Chronic Effects Of Insulin Research Articles

Interactions between insulin and diet on striatal dopamine uptake kinetics in rodent brain slices.

Diet influences dopamine transmission in motor- and reward-related basal ganglia circuitry. In part, this reflects diet-dependent regulation of circulating and brain insulin levels. Activation of striatal insulin receptors amplifies axonal dopamine release in brain slices, and regulates food preference invivo. The effect of insulin on dopamine release is indirect, and requires striatal cholinergic interneurons that express insulin receptors. However, insulin also acts directly on dopamine axons to increase dopamine uptake by promoting dopamine transporter (DAT) surface expression, counteracting enhanced dopamine release. Here, we determined the functional consequences of acute insulin exposure and chronic diet-induced changes in insulin on DAT activity after evoked dopamine release in striatal slices from adult ad-libitum fed (AL) rats and mice, and food-restricted (FR) or high-fat/high-sugar obesogenic (OB) diet rats. Uptake kinetics were assessed by fitting evoked dopamine transients to the Michaelis-Menten equationand extracting Cpeak and Vmax . Insulin (30nm) increased both parameters in the caudate putamen and nucleus accumbens core of AL rats in an insulin receptor- and PI3-kinase-dependent manner. A pure effect of insulin on uptake was unmasked using mice lacking striatal acetylcholine, in which increased Vmax caused a decrease in Cpeak . Dietalso influenced Vmax , which was lower in FR vs. AL. The effects of insulin on Cpeak and Vmax were amplified by FR but blunted by OB, consistent with opposite consequences of these diets on insulin levels and insulin receptor sensitivity. Overall, these data reveal acute and chronic effects of insulin and diet on dopamine release and uptake that will influence brain reward pathways.

Metformin treatment does not affect total leptin levels and free leptin index in obese patients with polycystic ovary syndrome

Leptin, particularly in its free form, is deeply involved in the regulation of energy balance. Insulin was suggested to influence plasmatic leptin levels and soluble leptin receptor in humans. However, this study indicates that metformin treatment, although improving insulin levels, does not exert a significant effect on either total leptin level or free leptin index in obese women with hyperinsulinemia and PCOS.

Acute and chronic regulation of leptin synthesis, storage, and secretion by insulin and dexamethasone in human adipose tissue

Serum leptin levels are upregulated in proportion to body fat and also increase over the short term in response to meals or insulin. To understand the mechanisms involved, we assessed leptin synthesis and secretion in samples of adipose tissue from subjects with a wide range of BMI. Tissue leptin content and relative rates of leptin biosynthesis, as determined by metabolic labeling, were highly correlated with each other and with BMI and fat cell size. To understand mechanisms regulating leptin synthesis in obesity, we used biosynthetic labeling to directly assess the effects of insulin and glucocorticoids (dexamethasone) on leptin synthesis and secretion in human adipose tissue. Chronic treatment (1-2 days in organ culture) with insulin increased relative rates of leptin biosynthesis without affecting leptin mRNA levels. In contrast, dexamethasone increased leptin mRNA and biosynthesis in parallel. Acute treatment with insulin or dexamethasone (added during 1-h preincubation and 45-min pulse labeling) did not affect relative rates of leptin biosynthesis, but pulse-chase studies showed that addition of insulin nearly doubled the release of [35S]leptin after a 1-h chase. We conclude that the higher leptin stores in adipose tissue of obese humans are maintained by chronic effects of insulin and glucocorticoids acting at pre- and posttranslational levels and that the ability of insulin to increase the release of preformed leptin may contribute to short-term variations in circulating leptin levels.

Characterization of the regulation of renal Na+/H+exchanger NHE3 by insulin

Insulin receptors are widely distributed in the kidney and affect multiple aspects of renal function. In the proximal tubule, insulin regulates volume and acid-base regulation through stimulation of the Na(+)/H(+) exchanger NHE3. This paper characterizes the signaling pathway by which insulin stimulates NHE3 in a cell culture model [opossum kidney (OK) cell]. Insulin has two distinct phases of action on NHE3. Chronic insulin (24 h) activates NHE3 through the classic phosphatidylinositol 3-kinase-serum- and glucocorticoid-dependent kinase 1 (PI3K-SGK1) pathway as insulin stimulates SGK1 phosphorylation and the insulin effect can be blocked by the PI3K inhibitor wortmannin or a dominant-negative SGK1. We showed that SGK1 transcript and protein are expressed in rat proximal tubule and OK cells. We previously showed that glucocorticoids augment the effect of insulin on NHE3 (Klisic J, Hu MC, Nief V, Reyes L, Fuster D, Moe OW, Ambuhl PM. Am J Physiol Renal Physiol 283: F532-F539, 2002). Part of this can be mediated via induction of SGK1 by glucocorticoids, and indeed the insulin effect on NHE3 can also be amplified by overexpression of SGK1. We next addressed the acute effect of insulin (1-2 h) on NHE3 by systematically examining the candidate signaling cascades and activation mechanisms of NHE3. We ruled out the PI3K-SGK1-Akt and TC10 pathways, increased surface NHE3, NHE3 phosphorylation, NHE3 association with calcineurin homologous protein 1 or megalin as mechanisms of acute activation of NHE3 by insulin. In summary, insulin stimulates NHE3 acutely via yet undefined pathways and mechanisms. The chronic effect of insulin is mediated by the classic PI3K-SGK1 route.

Cerebral ischemia-induced apoptosis and necrosis in normal and diabetic rats: Effects of insulin and C-peptide

Neuronal apoptosis has been demonstrated to be a significant factor in neurological deficiencies associated with diabetes, and these deficiencies are exaggerated following ischemia. Diabetic rats have an increased basal level of apoptosis compared to non-diabetics and it has been previously demonstrated that infarct volumes were greater in diabetic animals following middle cerebral artery occlusion (MCAO) when compared to non-diabetics. In this study, we evaluated both the acute and chronic effects of insulin and/or C-peptide on CNS necrosis and apoptosis in non-diabetic and streptozotocin-induced diabetic rats following MCAO with reperfusion. Two brain areas, the sensori-motor cortex (layers-5 and 6) and the CA1 and CA3 sectors (pyramidal cell layers) of the hippocampus, were analyzed for apoptosis using TUNEL and Caspase-3 immunoreactivity. The chronic administration of a low maintenance concentration of insulin (2 U/kg), or the acute administration of insulin (2 U/kg) with or without C-peptide, did not alter the lesion volume or basal levels of apoptosis or the apoptotic levels in animals subjected to 2-h MCAO followed by 24-h reperfusion. However, both the acute or chronic administration of a high concentration of insulin (12 U/kg) significantly decreased lesion volume and apoptosis subsequent to 2-h MCAO followed by 24-h reperfusion. High dose insulin treatment also decreased the basal level of apoptosis. We conclude that in diabetic rats subjected to ischemia and reperfusion chronic insulin treatment decreased the basal apoptotic level, and both acute and chronic insulin decreased the MCAO-induced lesion volume and apoptosis. Maintenance insulin concentrations with or without C-peptide were without effect.

Characterization of Multiple Signaling Pathways of Insulin in the Regulation of Vascular Endothelial Growth Factor Expression in Vascular Cells and Angiogenesis

The effects of insulin on vascular endothelial growth factor (VEGF) expression in cultured vascular cells and in angiogenesis were characterized. Insulin increased VEGF mRNA levels in mouse aortic smooth muscle cells from 10(-9) to 10(-7) m with an initial peak of 3.7-fold increases at 1 h and a second peak of 2.8-fold after 12 h. The first peak of VEGF expression was inhibited by LY294002, an inhibitor of phosphatidylinositol (PI) 3-kinase, and by the overexpression of dominant negative forms of p85 subunit of PI 3-kinase or Akt. Inhibitors of MEK kinase, PD98059, or overexpression of dominant negative forms of Ras was ineffective. In contrast, the chronic effect of insulin on VEGF expression was partially inhibited by both LY294002 or PD98059 as well as by the overexpression of dominant negatives of PI 3-kinase or Ras. The importance of PI 3-kinase-Akt pathway on VEGF expression was confirmed in mouse aortic smooth muscle cells isolated from insulin receptor substrate -1 knockout (IRS-1-/-) mice that showed parallel reductions of 46-49% in insulin-stimulated VEGF expression and PI 3-kinase-Akt activation. Insulin-induced activation of PI 3-kinase-Akt on hypoxia-induced VEGF expression and neovascularization was reduced by 40% in the retina of neonatal hypoxia model using IRS-1-/- mice. Thus, unlike other cells, insulin can regulate VEGF expression by both IRS-1/PI 3-kinase-Akt cascade and Ras-MAPK pathways in aortic smooth muscle cells. The in vivo results provide direct evidence that insulin can modulate hypoxia-induced angiogenesis via reduction in VEGF expression in vivo.

Intraventricular insulin suppresses the acoustic startle response in rats

We and others have previously reported that the hormone insulin alters brain noradrenergic function at the synaptic and molecular levels. In the present study, we examined the in vivo effect of insulin (administered chronically via osmotic minipumps at a dose of 5 mU/day into the third cerebral ventricle) on the acoustic startle response. Rats receiving chronic intraventricular insulin had a significantly reduced startle response relative to vehicle-treated controls (i.e., 47 ± 21% of baseline control startle response). Because our previous findings suggest that on an acute basis, insulin may enhance endogenous noradrenergic activity by inhibiting norepinephrine reuptake, we speculate here that the chronic effect of insulin is similar to that of the noradrenergic reuptake blocker, desipramine, which has been reported to decrease baseline startle performance.

Chronic Effect of Insulin on Monoamine Oxidase and Antioxidant Enzyme Activities in the Rat Brainstem

It was shown that hydrogen peroxide (H 2O 2) is a possible intracellular second messenger in specific insulin action. Because its concentration in the cell depends on the activity of both antioxidant enzymes and monoamine oxidase (MAO), we studied the influence of different insulin doses (0.4 and 4.0 IU/kg body mass, i.p., daily injected over 3 days) on the activity of MAO, types A and B, copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and catalase in the rat brainstem. Chronic insulin treatment significantly increased V max of MAO-A and B activities ( P < 0.05, P < 0.025, respectively) independent of the dose applied. CuZnSOD activity was also increased ( P < 0.025), but only when higher dose of hormone was injected. However, insulin had the opposite effect on MnSOD and catalase causing a decrease in their activities ( P < 0.005). The observed changes in the activities of the enzymes studied are possible compensations that potentially maintain an optimal H 2O 2 level in the brainstem, which might be important for insulin action.

Acute and Chronic Effect of Insulin on Leptin Production in Humans: Studies In Vivo and In Vitro

This study was undertaken to investigate the changes in obesity (OB) gene expression and production of leptin in response to insulin in vitro and in vivo under euglycemic and hyperglycemic conditions in humans. Three protocols were used: 1) euglycemic clamp with insulin infusion rates at 40, 120, 300, and 1,200 mU / m / min carried out for up to 5 h performed in 16 normal lean individuals, 30 obese individuals, and 31 patients with NIDDM; 2) 64-to 72-h hyperglycemic (glucose 12.6 mmol/l) clamp performed on 5 lean individuals; 3) long-term (96-h) primary culture of isolated abdominal adipocytes in the presence and absence of 100 nmol/l insulin. Short-term hyperinsulinemia in the range of 80 to > 10,000 microU/ml had no effect on circulating levels of leptin. During the prolonged hyperglycemic clamp, a rise in leptin was observed during the last 24 h of the study (P < 0.001). In the presence of insulin in vitro, OB gene expression increased at 72 h (P < 0.01), followed by an increase in leptin released to the medium (P < 0.001). In summary, insulin does not stimulate leptin production acutely; however, a long-term effect of insulin on leptin production could be demonstrated both in vivo and in vitro. These data suggest that insulin regulates OB gene expression and leptin production indirectly, probably through its trophic effect on adipocytes.

Acute and chronic effects of insulin on leptin production in humans: Studies in vivo and in vitro

Acute and chronic effects of insulin on leptin production in humans: Studies in vivo and in vitro

The Effects of Streptozotocin-induced Diabetes and Insulin Supplementation on Expression of the Glycogen Phosphorylase Gene in Rat Liver

We have previously observed that the chronic effects of streptozotocin-induced diabetes cause a decrease in the total hepatic glycogen phosphorylase activity with a corresponding reduction in the phosphorylase protein levels. These effects were normalized by insulin administration to diabetic rats. There was no change in the total glycogen synthase activity as a result of diabetes or insulin supplementation. These results are extended to examine the effects of diabetes and insulin administration to diabetic animals on the expression of phosphorylase and glycogen synthase enzymes. The expression (i.e. mRNA levels) of phosphorylase was down-regulated (45% of normal levels) in diabetic livers, and this was normalized by insulin supplementation to diabetic animals. Diabetes or insulin supplementation to diabetic rats showed no effect on the transcription rate of phosphorylase. As expected, diabetes (or insulin administration to diabetic animals) did not cause any alteration in the mRNA levels or in the transcription rate of hepatic glycogen synthase. The stability of phosphorylase mRNA was then examined using hepatocytes prepared from normal and diabetic rats. Diabetes caused a decrease in the half-life of phosphorylase mRNA from 14 h in normal hepatocytes to 6.5 h in diabetic hepatocytes. Insulin supplementation to the medium of diabetic hepatocytes increased the half-life of phosphorylase mRNA to a level comparable with normal values. This study indicates that the chronic effect of insulin on the activation of the total hepatic phosphorylase activity (and protein) is mediated through the stabilization of its mRNA levels.

Differential short- and long-term effects of insulin on ANG II action in human adrenal glomerulosa cells.

Insulin is known to modulate several functions in bovine adrenal cells, including steroidogenesis and growth. However, the role of insulin in regulating aldosterone synthesis in human adrenal glomerulosa cells has not been studied. In the present studies, we have examined the acute and chronic effects of insulin on angiotensin II (ANG II)-induced aldosterone synthesis in cultured normal and adenomatous human adrenal glomerulosa cells. Short-term insulin treatment (1.5 h) resulted in inhibition of ANG II-induced aldosterone synthesis. In contrast, chronic treatment (30 h) resulted in potentiation of ANG II action. The 12-lipoxygenase pathway of arachidonate metabolism has been shown to play a key role in ANG II-induced aldosterone synthesis. The acute inhibitory effects of insulin were in part mediated by inhibition of the 12-lipoxygenase pathway. The chronic stimulatory effect of insulin seemed to be due at least in part to the upregulation of cytochrome P-450 side-chain cleavage enzyme levels. These findings suggest that insulin has a differential temporal effect on ANG II action and therefore may be an important regulator of ANG II-induced aldosterone synthesis in the adrenal.

Effects of subbasal insulin infusion on resting and exercise-induced glucose turnover in depancreatized dogs

beta-Adrenergic blockade suppressed lipolysis and normalized the exercise-induced increments in glucose uptake (GlcU) and metabolic clearance rate (MCR) in alloxan-diabetic dogs with residual insulin, but not in insulin-deprived depancreatized dogs even when combined with methylpalmoxirate (MP), which suppresses fatty acid oxidation. The effects of a minimal amount of insulin (as in the alloxan-diabetic dog), were studied in depancreatized, 24-h insulin-deprived dogs during rest and treadmill exercise (6 km/h, 10% slope) using a 1/4 basal insulin infusion (50 microU.kg-1.min-1, insulin, n = 6) alone, or with MP (20 mg.kg-1.day orally, 2.5 days, MP+insulin, n = 6). At rest, insulin decreased circulating fatty acids (31%) and Glc (13%) and increased GlcU and MCR (86 and 72%). Glc production was unaffected. MP plus insulin markedly suppressed hepatic fatty acid oxidation, decreased Glc (44%) and Glc production (50%), and markedly increased MCR (128%). The exercise-induced increments in MCR were markedly improved only by MP plus insulin but were still lower than in the propranolol-treated alloxan-diabetic dogs. Plasma Glc inversely correlated with the exercise-induced increase in MCR (r = -0.86). We conclude that 1) acute infusion of subbasal insulin improved GlcU in depancreatized dogs at rest but not during exercise; 2) inhibition of fatty acid oxidation combined with subbasal insulin improved the exercise-induced increase in MCR; and 3) the difference in GlcU and MCR between the MP plus insulin-treated depancreatized dogs and the beta-blockade-treated alloxan-diabetic dogs suggests a difference between acute and chronic effects of insulin.

Multiple insulin-responsive elements regulate transcription of the GAPDH gene

Multiple elements in the upstream region of the GAPDH gene play a role in mediating the acute and chronic effect of insulin on GAPDH gene expression. The complexity of this regulation provides many layers of control. In differentiated tissues, the transcriptional response to insulin results from the additive effects of g/TRE, IRE-A and IRE-B. The gTRE may interact with newly synthesized c- fos/c- jun heterodimer to activate GAPDH gene transcription. Studies are underway to determine whether protein synthesis inhibitors affect the regulation of GAPDH. Because there are several elements that mediate the effect, it will be difficult to determine the significance of these findings until each cis-acting factor and its binding protein can be studied in isolation. IRE-A and IRE-B act together to promote a 5- to 8-fold insulin effect on HGAPDH-CAT in H35 hepatoma cells and a 3-fold effect in 3T3 adipoctyes. We have succeeded in detecting an insulin-sensitive DNA-binding protein referred to as IREA-BP with an element −480 to −435. Insulin treatment of differentiated 3T3 adipocytes for 1 hr results in a 4-fold increase in the amount of this binding protein, as estimated by the amount of 32P-labelled oligonucleotide retarded on non-denaturing PAGE (11). The effect of insulin on IRP-B is comparable. Furthermore, IREA-BP is induced during the process of fasting and refeeding rats, an important in vivo correlate with our tissue culture models (11). These observations imply that the binding proteins IREA-BP and IRP-B are essential components in the signal transduction pathway of insulin action on GAPDH gene expression in metabolically active tissues such as fat and liver. Differentiation-dependence and tissue-specificity are achieved through multiple regulatory elements and involve pre- and post-translational regulation of multiple transcription factors. IREA-BP is present in preadipocytes but activity is highly induced upon differentiation of preadipocytes to adipocytes. The IRE-B (−408 to −269) DNA binding protein is not detected in 3T3 preadipocytes. A gC/EBP like-protein takes part in the formation of this complex which may explain the inductive effect of differentiation on binding. Finally, footprint and cotransfection studies indicate that the differentiation-dependent protein C/EBP also regulates GAPDH gene transcription through a motif located within one hundred nucleotides of the promoter. We have begun to clone the IRE-A and IRE-B DNA binding proteins. An IRE-A binding protein that footprints the 3′ domain of the IRE-A has been cloned. Examination of the binding specificity of this protein has allowed us to postulate the existence of two or more domains in the IRE-A motif (see Fig. 1). We are in the process of raising antibodies to show that the protein which binds IRE-A in nuclear extracts is identical to IREA-BP. In the meantime we will explore the significance of the extensive homologies we have identified between this motif and elements in other insulin-sensitive genes.

Phorbol ester inhibition of insulin-stimulated deoxyribonucleic acid synthesis in BC3H-1 myocytes.

Insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA) each acutely stimulates hexose transport, amino acid uptake, and pyruvate dehydrogenase activity in the BC3H-1 myocyte in a nonadditive fashion, suggesting that the acute effects of insulin and TPA are mediated through a common mechanism of action. Here we have demonstrated that while chronic incubation with insulin stimulated DNA synthesis by 3- to 6-fold, TPA, in contrast, did not stimulate DNA synthesis and, indeed, caused a 70% inhibition of insulin-stimulated DNA synthesis in a dose-dependent fashion. In differentiated myocytes, insulin maximally stimulated hydroxyurea-sensitive [3H]thymidine incorporation into DNA at 200-400 nM with an ED50 of 5-8 nM, suggesting that insulin stimulates DNA synthesis via the insulin receptor rather than through growth factor receptors. Phorbol ester inhibition of insulin-stimulated DNA synthesis was specific for the active tumor-promoting phorbols and the synthetic diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol. Maximal TPA inhibition of insulin-stimulated DNA synthesis was observed at 100 nM with an ID50 of 30 nM TPA, values analogous to those required for TPA stimulation of hexose transport in the myocyte. Chronic incubation with TPA did not inhibit insulin-stimulated protein synthesis, acute K+ flux, K+ accumulation, cytosolic thymidine levels, or insulin binding, indicating that TPA inhibits a specific intracellular event mediating DNA synthesis and suggesting that the acute and chronic effects of insulin in BC3H-1 myocytes are regulated by distinct pathways.

Changes in the rate of carrier-mediated glucose transport by mouse mammary epithelial cells during ontogeny: hormone dependence delineated in vitro.

Epithelial cells were isolated from mammary glands of mice in various physiological states, and the rate of carrier-mediated glucose transport was determined with 3-O-methylglucose. The basal rate (in the absence of exogenous insulin) increases about 40-fold as the animal of origin progresses from the virgin to the midlactating state and declines precipitously during involution. Insulin does not acutely stimulate carrier-mediated transport by cells isolated from virgin or lactating mice and evokes only about a 50% enhancement of this transport rate in the cells derived from pregnant and early postlactational animals. However, culture of mammary explants from pregnant mice in the presence of insulin, cortisol, and PRL before isolation of the epithelial cells results in a 400% increase in the basal rate of carrier-mediated glucose transport in the absence of exogenous hormones; this effect requires 3 days of incubation. The enhanced rate is equivalent to that in cells freshly isolated from animals in early lactation. Insulin-like growth factor I can mimic the chronic effect of insulin, but multiplication-stimulating activity, epidermal growth factor, and 20% fetal calf serum do not supplant insulin in this system. The maximum velocity, but not the Km, of 3-O-methylglucose transport is markedly increased during ontogeny; this is compatible with an increase in the number of functional transporters during development. The results suggest that insulin and/or insulin like growth factor I are implicated in development of the high basal rate of carrier-mediated glucose transport in mammary cells from lactating mice.

β-Adrenergic desensitization by chronic insulin exposure in 3T3-L1 cultured adipocytes

Cultured 3T3-L1 cells provide a model system for studies of the long-term regulation of lipolysis. Insulin acutely inhibits isoproterenol-stimulated lipolysis primarily by decreasing the apparent affinity apparent Km for isoproterenol. In contrast, chronic insulin exposure inhibits lipolysis by a reduction in the maximal effect of isoproterenol Vmax. The decrease in Vmax can be observed with insulin concentrations that are as low as 10 −9 mol/L at the time of addition. The effect is stable to washing, and the cells' responsiveness to isoproterenol returns partially with continued culture. Chronic insulin exposure also markedly reduced dibutyryl-cAMP-stimulated lipolysis indicating an insulin-induced change distal to cAMP concentration in the cascade of reactions controlling lipolysis in these cells. Time course and insulin dose-response experiments indicate an additional proximal alteration. These results indicate that: (1) 3T3-L1 cells are a useful model for studying the long-term regulation of lipolysis. (2) Chronic insulin exposure inhibits lipolysis by a mechanism that differs from the acute effect of insulin. (3) The chronic effects of insulin may be mediated through changes at multiple levels in the lipolytic cascade.

Chronic effects of insulin and glucagon upon islet function.

Chronic administration of insulin to normal rats provokes a progressive reduction in both the pancreatic insulin content and the secretory responsiveness to glucose. The reduction of the secretory capacity is more marked than that of the stores of hormone. These results suggest that hyperinsulinism inhibits the secretory process within the beta-cell independently of its effects upon insulin synthesis. Prolonged treatment with zincglucagon also causes a reduction of the amount of insulin stored and secreted by the pancreas, but the ratio of insulin output to content is not significantly modified. The chronic influence of hyperglucagonism could thus correspond to a net catabolic effect upon the beta-cell. It is suggested that the reduced insulin-secretory capacity observed in experimental hyperinsulinism and hyperglucagonism could play a role in the impairment of glucose tolerance seen in the corresponding clinical conditions.