Dihydroxyacetone suppresses mTOR nutrient signaling and induces mitochondrial stress in liver cells.

Dihydroxyacetone (DHA) is the active ingredient in sunless tanning products and a combustion product from e-juices in electronic cigarettes (e-cigarettes). DHA is rapidly absorbed in cells and tissues and incorporated into several metabolic pathways through its conversion to dihydroxyacetone phosphate (DHAP). Previous studies have shown DHA induces cell cycle arrest, reactive oxygen species, and mitochondrial dysfunction, though the extent of these effects is highly cell-type specific. Here, we investigate DHA exposure effects in the metabolically active, HepG3 (C3A) cell line. Metabolic and mitochondrial changes were evaluated by characterizing the effects of DHA in metabolic pathways and nutrient-sensing mechanisms through mTOR-specific signaling. We also examined cytotoxicity and investigated the cell death mechanism induced by DHA exposure in HepG3 cells. Millimolar doses of DHA were cytotoxic and suppressed glycolysis and oxidative phosphorylation pathways. Nutrient sensing through mTOR was altered at both short and long time points. Increased mitochondrial reactive oxygen species (ROS) and mitochondrial-specific injury induced cell cycle arrest and cell death through a non-classical apoptotic mechanism. Despite its carbohydrate nature, millimolar doses of DHA are toxic to liver cells and may pose a significant health risk when higher concentrations are absorbed through e-cigarettes or spray tanning.

Successful treatment of aluminium phosphide poisoning by dihydroxyacetone: A two-case report study.

WHAT IS KNOWN AND OBJECTIVE: Aluminium phosphide (AlP) is an agricultural fumigant which produces phosphine gas in the presence of moisture. Phosphine inhibits oxidative phosphorylation and causes cell death by inhibiting cytochrome C oxidase. Clinical manifestations of AlP poisoning are refractory hypotension, tachycardia, low oxygen saturation and severe metabolic acidosis. CASE SUMMARY: Two cases received dihydroxyacetone (DHA) in addition to routine management of AlP poisoning. Administration of DHA (7 gr in 50 mL sodium bicarbonate, gavage) 2 times at a 1-hour interval improved the clinical signs. WHAT IS NEW AND CONCLUSION: This is the first case report to highlight the safe and successful treatment of AlP poisoning with DHA. However, more clinical studies are recommended to determine the precise mechanism of DHA action.

Metformin lowers glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation.

The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the carbohydrate-response element-binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a complex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3-3H]glucose relative to [2-3H]glucose; by an increase in the lactate m2/m1 isotopolog ratio from [1,2-13C2]glucose; by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1; and by marked G6P elevation by selective inhibition of phosphofructokinase-1; but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase-independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, Pi, and glycerol 3-phosphate.

Dihydroxyacetone Exposure Alters NAD(P)H and Induces Mitochondrial Stress and Autophagy in HEK293T Cells.

Dihydroxyacetone phosphate (DHAP) is the endogenous byproduct of fructose metabolism. Excess DHAP in cells can induce advanced glycation end products and oxidative stress. Dihydroxyacetone (DHA) is the triose precursor to DHAP. DHA is used as the active ingredient in sunless tanning products, including aerosolized spray tans, and is formed by the combustion of solvents found in electronic cigarettes. Human exposure to DHA has been increasing as the popularity of sunless tanning products and electronic cigarettes has grown. Topically applied DHA is absorbed through the viable layers of the skin and into the bloodstream. Exogenous exposure to DHA is cytotoxic in immortalized keratinocytes and melanoma cells with cell cycle arrest induced within 24 h and cell death occurring by apoptosis at consumer-relevant concentrations of DHA within 72 h. Less is known about systemic exposures to DHA that occur following absorption through skin, and now through inhalation of the aerosolized DHA used in spray tanning. In the present study, HEK293T cells were exposed to consumer-relevant concentrations of DHA to examine the cytotoxicity of systemic exposures. HEK293T cells were sensitive to consumer-relevant doses of DHA with an IC50 value of 2.4 ± 0.3 mM. However, cell cycle arrest did not begin until 48 h after DHA exposure. DHA-exposed cells showed altered metabolic activity with decreased mitochondrial function and decreased lactate and ATP production observed within 24 h of exposure. Autofluorescent imaging and NAD+ sensors also revealed an imbalance in the redox cofactors NAD+/NADH within 24 h of exposure. Cell death occurred by autophagy indicated by increases in LC3B and SIRT1. Despite DHA's ability to be converted to DHAP and integrated into metabolic pathways, the metabolic dysfunction and starvation responses observed in the HEK293T cells indicate that DHA does not readily contribute to the energetic pool in these cells.

Prevention of phosphine-induced cytotoxicity by nutrients in HepG2 cells.

BACKGROUND & OBJECTIVES: Phosphides used as an insecticide and rodenticide, produce phosphine (PH3) which causes accidental and intentional poisoning cases and deaths. There is no specific treatment or antidote available for PH3poisoning. It is suggested that PH3-induced toxicity is associated with adenosine triphosphate (ATP) depletion; therefore, in this study the effect of some nutrients was evaluated on PH3cytotoxicity in a cell culture model. METHODS: PH3was generated from reaction of zinc phosphide (10 mM) with water in the closed culture medium of HepG2 cells, and cytotoxicity was measured after one and three hours of incubation. ATP, glutathione (GSH) and lipid peroxidation were also assessed at one or three hours post-incubation. ATP suppliers including dihydroxyacetone, glyceraldehyde and fructose were added to the culture medium 10 min before PH3generation to prevent or reduce phosphine-induced cytotoxicity. RESULTS: Phosphine caused about 30 and 66 per cent cell death at one and three hours of incubation, respectively. ATP content of the cells was depleted to 14.7 per cent of control at one hour of incubation. ATP suppliers were able to prevent cytotoxicity and ATP depletion induced by PH3. Dihydroxyacetone, α-ketoglutarate, fructose and mannitol restored the ATP content of the cells from 14.7 per cent to about 40 , 34 , 32 and 30 per cent, respectively. Lipid peroxidation and GSH depletion were not significantly induced by zinc phosphide in this study. INTERPRETATION & CONCLUSIONS: The results supported the hypothesis that phosphine-induced cytotoxicity was due to decrease of ATP levels. ATP suppliers could prevent its toxicity by generating ATP through glycolysis. α-keto compounds such as dihydroxyacetone and α-ketoglutarate may bind to phosphine and restore mitochondrial respiration.

Interrelation between the inhibition of glycolytic flux by silibinin and the lowering of mitochondrial ROS production in perifused rat hepatocytes.

Silibinin, the most biologically active component of the polyphenolic extract from milk thistle seeds, is widely used to prevent many types of hepatobiliary disorders. Recent evidence suggests new applications for this ancient medication, notably for the treatment of type 2 diabetes owing to its antihyperglycemic properties. As we have lately demonstrated that silibinin lowered glucose production from various gluconeogenic substrates in perifused rat hepatocytes, the aim of this study was to examine the effect of silibinin on both oxidative glucose utilization and reactive oxygen species (ROS) generation since the release of ROS secondary to an increased mitochondrial metabolism may contribute to diabetic damage. We found that silibinin dose-dependently reduced glycolysis from carbohydrates in a cell perifusion system via an inhibitory effect targeted on pyruvate kinase activity. Furthermore, a dramatic effect upon oxidative phosphorylation was shown, as evidenced by a fall in ATP-to-ADP ratio, together with an increase in lactate-to-pyruvate ratio. The most attractive finding was that silibinin, at a concentration as low as 10 microM, fully mitigated the rise in metabolic flow-driven ROS formation. In addition, studies on isolated liver mitochondria revealed that this low dose of silibinin depressed ROS production linked to the electron transfer chain activity. From these results, one may tentatively suggest that interesting activities for silibinin, beyond its general antioxidant status, could be expected from its potential clinical use, especially in pathological conditions when mitochondrial ROS formation is severely enhanced.

Cytotoxicity of chloroquine in isolated rat hepatocytes.

Chloroquine is a synthetic quinoline being used as an antimalaria and antirheumatoid agent. Several cases of hepatotoxicity have been reported with the use of chloroquine. However, the mechanism(s) of its hepatotoxic effect is unknown. The purpose of this study was to investigate the cytotoxic mechanism of chloroquine. Cytotoxicity was studied using freshly isolated rat hepatocytes incubated in Krebs-Henseleit buffer under a flow of 95% O(2) and 5% CO(2). Chloroquine was toxic towards hepatocytes and caused cell death with an ED(50) of about 100 mm in 2 h. The events before cell death were rapid GSH depletion and lipid peroxidation. Cytochrome P-450 inhibitors, troleandromycine, cimetidine and quinidine increased the cytotoxicity of chloroquine. Antioxidants significantly prevented the cytotoxicity of chloroquine. Depleting the hepatocyte GSH beforehand increased the chloroquine cytotoxicity. Preventing chloroquine metabolism by specific P-450 inhibitors increased its toxicity, suggesting that a major part of its toxicity is mediated by chloroquine and not by its metabolites. A depletion of the antioxidant defense system is involved in the mechanism of cytotoxicity.

Identification of human and rat FAD-AMP lyase (cyclic FMN forming) as ATP-dependent dihydroxyacetone kinases.

Rat liver FAD-AMP lyase or FMN cyclase is the only known enzymatic source of the unusual flavin nucleotide riboflavin 4',5'-cyclic phosphate. To determine its molecular identity, a peptide-mass fingerprint of the purified rat enzyme was obtained. It pointed to highly related, mammalian hypothetical proteins putatively classified as dihydroxyacetone (Dha) kinases due to weaker homologies to biochemically proven Dha kinases of plants, yeasts, and bacteria. The human protein LOC26007 cDNA was used to design PCR primers. The product amplified from human brain cDNA was cloned, sequenced (GenBank Accession No. ), and found to differ from protein LOC26007 cDNA by three SNPs. Its heterologous expression yielded a protein active both as FMN cyclase and ATP-dependent Dha kinase, each activity being inhibited by the substrate(s) of the other. Cyclase and kinase activities copurified from rat liver extracts. Evidence supports that a single protein sustains both activities, probably in a single active center. Putative Dha kinases from other mammals are likely to be FMN cyclases too. Future work will profit from the availability of the structure of Citrobacter freundii Dha kinase, which contains substrate-interacting residues conserved in human Dha kinase/FMN cyclase.

Overexpression of short heterodimer partner recovers impaired glucose-stimulated insulin secretion of pancreatic beta-cells overexpressing UCP2.

The short heterodimer partner (SHP) (NR0B2) is an orphan nuclear receptor whose function in pancreatic beta-cells is unclear. Mitochondrial uncoupling protein (UCP2) in beta-cells is upregulated in obesity-related diabetes, causing impaired glucose-stimulated insulin secretion (GSIS). We investigated whether SHP plays a role in UCP2-induced GSIS impairment. We overexpressed SHP in normal islet cells and in islet cells overexpressing UCP2 by an adenovirus-mediated infection technique. We found that SHP overexpression enhanced GSIS in normal islets, and restored GSIS in UCP2-overexpressing islets. SHP overexpression increased the glucose sensitivity of ATP-sensitive K+ (KATP) channels and enhanced the ATP/ADP ratio. A peroxisome proliferator-activated receptor gamma (PPARgamma) antagonist, GW9662, did not block the SHP effect on GSIS. SHP overexpression also corrected the impaired sensitivity of UCP2-overexpressing beta-cells to methylpyruvate, another energy fuel that bypasses glycolysis and directly enters the Krebs cycle. KATP channel inhibition mediated by dihydroxyacetone, which gives reducing equivalents directly to complex II of the electron transport system, was similar in Ad-Null-, Ad-UCP2- and Ad-UCP2+Ad-SHP-infected cells. The mitochondrial metabolic inhibitor sodium azide totally blocked the effect of SHP overexpression on GSIS. These results suggest that SHP positively regulates GSIS in beta-cells and restores glucose sensitivity in UCP2-overexpressing beta-cells by enhancing mitochondrial glucose metabolism, independent of PPARgamma activation.

Sunless skin tanning with dihydroxyacetone delays broad-spectrum ultraviolet photocarcinogenesis in hairless mice.

Sunless tanning with dihydroxyacetone (DHA) is not considered to be a sunscreen although it does absorb parts of the ultraviolet (UV) spectrum. We investigated the protection with topical application of DHA against solar UV-induced skin carcinogenesis in lightly pigmented hairless hr/hr C3H/Tif mice. Broad-spectrum UV radiation, simulating the UV part of the solar spectrum was obtained from one Philips TL12 and five Bellarium-S SA-1-12 tubes. Three groups of mice were UV-exposed four times a week to a dose-equivalent of four times the standard erythema dose (SED), without or with application of 5 or 20% DHA only twice a week. Similarly, three groups of mice were treated with DHA and irradiated with a high UV dose (8 SED), simulating a skin burn. Two groups (controls) were not irradiated, but either left untreated or treated with 20% DHA alone. The UV-induced skin pigmentation by melanogenesis could easily be distinguished from DHA-induced browning and was measured by a non-invasive, semi-quantitative method. Application of 20% DHA reduced by 63% the pigmentation produced by 4 SED, however, only by 28% the pigmentation produced by 8 SED. Furthermore, topical application of 20% DHA significantly delayed the time to appearance of the first tumor >or=1mm (P=0.0012) and the time to appearance of the third tumor (P=2 x 10(-6)) in mice irradiated with 4 SED. However, 20% DHA did not delay tumor development in mice irradiated with 8 SED. Application of 5% DHA did not influence pigmentation or photocarcinogenesis. In conclusion, this is the first study to show that the superficial skin coloring generated by frequent topical application of DHA in high concentrations may delay skin cancer development in hairless mice irradiated with moderate UV doses.

Dihydroxyacetone-induced oscillations in cytoplasmic free Ca2+ and the ATP/ADP ratio in pancreatic beta-cells at substimulatory glucose.

Glucose stimulation of pancreatic beta-cells causes oscillatory influx of Ca2+, leading to pulsatile insulin secretion. We have proposed that this is due to oscillations of glycolysis and the ATP/ADP ratio, which modulate the activity of ATP-sensitive K+ channels. We show here that dihydroxyacetone, a secretagogue that feeds into glycolysis below the putative oscillator phosphofructokinase, could cause a single initial peak in cytoplasmic free Ca2+ ([Ca2+]i) but did not by itself cause repeated oscillations in [Ca2+]i in mouse pancreatic beta-cells. However, in the presence of a substimulatory concentration of glucose (4 mm), dihydroxyacetone induced [Ca2+]i oscillations. Furthermore, these oscillations correlated with oscillations in the ATP/ADP ratio, as seen previously with glucose stimulation. Insulin secretion in response to dihydroxyacetone was transient in the absence of glucose but was considerably enhanced and somewhat prolonged in the presence of a substimulatory concentration of glucose, in accordance with the enhanced [Ca2+]i response. These results are consistent with the hypothesized role of phosphofructokinase as the generator of the oscillations. Dihydroxyacetone may affect phosphofructokinase by raising the free concentration of fructose 1,6-bisphosphate to a critical level at which it activates the enzyme autocatalytically, thereby inducing the pulses of phosphofructokinase activity that cause the metabolic oscillations.

Mitochondrial metabolism sets the maximal limit of fuel-stimulated insulin secretion in a model pancreatic beta cell: a survey of four fuel secretagogues.

The precise metabolic steps that couple glucose catabolism to insulin secretion in the pancreatic beta cell are incompletely understood. ATP generated from glycolytic metabolism in the cytosol, from mitochondrial metabolism, and/or from the hydrogen shuttles operating between cytosolic and mitochondrial compartments has been implicated as an important coupling factor. To identify the importance of each of these metabolic pathways, we have compared the fates of four fuel secretagogues (glucose, pyruvate, dihydroxyacetone, and glycerol) in the INS1-E beta cell line. Two of these fuels, dihydroxyacetone and glycerol, are normally ineffective as secretagogues but are enabled by adenovirus-mediated expression of glycerol kinase. Comparison of these two particular fuels allows the effect of redox state on insulin secretion to be evaluated since the phosphorylated products dihydroxyacetone phosphate and glycerol phosphate lie on opposite sides of the NADH-consuming glycerophosphate dehydrogenase reaction. Based upon measurements of glycolytic metabolites, mitochondrial oxidation, mitochondrial matrix calcium, and mitochondrial membrane potential, we find that insulin secretion most tightly correlates with mitochondrial metabolism for each of the four fuels. In the case of glucose stimulation, the high control strength of glucose phosphorylation sets the pace of glucose metabolism and thus the rate of insulin secretion. However, bypassing this reaction with pyruvate, dihydroxyacetone, or glycerol uncovers constraints imposed by mitochondrial metabolism, each of which attains a similar maximal limit of insulin secretion. More specifically, we found that the hyperpolarization of the mitochondrial membrane, related to the proton export from the mitochondrial matrix, correlates well with insulin secretion. Based on these findings, we propose that fuel-stimulated secretion is in fact limited by the inherent thermodynamic constraints of proton gradient formation.

The FAMMM syndrome: epidemiology and surveillance strategies.

Research into the epidemiology of the melanoma-prone FAMMM syndrome, molecular genetics of the occurrences of melanoma, the photobiology of DNA damage/repair, diagnostic epiluminescence, microscopic/imaging techniques, and a new concept of photoprotection have altered melanoma strategies in surveillance and prevention. Molecular genetic research has implicated the importance of hereditary aspects of melanoma and associated malignancies. High-risk pedigrees can be identified through an informatic analysis of the occurrence patterns of melanoma and systemic cancers in kindreds. All ultraviolet radiation results in cutaneous DNA damage and in high-risk individuals may cause melanoma. We may reverse the epidemic trend in melanoma occurrences in these high-risk pedigrees if we are willing to change our cultural approach to sunlight exposure with restrictive sunlight behavior, wearing of ultraviolet protective clothes, the use of broad-spectrum ultraviolet protection from nightly topical dihydroxyacetone coupled with daytime UVB sunscreens, and periodic surveillance.

Reduced dihydroxyacetone sensitivity and normal sensitivity to glyceraldehyde and oxidizing agent of ATP-sensitive K+ channels of pancreatic beta cells in NIDDM rats.

The inhibition of ATP-sensitive K+(KATP) channels in pancreatic beta cells is a key step of insulin secretion induced by glucose. Glucose-induced insulin secretion from the beta cells is selectively impaired in patients with noninsulin-dependent diabetes mellitus (NIDDM) and in animal models of it. In order to clarify the site of this abnormal glucose response, we studied the effects of insulin secretagogues and sulfhydryl oxidizing agent, 2,2'-dithio-bis (5-nitropyridine) (DTBNP), on KATP channels in single beta cells of neonatally streptozotocin-induced NIDDM rats. We used the patch-clamp technique in cell-attached mode (Vpipette = 0 mV). The inhibitory response to glucose of KATP channels was lacking in NIDDM rats, indicating reduced sensitivity to glucose of the channels. Glyceraldehyde (2-5 mM) in the diabetic beta cells elicited the same KATP channel inhibition as that obtained in controls. In contrast, dihydroxyacetone (DHA, 2-10 mM) sensitivity of KATP channels was significantly reduced in the beta cells of NIDDM rats. KATP channels in the diabetic beta cells were rapidly inhibited by 50 microM DTBNP, just as in the normal beta cells, suggesting that KATP channel function was normal. This indicates that one of the sites responsible for impaired glucose-induced insulin secretion in the pancreatic beta cells of NIDDM rats is located in the glycerol phosphate shuttle.

Potentiation of hypoxic injury in cultured rabbit hepatocytes by the quinoxalinone anxiolytic, panadiplon.

The quinoxalinone anxiolytic, panadiplon, produces hepatic metabolic inhibition (mitochondrial impairment), microvesicular steatosis and centrilobular necrosis in rabbits. Metabolic inhibition occurs in cultured hepatocytes without cytotoxicity, suggesting that hepatic injury is influenced by additional factors. The present experiments were conducted to determine if metabolic inhibition by panadiplon predisposed hepatocytes to hypoxic injury. Injury (cell death) was evaluated by lactate dehydrogenase (LDH) release from cells; ATP and glycogen levels were also evaluated. Under hypoxic conditions, control cultures showed a 6.5-fold increase in LDH release compared to normoxic controls, with a coincident 80% decrease in ATP and 50% decrease in glycogen levels. Under normoxic conditions 10 microgram/ml panadiplon treatment for 48 h reduced ATP and glycogen levels by 40% but did not cause an increase in LDH leakage. Cells treated with panadiplon, then exposed to hypoxia conditions, showed a significant level of injury compared to normoxic control cultures, and a further reduction in ATP. No additional decrease in glycogen ws observed. In an attempt to prevent panadiplon-mediated injury, glycolytic substrates (dihydroxyacetone or pyruvate) were included during normoxic and hypoxic incubations. Both cotreatments reduced the level of LDH leakage produced by panadiplon during hypoxia. Cotreatment did not generally increase ATP or glycogen levels (compared to panadiplon treatment groups) during hypoxia, though individual experiments showed a slight increase in ATP levels. During normoxia both cotreatments with panadiplon resulted in significantly higher glycogen levels than in panadiplon cultures alone. These results suggest that cellular glycogen and subsequently ATP levels are reduced during panadiplon exposure, metabolically predisposing hepatocytes to hypoxic injury.

Oxygen dependence of hepatocyte susceptibility to mitochondrial respiratory inhibitors.

Most zone 3 specific hepatotoxins or their metabolites are mitochondrial toxins, and yet the susceptibility of hepatocytes to respiratory inhibitors at the low O2 concentrations found in zone 3 is not known. Potassium cyanide (CN) and antimycin A (AA) were found to be 5- and 2-fold more cytotoxic at 1% than at 95% O2, respectively. CN also inhibited the respiration of hepatocytes 36% more at 1% O2 than at 95% O2; however, AA inhibited the respiration to the same level at 1% and 95% O2. CN but not AA depleted ATP levels of hepatocytes more extensively at 1% than at 95% O2. The CN-trapping agents dihydroxyacetone, glyceraldehyde, alpha-ketoglutarate and pyruvate prevented CN-induced cytotoxicity more effectively at 95% O2 than at 1% O2. In contrast, thiosulfate was less effective in preventing CN toxicity at 95% than at 1% O2. Hepatocyte thiocyanate formation from CN and thiosulfate was much faster at 1% than at 95% O2, suggesting that rhodanese, the mitochondrial enzyme that forms thiocyanate from CN and thiosulfate, is more effective at 1% O2 than at 95% O2.

Reduced sensitivity of dihydroxyacetone on ATP-sensitive K+ channels of pancreatic beta cells in GK rats.

In the GK (Goto-Kakizaki) rat, a genetic model of non-insulin-dependent diabetes mellitus, glucose-induced insulin secretion is selectively impaired. In addition, it has been suggested by previous studies that impaired glucose metabolism in beta cells of the GK rat results in insufficient closure of ATP-sensitive K+ channels (KATP channels) and a consequent decrease in depolarization, leading to a decreased insulin release. We have recently reported that the site of disturbed glucose metabolism is probably located in the early stages of glycolysis or in the glycerol phosphate shuttle. In the present study, in order to identify the impaired metabolic step in diabetic beta cells, we have investigated insulin secretory capacity by stimulation with dihydroxyacetone (DHA), which is known to be directly converted to DHA-phosphate and to preferentially enter the glycerol phosphate shuttle. In addition, using the patch-clamp technique, we also have studied the sensitivity of DHA on the KATP channels of beta cells in GK rats. The insulin secretion in response to 5 mmol/l DHA with 2.8 mmol/l glucose was impaired, and DHA sensitivity of the KATP channels was reduced in beta cells of GK rats. From these results, we suggest that the intracellular site responsible for impaired glucose metabolism in pancreatic beta cells of GK rats is located in the glycerol phosphate shuttle.

Prevention of cyanide-induced cytotoxicity by nutrients in isolated rat hepatocytes.

The effects of various glycolytic substrates and keto acid metabolites on the cytotoxic effects of cyanide have been studied with isolated rat hepatocytes. The sequence of cytotoxic events with 2 mM cyanide was an immediate inhibition of respiration followed by ATP depletion. Disruption of the plasma membrane occurred when 85-90% of ATP levels had been depleted. Fructose, dihydroxyacetone, glyceraldehyde, pyruvate, and alpha-ketoglutarate prevented cyanide-induced cytotoxicity and ATP depletion. Hepatocyte respiration was also restored by all except fructose. Fructose, unlike the others, also did not prevent cytotoxicity if added 30-60 min after cyanide. Fluoride, an inhibitor of the glycolytic enzyme enolase, prevented protection by fructose but not dihydroxyacetone or glyceraldehyde, suggesting that dihydroxyacetone and glyceraldehyde are cytoprotective by trapping cyanide, thereby restoring cytochrome oxidase activity and cellular ATP levels. Fructose, on the other hand, may be cytoprotective by supplying ATP through glycolysis. Hepatocytes isolated from fasted rats were five- to sevenfold more susceptible to cyanide-induced cytotoxicity. Furthermore, all glycogenic and gluconeogenic amino acids and carbohydrates were cytoprotective against cyanide toxicity toward fasted hepatocytes, suggesting that cellular energy stores determine their resistance to cyanide.

Inhibition of glucose-6-phosphate phosphohydrolase by 3-mercaptopicolinate and two analogs is metabolically directive.

3-Mercaptopicolinae (3-MP) blocks gluconeogenesis from lactate, pyruvate, alanine, and other substrates through its inhibition of phosphoenolpyruvate carboxykinase. Nevertheless, we observed increased glycogenesis, net glucose uptake, and glucose-6-P levels in livers perfused with glucose in the presence of 3-MP. In perfusions with 20 mM dihydroxyacetone, increased glycogenesis and decreased glucose production were observed with 3-MP. These metabolic effects suggested additional site(s) of action of 3-MP. Further studies showed that 3-MP inhibits glucose-6-P phosphohydrolase activity of intact liver microsomes. Several compounds with structural similarities to 3-MP (2-mercaptonicotinic acid, picolinic acid, cysteine, reduced glutathione, nicotinic acid, quinolinic acid, tryptophan, and pyridine) were tested for their effect on glucose-6-P phosphohydrolase activity. Two of these compounds, 2-mercaptonicotinic acid and picolinic acid, were found to inhibit. In perfusions including 7.5 mM fructose, the addition of 3-MP, 2-mercaptonicotinic acid, or picolinic acid increased glycogenesis, decreased glucose production, and increased hepatic glucose-6-P concentrations. These observations indicate that the inhibition of glucose-6-P phosphohydrolase may play a role in enhanced glycogenesis from glucose, dihydroxyacetone, and fructose in isolated livers from 48-h fasted rats perfused with 3-MP or certain sulfhydryl-containing and sulfhydryl-devoid analogs.

Intracellular pH and the stimulus-secretion coupling in insulin-producing RINm5F cells.

The regulation of intracellular pH (pHi) and its role in the insulin-secretory process were evaluated, by using the clonal insulin-secreting cell line RINm5F. Glyceraldehyde, lactate and dihydroxyacetone decreased pHi, but only the first two released insulin. In the presence of extracellular Na+ the cells counteracted the acidification. Blocking the Na+/H+ exchange in acidic cells resulted in a drastic further lowering of pHi, an effect not obtained under basal conditions. Whereas glyceraldehyde depolarized the cells, lactate was without effect. Dihydroxyacetone hyperpolarized the cells in the presence of extracellular Na+, but this effect disappeared when Na+ was excluded from the medium. Stimulation with glyceraldehyde resulted in increased free cytoplasmic Ca2+ concentration ([Ca2+]i). Dihydroxyacetone and lactate had no effect on [Ca2+]i in the presence of Na+, but lactate induced a decrease in [Ca2+]i in Na(+)-deficient medium. In RINm5F cells the activity of the Na+/H+ antiport could not be augmented by activation of protein kinase C (PKC). Hence, in insulin-secreting cells a PKC-insensitive Na+/H+ antiport is the major mechanism restoring a decrease in pHi. Acidification itself does not affect membrane potential, but may directly interact with the mechanisms regulating exocytosis.