5-aminolevulinic acid combined with ferrous ion reduces adiposity and improves glucose tolerance in diet-induced obese mice via enhancing mitochondrial function.

BACKGROUND: Mitochondrial dysfunction is associated with obesity and various obesity-associated pathological conditions including glucose intolerance. 5-Aminolevulinic acid (ALA), a precursor of heme metabolites, is a natural amino acid synthesized in the mitochondria, and various types of cytochromes containing heme contribute to aerobic energy metabolism. Thus, ALA might have beneficial effects on the reduction of adiposity and improvement of glucose tolerance through its promotion of heme synthesis. In the present study, we investigated the effects of ALA combined with sodium ferrous citrate (SFC) on obesity and glucose intolerance in diet-induced obese mice. METHODS: We used 20-weeks-old male C57BL/6J diet-induced obesity (DIO) mice that had been fed high-fat diet from 4th week or wild-type C57BL/6J mice. The DIO mice were orally administered ALA combined with SFC (ALA/SFC) for 6 weeks. At the 4th and 5th week during ALA/SFC administration, mice were fasted for 5 h and overnight, respectively and used for oral glucose tolerance test. After the ALA/SFC administration, the plasma glucose levels, weight of white adipose tissue, and expression levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes were examined. Furthermore, the effects of ALA/SFC on lipid content and glucose uptake were examined in vitro. RESULTS: Oral administration of ALA/SFC for 6 weeks reduced the body weight by about 10% and the weight of white adipose tissues in these animals. In vitro, ALA/SFC reduced lipid content in mouse 3T3-L1 adipocytes in a dose dependent manner, and enhanced glucose uptake in 3T3-L1 adipocytes by 70-90% and rat L6 myoblasts by 30% at 6 h. Additionally, oral administration of ALA/SFC reduced plasma glucose levels and improved glucose tolerance in DIO mice. Furthermore, ALA/SFC enhanced the expression of OXPHOS complexes III, IV, and V by 40-70% in white adipose tissues of DIO mice, improving mitochondrial function. CONCLUSIONS: Our findings indicate that ALA/SFC is effective in the reduction of adiposity and improvement of glucose tolerance, and that the induction of mitochondrial OXPHOS complex III, IV, and V by ALA/SFC might be an essential component of the molecular mechanisms underlying these effects. ALA/SFC might be a useful supplement for obesity and obesity-related metabolic disease such as type 2 diabetes mellitus.

The Safety and Tolerability of 5-Aminolevulinic Acid Phosphate with Sodium Ferrous Citrate in Patients with Type 2 Diabetes Mellitus in Bahrain.

Type 2 diabetes mellitus is prevalent especially in Gulf countries and poses serious long-term risks to patients. A multifaceted treatment approach can include nutritional supplements with antioxidant properties such as 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC). This prospective, randomized, single-blind, placebo-controlled, dose escalating pilot clinical trial assessed the safety of 5-ALA with SFC at doses up to 200 mg 5-ALA/229.42 mg SFC per day in patients living in Bahrain with type 2 diabetes mellitus that was uncontrolled despite the use of one or more antidiabetic drugs. Fifty-three patients (n = 53) from 3 sites at one center were enrolled by Dr. Feryal (Site #01), Dr. Hesham (Site #02), and Dr. Waleed (Site #03) (n = 35, 5-ALA-SFC; n = 18, placebo). There was no significant difference in incidence of adverse events reported, and the most frequent events reported were gastrointestinal in nature, consistent with the known safety profile of 5-ALA in patients with diabetes. No significant changes in laboratory values and no difference in hypoglycemia between patients receiving 5-ALA and placebo were noted. Overall, the current results support that use of 5-ALA-SFC up to 200 mg per day taken as 2 divided doses is safe in patients taking concomitant oral antidiabetic medications and may offer benefits in the diabetic population. This trial is registered with ClinicalTrials.gov NCT02481141.

Combination of 5-aminolevulinic acid and ferrous ion reduces plasma glucose and hemoglobin A1c levels in Zucker diabetic fatty rats.

Mitochondrial dysfunction is associated with type 2 diabetes mellitus (T2DM). 5-Aminolevulinic acid (ALA), a natural amino acid produced only in the mitochondria, is a precursor of heme. Cytochromes that contain heme play an important role in aerobic energy metabolism. Thus, ALA may help reduce T2DM-associated hyperglycemia. In this study, we investigated the effect of ALA combined with sodium ferrous citrate (SFC) on hyperglycemia in Zucker diabetic fatty (ZDF) rats. We found that the gavage administration of ALA combined with SFC (ALA/SFC) for 6 weeks reduced plasma glucose and hemoglobin A1c (HbA1c) levels in rats without affecting plasma insulin levels. The glucose-lowering effect depended on the amount of ALA/SFC administered per day. Furthermore, the glucose tolerance was also significantly improved by ALA/SFC administration. Although food intake was slightly reduced in the rats administered ALA/SFC, there was no effect on their body weight. Importantly, ALA/SFC administration induced heme oxygenase-1 (HO-1) expression in white adipose tissue and liver, and the induced expression levels of HO-1 correlated with the glucose-lowering effects of ALA/SFC. Taken together, these results suggest that ALA combined with ferrous ion is effective in reducing hyperglycemia of T2DM without affecting plasma insulin levels. HO-1 induction may be involved in the mechanisms underlying the glucose-lowering effect of ALA/SFC.

[Successful treatment of neuromyelitis optica spectrum disorder by early initiation of plasma exchange].

A 39-year-old woman initially developed vomiting and intractable hiccup, followed by progressive dysphagia, dysarthria and hypoglossal nerve palsy. She was admitted to our department on the 30th day of illness. MRI-FLAIR images of the brain revealed a hyperintense lesion in the dorsal medulla. A diagnosis of neuromyelitis optica spectrum disorder (NMOSD) was entertained according to the clinical course and the MRI images. The dysphagia was intractable to methylprednisolone pulse therapy, and so a course of plasma exchange therapy was initiated on the 32nd day of illness. After the third plasma exchange, the symptoms began to improve. Thereafter the patient's serum on admission was reported as positive for anti-aquaporin-4 antibody. Considering the irreversible nature of NMOSD pathology, early initiation of plasma exchange therapy is recommended to minimize the lesion in the case of steroid-refractory NMOSD patients.

Alternately binding probe competitive PCR as a simple, cost-effective, and accurate quantification method for JAK2V617F allele burden in myeloproliferative neoplasms.

We developed a simple, cost-effective, and accurate JAK2 allele burden quantification method named alternately binding probe competitive PCR (ABC-PCR). ABC-PCR can be performed to quantify target JAK2 allele burdens in a single reaction. The throughput and running cost of ABC-PCR are markedly improved compared with those of allele-specific quantitative PCR (AS-qPCR). The quantification of samples with known JAK2 allele burdens revealed that ABC-PCR had a small assay-to-assay variation. The JAK2 allele burdens in the patients with myeloproliferative neoplasms measured by ABC-PCR and AS-qPCR showed a good fitting. ABC-PCR would be a powerful tool for quantifying target JAK2 allele burdens.

Urinary prostasin in humans: relationships among prostasin, aldosterone and epithelial sodium channel activity.

Prostasin, a membrane-bound serine protease, is known to increase the activity of the epithelial sodium channel (ENaC). Gene expression of prostasin was shown to be regulated by aldosterone, which increases the rate of sodium reabsorption through ENaC. To clarify the physiological relationships among prostasin, aldosterone and ENaC, we developed a specific radioimmunoassay (RIA) for human prostasin. Prostasin levels in urine were determined in 26 normotensive and 121 hypertensive subjects. Aldosterone content in urine and plasma, urinary Na/K ratio and other clinical parameters were also measured. We observed a highly significant correlation between prostasin and aldosterone concentration in urine (correlation coefficient: 0.673, P<0.0001). A significant correlation was also found between urinary prostasin and plasma aldosterone concentration (correlation coefficient: 0.229, P<0.05). In addition, urinary prostasin excretion was inversely correlated with urinary Na/K ratio (correlation coefficient: -0.425, P<0.0001). In conclusion, we developed a prostasin-specific RIA and applied it to the clinical study. Our findings suggest that urinary prostasin level is strongly correlated with urinary or plasma aldosterone level and may serve as a surrogate marker for ENaC activation in hypertensive patients. However, it is not clear, at the present time, whether endogenous aldosterone regulates prostasin expression or vice versa.

Structures and diverse functions of frog growth hormone-releasing peptide (fGRP) and its related peptides (fGRP-RPs): a review.

A new Arg-Phe-NH(2) (RFamide) peptide has been discovered in the amphibian hypothalamus. The cell bodies and terminals containing this peptide were localized in the suprachiasmatic nucleus and median eminence, respectively. This peptide was further revealed to have a considerable growth hormone (GH)-releasing activity in vitro and in vivo and hence designated as frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the fGRP precursor polypeptide revealed that it encodes fGRP and its putative gene-related peptides (fGRP-RP-1, -RP-2, and -RP-3). Subsequently, we identified these putative fGRP-RPs as mature peptides and analyzed their hypophysiotropic activities. Only fGRP-RP-2 stimulated the release of GH and prolactin (PRL) in vitro and in vivo. Thus, in addition to fGRP, fGRP-RP-2 acts as a hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.

Identification of immunoreactive plasma and stomach ghrelin, and expression of stomach ghrelin mRNA in the bullfrog, Rana catesbeiana.

In this study, we established a radioimmunoassay (RIA) specific for ghrelin from the bullfrog Rana catesbeiana using a novel antibody raised against the C-terminal amino acid sequence of bullfrog ghrelin [13-28]. We also examined the distribution of ghrelin-producing cells in the stomachs of bullfrogs using this antibody and a cRNA probe specific for the bullfrog ghrelin gene. Ghrelin levels in plasma and stomach extracts were approximately 150 fmol/ml and 83-135 fmol/mg wet tissue, respectively. Reverse-phase high performance liquid chromatographic analysis, combined with bullfrog ghrelin RIA, revealed that ghrelin immunoreactivity in the stomach was composed of non-acylated ghrelin (des-acyl ghrelin) and several acylated forms of ghrelin bearing different fatty acid modifications, which could induce increases in intracellular Ca2+ in cells expressing the rat GH secretagogue receptor. In the stomach, the major storage form was acylated ghrelin. In bullfrog plasma, however, the majority of ghrelin immunoreactivity was des-acyl ghrelin and C-terminal fragments of frog ghrelin. Acylated ghrelin forms comprised only minor peaks. Ghrelin-immunopositive and ghrelin mRNA-expressing cells were observed within the mucosal layer of the stomach. Following starvation, significant increases in plasma ghrelin levels and stomach ghrelin mRNA levels were observed as early as 10 days after starvation. These results indicate that ghrelin is present in the stomach and plasma of the bullfrog, which can be detected with our novel antibody. Interestingly, the primary storage form of ghrelin in the stomach differed from the circulating form dominating in the plasma. Furthermore, increases in ghrelin levels in plasma and mRNA levels in the stomach after starvation suggest the possible involvement of ghrelin in energy homeostasis in the bullfrog.

Development of radioimmunoassay for bullfrog thyroid-stimulating hormone (TSH): effects of hypothalamic releasing hormones on the release of TSH from the pituitary in vitro.

A bullfrog (Rana catesbeiana) thyroid-stimulating hormone (TSH) beta-subunit (TSHbeta) antiserum was produced by employing a C-terminal peptide synthesized on the basis of the amino acid sequence deduced from bullfrog TSHbeta cDNA. Immunohistochemical studies revealed that the bullfrog adenohypophyseal cells that immunologically reacted with the anti-bullfrog TSHbeta corresponded to those positively stained with an antiserum against human (h) TSHbeta. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of bullfrog TSH. The sensitivity of the RIA was 0.75+/-0.07ng TSH/100microl assay buffer. The interassay and intraassay coefficients of variation were 7.6 and 5.3%, respectively. Several dilutions of pituitary homogenates of larval and adult bullfrogs, or medium in which bullfrog pituitary cells were cultured, yielded dose-response curves that were parallel to the standard curve. Bullfrog prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and alpha-subunit derived from glycoprotein hormones did not react in this assay. Immunoassayable TSH in the pituitary culture medium was confirmed to exist in the form of TSHbeta coupled with the alpha-subunit by an immunoprecipitation experiment using the TSHbeta antiserum and an alpha-subunit antiserum. TSH released from pituitary cells into the medium was also confirmed to possess a considerable activity in stimulating the release of thyroxine from the thyroid glands of larval bullfrogs in vitro. The effects of hypothalamic hormones such as mammalian gonadotropin-releasing hormone (mGnRH), ovine corticotropin-releasing hormone (oCRH), and thyrotropin-releasing hormone (TRH) on the release of TSH by dispersed anterior pituitary cells of the bullfrog larvae and adults were also studied. CRH markedly stimulated the release of TSH from both adult and larval pituitary cells. Both TRH and GnRH moderately stimulated the release of TSH from adult pituitary cells but not from the larval cells. This is the first report on the development of an RIA for amphibian TSH, which has provided the direct evidence that the release of TSH from the amphibian pituitary is enhanced by the hypothalamic releasing hormones such as CRH, TRH, and GnRH.

Novel neuropeptides related to frog growth hormone-releasing peptide: isolation, sequence, and functional analysis.

We previously identified in the bullfrog a novel hypothalamic RFamide peptide (SLKPAANLPLRF-NH(2)) that stimulated GH release in vitro and in vivo and therefore was designated frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the deduced fGRP precursor polypeptide further revealed that it encodes fGRP and its related peptides (fGRP-RP-1, -RP-2, and -RP-3). In this study immunoaffinity purification using the antibody against fGRP was therefore conducted to determine whether these three putative fGRP-RPs exist as mature endogenous ligands in the frog brain. The mass peaks of the isolated immunoreactive substances were detected at 535.78, 1034.14, and 1079.71 m/z ([M+2H](2+)), and their sequences, SIPNLPQRF-NH(2), YLSGKTKVQSMANLPQRF-NH(2), and AQYTNHFVHSLDTLPLRF-NH(2), were revealed by the fragmentation, showing mature forms encoded in the cDNA sequences of fGRP-RP-1, -RP-2, and -RP-3, respectively. All of these fGRP-RPs contained a C-terminal -LPXRF-NH(2) (X = L or Q) sequence, such as fGRP. This study further analyzed hypophysiotropic activities of the identified endogenous fGRP-RPs. Only fGRP-RP-2 stimulated, in a dose-related way, the release of PRL from cultured frog pituitary cells; its threshold concentration ranged from less than 10(-7) M. A similar stimulatory action of fGRP-RP-2 on GH release was evident. It was ascertained that fGRP-RP-2 was also effective in elevating the circulating GH and PRL levels when administered systemically. In contrast, fGRP-RPs did not have any appreciable effect on the release of gonadotropins. Thus, fGRP-RP-2 may act as a novel hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.

A novel amphibian hypothalamic neuropeptide: isolation, localization, and biological activity.

Neuropeptides similar to the molluscan cardioexcitatory Phe-Met-Arg-Phe-NH2 have been identified in several vertebrates and characterized by the RFa motif at their C terminus (RFa peptides). In this study, we sought to identify an amphibian hypothalamic RFa peptide that may regulate secretion of hormones by the anterior pituitary gland. An acid extract of bullfrog hypothalami was passed through C-18 reversed-phase cartridges, and then the retained material was subjected to HPLC, initially using a C-18 reversed-phase column. RFa immunoreactivity was measured in the eluted fractions by a dot immunoblot assay employing an antiserum raised against RFa. Immunoreactive fractions were subjected to further cation exchange and reversed-phase HPLC purification. The isolated peptide was a novel RFa peptide and shown to have the sequence Ser-Leu-Lys-Pro-Ala-Ala-Asn-Leu-Pro-Leu-Arg-Phe-NH2. The cell bodies and terminals containing this peptide were localized immunohistochemically in the suprachiasmatic nucleus and median eminence, respectively. This RFa peptide stimulated, in a dose-related way, the release of GH from cultured pituitary cells, its threshold concentration ranging between 10(-9) and 10(-8) M. This peptide did not have any appreciable effect on the secretion of PRL and gonadotropins. It was ascertained that the peptide was also effective in elevating the circulating GH level when administered systemically. Thus, the amphibian hypothalamus was revealed to contain a novel functional RFa peptide that stimulates GH release. This peptide was designated frog GH-releasing peptide.