Insulin In Presence Research Articles

ANTIOXIDANTS ENHANCE CHROMIUM (VI) MEDIATED TOXICITY IN NEUROSPORA CRASSA

Chromium can be found in the environment in two main valence states: hexavalent Cr (VI) and trivalent Cr (III). Reduction activation of carcinogenic Cr (VI) is required for the induction of DNA damage and mutations. There is a controversy over antioxidants’ role in Cr (VI) toxicity and cell death in mammalian systems. In the present study we compared the effect of antioxidants and insulin on Cr (VI) induced genotoxicity in wall-less mutant of Neurospora crassa (slime), which contains insulin receptor on its cell surface as a model system. Analysis by comet assay indicated Potassium dichromate (K2 Cr2 O7 ) induced DNA damage in slime cells, as increase in comet tail moment and the effect was dose dependent. Simultaneous addition of N-acetyl cysteine (NAC) & Sodium ascorbate (ASC) increased the extent of DNA damage whereas insulin decreased the extent of DNA damage induced by Cr (VI). The simultaneous exposure of antioxidants and insulin in presence of Cr (VI) showed enhanced cytotoxicity as observed by MTT assay. The results obtained suggest that N-acetyl cysteine and Sodium ascorbate reduce Cr (VI) to Cr (III) enhancing the levels of reactive oxygen species. Absence of protective role of insulin in presence of antioxidants indicate that a differential route may be adopted by the cells in response to metallic stress.

Peptide Hormone Insulin Regulates Function, Expression, and SUMOylation of Organic Anion Transporter 3.

Organic anion transporter 3 (OAT3) plays an important role in the disposition of various anionic drugs which impacts the pharmacokinetics and pharmacodynamics of the therapeutics, thus influencing the pharmacological effects and toxicity of the drugs. In this study, we investigated the effect of insulin on the regulation of OAT3 function, expression, and SUMOylation. We demonstrated that insulin induced an increase in OAT3 transport activity through a dose- and time-dependent manner in COS-7 cells. The insulin-induced elevation in OAT3 function was blocked by PKA inhibitor H89, which correlated well with OAT3 protein expression. Moreover, both PKA activator Bt2-cAMP-induced increase and insulin-induced increase in OAT3 function were blocked by PKB inhibitor AKTi1/2. To further investigate the involvement of SUMOylation, we treated OAT3-expressing cells with insulin in presence or absence of H89 or AKTi1/2 followed by examining OAT3 SUMOylation. We showed that insulin enhanced OAT3 SUMOylation, and such enhancement was abrogated by H89 and AKTi1/2. Lastly, insulin increased OAT3 function and SUMOylation in rat kidney slice. In conclusion, our investigations demonstrated that insulin regulated OAT3 function, expression, and SUMOylation through PKA/PKB signaling pathway. Graphical abstract.

Folate-chitosan nanoparticles triggered insulin cellular uptake and improved in vivo hypoglycemic activity.

The aim of this study was to prove a prolonged control of glucose levels, in rats, by the oral use of insulin folate-chitosan nanoparticles (FA-CS NPs). It was possible to prepare positively charged NPs with an average particle size of 288 ± 5.11 nm and >80% entrapment efficiency. The system was able to enhance the stability of insulin in presence of GIT enzymes, with less than 10% release at pH 1.2 and an 8 hr released amount of 38.92 ± 4.52% in PBS pH 7.4. A 5 fold enhancement in insulin intestinal permeability and cellular uptake over insulin solution was proven. The cellular uptake pathways was found to occur by several mechanisms. Besides, cell compatibility and absence of histopathological alterations was also demonstrated. Finally, a controlled blood glucose level for 8 h in rats. These results anticipated FA-CS NPs as a promising oral insulin candidate.

The saturation degree of fatty acids and their derived acylcarnitines determines the direct effect of metabolically active thyroid hormones on insulin sensitivity in skeletal muscle cells.

Using differentiated rat L6 cells, we studied the direct effect of 3,5,3'-triiodo-l-thyronine (T3) and 3,5-diiodo-l-thyronine (T2) on the response to insulin in presence of fatty acids with a varying degree of saturation. We found that T3 and T2 both invert the response to insulin by modulating Akt Ser473 phosphorylation in the presence of palmitate and oleate. Both hormones prevented palmitate-induced insulin resistance, whereas increased insulin sensitivity in the presence of oleate was reduced, with normalization to (or, in the case of T3, even below) control levels. Both hormones effectively reduced intracellular acylcarnitine concentrations. Interestingly, insulin sensitization was lowered by incubation of the myotubes with relevant concentrations of palmitoylcarnitines (C16) and increased by oleylcarnitines and linoleylcarnitines (C18:1 and C18:2, respectively). The efficiency of mitochondrial respiration decreased in the order palmitate-oleate-linoleate; in the presence of palmitate, only T3 increased ATP synthesis-independent cellular respiration and mitochondrial respiratory complex activities. Both hormones modulated gene expression and enzyme activities related to insulin sensitivity, glucose metabolism, and lipid handling. Although T2 and T3 differentially regulated the expression of relevant genes involved in glucose metabolism, they equally stimulated related metabolic activities. T2 and T3 differentially modulated mitochondrial fatty acid uptake and oxidation in the presence of each fatty acid. The results show that T2 and T3 both invert the fatty acid-induced response to insulin but through different mechanisms, and that the outcome depends on the degree of saturation of the fatty acids and their derived acylcarnitines.-Giacco, A., delli Paoli, G., Senese, R., Cioffi, F., Silvestri, E., Moreno, M., Ruoppolo, M., Caterino, M., Costanzo, M., Lombardi, A., Goglia, F., Lanni, A., de Lange, P. The saturation degree of fatty acids and their derived acylcarnitines determines the direct effect of metabolically active thyroid hormones on insulin sensitivity in skeletal muscle cells.

Insulin–eukaryotic model membrane interaction: Mechanistic insight of insulin fibrillation and membrane disruption

Injection of exogenous insulin in the subcutaneous mass has been a proven therapy for type II diabetes. However, chronic administration of insulin often develops local amyloidosis at the injection site, pathologically known as “Insulin Ball”. This reduces the insulin bioavailability and exacerbates the disease pathology. Thus, the molecular interaction between insulin and the recipient's membrane surface plays a co-operative role in accelerating the amyloidosis. This interaction, however, is different from the molecular interaction of insulin with the native membranous environment of the pancreatic β-cells. The differential membrane mediated interaction that directly affects the aggregation kinetics of insulin remains elusive yet intriguing to understand the mechanism of pathological development. In this study we have characterized the interactions of insulin at different states with model eukaryotic membranes using high and low-resolution spectroscopic techniques in combination with microscopic investigation. Our results show that insulin amyloid intermediates are capable of interacting with model membranes with variable functional affinity towards the different compositions. Fluorescence correlation spectroscopy confirms the aggregation states of insulin in presence of the eukaryotic model membranes while solid-state NMR spectroscopy in conjugation with differential scanning calorimetry elucidates the molecular interaction of insulin intermediates with the lipid head groups along with the acyl chains. Additionally, dye leakage assays support the eukaryotic model membrane disruption by insulin intermediates, similar to hIAPP and Aβ40, as previously reported. Thus, the present study establishes the distinct mode of interactions of insulin amyloid with pancreatic β-cell and general mammalian cell mimicking membranes.

Thermodynamic and kinetic studies of As2O3 toxicological effects on human insulin in generation diabetes mellitus

The interaction of arsenic trioxide with human insulin was investigated by circular dichroism (CD), cyclic voltammetry and electrophoresis techniques. The interfacial behavior of insulin in presence of As2O3 onto the Ag electrode surface was studied at 310 K in phosphate buffer solution (PBS). According to Far-UV CD spectroscopy results, As2O3 caused to decrease in structural compactness and variety of alpha helix into beta structures. Near-UV CD indicated that As2O3 dissociates disulfide linkage in insulin structure. The kinetic parameters, including charge-transfer coefficient and apparent heterogeneous electron transfer rate constant were also determined. The thermodynamic parameters of insulin denaturation in presence of arsenic trioxide were calculated and reported. The obtained results indicated strong adsorption of insulin in presence of arsenic trioxide onto the Ag surface via chemisorptions.

A Comparative Study of Metformin alone, in Combination with Insulin or Metformin Plus Insulin in Presence of Simvastatin in Libyan Diabetic Patients

Type 2 of diabetes mellitus (DM) is associated with progressive failure of pancreatic B-cells to secrete insulin, decreased insulin action, due to reduced insulin receptors at the target sites. Dyslipidemia is a common risk of DM and is responsible to a large extent for cardiovascular disease-related morbidity and mortality. This study was planned to compare the effects of metformin alone and a combination of metformin and insulin in presence of simvastatin on glycemic and lipid levels in Libyan diabeticpatients. This retrospective study was conducted at Benghazi Diabetic Center (Libya) on 100 patients with type 2 DM of 40-60 years old. Patients were selected for follow up on basis of inclusion and exclusion criteria, and were divided into three groups. The first group (n=30), received metformin (1-2 gm/day). The second group (n=40) received metformin (1-2 gm/day) and insulin (mixtard 30/70, 30-60 units/day) and third group (n=30) received metformin (1-2 gm/day), insulin (30-60 units/day) plus simvastatin (40 mg/day). Glucose blood levels (FBG, PPBG and HBA1c), lipid profile (TC, TG, HDL-C, LDL-C and AI), hepatic function (ALT, AST, ALP and Bilirubin) and renal function (creatinine and urea) were measured for each patient. All the patients had a good glycemic control with significant decrease in HbAc1 of metformin plus insulin treated group. No significant differences in lipid profile of metformin treated group and metformin plus insulin treated group were observed. Date revealed both significant increase in HDL-C and decrease in TC, LDL-C and atherogenic index levels but without any change in TG in metformin and insulin treated group as compared with metformin, insulin plus simvastatin treated group. All the findings of hepatic and renal functions were within the normal range except for bilirubin which significantly increased in metformin, insulin plus simvastatin treated group compared with other treated groups.In conclusion, the efficacy of metformin in controlling hyperglycemia was enhanced with insulin without negative effects. Simvastatin was effective in controlling dyslipidemia associated with DM and produced a profound reduction in TC and LDL-C of the patients.

BM-MSCs Differentiated Insulin-Producing Cells Produce More Insulin in Presence of EGF than of FGF

Adult stem cells have the ability to differentiate into islet like cells for the hope of treating diabetes mellitus (DM). The most important part is the differentiation process from na?ve stem cells to fully differentiated fully functional islet cells. For this purpose, we wanted to optimize the current proposed differentiation media by replacing the FGF with EGF and measure insulin production. Bone marrow-derived MSCs were from mice long bones and expanded in cell culture before induction of differentiation. Stem cells surface markers were analyzed by immunocytochemistry. Cultured stem cells were negative for CD34 while they expressed high levels of CD90. Differentiated cells morphology was studied by using H & E stain. Differentiated cells were detected by studying protein expression of insulin as specific marker for IPC differentiation. Cells function was studied by measuring the insulin production in tissue culture supernatant in vitro and also insulin release in response to glucose challenge. Ditizone staining were both positive. Insulin was secreted by these cells in response to different concentrations of glucose stimulation in a regulated manner. Cells induced with formula contain EGF produced more insulin in the same formula but contain FGF instead, this prove that EGF is the best to use during differentiation process.

Effects of (6)-gingerol, ginger component on adipocyte development and differentiation in 3T3-L1

Purpose: The objective of this study was to investigate the effects of (6)-gingerol, ginger components proliferation and adipocyte differentiation from early to lately steps. Methods: 3T3-L1 preadipocytes were cultured. Differentiation of confluent cells was induced with dexamethasone, isobutylxanthin and insulin for 2 day and cells were cultured by medium with insulin in presence of various concentrations 0, 25, 50, 100 (μmol/L) of (6)-gingerol for 4 day. Cell viability was measured using the EZ Cytox assay kit. In addition, we examined the expression of mRNA levels associated with each adipocyte differentiation step by real time reverse transcription polymerase chain reaction. Results: (6)-Gingerol inhibited adipocyte proliferation in a dose and time dependent manner. Expression of C/EBPβ, associated with early differentiation step remained unchaged. However, intermmediate, late differentiation step and adipocytokines were effectively changed in dose-dependently manner in cell groups treated with (6)-gingerol. Conclusion: This study has shown that treatment with (6)-gingerol inhibited adipocyte proliferation as well as each adipocyte differentiation step. In particular, the (6)-gingerol more effectively inhibited adipocyte differentiation from intermmediate differentiation step.

A molecular simulation study of the protection of insulin bioactive structure by trehalose.

Biopharmaceuticals are proteins with a crucial role in the treatment of many diseases. However, these protein medicines are often thermally labile and therefore unsuitable for long-term application and storage, as they tend to lose their activity under ambient conditions. Desiccation is one approach to improving protein stability, but the drying process itself can cause irreversible damage. In the current study, insulin was chosen as an example of a thermally sensitive biopharmaceutical to investigate whether the disaccharide, trehalose, can prevent loss of structural integrity due to drying. The experiment was performed using replica exchange molecular simulation and Gromacs software with a Gromos96 (53a6) force field. The results indicate that trehalose preserves the bioactive structure of insulin during drying, consistent with the use of trehalose as a protectant for thermally sensitive biopharmaceuticals. For instance, at the water content of 1.77%, insulin without any protectants yields the highest RMSD value as 0.451 nm, then the RMSD of insulin in presence of trehalose only ca. 0.100 nm.

Poly(lactic-co-glycolic) acid loaded nano-insulin has greater potentials of combating arsenic induced hyperglycemia in mice: Some novel findings

Diabetes is a menacing problem, particularly to inhabitants of groundwater arsenic contaminated areas needing new medical approaches. This study examines if PLGA loaded nano-insulin (NIn), administered either intraperitoneally (i.p.) or through oral route, has a greater cost-effective anti-hyperglycemic potential than that of insulin in chronically arsenite-fed hyperglycemic mice. The particle size, morphology and zeta potential of nano-insulin were determined using dynamic light scattering method, scanning electronic and atomic force microscopies. The ability of the nano-insulin (NIn) to cross the blood–brain barrier (BBB) was also checked. Circular dichroic spectroscopic (CD) data of insulin and nano-insulin in presence or absence of arsenic were compared. Several diabetic markers in different groups of experimental and control mice were assessed. The mitochondrial functioning through indices like cytochrome c, pyruvate-kinase, glucokinase, ATP/ADP ratio, mitochondrial membrane potential, cell membrane potential and calcium-ion level was also evaluated. Expressions of the relevant marker proteins and mRNAs like insulin, GLUT2, GLUT4, IRS1, IRS2, UCP2, PI3, PPARγ, CYP1A1, Bcl2, caspase3 and p38 for tracking-down the signaling cascade were also analyzed. Results revealed that i.p.-injected nano-encapsulated-insulin showed better results; NIn, due to its smaller size, faster mobility, site-specific release, could cross BBB and showed positive modulation in mitochondrial signaling cascades and other downstream signaling molecules in reducing arsenic-induced-hyperglycemia. CD data indicated that nano-insulin had less distorted secondary structure as compared with that of insulin in presence of arsenic. Thus, overall analyses revealed that PLGA nano-insulin showed better efficacy in combating arsenite-induced-hyperglycemia than that of insulin and therefore, has greater potentials for use in nano-encapsulated form.

Insulin targets the Na+/K+ ATPase in enterocytes via PI3K, PKC, and MAPKS

The effect of insulin on intestinal Na+/K+ ATPase is till now undetermined, and it is still unclear whether insulin exerts any modulatory effect on glucose absorption by targeting the ATPase. This work attempted to address this question and to unravel the signaling pathway involved using Caco-2 cells as a model. After an overnight starvation, cells were treated with insulin in presence and absence of specific inhibitors of some known mediators. The activity of the pump was assayed by measuring the ouabain-inhibitable inorganic phosphate (Pi) released, whereas changes in its abundance were determined by western blot analysis. Insulin decreased the activity and abundance of the ATPase in a crude membrane homogenate. This effect disappeared completely upon inhibition of either phosphotidylinositol-3 kinase (PI3K) or protein kinase C (PKC), but was partially abolished when p38MAPK or MEK/ERK were inhibited separately. Activation of PKC with phorbol-12-myristate-13-acetate (PMA) imitated the effect of insulin and was not affected by inhibition of PI3K. The data suggest that PI3K and PKC are along the same pathway that branches into two separate ones involving each either p38MAP kinase or MEK/ERK. This hypothesis was confirmed by the data obtained from the treatment of Caco-2 cells with PMA, when p38MAPK and MEK/ERK were inhibited simultaneously. Concomitant inhibition of p38MAPK and MEK/ERK abrogated fully the effect of insulin, indicating that no other pathways are present in addition to the ones proposed above.

Sildenafil Reduces Insulin-Resistance in Human Endothelial Cells

BackgroundThe efficacy of Phosphodiesterase 5 (PDE5) inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS) proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro.Methodology/Principal FindingsHuman umbilical vein endothelial cells (HUVECs) were treated with insulin in presence of glucose 30 mM (HG) and glucosamine 10 mM (Gluc-N) with or without sildenafil. Insulin increased the expression of PDE5 and eNOS mRNA assayed by Real time-PCR. Cytofluorimetric analysis showed that sildenafil significantly increased NO production in basal condition. This effect was partially inhibited by the PI3K inhibitor LY 294002 and completely inhibited by the NOS inhibitor L-NAME. Akt-1 and eNOS activation was reduced in conditions mimicking insulin resistance and completely restored by sildenafil treatment. Conversely sildenafil treatment can counteract this noxious effect by increasing NO production through eNOS activation and reducing oxidative stress induced by hyperglycaemia and glucosamine.Conclusions/SignificanceThese data indicate that sildenafil might improve NOS activity of endothelial cells in insulin resistance conditions and suggest the potential therapeutic use of sildenafil for improving vascular function in diabetic patients.

SGK1 and insulin reduce the activity of mammalian electrogenic Na+/Monocarboxylate Transporters (SMCTe/Slc5a8)

SMCTe (SLC5A8) transports several monocarboxylates, including short‐chain fatty acids, ketone‐bodies, pyruvate, lactate, and keto‐isocaproic acid (KIC). We found SMCTe mRNA in zebrafish pancreas (in situ hybridization); other groups have demonstrated that SLC5A8 gene silencing is associated with pancreatic cancer. Extracellular KIC stimulates insulin secretion in pancreas, suggesting a potential role for SMCTe. However, the physiological role of SMCTe in pancreas is unknown. Using RT‐PCR with rat SMCTe‐specific primers, we found that SMCTe mRNA is expressed in rat pancreas. Using Western Blot analysis with specific SMCTe antibodies, we also detected SMCTe protein in rat pancreas (~75 kDa). We next tested the effect of insulin on SMCTe function using 22Na+ uptake in Xenopus oocytes injected with human SMCTe (hSMCTe) or co‐injected with hSGK1 cRNA. Specifically, we evaluated transport 22Na+ uptake elicited by 40 U insulin in presence or absence of KIC. Insulin reduced KIC‐mediated 22Na+ uptake transport by ~25% in hSMCTe‐oocytes. Human SGK1 also reduced KIC‐mediated 22Na+ uptake transport by ~25% in hSMCTe‐oocytes. Co‐expression of hSMCTe and hSGK1 in oocytes did not further reduce KIC‐mediated 22Na+ uptake. These results indicate that the signal transduction pathway by which insulin and SKG1 reduce SMCTe transport activity overlaps.CONACYT48709MJ(CP), EY017732(MFR), DK070756(DM), DK64635(GG)

Apolipoprotein A-V Modulates Insulin Secretion in Pancreatic β-cells Through its Interaction with Midkine

Apolipoprotein A-V is an important determinant of plasma triglyceride level in both humans and mice. This study showed the physiological impact of apoA-V on insulin secretion in rat pancreatic β-cells (INS-1 cells). In order to precise the mechanism of action, binding experiments coupled to mass spectrometry were performed to identify a potential membrane receptor. Results showed an interaction between apoA-V and midkine protein. Confocal microscopy confirmed the plasma membrane co-localisation of this two-proteins after the treatment of INS-1 cells with the apo-AV recombinant protein and indicated that the cell surface midkine could be involved in apoA-V endocytosis, since these two proteins were co-translocated at the plasma membrane or in the cytosol compartment. This co-localisation is correlated with an increase in insulin secretion in a dose dependant manner during short incubation period. Reduction of midkine expression by small interfering RNA duplexes revealed a decrease in the ability of these transfected cells to secrete insulin in presence of apoA-V. Competition experiments for the apoA-V-midkine binding at the cell surface using antibody directed against midkine is able to influence INS-1 cell function as insulin secretion. Our results showed apoA-V ability to enhance insulin secretion in β-cells and provide evidence of an internalization pathway involving the midkine as partner.

Insulin-egg yolk dispersions in self microemulsifying system

Formulation of insulin into a microemulsion very often presents a physicochemical instability during their preparation and storage. In order to overcome this lack of stability and facilitate the handling of these colloidal systems, stabilization of insulin in presence of hydrophobic components of a microemulsion appears as the most promising strategy. The present paper reports the use of egg yolk for stabilization of insulin in self microemulsifying dispersions. Insulin loaded egg yolk self microemulsifying dispersions were prepared by lyophilization followed by dispersion into self microemulsifying vehicle. The physicochemical characterization of selfmicroemulsifying dispersions includes such as insulin encapsulation efficiency, in vitro stability of insulin in presence of proteolytic enzymes and in vitro release. The biological activity of insulin from the dispersion was estimated by enzyme-linked immunosorbant assay and in vivo using Wistar diabetic rats. The particle size ranged 1.023±0.316 μm in diameter and insulin encapsulation efficiency was 98.2±0.9 %. Insulin hydrophobic self microemulsifying dispersions suppressed insulin release in pH 7.4 phosphate buffer and shown to protect insulin from enzymatic degradation in vitro in presence of chymotripsin. Egg yolk encapsulated insulin was bioactive, demonstrated through both in vivo and in vitro.

Insulin-Induced Arteriolar Dilation after Tyrosine Kinase and Nitric Oxide Synthase Inhibition in Hamster Cheek Pouch Microcirculation

We investigated the effects of tyrosine kinase (TK) and nitric oxide synthase (NOS) inhibition on insulin-induced dilation of arterioles. We determined the arteriolar diameter, red blood cell velocity (V_RBC) and blood flow changes in hamster cheek pouch microcirculation as affected by insulin in presence of TK and NOS inhibitors, genistein, piceatannol and N^G-monomethyl-L-arginine (L-NMMA). Microvessels were visualized by a fluorescent microscopy technique. Arteriolar diameter and V_RBC were measured after topical application of insulin and genistein or piceatannol or L-NMMA. Insulin (10 µU/ml) induced diameter and V_RBC increase in A3 and A2 arterioles by 30 ± 5 and 123 ± 4%, 16 ± 4 and 102 ± 3%, as percent of baseline values, respectively. After genistein or piceatannol prior to insulin A3 and A2 arterioles dilated by 10 ± 4, 5 ± 2% and 9 ± 4, 2 ± 1%, respectively. After L-NMMA prior to insulin A2 and A3, arteriole diameters increased by 12 ± 3 and 7 ± 2%, respectively. V_RBC increased significantly in all the cases. TK and NOS inhibitors applied together abolished insulin-induced dilation with a reduction in V_RBC and blood flow. In conclusion, full insulin-induced dilation of hamster cheek pouch arterioles requires TK signaling pathways. Furthermore, activation of insulin receptors, as well as other TK receptors, appears to be required for vasomotor tone regulation.

Differentiation of Two Classes of "A" System Amino Acid Transporters

Injection of mRNA from GF-14 cells (a mouse lymphocyte cell line) into Xenopus oocytes led to the expression of a transport activity characteristic of amino acid transport system A, i.e., Na + dependent and recognizing aminoisobutyric acid and its methyl derivative (AIB and meAIB). Sucrose density gradient fractionation of GF-14 mRNA showed that the maximum level of expression of activity was associated with a 2.2-kb RNA fraction. Pretreatment of GF-14 cells with insulin caused a twofold increase in system A transport activity in the cells themselves and the mRNA from the insulin-treated cells induced a comparable increase in A system transport over control mRNA when expressed in oocytes. mRNA from cells treated with insulin in presence of actinomycin D did not show a response to insulin, suggesting that GF-14 cells synthesized new transporter in response to insulin. Similar experiments with mRNA from the Ehrlich ascites cells, showed that expression of system A type transport was associated chiefly with a 4.2-kb mRNA fraction. Although insulin treatment of Ehrlich cells also caused an increase in A system transport activity in the cells themselves (nearly twofold), the response to insulin was not blocked by actinomycin D or cycloheximide. Moreover, mRNA from insulin-treated and untreated Ehrlich cells gave the same level of expression in oocytes of A type amino acid transport. In contrast to GF-14 cells, Western blots of plasma membranes showed that insulin treatment of Ehrlich cells increased the amount therein of a 120- to 130-kDa peptide, previously associated with amino acid transport ( Proc. Natl. Acad. Sci. USA 85, 7877, 1988), suggesting that in the Ehrlich cells, but not in GF-14, cells this polypeptide was translocated from intracellular sites in response to insulin. The data associate two distinctly different responses to insulin with A-type amino acid transporters and are consistent with the existence of more than a single A type amino acid transporter in mouse cells.

Communications: Effects of Formulation Variables on the Pulmonary Delivery of Insulin

AbstractThe effects of some formulation variables on the pulmonary absorption of insulin in presence of a bile salt, sodium glycocholate (NaGC), have been described in this report. Relationship between intratracheally administered insulin dose and pharmacodynamic response has been established. The data obtained from this study revealed that an increase in the viscosity of an aqueous formulation administered to pulmonary sacs can facilitate insulin absorption probably due to reduced mucocilliary clearance. A hypertonic formulation produced similar overall hypoglycemic effect to the isotonic solution. On the other hand, hypotonicity elicited significantly improved hypoglycemic response, probably due to membrane damage. The cumulative hypoglycemic effect of intratracheally administered insulin has been linearly correlated with the logarithmic doses.

Insulin signalling and regulation of glucokinase gene expression in cultured hepatocytes.

In cultured rat hepatocytes, transcription of the glucokinase gene is turned on by insulin and turned off by glucagon/cAMP, the latter being the dominant effector system. It is thus possible that in the absence of hormones the gene is maintained in a repressed state by the basal level of cAMP and that insulin turns on transcription by relieving cAMP repression, for instance via activation of a cyclic-nucleotide phosphodiesterase. Three inhibitors of this class of enzymes were tested for their effect on the insulin-dependent induction of the glucokinase gene in hepatocytes. Isobutyl methylxanthine, the prototype inhibitor, abrogated the gene response to insulin, as shown by run-on transcription assay. Among the drugs investigated, Ly186126, a preferential inhibitor of type-III phosphodiesterase, proved the most potent in inhibiting insulin-induced accumulation of glucokinase mRNA. Type-III phosphodiesterase is inhibited by cGMP. Induction of glucokinase mRNA was prevented in hepatocytes challenged with insulin in presence of 8-bromoguanosine-3',5'-phosphate. These results are consistent with the involvement of type-III phosphodiesterase in transduction of the insulin signal to the glucokinase gene. However, we were unable to detect significant decreases in total cellular cAMP level or cAMP-dependent-protein-kinase ratio after the addition of insulin to hepatocytes. Many effects of glucagon are mediated via cAMP-dependent protein-kinase phosphorylation of regulatory proteins and, conversely, insulin effects are often accompanied by protein dephosphorylation. A specific inhibitor of protein phosphatases PP1 and PP2A, okadaic acid, was shown to abolish the transcriptional response of the glucokinase gene to insulin. Thus, interference of insulin with the cAMP signal transduction pathway at several steps may be a critical aspect of insulin action on hepatic glucokinase gene expression. In addition, insulin induction of glucokinase mRNA was suppressed by inhibitors of protein synthesis. The underlying mechanism was a severe inhibition of the transcriptional effect of insulin, rather than mRNA destabilization, as demonstrated by run-on transcription assays with nuclei from cycloheximide-treated or pactamycin-treated cells. Transcription of the glucokinase gene may therefore depend on de novo synthesis of the product of an early-response gene induced by insulin, or may require a short-lived trans-acting or accessory factor of transcription. Alternatively, insulin signalling may be compromised in hepatocytes by a mechanism indirectly related to the arrest of protein synthesis.