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Synthesis and biological activities of D-chiro-inositol analogues with insulin-like actions
Abstract
D-chiro-inositol (DCI, 1) evokes therapeutic actions in diabetes and insulin resistance but has sub-optimal pharmacokinetic profiles. To investigate what positions on the DCI cyclohexanol ring may be amenable to modification to improve pharmaceutical formulations, a series of analogues based on DCI were synthesised. These compounds were then evaluated for their ability to stimulate glucose transport using 3T3-L1 adipocytes as a model system. Positional analyses indicate that the hydroxyl group at position 1 is not essential for activity and can be modified without affecting glucose uptake. Removal of the hydroxyl at position 3 also had minimal effect on activity but this group is sensitive to modification. By comparison, the oxygen at position 2 is crucial to the potency of DCI, although this group can withstand modification without fundamentally affecting activity. These data reveal that positions 1 and 2 on the cyclohexanol ring of DCI offer further scope for modification to develop DCI analogues with desirable pharmacokinetic profiles for the potential treatment of metabolic disease.
... T2DM was first described in 1988 as a part of the metabolic syndrome. T2DM (formerly known as diabetes without insulin) is the most common diabetes' form, characterized by hyperglycemia, insulin resistance, and relative insulin deficiency ( Figure 2) [52,53]. ...
... It is estimated that 439 million patients will have T2DM before 2030. The frequency of T2DM varies according to geographical region and risk factors related with the lifestyle and environment impact [52,54]. An increase in the number of T2DM cases is expected over the next two decades and a significant part of this increase will occur in developing countries where the majority of patients are between 45 and 64 years old [53]. ...
... Many of them are also associated with human lifestyle such as: Lack of physical activity, sedentary lifestyle, smoking, and excessive alcohol consumption. It has been shown that obesity contributes to approximately 55% of T2DM cases [51,52,55]. Moreover, additional factors that increase the risk of T2DM are aging and high-fat diet [51]. ...
Cyclitols play a particularly important role in cell functioning because they are involved in ion channel physiology, phosphate storage, signal transduction, cell wall formation, membrane biogenesis, osmoregulation and they have antioxidant activity. They are involved in the cell membranes as a phosphatidyl myo-inositol, an inositol triphosphate precursor, which acts as a transmitter that regulates the activity of several hormones, such as follicle-stimulating hormone, thyrotropin, and insulin. The aim of this paper is to characterize the selected cyclitols: myo-inositol, D-chiro-inositol, and D-pinitol in type-2 metabolic syndrome and diabetes treatment. Results and discussion: Cyclitols have certain clinical applications in the treatment of metabolic syndromes and are considered to be an option as a dietary supplement for the treatment or prevention of gestational diabetes mellitus and type-2 diabetes. Improved metabolic parameters observed after using cyclitols, like myo-inositol, in the treatment of polycystic ovary syndrome and type-2 diabetes suggest that they may have a protective effect on the cardiovascular system. Pinitol, together with myo-inositol,maybe responsible for improving lipid profiles by reducing serum triglyceride and total cholesterol. Pinitol is also well-researched and documented for insulin-like effects. Myo-inositol, D-chiro-inositol, and D-pinitol indicate a number of therapeutic and health-promoting properties.
... These compounds, acting synergistically, contribute significantly to the plant's medicinal properties, such as antioxidant, anthelmintic, anti-inflammatory, antidiabetic, antimicrobial, and analgesic activities [31,32,36,37]. Additionally, the isolated compound 2, deoxychiro-inositol, has been reported to exhibit insulin-like actions [38]. Nanoparticle synthesis: Over the last few decades, there has been a rise in the fabrication of therapeutic and diagnostic agents based on nanoparticles for various medical uses, with plants and herbs emerging as promising sources for nanoparticle synthesis [39,40]. ...
This review comprehensively explores I. staphylina's traditional uses, diverse applications, and pharmacological activities. Extensively used in traditional medicine, this plant addresses a range of ailments, including stomach disorders, respiratory issues, and rheumatism. Research has highlighted its significant antimicrobial, anti-inflammatory, antioxidant, antidiabetic, anthelmintic, and analgesic properties. Notably, its antiulcer activity highlights its potential as a novel antiulcer agent, while hepatoprotective and nephroprotective effects suggest therapeutic applications in liver and kidney disorders. Studies on its anti-diabetic potential show significant reductions in blood glucose levels and positive impacts on biochemical markers. The plant's anti-mutagenic activity against base-pair mutations expands its potential applications. The review also discusses the isolation and pharmacological applications of pure compounds identified through LC-MS and NMR analyses. This review identifies I. staphylina as a promising source of bioactive compounds with therapeutic potential, emphasizing the need for further research to isolate and characterize its active constituents.
... Bioactive and functional properties of beer intended for a special group of consumers (gluten-free beer) can be improved by using such unconventional ingredients in the malting process. An additional aspect of using buckwheat is the high content of bioactive inositols, namely D-chiro-inositol (DCI) and myo-inositol (MI), which have potential positive effects on the glycaemic index, as well as immunomodulatory, signalling, and cell-growth stimulating properties (Gillaspy 2011, Yao et al., 2011, Rendle et al., 2016, Owczarczyk-Saczonek et al., 2018. Phosphoglycans P-type that contain D-chiroinositol and galactosamine may act as secondary relay mimicking insulin or amplifying the signal generated by this blood sugar regulator (Larner et al., 2010). ...
Relatively high levels of phytates in buckwheat malt and the low activity of endogenous phytases that limit the effective use of substrates for fermentation and yeast metabolism (starch, proteins, minerals) are an argument for using phytases in beer production technology. Two mash-in programs were applied: (1) the Congress program, typical for basic raw materials, (2) a program with temperature optimized for phytase activity. Commercial preparations of 3-phytase (Finase P) and 6-phytase (Ronozyme) were used in the study. Monitored levels of selected fermentable sugars indicates a statistically significant effect of phytase addition on the glucose content in both mash-in programs used. The SEC-HPLC chromatography allowed to select a key polypeptide with an estimated molecular weight of 40 kDa, whose relative peak area decreases as a result of the applied mash-increase treatment with phosphorolytic enzymes, although this relation was not statistically confirmed in the analysis of free amino acids content. The analyses carried out also indicate that apart from the target molecules, namely phytate and inositol, the use of phytases in the process of buckwheat wort preparation slightly changes the profile of fermentable sugars and causes significant changes in the polypeptide profile of the final mash.
... ДХИ принимает непосредственное участие в реализации биологических эффектов инсулина [14]. Более того, схожие с ДХИ молекулы характеризуются инсулиноподобным действием [15]. ДХИ ускоряет дефосфорилирование гликогенсинтазы и пируватдегидрогеназы, ограничивающей скорость ферментов метаболизма глюкозы. ...
D-chiroinositol (DCI) is one of the 9 inositol isomers that forms inositol phosphoglycans, which are mediators of the action of insulin. The metabolism of DCI and myo-inositol (MI) is impaired against the background of insulin resistance, including the patients with polycystic ovary syndrome (PCOS).
Aim. Highlight the most characteristic pharmacological properties of DCI.
Materials and methods. Systematic computer analysis of 45 600 publications on the biological roles of inositol by methods of the topological theory of pattern recognition and systems biology analysis of human proteome.
Results. A complex of interactions between metabolic disorders of DCI, PCOS, ovulation disorders, obesity associated with numerous hormonal disorders is described. Supplements with DCI and MI increase the sensitivity of cells to insulin and normalize the metabolism of androgens. An important difference between DCI and MI is the presence of DCI in the composition of inositol phosphoglycans that mediate the action of insulin on cells, as well as its participation in the realization of the therapeutic effects of metformin. The use of the MI+DCI combination allows achieving positive dynamics in reducing excess body weight, normalizing blood lipids, glucose and insulin levels, restoring the ovulatory menstrual cycle, improving oocyte quality, and helps prevent gestational diabetes in pregnant women and macrosomia of the fetus. DCI is more effective than MI in reducing the risk of folate-resistant neural tube defects.
Conclusion. The therapeutic potential of DCI in combination with MI for treating PCOS and hyperandrogenism is evident. Depending on the therapeutic expediency, various ratios of MI:DCI can be used for the treatment of PCOS and disorders of carbohydrate and lipid metabolism.A
... These compounds have been found to have a positive impact on the glycemic index, immunomodulatory, signaling, and cell growth-stimulating properties. [4][5][6] P-type phosphoglycans containing Dchiro-inositol and galactosamine can act as secondary messengers that mimic the action of insulin or amplify the signal generated by this regulator of blood sugar levels. [7,8] Myo-inositol, on the other hand, is a hexahydroxylic cyclohexane derivative present in all types of living cells, in concentrations varying from 0.03 mM (blood plasma) to 3 mM (brain tissue, cerebrospinal fluid, plasma, fetal, and neonatal human milk). ...
The impact of two commercial enzyme preparations (Mats L Classic, Brewers Compass) used for mashing buckwheat malt was studied to assess their efficacies in releasing D-chiro- and myo-inositols, fermentable sugars and low molecular weight proteins and peptides. Concentrations of these compounds were determined in buckwheat malt, wort, and beer using sensitive ion-exchange and size exclusion chromatography techniques. These were preliminary studies whose general aim was to research the possibilities of functional beer production that could be dedicated for patients with coeliac disease. The application of enzyme preparations had no significant (Brewers Compass) or only little impact (Mats L Classic) on the recovery of D-chiro-inositol (DCI) and myo-inositol (MI) present in the buckwheat malt. In contrast to DCI, whose concentration in the buckwheat beer was 0.3 mg/mL, a minor fraction of MI (11–14.8%) was released into the beverage. In comparison to a barley beer, the oligosaccharide profile of the buckwheat beer was characterized by a relatively high proportion of residual sugars with a degree of polymerization above 5. SEC–HPLC chromatography showed significant changes within the polypeptide fractions after mashing and malting, with a marked increase in the proportion of low molecular weight peptides.
... This shows a potential for the development and use of engineered DCI analogies with specific pharmacokinetic and dynamic properties. [18] Currently, improved gel-capsule versions of MI and DCI are available which allow enhanced bioavailability as compared to powered formulations. The ratios of MI and DCI vary and are a subject of debate. ...
This review describes the mechanistic, animal, and clinical data related to the use of inositols in midlife. It covers studies related to the mechanism of action of myo-inositol and D-chiro-inositol and randomized controlled trials conducted in postmenopausal women with metabolic syndrome and supports these data with the results of in vitro and animal studies on inositol in nephropathy and other related conditions. Recent advances related to biochemistry, pharmaceutical science, and genetics are discussed. It concludes that inositols have a potential role to play in maintaining metabolic health in postmenopausal women.
This review describes applications of radical reactions developed in the authors’ laboratory for the modification of carbohydrates. It focuses on tin‐free variation on the Barton‐McCombie deoxygenation; on reductive de‐iodination; on intermolecular radical additions of xanthates; on allylations and vinylations of xanthates and iodides, with emphasis on allylations using allylic alcohols activated as fluoropyridyl ethers; and, finally, on the synthesis of mercaptosugars. @font‐face {font‐family:"MS Mincho"; panose‐1:2 2 6 9 4 2 5 8 3 4; mso‐font‐alt:"ＭＳ 明朝"; mso‐font‐charset:128; mso‐generic‐font‐family:modern; mso‐font‐pitch:fixed; mso‐font‐signature:‐536870145 1791491579 134217746 0 131231 0;}@font‐face {font‐family:"Cambria Math"; panose‐1:2 4 5 3 5 4 6 3 2 4; mso‐font‐charset:0; mso‐generic‐font‐family:roman; mso‐font‐pitch:variable; mso‐font‐signature:‐536870145 1107305727 0 0 415 0;}@font‐face {font‐family:"Myriad Pro"; panose‐1:2 11 6 4 2 2 2 2 2 4; mso‐font‐alt:Corbel; mso‐font‐charset:0; mso‐generic‐font‐family:swiss; mso‐font‐pitch:variable; mso‐font‐signature:‐1610612049 1342185547 0 0 415 0;}@font‐face {font‐family:"\@MS Mincho"; panose‐1:2 2 6 9 4 2 5 8 3 4; mso‐font‐charset:128; mso‐generic‐font‐family:modern; mso‐font‐pitch:fixed; mso‐font‐signature:‐536870145 1791491579 134217746 0 131231 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso‐style‐unhide:no; mso‐style‐parent:""; margin:0cm; line‐height:150%; mso‐pagination:widow‐orphan; font‐size:8.0pt; mso‐bidi‐font‐size:12.0pt; font‐family:"Myriad Pro",sans‐serif; mso‐fareast‐font‐family:"MS Mincho"; mso‐bidi‐font‐family:"Times New Roman"; mso‐ansi‐language:DE; mso‐fareast‐language:JA;}p.Helv‐Abstract, li.Helv‐Abstract, div.Helv‐Abstract {mso‐style‐name:Helv‐Abstract; mso‐style‐unhide:no; mso‐style‐qformat:yes; margin‐top:6.0pt; margin‐right:0cm; margin‐bottom:6.0pt; margin‐left:0cm; text‐align:justify; text‐justify:inter‐ideograph; line‐height:150%; mso‐pagination:widow‐orphan; font‐size:8.0pt; mso‐bidi‐font‐size:10.0pt; font‐family:"Myriad Pro",sans‐serif; mso‐fareast‐font‐family:"MS Mincho"; mso‐bidi‐font‐family:"Times New Roman"; mso‐ansi‐language:EN‐US; mso‐fareast‐language:JA;}.MsoChpDefault {mso‐style‐type:export‐only; mso‐default‐props:yes; font‐size:10.0pt; mso‐ansi‐font‐size:10.0pt; mso‐bidi‐font‐size:10.0pt; mso‐fareast‐font‐family:"MS Mincho";}div.WordSection1 {page:WordSection1;}
Previously unknown entities in the form of 1,2,3,4,5,6,7,8,9,10‐decahydroxydecalins (DHDs) were conceptualized, and the first member of this class, an inosito‐inositol, was synthesized via a series of sequentially executed oxyfunctionalization maneuvers with attendant regio‐ and stereocontrol. The DHD accessed here was subjected to preliminary in silico evaluation with β‐amyloid and is expected to hold promise in Alzheimer's disease therapeutics. image
The improvements in the crystal structure refinement program SHELXL have been closely coupled with the development and increasing importance of the CIF (Crystallographic Information Framework) format for validating and archiving crystal structures. An important simplification is that now only one file in CIF format (for convenience, referred to simply as ‘a CIF’) containing embedded reflection data and SHELXL instructions is needed for a complete structure archive; the program SHREDCIF can be used to extract the .hkl and .ins files required for further refinement with SHELXL. Recent developments in SHELXL facilitate refinement against neutron diffraction data, the treatment of H atoms, the determination of absolute structure, the input of partial structure factors and the refinement of twinned and disordered structures. SHELXL is available free to academics for the Windows, Linux and Mac OS X operating systems, and is particularly suitable for multiple-core processors.
The new computer program SHELXT employs a novel dual-space algorithm to solve the phase problem for single-crystal reflection data expanded to the space group P1. Missing data are taken into account and the resolution extended if necessary. All space groups in the specified Laue group are tested to find which are consistent with the P1 phases. After applying the resulting origin shifts and space-group symmetry, the solutions are subject to further dual-space recycling followed by a peak search and summation of the electron density around each peak. Elements are assigned to give the best fit to the integrated peak densities and if necessary additional elements are considered. An isotropic refinement is followed for non-centrosymmetric space groups by the calculation of a Flack parameter and, if appropriate, inversion of the structure. The structure is assembled to maximize its connectivity and centred optimally in the unit cell. SHELXT has already solved many thousand structures with a high success rate, and is optimized for multiprocessor computers. It is, however, unsuitable for severely disordered and twinned structures because it is based on the assumption that the structure consists of atoms.
Diabetic nephropathy is a serious complication of diabetes mellitus with a pressing need for effective metabolic markers to detect renal impairment. Of potential significance are the inositol compounds, myo-inositol (MI), and the less abundant stereoisomer, D-chiro-inositol (DCI), which are excreted at increased levels in the urine in diabetes mellitus, a phenomenon known as inosituria. There is also a selective urinary excretion of DCI compared to MI. As the biological origins of altered inositol metabolism in diabetes mellitus are unknown, the aim of this study was to determine whether the diabetic kidney was directly responsible. Kidneys isolated from four-week streptozotocin-induced diabetic rats were characterized by a 3-fold reduction in glomerular filtration rate (GFR) compared to matched non-diabetic kidneys. When perfused with fixed quantities of MI (50 µM) and DCI (5 µM) under normoglycemic conditions (5 mM glucose), GFR-normalized urinary excretion of MI was increased by 1.7-fold in diabetic vs. non-diabetic kidneys. By comparison, GFR-normalized urinary excretion of DCI was increased by 4-fold. Perfusion conditions replicating hyperglycemia (20 mM glucose) potentiated DCI but not MI urinary excretion in both non-diabetic and diabetic kidneys. Overall, there was a 2.4-fold increase in DCI urinary excretion compared to MI in diabetic kidneys that was independent of glucose ambience. This increased urinary excretion of DCI and MI in diabetic kidneys occurred despite increased renal expression of the inositol transporters, sodium myo-inositol transporter subtype 1 and 2 (SMIT1 and SMIT2). These findings show that the diabetic kidney primarily mediates inosituria and altered urinary partitioning of MI and DCI. Urinary inositol levels might therefore serve as an indicator of impaired renal function in diabetes mellitus with wider implications for monitoring chronic kidney disease.
d-chiro-Inositol (DCI) is a cyclic sugar alcohol that evokes both antidiabetic and insulin sensitizing effects. Pharmacological administration of DCI has been shown to lower blood glucose in rat models of diabetes mellitus and enhance insulin sensitivity in humans with polycystic ovary syndrome (PCOS). We hypothesised that the antidiabetic effects of DCI could be due to inhibition of hepatic glucose output (HGO). To test this hypothesis, we perfused isolated rat livers either with buffer, myo-inositol, DCI, or insulin, and investigated their respective effects on the stimulation of HGO by epinephrine. We found that perfusion with 200 μM DCI attenuated epinephrine-stimulated HGO by 35% over 30 min as compared to the buffer control perfusion (p=0.05). By comparison, perfusion with 1 nM insulin attenuated epinephrine-stimulated HGO by 57% (p<0.0001). The glucose-lowering effects by DCI occurred independently of insulin and were specific to the DCI stereoisomer as 200 μM myo-inositol had no effect. These findings suggest that DCI could evoke its antidiabetic effects in vivo by inhibition of HGO. Further identification of the protein targets involved could open up new avenues to regulate hyperglycaemia with wider implications for the treatment of hepatic insulin resistance in PCOS.
PCOS is the main cause of infertility due to metabolic, hormonal and ovarian dysfunctions. Women affected by PCOS often suffer of insulin resistance and of a compensatory hyperinsulinemia. These conditions put the patients at risk of developing several metabolic disorders. Both myo-inositol (MI) and D-chiro inositol (DCI) glycans administration has been reported to exert beneficial effects at metabolic, hormonal and ovarian level. Beside these common features, MI and DCI are indeed different molecules: they belong to two different signal cascades and regulate different biological processes.
In this study, we aim to verify whether the two molecules have a synergistic action by acting on their specific cellular pathways. The effectiveness in reducing the risk of metabolic syndrome as well as in enhancing the ovarian functions of a combined therapy with MI and DCI was compared to a mono therapy in a randomized controlled trial.
Fifty overweight women with PCOS were enrolled and divided in two groups to receive MI and DCL (MI+DCI group) or MI alone (MI group) for a period of six months. Baseline measurements were repeated at three months (T1) and at the end of the treatment (T2).
At the end of the treatment, both MI and MI+DCI groups showed an improvement of the metabolic parameters and no significant differences were found. As expected, the combined supplementation with MI and DCI resulted to be more effective, compared to the MI group, after three months of treatment.
The combined administration of MI and DCI in physiological plasma ratio (40:1) should be considered as the first line approach in PCOS overweight patients, being able to reduce the metabolic and clinical alteration of PCOS and, therefore, reduce the risk of metabolic syndrome.
Dose-dependent side effects related to myo-inositol (MI) oral administration represent a significant shortcoming for its clinical use. Aiming to search for a pharmaceutical form able to be better absorbed, the pharmacokinetic (PK) profile of the new manufactured MI soft gelatin capsule form was evaluated and compared with the commercially available MI powder.
A single-dose relative trial, consisting of four phases, was performed on 20 healthy volunteers who received different doses of MI powder and MI soft gelatin capsules. PK profiles related to the two pharmaceutical forms were obtained by analysis of MI plasma concentration, and the respective MI bioavailability was compared.
The administration of MI powder and MI soft gelatin capsules resulted in a different bioavailability. MI soft gelatin capsule form showed similar PK parameters compared with three times higher doses of MI in powder form.
MI soft gelatin capsules displayed an improved bioavailability, allowing to substantially reduce the administered dose and to minimize the dose-dependent side effects. Considering the number of conditions in which MI supplementation is recommended, this evidence could support a broader use of MI in clinical practice.
Classical actions of insulin involve increased glucose uptake from the bloodstream and its metabolism in peripheral tissues, the most important and relevant effects for human health. However, nonoxidative and oxidative glucose disposal by activation of glycogen synthase (GS) and mitochondrial pyruvate dehydrogenase (PDH) remain incompletely explained by current models for insulin action. Since the discovery of insulin receptor Tyr kinase activity about 25 years ago, the dominant paradigm for intracellular signaling by insulin invokes protein phosphorylation downstream of the receptor and its primary Tyr phosphorylated substrates-the insulin receptor substrate family of proteins. This scheme accounts for most, but not all, intracellular actions of insulin. Essentially forgotten is the previous literature and continuing work on second messengers generated in cells in response to insulin. Treatment and even prevention of diabetes and metabolic syndrome will benefit from a more complete elucidation of cellular-signaling events activated by insulin, to include the actions of second messengers such as glycan molecules that contain D-chiro-inositol (DCI). The metabolism of DCI is associated with insulin sensitivity and resistance, supporting the concept that second messengers have a role in responses to and resistance to insulin.
Exposure of insulin-responsive, differentiated 3T3-L1 cells (adipocytes) to 0.1 to 1.0 microgram/ml of insulin for 3 to 48 h resulted in a persistent state of enhanced 2-deoxy-D-glucose and 3-O-methyl-D-glucose uptake. Elevated basal transport activity was retained under conditions where 125I-insulin binding activity remained unchanged and exchangeable insulin was dissociated from cell surface receptors. The appearance of enhanced hexose transport activity was prevented by cycloheximide and could be distinguished from the activation of the glucose transport system observed in these and other cells during glucose deprivation. Rigorously washed adipocytes, exhibiting insulin-induced elevations in basal transport activity, were refractory to further stimulation by insulin during hexose uptake assays. Insulin-unresponsive 3T3-L1 preadipocytes failed to increase hexose uptake activity when treated under conditions that elicited an optimal response in adipocytes.
Some of the acute actions of insulin may be mediated by an enzyme-modulating inositol phosphate glycan, produced by the insulin-sensitive hydrolysis of glycosyl-phosphatidylinositol (GPI) that is structurally similar to a membrane protein anchor. An inositol glycan fragment from the structurally characterized Trypanosoma brucei variant surface glycoprotein GPI anchor is evaluated for insulin-mimetic antilipolytic activity. The fragment specifically and dose-dependently inhibits isoproterenol-stimulated lipolysis. Like the effect of insulin, glycan-induced antilipolysis is blocked by the low Km cAMP phosphodiesterase inhibitor imazodan (CI-914) and the serine/threonine phosphatase inhibitor, okadaic acid, suggesting that the activation of both cAMP phosphodiesterase and serine/threonine protein phosphatases are necessary. Moreover, this fragment causes a specific and dose-dependent inhibition of both microsomal glucose-6-phosphatase (EC 3.1.3.9) and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activity. Additionally, direct addition of the glycan to hepatocytes caused marked inhibition of glucose production from pyruvate. These results suggest that the direct modification of the activities of these two gluconeogenic enzymes by an inositol glycan may play a role in the inhibition of glucose output by insulin and provide the first evidence for the insulin-mimetic properties of a chemically characterized inositol glycan.
Addition of isoproterenol to isolated rat adipocytes prelabeled with [32P]phosphate caused an increase in the phosphorylation and activation of phospholipid methyltransferase. 32P-Labeled phospholipid methyltransferase was recovered by immunoprecipitation and gel electrophoresis. Analysis of 32P-labeled peptides revealed one site of phosphorylation regulated by isoproterenol, and analysis of phosphoamino acids demonstrated that the incorporation of [32P]phosphate was on phosphoserine. Incubation of adipocytes with isoproterenol in the presence of insulin or a phospho-oligosaccharide inhibited the phosphorylation and activation of this enzyme. The inhibitory effect of insulin on the phosphorylation of phospholipid methyltransferase was reversible, and it was mimicked by a phospho-oligosaccharide. The phospho-oligosaccharide was generated by hydrolysis of an isolated glycophospholipid with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. The insulin-like effect of this phospho-oligosaccharide on the phosphorylation of phospholipid methyltransferase was demonstrated in isolated adipocytes, and the effect was abolished by treatment of the phospho-oligosaccharide with 10% NH4OH, nitrous acid, or sodium periodate. These data suggest that in intact adipocytes the effect of insulin to inhibit the phosphorylation/activation of phospholipid methyltransferase is mediated by a phospho-oligosaccharide generated by a phosphatidylinositol-specific phospholipase C.
3T3-L1 preadipocytes, cloned from 3T3 mouse embryo fibroblasts, differentiate in monolayer culture into cells with morphological and biochemical characteristics of adipocytes. Deposition of cytoplasmic triglyceride is associated with an increased lipogenic rate and a coordinate rise in the activities of many lipogenic enzymes (Mackall, J.C., Student, A.K., Polakis, S.E., and Lane, M.D. (1976) J. Biol. Chem. 251, 6462-6464). During differentiation induced by a 48-h treatment of postconfluent cells with methylisobutylxanthine, dexamethasone, and insulin, fatty acid synthetase activity increased to a level 19.5-fold higher than that of undifferentiated 3T3-L1 cells or nondifferentiating 3T3-C2 cells. The rate of [3H]leucine incorporation into immunoadsorbable fatty acid synthetase rose to a maximum and then declined to a new level 12.5-fold higher in differentiated than in undifferentiated 3T3-L1 cells. The kinetics of the changing [3H]leucine incorporation rate was reflected in the kinetics of the rise in fatty acid synthetase activity. The rate of degradation of fatty acid synthetase, determined by pulse-chase experiments, was unaffected by differentiation, the t1/2 remaining constant at 1.4 days. It is concluded that the higher level of fatty acid synthetase activity in differentiated 3T3-L1 cells can be attributed entirely to an increased rate of enzyme synthesis. The rate of total cellular protein synthesis also increases early early in differentiation, lending support to a model in which the synthesis of a large number of "differentiated proteins" is coordinately induced.
D-chiro-inositol is a rare inositol isomer present in inositol phosphoglycans which are proposed mediators of insulin action. To study D-chiro-inositol metabolism in diabetes mellitus, a sensitive and specific assay was developed using negative-ion chemical ionization gas chromatography/mass spectrometry. Median urinary D-chiro-inositol excretion, which was 2.1 mumol/day in nondiabetics, was substantially increased to 12 mumol/day in non-insulin-dependent diabetes (P < 0.0001) and to 74 mumol/day in insulin-dependent diabetes (P < 0.0001). Urinary D-chiro-inositol was strongly correlated with fasting plasma glucose (r = 0.568, P < 0.0001), glycated hemoglobin (r = 0.529, P < 0.0001), and urinary glucose (r = 0.368, P = 0.01). The renal clearance of D-chiro-inositol was selectively elevated in both non-insulin-dependent and insulin-dependent diabetes when compared with the clearances of L-chiro-inositol or myo-inositol and exceeded the glomerular filtration rate in 71% of the diabetics but in none of the nondiabetics. In poorly controlled diabetic patients insulin treatment reduced urinary D-chiro-inositol losses by 63% and increased plasma levels by 8.8-fold. The metabolism of D-chiro-inositol is abnormal in diabetes and appears to be influenced by short- and long-term metabolic control.
The acute effects of administration of D-chiroinositol (D-CI), a component of a putative mediator of insulin action, on plasma glucose were examined in low dose streptozotocin-treated rats and normal rats given a glucose load and the effects on plasma glucose and insulin were determined in five obese rhesus monkeys with varying degrees of spontaneous insulin resistance. Single dose intragastric D-CI (10 mg/kg) administered to streptozotocin-treated rats produced a 30-40% decrease in plasma glucose (P < 0.05) at 30-120 min. Single dose intragastric D-CI (2-15 mg/kg) administered to normal rats 2 h before ip glucose produced a 30-50% decrease (P < 0.05) in plasma glucose. D-CI (10 mg/kg) caused a 50% increase (P < 0.05) in glucose disappearance rates in these rats. Myoinositol (10 mg/kg) was without effect. Intravenously administered single dose D-CI (100 mg/kg) increased both the glucose and insulin disappearance rates by 129 +/- 41% (mean +/- SE; P < 0.06) and 89 +/- 39% (P = 0.01), respectively, in all monkeys between 0-30 min compared to control values. D-CI administration, therefore, lowered elevated plasma glucose in streptozotocin-treated hyperglycemic rats, normal rats given a glucose load, and spontaneously insulin-resistant monkeys with or without noninsulin-dependent diabetes mellitus. Intravenous D-CI also lowered plasma insulin in these monkeys.
A novel phosphoinositolglycan-peptide (PIG-P) from the yeast Saccharomyces cerevisiae potently mimicks insulin action on glucose transport and metabolism in rat muscle and adipose tissue. The aim of the present study was to elucidate the cellular signalling pathways of this insulin-mimetic compound. Rapid onset and reversibility of PIG-P action on glucose transport were observed in isolated adipocytes with a half-time of transport stimulation of 6-8 min (insulin less than 5 min). Combined treatment with PIG-P and insulin indicated additive stimulation of glucose transport at submaximal concentrations and non-additive action of both agents at maximal doses. The tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was markedly increased in response to PIG-P in rat cardiomyocytes without any effect on the tyrosine phosphorylation of the insulin receptor beta-subunit. PIG-P action in these cells was accompanied by phosphorylation/dephosphorylation of several proteins with molecular masses of 15-30 kDa, a response not detected with insulin. Downstream signalling of IRS-1 was then analysed by monitoring IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity in cardiomyocytes. A stable (2 and 15 min incubation with PIG-P) 7-fold stimulation corresponding to about 50% of insulin action could be detected. Increased tyrosine phosphorylation of IRS-1 and enhanced PI 3-kinase activity in response to PIG-P independent of the insulin receptor was also observed in isolated adipocytes. Involvement of PI 3-kinase in PIG-P action was subsequently confirmed by the dose-dependent inhibition of PIG-P-activated glucose transport in rat diaphragm and adipocytes by the PI 3-kinase inhibitors wortmannin and LY294002. These data suggest divergent upstream signalling by insulin and PIG-P involving phosphoproteins not affected by insulin. However, PIG-P and insulin action converge at the level of IRS-1 inducing insulin-independent PI 3-kinase-mediated signalling to glucose transport.
Phosphoinositolglycan molecules isolated from insulin-sensitive mammalian tissues have been demonstrated in numerous in vitro studies to exert partial insulin-mimetic activity on glucose and lipid metabolism in insulin-sensitive cells. However, their ill-defined structures, heterogeneous nature, and limited availability have prohibited the analysis of the underlying molecular mechanism. Phosphoinositolglycan-peptide (PIG-P) of defined and homogeneous structure prepared in large scale from the core glycan of a glycosyl-phosphatidylinositol-anchored membrane protein from Saccharomyces cerevisiae has recently been shown to stimulate glucose transport as well as a number of glucose-metabolizing enzymes and pathways to up to 90% (at 2 to 10 microns) of the maximal insulin effect in isolated rat adipocytes, cardiomyocytes, and diaphragms (G. Müller et al., 1997, Endocrinology 138: 3459-3476). Consequently, we used this PIG-P for the present study in which we compare its intracellular signaling with that of insulin. The activation of glucose transport by both PIG-P and insulin in isolated rat adipocytes and diaphragms was found to require stimulation of phosphatidylinositol (PI) 3-kinase but to be independent of functional p70S6kinase and mitogen-activated protein kinase. The increase in glycerol-3-phosphate acyltransferase activity in rat adipocytes in response to PIG-P and insulin was dependent on both PI 3-kinase and p70S6kinase. This suggest that the signaling pathways for PIG-P and insulin to glucose transport and metabolism converage at the level of PI 3-kinase. A component of the PIG-P signaling pathway located up-stream of PI 3-kinase was identified by desensitization of isolated rat adipocytes for PIG-P action by combined treatment with trypsin and NaCl under conditions that preserved cell viability and the insulin-mimetic activity of sodium vanadate but completely blunted the insulin response. Incubation of the cells with either trypsin or NaCl alone was ineffective. The desensitized adipocytes were reconstituted for stimulation of lipogenesis by PIG-P by addition of the concentrated trypsin/salt extract. The reconstituted adipocytes exhibited 65-75% of the maximal PIG-P response and similar EC50 values for PIG-P (2 to 5 microns) compared with control cells. A proteinaceous N-ethylmaleimide (NEM)-sensitive component contained in the trypsin/salt extract was demonstrated to bind in a functional manner to the adipocyte plasma membrane of desensitized adipocytes via bipolar interactions. An excess of trypsin/salt extract inhibited PIG-P action in untreated adipocytes in a competitive fashion compatible with a receptor function for PIG-P of this protein. The presence of the putative PIG-P receptor protein in detergent-insoluble complexes prepared from isolated rat adipocytes suggests that caveolae/detergent-insoluble complexes of the plasma membrane may play a role in insulin-mimetic signaling by PIG-P. Furthermore, treatment of isolated rat diaphragms and adipocytes with PIG-P as well as with other agents exerting partially insulin-mimetic activity, such as PI-specific phospholipase C (PLC) and the sulfonylurea glimepiride, triggered tyrosine phosphorylation of the caveolar marker protein caveolin, which was apparently correlated with stimulation of lipogenesis. Strikingly, in adipocytes subjected to combined trypsin/salt treatment, PIG-P, PI-specific PLC, and glimepiride failed completely to provoke insulin-mimetic effects. A working model is presented for a signaling pathway in insulin-sensitive cells used by PIG(-P) molecules which involves GPI structures, the trypsin/salt- and NEM-sensitive receptor protein for PIG-P, and additional proteins located in caveolae/detergent-insoluble complexes.
Women with the polycystic ovary syndrome have insulin resistance and hyperinsulinemia, possibly because of a deficiency of a D-chiro-inositol-containing phosphoglycan that mediates the action of insulin. We hypothesized that the administration of D-chiro-inositol would replenish stores of the mediator and improve insulin sensitivity.
We measured steroids in serum and performed oral glucose-tolerance tests before and after the oral administration of 1200 mg of D-chiro-inositol or placebo once daily for six to eight weeks in 44 obese women with the polycystic ovary syndrome. The serum progesterone concentration was measured weekly to monitor for ovulation.
In the 22 women given D-chiro-inositol, the mean (+/-SD) area under the plasma insulin curve after the oral administration of glucose decreased from 13,417+/-11,572 to 5158+/-6714 microU per milliliter per minute (81+/-69 to 31+/-40 nmol per liter per minute) (P=0.007; P=0.07 for the comparison of this change with the change in the placebo group); glucose tolerance did not change significantly. The serum free testosterone concentration in these 22 women decreased from 1.1+/-0.8 to 0.5+/-0.5 ng per deciliter (38+/-7 to 17+/-3 pmol per liter) (P=0.006 for the comparison with the change in the placebo group). The women's diastolic and systolic blood pressure decreased by 4 mm Hg (P<0.001 and P=0.05, respectively, for the comparisons with the changes in the placebo group), and their plasma triglyceride concentrations decreased from 184+/-88 to 110+/-61 mg per deciliter (2.1+/-0.2 to 1.2+/-0.1 mmol per liter) (P=0.002 for the comparison with the change in the placebo group). None of these variables changed appreciably in the placebo group. Nineteen of the 22 women who received D-chiro-inositol ovulated, as compared with 6 of the 22 women in the placebo group (P<0.001).
D-Chiro-inositol increases the action of insulin in patients with the polycystic ovary syndrome, thereby improving ovulatory function and decreasing serum androgen concentrations, blood pressure, and plasma triglyceride concentrations.
Insulin induces a broad spectrum of effects over a wide time interval. It also stimulates the phosphorylation of some cellular proteins, while decreasing the state of phosphorylation of others. These observations indicate the presence of different, but not necessarily mutually exclusive, pathways of insulin action. One well-known pathway represents a phosphorylation cascade initiated by the tyrosine kinase activity of the insulin receptor followed by involvement of different MAP-kinases. Another pathway suggests the existence of low molecular weight insulin mediators whose synthesis and/or release is initiated by insulin. Comparable analysis of two kinds of insulin mediators, namely inositolphosphoglycans and prostaglandylinositol cyclic phosphate (cPIP), has been carried out. It has been shown that the expression of a number of enzymes, such as phospholipase A(2), phospholipase C, cyclo-oxygenase and IRS-1-like enzyme, could regulate the biosynthesis of cPIP in both normal and diabetes-related conditions. Data on the activity of a key enzyme of cPIP biosynthesis termed cPIP synthase (IRS-1-like enzyme) in various monkey tissues before and twice during an euglycemic hyperinsulinemic clamp have been presented. It has been concluded that in vivo insulin increases cPIP synthase activity in both liver and subcutaneous adipose tissue of lean normal monkeys. It has been also suggested that abnormal production of cPIP could be related to several pathologies including glucocorticoid-induced insulin resistance and diabetic embryopathy. Further studies on cPIP and other types of insulin mediators are necessary to aid our understanding of insulin action.
In this review we discuss the biological significance of D-chiro-inositol, originally discovered as a component of a putative mediator of intracellular insulin action, where as a putative mediator, it accelerates the dephosphorylation of glycogen synthase and pyruvate dehydrogenase, rate limiting enzymes of non-oxidative and oxidative glucose disposal.
Early studies demonstrated a linear relationship between its decreased urinary excretion and the degree of insulin resistance present. When tissue contents, including muscle, of type 2 diabetic subjects were assayed, they demonstrated a more general body deficiency. Administration of D-chiro-inositol to diabetic rats, Rhesus monkeys and now to humans accelerated glucose disposal and sensitized insulin action.
A defect in vivo in the epimerization of myoinositol to chiro-inositol in insulin sensitive tissues of the GK type 2 diabetic rat has been elucidated. Thus, administered D-chiro-inositol may act to bypass a defective normal epimerization of myo-inositol to D-chiro-inositol associated with insulin resistance and act to at least partially restore insulin sensitivity and glucose disposal.
It has recently been shown that the fat-derived hormone adiponectin has the ability to decrease hyperglycemia and to reverse insulin resistance. However, bacterially produced full-length adiponectin is functionally inactive. Here, we show that endogenous adiponectin secreted by adipocytes is post-translationally modified into eight different isoforms, as shown by two-dimensional gel electrophoresis. Carbohydrate detection revealed that six of the adiponectin isoforms are glycosylated. The glycosylation sites were mapped to several lysines (residues 68, 71, 80, and 104) located in the collagenous domain of adiponectin, each having the surrounding motif of GXKGE(D). These four lysines were found to be hydroxylated and subsequently glycosylated. The glycosides attached to each of these four hydroxylated lysines are possibly glucosylgalactosyl groups. Functional analysis revealed that full-length adiponectin produced by mammalian cells is much more potent than bacterially generated adiponectin in enhancing the ability of subphysiological concentrations of insulin to inhibit gluconeogenesis in primary rat hepatocytes, whereas this insulin-sensitizing ability was significantly attenuated when the four glycosylated lysines were substituted with arginines. These results indicate that full-length adiponectin produced by mammalian cells is functionally active as an insulin sensitizer and that hydroxylation and glycosylation of the four lysines in the collagenous domain might contribute to this activity.
The trans-diequatorial 3,4-diol of 2,5-di-O-benzyl-D-chiro-inositol cleaved selectively with the periodate ion in the presence of the trans-diaxial 1,6-diol to give a dialdehyde (dialdose) from which 3,6-di-O-benzyl-D-manno-tetrahydroxyazepane (1) was made. The trans-diaxial 1,6-diol of 3,4-di-O-allyl-2,5-di-O-benzyl-D-chiro-inositol was not cleaved satisfactorily by periodate, but replacement of the allyl substituents with tert-butyldimethylsilyl groups caused conformational inversion of the inositol ring, and the resulting trans-diequatorial 1,6-diol cleaved efficiently to give a dialdehyde from which 3,6-di-O-benzyl-L-ido-tetrahydroxyazepane (2) can be prepared.
To determine whether the administration of D-chiro-inositol, a putative insulin-sensitizing drug, would affect the concentration of circulating insulin, the levels of serum androgens, and the frequency of ovulation in lean women with the polycystic ovary syndrome.
In 20 lean women (body mass index, 20.0 to 24.4 kg/m 2) who had the polycystic ovary syndrome, treatment was initiated with either 600 mg of D-chiro-inositol or placebo orally once daily for 6 to 8 weeks. We performed oral glucose tolerance tests and measured serum sex steroids before and after therapy. To monitor for ovulation, we determined serum progesterone concentrations weekly.
In the 10 women given D-chiro-inositol, the mean (+/- standard error) area under the plasma insulin curve after oral administration of glucose decreased significantly from 8,343 +/- 1,149 mU/mL per min to 5,335 +/- 1,792 mU/mL per min in comparison with no significant change in the placebo group (P = 0.03 for difference between groups). Concomitantly, the serum free testosterone concentration decreased by 73% from 0.83 +/- 0.11 ng/dL to 0.22 +/- 0.03 ng/dL, a significant change in comparison with essentially no change in the placebo group (P = 0.01). Six of the 10 women (60%) in the D-chiro-inositol group ovulated in comparison with 2 of the 10 women (20%) in the placebo group (P = 0.17). Systolic (P = 0.002) and diastolic (P = 0.001) blood pressures, as well as plasma triglyceride concentrations (P = 0.001), decreased significantly in the D-chiro-inositol group in comparison with the placebo group, in which these variables either increased (blood pressure) or decreased minimally (triglycerides).
We conclude that, in lean women with the polycystic ovary syndrome, D-chiro-inositol reduces circulating insulin, decreases serum androgens, and ameliorates some of the metabolic abnormalities (increased blood pressure and hypertriglyceridemia) of syndrome X.
Endothelial dysfunction (ED) is an early feature of cardiovascular risk and diabetes. Hyperglycemia and hyperlipidemia are causative factors. Excessive endothelial mitochondrial superoxide (ROS) production with hyperglycemia and hyperlipidemia is a key mechanism. Inositol components of an insulin inositol glycan mediator, d-chiro-inositol (DCI) and 3-O-methyl DCI (pinitol), decrease hyperglycemia and hyperlipidemia. We tested whether these, myoinositol and dibutyryl DCI (db-DCI), would prevent or reverse ED in diabetic rats and rabbits. Oral inositols reduced hyperglycemia and hypertriglyceridemia with different potencies and prevented ED in rat aortic rings and mesenteric beds. Inositols added in vitro to five diabetic tissues reversed ED. Relaxation by Ach, NO, and electrical field stimulation was potentiated by inositols in vitro in rabbit penile corpus cavernosa. Inositols in vitro restored impaired contraction by the eNOS inhibitor l-NAME and increased NO effectiveness. DCI and db-DCI decreased elevated ROS in endothelial cells in high glucose and db-DCI reduced PKC activation, hexosamine pathway activity, and advanced glycation end products to basal levels. Xanthine/xanthine oxidase generated superoxide was reduced by superoxide dismutase or inositols, with db-DCI efficacious in a mechanism requiring chelated Fe³⁺. Histochemical examination of rat aortic rings for protein SNO demonstrated a decrease in diabetic rings with restoration by inositols. In summary, inositols prevented and reversed ED in rat and rabbit vessels, reduced elevated ROS in endothelial cells, potentiated nitrergic or vasculo-myogenic relaxations, and preserved NO signaling. These effects are related to their metabolic actions, direct superoxide scavenging, and enhancing and protecting NO signaling. Of the inositols tested, db-DCI was most effective.
• d-chiro-inositol
• diabetes
• NO
• insulin
• mimetic
Two insulin mediators, inositol phosphoglycans, were isolated from bovine liver by methods previously developed for rat liver, i.e. chromatography on an AG 1 x 8 ion exchange column and selective elution with HCl at pH 2.0 and 1.3. The pH 2.0 mediator containing D-chiroinositol stimulated pyruvate dehydrogenase phosphatase, whereas the pH 1.3 mediator containing myo-inositol inhibited cAMP-dependent protein kinase. Each mediator was further purified by thin layer and Bio-Gel P4 column chromatography and injected ip into normal fed rats together with [U-14C]glucose. After 2.5 h, diaphragms were removed, and glycogen isolated. Insulin mediators, like insulin, stimulated [U-14C]glucose incorporation into glycogen by 150-160% in a dose-dependent manner in the nanomolar range. Mediators injected iv in the nanomolar range into low dose streptozotocin-diabetic rats decreased plasma glucose 30-45% in 30-60 min, with a return to basal concentrations after 150-180 min. These in vivo insulin-like effects of mediator were observed without changes in serum insulin concentrations. The pH 2.0 mediator was 50-100 times more active (per nmol organic phosphate) than the pH 1.3 mediator in the ip diaphragm glycogenesis assay. Mediator effects on diaphragm were completely blocked by preincubation with an immunopurified inositol phosphoglycan antibody. Both mediators were equally active iv in lowering plasma glucose (per nmol inositol) at concentrations comparable to those of insulin.
Cell signaling via inositol phosphates, in particular via the second messenger myo-inositol 1,4,5-trisphosphate, and phosphoinositides comprises a huge field of biology. Of the nine 1,2,3,4,5,6-cyclohexanehexol isomers, myo-inositol is pre-eminent, with "other" inositols (cis-, epi-, allo-, muco-, neo-, L-chiro-, D-chiro-, and scyllo-) and derivatives rarer or thought not to exist in nature. However, neo- and D-chiro-inositol hexakisphosphates were recently revealed in both terrestrial and aquatic ecosystems, thus highlighting the paucity of knowledge of the origins and potential biological functions of such stereoisomers, a prevalent group of environmental organic phosphates, and their parent inositols. Some "other" inositols are medically relevant, for example, scyllo-inositol (neurodegenerative diseases) and D-chiro-inositol (diabetes). It is timely to consider exploration of the roles and applications of the "other" isomers and their derivatives, likely by exploiting techniques now well developed for the myo series.
The neuropeptide, α-calcitonin gene-related peptide (α-CGRP), is expressed from sensory nerves that innervate fat. However, how α-CGRP may act in adipose tissue is unclear. Using 3T3-L1 adipocytes we observed that rat α-CGRP (rαCGRP) evoked either a biphasic or monophasic reduction in intracellular free fatty acid (FFA) content. cAMP production was always monophasic and occurred when FFA responses were absent. Taken together with the observed potencies, these findings suggest that adipose tissue is a physiological target for α-CGRP. However, uncoupling of the FFA and CGRP-signalling responses with increasing passage number limits 3T3-L1 adipocytes as a suitable cellular model.
The polyhydroxylated azepanes (3R,4R,5R,6R)-tetrahydroxyazepane (−)-1 and (3S,4S,5S,6S)-tetrahydroxyazepane (+)-1 are synthesised from D-and L-chiro-inositol, respectively. Key transformations in the reaction sequence include a glycol-fission reaction with sodium metaperiodate supported on silica gel and the double reductive amination of a manno-1,6-dialdehyde derivative. This work represents the first synthesis of tetrahydroxyazepanes from inositols.
The CO bond in xanthates derived from carbohydrates can be reductively cleaved by heating in 2-propanol in the presence of equimolar amounts of dilauroyl peroxide, added in small portions; if benzene is used as the solvent, an O- to S- rearrangement of the xanthate occurs.
The highly stereoselective synthesis of (−)-epi-, (−)-allo- and neo-quercitols as well as stereospecific synthesis of (−)-talo- and (+)-gala-quercitols have been achieved. The general strategy is employing dihydroxylation of the isolated double bond of various kinds of protected chiral (1,4,5)-cyclohex-2-ene-triols, which are derived from d-(−)-quinic acid. The choosing of protecting groups from either BBA (butane 2,3-bisacetal) or acetyl groups will result in the various degrees of stereoselectivity of dihydroxylation. On the other hand, the cyclohexylidene acetal moiety is attributed to the stereospecificity during dihydroxylation to afford the request molecules.
Several cyclohexanepentols have been synthesized from inositols by blocking all but one hydroxyl group, converting the free hydroxyl group into its S-methyl dithiocarbonate, and treating it with tributylstannane. Suitable blocking-groups are methyl, benzyl, and methylthiomethyl ethers, and acetals. One cyclohexanepentol was prepared by the reductive deamination of an aminodeoxyinositol.
A crystalline substituted hexose dialdehyde with the D-manno configuration, 2,3-4,5-diisopropylidene-D-mannohexodial-dose (II) has been obtained by the lead tetraacetate oxidation of 1,2-5,6-diisopropylidene-D-inositol. Deacetonation of II gave amorphous D-mannohexodialdose, characterized as the tetraethyl-1,2-3,4-mercaptal, while reduction of II gave 2,3-4,5-diisopropylidene-D-mannitol. Derivatives of the substituted dialdehyde prepared include the bisphenylhydrazone and the bis-pnitrophenylhydrazone.
Efficient synthetic routes to several fluorinated isosteres of inositol have been developed that are based upon the unexpected selectivity observed in the (diethylamido)sulfur trifluoride reaction of polyhydroxylated cyclohexane derivatives. The conversion of D-pinitol to 1 D-1,5-dideoxy-1,5-difluoro-neo-inositol and to 1 D-1-deoxy-1-fluoro-myo-inositol is reported along with a mechanistic rationale for their formation. Furthermore, the cell growth inhibitory properties of three fluorinated inositol analogues on NIH 3T3 (normal fibroblasts) and v-sis-transformed NIH 3T3 cells are described. These inositol isosteres hold promise as tools for furthering our understanding of the phosphatidylinositol cascade and may also offer a new strategy in the treatment of neoplastic diseases.
L'hydrogénation catalytique du d-inosose en milieu fortement acide fournit du d-viburnitol. Sous l'action d'Acetobacter suboxydans, les d- et l-viburnitols se transforment en deux tétrahydroxy-cyclohexanones énantiomorphes.
In this note, we present a detailed procedure for highly effective and reproducible 3T3-L1 cell differentiation. Due to their potential to differentiate from fibroblasts to adipocytes, 3T3-L1 cells are widely used for studying adipogenesis and the biochemistry of adipocytes. However, using different kits and protocols published so far, we were not able to obtain full differentiation of the currently available American Type Culture Collection (ATCC) 3T3-L1 cell lots. Using rosiglitazone (2 μM) as an additional prodifferentiative agent, we achieved apparently complete differentiation of 3T3-L1 cells within 10 to 12 days that persisted for at least up to cell culture passage 10.
The glycan beta-galactosamine-(1-4)-3-O-methyl-D-chiro-inositol, called INS-2, was previously isolated from liver as a putative second messenger-modulator for insulin. Synthetic INS-2 injected intravenously in rats is both insulin-mimetic and insulin-sensitizing. This bioactivity is attributed to allosteric activation of pyruvate dehydrogenase phosphatase (PDHP) and protein phosphatase 2Calpha (PP2Calpha). Towards identification of potentially metabolically stable analogues of INS-2 and illumination of the mechanism of enzymatic activation, C-INS-2, the exact C-glycoside of INS-2, and C-INS-2-OH the deaminated analog of C-INS-2, were synthesized and their activity against these two enzymes evaluated. C-INS-2 activates PDHP comparable to INS-2, but failed to activate PP2Calpha. C-INS-2-OH was inactive against both phosphatases. These results and modeling of INS-2, C-INS-2 and C-INS-2-OH into the 3D structure of PDHP and PP2Calpha, suggest that INS-2 binds to distinctive sites on the two different phosphatases to activate insulin signaling. Thus the carbon analog could selectively favor glucose disposal via oxidative pathways.
Some of the actions of insulin may be mediated by the intracellular generation of an inositol phosphate glycan that modulates the activities of certain metabolic enzymes. The actions of this molecule were evaluated on glucose utilization in intact rat adipocytes. The inositol glycan led to the dose-dependent stimulation of glucose oxidation and lipogenesis. The extent of stimulation was similar to that observed for insulin. The stimulation of lipogenesis was seen only at high concentrations of glucose, suggesting regulation of processes distal to glucose uptake. The effects of the inositol glycan on intact adipocytes were specifically attenuated with inositol monophosphate in a dose dependent manner. These results further support a role for this substance as a second messenger for some of the actions of insulin, and indicate that the cellular uptake of the inositol glycan may occur by a specific transport system.
Two insulin mediators, inositol phosphoglycans, were isolated from bovine liver by methods previously developed for rat liver, i.e. chromatography on an AG 1 x 8 ion exchange column and selective elution with HCl at pH 2.0 and 1.3. The pH 2.0 mediator containing D-chiroinositol stimulated pyruvate dehydrogenase phosphatase, whereas the pH 1.3 mediator containing myo-inositol inhibited cAMP-dependent protein kinase. Each mediator was further purified by thin layer and Bio-Gel P4 column chromatography and injected ip into normal fed rats together with [U-14C]glucose. After 2.5 h, diaphragms were removed, and glycogen isolated. Insulin mediators, like insulin, stimulated [U-14C]glucose incorporation into glycogen by 150-160% in a dose-dependent manner in the nanomolar range. Mediators injected iv in the nanomolar range into low dose streptozotocin-diabetic rats decreased plasma glucose 30-45% in 30-60 min, with a return to basal concentrations after 150-180 min. These in vivo insulin-like effects of mediator were observed without changes in serum insulin concentrations. The pH 2.0 mediator was 50-100 times more active (per nmol organic phosphate) than the pH 1.3 mediator in the ip diaphragm glycogenesis assay. Mediator effects on diaphragm were completely blocked by preincubation with an immunopurified inositol phosphoglycan antibody. Both mediators were equally active iv in lowering plasma glucose (per nmol inositol) at concentrations comparable to those of insulin.
We compared the effects of infusing a chemically defined chiro-inositol glycan putative insulin mediator with an equivalent dose of insulin in low-dose (45 mg/kg) streptozotocin diabetic rats. Insulin decreased plasma glucose levels from 17.32 +/- 0.17 to 3.96 +/- 0.064 mmol/l (p < 0.0002) in 120 min, a decrease of 77.13%, while the putative mediator promoted a decrease in plasma glucose from 14.85 +/- 0.084 to 7.22 +/- 0.13 mmol/l (p < 0.007) in 60 min. The putative mediator maintained euglycaemia over the ensuing 60 min with a plasma glucose level of 7.01 +/- 0.10 mmol/l at 120 min. Thus, insulin further reduced the plasma glucose from euglycaemia at 60 min to produce hypoglycaemia at 120 min. The lack of production of hypoglycaemia by the putative mediator can be explained by its inhibition of glucose-stimulated insulin secretion by the islet beta cells, thus providing a potential negative feedback regulatory mechanism; or by its selective action on muscle to increase glycogen synthesis. The significance of these results in terms of future directions in drug design is herein considered.
Extracts of human liver were found to contain activities which copurified and coeluted with the two major subtypes of mediators (type A and type P) isolated from insulin-stimulated rat liver. The putative type A mediator from human liver inhibited cAMP-dependent protein kinase from bovine heart, decreased phosphoenolypyruvate carboxykinase mRNA levels in rat hepatoma cells, and stimulated lipogenesis in rat adipocytes. The putative type P mediator stimulated bovine heart pyruvate dehydrogenase phosphatase. Both fractions were able to stimulate proliferation of EGFR T17 fibroblasts and the type A was able to support growth in organotypic cultures of chicken embryo cochleovestibular ganglia. Both activities were resistant to Pronase treatment and the presence of carbohydrates, phosphate, and free-amino groups were confirmed in the two fractions. These properties are consistent with the structure/ function characteristics of the type A and P inositolphosphoglycans (IPG) previously characterized from rat liver. Further, the ability of the human-derived mediators to interact with rat adipocytes and bovine-derived metabolic enzymes suggests similarity in structure between the mediators purified from different species. Galactose oxidase-susceptible membrane-associated glycosylphosphatidylinositols (GPI) have been proposed to be the precursors of IPG. GPI was purified from human liver membranes followed by treatment with galactose oxidase and reduction with NaB3H4. Serial t.l.c. revealed three radiolabeled bands which comigrated with the putative GPI precursors found in rat liver. These galactose-oxidase-reactive lipidic compounds, however, were only partially susceptible to hydrolysis with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis and were resistant to glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei. These data indicate that IPG molecules with insulin-like biological activities are present in human liver.
The 1D and 1L enantiomers of 1,2-anhydro-myo-inositol (conduritol B epoxide) were synthesised from 1D-pinitol and 1L-quebrachitol, respectively, and their activities were compared in selected glycosidase inhibition assays. The 1D enantiomer was found to be the active isomer, functioning as an irreversible inhibitor of sweet almond beta-D-glucosidase. Neither isomer was active against the alpha-D-glucosidase from Bacillus stearothermophilus or the beta-D-galactosidase from Aspergillus oryzae.
In recent years, a number of cross-talk systems have been identified which feed into the insulin signalling cascade at the level of insulin receptor substrate (IRS) tyrosine phosphorylation, e.g., receptor and non-receptor tyrosine kinases and G-protein-coupled receptors. At the molecular level, a number of negative modulator and feedback systems somehow interacting with the beta-subunit (catecholamine-, phorbolester-, or tumor necrosis factor-alpha-induced serine/threonine phosphorylation, carboxy-terminal trimming by a thiol-dependent protease, association of inhibitory/regulatory proteins such as RAD, PC1, PED, alpha2-HS-glycoprotein) have been identified as candidate mechanisms for the impairment of insulin receptor function by elevations in the activity and/or amount of the corresponding modification enzymes/inhibitors. Both decreased responsiveness and sensitivity of the insulin receptor beta-subunit for insulin-induced tyrosine autophosphorylation have been demonstrated in several cellular and animal models of metabolic insulin resistance as well as in the adipose tissue and skeletal muscle of diabetic patients and obese Pima Indians compared to non-obese subjects. Therefore, induction of the insulin signalling cascade by bypassing the defective insulin receptor kinase may be useful for the therapy of non-insulin dependent diabetes mellitus. During the past two decades, phosphoinositolglycans (PIGs) of various origin have been demonstrated to exert potent insulin-mimetic metabolic effects upon incubation with cultured or isolated muscle and adipose cells. However, it remained to be elucidated whether these compounds actually manage to trigger insulin signalling and if so at which level of hierarchy within the signalling cascade the site of interference is located. Recent studies using isolated rat adipocytes and chemically synthesized PIG compounds point to IRS1/3 tyrosine phosphorylation by p59Lyn kinase as the site of cross-talk, the negative regulation of which by interaction with caveolin is apparently abrogated by PIG. This putative mechanism is thus compatible with the recently formulated caveolin signalling hypothesis, the supporting data for which are reviewed here. Though we have not obtained experimental evidence for the involvement of PIG in physiological insulin action, the potential cross-talk between insulin and PIG signalling, including the caveolae/detergent-insoluble glycolipid-enriched rafts as the compartments where the corresponding signalling components are concentrated, thus represent novel targets for signal transduction therapy.
The general problem of structural assignment of complex carbohydrate structures by NMR spectroscopy was reviewed. It was claimed that carbohydrates contains the hidden code to biological recognition. It was shown that the actual sensitivity obtained in a nanoprobe operating at 500 MHz was similar to the sensitivity obtained in a 3-mm probe on an 800MHz instrument, off course without the same spectral dispersion. Therefore, for many oligosaccharide samples it is more advantageous to use the new probe technology and the many new pulse sequences than going to higher field strength with the associated substantially much higher expense both in investment and running cost.
We isolated from beef liver a putative insulin mediator termed INS-2, 1. Its structure was determined to be a novel inositol glycan pseudo-disaccharide Mn(2+) chelate containing D-chiro-inositol 2a (as pinitol) and galactosamine. Purification methods were scaled up from those previously reported to isolate an inositol glycan with similar composition from rat liver.(1) Structure of the beef liver glycan was determined by degradative chemistry and 2D NMR spectroscopy and confirmed by chemical synthesis. Its structure is 4-O-(2-amino-2-deoxy-beta-D-galactopyranosyl)-3-O-methyl-D-chiro-inositol 1 (INS-2, Figure 1). Its role as an insulin mimetic was demonstrated by its action in vivo to decrease elevated blood glucose injected to low-dose streptozotocin diabetic rats in a stereospecific and dose-dependent manner. The pseudo-disaccharide also stimulated [(14)C]glucose incorporation into [(14)C]glycogen in a dose-dependent manner in H4IIE hepatoma cells in the presence of insulin, thus enhancing insulin action. Only when chelated to Mn(2+) did it activate pyruvate dehydrogenase phosphatase in vitro in a dose-dependent manner. To our knowledge, this is the first example of a beta-1,4-linked inositol glycan consisting of D-chiro-inositol and galactosamine isolated from animal tissues with insulin mimetic actions.
The antihyperglycemic effects of chemically synthesized d-chiro-inositol (d-CI), a component of an insulin mediator, have been demonstrated in rats. Buckwheat contains relatively high levels of d-CI: thus, it has been proposed as a source of d-CI for reducing serum glucose concentrations in diabetics. The present study evaluates the effects of a buckwheat concentrate, containing d-CI, on hyperglycemia and glucose tolerance in streptozotocin (STZ) rats. In fed STZ rats, both doses of the buckwheat concentrate (containing 10 and 20 mg of d-CI/kg of body weight) were effective for lowering serum glucose concentrations by 12-19% at 90 and 120 min after administration. Findings from this study demonstrate that a buckwheat concentrate is an effective source of d-CI for lowering serum glucose concentrations in rats and therefore may be useful in the treatment of diabetes.
Two pairs of C(2)-symmetric tetrahydroxyazepanes [(-), (+)-1 and (-), (+)-2] have been synthesized from the enantiomeric chiro-inositols and evaluated as glycosidase inhibitors. Alternative syntheses of ido-tetrahydroxyazepanes (-)- and (+)-2 from myo-inositol were also developed. The key synthetic transformations were glycol fission and cyclization of the derived dialdehydes by double reductive amination. The D-manno-tetrahydroxyazepane [(-)-1] showed selective inhibition of alpha-L-fucosidase and beta-D-galactosidase, while the enantiomer [(+)-1] was a selective inhibitor of an alpha-D-galactosidase. In contrast, the L-ido-tetrahydroxyazepane (+)-2 was a broad spectrum hexosidase inhibitor, but showed none of the reported hexosaminidase inhibition. Its enantiomer (-)-2 is a poor hexosidase inhibitor.
Insulin-stimulated glucose disposal in skeletal muscle proceeds predominantly through a nonoxidative pathway with glycogen synthase as a rate-limiting enzyme, yet the mechanisms for insulin activation of glycogen synthase are not understood despite years of investigation. Isolation of putative insulin second messengers from beef liver yielded a pseudo-disaccharide consisting of pinitol (3-O-methyl-d-chiro-inositol) beta-1,4 linked to galactosamine chelated with Mn(2+) (called INS2). Here we show that chemically synthesized INS2 has biological activity that significantly enhances insulin reduction of hyperglycemia in streptozotocin diabetic rats. We used computer modeling to dock INS2 onto the known three-dimensional crystal structure of protein phosphatase 2C (PP2C). Modeling and FlexX/CScore energy minimization predicted a unique favorable site on PP2C for INS2 in a surface cleft adjacent to the catalytic center. Binding of INS2 is predicted to involve formation of multiple H-bonds, including one with residue Asp163. Wild-type PP2C activity assayed with a phosphopeptide substrate was potently stimulated in a dose-dependent manner by INS2. In contrast, the D163A mutant of PP2C was not activated by INS2. The D163A mutant and wild-type PP2C in the absence of INS2 had the same Mn(2+)-dependent phosphatase activity with p-nitrophenyl phosphate as a substrate, showing that this mutation did not disrupt the catalytic site. We propose that INS2 allosterically activates PP2C, fulfilling the role of a putative mediator mimetic of insulin signaling to promote protein dephosphorylation and metabolic responses.
Evidence suggests that some actions of insulin are effected by inositolphosphoglycan (IPG) mediators. We hypothesize that a deficiency in D-chiro-inositol (DCI) and/or a DCI-containing IPG (DCI-IPG) may contribute to insulin resistance in humans.
To assess this possibility in polycystic ovary syndrome (PCOS), we determined insulin sensitivity (Si by frequently sampled intravenous glucose tolerance test), plasma and urinary DCI and myo-inositol (MYO) levels (by gas chromatography/mass spectrometry), and the release of insulin and DCI-IPG during the oral glucose tolerance test (area under the curve [AUC]) in 23 women with PCOS and 26 normal women.
Women with PCOS were heavier than control subjects (P = 0.002 for BMI), but also had decreased Si (P < 0.001) and increased AUC(insulin) (P < 0.001) compared with normal women, even when corrected for BMI. The urinary clearance of DCI (uCl(DCI)) was increased almost sixfold in PCOS compared with normal women (P = 0.001), but not MYO clearance (P = 0.10). uCl(DCI) correlated inversely with Si when all women were analyzed together (n = 49, r = -0.50, P < 0.001) and was one of the three best independent parameters predicting Si. Finally, the ratio of AUC(DCI-IPG) to AUC(insulin) was decreased threefold in women with PCOS (P < 0.001).
uCl(DCI) is inversely correlated with insulin sensitivity in women and is a strong independent predictor of insulin resistance in multivariate models. PCOS, which is characterized by insulin resistance, is associated with a selective increase in uCl(DCI) and impaired DCI-IPG release in response to insulin. These findings are consistent with a defect in tissue availability or utilization of DCI in PCOS that may contribute to the insulin resistance of the syndrome.
Infusion of pH 2.0 d-chiro-inositol glycan insulin putative mediator normalizes plasma glucose in streptozotocin diabetic rats at a dose equivalent to insulin without inducing hypoglycaemia
- Fonteles
Chiroinositol deficiency and insulin resistance. II. Acute effects of d-chiroinositol administration in streptozotocin-diabetic rats, normal rats given a glucose load, and spontaneously insulin-resistant rhesus monkeys
- Ortmeyer