Glucose-raising genetic variants in MADD and ADCY5 impair conversion of proinsulin to insulin

Introduction

Recent meta-analyses of genome-wide association studies revealed new genetic loci associated with fasting glycemia. For several of these loci, the mechanism of action in glucose homeostasis is unclear. The objective of the study was to establish metabolic phenotypes for these genetic variants to deliver clues to their pathomechanism.

Methods

In this cross-sectional study 1782 non-diabetic volunteers at increased risk for type 2 diabetes underwent an oral glucose tolerance test. Insulin, C-peptide and proinsulin were measured and genotyping was performed for 12 single nucleotide polymorphisms (SNP) in or near the genes GCK (rs4607517), DGKB (rs2191349), GCKR (rs780094), ADCY5 (rs11708067), MADD (rs7944584), ADRA2A (rs10885122), FADS1 (rs174550), CRY2 (rs11605924), SLC2A2 (rs11920090), PROX1 (rs340874), GLIS3 (rs7034200) and C2CD4B (rs11071657). Parameters of insulin secretion (AUC Insulin_0–30/AUC Glucose_0–30, AUC C-peptide_0–120/AUC Glucose_0–120), proinsulin-to-insulin conversion (fasting proinsulin, fasting proinsulin/insulin, AUC Proinsulin_0–120/AUCInsulin_0–120) and insulin resistance (HOMA-IR, Matsuda-Index) were assessed.

Results

After adjustment for confounding variables, the effect alleles of the ADCY5 and MADD SNPs were associated with an impaired proinsulin-to-insulin conversion (p = 0.002 and p = 0.0001, respectively). GLIS3 was nominally associated with impaired proinsulin-to-insulin conversion and insulin secretion. The diabetogenic alleles of DGKB and PROX1 were nominally associated with reduced insulin secretion. Nominally significant effects on insulin sensitivity could be found for MADD and PROX1.

Discussion

By examining parameters of glucose-stimulated proinsulin-to-insulin conversion during an OGTT, we show that the SNP in ADCY5 is implicated in defective proinsulin-to-insulin conversion. In addition, we confirmed previous findings on the role of a genetic variant in MADD on proinsulin-to-insulin conversion. These effects may also be related to neighboring regions of the genome.     

Effects of 16 genetic variants on fasting glucose and type 2 diabetes in South Asians: ADCY5 and GLIS3 variants may predispose to type 2 diabetes

BackgroundThe Meta-Analysis of Glucose and Insulin related traits Consortium (MAGIC) recently identified 16 loci robustly associated with fasting glucose, some of which were also associated with type 2 diabetes. The purpose of our study was to explore the role of these variants in South Asian populations of Punjabi ancestry, originating predominantly from the District of Mirpur, Pakistan.Conclusions/SignificanceAlthough only the SLC30A8 rs11558471 SNP was nominally associated with fasting glucose in our study, the finding that 12 out of 16 SNPs displayed a direction of effect consistent with European studies suggests that a number of these variants may contribute to fasting glucose variation in individuals of South Asian ancestry. We also provide evidence for the first time in South Asians that alleles of SNPs in GLIS3 and ADCY5 may confer risk of type 2 diabetes.     

Effects of epinephrine, glucagon and insulin on glucose metabolism of planaria

Epinephrine and insulin increased glucose uptake in Planaria, but epinephrine did so to a much grater extent. Glucagon proved to be without effect. The experiments support earlier results according to which in unicellular and invertebrate organisms membrane patterns can be found, which are similar to those of higher organisms and behave like receptors.     

Effects of exercise on insulin binding and glucose metabolism in muscle

To elucidate the mechanism of enhanced insulin sensitivity by muscle after exercise, we studied insulin binding, 2-deoxy-D-[1-14C]glucose (2-DOG) uptake and [5-3H]glucose utilization in glycolysis and glycogenesis in soleus and extensor digitorum longus (EDL) muscles of mice after 60 min of treadmill exercise. In the soleus, glycogenesis was increased after exercise (P less than 0.05) and remained sensitive to the action of insulin. Postexercise insulin-stimulated glycolysis was also increased in the soleus (P less than 0.05). In the EDL, glycogenesis was increased after exercise (P less than 0.05). However, this was already maximal in the absence of insulin and was not further stimulated by insulin (0.1-4 nM). The disposal of glucose occurred primarily via the glycolytic pathway (greater than 60%) in the soleus and EDL at rest and after exercise. The uptake of 2-DOG uptake was not altered in the soleus after exercise (4 h incubation at 18 degrees C). However, with 1-h incubations at 37 degrees C, a marked increase in 2-DOG uptake after exercise was observed in the soleus (P less than 0.05) in the absence (0 nM) and presence of insulin (0.2-4 nM) (P less than 0.05). A similar postexercise increase in 2-DOG uptake occurred in EDL. Despite the marked increase in glucose uptake and metabolism, no changes in insulin binding were apparent in either EDL or soleus at 37 degrees C or 18 degrees C. This study shows that the postexercise increase of glucose disposal does not appear to be directly attributable to increments in insulin binding to slow-twitch and fast-twitch muscles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hormone and glucose administration on hepatic glucose and glycogen metabolism in vivo. A 13C NMR study

Effects of peripheral venous injection of glucagon and insulin on [1-13C]glucose incorporation into hepatic glycogen of rats were studied by 13C NMR in vivo. Each animal was given a continuous somatostatin infusion and a 100-mg intravenous injection of [1-13C] glucose in NMR experiments or unlabeled glucose in parallel experiments for determination of serum glucose. Insulin administration caused serum glucose to fall below basal levels and accelerated the loss of hepatic [1-13C]glucose; these effects were counteracted by the addition of glucagon. Glucagon administration alone did not affect serum glucose or hepatic [1-13C] glucose but caused the loss of [1-13C]glucose from glycogen and inhibited [1-13C]glucose incorporation into glycogen. Insulin did not alter [1-13C]glucose incorporation into glycogen when given alone or in combination with glucagon. The data are consistent with a model in which liver glycogen synthesis increases linearly with hepatic glucose concentration above a threshold glucose concentration. Insulin did not alter the rate constant or the threshold for synthesis.     

Effects of glucose and insulin on HepG2‐C3A cell metabolism

HepG2, hepatocellular carcinoma cells, are used in drug toxicity studies and have also been explored for bioartificial livers. For these applications, the cells are under variable levels of nutrients and hormones, the effects of which on metabolism are poorly understood. In this study, HepG2‐C3A cells were cultured under varying levels of glucose (high, low, and glucose‐free) and insulin (without and with physiological levels of insulin) for 5 days. Cell growth was found to be comparable between high and low glucose media and lowest for glucose‐free medium. Several features of central metabolism were affected profoundly by the medium glucose levels. Glucose consumption was greater for low glucose medium compared to high glucose medium, consistent with known glucose feedback regulation mechanisms. Urea productivity was highest in glucose‐free medium. Further, it was seen that lactate acted as an alternative carbon source in the absence of glucose, whereas it acted as a sink for the high and low glucose media. Using a metabolic network flexibility analysis (MNFA) framework with stoichiometric and thermodynamic constraints, intracellular fluxes under varying levels of glucose and insulin were evaluated. The analysis indicates that urea production in HepG2‐C3A cells arises via the arginase II pathway rather than from ammonia detoxification. Further, involvement of the putrescine metabolism with glutamine metabolism caused higher urea production in glucose‐free medium consistent with higher glutamine uptake. MNFA indicated that in high and low glucose media, glycolysis, glutaminolysis, and oxidative phosphorylation were the main sources of energy (NADH, NADPH, and ATP). In the glucose‐free medium, due to very low glycolytic flux, higher malate to pyruvate glutaminolytic flux and TCA cycle contributed more significantly to energy metabolism. The presence of insulin lowered glycerol uptake and corresponding fluxes involved in lipid metabolism for all glucose levels but otherwise exerted negligible effect on metabolism. HepG2‐C3A cells thus show distinct differences from primary hepatocytes in terms of energy metabolism and urea production. This knowledge can be used to design media supplements and metabolically engineer cells to restore necessary hepatic functions to HepG2‐C3A cells for a range of applications. Biotechnol. Bioeng. 2010;107: 347–356. © 2010 Wiley Periodicals, Inc.     

Effects of age on various aspects of glucose and insulin metabolism

Summary Age is known to be associated with the development of glucose intolerance. In this review an effort has been made to differentiate between the effects of age per se on glucose tolerance, as distinguished from those of such age-related variables as obesity, diet, development of frank diabetes, etc. At the same time, an attempt was made to evaluate the evidence implicating abnormalities of insulin secretaion and/or insulin action in the development of glucose intolerance with age. It is concluded that the questions being asked are far from simple, and that available data do not provide unequivocal answers.     

雷帕霉素对小鼠葡萄糖代谢水平的影响

目的探讨雷帕霉素对葡萄糖代谢水平影响的特点、机制。方法选择4周龄、雄性C57BL/6小鼠,高热量、高脂饮食喂养8周后为肥胖组(HF,n=18),普通饲料喂养为正常组(NC,n=18)。两组小鼠分别给予安慰剂(n=6)、腹腔注射雷帕霉素(2 mg/kg,隔日1次,n=6)、喂饮2.37%亮氨酸水(n=6),2周后分别行灌胃葡萄糖耐量试验(glucose tolerance test,GTT)、胰岛素耐受性试验(insulin tolerance test,ITT)以及胰岛组织病理学检查。结果正常组小鼠腹腔注射雷帕霉素后葡萄糖负荷30min血糖水平显著升高(与安慰剂组比P=0.038,与亮氨酸组比P=0.035)。肥胖组小鼠腹腔注射雷帕霉素后空腹血糖水平显著高于安慰剂组(P=0.031),葡萄糖负荷30 min血糖显著高于安慰剂组(P=0.013)、亮氨酸组(P=0.041)。仅正常组小鼠胰岛素敏感性与安慰剂组相比显著降低(P=0.039)。雷帕霉素干预后腹腔脂肪量显著减少(正常组与安慰剂组比P0.001,肥胖组与安慰剂组比P=0.013)。结论雷帕霉素对哺乳动物糖代谢水平有显著影响,正常小鼠与机体胰岛素敏感性下降有关;肥胖小鼠与胰岛素分泌功能受损、胰岛素抵抗相关。     

Effects of insulin on glucose metabolism in isolated human fat cells

Isolated fat cells were used for the study of in vitro effects of insulin on glucose metabolism in human and rat adipose tissue. In human subcutaneous fat cells, effects of insulin could be detected at concentrations of glucose in the medium from 1 to 10 micro moles/ml. Cellular responsiveness was inversely proportional to the glucose level. At a constant concentration of 6 micro moles of glucose per ml, the effects of insulin at various concentrations up to 500 micro U/ml were investigated. At the highest concentration, which gave the maximal response, there was a 100% increase in the conversion of glucose-U-(14)C to glyceride-glycerol and a 40% increase in glucose oxidation. The dose-response curve was steepest between 2 and 20 micro U/ml. Rat epididymal fat cells were much more responsive to insulin. Glucose lipogenesis and pentose cycle activity could also be demonstrated in rat cells, whereas these activities could not be shown in fat cells from human omental and subcutaneous tissue. The findings for human cells are attributed to changes in cellular activity during preparation.     

Effects of growth hormone therapy on glucose metabolism and insulin sensitivity indices in prepubertal children with Prader-Willi syndrome

In Prader-Willi syndrome (PWS) growth hormone therapy (GHT) improves height, body composition, agility and muscular strength. In such patients it is necessary to consider the potential diabetogenic effect of GHT, since they tend to develop type 2 diabetes, particularly after the pubertal age. The aim of our study was to investigate the effects of GHT on glucose and insulin homeostasis in PWS children. An oral glucose tolerance test (OGTT) was performed in 24 prepubertal PWS children (15 male, 9 female, age: 5.8 +/- 2.8 years), 16 were obese (group A) and 8 had normal weight (group B), before and after 2.7 +/- 1.3 years GHT (0.22 +/- 0.03 mg/kg/week) and, only at baseline, in 35 prepubertal children with simple obesity (19 male, 16 female) (group C). Fasting glucose and insulin, glucose tolerance, insulin sensitivity index (ISI), homeostasis model assessment of insulin resistance (HOMA-IR), quick insulin check index (QUICKI), area under the curves (AUC) of glucose and insulin were estimated. At the start of GHT, all PWS children were normoglycaemic and normotolerant but two developed impaired glucose tolerance after 2.2 and 1.9 years of therapy, respectively. At baseline, group A showed lower fasting insulin levels, HOMA-IR and AUC of insulin, higher ISI, QUICKI and AUC of glucose than group C. Comparing groups A and B, AUC of insulin was higher and ISI lower in group A. During GHT, a significant increase of fasting insulin and glucose, a worsening of insulin resistance (HOMA-IR) and insulin sensitivity (QUICKI) was found only in group A while ISI did not change. The AUC of glucose decreased in both groups instead AUC of insulin did not change. BMI-SDS decreased in group A and increased in group B. The increased insulin resistance and decreased insulin sensitivity in obese PWS patients, as well as the occurrence of impaired glucose tolerance during GHT, suggest that a close monitoring of glucose and insulin homeostasis is mandatory, especially in treated obese PWS children.     

Effects of insulin and norepinephrine on glucose transport and metabolism in rat brown adipocytes. Potentiation by insulin of norepinephrine-induced glucose oxidation

Glucose is an important fuel for rat brown adipose tissue in vivo and its utilization is highly sensitive to insulin. In this study, the different glucose metabolic pathways and their regulation by insulin and norepinephrine were examined in isolated rat brown adipocytes, using [6-14C]glucose as a tracer. Glucose utilization was stimulated for insulin concentrations in the range of 40-1000 microU/ml. Furthermore, the addition of adenosine deaminase (200 mU/ml) or adenosine (10 microM) did not alter insulin sensitivity of glucose metabolism. The major effect of insulin (1 mU/ml) was a respective 7-fold and 5-fold stimulation of lipogenesis and lactate synthesis, whereas glucose oxidation remained very low. The 5-fold stimulation of total glucose metabolism by 1 mU/ml of insulin was accompanied by an 8-fold increase in glucose transport. In the presence of norepinephrine (8 microM), total glucose metabolism was increased 2-fold. This was linked to a 7-fold increase of glucose oxidation, whereas lipogenesis was greatly inhibited (by 72%). In addition, norepinephrine alone did not modify glucose transport. The addition of insulin to adipocytes incubated with norepinephrine, induced a potentiation of glucose oxidation, while lipogenesis remained very low. In conclusion, in the presence of insulin and norepinephrine glucose is a oxidative substrate for brown adipose tissue. However the quantitative importance of glucose as oxidative fuel remains to be determined.     

Effects of GH on urea,glucose and lipid metabolism,and insulin sensitivity during fasting in GH-deficient patients

Fasting-related states of distress pose major health problems, and growth hormone (GH) plays a key role in this context. The present study was designed to assess the effects of GH on substrate metabolism and insulin sensitivity during short-term fasting. Six GH-deficient adults underwent 42.5 h of fasting on two occasions, with and without concomitant GH replacement. Palmitate and urea fluxes were measured with the steady-state isotope dilution technique after infusion of [9,10-3H]palmitate and [13C]urea. During fasting with GH replacement, palmitate concentrations and fluxes increased by 50% [palmitate: 378 +/- 42 (GH) vs. 244 +/- 12 micromol/l, P < 0.05; palmitate: 412 +/- 58 (GH) vs. 276 +/- 42 microM, P = 0.05], and urea turnover and excretion decreased by 30-35% [urea rate of appearance: 336 +/- 22 (GH) vs. 439 +/- 43 micromol. kg-1. h-1, P < 0.01; urea excretion: 445 +/- 43 (GH) vs. 602 +/- 74 mmol/24 h, P < 0.05]. Insulin sensitivity (determined by a euglycemic hyperinsulinemic clamp) was significantly decreased [M value: 1.26 +/- 0.06 (GH) vs. 2.07 +/- 0.22 mg. kg-1. min-1, P < 0.01] during fasting with GH replacement. In conclusion, continued GH replacement during fasting in GH-deficient adults decreases insulin sensitivity, increases lipid utilization, and conserves protein.     

Effects of insulin on glucose metabolism in isolated heart myocytes from adult rats

The study examined the effect of insulin on glucose metabolism in freshly isolated calcium-tolerant heart myocytes from adult rats. The uptake of 2-deoxyglucose demonstrated an initial lag in response to insulin and the maximal insulin effect was not attained until after 3 min preincubation with the hormone. A dose-response study of 14CO2 production from [14C]glucose revealed that the maximum insulin stimulation of glucose utilization occurred with 5 mU/ml. Both the uptake and the oxidation of glucose proceeded at a linear rate in the absence and presence of insulin. However, insulin exerted a greater effect on the uptake (42-54%) than on the oxidation (17-22%) of exogenous glucose. Incorporation of glucose into glycogen was markedly increased by insulin and resulted in the myocyte glycogen concentration returning to in vivo levels. In the absence of insulin, glucose incorporation plateaued within 10 min of incubation and the glycogen concentration was not altered. Our findings also indicate that at equilibrium, insulin-treated cells exhibited a higher glycogen turnover rate. It thus appears that insulin exerts a differential effect on the different pathways in glucose metabolism in the isolated cardiac cells. This may be related in part to their quiescent state and lower energy demand.     

Effects of troglitazone on cellular differentiation, insulin signaling, and glucose metabolism in cultured human skeletal muscle cells

To determine the immediate effect of thiazolidinediones on human skeletal muscle, differentiated human myotubes were acutely (1 day) and myoblasts chronically (during the differentiation process) treated with troglitazone (TGZ). Chronic TGZ treatment resulted in loss of the typical multinucleated phenotype. The increase of muscle markers typically observed during differentiation was suppressed, while adipocyte markers increased markedly. Chronic TGZ treatment increased insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and membranous protein kinase B/Akt (PKB/Akt) Ser-473 phosphorylation more than 4-fold. Phosphorylation of p42/44 mitogen-activated protein kinase (42/44 MAPK/ERK) was unaltered. Basal glucose uptake as well as both basal and insulin-stimulated glycogen synthesis increased approximately 1.6- and approximately 2.5-fold after chronic TGZ treatment, respectively. A 2-fold stimulation of PI 3-kinase but no other significant TGZ effect was found after acute TGZ treatment. In conclusion, chronic TGZ treatment inhibited myogenic differentiation of that human muscle while inducing adipocyte-specific gene expression. The effects of chronic TGZ treatment on basal glucose transport may in part be secondary to this transdifferentiation. The enhancing effect on PI 3-kinase and PKB/Akt involved in both differentiation and glycogen synthesis appears to be pivotal in the cellular action of TGZ.     

Effects of insulin and histamine in themselves and in combination on the glucose metabolism of Tetrahymena

The glucose metabolism and the related intracellular processes of Tetrahymena pyriformis GL cells are measurably influenced by several hormones of higher organisms, among others by the endocytosis stimulating hormone histamine, and the glucose metabolism regulating hormone insulin. Histamine does not interfere with the glucose metabolizing action of insulin, but markedly enhances the utilization of glucose, to judge from a significant decrease in the PAS-positive (hexose) component of histamine-exposed Tetrahymena pyriformis GL cells.     

Effects of acetazolamide on insulin release, serum glucose and insulin, glucose tolerance, and alloxan sensitivity of mice

Isolated pancreatic islets exposed to 100 mM acetazolamide (AZM) and low glucose concentration exhibited increased insulin release, whereas those subjected to AZM and high glucose concentration exhibited decreased secretion of insulin. A slight transient hyperglycaemia was found 24 h after administration of 1.5 g/kg b.wt. of AZM to fed mice, whereas no such response was seen in starved mice. The serum insulin concentration was increased in the 24 h after AZM injection. Pretreatment with AZM caused decreased glucose tolerance and protection against alloxan toxicity. Inhibited carbonic anhydrase activity and ionic alterations might have played a role in the development of these effects of AZM in mice.     

Assessment of hepatic glucose metabolism by indirect calorimetry in combination with a non-invasive technique using naturally enriched 13C glucose in healthy children and adolescents

The metabolic fate of hepatic glucose can be best studied using invasive techniques such as tracer infusions and frequent blood sampling which have been revealed to be impractical in the pediatric age group. The aim of this study was to develop a non-invasive method based on indirect calorimetry and expired 13CO2 monitoring in order to gain insight into the mechanisms leading to impaired glucose tolerance in children and teenagers. As a first step, net glucose oxidation (NGO) and energy expenditure (EE) were measured in 47 subjects (range 7.5-17.3 years) of whom 18 were prepubertal (P1), 11 in early puberty (P2-P3) and 18 in late puberty (P4-P5) after 3-hourly loads of 180 mg/kg of oral maize glucose containing naturally enriched 13C. Isotope analysis allowed to calculate exogenous and endogenous glucose oxidation (EXGO, ENGO) and, hence, to derive TGS and NGS, that is glycogen turnover. NGO and EE decreased significantly with pubertal progression, reflecting higher metabolism at younger ages, whereas EXGO remained constant. TGS did not change significantly whereas NGS showed a significant negative correlation with pubertal progression: this can be explained by the fact that glycogenolysis exceeded glycogen synthesis in this experimental setting. This non-invasive method appears to be a promising tool to study the fate of hepatic glucose and therefore glycogen turnover in children at risk of developing glucose intolerance and/or type 2 diabetes.