The aetiology of insulin resistance is still an enigma. Mouse models are frequently employed to study the underlying pathology. The most commonly used methods to monitor insulin resistance are the HOMA-IR, glucose or insulin tolerance tests and the hyperinsulinemic euglycaemic clamp (HIEC). Unfortunately, these tests disturb steady state glucose metabolism. Here we describe a method in which blood glucose kinetics can be determined in fasted mice without noticeably perturbing glucose homeostasis. The method involves an intraperitoneal injection of a trace amount of [6,6-(2)H2]glucose and can be performed repeatedly in individual mice. The validity and performance of this novel method was tested in mice fed on chow or high-fat diet for a period of five weeks. After administering the mice with [6,6-(2)H2]glucose, decay of the glucose label was followed in small volumes of blood collected by tail tip bleeding during a 90-minute period. The total amount of blood collected was less than 120 μL. This novel approach confirmed in detail the well-known increase in insulin resistance induced by a high-fat diet. The mice showed reduced glucose clearance rate, and reduced hepatic and peripheral insulin sensitivity. To compensate for this insulin resistance, β-cell function was slightly increased. We conclude that this refinement of existing methods enables detailed information of glucose homeostasis in mice. Insulin resistance can be accurately determined while mechanistic insight is obtained in underlying pathology. In addition, this novel approach reduces the number of mice needed for longitudinal studies of insulin sensitivity and glucose metabolism.

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Objective Roux-en-Y gastric bypass (RYGB) is an effective way to induce sustainable weight loss and can be complicated by postprandial hyperinsulinaemic hypoglycaemia (PHH). To study the prevalence and the mechanisms behind the occurrence of hypoglycaemia after a mixed meal tolerance test (MMTT) in patients with primary RYGB. Design This is a cross-sectional study of patients 4 years after primary RYGB. Methods From a total population of 550 patients, a random sample of 44 patients completed the total test procedures. A standardized mixed meal was used as stimulus. Venous blood samples were collected at baseline, every 10 min during the first half hour and every 30 min until 210 min after the start. Symptoms were assessed by questionnaires. Hypoglycaemia is defined as a blood glucose level below 3.3 mmol/L. Results The prevalence of postprandial hypoglycaemia was 48% and was asymptomatic in all patients. Development of hypoglycaemia was more frequent in patients with lower weight at surgery (P = 0.045), with higher weight loss after surgery (P = 0.011), and with higher insulin sensitivity calculated by the homeostasis model assessment indexes (HOMA2-IR, P = 0.014) and enhanced beta cell function (insulinogenic index at 20 min, P = 0.001). Conclusion In a randomly selected population 4 years after primary RYGB surgery, 48% of patients developed a hypoglycaemic event during an MMTT without symptoms, suggesting the presence of hypoglycaemia unawareness in these patients. The findings in this study suggest that the pathophysiology of PHH is multifactorial.

Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects.

Chronic glucocorticoid use for treatment of inflammatory diseases is accompanied by severe side effects in humans (e.g. hyperglycemia and insulin resistance). The present studies were conducted to characterize consequences of chronic treatment with the synthetic glucocorticoid prednisolone on insulin sensitivity and blood glucose kinetics in mice. Prednisolone treatment increased fasting blood glucose and plasma insulin concentrations, but this apparently reduced insulin sensitivity could not be confirmed in hyperinsulinemic euglycemic clamp studies. Therefore, a novel method to study whole body glucose kinetics was used. This method revealed that prednisolone-treated mice show an increased hepatic glucose production (HGP). The increased HGP was accompanied by elevated plasma insulin concentrations, indicating reduced insulin sensitivity of hepatic glucose metabolism in prednisolone-treated mice. Compared with vehicle, prednisolone-treated mice had lower blood glucose concentrations, higher plasma free fatty acids, and higher plasma fibroblast growth factor-21 concentrations in the fed condition, i.e. mimicking a fasting situation. Next, the effects of 24-h fasting on energy metabolism were studied. Compared with controls, fasted prednisolone-treated mice had higher blood glucose concentrations and lower plasma beta-hydroxybutyrate concentrations. In conclusion, these results indicate that chronic prednisolone treatment reduces insulin sensitivity of HGP, induces a fasting-like phenotype in fed mice, and perturbs the fed-to-fasting transition.

The majority of secretory proteins are translocated into and across hydrophobic membranes via the universally conserved Sec pore. Accessory proteins, including the SecDF-YajC Escherichia coli membrane complex, are required for efficient protein secretion. E. coli SecDF-YajC has been proposed to be involved in the membrane cycling of SecA, the cytoplasmic bacterial translocation ATPase, and in the stabilizing of SecG, a subunit of the Sec pore. While there are no identified archaeal homologs of either SecA or SecG, many archaea possess homologs of SecD and SecF. Here, we present the first study that addresses the function of archaeal SecD and SecF homologs. We show that the SecD and SecF components in the model archaeon Haloferax volcanii form a cytoplasmic membrane complex in the native host. Furthermore, as in E. coli, an H. volcanii ⌬secFD mutant strain exhibits both severe cold sensitivity and a Sec-specific protein translocation defect. Taken together, these results demonstrate significant functional conservation among the prokaryotic SecD and SecF homologs despite the distinct composition of their translocation machineries.

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