Keld Fosgerau

Astrocyte glycogen metabolism is required for neural activity during aglycemia or intense stimulation in mouse white matter

We tested the hypothesis that inhibiting glycogen degradation accelerates compound action potential (CAP) failure in mouse optic nerve (MON) during aglycemia or high‐intensity stimulation. Axon function was assessed as the evoked CAP, and glycogen content was measured biochemically. Isofagomine, a novel inhibitor of central nervous system (CNS) glycogen phosphorylase, significantly increased glycogen content under normoglycemic conditions. When MONs were bathed in artificial cerebrospinal fluid (aCSF) containing 10 mM glucose, the CAP failed 16 min after exposure to glucose‐free aCSF. MONs bathed in aCSF plus isofagomine displayed accelerated CAP failure on glucose removal. Similar results were obtained in MONs bathed in 30 mM glucose, which increased baseline glycogen concentration. The ability of isofagomine to increase glycogen content thus was not translated into delayed CAP failure. This is likely due to the inability of the tissue to metabolize glycogen in the presence of isofagomine, highlighting the importance of glycogen in sustaining neural function during aglycemia. The hypothesis that glycogen breakdown supports intense neural activity was tested by blocking glycogen breakdown during periods of high‐frequency stimulation. The CAP area declined more rapidly when glycogen metabolism was inhibited by isofagomine, explicitly showing an important physiological role for glycogen metabolism during neural activity.

Long-term characterization of the diet-induced obese and diet-resistant rat model: a polygenetic rat model mimicking the human obesity syndrome.

The availability of useful animal models reflecting the human obesity syndrome is crucial in the search for novel compounds for the pharmacological treatment of obesity. In the current study, we have performed an extensive characterization of the obesity syndrome in a polygenetic animal model, namely the selectively bred diet-induced obese (DIO) and diet-resistant (DR) rat strains. We show that they constitute useful models of the human obesity syndrome. DIO and DR rats were fed either a high-energy (HE) or a standard chow (Chow) diet from weaning to 9 months of age. Metabolic characterization including blood biochemistry and glucose homeostasis was examined at 2, 3, 6, and 9 months of age. Furthermore, in 6-month-old HE-fed DIO rats, the anti-obesity effects of liraglutide and sibutramine were examined in a 28-day study. Only HE-fed DIO rats developed visceral obesity, hyperleptinemia, hyperinsulinemia, and dyslipidemia, and showed a worsening of glucose tolerance over time. In line with the hyperlipidemic profile, a severe hepatic fat infiltration was observed in DIO rats at 6 months of age. The effects of liraglutide and sibutramine were tested in 6-month-old DIO rats. Both compounds effectively reduced food intake and body weight in DIO rats. Liraglutide furthermore improved glucose tolerance when compared with sibutramine. Our data highlights the usefulness of a polygenetic animal model for screening of compounds affecting food intake, body weight, and glucose homeostasis. Furthermore, the results underscore the effectiveness of GLP-1 mimetics both as anti-diabetes and anti-obesity agents.

Compartmentation of lactate originating from glycogen and glucose in cultured astrocytes.

Brain glycogen metabolism was investigated by employing isofagomine, an inhibitor of glycogen phosphorylase. Cultured cerebellar and neocortical astrocytes were incubated in medium containing [U-13C]glucose in the absence or presence of isofagomine and the amounts and percent labeling of intra- and extracellular metabolites were determined by mass spectrometry (MS). The percent labeling in glycogen was markedly decreased in the presence of isofagomine. Surprisingly, the percent labeling of intracellular lactate was also decreased demonstrating the importance of glycogen turnover. The decrease was limited to the percent labeling in the intracellular pool of lactate, which was considerably lower compared to that observed in the medium in which it was close to 100%. These findings indicate compartmentation of lactate derived from glycogenolysis and that derived from glycolysis. Inhibiting glycogen degradation had no effect on the percent labeling in citrate. However, the percent labeling of extracellular glutamine was slightly decreased in neocortical astrocytes exposed to isofagomine, indicating an importance of glycogen turnover in the synthesis of releasable glutamine. In conclusion, the results demonstrate that glycogen in cultured astrocytes is continuously synthesized and degraded. Moreover, it was found that lactate originating from glycogen is compartmentalized from that derived from glucose, which lends further support to a compartmentalized metabolism in astrocytes.

The novel GLP-1-gastrin dual agonist, ZP3022, increases β-cell mass and prevents diabetes in db/db mice

Diabetes is characterized by β‐cell deficiency, and therefore restoration of β‐cell function has been suggested as a potential therapy. We hypothesized that a novel glucagon‐like peptide‐1 (GLP‐1)‐gastrin dual agonist, ZP3022, improves glycaemic control via improvement of β‐cell status in db/db mice.

Characterisation of Age-Dependent Beta Cell Dynamics in the Male db/db Mice

Aim To characterise changes in pancreatic beta cell mass during the development of diabetes in untreated male C57BLKS/J db/db mice. Methods Blood samples were collected from a total of 72 untreated male db/db mice aged 5, 6, 8, 10, 12, 14, 18, 24 and 34 weeks, for measurement of terminal blood glucose, HbA1c, plasma insulin, and C-peptide. Pancreata were removed for quantification of beta cell mass, islet numbers as well as proliferation and apoptosis by immunohistochemistry and stereology. Results Total pancreatic beta cell mass increased significantly from 2.1 ± 0.3 mg in mice aged 5 weeks to a peak value of 4.84 ± 0.26 mg (P < 0.05) in 12-week-old mice, then gradually decreased to 3.27 ± 0.44 mg in mice aged 34 weeks. Analysis of islets in the 5-, 10-, and 24-week age groups showed increased beta cell proliferation in the 10-week-old animals whereas a low proliferation is seen in older animals. The expansion in beta cell mass was driven by an increase in mean islet mass as the total number of islets was unchanged in the three groups. Conclusions/Interpretation The age-dependent beta cell dynamics in male db/db mice has been described from 5-34 weeks of age and at the same time alterations in insulin/glucose homeostasis were assessed. High beta cell proliferation and increased beta cell mass occur in young animals followed by a gradual decline characterised by a low beta cell proliferation in older animals. The expansion of beta cell mass was caused by an increase in mean islet mass and not islet number.

The Novel GLP-1–Gastrin Dual Agonist ZP3022 Improves Glucose Homeostasis and Increases β -Cell Mass without Affecting Islet Number in db/db Mice

Antidiabetic treatments aiming to preserve or even to increase β-cell mass are currently gaining increased interest. Here we investigated the effect of chronic treatment with the novel glucagon-like peptide-1 (GLP-1)–gastrin dual agonist ZP3022 (HGEGTFTSDLSKQMEEEAVRLFIEWLKN-8Ado-8Ado-YGWLDF-NH2) on glycemic control, β-cell mass and proliferation, and islet number. Male db/db mice were treated with ZP3022, liraglutide, or vehicle for 2, 4, or 8 weeks, with terminal assessment of hemoglobin A1c, basal blood glucose, and plasma insulin concentrations. Pancreata were removed for immunohistochemical staining and stereological quantification of β-cell mass, islet numbers, proliferation, and apoptosis. Treatment with ZP3022 or liraglutide led to a significant improvement in glycemic control. ZP3022 treatment resulted in a sustained increase in β-cell mass after 4 and 8 weeks of treatment, whereas the effect of liraglutide was transient. The expansion in β-cell mass observed in the ZP3022-treated mice appeared to be driven by increased β-cell proliferation in existing islets rather than by formation of new islets, as mean islet mass increased but the number of islets remained constant. Our data demonstrate that the GLP-1–gastrin dual agonist ZP3022 causes a sustained improvement in glycemic control accompanied by an increase in β-cell mass, increased proliferation, and increased mean islet mass. The results highlight that the GLP-1–gastrin dual agonist increases β-cell mass more than liraglutide and that dual agonists could potentially be developed into a new class of antidiabetic treatments.

Evidence against Glycogen Cycling of Gluconeogenic Substrates in Various Liver Preparations

The effect of inhibition of glycogen phosphorylase by 1,4-dideoxy-1,4-imino-d-arabinitol on rates of gluconeogenesis, gluconeogenic deposition into glycogen, and glycogen recycling was investigated in primary cultured hepatocytes, in perfused rat liver, and in fed or fasted rats in vivo clamped at high physiological levels of plasma lactate. 1,4-Dideoxy-1,4-imino-d-arabinitol did not alter the synthesis of glycerol-derived glucose in hepatocytes or lactate-derived glucose in perfused liver or fed or fasted rats in vivo. Thus, 1,4-dideoxy-1,4-imino-d-arabinitol inhibited hepatic glucose output in the perfused rat liver (0.77 ± 0.19versus 0.33 ± 0.09, p < 0.05), whereas the rate of lactate-derived gluconeogenesis was unaltered (0.22 ± 0.09 versus 0.18 ± 0.08,p = not significant) (1,4-dideoxy-1,4-imino-d-arabinitol versusvehicle, μmol/min * g). Overall, the data suggest that 1,4-dideoxy-1,4-imino-d-arabinitol inhibited glycogen breakdown with no direct or indirect effects on the rates of gluconeogenesis. Total end point glycogen content (μmol of glycosyl units/g of wet liver) were similar in fed (235 ± 19versus 217 ± 22, p = not significant) or fasted rats (10 ± 2 versus 7 ± 2, p = not significant) with or without 1,4-dideoxy-1,4-imino-d-arabinitol, respectively. The data demonstrate no glycogen cycling under the investigated conditions and no effect of 1,4-dideoxy-1,4-imino-d-arabinitol on gluconeogenic deposition into glycogen. Taken together, these data also suggest that inhibition of glycogen phosphorylase may prove beneficial in the treatment of type 2 diabetes.

Interleukin-6 autoantibodies are involved in the pathogenesis of a subset of Type 2 diabetes

Interleukin-6 (IL6) is critically involved in inflammation and metabolism. About 1% of people produce IL6 autoantibodies (aAb-IL6) that impair IL6 signaling in vivo. We tested the hypothesis that the prevalence of such aAb-IL6 is increased in type 2 diabetic patients and that aAb-IL6 plays a direct role in causing hyperglycemia. In humans, the prevalence of circulating high-affinity neutralizing aAb-IL6 was 2.5% in the type 2 diabetic patients and 1% in the controls (odds ratio 2.5, 95% confidence interval 1.2-4.9, P=0.01). To test for the role of aAb-IL6 in causing hyperglycemia, such aAb-IL6 were induced in mice by a validated vaccination procedure. Mice with plasma levels of aAb-IL6 similar to the 2.5% type 2 diabetic patients developed obesity and impaired glucose tolerance (area under the curve (AUC) glucose, 2056+/-62 vs 1793+/-62, P=0.05) as compared with sham-vaccinated mice, when challenged with a high-fat diet. Mice with very high plasma levels of aAb-IL6 developed elevated fasting plasma glucose (mM, 4.8+/-0.4 vs 3.3+/-0.1, P<0.001) and impaired glucose tolerance (AUC glucose, 1340+/-38 vs 916+/-25, P<0.001) as compared with sham-control mice on normal chow. In conclusion, the prevalence of plasma aAb-IL6 at levels known to impair IL6 signaling in vivo is increased 2.5-fold in people with type 2 diabetes. In mice, matching levels of aAb-IL6 cause obesity and hyperglycemia. These data suggest that a small subset of type 2 diabetes may in part evolve from an autoimmune attack against IL6.

The anti-diabetic effects of GLP-1-gastrin dual agonist ZP3022 in ZDF rats.

AIMS/HYPOTHESIS Combination treatment with exendin-4 and gastrin has proven beneficial in treatment of diabetes and preservation of beta cell mass in diabetic mice. Here, we examined the chronic effects of a GLP-1-gastrin dual agonist ZP3022 on glycemic control and beta cell dysfunction in overtly diabetic Zucker Diabetic Fatty (ZDF) rats. METHODS ZDF rats aged 11 weeks were dosed s.c., b.i.d. for 8 weeks with vehicle, ZP3022, liraglutide, exendin-4, or gastrin-17 with or without exendin-4. Glycemic control was assessed by measurements of HbA1c and blood glucose levels, as well as glucose tolerance during an oral glucose tolerance test (OGTT). Beta cell dynamics were examined by morphometric analyses of beta and alpha cell fractions. RESULTS ZP3022 improved glycemic control as measured by terminal HbA1c levels (6.2±0.12 (high dose) vs. 7.9±0.07% (vehicle), P<0.001), as did all treatments, except gastrin-17 monotherapy. In contrast, only ZP3022, exendin-4 and combination treatment with exendin-4 and gastrin-17 significantly improved glucose tolerance and increased insulin levels during an OGTT. Moreover, only ZP3022 significantly enhanced the beta cell fraction in ZDF rats, a difference of 41%, when compared to the vehicle group (0.31±0.03 vs. 0.22±0.02%, respectively, P<0.05). CONCLUSION These data suggest that ZP3022 may have therapeutic potential in the prevention/delay of beta cell dysfunction in type 2 diabetes.

Mouse models of nonalcoholic steatohepatitis in preclinical drug development

Nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease in the Western world. NAFLD is a complex spectrum of liver diseases ranging from benign hepatic steatosis to its more aggressive necroinflammatory manifestation, nonalcoholic steatohepatitis (NASH). NASH pathogenesis is multifactorial and risk factors are almost identical to those of the metabolic syndrome. This has prompted substantial efforts to identify novel drug therapies for correcting underlying metabolic deficits, and to prevent or alleviate hepatic fibrosis in NASH. Available mouse models of NASH address different aspects of the disease, have varying clinical translatability, and, therefore, also show different utility in drug discovery.