Tetsuji Okawa

Beneficial Effects of Exendin-4 on Experimental Polyneuropathy in Diabetic Mice

OBJECTIVE The therapeutic potential of exendin-4, an agonist of the glucagon-like peptide-1 receptor (GLP-1R), on diabetic polyneuropathy (DPN) in streptozotocin (STZ)-induced diabetic mice was investigated. RESEARCH DESIGN AND METHODS The presence of the GLP-1R in lumbar dorsal root ganglion (DRG) was evaluated by immunohistochemical analyses. DRG neurons were dissected from C57BL6/J mice and cultured with or without Schwann cell–conditioned media in the presence or absence of GLP-1 (7–37) or exendin-4. Then neurite outgrowth was determined. In animal-model experiments, mice were made diabetic by STZ administration, and after 12 weeks of diabetes, exendin-4 (10 nmol/kg) was intraperitoneally administered once daily for 4 weeks. Peripheral nerve function was determined by the current perception threshold and motor and sensory nerve conduction velocity (MNCV and SNCV, respectively). Sciatic nerve blood flow (SNBF) and intraepidermal nerve fiber densities (IENFDs) also were evaluated. RESULTS The expression of the GLP-1R in DRG neurons was confirmed. GLP-1 (7–37) and exendin-4 significantly promoted neurite outgrowth of DRG neurons. Both GLP-1R agonists accelerated the impaired neurite outgrowth of DRG neurons cultured with Schwann cell–conditioned media that mimicked the diabetic condition. At the doses used, exendin-4 had no effect on blood glucose or HbA1c levels. Hypoalgesia and delayed MNCV and SNCV in diabetic mice were improved by exendin-4 without affecting the reduced SNBF. The decreased IENFDs in sole skins of diabetic mice were ameliorated by exendin-4. CONCLUSIONS Our findings indicate that exendin-4 ameliorates the severity of DPN, which may be achieved by its direct actions on DRG neurons and their axons.

Mesenchymal stem cells ameliorate impaired wound healing through enhancing keratinocyte functions in diabetic foot ulcerations on the plantar skin of rats

AIMS/HYPOTHESIS Although the initial healing stage involves a re-epithelialization in humans, diabetic foot ulceration (DFU) has been investigated using rodent models with wounds on the thigh skin, in which a wound contraction is initiated. In this study, we established a rodent model of DFU on the plantar skin and evaluated the therapeutic efficacy of bone-marrow-derived mesenchymal stem cells (BM-MSCs) in this model. METHODS The wounds made on the hind paws or thighs of streptozotocin induced diabetic or control rats were treated with BM-MSCs. Expression levels of phosphorylated focal adhesion kinase (pFAK), matrix metaroprotease (MMP)-2, EGF, and IGF-1, were evaluated in human keratinocytes, which were cultured in conditioned media of BM-MSCs (MSC-CM) with high glucose levels. RESULTS Re-epithelialization initiated the healing process on the plantar, but not on the thigh, skin. The therapy utilizing BM-MSCs ameliorated the delayed healing in diabetic rats. In the keratinocytes cultured with MSC-CM, the decreased pFAK levels in the high glucose condition were restored, and the MMP2, EGF, and IGF-1 levels increased. CONCLUSIONS/INTERPRETATION Our study established a novel rat DFU model. The impaired healing process in diabetic rats was ameliorated by transplantation of BM-MSCs. This amelioration might be accounted for by the modification of keratinocyte functions.

Mesenchymal Stem Cell-Like Cells Derived from Mouse Induced Pluripotent Stem Cells Ameliorate Diabetic Polyneuropathy in Mice

Background. Although pathological involvements of diabetic polyneuropathy (DPN) have been reported, no dependable treatment of DPN has been achieved. Recent studies have shown that mesenchymal stem cells (MSCs) ameliorate DPN. Here we demonstrate a differentiation of induced pluripotent stem cells (iPSCs) into MSC-like cells and investigate the therapeutic potential of the MSC-like cell transplantation on DPN. Research Design and Methods. For induction into MSC-like cells, GFP-expressing iPSCs were cultured with retinoic acid, followed by adherent culture for 4 months. The MSC-like cells, characterized with flow cytometry and RT-PCR analyses, were transplanted into muscles of streptozotocin-diabetic mice. Three weeks after the transplantation, neurophysiological functions were evaluated. Results. The MSC-like cells expressed MSC markers and angiogenic/neurotrophic factors. The transplanted cells resided in hindlimb muscles and peripheral nerves, and some transplanted cells expressed S100β in the nerves. Impairments of current perception thresholds, nerve conduction velocities, and plantar skin blood flow in the diabetic mice were ameliorated in limbs with the transplanted cells. The capillary number-to-muscle fiber ratios were increased in transplanted hindlimbs of diabetic mice. Conclusions. These results suggest that MSC-like cell transplantation might have therapeutic effects on DPN through secreting angiogenic/neurotrophic factors and differentiation to Schwann cell-like cells.

The Role of S100B in the Interaction Between Adipocytes and Macrophages

The S100 calcium binding protein B (S100B) implicated in brain inflammation acts via the receptor of advanced glycation end products (RAGE) and is also secreted from adipocytes. We investigated the role of S100B in the interaction between adipocytes and macrophages using a cell‐culture model.

Transplantation of Neural Crest-Like Cells Derived from Induced Pluripotent Stem Cells Improves Diabetic Polyneuropathy in Mice:

Impaired vascularity and nerve degeneration are the most important pathophysiological abnormalities of diabetic polyneuropathy (DPN). Therefore, regeneration of both the vascular and nervous systems is required for the treatment of DPN. The neural crest (NC) is a transient embryonic structure in vertebrates that differentiates into a vast range of cells, including peripheral neurons, Schwann cells, and vascular smooth muscle cells. In this study, we investigated the ability of transplantation of NC-like (NCL) cells derived from aged mouse induced pluripotent stem (iPS) cells in the treatment of DPN. iPS cells were induced to differentiate into neural cells by stromal cell-derived inducing activity (SDIA) and subsequently supplemented with bone morphogenetic protein 4 to promote differentiation of NC lineage. After the induction, p75 neurotrophin receptor-positive NCL cells were purified using magnetic-activated cell sorting. Sorted NCL cells differentiated to peripheral neurons, glial cells, and smooth muscle cells by additional SDIA. NCL cells were transplanted into hind limb skeletal muscles of 16-week streptozotocin-diabetic mice. Nerve conduction velocity, current perception threshold, intraepidermal nerve fiber density, sensitivity to thermal stimuli, sciatic nerve blood flow, plantar skin blood flow, and capillary number-to-muscle fiber ratio were evaluated. Four weeks after transplantation, the engrafted cells produced growth factors: nerve growth factor, neurotrophin 3, vascular endothelial growth factor, and basic fibroblast growth factor. It was also confirmed that some engrafted cells differentiated into vascular smooth muscle cells or Schwann cell-like cells at each intrinsic site. The transplantation improved the impaired nerve and vascular functions. These results suggest that transplantation of NCL cells derived from iPS cells could have therapeutic effects on DPN through paracrine actions of growth factors and differentiation into Schwann cell-like cells and vascular smooth muscle cells.

Therapeutic efficacy of bone marrow‐derived mononuclear cells in diabetic polyneuropathy is impaired with aging or diabetes

Recent studies have shown that cell transplantation therapies, such as endothelial precursor cells, bone marrow‐derived mononuclear cells (BM‐MNCs) and mesenchymal stem cells, are effective on diabetic polyneuropathy through ameliorating impaired nerve blood flow in diabetic rats. Here, we investigated the effects of BM‐MNCs transplantation in diabetic polyneuropathy using BM‐MNCs derived from adult (16‐week‐old) diabetic (AD), adult non‐diabetic (AN) or young (8‐week‐old) non‐diabetic (YN) rats.

Angioblast Derived from ES Cells Construct Blood Vessels and Ameliorate Diabetic Polyneuropathy in Mice.

Background. Although numerous reports addressing pathological involvements of diabetic polyneuropathy have been conducted, a universally effective treatment of diabetic polyneuropathy has not yet been established. Recently, regenerative medicine studies in diabetic polyneuropathy using somatic stem/progenitor cell have been reported. However, the effectiveness of these cell transplantations was restricted because of their functional and numerical impairment in diabetic objects. Here, we investigated the efficacy of treatment for diabetic polyneuropathy using angioblast-like cells derived from mouse embryonic stem cells. Methods and Results. Angioblast-like cells were obtained from mouse embryonic stem cells and transplantation of these cells improved several physiological impairments in diabetic polyneuropathy: hypoalgesia, delayed nerve conduction velocities, and reduced blood flow in sciatic nerve and plantar skin. Furthermore, pathologically, the capillary number to muscle fiber ratios were increased in skeletal muscles of transplanted hindlimbs, and intraepidermal nerve fiber densities were ameliorated in transplanted plantar skin. Transplanted cells maintained their viabilities and differentiated to endothelial cells and smooth muscle cells around the injection sites. Moreover, several transplanted cells constructed chimeric blood vessels with recipient cells. Conclusions. These results suggest that transplantation of angioblast like cells induced from embryonic stem cells appears to be a novel therapeutic strategy for diabetic polyneuropathy.

Sensory and motor physiological functions are impaired in gastric inhibitory polypeptide receptor‐deficient mice

Gastric inhibitory polypeptide (GIP) is an incretin secreted from the gastrointestinal tract after an ingestion of nutrients, and stimulates an insulin secretion from the pancreatic islets. Additionally, GIP has important roles in extrapancreatic tissues: fat accumulation in adipose tissue, neuroprotective effects in the central nervous system and an inhibition of bone resorption. In the current study, we investigated the effects of GIP signaling on the peripheral nervous system (PNS).

A novel loss-of-function mutation of GATA3 (p.R299Q) in a Japanese family with Hypoparathyroidism, Deafness, and Renal Dysplasia (HDR) syndrome

BackgroundHypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is a rare autosomal dominant disorder caused by mutations in the zinc finger transcription factor gene, GATA3. GATA3 has 2 zinc finger domains, which play an important role in the increase in target gene transcription activity.Case presentationA 50-year-old woman and her 27-year-old daughter were followed up because of hypoparathyroidism. They had bilateral sensorineural deafness. Abdominal computed tomography scanning revealed renal dysplasia in the mother, but no renal anomaly in the daughter. Direct sequencing of GATA3 gene revealed a novel heterozygous missense mutation at codon 299 (p.R299Q) in exon 4. This mutation is located at the junction between the 2 zinc fingers. The structure prediction showed that it caused a conformation change in this junction area, affecting the spatial position of the zinc fingers. Additionally, a more marked conformation change was observed in the N-terminal zinc finger region compared to that in the C-terminal region. Functional analysis of this mutant protein using an in vitro luciferase reporter assay system confirmed that the mutation abolished the enhancing effects of wild-type GATA3 on the promoter activity of the consensus GATA responsive element and that of human PTH gene.ConclusionWe identified a novel R299Q mutation in GATA3 in a Japanese family with HDR syndrome. We confirmed that R299Q is a loss-of-function mutation, due to the extensive conformational change in the zinc fingers of GATA3.

Deceptive HbA1c in a patient with pure red cell aplasia

In September, 2008, a 59-year-old Japanese woman with pure red cell aplasia was diagnosed with diabetes mellitus by a haematologist, on the basis of a high concentration of HbA1c (8·2%). At the time of diagnosis, her fasting plasma glucose was 5·8 mmol/L. She immediately began taking acarbose, 300 mg daily. About 3 years later, she began sitagliptin, a dipeptidyl peptidase-4 inhibitor, at a daily dose of 50 mg. Her blood haemoglobin concentration remained low (50–70 g/L). Despite additional treatment with longterm blood transfusions (but not recombinant human erythropoietin), her HbA1c remained high (8·0%). Therefore, the haematologist deemed her glycaemic control to be a more serious problem than her HbA1c concentration. In May, 2012, this patient was referred to our department for help to control her diabetes mellitus. High-performance liquid chromatography (HPLC) and immunoassay both indicated similar HbA1c con centrations, and the HPLC chromatogram contained no abnormal haemoglobin peaks. Despite her high HbA1c level, fasting plasma glucose (6·5 mmol/L), 1-h postprandial glucose (6·9 mmol/L), glycated albumin (14·5%), and 1,5-anhydroglucitol (67·0 μmol/L) showed only mild glucose intolerance. Thus, we judged this patient to have near-normal glucose tolerance and recommended that she stop taking oral hypoglycaemic agents. After stopping these agents, in her fi nal follow-up in November, 2012, fasting plasma glucose, glycated albumin, and 1,5-anhydroglucitol were still stable (6·6 mmol/L, 16·0%, and 112·0 μmol/L, respec tively), but her HbA1c was inappropriately high (7·8%). HbA1c tests are more stable and convenient than are tests for fasting plasma glucose and oral glucose tolerance. For this and other reasons, in 2009 an International aExpert Committee recommended that these tests be used to diagnose diabetes. Moreover, HbA1c tests are widely used for glycaemic management, since HbA1c is a pivotal biomarker whose high concentrations strongly predict complications of diabetes. However, some physicians mistakenly believe that a patient’s HbA1c concentration always reveals that patient’s diabetic status. Certain types of anaemia tend to make an HbA1c test inaccurate. For example, anaemia with abnormal erythrocyte turn over, such as haemolytic anaemia, nephrogenic anaemia treated with e rythropoietin, blood transfusions, and secondary anaemia due to liver cirrhosis all lower HbA1c, whereas iron defi ciency anaemia increases it (panel). In addition, in people with abnormal haemo globin, a condition sometimes associated with anaemia, the HbA1c con centration diff ers dependent on the method used to measure it. Thus, use of HbA1c concentrations to diagnose diabetes in patients with anaemia can lead to overdiagnosis or underdiagnosis in such patients. To the best of our knowledge, our report is the fi rst to show that pure red cell aplasia, a form of aplastic anaemia, can raise HbA1c to an inappropriate concentration. High HbA1c in patients with pure red cell aplasia might be due to slow erythrocyte turnover such as iron defi ciency anaemia that increases the mean age of circulating erythrocytes in the setting of decreased erythropoiesis and raises a proportion of highly glycated haemoglobin, although further studies are needed. In any case, tests measuring serum glycated albumin and 1,5-anhydroglucital, which are usually unaff ected by pure red cell aplasia, should be used instead of HbA1c tests to monitor glycaemic control in patients with pure red cell aplasia.