PPARα activation reverses adverse effects induced by high-saturated-fat feeding on pancreatic β-cell function in late pregnancy

We examined whether the additional demand for insulin secretion imposed by dietary saturated fat-induced insulin resistance during pregnancy is accommodated at late pregnancy, already characterized by insulin resistance. We also assessed whether effects of dietary saturated fat are influenced by PPARα activation or substitution of 7% of dietary fatty acids (FAs) with long-chain ω-3 FA, manipulations that improve insulin action in the nonpregnant state. Glucose tolerance at day 19 of pregnancy in the rat was impaired by high-saturated-fat feeding throughout pregnancy. Despite modestly enhanced glucose-stimulated insulin secretion (GSIS) in vivo, islet perifusions revealed an increased glucose threshold and decreased glucose responsiveness of GSIS in the saturated-fat-fed pregnant group. Thus, insulin resistance evoked by dietary saturated fat is partially countered by augmented insulin secretion, but compensation is compromised by impaired islet function. Substitution of 7% of saturated FA with long-chain ω-3 FA suppressed GSIS in vivo but did not modify the effect of saturated-fat feeding to impair GSIS by perifused islets. PPARα activation (24 h) rescued impaired islet function that was identified using perifused islets, but GSIS in vivo was suppressed such that glucose tolerance was not improved, suggesting modification of the feedback loop between insulin action and secretion.

Insulin secretion in the conscious mouse is biphasic and pulsatile

Islets in most species respond to increased glucose with biphasic insulin secretion, marked by a sharp first-phase peak and a slowly rising second phase. Mouse islets in vitro, however, lack a robust second phase. To date, this observation has not been extended in vivo. We thus compared insulin secretion from conscious mice with isolated mouse islets in vitro. The arterial plasma insulin response to a hyperglycemic clamp was measured in conscious mice 1 wk after surgical implantation of carotid artery and jugular vein catheters. Mice were transfused using clamps with blood from a donor mouse to maintain blood volume, allowing frequent arterial sampling. When plasma glucose in vivo was raised from ∼5 to ∼13 mM, insulin rose to a first-phase peak of 403 ± 73% above basal secretion ( n = 5), followed by a rising second phase of mean 289 ± 41%. In contrast, perifused mouse islets (∼75 islets/trial) responded with a similar first phase of 508 ± 94% ( n = 4) but a smaller and virtually flat second phase of 169 ± 9% ( n = 4, P < 0.05). Furthermore, the slope of the second-phase response differed significantly from zero in mice (2.63 ± 0.39%/min, P < 0.01), in contrast to perifused islets (0.18 ± 0.14%/min, P > 0.30). Mice also displayed pulsatile patterns in insulin concentration (period: 4.2 ± 0.4 min, n = 8). Conscious mice thus responded to increased glucose with biphasic and pulsatile insulin secretion, as in other species. The robust second phase observed in vivo suggests that the processes needed to generate second-phase insulin secretion may be abrogated by islet isolation.

Acute (24 h) activation of peroxisome proliferator-activated receptor-alpha (PPARalpha) reverses high-fat feeding-induced insulin hypersecretion in vivo and in perifused pancreatic islets

Abnormal depletion or accumulation of islet lipid may be important for the development of pancreatic beta cell failure. Long-term lipid sensing by beta cells may be co-ordinated via peroxisome proliferator-activated receptors (PPARs). We investigated whether PPARalpha activation in vivo for 24 h affects basal and glucose-stimulated insulin secretion in vivo after intravenous glucose administration and ex vivo in isolated perifused islets. Insulin secretion after intravenous glucose challenge was greatly increased by high-fat feeding (4 weeks) but glucose tolerance was minimally perturbed, demonstrating insulin hypersecretion compensated for insulin resistance. The effect of high-fat feeding to enhance glucose-stimulated insulin secretion was retained in perifused islets demonstrating a stable, long-term effect of high-fat feeding to potentiate islet glucose stimulus-secretion coupling. Treatment of high-fat-fed rats with WY14,643 for 24 h reversed insulin hypersecretion in vivo without impairing glucose tolerance, suggesting improved insulin action, and ex vivo in perfused islets. PPARalpha activation only affected hypersecretion of insulin since glucose-stimulated insulin secretion was unaffected by WY14,643 treatment in vivo in control rats or in perifused islets from control rats. Our data demonstrate that activation of PPARalpha for 24 h can oppose insulin hypersecretion elicited by high-fat feeding via stable long-term effects exerted on islet function. PPARalpha could, therefore, participate in ameliorating abnormal glucose homeostasis and hyperinsulinaemia in dietary insulin resistance via modulation of islet function, extending the established requirement for PPARalpha for normal islet lipid homeostasis.

Glucagon-like peptide 1 and fatty acids amplify pulsatile insulin secretion from perifused rat islets

Glucose-induced insulin secretion from isolated, perifused rat islets is pulsatile with a period of about 5—10min, similar to the insulin oscillations that are seen in healthy humans but which are impaired in Type II diabetes. We evaluated the pattern of enhancement by the potent incretin, glucagon-like peptide 1 (GLP-1). GLP-1 increased the amplitude of pulses and the magnitude of insulin secretion from the perifused islets, without affecting the average time interval between pulses. Forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine had the same effect, suggesting that the effect was due to elevated cAMP levels. The possibility that cAMP might enhance the amplitude of pulses by reducing phosphofructo-2-kinase (PFK-2) activity was eliminated when the liver isoform of PFK-2 was shown to be absent from β-cells. The possibility that cAMP enhanced pulsatile secretion, at least in part, by stimulating lipolysis was supported by the observations that added oleate had a similar effect on secretion, and that the incretin effect of GLP-1 was inhibited by the lipase inhibitor orlistat. These data show that the physiological incretin GLP-1 preserves and enhances normal pulsatile insulin secretion, which may be essential in proposed therapeutic uses of GLP-1 or its analogues.

Effect of Modified Diabetic Splenocytes on Mice Injected with Multiple Low-Dose Streptozotocin

This work reports the effects of a previous injection of mitomycin-modified splenocytes from multiple-low dose streptozotocin-treated mice (mld-sz) on autoimmune diabetes produced by mld-sz. Our work shows that a previous inoculation of modified mononuclear splenocytes from mld-sz mice prevents alterations in glycemia, in insulin secretion (IS) pattern from isolated perifused islets, and in mass of pancreatic islets. Immunohistochemistry showed an alteration in the number of beta, but not of alpha or delta cells. While a mononuclear intra-islet infiltration was observed in mld-sz mice, a predominantly polar or peri-islet infiltration was seen in vaccinated mice. Islet-associated mononuclear cells from mld-sz mice produced diabetes and induced a diminished IS when transferred to normal receptors. Those cells from previously vaccinated mld-sz mice had no effect when injected into normal receptors. In addition, they also inhibited the damage induced in normal receptors by the islet-associated mononuclear cells from mld-sz animals. Cellular death was also prevented by previous vaccination. Our results suggest that vaccination with modified splenocytes from mld-sz mice is capable of shifting the islet cells infiltration pattern from an aggressive one toward a protective one and thus preventing the ß cell destruction observed in mld-sz mice.

Role of glucose in chronic desensitization of isolated rat islets and mouse insulinoma (betaTC-3) cells to glucose-dependent insulinotropic polypeptide

It is well documented that the release of insulin from isolated perifused islets attenuates over time, despite a continued glucose stimulation. In the current study we have shown that potentiation of insulin release by the intestinal hormone glucose-dependent insulinotropic polypeptide (GIP) is also attenuated after its continuous application. In less than 20 h of maintained stimulus with either hyperglycaemia (11.0 mM glucose) or GIP (10 nM) under hyperglycaemic conditions, insulin release returned to basal values. This was not due to loss of islet viability or reduction in the releasable pool of insulin granules, as 1 mM isobutylmethylxanthine was able to stimulate equivalent insulin release under both conditions. Further examination of chronic GIP desensitization was examined in cultured mouse insulinoma (betaTC-3) cells. GIP-stimulated cAMP production was not greatly affected by the prevailing glucose conditions, suggesting that the glucose dependence of GIP-stimulated insulin release occurs distally to the increase in intracellular cAMP in betaTC-3 cells. The GIP-stimulated cAMP response curve after desensitization was of similar magnitude at all glucose concentrations, but GIP pretreatment did not affect forskolin-stimulated cAMP production. Desensitization of the cAMP response in betaTC-3 cells was shown not to involve induction of dipeptidyl peptidase IV or pertussis toxin-sensitive G-proteins, activation of protein kinase C or protein kinase A, or modulation of phosphodiesterase activity. Homologous desensitization of the insulin-potentiating activity of GIP was found to affect both GIP-stimulated and forskolin-stimulated insulin release, indicating desensitization of distal steps in the stimulus-exocytosis cascade.

Insulinotropic action of β-l-glucose pentaacetate

The metabolism of β-l-glucose pentaacetate and its interference with the catabolism ofl-[U-14C]glutamine, [U-14C]palmitate,d-[U-14C]glucose, andd-[5-3H]glucose were examined in rat pancreatic islets. Likewise, attention was paid to the effects of this ester on the biosynthesis of islet peptides, the release of insulin from incubated or perifused islets, the functional behavior of individual B cells examined in a reverse hemolytic plaque assay of insulin secretion, adenylate cyclase activity in a membrane-enriched islet subcellular fraction, cAMP production by intact islets, tritiated inositol phosphate production by islets preincubated with myo-[2-3H]inositol, islet cell intracellular pH, 86Rb and 45Ca efflux from prelabeled perifused islets, and electrical activity in single isolated B cells. The results of these experiments were interpreted to indicate that the insulinotropic action of β-l-glucose pentaacetate is not attributable to any nutritional value of the ester but, instead, appears to result from a direct effect of the ester itself on a yet unidentified receptor system, resulting in a decrease in K+ conductance, plasma membrane depolarization, and induction of electrical activity.

Chronic exposure to TPA depletes PKCα and augments Ca-dependent insulin secretion from cultured rat islets

The insulin secretory responses of rat islets to glucose (15 mM), 12- O-tetradecanoylphorbol 13-acetate (TPA; 500 nM), and potassium (30 mM) were determined from perifused islets cultured for 22–24 h in CMRL-1066 medium (control cultured) or islets cultured in the additional presence of 500 nM TPA. Islet content of protein kinase C α (PKCα) and serine and threonine phosphoprotein patterns were also monitored after the culture period. Compared with freshly isolated islets, culturing alone had no adverse effect on the capacity of TPA or 30 mM potassium to stimulate secretion or on the islet content of PKCα. In agreement with previous studies, culturing in TPA reduced the islet content of immunoreactive PKCα by >95% and abolished the capacity of the phorbol ester to stimulate secretion during a subsequent dynamic perifusion. Culturing in TPA slightly improved the insulin secretory response to 15 mM glucose compared with control-cultured islets; however, sustained rates of 15 mM glucose-induced secretion from these islets were significantly less than the responses of freshly isolated islets. Islets cultured in TPA responded to 30 mM potassium with a markedly amplified insulin secretory response that was abolished by nitrendipine. Enhanced phosphorylation of several islet proteins was also observed in TPA-cultured islets compared with control-cultured islets. These findings demonstrate that culturing alone impairs glucose-induced secretion, a response that is improved but still subnormal compared with freshly isolated islet responses, if TPA is included in the culture medium. Sustained phosphorylation of several islet proteins in TPA-cultured islets may account, at least in part, for augmented calcium-dependent secretion.

Insulin release transduction mechanism through acid glucan 1,4-α-glucosidase activation is Ca2+ regulated

An important signal involved in glucose-stimulated insulin secretion is transduced through the action of a lysosomal acid, glucan 1,4-α-glucosidase. We investigated the Ca2+ dependency of this enzyme activity in relation to insulin release. In isolated islets, increased levels of extracellular Ca2+induced a large increase in acid glucan 1,4-α-glucosidase activity accompanied by a similar increase in insulin release at both substimulatory and stimulatory concentrations of glucose. At low glucose the Ca2+ “inflow” blocker nifedipine unexpectedly stimulated enzyme activity without affecting insulin release. However, nifedipine suppressed45Ca2+outflow from perifused islets at low glucose and at Ca2+ deficiency when intracellular Ca2+ was mobilized by carbachol. This nifedepine-induced retention of Ca2+ was reflected in increased acid glucan 1,4-α-glucosidase activity. Adding different physiological Ca2+ concentrations or nifedipine to islet homogenates did not increase enzyme activity. Neither selective glucan 1,4-α-glucosidase inhibition nor the ensuing suppression of glucose-induced insulin release was overcome by a maximal Ca2+ concentration. Hence, Ca2+-induced changes in acid glucan 1,4-α-glucosidase activity were intimately coupled to similar changes in Ca2+-glucose-induced insulin release. Ca2+ did not affect the enzyme itself but presumably activated either glucan 1,4-α-glucosidase-containing organelles or closely interconnected messengers.