This study examined the effects of glucagon-like peptide-1 (GLP-1) on insulin secretion alone and in combination with sulphonylureas or nateglinide, with particular attention to K_ATP channel-independent insulin secretion. In depolarised cells, GLP-1 significantly augmented glucose-induced K_ATP channel-independent insulin secretion in a glucose concentration-dependent manner. GLP-1 similarly augmented the K_ATP channel-independent insulin-releasing effects of tolbutamide, glibenclamide or nateglinide. Downregulation of protein kinase A (PKA)- or protein kinase C (PKC)-signalling pathways in culture revealed that the K_ATP channel-independent effects of sulphonylureas or nateglinide were critically dependent upon intact PKA and PKC signalling. In contrast, GLP-1 exhibited a reduced but still significant insulin-releasing effect following PKA and PKC downregulation, indicating that GLP-1 can modulate K_ATP channel-independent insulin secretion by protein kinase-dependent and -independent mechanisms. The synergistic insulin-releasing effects of combinatorial GLP-1 and sulphonylurea/nateglinide were lost following PKA- or PKC-desensitisation, despite GLP-1 retaining an insulin-releasing effect, demonstrating that GLP-1 can induce insulin release under conditions where sulphonylureas and nateglinide are no longer effective. Our results provide new insights into the mechanisms of action of GLP-1, and further highlight the promise of GLP-1 or similarly acting analogues alone or in combination with sulphonylureas or meglitinide drugs in type 2 diabetes therapy.

11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD-1), a regulator of intrahepatocellular glucocorticoid activity, is bidirectional in homogenates but catalyses 11 beta-reduction (regenerating glucocorticoid) in intact primary hepatocytes in culture. To examine this discrepancy at the whole-organ level, we examined 11 beta-HSD-1 activity in the intact bivascularly perfused rat liver. On a single pass through male rat liver, 44+/-5% of 11-dehydrocorticosterone (11-DHC) recovered was 11 beta-reduced to corticosterone, whereas 10+/-1% of corticosterone was 11 beta-dehydrogenated to 11-DHC. 11 beta-Reduction was less in female liver (21+/-2%, P<0.01) and was significantly greater with perfusion of all substrate via the portal vein (50+/-3%) than via the hepatic artery (30+/-2%, P<0.05). 11 beta-Reductase activity was not saturated by 11-DHC (10(-)(9)-10(-)(6) M). Perfusion with carbenoxolone (CBX, 10(-)(6)-10(-)(3 )M) did not alter 11 beta-reduction of 11-DHC. In contrast, pretreatment with CBX in vivo (10 mg/day) for 7 days inhibited 11 beta-reductase (19+/-4% conversion, P<0.01). Concentrations of 11-DHC in male rat plasma were 44+/-6 nM. Thus 11 beta-HSD-1 is predominantly an 11 beta-reductase in the intact rat liver and is only inhibited by chronic administration of CBX. The substantial concentrations of plasma 11-DHC as substrate suggest that 11 beta-HSD-1 activity and its potential selective inhibition could modify glucocorticoid action in vivo.

Platelets play a critical role in both the initiation and progression of atherosclerosis, and even more so in the ensuing atherothrombotic complications. Low-dose aspirin remains the mainstay of antiplatelet therapy in high-risk patients by reducing the risk of myocardial ischemia, stroke or death due to cardiovascular disease. However, antiplatelet therapies lose their efficacy in people with diabetes mellitus, increasing the risk of future atherothrombotic events. The molecular mechanisms that promote platelet hyperactivity remain unclear but could be due to glycation-induced conformational changes of platelet membranes resulting in impaired aspirin entry or less-efficient acetylation/compensatory increase in COX-2 expression in newborn platelets. Emerging evidence from our laboratory and elsewhere suggest that enhanced platelet turnover (thrombopoiesis), particularly the production of immature reticulated platelets from the bone marrow, could contribute to atherosclerotic complications. We have identified a major role for neutrophil-derived S100A8/A9, a damage-associated molecular pattern, in driving reticulated thrombopoiesis by directly interacting with its receptors on Kupffer cells in the liver. In this review, we discuss the role of hepatic inflammation in driving reticulated platelet production and suggest potential targets to control their production, improve efficacy of current antiplatelet therapies and reduce the risk of atherothrombotic complications.

The high-fat, high-sucrose diet (HFSD)–fed C57Bl/6 mouse is a widely used model of prediabetes. However, studies typically implement a relatively short dietary intervention lasting between 4 and 16 weeks; as a result, little is known about how a long-term HFSD influences the metabolic profile of these mice. Therefore, the aim of this investigation was to examine the effects of consuming a HFSD for 42 weeks on the development of hyperinsulinaemia and glucose intolerance in male C57Bl/6 mice. Two cohorts of HFSD mice were studied at independent institutes and they underwent an oral glucose tolerance test (OGTT) with measures of plasma insulin and free fatty acids (FFA). Age-matched chow-fed control mice were also studied. The HFSD-fed mice were hyperinsulinaemic and grossly obese, being over 25 g heavier than chow-fed mice, which was due to a marked expansion of subcutaneous adipose tissue. This was associated with a 3-fold increase in liver lipid content. Glucose tolerance, however, was either the same or better than control mice due to the preservation of glucose disposal as revealed by a dynamic stable isotope-labelled OGTT. In addition, plasma FFAs were suppressed to lower levels in HFSD mice during the OGTT. In conclusion, we have made the paradoxical observation that long-term HFSD feeding results in the resolution of glucose intolerance in the C57Bl/6 mouse. Mechanistically, we propose that the gross expansion of subcutaneous adipose tissue increases the glucose disposal capacity of the HFSD-fed mouse, which overcomes the prevailing insulin resistance to improve glucose tolerance.

Targeted deletion of VGF, a neuronal and endocrine secreted protein and neuropeptide precursor, produces a lean, hypermetabolic mouse that is resistant to diet-, lesion-, and genetically induced obesity and diabetes. We hypothesized that increased sympathetic nervous system activity in Vgf−/Vgf− knockout mice is responsible for increased energy expenditure and decreased fat storage and that increased β-adrenergic receptor stimulation induces lipolysis in white adipose tissue (WAT) of Vgf−/Vgf− mice. We found that fat mass was markedly reduced in Vgf−/Vgf− mice. Within knockout WAT, phosphorylation of protein kinase A substrate increased in males and females, phosphorylation of hormone-sensitive lipase (HSL) (ser563) increased in females, and levels of adipose triglyceride lipase, comparative gene identification-58, and phospho-perilipin were higher in male Vgf−/Vgf− WAT compared with wild-type, consistent with increased lipolysis. The phosphorylation of AMP-activated protein kinase (AMPK) (Thr172) and levels of the AMPK kinase, transforming growth factor β-activated kinase 1, were decreased. This was associated with a decrease in HSL ser565 phosphorylation, the site phosphorylated by AMPK, in both male and female Vgf−/Vgf− WAT. No significant differences in phosphorylation of CREB or the p42/44 MAPK were noted. Despite this evidence supporting increased cAMP signaling and lipolysis, lipogenesis as assessed by fatty acid synthase protein expression and phosphorylated acetyl-CoA carboxylase was not decreased. Our data suggest that the VGF precursor or selected VGF-derived peptides dampen sympathetic outflow pathway activity to WAT to regulate fat storage and lipolysis.

In obese humans, metabolism of glucocorticoids by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and A-ring reduction (by 5α- and 5β-reductases) is dysregulated in a tissue specific manner. These changes have been recapitulated in leptin resistant obese Zucker rats but were not observed in high-fat fed Wistar rats. Recent data from mouse models suggest that such discrepancies may reflect differences in leptin signalling. We therefore compared glucocorticoid metabolism in murine models of leptin deficiency and resistance. Male ob/ob and db/db mice and their respective littermate controls (n=10–12/group) were studied at the age of 12 weeks. Enzyme activities and mRNA expression were quantified in snap-frozen tissues. The patterns of altered pathways of steroid metabolism in obesity were similar in ob/ob and db/db mice. In liver, 5β-reductase activity and mRNA were increased and 11β-HSD1 decreased in obese mice, whereas 5α-reductase 1 (5αR1) mRNA was not altered. In visceral adipose depots, 5β-reductase was not expressed, 11β-HSD1 activity was increased and 5αR1 mRNA was not altered in obesity. By contrast, in subcutaneous adipose tissue 11β-HSD1 and 5αR1 mRNA were decreased. Systematic differences were not found between ob/ob and db/db murine models of obesity, suggesting that variations in leptin signalling through the short splice variant of the Ob receptor do not contribute to dysregulation of glucocorticoid metabolism.

Fetuses with intrauterine growth restriction (IUGR) have high concentrations of catecholamines, which lowers the insulin secretion and glucose uptake. Here, we studied the effect of hypercatecholaminemia on glucose metabolism in sheep fetuses with placental insufficiency-induced IUGR. Norepinephrine concentrations are elevated throughout late gestation in IUGR fetuses but not in IUGR fetuses with a bilateral adrenal demedullation (IAD) at 0.65 of gestation. Euglycemic (EC) and hyperinsulinemic–euglycemic (HEC) clamps were performed in control, intact-IUGR, and IAD fetuses at 0.87 of gestation. Compared to controls, basal oxygen, glucose, and insulin concentrations were lower in IUGR groups. Norepinephrine concentrations were five-fold higher in IUGR fetuses than in IAD fetuses. During the EC, rates of glucose entry (GER, umbilical + exogenous), glucose utilization (GUR), and glucose oxidation (GOR) were greater in IUGR groups than in controls. In IUGR and IAD fetuses with euglycemia and euinsulinemia, glucose production rates (GPR) remained elevated. During the HEC, GER and GOR were not different among groups. In IUGR and IAD fetuses, GURs were 40% greater than in controls, which paralleled the sustained GPR despite hyperinsulinemia. Glucose-stimulated insulin concentrations were augmented in IAD fetuses compared to IUGR fetuses. Fetal weights were not different between IUGR groups but were less than controls. Regardless of norepinephrine concentrations, IUGR fetuses not only develop greater peripheral insulin sensitivity for glucose utilization but also develop hepatic insulin resistance because GPR was maintained and unaffected by euglycemia or hyperinsulinemia. These findings show that adaptation in glucose metabolism of IUGR fetuses are independent of catecholamines, which implicate that hypoxemia and hypoglycemia cause the metabolic responses.

Mutations in the gene encoding hepatocyte nuclear factor (HNF)1β result in maturity-onset diabetes of the young-(MODY)5, by impairing insulin secretory responses and, possibly, by reducing β-cell mass. The functional role of HNF1β in normal β-cells is poorly understood; therefore, in the present study, wild-type (WT) HNF1β, or one of two naturally occurring MODY5 mutations (an activating mutation, P328L329del, or a dominant-negative form, A263insGG) were conditionally expressed in the pancreatic β-cell line, insulin-1 (INS-1), and the functional consequences examined. Surprisingly, overexpression of the dominant-negative mutant did not modify any of the functional properties of the cells studied (including insulin secretion, cell growth and viability). By contrast, expression of WT HNF1β was associated with a time- and dose-dependent inhibition of INS-1 cell proliferation and a marked increase in apoptosis. Induction of WT HNF1β also inhibited the insulin secretory response to nutrient stimuli, membrane depolarisation or activation of protein kinases A and C and this correlated with a significant decrease in pancrease-duodenum homeobox-1 protein levels. The attenuation of insulin secretion was, however, dissociated from the inhibition of proliferation and loss of viability, since expression of the P328L329del mutant led to a reduced rate of cell proliferation, but failed to induce apoptosis or to alter insulin secretion. Taken together, the present results suggest that mature rodent β-cells are sensitive to increased expression of WT HNF1β and they imply that the levels of this protein are tightly regulated to maintain secretory competence and cell viability.

5α-Reductases irreversibly catalyse A-ring reduction of pregnene steroids, including glucocorticoids and androgens. Genetic disruption of 5α-reductase 1 in male mice impairs glucocorticoid clearance and predisposes to glucose intolerance and hepatic steatosis upon metabolic challenge. However, it is unclear whether this is driven by changes in androgen and/or glucocorticoid action. Female mice with transgenic disruption of 5α-reductase 1 (5αR1-KO) were studied, representing a ‘low androgen’ state. Glucocorticoid clearance and stress responses were studied in mice aged 6 months. Metabolism was assessed in mice on normal chow (aged 6 and 12 m) and also in a separate cohort following 1-month high-fat diet (aged 3 m). Female 5αR1-KO mice had adrenal suppression (44% lower AUC corticosterone after stress), and upon corticosterone infusion, accumulated hepatic glucocorticoids (~27% increased corticosterone). Female 5αR1-KO mice aged 6 m fed normal chow demonstrated insulin resistance (~35% increased area under curve (AUC) for insulin upon glucose tolerance testing) and hepatic steatosis (~33% increased hepatic triglycerides) compared with controls. This progressed to obesity (~12% increased body weight) and sustained insulin resistance (~38% increased AUC insulin) by age 12 m. Hepatic transcript profiles supported impaired lipid β-oxidation and increased triglyceride storage. Female 5αR1-KO mice were also predisposed to develop high-fat diet-induced insulin resistance. Exaggerated predisposition to metabolic disorders in female mice, compared with that seen in male mice, after disruption of 5αR1 suggests phenotypic changes may be underpinned by altered metabolism of glucocorticoids rather than androgens.

Chronic elevations in interleukin (IL)-6 have been associated with insulin resistance, but acute IL-6 administration can enhance insulin sensitivity. Our aim was to exogenously administer IL-6 to rats to elicit either chronic or repeated acute elevations in systemic IL-6. We hypothesized that a continuous elevation of IL-6 would inhibit glucose tolerance and insulin sensitivity while acute intermittent elevations would improve it. Male Wistar rats were treated for 14d with recombinant human IL-6 (2.4 μg/day) or saline administered either by miniosmotic pump (continuous IL-6) or via twice-daily injection (intermittent IL-6). Glucose and insulin tolerance tests were performed following 14-d treatment and 24 h later rats were administered a bolus of insulin (150 mU/g) or saline intraperitoneally. Approximately, 10 min after insulin injection soleus, gastrocnemius and liver were excised and rapidly frozen in liquid nitrogen for subsequent metabolic measures. Irrespective of the mode of delivery, IL-6 treatment increased basal insulin sensitivity, as measured by the homeostatic model assessment of insulin resistance, and enhanced glucose clearance during an i.p. glucose tolerance test. IL-6 increased circulating fatty acids, but did not increase triglyceride accumulation in either skeletal muscle or liver, while it increased the protein expression of both PPARα and UCP2 in skeletal muscle, suggesting that IL-6 can enhance fat oxidation via mitochondrial uncoupling. These data demonstrate that, irrespective of the mode of delivery, IL-6 administration over 2 weeks enhances glucose tolerance. Our results do not support the notion that prolonged chronically elevated IL-6 impairs insulin action in vivo.