Metabolism of Insulin Glargine After Repeated Daily Subcutaneous Injections in Subjects With Type 2 Diabetes

OBJECTIVE

To investigate concentration of plasma insulin glargine after its subcutaneous dosing compared with concentration of its metabolites 1 (M1) and 2 (M2) in subjects with type 2 diabetes.

RESEARCH DESIGN AND METHODS

Nine subjects underwent a 32-h euglycemic glucose clamp study (0.4 units/kg glargine after 1 week of daily glargine administration). Glargine, M1, and M2 were measured by a specific liquid chromatography-tandem mass spectrometry assay.

RESULTS

Glargine was detected in only five of the nine subjects, at few time points, and at negligible concentrations. M1 was detected in all subjects and exhibited the same pattern as traditional radioimmunoassay-measured plasma insulin. M2 was not detected at all.

CONCLUSIONS

After subcutaneous injection, glargine was minimally detectable in blood, whereas its metabolite M1 accounted for most (>90%) of the plasma insulin concentration and metabolic action of the injected glargine.After subcutaneous injection in vivo, the long-acting insulin analog glargine undergoes a sequential cleavage of the COOH terminus of the B-chain–forming metabolites M1 and M2 (1,2). M1 and M2 fully retain the same metabolic properties as human insulin (HI), but in contrast to glargine, they do not differ from HI in affinity for IGF-1 receptor (IGF-1R) and mitogenesis (3). In the companion article in this issue (Bolli et al. [4]), glargine metabolism has been studied in subjects with type 1 diabetes, but there are no data on type 2 diabetes. The aim of the current study was to shed light on this question.     

Dissecting the genetic make-up of North-East Sardinia using a large set of haploid and autosomal markers

Sardinia has been used for genetic studies because of its historical isolation, genetic homogeneity and increased prevalence of certain rare diseases. Controversy remains concerning the genetic substructure and the extent of genetic homogeneity, which has implications for the design of genome-wide association studies (GWAS). We revisited this issue by examining the genetic make-up of a sample from North-East Sardinia using a dense set of autosomal, Y chromosome and mitochondrial markers to assess the potential of the sample for GWAS and fine mapping studies. We genotyped individuals for 500K single-nucleotide polymorphisms, Y chromosome markers and sequenced the mitochondrial hypervariable (HVI-HVII) regions. We identified major haplogroups and compared these with other populations. We estimated linkage disequilibrium (LD) and haplotype diversity across autosomal markers, and compared these with other populations. Our results show that within Sardinia there is no major population substructure and thus it can be considered a genetically homogenous population. We did not find substantial differences in the extent of LD in Sardinians compared with other populations. However, we showed that at least 9% of genomic regions in Sardinians differed in LD structure, which is helpful for identifying functional variants using fine mapping. We concluded that Sardinia is a powerful setting for genetic studies including GWAS and other mapping approaches.     

FZD6 is a novel gene for human neural tube defects

Neural tube defects (NTDs) are severe malformations of the central nervous system, affecting 1 of 1,000 live births. Mouse models were instrumental in defining the signaling pathways defective in NTDs, including the planar cell polarity (PCP), also called noncanonical Frizzled/Disheveled pathway. Based on the highly penetrant occurrence of NTDs in double Fzd3/Fzd6(-/-) mutant mice, we investigated the role of the human orthologues, FZD3 and FZD6, by resequencing a cohort of 473 NTDs patients and 639 ethnically matched controls. While we could not demonstrate a significant contribution of FZD3 gene, we identified five rare FZD6 variants that were absent in all controls and predicted to have a functional effect by computational analysis: one de novo frameshift mutation (c.1843_1844insA), three missense changes (p.Arg405Gln, p.Arg511Cys p.Arg511His), and one substitution (c.*20C>T) affecting the 3'-untranslated region (UTR) of the gene. The overall rate of predicted deleterious variants of FZD6 was 5.1-fold higher in cases compared to controls, resulting in a significantly increased NTDs mutation burden. This study demonstrates that rare nonsynonymous variants in FZD6 may contribute to NTDs in humans and enlarges the spectrum of mutations that link PCP pathway to NTDs.     

Acanthamoeba polyphaga, a potential environmental vector for the transmission of food-borne and opportunistic pathogens

The endosymbiotic relationship could represent for many bacteria an important condition favouring their spread in the environment and in foods. For this purpose we studied the behaviour of some food-borne and opportunistic pathogens (Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis, Salmonella enterica serovar Enteritidis, Aeromonas hydrophila, Yersinia enterocolitica) when internalized in Acanthamoeba polyphaga. Our results confirm the capability of the bacteria tested to grow within amoebal hosts. We can observe two types of interactions of the bacteria internalized in A. polyphaga. The first type, showed by Y. enterocolitica and A. hydrophila, was characterized by an early replication, probably followed by the killing and digestion of the bacteria. The second type, showed by E. faecalis and S. aureus was characterized by the persistence and grow inside the host without lysis. Lastly, when amoebae were co-cultured with L. monocytogenes and S. Enteritidis, an eclipse phase followed by an active intracellular growth was observed, suggesting a third type of predator-prey trend. The extracellular count in presence of A. polyphaga, as a result of an intracellular multiplication and subsequent release, was characterized by an increase of E. faecalis, S. aureus, L. monocytogenes and S. Enteritidis, and by a low or absent cell count for Y. enterocolitica and A. hydrophila. Our study suggests that the investigated food-borne and opportunistic pathogens are, in most cases, able to interact with A. polyphaga, to intracellularly replicate and, lastly, to be potentially spread in the environment, underlining the possible role of this protozoan in food contamination.     

C9ORF72 hexanucleotide repeat expansions in the Italian sporadic ALS population

It has been recently reported that a large proportion of patients with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are associated with a hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72. We have assessed 1757 Italian sporadic ALS cases, 133 from Sardinia, 101 from Sicily, and 1523 from mainland Italy. Sixty (3.7%) of 1624 mainland Italians and Sicilians and 9 (6.8%) of the 133 Sardinian sporadic ALS cases carried the pathogenic repeat expansion. None of the 619 regionally matched control samples (1238 chromosomes) carried the expansion. Twenty-five cases (36.2%) had behavioral FTD in addition to ALS. FTD or unspecified dementia was also detected in 19 pedigrees (27.5%) in first-degree relatives of ALS patients. Cases carrying the C9ORF72 hexanucleotide expansion survived 1 year less than cases who did not carry this mutation. In conclusion, we found that C9ORF72 hexanucleotide repeat expansions represents a sizeable proportion of apparent sporadic ALS in the Italian and Sardinian population, representing by far the most common mutation in Italy and the second most common in Sardinia.     

Tocopherols and tocotrienols plasma levels are associated with cognitive impairment

Vitamin E includes 8 natural compounds (4 tocopherols, 4 tocotrienols) with potential neuroprotective activity. α-Tocopherol has mainly been investigated in relation to cognitive impairment. We examined the relation of all plasma vitamin E forms and markers of vitamin E damage (α-tocopherylquinone, 5-nitro-γ-tocopherol) to mild cognitive impairment (MCI) and Alzheimer's disease (AD). Within the AddNeuroMed-Project, plasma tocopherols, tocotrienols, α-tocopherylquinone, and 5-nitro-γ-tocopherol were assessed in 168 AD cases, 166 MCI, and 187 cognitively normal (CN) people. Compared with cognitively normal subjects, AD and MCI had lower levels of total tocopherols, total tocotrienols, and total vitamin E. In multivariable-polytomous-logistic regression analysis, both MCI and AD cases had 85% lower odds to be in the highest tertile of total tocopherols and total vitamin E, and they were, respectively, 92% and 94% less likely to be in the highest tertile of total tocotrienols than the lowest tertile. Further, both disorders were associated with increased vitamin E damage. Low plasma tocopherols and tocotrienols levels are associated with increased odds of MCI and AD.     

Short-Term Effects of Nitrogen Dioxide on Mortality and Susceptibility Factors in 10 Italian Cities: The EpiAir Study

Background: Several studies have shown an association between nitrogen dioxide (NO₂) and mortality. In Italy, the EpiAir multicentric study, “Air Pollution and Health: Epidemiological Surveillance and Primary Prevention,” investigated short-term health effects of air pollution, including NO₂.Objectives: To study the individual susceptibility, we evaluated the association between NO₂ and cause-specific mortality, investigating individual sociodemographic features and chronic/acute medical conditions as potential effect modifiers.Methods: We considered 276,205 natural deaths of persons > 35 years of age, resident in 10 Italian cities, and deceased between 2001 and 2005. We chose a time-stratified case-crossover analysis to evaluate the short-term effects of NO₂ on natural, cardiac, cerebrovascular, and respiratory mortality. For each subject, we collected information on sociodemographic features and hospital admissions in the previous 2 years. Fixed monitors provided daily concentrations of NO₂, particulate matter ≤ 10 μm in aerodynamic diameter (PM₁₀) and ozone (O₃).Results: We found statistically significant associations with a 10-μg/m³ increase of NO₂ for natural mortality [2.09% for lag 0–5; 95% confidence interval (CI), 0.96–3.24], for cardiac mortality (2.63% for lag 0–5; 95% CI, 1.53–3.75), and for respiratory mortality (3.48% for lag 1–5; 95% CI, 0.75–6.29). These associations were independent from those of PM₁₀ and O₃. Stronger associations were estimated for subjects with at least one hospital admission in the 2 previous years and for subjects with three or more specific chronic conditions. Some cardiovascular conditions (i.e., ischemic heart disease, pulmonary circulation impairment, heart conduction disorders, heart failure) and diabetes appeared to confer a strong susceptibility to air pollution.Conclusions: Our results suggest significant and likely independent effects of NO₂ on natural, cardiac, and respiratory mortality, particularly among subjects with specific cardiovascular preexisting chronic conditions and diabetes.