UCN2: a new candidate influencing pancreatic β-cell adaptations in pregnancy

The corticotropin-releasing hormone (CRH) family of peptides, including urocortin (UCN) 1, 2 and 3, are established hypothalamic neuroendocrine peptides, regulating the physiological and behaviour responses to stress indirectly, via the hypothalamic-pituitary-adrenal (HPA) axis. More recently, these peptides have been implicated in diverse roles in peripheral organs through direct signalling, including in placental and pancreatic islet physiology. CRH has been shown to stimulate insulin release through activation of its cognate receptors, CRH receptor 1 (CRHR1) and 2. However, the physiological significance of this is unknown. We have previously reported that during mouse pregnancy, expression of CRH peptides increase in mouse placenta suggesting that these peptides may play a role in various biological functions associated with pregnancy, particularly the pancreatic islet adaptations that occur in the pregnant state to compensate for the physiological increase in maternal insulin resistance. In the current study, we show that mouse pregnancy is associated with increased circulating levels of UCN2 and that when we pharmacologically block endogenous CRHR signalling in pregnant mice, impairment of glucose tolerance is observed. This effect on glucose tolerance was comparable to that displayed with specific CRHR2 blockade and not with specific CRHR1 blockade. No effects on insulin sensitivity or the proliferative capacity of β-cells were detected. Thus, CRHR2 signalling appears to be involved in β-cell adaptive responses to pregnancy in the mouse, with endogenous placental UCN2 being the likely signal mediating this.

Acetoacetate and β-hydroxybutyrate in combination with other metabolites release insulin from INS-1 cells and provide clues about pathways in insulin secretion

Mitochondrial anaplerosis is important for insulin secretion, but only some of the products of anaplerosis are known. We discovered novel effects of mitochondrial metabolites on insulin release in INS-1 832/13 cells that suggested pathways to some of these products. Acetoacetate, β-hydroxybutyrate, α-ketoisocaproate (KIC), and monomethyl succinate (MMS) alone did not stimulate insulin release. Lactate released very little insulin. When acetoacetate, β-hydroxybutyrate, or KIC were combined with MMS, or either ketone body was combined with lactate, insulin release was stimulated 10-fold to 20-fold the controls (almost as much as with glucose). Pyruvate was a potent stimulus of insulin release. In rat pancreatic islets, β-hydroxybutyrate potentiated MMS- and glucose-induced insulin release. The pathways of their metabolism suggest that, in addition to producing ATP, the ketone bodies and KIC supply the acetate component and MMS supplies the oxaloacetate component of citrate. In line with this, citrate was increased by β-hydroxybutyrate plus MMS in INS-1 cells and by β-hydroxybutyrate plus succinate in mitochondria. The two ketone bodies and KIC can also be metabolized to acetoacetyl-CoA and acetyl-CoA, which are precursors of other short-chain acyl-CoAs (SC-CoAs). Measurements of SC-CoAs by LC-MS/MS in INS-1 cells confirmed that KIC, β-hydroxybutyrate, glucose, and pyruvate increased the levels of acetyl-CoA, acetoacetyl-CoA, succinyl-CoA, hydroxymethylglutaryl-CoA, and malonyl-CoA. MMS increased incorporation of 14C from β-hydroxybutyrate into citrate, acid-precipitable material, and lipids, suggesting that the two molecules complement one another to increase anaplerosis. The results suggest that, besides citrate, some of the products of anaplerosis are SC-CoAs, which may be precursors of molecules involved in insulin secretion.

Glibenclamide inhibits islet carnitine palmitoyltransferase 1 activity, leading to PKC-dependent insulin exocytosis

Hypoglycemic sulfonylureas such as glibenclamide have been widely used to treat type 2 diabetic patients for 40 yr, but controversy remains about their mode of action. The widely held view is that they promote rapid insulin exocytosis by binding to and blocking pancreatic β-cell ATP-dependent K+ (KATP) channels in the plasma membrane. This event stimulates Ca2+ influx and sets in motion the exocytotic release of insulin. However, recent reports show that >90% of glibenclamide-binding sites are localized intracellularly and that the drug can stimulate insulin release independently of changes in KATP channels and cytoplasmic free Ca2+. Also, glibenclamide specifically and progressively accumulates in islets in association with secretory granules and mitochondria and causes long-lasting insulin secretion. It has been proposed that nutrient insulin secretagogues stimulate insulin release by increasing formation of malonyl-CoA, which, by blocking carnitine palmitoyltransferase 1 (CPT-1), switches fatty acid (FA) catabolism to synthesis of PKC-activating lipids. We show that glibenclamide dose-dependently inhibits β-cell CPT-1 activity, consequently suppressing FA oxidation to the same extent as glucose in cultured fetal rat islets. This is associated with enhanced diacylglycerol (DAG) formation, PKC activation, and KATP-independent glibenclamide-stimulated insulin exocytosis. The fat oxidation inhibitor etomoxir stimulated KATP-independent insulin secretion to the same extent as glibenclamide, and the action of both drugs was not additive. We propose a mechanism in which inhibition of CPT-1 activity by glibenclamide switches β-cell FA metabolism to DAG synthesis and subsequent PKC-dependent and KATP-independent insulin exocytosis. We suggest that chronic CPT inhibition, through the progressive islet accumulation of glibenclamide, may explain the prolonged stimulation of insulin secretion in some diabetic patients even after drug removal that contributes to the sustained hypoglycemia of the sulfonylurea.

Insulinotropic action of α-d-glucose pentaacetate: functional aspects

The functional determinants of the insulinotropic action of α-d-glucose pentaacetate were investigated in rat pancreatic islets. The ester mimicked the effect of nutrient secretagogues by recruiting individual B cells into an active secretory state, stimulating proinsulin biosynthesis, inhibiting86Rb outflow, and augmenting45Ca efflux from prelabeled islets. The secretory response to the ester was suppressed in the absence of Ca2+ and potentiated by theophylline or cytochalasin B. The generation of acetate from the ester apparently played a small role in its insulinotropic action. Thus acetate, methyl acetate, ethyl acetate, α-d-galactose pentaacetate, and β-d-galactose pentaacetate all failed to stimulate insulin release. The secretory response to α-d-glucose pentaacetate was reproduced by β-d-glucose pentaacetate and, to a lesser extent, by β-l-glucose pentaacetate. It differed from that evoked by unesterifiedd-glucose by its resistance to 3- O-methyl-d-glucose,d-mannoheptulose, and 2-deoxy-d-glucose. It is concluded that the insulinotropic action of α-d-glucose pentaacetate, although linked to the generation of the hexose from its ester, entails a coupling mechanism that is not identical to that currently implied in the process of glucose-induced insulin release.