Safety and effectiveness of insulin detemir in combination with oral antidiabetic agents in an outpatient specialist setting: results of the Italian SOLVE™ observational study.

AIM: The addition of basal insulin to oral antidiabetics (OADs) is described by a large number of guidelines and commonly used in clinical practice as a way to start insulin therapy in patients with type 2 diabetes mellitus in order to maximize compliance and minimise the impact of side effects (mainly hypoglycemia and body weight increase). METHODS: SOLVE™ was a 24-week international observational study conducted in 10 countries (including Italy) for the evaluation of the safety and effectiveness of once-daily insulin detemir as add-on therapy in patients with type 2 diabetes mellitus (T2DM) already treated with one or more OADs. The Italian arm of the Solve™ Study aimed to evaluate the safety and the effectiveness of once-daily insulin detemir in combination with OAD agents for the treatment of patients with T2DM in the Italian outpatient specialist setting. The primary endpoint was to assess the incidence of serious adverse drug reactions (SADRs) including in the specific major hypoglycemic events during 24 weeks of once-daily insulin detemir treatment. RESULTS: A total of 4625 patients were enrolled in the study by 223 Italian centres for diabetes care. At baseline the mean (±SD) demographic characteristics of the patients were: age 66.5 (±10.0) years, duration of diabetes 13.25 (±8.14) years, weight 78.95 (±15.86) kg and BMI 29.5 (±5.0) kg/m2. At the end of the study, 3 SADRs (of which 2 were major hypoglycemia) were reported in 2 patients (<0.1%). The percentage of patients with at least 1 minor hypoglycemic event during the 4 weeks preceding insulin initiation was 3.6%. Following insulin initiation, 5.7% (as recorded at baseline visit) had at least 1 minor hypoglycemic event, which decreased slightly by the end of the study compared to baseline (4.8%). In addition, before insulin initiation the mean (±SD) glycemic control values were: fasting plasma glucose (FPG) 11.43 (±3.2) mmol/L and HbA1c 9.16% (±1.46). At the end of the study, HbA1c was reduced by 1.35% (±1.57) (P<0.001), FPG was reduced by 3.34 mmol/L (P<0.001) and the percentage of patients with HbA1c<7% was 21.9%. A mean reduction of 0.52 kg of body weight (P<0.001) was observed compared to before insulin initiation; the body weight reduction was more pronounced in patients with higher BMI before insulin initiation (-1.0 kg for 30

Endosome to Golgi transport of ricin is independent of clathrin and of the Rab9- and Rab11-GTPases.

The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.

Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae.

Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

Ricin transport into cells: studies of endocytosis and intracellular transport.

The plant toxin ricin binds to both glycoproteins and glycolipids with terminal galactose, and the toxin will therefore be endocytosed by the different mechanisms operating in a given cell. After endocytosis the toxin is transported to the Golgi apparatus by a process that differs from the Rab9-dependent transport of mannose-6-phosphate receptors. Retrograde toxin transport from the Golgi apparatus to the endoplasmic reticulum (ER) seems to be a requirement for subsequent toxin translocation to the cytosol where the toxin inhibits protein synthesis enzymatically. By using ricin we have characterized different types of endocytosis and the transport steps used by this toxin.

Rab7: a key to lysosome biogenesis.

The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.

Role for dynamin in late endosome dynamics and trafficking of the cation-independent mannose 6-phosphate receptor.

It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a perinuclear cluster of cation-independent mannose 6-phosphate receptor (CI-MPR)-containing structures. By electron microscopy (EM), it was shown that these structures were late endosomes and that the endogenous dynamin was preferentially localized to tubulo-vesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutant-expressing cells. Quantitative EM analysis of the ratio of CI-MPR to lysosome-associated membrane protein-1 in endosome profiles revealed a higher colocalization of the two markers in dynK44A-expressing cells than in control cells. Western blot analysis showed that dynK44A-expressing cells had an increased cellular procathepsin D content. Finally, EM revealed that in dynK44A-expressing cells, endosomal tubules containing CI-MPR were formed. These results are in contrast to recent reports that dynamin-2 is exclusively associated with endocytic structures at the plasma membrane. They suggest instead that endogenous dynamin also plays an important role in the molecular machinery behind the recycling of the CI-MPR from endosomes to the trans-Golgi network, and we propose that dynamin is required for the final scission of vesicles budding from endosome tubules.