An Experimental model of phenformin-induced lactic acidosis in rats

Summary An experimental model of phenformininduced lactic acidosis was established in rats. Following a subtotal nephrectomy, renal failure developed (serum creatinine 4.5±0.1mg/100ml and 2.8±0.1 mg/100 ml on the 1st and 8th postoperative days respectively). Immediately after nephrectomy intra-peritoneal phenformin treatment, 16 mg/day, was commenced. Lactic acidosis developed progressively within 8 days, or earlier in the rats with the most severe renal insufficiency. The metabolic pattern was very similar to that observed in diabetic patients with a biguanide-induced lactic acidosis: on the 8th day, 2 h after the last phenformin injection, blood lactate was 10.8±1.0 mmol/1 (controls: 1.50±0.03); pyruvate was 0.56±0.06 mmol/1 (controls: 0.10±0.01) and blood pH: 7.00 ± 0.02 (vs 7.34±0.02); 3-hydroxybutyrate was 1.41±0.37 mmol/1 (vs 0.32 ±0.03); acetoacetate: 0.51±0.15 mmol/1 (vs 0.17 ±0.01), and free glycerol: 0.63 ±0.07 mmol/1 (vs 0.14 ±0.02). Increased concentrations of alanine (1.66±0.26 mmol/1, vs 0.48 ± 0.04 in controls) and low blood glucose levels (23± 8 mg/ 100 ml vs 70 ± 2, after a 12 hours fast) accompanied the lactic acidosis in spite of high glucagon levels (2030±170 pg/ml vs 108±10 in controls) and low insulin/glucagon molar ratio (0.19 vs 6.9 in controls). Normal rats, treated with phenformin at same doses, and nephrectomized rats injected with saline served as controls and remained free of lactic acidosis. Hydroxyphenformin (16 mg/day) injected in nephrectomized rats, was biologically inactive. Glucose production from¹⁴C-lactate was 425 ±85 mol/100 g body wt/h, vs 1050 ± 90 in control animals. Blood lactate specific activity declined more slowly in the lactic acidotic rats than in controls, suggesting that a decrease in lactate utilization contributed to hyperlactataemia more than an increased lactate production.     

Metformin,phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis

Metformin exerts its plasma glucose-lowering therapeutic effect primarily through inhibition of hepatic gluconeogenesis. However, the precise molecular mechanism by which metformin inhibits hepatic gluconeogenesis is still unclear. Although inhibition of mitochondrial complex I is frequently invoked as metformin’s primary mechanism of action, the metabolic effects of complex I inhibition have not been thoroughly evaluated in vivo. Here, we show that acute portal infusion of piericidin A, a potent and specific complex I inhibitor, does not reduce hepatic gluconeogenesis in vivo. In contrast, we show that metformin, phenformin, and galegine selectively inhibit hepatic gluconeogenesis from glycerol. Specifically, we show that guanides/biguanides interact with complex IV to reduce its enzymatic activity, leading to indirect inhibition of glycerol-3-phosphate (G3P) dehydrogenase (GPD2), increased cytosolic redox, and reduced glycerol-derived gluconeogenesis. We report that inhibition of complex IV with potassium cyanide replicates the effects of the guanides/biguanides in vitro by selectively reducing glycerol-derived gluconeogenesis via increased cytosolic redox. Finally, we show that complex IV inhibition is sufficient to inhibit G3P-mediated respiration and gluconeogenesis from glycerol. Taken together, we propose a mechanism of metformin action in which complex IV–mediated inhibition of GPD2 reduces glycerol-derived hepatic gluconeogenesis.

Metformin (1,1-dimethylbiguanide) is the standard first-line pharmaceutical intervention for type 2 diabetes mellitus (T2D) and is one of the most widely prescribed drugs worldwide (1, 2). Metformin and other more potent synthetic guanide/biguanide derivatives, such as phenformin (N-phenethylbiguanide), have glucose-lowering effects in patients with T2D. Following oral administration, metformin accumulates to a high degree within the liver due to first-pass uptake in the portal vein following absorption from the gut, and the presence of the organic cation transporter 1 (OCT1) in the sinusoidal endothelial cells of the liver (3–7). This is in contrast to skeletal and cardiac muscle, where OCT1 is not highly expressed. The observed glucose-lowering effects in individuals with poorly controlled T2D can mostly be attributed to inhibition of hepatic gluconeogenesis, as opposed to altering insulin sensitivity or secretion (8–13); however, despite the extensive literature spanning several decades examining metformin’s effects in vivo and in vitro, a consensus on metformin’s precise mechanism of action still does not exist.The most well-studied mechanism is complex I inhibition, which is central to several frequently invoked mechanisms of metformin action, including adenosine monophosphate (AMP)-activated protein kinase activation, decreased energy charge ([adenosine triphosphate {ATP}]:[adenosine diphosphate {ADP}] and [ATP]:[AMP] ratios), and AMP inhibition of fructose 1,6-bisphosphatase, among others (14–18). Yet, complex I inhibition is only observed at suprapharmacological concentrations (>1 mM) of metformin, which is severalfold higher than concentrations achieved in vivo (3, 7, 19, 20). Furthermore, no study to date has convincingly demonstrated that complex I inhibition can, in fact, replicate metformin’s glucose-lowering effects in vivo. To address this question, we sought to specifically inhibit complex I activity in vivo to determine whether the metabolic effects of impaired complex I activity resemble those observed with metformin.We and others have previously proposed an alternative mechanism of metformin action, in which alterations in hepatic redox state and inhibition of glycerol-3-phosphate dehydrogenase (GPD2) potentiate metformin’s glucose-lowering effects (20–22). GPD2 is central to the α-glycerophosphate shuttle, one of two redox shuttles, which transfers reducing equivalents from the cytosol to the mitochondrial matrix. Specifically, GPD2 transfers electrons to mitochondrial ubiquinone, generating ubiquinol that is reoxidized by complex III (ubiquinol cytochrome c reductase) of the electron transport chain (ETC) (23). Thus, excess mitochondrial ubiquinol decreases GPD2 activity and increases reducing equivalents in the cytosol, which can be experimentally represented by an increased [lactate]:[pyruvate] ratio (referred to here as increased cytosolic redox state). Increased cytosolic redox is predicted to selectively reduce gluconeogenesis from reduced substrates (e.g., lactate and glycerol), while gluconeogenesis from nonreduced substrates (e.g., alanine, dihydroxyacetone phosphate [DHAP], and pyruvate) is unaffected (24), which is in contrast to a complex I-dependent mechanism of metformin action. This is consistent with metformin’s effects in both humans and rodents (12, 20, 25).Clinical studies have shown that the glucose-lowering effects of metformin in patients with fasting hyperglycemia, due to poorly controlled T2D, can mostly be attributed to reductions in hepatic glucose production (HGP); however, these effects are not consistently observed in normoglycemic individuals (8, 26, 27). These paradoxical effects provide insights into potential mechanisms of metformin action in humans: Individuals with poorly controlled T2D have dysregulated white adipose tissue (WAT) lipolysis, leading to increased flux of fatty acids and glycerol delivery to the liver, the latter of which increases hepatic gluconeogenesis through a substrate push mechanism (28–33). Accordingly, selective inhibition of glycerol-derived gluconeogenesis, due to increased cytosolic redox, may explain metformin’s paradoxical effects, but this has not yet been demonstrated in vivo.Here, we examined whether targeted inhibition of complex I using piericidin A, a specific and irreversible inhibitor of mitochondrial nicotinamide adenine dinucleotide reduced (NADH)–ubiquinone oxidoreductase (complex I), is sufficient to mediate metformin’s glucose-lowering effects in vitro and in vivo. We also examine the effects of metformin, two more potent guanides/biguanides (phenformin and galegine [isoamylene guanidine]), and piericidin A on glycerol-derived gluconeogenesis and cytosolic redox state in liver slices, as well as their effects on [¹³C₃]glycerol incorporation into [¹³C₃]glucose in awake rats with indwelling intraportal catheters. Finally, we investigated a mechanism of metformin/phenformin/galegine action through cytochrome c oxidase (complex IV)-mediated inhibition of GPD2 activity, which, in turn, can explain guanide/biguanide effects to increase the hepatic cytosolic redox state and reduce glycerol-derived hepatic gluconeogenesis and glycerol-3-phosphate (G3P)-dependent mitochondrial oxidation.     

Cholesterol Lowering Effect of Metformin in Combined Hyperlipidemia: Placebo Controlled Double Blind Trial

Metformin, an antidiabetic biguanide derivative, prevents experimental atherosclerosis and induces structural changes in lipoproteins in experimental animals. In the present study we investigated the effect of metformin on serum lipoproteins and platelet function in 24 non-diabetic patients with type II B hyperlipidemia. The patients were randomly given metformin in two dosage levels (1.0 g/day and 2.0 g/day) and placebo for periods of nine weeks in a crossover trial. Metformin caused a dose dependent fall in the concentrations of total serum cholesterol and of LDL-cholesterol. The average concentration of total cholesterol was 8.54 ± 0.22 (SE) mmol/l, 8.12 ± 0.19 mmol/l and 7.79 ± 0.15 mmol/l during placebo, metformin 1.0 g/day and 2.0 g/day treatments, respectively. Both metformin values differed significantly (P < 0.05) from the placebo value. Thus there was an average fall of 8.1% in total cholesterol after the higher metformin dose. LDL-cholesterol was 5.25 ± 0.23 mmol/l after placebo, falling by 3.1% and 9.6% after metformin doses of 1.0 g/day and 2.0 g/day, respectively. The concentrations of HDL-cholesterol and total serum triglycerides showed no significant changes. Body weight, blood glucose, plasma insulin, blood lactate, platelet function and urinary excretion of prostanoids remained unchanged during the study. The reduction of total-and LDL-cholesterol levels may be a welcome additional consequence of metformin during treatment of diabetic patients with hypercholesterolemia.     

Orthokeratology lens-related Acanthamoeba keratitis: case report and analytical review

Acanthamoeba keratitis (AK) is a rare but severe ocular infection with a significant risk of vision loss. Contact lens use is the main risk factor for AK. The orthokeratology (OK) lens, a specially designed contact lens, has been used worldwide as an effective method of myopia control. However, the OK lens is associated with an increased risk of Acanthamoeba infection. Many primary practitioners are concerned about this infection because of its relative rarity, the lack of promising therapeutic medications, and the need for referral. We herein report two cases of AK associated with OK lenses, present a systematic review of such cases, and discuss the possible reasons for the higher incidence rate of this infection in patients who wear OK lenses. We combined the clinical knowledge and skills of corneal specialists and lens experts with the sole objective of addressing these OK lens-related AK cases. We found that the most common risk factors were rinsing the lenses or lens cases with tap water. Prompt and accurate diagnosis along with adequate amoebicidal treatment are essential to ensure desirable outcomes for OK lens wearers who develop AK. Appropriate OK lens parameters and regular checkups are also important.     

Diabetes pharmacotherapy and effects on the musculoskeletal system

Persons with type 1 or type 2 diabetes have a significantly higher fracture risk than age‐matched persons without diabetes, attributed to disease‐specific deficits in the microarchitecture and material properties of bone tissue. Therefore, independent effects of diabetes drugs on skeletal integrity are vitally important. Studies of incretin‐based therapies have shown divergent effects of different agents on fracture risk, including detrimental, beneficial, and neutral effects. The sulfonylurea class of drugs, owing to its hypoglycemic potential, is thought to amplify the risk of fall‐related fractures, particularly in the elderly. Other agents such as the biguanides may, in fact, be osteo‐anabolic. In contrast, despite similarly expected anabolic properties of insulin, data suggests that insulin pharmacotherapy itself, particularly in type 2 diabetes, may be a risk factor for fracture, negatively associated with determinants of bone quality and bone strength. Finally, sodium‐dependent glucose co‐transporter 2 inhibitors have been associated with an increased risk of atypical fractures in select populations, and possibly with an increase in lower extremity amputation with specific SGLT2I drugs. The role of skeletal muscle, as a potential mediator and determinant of bone quality, is also a relevant area of exploration. Currently, data regarding the impact of glucose lowering medications on diabetes‐related muscle atrophy is more limited, although preclinical studies suggest that various hypoglycemic agents may have either aggravating (sulfonylureas, glinides) or repairing (thiazolidinediones, biguanides, incretins) effects on skeletal muscle atrophy, thereby influencing bone quality. Hence, the therapeutic efficacy of each hypoglycemic agent must also be evaluated in light of its impact, alone or in combination, on musculoskeletal health, when determining an individualized treatment approach. Moreover, the effect of newer medications (potentially seeking expanded clinical indication into the pediatric age range) on the growing skeleton is largely unknown. Herein, we review the available literature regarding effects of diabetes pharmacotherapy, by drug class and/or by clinical indication, on the musculoskeletal health of persons with diabetes.     

双胍类隐形眼镜护理液对白色念珠菌的杀灭效果影响因素研究

目的研究双胍类隐形眼镜护理液对白色念珠菌的杀灭效果及其影响因素。方法采用悬液定量试验进行了实验室观察。结果含聚六亚甲基胍1.0mg/L的该护理液原液于常温下作用6h,对白色念珠菌国内株和美国株的杀灭率分别为78.21%和95.27%。将护理液分别置于玻璃管、聚丙烯塑料管、聚苯乙烯塑料管内浸泡作用均为6h,对白色念珠菌平均杀灭率分别为64.55%、76.41%、95.73%。将护理液分别置于不同形状容器即尖底聚丙烯塑料管、圆底聚丙烯塑料管内浸泡作用6h,对白色念珠菌平均杀灭率分别为77.41%和78.89%。混合菌悬液用或不用玻璃珠进行振荡混合,用于杀菌试验,计算出平均杀灭率分别为78.21%和78.94%;菌悬液振荡混合90s和20s,用于杀菌试验,计算出平均杀灭率分别为80.18%和79.27%。结论该护理液对相同菌株不同来源的白色念珠菌杀灭效果存在差异,不同材质的盛放容器对该护理液杀菌效果有显著影响。     

Inhibition of bile salt absorption by blood-sugar lowering biguanides

Summary The effect of blood sugar lowering biguanides (phenethyl-, butyl- and dimethylbiguanide) upon jejunal and ileal transport of bile salts (tauro- and glycocholate) was tested in rat small intestine by an in vitro technique. Biguanides inhibited active transport of bile salts in the ileum, but did not affect diffusional absorption of bile salts in the jejunum. The inhibitory effect was time-dependent and not reversible under in vitro incubation conditions, suggesting that biguanides must enter intestinal mucosal cells in order to exert their inhibitory action on active transport of glucose analogues, amino acids, calcium and bile salts. Since biguanides achieve high tissue concentrations in the small intestine even after parenteral administration, inhibition of ileal bile salt reabsorption by biguanides could possibly explain the lipid- and cholesterol-lowering effect of these oral antidiabetic drugs.     

儿童糖尿病酮症酸中毒及双胍类药物治疗中乳酸酸中毒问题的研究

目的：探讨１型糖尿病儿童酮症酸中毒时乳酸酸中毒发生情况,乳酸酸中毒与双胍类药物副作用的关系,双胍类药物在治疗１型糖尿病病人时的乳酸水平。方法：测定１９９６年２月－１９９７年１０月期间我院住院的１型糖尿病病人入院时乳酸水平,及纠正酸中毒后,双胍药治疗前后乳酸水平。结果：３３例病人入院时发生酮症酸中毒的有２３例,乳酸酸中毒１例。