Biological Evaluation of Antibody-Drug Conjugates Produced by Tag-Free Lipoate Ligase A Modification.

The concept of tag-free protein modification has attracted considerable interest in chemical biology because of its flexible and straightforward reaction process. In 2021, a groundbreaking approach using lipoate ligase A (LplA) for tag-free enzymatic modification of antibodies was unveiled, demonstrating its potential for the generation of precise antibody conjugates. In this study, to further explore LplA-mediated antibody-drug conjugate (ADC) synthesis, we performed initial biological evaluations of ADCs synthesized using LplA. Using the anti-HER2 antibody trastuzumab, we introduced octanoic acid azide using LplA and subsequently obtained an ADC using click chemistry with the drug DBCO-VC-PAB-MMAE. The bioactivity of the synthesized anti-HER2-ADC was evaluated using HER2-positive SKBR-3 and HER2-negative MCF7 cells. Its toxicity and selectivity were found to be comparable to those of the FDA-approved Kadcyla. In addition, a stability study involving rat and human plasma demonstrated the stability of the LplA-mediated ADC. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. These preliminary in vitro evaluations suggested that LplA-derived ADCs can have considerable pharmaceutical potential. Our results can set the stage for further in vivo evaluations and safety assessments. We suggest that the integration of tag-free LplA methods into the production of ADCs can offer a novel and promising approach for biopharmaceutical manufacturing.

Trastuzumab deruxtecan in patients with locally advanced or metastatic HER2-positive gastric cancer: a multicenter, open-label, expanded-access study.

BACKGROUND: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate that consists of an anti-human epidermal growth factor receptor 2 (HER2) antibody bound by a cleavable tetrapeptide-based linker to a cytotoxic topoisomerase I inhibitor. Prior to marketing approval in Japan in September 2020, this expanded-access study was conducted to provide T-DXd to previously treated patients with locally advanced or metastatic HER2-positive gastric or gastroesophageal junction adenocarcinomas. METHODS: This multicenter, open-label, expanded-access study was conducted between March 25 and September 25, 2020 at 17 Japanese sites. Previously treated patients with locally advanced or metastatic HER2-positive gastric or gastroesophageal junction adenocarcinomas received T-DXd 6.4 mg/kg via intravenous infusions at 3-week intervals. Serious adverse events (SAEs), all potential cases of interstitial lung disease (ILD)/pneumonitis, all liver-related events potentially meeting Hy's Law criteria, and all cases of overdose were reported on the case report forms. RESULTS: A total of 64 patients were treated with T-DXd. Among the 17 (26.6%) patients with reported SAEs, 10 (15.6%) had SAEs related to T-DXd treatment. Febrile neutropenia was the most common SAE (n = 6). SAEs led to death in six patients; drug-related SAEs (sepsis and febrile neutropenia) led to death in one patient. Drug-related ILD, as determined by the external Adjudication Committee, occurred in three patients (Grade 1, Grade 2, and Grade 3: all n = 1). CONCLUSION: This expanded-access study provided T-DXd to a broader population of Japanese patients prior to marketing approval in Japan, bridging the gap between clinical trials and drug approval. No new safety concerns were identified.

Analytical studies on the conjugation site specificity of trastuzumab modified by Escherichia coli lipoate ligase A: multiple-enzyme digestion approach for peptide mapping.

Tag-free protein modification has received considerable attention in the field of chemical biology owing to the versatility and simplicity of the reaction sequence. In 2021, a novel tag-free enzymatic modification of antibodies utilizing lipoate ligase A (LplA) was reported to reveal its potential in the production of site-specific antibody conjugates. Primary peptide mapping analysis revealed the biased site specificity of antibodies modified by LplA; however, quantitative analysis remains challenging because of the complicated heterogeneity derived from biased selective modification. In an effort to further understand the site occupancy of LplA-modified antibodies, this study employed numerous unconventional techniques and strategies. Optimization of HPLC conditions and utilization of enzymes such as trypsin, Glu-C, and chymotrypsin significantly increased sequence data coverage. The transition from traditional spectral counting to a more accurate peak area-based label-free quantification helped better analyze peptide modification levels. The results obtained indicate that LplA-induced modifications are specific lysines, particularly the light chain Lys188/190 site, which have an increased modification rate compared to chemically induced modifications. This study not only contributes to the understanding of peptide modification, but also presents an improved methodology that promises to stimulate further research in this field.

Lipoate-acid ligase a modification of native antibody: Synthesis and conjugation site analysis.

Bioconjugation is an important chemical biology research focus, especially in the development of methods to produce pharmaceutical bioconjugates and antibody-drug conjugates (ADCs). In this report, an enzyme-catalyzed conjugation method combined with a chemical reaction was used to modify a native antibody under mild reaction conditions. Our investigation revealed that lipoic-acid ligase (LplA) modifies native IgG1 with biased site-specificity. An intact mass analysis revealed that 98.3% of IgG1 was modified by LplA and possessed at least one molecule of octanocic acid. The average number of modifications per antibody was calculated to be 4.6. Peptide mapping analysis revealed that the modified residues were K225, K249 and K363 in the Fc region, and K30, K76 and K136 in the heavy chain and K39/K42, K169, K188 and K190 in the light chain of the Fab region. Careful evaluation including solvent exposed amino acid analysis suggested that these conjugate sites were not only solvent exposed but also biased by the site-specificity of LplA. Furthermore, antibody fragment conjugation may be able to take advantage of this enzymatic approach. This feasibility study serves as a demonstration for preparing enzymatically modified antibodies with conjugation site analysis.

Effects of metformin on compensatory pancreatic ß-cell hyperplasia in mice fed a high-fat diet.

Metformin has been widely used for the treatment of type 2 diabetes. However, the effect of metformin on pancreatic ß-cells remains controversial. In this study, we investigated the impacts of treatment with metformin on pancreatic ß-cells in a mouse model fed a high-fat diet (HFD), which triggers adaptive ß-cell replication. An 8-wk treatment with metformin improved insulin resistance and suppressed the compensatory ß-cell hyperplasia induced by HFD-feeding. In contrast, the increment in ß-cell mass arising from 60 wk of HFD feeding was similar in mice treated with and those treated without metformin. Interestingly, metformin suppressed ß-cell proliferation induced by 1 wk of HFD feeding without any changes in insulin resistance. Metformin directly suppressed glucose-induced ß-cell proliferation in islets and INS-1 cells in accordance with a reduction in mammalian target of rapamycin phosphorylation. Taken together, metformin suppressed HFD-induced ß-cell proliferation independent of the improvement of insulin resistance, partly via direct actions.

Quercetin-3-O-glucuronide inhibits noradrenaline-promoted invasion of MDA-MB-231 human breast cancer cells by blocking ß2-adrenergic signaling.

Endogenous catecholamines such as adrenaline (A) and noradrenaline (NA) are released from the adrenal gland and sympathetic nervous system during exposure to stress. The adrenergic system plays a central role in stress signaling, and excessive stress was found to be associated with increased production of reactive oxygen species (ROS). Overproduction of ROS induces oxidative damage in tissues and causes the development of diseases such as cancer. In this study, we investigated the effects of quercetin-3-O-glucuronide (Q3G), a circulating metabolite of quercetin, which is a type of natural flavonoid, on the catecholamine-induced ß2-adrenergic receptor (ß2-AR)-mediated response in MDA-MB-231 human breast cancer cells expressing ß2-AR. Treatment with A or NA at concentrations above 1µM generated significant levels of ROS, and NA treatment induced the gene expression of heme oxygenase-1 (HMOX1), and matrix metalloproteinase-2 (MMP-2) and -9 (MMP9). Inhibitors of p38 MAP kinase (SB203580), cAMP-dependent protein kinase (PKA) (H-89), activator protein-1 (AP-1) transcription factor (SR11302), and NF-κB and AP-1 (Tanshinone IIA) decreased MMP2 and MMP9 gene expression. NA also enhanced cAMP induction, RAS activation and phosphorylation of ERK1/2. These results suggested that the cAMP-PKA, MAPK, and ROS-NF-κB pathways are involved in ß2-AR signaling. Treatment with 0.1µM Q3G suppressed ROS generation, cAMP and RAS activation, phosphorylation of ERK1/2 and the expression of HMOX1, MMP2, and MMP9 genes. Furthermore, Q3G (0.1µM) suppressed invasion of MDA-MB-231 breast cancer cells and MMP-9 induction, and inhibited the binding of [(3)H]-NA to ß2-AR. These results suggest that Q3G may function to suppress invasion of breast cancer cells by controlling ß2-adrenergic signaling, and may be a dietary chemopreventive factor for stress-related breast cancer.

Using miglitol at 30 min before meal is effective in hyperinsulinemic hypoglycemia after a total gastrectomy.

A 45-year-old woman who had undergone total gastrectomy for gastric cancer presented with a history of postprandial hypoglycemic episodes with loss of consciousness after meals. Laboratory findings revealed marked hyperinsulinemia and hypoglycemia after a meal. We first treated the patient with octreotide; however, she was unable to continue the treatment because of adverse effects of the drug, such as nausea and headache. Diazoxide was used next for preventing hyperinsulinemia; however, this was not effective for suppressing the postprandial insulin secretion. Since hypoglycemia following gastrectomy is thought to be caused by rapid delivery of nutrients into the duodenum, we performed a meal tolerance test while varying the timing of administration of miglitol in relation to the meal. Miglitol was administered 30 min before, just before, or both 30 min and just before a meal. In the case of administration just before a meal, insulin secretion was suppressed, although hypoglycemia was not prevented. Administration of the drug 30 min before a meal prevented postprandial hypoglycemia by slowing the increase of the blood glucose and serum insulin levels following the meal to a greater degree than administration just before a meal. Miglitol administration both 30 min and just before a meal caused an even smoother increase in blood glucose and serum insulin levels following the meal. In this report, we propose a new therapeutic approach for reactive hypoglycemia after gastrectomy, namely, administration of miglitol 30 min before meals.

Second-line treatments for dyslipidemia in patients at risk of cardiovascular disease.

Previous studies have shown that approximately 50% patients at risk of cardiovascular disease do not achieve lipid management goals. Thus, improvements dyslipidemia management are needed. We investigated the clinical choice and efficacy of second-line treatments for dyslipidemia in the Japanese clinical setting. Using a retrospective cohort design, we collected lipid profile data from patients who had been treated with hypolipidemic agents at a stable dosage for at least 12 weeks. These patients had then been administered a second-line treatment for dyslipidemia because they had not achieved the low-density lipoprotein cholesterol (LDL-C) management goals. We included data from 641 patients in our analysis. The top three choices for second-line treatment were adding ezetimibe, switching to strong statins (statin switching), and doubling the original statin dosage (statin doubling). Adding ezetimibe, statin switching, and statin doubling decreased LDL-C levels by 28.2 ± 14.5%, 23.2 ± 24.4%, and 23.5 ± 17.2%, respectively. Among these three strategies, adding ezetimibe decreased LDL-C levels to the maximum extent. In patients with dysglycemia, baseline-adjusted change in hemoglobin A1c (HbA1c) levels decreased slightly in the adding-ezetimibe, statin-switching, and statin-doubling groups, but the differences were not statistically significant among the groups (-0.10 ± 0.62%, -0.22 ± 0.54%, and -0.12 ± 0.52%, p = 0.19). In conclusion, the most common second-line treatment options for dyslipidemia were adding ezetimibe, statin switching, or statin doubling. Adding ezetimibe resulted in the highest reduction in LDL-C levels. These strategies did not increase HbA1c levels when administered with conventional diabetes treatment.

Tag-free protein modification by lipoate ligase A: exploring substrate tolerance.

This study delves into the functional intricacies of lipoate ligase A (LplA), an enzyme showing great promise in bioconjugation due to its unique capacity for introducing azido groups into proteins without requiring a genetic tag. We aimed to enhance the understanding of LplA's functionality, particularly its substrate tolerance and the reliability of various analytical techniques. A pivotal aspect of our approach was incorporating azido groups into a range of proteins, followed by the addition of the fluorescent molecule Cy3 via click chemistry. Analysis of fluorescent intensity in the altered proteins indicated varying degrees of conjugation. Additionally, phenyl resin-based RP-HPLC facilitated effective separation of modified proteins, unmodified proteins, and remaining fluorescent tags post-separation. SASA analysis provided insights into conjugation trends, guiding the identification of proteins amenable to LplA's tag-free modification. Our findings demonstrate LplA's broad substrate tolerability for protein modification.

Diurnal rhythmicity in biological processes involved in bioavailability of functional food factors.

In the past few decades, many types of functional factors have been identified in dietary foods; for example, flavonoids are major groups widely distributed in the plant kingdom. However, the absorption rates of the functional food factors are usually low, and many of these are difficult to be absorbed in the intact forms because of metabolization by biological processes during absorption. To gain adequate beneficial effects, it is therefore mandatory to know whether functional food factors are absorbed in sufficient quantity, and then reach target organs while maintaining beneficial effects. These are the reasons why the bioavailability of functional food factors has been well investigated using rodent models. Recently, many of the biological processes have been reported to follow diurnal rhythms recurring every 24 h. Therefore, absorption and metabolism of functional food factors influenced by the biological processes may vary with time of day. Consequently, the evaluation of the bioavailability of functional food factors using rodent models should take into consideration the timing of consumption. In this review, we provide a perspective overview of the diurnal rhythm of biological processes involved in the bioavailability of functional food factors, particularly flavonoids.

Interstitial deletion of the Apc locus in ß-catenin-overexpressing cells is a signature of radiation-induced intestinal tumors in C3B6F1 ApcMin/+ mice†.

Recent studies have identified interstitial deletions in the cancer genome as a radiation-related mutational signature, although most of them do not fall on cancer driver genes. Pioneering studies in the field have indicated the presence of loss of heterozygosity (LOH) spanning Apc in a subset of sporadic and radiation-induced intestinal tumors of ApcMin/+ mice, albeit with a substantial subset in which LOH was not detected; whether copy number losses accompany such LOH has also been unclear. Herein, we analyzed intestinal tumors of C3B6F1 ApcMin/+ mice that were either left untreated or irradiated with 2 Gy of γ-rays. We observed intratumor mosaicism with respect to the nuclear/cytoplasmic accumulation of immunohistochemically detectable ß-catenin, which is a hallmark of Apc+ allele loss. An immunoguided laser microdissection approach enabled the detection of LOH involving the Apc+ allele in ß-catenin-overexpressing cells; in contrast, the LOH was not observed in the non-overexpressing cells. With this improvement, LOH involving Apc+ was detected in all 22 tumors analyzed, in contrast to what has been reported previously. The use of a formalin-free fixative facilitated the LOH and microarray-based DNA copy number analyses, enabling the classification of the aberrations as nondisjunction/mitotic recombination type or interstitial deletion type. Of note, the latter was observed only in radiation-induced tumors (nonirradiated, 0 of 8; irradiated, 11 of 14). Thus, an analysis considering intratumor heterogeneity identifies interstitial deletion involving the Apc+ allele as a causative radiation-related event in intestinal tumors of ApcMin/+ mice, providing an accurate approach for attributing individual tumors to radiation exposure.

Comparison of the Effectiveness of Once-Daily Alogliptin/Metformin and Twice-Daily Anagliptin/Metformin Combination Tablet in a Randomized, Parallel-Group, Open-Label Trial in Japanese Patients with Type 2 Diabetes.

INTRODUCTION: The combination tablets of dipeptidyl peptidase-4 (DPP-4) inhibitors and metformin are used for both once-daily and twice-daily agents in Japan. If there is no difference in effectiveness between the once-daily and twice-daily DPP-4 inhibitor/metformin combination tablets, the once-daily agent is advantageous in terms of frequency of administration. The aim of this study was to compare the effectiveness of once-daily alogliptin/metformin combination tablet (alogliptin 25 mg/metformin 500 mg) and twice-daily anagliptin/metformin combination tablet low dose (LD) (anagliptin 100 mg/metformin 250 mg). METHODS: Forty-eight Japanese patients with type 2 diabetes whose metformin administration of 250 mg twice daily had remained unchanged for at least 8 weeks, except when using DPP-4 inhibitors, glucagon-like peptide-1 receptor agonists, or insulin, were randomized to either the once-daily alogliptin/metformin combination tablet group or the twice-daily anagliptin/metformin combination tablet LD group. The primary endpoint was the difference in glycosylated hemoglobin (HbA1c) levels from baseline to week 12 of administration, whereas the secondary endpoints were fasting blood glucose, body mass index (BMI), and adherence. RESULTS: Forty-four patients completed the study, and intention-to-treat analyses were performed. The adjusted mean value (standard error) for the change in HbA1c from week 0 to 12, was - 0.75 (0.109)% for the once-daily alogliptin/metformin combination tablet group and - 0.65 (0.109)% for the twice-daily anagliptin/metformin combination tablet LD group, with an intergroup difference of - 0.10% (95% confidence interval, CI - 0.407, 0.215). The upper limit of the bilateral 95% CI was 0.215%, below the 0.40% pre-defined as the non-inferiority margin. Fasting blood glucose, BMI, and adherence were not significantly different between the groups. CONCLUSIONS: The once-daily alogliptin/metformin combination tablet was non-inferior to the twice-daily anagliptin/metformin combination tablet LD in Japanese patients with type 2 diabetes. TRIAL REGISTRATION: University Hospital Medical Information Network Clinical Trial Registry (UMIN-CTR) (registration number: UMIN000034951).

Lung-Cancer Risk in Mice after Exposure to Gamma Rays, Carbon Ions or Neutrons: Egfr Pathway Activation and Frequent Nuclear Abnormality.

Lung is one of the high-risk organs for radiation-induced carcinogenesis, but the risk of secondary lung-cancer development after particle-beam therapy and the underlying mechanism(s) remain to be elucidated. To investigate the effects of particle-beam radiation on adjacent normal tissues during cancer therapy, 7-week-old male and female B6C3F1 mice were irradiated with 0.2-4 Gy of gamma rays (for comparison), carbon ions (290 MeV/u, linear energy transfer 13 keV/µm), or fast neutrons (0.05-1 Gy, mean energy, ∼2 MeV), and lung-tumor development was assessed by histopathology. Mice irradiated with ≥2 Gy of carbon ions or ≥0.2 Gy of neutrons developed lung adenocarcinoma (AC) significantly sooner than did non-irradiated mice. The relative biological effectiveness values for carbon ions for lung AC development were 1.07 for male mice and 2.59 for females, and the corresponding values for neutrons were 4.63 and 4.57. Genomic analysis of lung ACs revealed alterations in genes involved in Egfr signaling. Hyperphosphorylation of Erk and a frequent nuclear abnormality (i.e., nuclear groove) were observed in lung ACs of mice irradiated with carbon ions or neutrons compared with ACs from non-irradiated or gamma-ray-irradiated groups. Our data indicate that the induction of lung AC by carbon ions occurred at a rate similar to that for gamma rays in males and approximately 2-to 3-fold greater than that for gamma rays in females. In contrast, the effect of neutrons on lung AC development was approximately 4- to 5-fold greater than that of gamma rays. Our results provide valuable information concerning risk assessment of radiation-induced lung tumors after particle-beam therapy and increase our understanding of the molecular basis of tumor development.

Calorie Restriction Suppresses the Progression of Radiation-Induced Intestinal Tumours in C3B6F1 Apc Min/+ Mice.

BACKGROUND/AIM: Progress in cancer treatment and diagnosis has made second cancer after medical radiation exposure a particular concern among childhood cancer survivors. Calorie restriction (CR) is a broadly effective cancer prevention strategy, although its effects on radiation-induced intestinal tumours are unclear. Here we examined the cancer-preventative efficacy of a CR diet at different starting ages on radiation induction of intestinal tumours in mice. MATERIALS AND METHODS: Male C3B6F1 ApcMin/+ mice were irradiated with 0 or 2 Gy of X-rays at 2 weeks of age. After an interval of 2, 8 or 18 weeks, mice were fed with a non-CR (95 kcal/week/mouse) or CR (65 kcal/week/mouse) diet. Intestinal tumours were evaluated for number, size distribution and malignancy. RESULTS: CR suppressed the size and progression of both spontaneous and radiation-induced intestinal tumours depending on age at starting of CR. CR diets were effective even administered to adult mice. CONCLUSION: CR was effective for suppression of tumour progression, which was accelerated by radiation exposure. Use of CR might be a useful cancer-prevention strategy for radiation-induced tumours of the intestinal tract.

Fermentative production of sulfur-containing amino acid with engineering putative l-cystathionine and l-cysteine uptake systems in Escherichia coli.

Here, proteins involved in sulfur-containing amino acid uptake in Escherichia coli strains were investigated with the aim of applying the findings in fermentative amino acid production. A search of genes in an l-methionine auxotrophic strain library suggested YecSC as the putative transporter of l-cystathionine. l-Methionine production increased by 15% after amplification of yecSC in producer strains. A candidate protein responsible for l-cysteine uptake was also found by experimentation with multicopy suppressor E. coli strains that recovered from growth defects caused by l-cysteine auxotrophy. Based on the results of an uptake assay, growth using l-cysteine as a sole sulfur source, and sensitivity to l-cysteine toxicity, we proposed that YeaN is an l-cysteine transporter. l-Cysteine production increased by 50% as a result of disrupting yeaN in producer strain. The study of amino acid transporters is valuable to industrialized amino acid production and also sheds light on the role of these transporters in sulfur assimilation.

Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets.

Glucokinase-mediated glucose signaling induces insulin secretion, proliferation, and apoptosis in pancreatic ß-cells. However, the precise molecular mechanisms underlying these processes are not clearly understood. Here, we demonstrated that glucokinase activation using a glucokinase activator (GKA) significantly upregulated the expression of Fibulin-5 (Fbln5), a matricellular protein involved in matrix-cell signaling, in isolated mouse islets. The islet Fbln5 expression was induced by ambient glucose in a time- and dose-dependent manner and further enhanced by high-fat diet or the deletion of insulin receptor substrate 2 (IRS-2), whereas the GKA-induced increase in Fbln5 expression was diminished in Irs-2-deficient islets. GKA-induced Fbln5 upregulation in the islets was blunted by a glucokinase inhibitor, KATP channel opener, Ca2+ channel blocker and calcineurin inhibitor, while it was augmented by harmine, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1 A inhibitor. Although deletion of Fbln5 in mice had no significant effects on the glucose tolerance or ß-cell functions, adenovirus-mediated Fbln5 overexpression increased glucose-stimulated insulin secretion in INS-1 rat insulinoma cells. Since the islet Fbln5 expression is regulated through a glucokinase/KATP channel/calcineurin/nuclear factor of activated T cells (NFAT) pathway crucial for the maintenance of ß-cell functions, further investigation of Fbln5 functions in the islets is warranted.

Metabolic recovery of lipodystrophy, liver steatosis, and pancreatic ß cell proliferation after the withdrawal of OSI-906.

Growth factor signaling via insulin receptor (IR) and IGF-1 receptor (IGF1R) plays several important roles in the pathogenesis of metabolic syndrome and diabetes. OSI-906 (linsitinib), an anti-tumor drug, is an orally bioavailable dual inhibitor of IR and IGF1R. To investigate the recovery from metabolic changes induced by the acute inhibition of IR and IGF1R in adult mice, mice were treated with OSI-906 or a vehicle for 7 days and the results were analyzed on the last day of injection (Day 7) or after 7 or 21 days of withdrawal (Day 14 or Day 28). On day 7, the visceral white fat mass was significantly reduced in mice treated with OSI-906 accompanied by a reduced expression of leptin and an increased expression of the lipolysis-related genes Lpl and Atgl. Interestingly, the lipoatrophy and the observed changes in gene expression were completely reversed on day 14. Similarly, liver steatosis and ß cell proliferation were transiently observed on day 7 but had disappeared by day 14. Taken together, these results suggest that this model for the acute inhibition of systemic IR/IGF1R signaling may be useful for investigating the recovery from metabolic disorders induced by impaired growth factor signaling.

ydjN encodes an S-sulfocysteine transporter required by Escherichia coli for growth on S-sulfocysteine as a sulfur source.

Sulfur is an essential element for growth and many physiological functions. As sulfur sources for Escherichia coli and related bacteria, specific transporters import various sulfur-containing compounds from the environment. In this study, we identified and characterized an alternative function of the cystine transporter YdjN in E. coli as a transporter of S-sulfocysteine, a sulfur-containing intermediate in the assimilatory cysteine biosynthesis that is used as a sulfur source for the growth of E. coli We also demonstrated that the transport of S-sulfocysteine via YdjN depends on the transcriptional regulator CysB, a master regulator that controls most of the genes involved in sulfur assimilation and cysteine metabolism. We found that the use of S-sulfocysteine as a sulfur source depends on glutathione because mutations in glutathione biosynthetic genes abolish growth when S-sulfocysteine is used as a sole sulfur source, thereby supporting the previous findings that the conversion of S-sulfocysteine to cysteine is catalyzed by glutaredoxins. To the best of our knowledge, this is the first report of a functional S-sulfocysteine transporter across organisms, which strongly supports the hypothesis that S-sulfocysteine is not only a metabolic intermediate but also a physiologically significant substance in specific natural environments.

DPP-4 inhibition improves early mortality, ß cell function, and adipose tissue inflammation in db/db mice fed a diet containing sucrose and linoleic acid.

BACKGROUND: Diabetes therapy that not only lowers glucose levels but also lengthens life spans is required. We previously demonstrated that DPP-4 inhibition ameliorated ß cell apoptosis and adipose tissue inflammation in ß cell-specific glucokinase haploinsufficient mice fed a diet containing a combination of sucrose and linoleic acid (SL). METHODS: In this study, we investigated the effects of DPP-4 inhibition in obese diabetic db/db mice fed an SL diet or a control diet containing sucrose and oleic acid (SO). We also examined the effects of DPP-4 inhibition in IRS-1-deficient mice fed an SL or SO diet as a model of insulin resistance. RESULTS: DPP-4 inhibition efficiently increases the active GLP-1 levels in db/db mice. Unexpectedly, the SL diet, but not the SO diet, markedly increases mortality in the db/db mice. DPP-4 inhibition reduces the early lethality in SL-fed db/db mice. DPP-4 inhibition improves glucose tolerance, ß cell function, and adipose tissue inflammation in db/db mice fed either diet. No significant changes in glycemic control or ß cell mass were observed in any of the IRS-1-deficient mouse groups. CONCLUSIONS: A diet containing a combination of sucrose and linoleic acid causes early lethality in obese diabetic db/db mice, but not in lean and insulin resistant IRS-1 knockout mice. DPP-4 inhibition has protective effects against the diet-induced lethality in db/db mice.

Evaluation of the appropriateness of using glucometers for measuring the blood glucose levels in mice.

Glucometers are also widely used in diabetes research conducted using animal models. However, the appropriateness of measuring blood glucose levels using glucometers in animal models remains unclear. In this study, we evaluated the consistency between the blood glucose levels measured by 11 models of glucometers and plasma glucose levels measured by a laboratory biochemical test in blood samples collected by retro-orbital sinus puncture or tail-tip amputation. In both blood samples obtained by retro-orbital sinus puncture and those obtained by tail-tip amputation, 10 of the 11 models of glucometers yielded higher glucose values, while 1 yielded lower glucose values, than the plasma glucose values yielded by the laboratory test, the differences being in direct proportion to the plasma glucose values. Most glucometers recorded higher blood glucose levels after glucose loading and lower blood glucose levels after insulin loading in retro-orbital sinus blood as compared to tail vein blood. Our data suggest that the blood glucose levels measured by glucometers in mice tended to be higher than the plasma glucose levels yielded by the biochemical test under the hyperglycemic state, and that differences in the measured levels were observed according to the blood collection method depending on the glycemia status.