMPK) activity could be mediated by SIRT1, an NAD(+)-dependent histone/protein deacetylase that has been linked to the increase in longevity caused by caloric restriction. Incubation with 25 vs. 5mM glucose for 6h concurrently diminished the phosphorylation of AMPK (Thr 172) and ACC (Ser 79), increased lactate release, and decreased the abundance and activity of SIRT1. In contrast, incubation with pyruvate (0.1 and 1mM) for 2h increased AMPK phosphorylation and SIRT1 abundance and activity. The putative SIRT1 activators resveratrol and quercetin also increased AMPK phosphorylation. None of the tested compounds (low or high glucose, pyruvate, and resveratrol) significantly altered the AMP/ATP ratio. Collectively, these findings raise the possibility that glucose-induced changes in AMPK are linked to alterations in SIRT1 abundance and activity and possibly cellular redox state.

α-Adrenergic Receptor–Stimulated Hypertrophy in Adult Rat Ventricular Myocytes Is Mediated via Thioredoxin-1–Sensitive Oxidative Modification of Thiols on Ras

Background—&agr;-Adrenergic receptor (&agr;AR)–stimulated hypertrophy in adult rat ventricular myocytes is mediated by reactive oxygen species–dependent activation of the Ras-Raf-MEK1/2-ERK1/2 signaling pathway. Because Ras is known to have redox-sensitive cysteine residues, we tested the hypothesis that &agr;AR-stimulated hypertrophic signaling is mediated via oxidative modification of Ras thiols. Methods and Results—The effect of &agr;AR stimulation on the number of free thiols on Ras was measured with biotinylated iodoacetamide labeling. &agr;AR stimulation caused a 48% decrease in biotinylated iodoacetamide–labeled Ras that was reversed by dithiothreitol (10 mmol/L), indicating a decrease in the availability of free thiols on Ras as a result of an oxidative posttranslational modification. This effect was abolished by adenoviral overexpression of thioredoxin-1 (TRX1) and potentiated by the TRX reductase inhibitor azelaic acid. Likewise, &agr;AR-stimulated Ras activation was abolished by TRX1 overexpression and potentiated by azelaic acid. TRX1 overexpression inhibited the &agr;AR-stimulated phosphorylation of MEK1/2, ERK1/2, and p90RSK and prevented cellular hypertrophy, sarcomere reorganization, and protein synthesis (versus &bgr;-galactosidase). Azelaic acid potentiated &agr;AR-stimulated protein synthesis. Although TRX1 can directly reduce thiols, it also can scavenge ROS by increasing peroxidase activity. To examine this possibility, peroxidase activity was increased by transfection with catalase, and intracellular reactive oxygen species were measured with dichlorofluorescein diacetate fluorescence. Although catalase increased peroxidase activity ≈20-fold, TRX1 had no effect. Likewise, the &agr;AR-stimulated increase in dichlorofluorescein diacetate fluorescence was abolished with catalase but retained with TRX1. Conclusions—&agr;AR-stimulated hypertrophic signaling in adult rat ventricular myocytes is mediated via a TRX1-sensitive posttranslational oxidative modification of thiols on Ras.

Effects of Long-Term Aldose Reductase Inhibition on Development of Experimental Diabetic Neuropathy: Ultrastructural and Morphometric Studies of Sural Nerve in Streptozocin-Induced Diabetic Rats

There is controversy over the efficacy of aldose reductase inhibitors in preventing the development of peripheral nerve lesions in experimental diabetes. This study was designed to show whether long-term (28-wk) inhibition of aldose reductase by ponalrestat influences structural changes in peripheral sensory nerve in rats with chronic streptozocin-induced diabetes. Sciatic nerve levels of sorbitol and fructose were significantly reduced but not completely normalized by ponalrestat treatment, myo-lnositol levels, which tended to decrease in diabetic rats, were significantly increased by ponalrestat treatment and exceeded the level in nondiabetic control rats (P < 0.01). Ponalrestat treatment significantly increased nerve conduction velocity over the 28 wk of treatment (P < 0.05), but levels remained well below those of control rats. Structural analysis of sural nerve of diabetic rats disclosed significant preventive effects of ponalrestat on the reduction in myelinated nerve fiber size and fiber occupancy. Axon-fiber size ratio was also preserved in the ponalrestat-treated group. However, diffuse deposition of glycogen and increased glycogenosomes within axons were not influenced by ponalrestat treatment. In contrast to the effect on myelinated nerve fibers, morphometry of unmyelinated nerve fibers did not reveal a significant effect of ponalrestat treatment. These results suggest that chronic treatment with an aldose reductase inhibitor has beneficial effects on the peripheral sensory nerve of experimentally diabetic rats. The effects were primarily on myelinated rather than unmyelinated nerve fibers.

S-glutathiolation by peroxynitrite of p21ras at cysteine-118 mediates its direct activation and downstream signaling in endothelial cells

The highly reactive species, peroxynitrite, is produced in endothelial cells in pathological states in which the production of superoxide anion and NO is increased. Here, we show that peroxynitrite added exogenously or generated endogenously in response to exposure to an NO donor or oxidized low‐density lipoproteins (oxLDL) increases p21ras activity in bovine aortic endothelial cells. The activation is not dependent on upstream elements but rather is due to direct targeting of p21ras by reversible S‐glutathiolation of cysteine thiols as demonstrated by biotin‐labeling techniques. The time course of p21ras S‐glutathiolation following peroxynitrite corresponds to the increase in its Raf‐1 binding activity and translocation to the membrane. Moreover, p21ras S‐glutathiolation and activation can be reversed by dithiothreitol, confirming the importance of a disulfide bond. S‐glutathiolation also promoted guanine nucleotide exchange of recombinant p21ras. In addition, the oxidant‐induced activation of Mek/Erk and PI3 kinase/Akt was abrogated by dominant‐negative and Cys‐118 p21ras mutants, and the latter also prevented S‐glutathiolation of p21ras. These results indicate that peroxynitrite arising from NO donors or pathological stimuli such as oxLDL triggers direct S‐glutathiolation of p21ras Cys118, which increases p21ras activity and mediates downstream signaling.

Hyperglycemia and insulin resistance: possible mechanisms.

Abstract: Sustained hyperglycemia impairs insulin‐stimulated glucose utilization and glycogen synthesis in human and rat skeletal muscles, a phenomenon referred to clinically as glucose toxicity. In rat extensor digitorum longus (EDL) muscle preparations preincubated for 2‐4 h in a hyperglycemic medium (25 mM vs. 0 mM glucose), we have shown that the ability of insulin to stimulate glucose incorporation into glycogen is impaired. Interestingly, this was associated with a decreased activation of Akt/PKB, but not its upstream regulator, PI3‐kinase. A similar pattern of signaling abnormalities has been observed in adipocytes, L6 muscle cells, C2C12 cells, and (as reported here) EDL incubated with C2‐ceramide. On the other hand, no increase was observed in ceramide mass in EDL incubated with 25 mM glucose. Hyperglycemia‐induced insulin resistance also has been described in adipocytes, where it has been linked to activation of novel and conventional protein kinase C isoforms that phosphorylate the insulin receptor and IRS. In addition, we have recently shown that hyperglycemia causes insulin resistance in cultured human umbilical vein endothelial cells (HUVEC). Here, it was associated with an increased propensity to apoptosis and, as in muscle, with an impaired ability of insulin to activate Akt. Interestingly, these effects of hyperglycemia and an increase in diacylglycerol synthesis, which is also caused, were prevented by adding AICAR, an activator of AMP‐activated protein kinase (AMPK), to the incubation medium. These results suggest that hyperglycemia causes insulin resistance in cells other than those in classic insulin target tissues. Whether AMPK activation can reverse or prevent insulin resistance in all of these cells remains to be determined.