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Oxytocin (OXT) analogues have been designed to overcome the limitation of the short half-life of the native OXT peptide. Here, we tested ASK2131 on obesity related outcomes in diet-induced obese (DIO) Sprague Dawley rats. In vitro function assays were conducted. The effects of daily subcutaneous injections of ASK2131 vs. OXT and pair-feeding were assessed on food intake and body weight in vivo. ASK2131 is a longer-lasting OXT analog with improved pharmacokinetics compared to OXT (T1/2: 2.3 vs. 0.12 h). In chronic 22-day administration, ASK2131 was administered at 50 nmol/kg, while OXT doses were titrated up to 600 nmol/kg because OXT appeared to be less effective at reducing energy intake relative to ASK2131 at equimolar doses. After 22 days, vehicle-treated animals gained 4.5% body weight, OXT rats maintained their body weight, while those treated with ASK2131 declined in weight continuously over the 22-day period, leading to a 6.6 ± 1.3% reduction (mean ± standard error) compared to baseline. Compared to their pair-fed counterparts, ASK2131-treated rats showed a more pronounced reduction in body weight through most of the study. In summary, ASK2131 is a promising OXT-based therapeutic, with extended in vivo stability and improved potency leading to a profound reduction in body weight partly explained by reduced food intake. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.12.009 Byline: Wataru Nishimura (a)(b), Sheldon Rowan (b)(c), Therese Salameh (a), Richard L. Maas (b)(c), Susan Bonner-Weir (a)(b), Susan M. Sell (d), Arun Sharma (a)(b) Keywords: MafB; MafA; Pax6; Sey; Kreisler; Insulin gene transcription factor; Pancreatic development; Endocrine differentiation; Pancreatic islets Abstract: During pancreatic development insulin.sup.+ cells co-express the transcription factors MafB and Pax6, and transition from a MafA.sup.- to MafA.sup.+ state. To examine the role of Pax6 and MafB in the development of [beta]-cells, we analyzed embryonic pancreata from Pax6- and MafB-deficient mice. Pax6 deficiency, as manifest in the Pax6.sup.Sey-Neu allele, reduced not only the number of cells expressing insulin or glucagon, but also the number of MafB, PDX-1 and MafA expressing cells. We show that MafB can directly activate expression of insulin and glucagon, and a MafB protein engineered to contain N248S mutation in the MafB (kr.sup.ENU) results in significantly reduced activation. Furthermore, pancreata from MafB deficient (kr.sup.ENU /kr.sup.ENU) mice exhibited reduced number of cells expressing insulin, glucagon, PDX-1 and MafA, with only a minor reduction in MafB expressing cells. MafB deficiency does not affect endocrine specification but does affect the lineage commitment of the endocrine cells and their maturation. Similar to Pax6 deficient mice, MafB deficient mice showed reductions both in insulin and glucagon expressing cells and in the ability of MafB and PDX-1 expressing cells to activate expression of these hormones. However, MafB deficient mice exhibited no effect on Pax6 expression. These results suggest that MafB may function as a downstream mediator of Pax6 in regulating the specification of insulin and glucagon expressing cells. Interestingly, the remaining insulin.sup.+ cells in these knockouts preferentially express Hb9, suggesting the existence of an alternate pathway for the generation of insulin expressing cells, even in the absence of Pax6 and MafB function. Thus, Pax6 acts upstream of MafB, which in turn may trigger the expression of insulin and regulate the PDX-1 and MafA expression required for [beta]-cell maturation. Author Affiliation: (a) Section of Islet Transplantation and Cell Biology, Joslin Diabetes Center, USA (b) Department of Medicine, Harvard Medical School, Boston, MA 02215, USA (c) Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02215, USA (d) Bioinformatics Research Center and Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA Article History: Received 30 August 2007; Revised 6 December 2007; Accepted 10 December 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.02.028 Byline: Wataru Nishimura (a)(b), Takuma Kondo (a)(b), Therese Salameh (a), Ilham El Khattabi (a)(b), Rikke Dodge (a)(b), Susan Bonner-Weir (a)(b), Arun Sharma (a)(b) Keywords: MafA; MafB; Maf factors; Insulin gene transcription factor; Pancreatic development; Endocrine differentiation; Pancreatic islets Abstract: Major insulin gene transcription factors, such as PDX-1 or NeuroD1, have equally important roles in pancreatic development and the differentiation of pancreatic endocrine cells. Previously, we identified and cloned another critical insulin gene transcription factor MafA (RIPE3b1) and reported that other Maf factors were expressed in pancreatic endocrine cells. Maf factors are important regulators of cellular differentiation; to understand their role in differentiation of pancreatic endocrine cells, we analyzed the expression pattern of large-Maf factors in the pancreas of embryonic and adult mice. Ectopically expressed large-Maf factors, MafA, MafB, or cMaf, induced expression from insulin and glucagon reporter constructs, demonstrating a redundancy in their function. Yet in adult pancreas, cMaf was expressed in both [alpha]- and [beta]-cells, and MafA and MafB showed selective expression in the [beta]- and [alpha]-cells, respectively. Interestingly, during embryonic development, a significant proportion of MafB-expressing cells also expressed insulin. In embryos, MafB is expressed before MafA, and our results suggest that the differentiation of [beta]-cells proceeds through a MafB.sup.+ MafA.sup.- Ins.sup.+ intermediate cell to MafB.sup.- MafA.sup.+ Ins.sup.+ cells. Furthermore, the MafB to MafA transition follows induction of PDX-1 expression (Pdx-1.sup.high) in MafB.sup.+ Ins.sup.+ cells. We suggest that MafB may have a dual role in regulating embryonic differentiation of both [beta]- and [alpha]-cells while MafA may regulate replication/survival and function of [beta]-cells after birth. Thus, this redundancy in the function and expression of the large-Maf factors may explain the normal islet morphology observed in the MafA knockout mice at birth. Author Affiliation: (a) Section of Islet Transplantation and Cell Biology, Joslin Diabetes Center, Boston, MA 02215, USA (b) Department of Medicine, Harvard Medical School, Boston, MA 02132, USA Article History: Received 9 December 2005; Revised 27 January 2006; Accepted 16 February 2006 Article Note: (footnote) [star] Maf factors are important for pancreatic differentiation.

Change from MafB to MafA expression coming with distinction to pancreatic endocrine cells is studied.

Abstract Central nervous system (CNS) insulin resistance is a condition in which the cells within the CNS do not respond to insulin appropriately and is often linked to aberrant CNS insulin levels. CNS insulin is primarily derived from the periphery. Aberrant CNS insulin levels can arise due to various factors including i) decreased endogenous insulin transport into the brain, across the blood-brain barrier (BBB), ii) reduced CNS sequestration of insulin, and iii) increased CNS degradation. While the sole route of endogenous insulin transport into the brain is via the BBB, there are multiple therapeutic routes of administration that have been investigated to deliver exogenous insulin to the CNS. These alternative administrative routes can be utilized to increase the amount of CNS insulin and aid in overcoming CNS insulin resistance. This review focuses on the intravenous, intracerebroventricular, intranasal, ocular, and intrathecal routes of administration and compares the impact of insulin delivery. [ABSTRACT FROM AUTHOR]

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has two active forms, PACAP1-27 and PACAP1-38. Among the well-established actions are PACAP's neurotrophic and neuroprotective effects, which have also been proven in models of different retinopathies. The route of delivery is usually intravitreal in studies proving PACAP's retinoprotective effects. Recently, we have shown that PACAP1-27 delivered as eye drops in benzalkonium-chloride was able to cross the ocular barriers and exert retinoprotection in ischemia. Since PACAP1-38 is the dominant form of the naturally occurring PACAP, our aim was to investigate whether the longer form is also able to cross the barriers and exert protective effects in permanent bilateral common carotid artery occlusion (BCCAO), a model of retinal hypoperfusion. Our results show that radioactive PACAP1-38 eye drops could effectively pass through the ocular barriers to reach the retina. Routine histological analysis and immunohistochemical evaluation of the Müller glial cells revealed that PACAP1-38 exerted retinoprotective effects. PACAP1-38 attenuated the damage caused by hypoperfusion, apparent in almost all retinal layers, and it decreased the glial cell overactivation. Overall, our results confirm that PACAP1-38 given in the form of eye drops is a novel protective therapeutic approach to treat retinal diseases. [ABSTRACT FROM AUTHOR]

Background: Disruption of the blood-brain barrier (BBB) occurs in many diseases and is often mediated by inflammatory and neuroimmune mechanisms. Inflammation is well established as a cause of BBB disruption, but many mechanistic questions remain.Methods: We used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in mice. BBB disruption was measured using (14)C-sucrose and radioactively labeled albumin. Brain cytokine responses were measured using multiplex technology and dependence on cyclooxygenase (COX) and oxidative stress determined by treatments with indomethacin and N-acetylcysteine. Astrocyte and microglia/macrophage responses were measured using brain immunohistochemistry. In vitro studies used Transwell cultures of primary brain endothelial cells co- or tri-cultured with astrocytes and pericytes to measure effects of LPS on transendothelial electrical resistance (TEER), cellular distribution of tight junction proteins, and permeability to (14)C-sucrose and radioactive albumin.Results: In comparison to LPS-induced weight loss, the BBB was relatively resistant to LPS-induced disruption. Disruption occurred only with the highest dose of LPS and was most evident in the frontal cortex, thalamus, pons-medulla, and cerebellum with no disruption in the hypothalamus. The in vitro and in vivo patterns of LPS-induced disruption as measured with (14)C-sucrose, radioactive albumin, and TEER suggested involvement of both paracellular and transcytotic pathways. Disruption as measured with albumin and (14)C-sucrose, but not TEER, was blocked by indomethacin. N-acetylcysteine did not affect disruption. In vivo, the measures of neuroinflammation induced by LPS were mainly not reversed by indomethacin. In vitro, the effects on LPS and indomethacin were not altered when brain endothelial cells (BECs) were cultured with astrocytes or pericytes.Conclusions: The BBB is relatively resistant to LPS-induced disruption with some brain regions more vulnerable than others. LPS-induced disruption appears is to be dependent on COX but not on oxidative stress. Based on in vivo and in vitro measures of neuroinflammation, it appears that astrocytes, microglia/macrophages, and pericytes play little role in the LPS-mediated disruption of the BBB. [ABSTRACT FROM AUTHOR]

Intranasal insulin has shown efficacy in patients with Alzheimer's disease (AD), but there are no preclinical studies determining whether or how it reaches the brain. Here, we showed that insulin applied at the level of the cribriform plate via the nasal route quickly distributed throughout the brain and reversed learning and memory deficits in an AD mouse model. Intranasal insulin entered the blood stream poorly and had no peripheral metabolic effects. Uptake into the brain from the cribriform plate was saturable, stimulated by PKC inhibition, and responded differently to cellular pathway inhibitors than did insulin transport at the blood-brain barrier. In summary, these results show intranasal delivery to be an effective way to deliver insulin to the brain. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) in its various forms has emerged as a major problem for modern society. Acute TBI can transform into a chronic condition and be a risk factor for neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases, probably through induction of oxidative stress and neuroinflammation. Here, we examined the ability of the antioxidant molecular hydrogen given in drinking water (molecular hydrogen water; mHW) to alter the acute changes induced by controlled cortical impact (CCI), a commonly used experimental model of TBI. We found that mHW reversed CCI-induced edema by about half, completely blocked pathological tau expression, accentuated an early increase seen in several cytokines but attenuated that increase by day 7, reversed changes seen in the protein levels of aquaporin-4, HIF-1, MMP-2, and MMP-9, but not for amyloid beta peptide 1–40 or 1–42. Treatment with mHW also reversed the increase seen 4 h after CCI in gene expression related to oxidation/carbohydrate metabolism, cytokine release, leukocyte or cell migration, cytokine transport, ATP and nucleotide binding. Finally, we found that mHW preserved or increased ATP levels and propose a new mechanism for mHW, that of ATP production through the Jagendorf reaction. These results show that molecular hydrogen given in drinking water reverses many of the sequelae of CCI and suggests that it could be an easily administered, highly effective treatment for TBI. [ABSTRACT FROM AUTHOR]

Since current cancer therapies do not work effectively for all patients, there is a pressing need for alternate, affordable, yet effective treatments for inoperable and chemo-resistant tumors. Presented in this paper are our investigations on the application of short duration (milli and micro seconds) electrical voltage pulses for the treatment of breast carcinoma. For this purpose, electroporation, a pulse power technology, was evaluated for its effect upon the viability and proliferation of human breast cancer cells using a number of voltages and pulse durations. The results indicate the promising potential of electrical pulses to effectively and economically enhance drug transport across the normally impermeable plasma cell membranes and to reduce proliferation. [ABSTRACT FROM PUBLISHER]

Among the more promising treatments proposed for Alzheimer's disease (AD) and Parkinson's disease (PD) are those reducing brain insulin resistance. The antidiabetics in the class of incretin receptor agonists (IRAs) reduce symptoms and brain pathology in animal models of AD and PD, as well as glucose utilization in AD cases and clinical symptoms in PD cases after their systemic administration. At least 9 different IRAs are showing promise as AD and PD therapeutics, but we still lack quantitative data on their relative ability to cross the blood–brain barrier (BBB) reaching the brain parenchyma. We consequently compared brain uptake pharmacokinetics of intravenous 125I-labeled IRAs in adult CD-1 mice over the course of 60 min. We tested single IRAs (exendin-4, liraglutide, lixisenatide, and semaglutide), which bind receptors for one incretin (glucagon-like peptide-1 [GLP-1]), and dual IRAs, which bind receptors for two incretins (GLP-1 and glucose-dependent insulinotropic polypeptide [GIP]), including unbranched, acylated, PEGylated, or C-terminally modified forms (Finan/Ma Peptides 17, 18, and 20 and Hölscher peptides DA3-CH and DA-JC4). The non-acylated and non-PEGylated IRAs (exendin-4, lixisenatide, Peptide 17, DA3-CH and DA-JC4) had significant rates of blood-to-brain influx (K i), but the acylated IRAs (liraglutide, semaglutide, and Peptide 18) did not measurably cross the BBB. The brain influx of the non-acylated, non-PEGylated IRAs were not saturable up to 1 μg of these drugs and was most likely mediated by adsorptive transcytosis across brain endothelial cells, as observed for exendin-4. Of the non-acylated, non-PEGylated IRAs tested, exendin-4 and DA-JC4 were best able to cross the BBB based on their rate of brain influx, percentage reaching the brain that accumulated in brain parenchyma, and percentage of the systemic dose taken up per gram of brain tissue. Exendin-4 and DA-JC4 thus merit special attention as IRAs well-suited to enter the central nervous system (CNS), thus reaching areas pathologic in AD and PD. [ABSTRACT FROM AUTHOR]

All forms of diabetes mellitus are characterized by chronic hyperglycemia resulting in the development of a number of microvascular and macrovascular pathologies. Diabetes is also associated with changes in brain microvasculature leading to dysfunction and disruption of the blood-brain barrier (BBB). These changes are correlated with a decline in cognitive function. The BBB is a regulatory interface between brain and blood, preventing the unrestricted leakage of plasma proteins into the central nervous system (CNS) and performing nutritive, homeostatic, and communication roles. Studies have shown that in a streptozotocin-induced mouse model of type 1 diabetes, where insulin and leptin levels are low, BBB dysfunction occurs in five brain regions: frontal cortex, occipital cortex, parietal cortex, midbrain, and thalamus. In contrast, in a diet induced obese (DIO) model of type 2 diabetes, where insulin and leptin levels are high, BBB dysfunction did not occur in those five regions but did occur in the hippocampus and hypothalamus. In both models, all mice were hyperglycemic, indicating that this is not the only mechanism by which BBB dysfunction occurs. To further investigate the role of the hormones leptin and insulin in BBB dysfunction, we utilized the BTBR.Cg-Lepob/WiscJ ob/ob mice, a transgenic model of type 2 diabetes where insulin is high and no leptin is present. Using radioactively labeled ligands, we have demonstrated that loss of leptin leads to BBB dysfunction in all brain regions as early as four weeks of age. Importantly, these mice are not born with this dysfunction as studies in two-day old pups demonstrate an intact BBB. Replenishment of leptin to these BTBR.Cg-Lepob/WiscJ ob/ob mice from birth was able to attenuate BBB dysfunction observed at four-weeks of age. These studies indicate that leptin is a key component in maintaining BBB integrity in diabetes. Disclosure: T.S. Salameh: None. W. Mortell: None. W.A. Banks: None. Funding: National Institute of Neurological Disorders and Stroke (1R21NS093368-01A1) [ABSTRACT FROM AUTHOR]

During pancreatic development insulin+ cells transition from a MafA- to MafA+ state. Our previous results suggested that the expression of transcription factor MafB in insulin+ cells precedes this maturation step. To directly examine the role of MafB in this process, we analyzed embryonic pancreas from mice carrying a kreisler mutation in the mafBkr/kr gene. We show that this kr allele, for which serine is replaced by asparagine in the conserved, DNA-binding domain found in all bZIP family members, reduced the ability of MafB to activate both insulin and glucagon gene expression and that mice with this mutation had a significant reduction in the insulin and glucagon expressing cells. Mutation also resulted in the reduction of PDX-1+ and MafA+ cells with only a minor reduction in MafB+ cells. Interestingly, the proportion of MafB+ or PDX-1+ cells that also expressed insulin was reduced. However mutation in MafB did not affect the expression of Nkx2.2, Nkx6.1, Ngn3 and Pax6 suggesting that MafB does not affect the endocrine specification but rather plays a role in the maturation of insulin+ cells. Examination of Pax6 sey/sey mutant mice demonstrated a reduction in the insulin, glucagon, MatB, PDX-1 and MafA expressing cells. Since Pax6 expression was not altered in kreisler mice, our results suggest a role of MafB in the reduction of insulin and glucagon expressing cells in sey/sey mice. Furthermore, as in kreisler mice, the proportion of MafB+ or PDX-1+ cells expressing insulin was reduced in sey/sey mice, suggesting roles for both Pax6 and MafB in regulating the efficiency of terminal differentiation/maturation of insulin+ cells. Interestingly, unlike MafB and PDX-1 that were also expressed in some insulin- cells, all MafA+ cells in these mutant mice expressed insulin. Thus, our results indicate that MafA is a marker of terminally differentiated β-cell, and that Pax6 acts upstream of MafB, which in turn contributes to the expression of PDX-1 and MafA in differentiating insulin+ cells. [ABSTRACT FROM AUTHOR]

Abstract: During pancreatic development insulin+ cells co-express the transcription factors MafB and Pax6, and transition from a MafA− to MafA+ state. To examine the role of Pax6 and MafB in the development of β-cells, we analyzed embryonic pancreata from Pax6- and MafB-deficient mice. Pax6 deficiency, as manifest in the Pax6 Sey–Neu allele, reduced not only the number of cells expressing insulin or glucagon, but also the number of MafB, PDX-1 and MafA expressing cells. We show that MafB can directly activate expression of insulin and glucagon, and a MafB protein engineered to contain N248S mutation in the MafB (kr ENU ) results in significantly reduced activation. Furthermore, pancreata from MafB deficient (kr ENU /kr ENU ) mice exhibited reduced number of cells expressing insulin, glucagon, PDX-1 and MafA, with only a minor reduction in MafB expressing cells. MafB deficiency does not affect endocrine specification but does affect the lineage commitment of the endocrine cells and their maturation. Similar to Pax6 deficient mice, MafB deficient mice showed reductions both in insulin and glucagon expressing cells and in the ability of MafB and PDX-1 expressing cells to activate expression of these hormones. However, MafB deficient mice exhibited no effect on Pax6 expression. These results suggest that MafB may function as a downstream mediator of Pax6 in regulating the specification of insulin and glucagon expressing cells. Interestingly, the remaining insulin+ cells in these knockouts preferentially express Hb9, suggesting the existence of an alternate pathway for the generation of insulin expressing cells, even in the absence of Pax6 and MafB function. Thus, Pax6 acts upstream of MafB, which in turn may trigger the expression of insulin and regulate the PDX-1 and MafA expression required for β-cell maturation. [Copyright &y& Elsevier]

Abstract: Major insulin gene transcription factors, such as PDX-1 or NeuroD1, have equally important roles in pancreatic development and the differentiation of pancreatic endocrine cells. Previously, we identified and cloned another critical insulin gene transcription factor MafA (RIPE3b1) and reported that other Maf factors were expressed in pancreatic endocrine cells. Maf factors are important regulators of cellular differentiation; to understand their role in differentiation of pancreatic endocrine cells, we analyzed the expression pattern of large-Maf factors in the pancreas of embryonic and adult mice. Ectopically expressed large-Maf factors, MafA, MafB, or cMaf, induced expression from insulin and glucagon reporter constructs, demonstrating a redundancy in their function. Yet in adult pancreas, cMaf was expressed in both α- and β-cells, and MafA and MafB showed selective expression in the β- and α-cells, respectively. Interestingly, during embryonic development, a significant proportion of MafB-expressing cells also expressed insulin. In embryos, MafB is expressed before MafA, and our results suggest that the differentiation of β-cells proceeds through a MafB+ MafA− Ins+ intermediate cell to MafB− MafA+ Ins+ cells. Furthermore, the MafB to MafA transition follows induction of PDX-1 expression (Pdx-1high) in MafB+ Ins+ cells. We suggest that MafB may have a dual role in regulating embryonic differentiation of both β- and α-cells while MafA may regulate replication/survival and function of β-cells after birth. Thus, this redundancy in the function and expression of the large-Maf factors may explain the normal islet morphology observed in the MafA knockout mice at birth. [Copyright &y& Elsevier]

Purpose: Pituitary adenylate cyclase activating polypeptide (PACAP) is neuroprotective in neuronal injuries. Bilateral common carotid artery occlusion (BCCAO) causes chronic hypoperfusion-induced degeneration in the rat retina, where we proved the retinoprotective effect of intravitreal PACAP. Although this route of administration is a common clinical practice in several diseases, easier routes are clinically important. Our aim was to investigate the potential retinoprotective effects of PACAP eye drops in BCCAO-induced ischemic retinopathy., Methods: After performing BCCAO in rats, the right eyes were treated with PACAP1-27 eye drops (1 μg/drop, 2 × 1 drops/day for 5 days), containing different vehicles: saline, water for injections, thiomersal or benzalkonium solution for ophthalmic use (SOCB). Histology and immunohistochemistry were performed 2 weeks after surgery, while molecular analysis was performed 24 hours after BCCAO. Passage of PACAP1-27 through the ocular layers was tested with radioactive PACAP-SOCB in mice., Results: Bilateral common carotid artery occlusion led to a severe degeneration of all retinal layers. Solution for ophthalmic use was the most effective vehicle for delivering PACAP (PACAP-SOCB), significantly ameliorating BCCAO-induced damage. The massive upregulation of GFAP was not observed in retinas treated with PACAP-SOCB eye drops. PACAP-SOCB treatment also increased activation of the protective Akt and ERK1/2 in hypoperfused retinas. The cytokine profile showing upregulation in different cytokines was attenuated by PACAP-SOCB. Radioactive PACAP reached the retina when delivered in SOCB-containing eye drops., Conclusions: PACAP1-27, delivered in the SOCB vehicle as eye drops, was retinoprotective in ischemic retinopathy, providing the basis for future therapeutic administration.

All forms of diabetes mellitus are characterized by chronic hyperglycemia, resulting in the development of a number of microvascular and macrovascular pathologies. Diabetes is also associated with changes in brain microvasculature, leading to dysfunction and ultimately disruption of the blood-brain barrier (BBB). These changes are correlated with a decline in cognitive function. In diabetes, BBB damage is associated with increased oxidative stress and reactive oxygen species. This occurs because of the increased oxidative metabolism of glucose caused by hyperglycemia. Decreasing the production of bicarbonate with the use of a mitochondrial carbonic anhydrase inhibitor (mCAi) limits oxidative metabolism and the production of reactive oxygen species. In this study, we have demonstrated that 1) streptozotocin-induced diabetes resulted in BBB disruption, 2) ultrastructural studies showed a breakdown of the BBB and changes to the neurovascular unit (NVU), including a loss of brain pericytes and retraction of astrocytes, the two cell types that maintain the BBB, and 3) treatment with topiramate, a mCAi, attenuated the effects of diabetes on BBB disruption and ultrastructural changes in the neurovascular unit., (U.S. Government work not protected by U.S. copyright.)

A variety of compounds will distribute into the brain when placed at the cribriform plate by intranasal (i.n.) administration. In this study, we investigated the ability of albumin, a protein that can act as a drug carrier but is excluded from brain by the blood-brain barrier, to distribute into the brain after i.n. administration. We labeled bovine serum albumin with [(125)I] ([(125)I]Alb) and studied its uptake into 11 brain regions and its entry into the blood from 5 minutes to 6 hours after i.n. administration. [(125)I]Alb was present throughout the brain at 5 minutes. Several regions showed distinct peaks in uptake that ranged from 5 minutes (parietal cortex) to 60 minutes (midbrain). About 2-4% of the i.n. [(125)I]Alb entered the bloodstream. The highest levels occurred in the olfactory bulb and striatum. Distribution was dose-dependent, with less taken up by whole brain, cortex, and blood at the higher dose of albumin. Uptake was selectively increased into the olfactory bulb and cortex by the fluid-phase stimulator PMA (phorbol 12-myristate 13-acetate), but inhibitors to receptor-mediated transcytosis, caveolae, and phosphoinositide 3-kinase were without effect. Albumin altered the distribution of radioactive leptin given by i.n. administration, decreasing uptake into the blood and by the cerebellum and increasing uptake by the hypothalamus. We conclude that [(125)I]Alb administered i.n. reaches all parts of the brain through a dose-dependent mechanism that may involve fluid-phase transcytosis and, as illustrated by leptin, can affect the delivery of other substances to the brain after their i.n. administration., (U.S. Government work not protected by U.S. copyright.)

Peptides and proteins have potent effects on the brain after their peripheral administration, suggesting that they may be good substrates for the development of CNS therapeutics. Major hurdles to such development include their relation to the blood-brain barrier (BBB) and poor pharmacokinetics. Some peptides cross the BBB by transendothelial diffusion and others cross in the blood-to-brain direction by saturable transporters. Some regulatory proteins are also transported across the BBB and antibodies can enter the CNS via the extracellular pathways. Glycoproteins and some antibody fragments can be taken up and cross the BBB by mechanisms related to adsorptive endocytosis/transcytosis. Many peptides and proteins are transported out of the CNS by saturable efflux systems and enzymatic activity in the blood, CNS, or BBB are substantial barriers to others. Both influx and efflux transporters are altered by various substances and in disease states. Strategies that manipulate these interactions between the BBB and peptides and proteins provide many opportunities for the development of therapeutics. Such strategies include increasing transendothelial diffusion of small peptides, upregulation of saturable influx transporters with allosteric regulators and other posttranslational means, use of vectors and other Trojan horse strategies, inhibition of efflux transporters including with antisense molecules, and improvement in pharmacokinetic parameters to overcome short half-lives, tissue sequestration, and enzymatic degradation., (© 2014 Elsevier Inc. All rights reserved.)

The transcription factor MafA/RIPE3b1 is an important regulator of insulin gene expression. MafA binds to the insulin enhancer element RIPE3b (C1-A2), now designated as insulin MARE (Maf response element). The insulin MARE element shares an overlapping DNA-binding region with another insulin enhancer element A2. A2.2, a beta-cell-specific activator, like the MARE-binding factor MafA, binds to the overlapping A2 element. Our previous results demonstrated that two nucleotides in the overlapping region are required for the binding of both factors. Surprisingly, instead of interfering with each other's binding activity, the MafA and the A2-binding factors co-operatively activated insulin gene expression. To understand the molecular mechanisms responsible for this functional co-operation, we have determined the nucleotides essential for the binding of the A2.2 factor. Using this information, we have constructed non-overlapping DNA-binding elements and their derivatives, and subsequently analysed the effect of these modifications on insulin gene expression. Our results demonstrate that the overlapping binding site is essential for maximal insulin gene expression. Furthermore, the overlapping organization is critical for MafA-mediated transcriptional activation, but has a minor effect on the activity of A2-binding factors. Interestingly, the binding affinities of both MafA and A2.2 to the overlapping or non-overlapping binding sites were not significantly different, implying that the overlapping binding organization may increase the activation potential of MafA by physical/functional interactions with A2-binding factors. Thus our results demonstrate a novel mechanism for the regulation of MafA activity, and in turn beta-cell function, by altering expression and/or binding of the A2.2 factor. Our results further suggest that the major downstream targets of MafA will in addition to the MARE element have a binding site for the A2.2 factor.