Administration of heme arginate ameliorates murine type 2 diabetes independently of heme oxygenase activity

Amelioration of rodent type 2 diabetes by hemin has been linked to increased heme oxygenase (HO) activity, however alternative mechanisms have recently been proposed for its anti-diabetic effect. We sought to determine the anti-diabetic efficacy of heme arginate (HA), a clinically licensed preparation of heme, and whether its predominant mode of action is via increased HO activity. Intravenous administration of HA reduced hyperglycemia in diabetic (db/db) mice. Co-administration of the HO inhibitor stannous (IV) mesoporphyrin IX dichloride (SM) resulted unexpectedly in a further improvement in glycaemic control despite restoring HO activity to baseline levels. The anti-diabetic effects of HA±SM were associated with increased adiposity, increased serum adiponectin levels, reduced adipose tissue and islet inflammation and preservation of islet β-cell function. HO activity independent effects of HA on adipogenesis and β-cell inflammation were further confirmed in cell culture models using the 3T3-L1 pre-adipocyte and MIN6 β-cell lines, respectively. In conclusion, our work demonstrates that the heme component of HA ameliorates experimental type 2 diabetes by promoting metabolically favourable adipogenesis and preserving islet β-cell function, but this is not mediated via increased HO activity

Behavioral Problems and Intelligence Quotient Changes in Pediatric Epilepsy: A Case–Control Study

Background: Epilepsy is the most common chronic neurological disease and has neurological impairment as an important comorbidity. Objective: To find behavioral problems and intelligence quotient (IQ) changes associated with epilepsy and to know the association of variables such as frequency, type of seizures, and duration of disease with cognitive impairment. Materials and Methods: A descriptive cross sectional study, consisting of 50 cases (patients of epilepsy) and 50 controls (other patients of same socioeconomic status) was conducted at S.P. Medical College, Bikaner. The patients were subjected to detailed clinical history, thorough examination, Pediatric Symptom Checklist, and Bhatia's Battery of Performance intelligence Test. Data analysis was carried out with the help of SPSS 22 software. Results: The prevalence of behavioral problems in generalized and partial seizure group was high (42% and 53.8%) as compared to control group (9%). Low IQ was present more in the patients (44%) of generalized and partial seizure group as compared with the control group, and results were statistically significant. Furthermore, behavioral problems were more in patients who were having more number of seizures (≥3 per year) with significant P values (χ2 = 5.067, P = 0.024). Conclusion: We conclusively found that behavioral problems and cognitive factors, apart from control of seizures, must be kept in mind to determine how well a child with epilepsy progresses toward independence

HA+SM increases islet area but reduces islet macrophage infiltration and expression of iNOS protein.

<p><b>A.</b> Representative images of the islets from each treatment group. β-cells are visualised by staining with anti-insulin antibody (green), with either F4/80⁺ macrophages (upper panel) or iNOS positive cells (lower panel) stained in brown. Scale bars represent 50 µM. Quantification of <b>B.</b> islet cross-sectional surface area; and number of <b>C.</b> F4/80⁺ macrophages or <b>D.</b> iNOS⁺ cells per unit islet area in each treatment group. ^‡P<0.05,^‡‡‡P<0.001 v lean PBS, *P<0.05, **P<0.01, ***P<0.001 v db/db PBS, ^††P<0.01 v db/db HA. P-values derived from Bonferroni’s multiple comparison post-test when P<0.05 by one-way analysis of variance. The data are mean ± s.e.m from n>25 islets/mouse.</p

HA±SM improved glycaemic control and β-cell function despite a significant increase in body weight in db/db mice.

<p><b>A.</b> Fasting glucose, <b>B.</b> Body weight, <b>C.</b> HOMA–IR as measure of insulin resistance and <b>D.</b> HOMA-β as measure β-cell function during administration of PBS (n = 4), HA (n = 4), SM (n = 5) and HA+SM (n = 8) to db/db mice for 8 weeks. ^†P<0.05: HA v PBS; *P<0.05, **P<0.01, ***P<0.001: HA+SM v PBS, ^#P<0.05, ^##P<0.01: HA+SM v HA. P-values derived by 2-way ANOVA with Bonferroni’s multiple comparison post-test. Data given are means ± s.e.m.</p

HA±SM reduces cytokine mix (CM) induced pro-inflammatory response in MIN6 β-cell line.

<p>Relative gene expression of <b>A.</b> iNOS, <b>B.</b> IL-1β, C. MCP-1α, <b>D.</b> Cxcl-1 in MIN6 β-cells pre-treated with either PBS, HA, SM or HA+SM prior to stimulation with cytokine mix. <b>E.</b> HO activity in MIN6 β-cells. *P<0.05, **P<0.01, ***P<0.001. P-values derived from Bonferroni’s multiple comparison post-test when P<0.05 by one-way analysis of variance. The data are means ± s.e.m from n = 4 independent experiments/group.</p

Clinical parameters in the pilot study examining the efficacy of heme arginate (HA) and L-arginine (LA) in the db/db mouse model of type 2 diabetes.

<p>Clinical parameters in the pilot study examining the efficacy of heme arginate (HA) and L-arginine (LA) in the db/db mouse model of type 2 diabetes.</p

Administration of SM further augmented HO-1 protein expression but abrogated the increase in HO activity induced by HA.

<p>Representative immuno-blots of HO-1 expression in <b>A.</b> Epididymal fat and <b>B.</b> Liver. <b>C.</b> Hepatic HO activity in each treatment group. ***P<0.001, P-values derived from Bonferroni’s multiple comparison post-test where P<0.05 by one-way analysis of variance. Data given are means ± s.e.m.</p

Concomitant administration of SM accentuates adipogenic potential of HA <i>in vitro</i>.

<p><b>A.</b> HO activity, <b>B.</b> Heme concentrations, <b>C.</b> Quantification of Oil Red staining and <b>D.</b> Representative light microscopic (x100) image of Oil Red uptake in differentiated adipocytes following 10 days treatment of 3T3 cells with an adipogenic differentiation cocktail alone or with the addition of protoporphyrins. B, C: *P<0.05, **P<0.01, ***P<0.001 v PBS; ^†P<0.05, ^††P<0.01 v HA. <b>E.</b> Time course changes in gene expression of adipogenic genes including PPAR-γ, Adipoq, FASN in 3T3-L1 cells treated with an adipogenic differentiation cocktail alone or with the addition of 20 µM HA, 20 µM SM or HA+SM. ^†††P<0.001: HA v PBS; ^‡P<0.05,^‡‡‡P<0.001: SM v PBS, ***P<0.001: HA+SM v PBS; ^#P<0.05, ^##P<0.01: HA+SM v HA. P-values derived by 2-way ANOVA with Bonferroni’s multiple comparison post-test. The data are mean ± s.e.m from n = 4/group. F. Representative (200x) images of Oil Red staining of stromal vascular fraction cells from epididymal fat pads of HO-1^+/+ (left) and HO-1^+/− (right) mice either undifferentiated or following differentiation by an adipogenic cocktail in the presence of HA.</p