Antihyperglycemic effect of aqueous and ethanolic extracts of Gongronema latifolium leaves on glucose and glycogen metabolism in livers of normal and streptozotocin-induced diabetic rats

The present study was designed to investigate the antihyperglycemic effects of aqueous and ethanolic extracts from Gongronema latifolium leaves on glucose and glycogen metabolism in livers of non-diabetic and streptozotocin-induced diabetic rats. To investigate the effects of aqueous or ethanolic leaf extracts of G. latifolium, non-diabetic and STZ diabetic rats were treated twice daily (100 mg/Kg) for two weeks. Diabetic rats showed a significant decrease in the activities of hepatic hexokinase (HK), phosphofructokinase (PFK) and glucose-6-phosphate dehydrogenase (G6PDH) and an increase in glucokinase (GK) activity. The levels of hepatic glycogen and glucose were also increased in diabetic rats. However, there were no significant differences in the activities of glucose-6-phosphatase (G6Pase) in treated and untreated diabetic rats. The ethanolic extract significantly increased the activities of HK (p<0.01), PFK (p<0.001) and G6PDH (p<0.01) in diabetic rats, decreased the activity of GK (p<0.05) and the levels of hepatic glycogen (p<0.01) and both hepatic (p<0.001) and blood glucose (40%). The aqueous extract of G. latifolium was only able to significantly increase the activities of HK and decrease the activities of GK but did not produce any significant change in the hepatic glycogen and both hepatic and blood glucose content of diabetic rats. Our data show that the ethanolic extract from G. latifolium leaves has antihyperglycemic potency, which is thought to be mediated through the activation of HK, PFK, G6PDH and inhibition of GK in the liver. The ethanolic extract is under further investigation to determine the chemical structure of the active compound(s) and its/their mechanism of action.     

Dosage effect of streptozotocin on rat tissue enzyme activities and glycogen concentration

The effect of different dosages of streptozotocin (STZ) on selected rat tissue enzyme activities and glycogen concentration were investigated. The rats were administered STZ intravenously at 60 (STZ-60), 80 (STZ-80), 100 (STZ-100), and 150 (STZ-150) mg/kg body weight. They were used 3 weeks postinjection. Mortality prior to kill occurred only in the STZ-100 and STZ-150 rats. All diabetic rats showed reduced growth rate, hyperglycemia, hypoinsulinemia, and hyperlipemia. Phosphofructokinase (PFK) and succinate dehydrogenase (SDH) activities were significantly reduced in the red gastrocnemius muscle of all diabetic rats, and in the white gastrocnemius and soleus of STZ-100 and STZ-150 groups. PFK activity in the heart remained unaltered, but SDH activity was below normal. Liver SDH activity was not affected by insulin deficiency. Glycogen content was markedly increased in the heart and decreased in the liver of all diabetic rats. Glycogen content in the skeletal muscle was similar to the controls, except for the lower values in the soleus of STZ-100 and STZ-150 rats. When STZ-80 and STZ-150 rats were given insulin therapy, the STZ-80 rats showed a greater response to the treatment. Despite similar levels of plasma immunoreactive insulin among all groups of diabetic rats, the STZ-100 and STZ-150 rats had higher mortality, greater loss in body weight, and alterations in enzyme activities and glycogen content in the tissues studied.     

Control of glycogen deposition

Traditionally, glycogen synthase (GS) has been considered to catalyze the key step of glycogen synthesis and to exercise most of the control over this metabolic pathway. However, recent advances have shown that other factors must be considered. Moreover, the control of glycogen deposition does not follow identical mechanisms in muscle and liver. Glucose must be phosphorylated to promote activation of GS. Glucose-6-phosphate (Glc-6-P) binds to GS, causing the allosteric activation of the enzyme probably through a conformational rearrangement that simultaneously converts it into a better substrate for protein phosphatases, which can then lead to the covalent activation of GS. The potency of Glc-6-P for activation of liver GS is determined by its source, since Glc-6-P arising from the catalytic action of glucokinase (GK) is much more effective in mediating the activation of the enzyme than the same metabolite produced by hexokinase I (HK I). As a result, hepatic glycogen deposition from glucose is subject to a system of control in which the 'controller', GS, is in turn controlled by GK. In contrast, in skeletal muscle, the control of glycogen synthesis is shared between glucose transport and GS. The characteristics of the two pairs of isoenzymes, liver GS/GK and muscle GS/HK I, and the relationships that they establish are tailored to suit specific metabolic roles of the tissues in which they are expressed. The key enzymes in glycogen metabolism change their intracellular localization in response to glucose. The changes in the intracellular distribution of liver GS and GK triggered by glucose correlate with stimulation of glycogen synthesis. The translocation of GS, which constitutes an additional mechanism of control, causes the orderly deposition of hepatic glycogen and probably represents a functional advantage in the metabolism of the polysaccharide.     

Liver glycogen synthase but not the muscle isoform differentiates between glucose 6-phosphate produced by glucokinase or hexokinase

Using adenovirus-mediated gene transfer into FTO-2B cells, a rat hepatoma cell line, we have overexpressed hexokinase I (HK I), glucokinase (GK), liver glycogen synthase (LGS), muscle glycogen synthase (MGS), and combinations of each of the two glucose-phosphorylating enzymes with each one of the GS isoforms. FTO-2B cells do not synthesize glycogen even when incubated with high doses of glucose. Adenovirus-induced overexpression of HK I and/or LGS, two enzymes endogenously expressed by these cells, did not produce a significant increase in the levels of active GS and the total glycogen content. In contrast, GK overexpression led to the glucose-dependent activation of endogenous or overexpressed LGS and to the accumulation of glycogen. Similarly overexpressed MGS was efficiently activated by the glucose-6-phosphate (Glc-6-P) produced by either endogenous or overexpressed HK I and by overexpressed GK. These results indicate the existence of at least two pools of Glc-6-P in the cell, one of them is accessible to both isoforms of GS and is replenished by the action of GK, whereas LGS is excluded from the cellular compartment where the Glc-6-P produced by HK I is directed. These findings are interpreted in terms of the metabolic role that the two pairs of enzymes, HK I-MGS in the muscle and GK-LGS in the hepatocyte, perform in their respective tissues.     

Metformin reverses hexokinase and 6-phosphofructo-1-kinase inhibition in skeletal muscle, liver and adipose tissues from streptozotocin-induced diabetic mouse

The present work describes the effects of metformin on hexokinase (HK) and phosphofructokinase (PFK) activities and localization in different tissues from streptozotocin-induced diabetic mice. Diabetic mice present lower HK and PFK activities (50%) in skeletal muscle, liver and adipose tissue, as compared with control (P < 0.05). Treatment with 250 mg/kg metformin reverses this pattern of enzyme inhibition with concomitant reversal of hyperglycemia and hypolactacidemia. Furthermore, the treatment increases the cytoskeleton-associated PFK activity in skeletal muscle; this activity has been described as an important mechanism for the enzyme activation. This effect might be due to the increased phosphorylation of serine residues in the enzyme, a modification which has been described to increase the interaction of PFK with f-actin. The current work supports the hypothesis that metformin hypoglycemic effects involve the activation of glycolysis through its regulatory enzymes, which may be potential targets for the development of new hypoglycemic drugs.     

Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis

Summary The mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P₂) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M₁-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M₂ isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P₂ may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.Abbreviations ADP adenosine diphosphate - AMP adenosine monophosphate - ATP adenonine triphoshate - EDTA ethylenediaminetetra-acetic acid - EGTA ethylene glycol tetra-acetic acid - F-6-P fructose 6-phosphate - F-1,6-P₂ fructose 1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate - K _a activation coefficient - I₅₀ concentration of inhibitor which reduces control activity by 50% - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PFK 6-phosphofructo-1-kinase - PK pyruvate kinase     

Elevated expression and activity of protein-tyrosine phosphatase 1B in skeletal muscle of insulin-resistant type II diabetic Goto-Kakizaki rats

We investigated the cellular mechanism(s) of insulin resistance associated with non-insulin dependent diabetes mellitus (NIDDM) using skeletal muscles isolated from non-obese, insulin resistant type II diabetic Goto-Kakizaki (GK) rats, a well known genetic rat model for type II diabetic humans. Relative to non-diabetic control rats (WKY), insulin-stimulated insulin receptor (IR) autophosphorylation and insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation were significantly inhibited in GK skeletal muscles. This may be due to increased dephosphorylation by a protein tyrosine phosphatase (PTPase). Therefore, we measured skeletal muscle total PTPase and PTPase 1B activities in the skeletal muscles isolated from control rats (WKY) and diabetic Goto-Kakizaki (GK) rats. PTPase activity was measured using a synthetic phosphopeptide, TRDIY(P)ETDY(P)Y(P)RK, as the substrate. Basal PTPase activity was 2-fold higher (P < 0.001) in skeletal muscle of GK rats when compared to WKY. Insulin infusion inhibited skeletal muscle PTPase activity in both control (26.20% of basal, P < 0.001) and GK (25.35% of basal, P < 0.001) rats. However, PTPase activity in skeletal muscle of insulin-stimulated GK rats was 200% higher than hormone-treated WKY controls (P < 0.001). Immunoprecipitation of PTPase 1B from skeletal muscle lysates and analysis of the enzyme activity in immunoprecipitates indicated that both basal and insulin-stimulated PTPase 1B activities were significantly higher (twofold, P < 0.001) in skeletal muscle of diabetic GK rats when compared to WKY controls. The increase in PTPase 1B activity in diabetic GK rats was associated with an increased expression of the PTPase 1B protein. We concluded that insulin resistance of GK rats is accompanied atleast by an abnormal regulation of PTPase 1B. Elevated PTPase 1B activity through enhanced tyrosine dephosphorylation of the insulin receptor and its substrates, may lead to impaired glucose tolerance and insulin resistance in GK rats.     

Lactate downregulates the glycolytic enzymes hexokinase and phosphofructokinase in diverse tissues from mice

We examined the effects of lactate on the enzymatic activity of hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) in various mouse tissues. Our results showed that lactate inhibited PFK activity in all the analyzed tissues. This inhibitory effect was observed in skeletal muscle even in the presence of insulin. Lactate directly inhibited the phosphorylation of PFK tyrosine residues in skeletal muscle, an important mechanism of the enzyme activation. Moreover, lactate indirectly inhibited HK activity, which resulted from its cellular redistribution, here attributed to alterations of HK structure. PK activity was not affected by lactate. The activity of HK and PFK is directly related to glucose metabolism. Thus, it is conceivable that lactate exposure can induce inhibition of glucose consumption in tissues.     

Seasonal changes in the activities of glycolytic enzymes from liver and skeletal muscle of Rana perezi

Glycogen phosphorylase (GP), Hexokinase (HK), Phosphofructokinase (PFK), Pyruvate kinase (PK) and Lactate dehydrogenase (LDH) activities from skeletal muscle and liver were measured in Rana perezi for the four seasons of the year. Skeletal muscle showed a decrease in PFK, PK and LDH activity during winter and summer. Liver displayed an increase in GP activity in spring and in PK and LDH in autumn.     

红景天提取物提高高强度跑台运动小鼠的抗氧化能力

为了考察红景天提取物对高强度跑台运动小鼠的抗氧化能力的影响,本研究将30只昆明小鼠随机分成对照组、模型组和红景天提取物组,每组10只。红景天提取物组小鼠按照500 mg/kg bw的剂量灌胃红景天提取液(2 m L)。对照组和模型组小鼠灌胃等体积的蒸馏水,共灌胃4周。采用硫酸蒽酮比色法检测小鼠肝脏和肌肉组织糖原的含量;采用硫代巴比妥酸比色法检测小鼠骨骼肌组织中的丙二醛(MDA)水平;采用RT-PCR和Western blotting检测骨骼肌组织中的超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)的表达水平;采用苏木精和伊红(HE)染色评价骨骼肌病理改变。研究显示,模型组小鼠的跑台运动时间显著低于红景天提取物组(61.32 min vs 83.22 min,p<0.05);与模型组相比,红景天提取物组小鼠骨骼肌的炎性细胞浸润明显减轻,肌纤维排列明显改善;红景天提取物组的肝糖原和肌糖原含量均显著高于模型组;红景天提取物组小鼠骨骼肌组织中的MDA水平显著低于模型组;红景天提取物组的SOD、GSH-Px和CAT m RNA和蛋白表达水平均显著高于模型组;红景天提取物可通过上调抗氧化酶表达来增加抗氧化能力,减弱骨骼肌损伤,并增加机体的抗疲劳能力。     

Effects of maternal exercise on liver and skeletal muscle glycogen storage in pregnant rats.

To examine the effects of maternal exercise on liver and skeletal muscle glycogen storage, female Sprague-Dawley rats were randomly divided into control, nonpregnant runner, pregnant nonrunning control, pregnant runner, and prepregnant exercised control groups. The exercise consisted of treadmill running at 30 m/min on a 10 degree incline for 60 min, 5 days/wk. Pregnancy alone, on day 20 of gestation, decreased maternal liver glycogen content and increased red and white gastrocnemius muscle glycogen storage above control values (P less than 0.05). In contrast, exercise in nonpregnant animals augmented liver glycogen storage and also increased red and white gastrocnemius glycogen content (P less than 0.05). By combining exercise and pregnancy, the decrease in liver glycogen storage in the pregnant nonexercised condition was prevented in the pregnant runner group and more glycogen was stored in both the red and white portions of the gastrocnemius than all other groups (P less than 0.05). Fetal body weight was greatest (P less than 0.05) in the pregnant runner group and lowest (P less than 0.05) in the prepregnant exercise control group. These results demonstrate that chronic maternal exercise may change maternal glycogen storage patterns in the liver and skeletal muscle with some alteration in fetal outcome.     

The effects of swimming and running regimens on skeletal muscle glycogen in the rat.

Endurance capacity and the effects of different post-exercise states on skeletal muscle glycogen have been studied in rats trained by swimming or running and in sedentary controls. Regular endurance exercise resulted in increased skeletal muscle glycogen stores. A greater depletion was observed in trained animals than in non-trained animals after a training bout or exhaustive exercise. While muscle glycogen levels did not reflect a differential training stimulus (running vs swimming), swimming as a measure of exhaustive exercise was deemed invalid because of the ability of trained swimmers to avoid stenuous exercise by an alteration of swimming pattern.     

Heterogeneity of glycogen synthesis upon refeeding following starvation.

1. Starvation of rats for 40 hr decreased the body weight, liver weight and blood glucose concentration. The hepatic and skeletal muscle glycogen concentrations were decreased by 95% (from 410 mumol/g tissue to 16 mumol/g tissue) and 55% (from 40 mumol/g tissue to 18.5 mumol/g tissue), respectively. 2. Fine structural analysis of glycogen purified from the liver and skeletal muscle of starved rats suggested that the glycogenolysis included a lysosomal component, in addition to the conventional phosphorolytic pathway. In support of this the hepatic acid alpha-glucosidase activity increased 1.8-fold following starvation. 3. Refeeding resulted in liver glycogen synthesis at a linear rate of 40 mumol/g tissue per hr over the first 13 hr of refeeding. The hepatic glycogen store were replenished by 8 hr of refeeding, but synthesis continued and the hepatic glycogen content peaked at 24 hr (approximately 670 mumol/g tissue). 4. Refeeding resulted in skeletal muscle glycogen synthesis at an initial rate of 40 mumol/g tissue per hr. The muscle glycogen store was replenished by 30 min of refeeding, but synthesis continued and the glycogen content peaked at 13 hr (approximately 50 mumol/g tissue). 5. Both liver and skeletal muscle glycogen synthesis were inhomogeneous with respect to molecular size; high molecular weight glycogen was initially synthesised at a faster rate than low molecular weight glycogen. These observations support suggestions that there is more than a single site of glycogen synthesis.     

不同糖源及糖水平对大菱鲆糖代谢酶活性的影响

采用34双因素实验设计, 以初始质量为(8.060.08) g的大菱鲆幼鱼(Scophthalmus maximus L.)为对象, 研究在饲料中添加3种糖源(葡萄糖、蔗糖和糊精)及4个水平(0、5%、15%、28%)对大菱鲆肝脏糖酵解关键酶己糖激酶(HK)、葡萄糖激酶(GK)、磷酸果糖激酶(PFK)、丙酮酸激酶(PK)和糖异生关键酶磷酸烯醇式丙酮酸羧激酶(PEPCK)、1, 6-二磷酸果糖酶(FBPase)活性的影响。结果表明: 饲料糖添加量从0升高到15%时, 大菱鲆的糖酵解酶GK和PK活性随饲料葡萄糖或糊精含量的增加而增加; 当饲料中葡萄糖或糊精含量为28%时, GK和PK活性有下降的趋势。3种糖源的4个添加水平对HK和PFK活性均无显著影响(P 0.05)。添加不同水平的葡萄糖对大菱鲆糖异生途径的PEPCK活性无显著影响(P 0.05), 但在饲料中葡萄糖添加量为5%时显著促进了FBPase活性(P 0.05), 当葡萄糖添加量升高为15%或28%时, FBPase活性与对照组无显著差异(P 0.05)。糊精作为饲料糖源时抑制了大菱鲆肝脏FBPase和PEPCK的活性, 而添加不同水平的蔗糖对FBPase和PEPCK活性的影响均不显著(P 0.05)。总的来说, 从大菱鲆幼鱼肝脏糖代谢角度而言, 在饲料中添加15%的葡萄糖或糊精时, 可以有效促进大菱鲆肝脏糖酵解能力; 较添加葡萄糖, 糊精在促进大菱鲆肝脏糖酵解的同时对糖异生存在一定程度的抑制。蔗糖作为饲料糖源时, 仅在添加量为28%时显著促进糖酵解酶GK活性, 糖酵解其他酶活性以及糖异生酶活性均不受蔗糖水平的显著影响。       

Hepatic glycogen synthesis in the absence of glucokinase: the case of embryonic liver

Glucokinase (GK, hexokinase type IV) is required for the accumulation of glycogen in adult liver and hepatoma cells. Paradoxically, mammalian embryonic livers store glycogen successfully in the absence of GK. Here we address how mammalian embryonic livers, but not adult livers or hepatoma cells, manage to accumulate glycogen in the absence of this enzyme. Hexokinase type I or II (HKI, HKII) substitutes for GK in hepatomas and in embryonic livers. We engineered FTO2B cells, a hepatoma cell line in which GK is not expressed, to unveil the modifications required to allow them to accumulate glycogen. In the light of these results, we then examined glycogen metabolism in embryonic liver. Glycogen accumulation in FTO2B cells can be triggered through elevated expression of HKI or either of the protein phosphatase 1 regulatory subunits, namely PTG or G L. Between these two strategies to activate glycogen deposition in the absence of GK, embryonic livers choose to express massive levels of HKI and HKII. We conclude that although the GK/liver glycogen synthase tandem is ideally suited to store glycogen in liver when blood glucose is high, the substitution of HKI for GK in embryonic livers allows the HKI/liver glycogen synthase tandem to make glycogen independently of the glucose concentration in blood, although it requires huge levels of HK. Moreover, the physiological consequence of the HK isoform switch is that the embryonic liver safeguards its glycogen deposits, required as the main source of energy at birth, from maternal starvation.     

Insulin Resistance Is Not Associated with an Impaired Mitochondrial Function in Contracting Gastrocnemius Muscle of Goto-Kakizaki Diabetic Rats In Vivo

Insulin resistance, altered lipid metabolism and mitochondrial dysfunction in skeletal muscle would play a major role in type 2 diabetes mellitus (T2DM) development, but the causal relationships between these events remain conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated throughout a multidisciplinary approach combining in vivo and in vitro measurements in Goto-Kakizaki (GK) rats, a non-obese T2DM model developing peripheral insulin resistant without abnormal level of plasma non-esterified fatty acids (NEFA). Wistar rats were used as controls. Mechanical performance and energy metabolism were assessed strictly non-invasively using magnetic resonance (MR) imaging and 31-phosphorus MR spectroscopy (³¹P-MRS). Compared with control group, plasma insulin and glucose were respectively lower and higher in GK rats, but plasma NEFA level was normal. In resting GK muscle, phosphocreatine content was reduced whereas glucose content and intracellular pH were both higher. However, there were not differences between both groups for basal oxidative ATP synthesis rate, citrate synthase activity, and intramyocellular contents for lipids, glycogen, ATP and ADP (an important in vivo mitochondrial regulator). During a standardized fatiguing protocol (6 min of maximal repeated isometric contractions electrically induced at a frequency of 1.7 Hz), mechanical performance and glycolytic ATP production rate were reduced in diabetic animals whereas oxidative ATP production rate, maximal mitochondrial capacity and ATP cost of contraction were not changed. These findings provide in vivo evidence that insulin resistance is not caused by an impairment of mitochondrial function in this diabetic model.     

Glycogen synthase,glycogen phosphorylase and α-amylase activity in homogenates of islets of GK rats: Comparison with hepatic and pancreatic extracts

Glycogen accumulation in pancreatic islet cells in situations of sustained hyperglycaemia may participate in the phenomenon of so-called B-cell glucotoxicity. Unexpectedly, however, previously little if any glycogen was found in islet cells of non-insulin-dependent diabetic Goto-Kakizaki rats (GK rats). Therefore, the activities of glycogen synthase, glycogen phosphorylase and α-amylase were measured in islets of control and GK rats. No significant difference in enzymatic activity was observed between the control and diabetic animals. In the liver, the activity of glycogen synthase appeared even somewhat higher in GK rats than in control animals. It is concluded that the diabetic syndrome in the GK rats does not involve any major anomaly of glycogen synthase and glycogen phosphorylase activity in the liver of these animals, as well as α-amylase, in pancreatic islets.