Deficient Leptin Cellular Signaling Plays a Key Role in Brain Ultrastructural Remodeling in Obesity and Type 2 Diabetes Mellitus

Abstract

The triad of obesity, metabolic syndrome (MetS), Type 2 diabetes mellitus (T2DM) and advancing age are currently global societal problems that are expected to grow over the coming decades. This triad is associated with multiple end-organ complications of diabetic vasculopathy (maco-microvessel disease), neuropathy, retinopathy, nephropathy, cardiomyopathy, cognopathy encephalopathy and/or late-onset Alzheimer's disease. Further, obesity, MetS, T2DM and their complications are associated with economical and individual family burdens. This review with original data focuses on the white adipose tissue-derived adipokine/hormone leptin and how its deficient signaling is associated with brain remodeling in hyperphagic, obese, or hyperglycemic female mice. Specifically, the ultrastructural remodeling of the capillary neurovascular unit, brain endothelial cells (BECs) and their endothelial glycocalyx (ecGCx), the blood-brain barrier (BBB), the ventricular ependymal cells, choroid plexus, blood-cerebrospinal fluid barrier (BCSFB), and tanycytes are examined in female mice with impaired leptin signaling from either dysfunction of the leptin receptor (DIO and db/db models) or the novel leptin deficiency (BTBR ob/ob model).

Keywords: adipose tissue; aging; blood-brain barrier; blood-cerebrospinal fluid barrier; endothelial cell; endothelial glycocalyx; insulin resistance; leptin resistance; microglia; neurovascular unit; obesity; permeability; ultrastructure.

Figure 1

Timeline for discovery and development of key mouse models in obesity and Type 2 diabetes mellitus (T2DM) research. The dashed timeline illustrates important discoveries and models from 1949 to date. References are inserted below the dates of each model. The central importance of leptin is emphasized. FFA = free fatty acids; VAT = visceral adipose tissue; WAT = white adipose tissue.

Figure 2

Brain ultrastructural remodeling in obesity models studied to date with insulin and leptin levels for comparison. Panel A depicts the importance of obesity, insulin resistance-deficient leptin cellular signaling in regards to the development of age-related diseases such as late onset Alzheimer’s disease (LOAD) and neurodegeneration (left-hand side blue coloration). Boxes 1, 3, and 4 (right-hand side) all share deficient cellular leptin signaling. Box 2 depicts the streptozotocin induced insulinopenic diabetes mellitus (DM). Panel B illustrates the increased (upward arrows) and decreased (downward arrows) of insulin and leptin in each model discussed in Panel A. AC = astrocyte; AJ = adherens junction; AKT = protein kinase B; EC = endothelial cell; IFG = impaired fasting glucose; IGT = impaired glucose tolerance; i.p. = intraperitoneal; IRS−1 = insulin receptor substrate−1; LAN = lanthanum nitrate; NVU = neurovascular unit; PI3Kinase = phosphoinositide 3-kinase; T2DM = Type 2 diabetes mellitus; SQ = subcutaneous fat; TJ = tight junction; VAT = visceral adipose tissue; WAT = white adipose tissue.

Figure 3

Venn diagram illustrates the shared importance of leptin in diet-induced obesity (DIO), Western, db/db and BTBR ob/ob models. Deficient cellular leptin signaling is common in all three models, but through different mechanisms. All models are obese and there is dysglycemia in the diet induced obesity (DIO) model, overt T2DM in the db/db, and elevated blood glucose levels in the BTBR ob/ob. IGT = impaired glucose tolerance; IR = insulin resistance; LR = leptin resistance; T2DM = Type 2 diabetes mellitus.

Figure 4

The neurovascular unit (NVU) in healthy control C57BL/6J mice at 20 weeks of age from cortical Layer III. Panel A astrocyte foot processes (AC) are pseudo-colored golden to emphasize their important anatomical location. Note the ramified microglial cell (rMGC) (yellow arrow) surveilling the NVU in Panel A. Panel B illustrates a horizontal image of the NVU (contrasting with the cross-section image in Panel A) to better illustrate the electron dense tight and adherens junctions (TJ/AJ) (yellow arrows) within the paracellular regions and emphasize the clear zone or corona of electron lucent AC foot processes (ACfp). Panel C depicts a NVU within the hippocampus where a myelinated axon bundle is present. Additionally, oligodendrocytes may exist in the NVU basement membrane (BM) especially in the subcortical and white matter regions (not shown). Note that the BM splits to encompass the pericyte foot processes (Pcfp). Magnification ×4000; scale bar = 1 µm. Panels A and B are adapted with permission from reference [39]. AC = astrocyte; ACfp = astrocyte foot processes; BM = basement membrane; Cap L and CL = capillary Lumen of the NVU; CKC = control C57BL/6J model; EC = endothelial cell; Mt = mitochondria; rMGC = ramified MGC; Pc = pericyte; Pcfp = pericyte foot processes.

Figure 5

The endothelial glycocalyx as the first of component of the “tripartite BBB”. Normal components of the endothelial glycocalyx (ecGCx): a unique extracellular matrix. The ecGCx is composed of two classes of proteins that are mostly anchored [proteoglycan(s) (PGN) (purple), glycoprotein(s) (GP) (green)] and of hyaluronic acid (HA) hyaluronan (an exceedingly long polymer of disaccharides non-sulfated glycosaminoglycan). (HA) (blue) may be either unattached (free floating), attached to CD44 brain endothelial cell(s) (BEC) plasma membrane, or form HA-HA complexes. Non-sulfated HAs not anchored to the BECs may reversibly interact at the lumen with plasma-derived albumin, fibrinogen and soluble PGNs. The PGNs and glycoproteins side chains consisting of glycosaminoglycans (GAGs) covalently bound to core proteins are highly sulfated (red dots). The two primary PGNs are the syndecans and glypicans. The glycoproteins consist primarily of selectins (P and E), integrins (alpha v and beta 3) and immunoglobulin superfamily of ICAM-1, VE-CAM and PE CAM-1. Caveolae are invaginations of lipid rafts of the endothelial plasma membrane and contain CD44 important which anchors hyaluronan to the BEC plasma membrane and glycosylphosphatidylinositol (GPI) which anchors glypican-1. The GPI/glypican-1 interaction is thought to activate endothelial nitric oxide synthase (eNOS) to produce bioavailable nitric oxide (NO) via the calcium calmodulin dependent Caveolin-1 (Cav-1) protein.

Figure 6

The properties of the endothelial cell glycocalyx (ecCGx) shield and mechanotransduction. These images of the aorta EC illustrate relations among the caveolae, the plasma membrane (pm, lined for clarity), and the ecGCx, with its glypican-1 (GLP-I) anchored to the lipid rafts of the caveolae. Note that the ecCGx extends into the caveolae. The Cav-1 protein is responsible for the omega shape (Ω) of the caveolae (yellow icon) (dashed arrow). The shear stress of laminar flow through the ecGCx induces mechanotranduction and release of NO, resulting in vasodilation (open arrow). The ecGCx component glypican-1 is the primary mechano-sensor for shear stress-induced NO production. Scale bar on the left panel is 20 nm and the right-hand panel is an exploded view of left panel. BH4 = tetrahydrobiopterin; Ca++/CaM = calcium calmodulin; Cav-1 = caveolin-1; GIP = glycosylphosphatidylinositol; GLP-1 = glypican-1; pm = plasma membrane.

Figure 7

Lanthanum nitrate (LaN) staining of the endothelial glycocalyx (ecGCx) showing attenuation and loss in the diabetic obese BTBR ob/ob model: cortical Layer III at 20 weeks of age. Panels A and B demonstrate in the wild type control an intense electron dense LaN staining reflecting an intact continuous ecGCx or endothelial surface layer (arrows) of the brain endothelial cell(s) (BEC) at ×4000, ×8000; scale bars = 0.5, 0.2 µm respectively. In addition, note the intact astrocyte foot processes (ACfp), pericyte foot processes (Pcfp), and intact mito-chondria (Mt). Panels C and D depict the attenuation, shedding, and/or loss of the continuous ecGCx (arrows) with clumping of remaining LaN positive ecGCx (arrows) at ×4000, ×8000; 0.5 and 0.2 µm respectively in the BTBR ob/ob obese diabetic models. Furthermore, note the intact ACfp, Pcfp and intact Mt. Scale bars lower left corner of each panel and magnification in the upper right. CL = capillary neurovascular lumen; EC = endothelial cells.

Figure 8

Lanthanum nitrate (LaN) staining of the endothelial glycocalyx (ecGCx) showing attenuation and loss in the obese diabetic BTBR ob/ob model: hippocampus CA-1 regions. Note the highly decorated endothelium by LaN staining in the control wild type (WT) hippocampus (HC) model with intact ecGCx (left-hand figure). In the obese diabetic BTBR ob/ob model, the ecGCx (arrows) when present is markedly attenuated and very thinned (right-hand figure). Magnification ×10,000; bar = 0.2 µm. ACfp = astrocyte foot process; CL = capillary lumen; EC = endothelial cell; HC = hippocampus; WT = wild type control.

Figure 9

Leptin replacement in the obese diabetic BTBR ob/ob protects the brain endothelial cell glycocalyx (ecGCx) in cortical Layer III and hippocampus. Panel A illustrates the continuous decoration of the ecGCx with lanthanum nitrate (LaN) staining in the heterozygous non-diabetic control model cortical Layer III (arrows). Panel B depicts the marked attenuation and/or loss of the ecGCx in the obese diabetic BTBR ob/ob model cortical Layer III. Note when the ecGCx is present it is clumped and discontinuous (arrows). Panel C also illustrates the continuous decoration of the ecGCx in hippocampus CA-1 regions of the BTBR ob/ob models that were treated with intraperitoneal leptin for 16 weeks and stained with LaN (arrows). The ecGCx is continuous and comparable to the control model in Panel A. Panel D depicts the complete loss of the ecGCx by LaN staining in the hippocampus CA-1 regions of the BTBR ob/ob. Note that the tight and adherens junction (TJ/AJ) remain intact (yellow arrows). Magnification ×4000; scale bar = 0.5 μm in Panels A and B. Magnification ×10,000; scale bar = 0.2 µm in Panels C and D. ACfp = astrocyte foot process; Cl = capillary lumen; BEC = brain endothelial cell; HIP and HC = hippocampus CA-1 regions; LAN—LaN = lanthanum nitrate stained.

Figure 10

Endothelial and microglial activation in cortical and hippocampal NVUs of the BTBR ob/ob model. Panels A and B depict swollen electron lucent activated endothelial cells (aECs) and thickened basement membranes of cortical Layer III. Note the vacuolization of the BM in Panels A and B (asterisks). Magnification ×2000 and ×6000 respectively. Panels C through F depict an aMGC encroaching between the BEC and the rest of the NVU. Panels C and D depict an aMGC with loss of pericytes and astrocytes. Panel D (a lower magnification of Panel C) illustrates the aMGC pseudo-colored red and its relation to a pyramidal cell (PYR). Panel E depicts aMGC cytoplasmic processes (pseudo-colored red) directly abutting the ECs basement membrane and is a higher magnification of boxed in region in Panel D. Furthermore, note the exosomes within the capillary lumen that are between 50–75 nm in diameter, which may indicate stressed EC activation. Panel F illustrates that while these endothelial changes are occurring to the left and inferior NVU, the right or opposite side of the endothelial lining depicts the tight and adherens junction (TJ/AJ) remaining intact (arrows). Various magnifications labeled upper right and scale bars in each panel in lower left. ACfp = astrocyte foot processes; aMGC = activated microglial cell; CL = capillary lumen; iBM = inner basement membrane; M = myelin; n = nucleus; oBM = outer basement membrane; PYR = pyramidal cell; RBC = red blood cell. TJ/AJ = tight junctions/adherens junctions.

Figure 11

Comparison of ramified microglia (rMGC) in control mice to activated microglia (aMGC) in obese Type 2 diabetes mellitus (T2DM) db/db model. Panel A illustrates the normal appearance of ramified microglia (rMGC-pseudo-colored green) (see Figure 5A,C). In addition, note the insert in Panel A and the presence of cristae in rMGC at higher magnification ×6000). Panels B and C depict aMGCs (pseudo-colored red) with swollen electron lucent aberrant mitochondria (aMt) (pseudo-colored yellow) the db/db diabetic mice. Magnification ×2000; scale bar = 1 µm in A and B and 2 µm in Panel C. These images are adapted with permission from references [3,39]. CC = chromatin condensation; CKC = control C57BL/6J female non-diabetic model; DBC = female diabetic db/db model.

Figure 12

Gross brain atrophy in the db/db model but not in the diabetic BTBR ob/ob model. Panel B illustrates the brain atrophy at the time of surgical removal of the diabetic db/db models as compared to control models in Panel A. Panel B depicts the marked atrophy or loss in the cortical-parietal-hippocampal regions (outlined by the yellow dashed lines) in comparison to the control in Panel A at 26-weeks. Panel C demonstrates the BTBR ob/ob model at 20-weeks without atrophy in the parietal-hippocampal regions of the whole brain when compared to the db/db model in Panel B at 26-weeks. This may correlate with the less severe ultrastructural remodeling of the neurovascular unit and less microglial neuroinflammation in BTBR ob/ob mice as compared to db/db mice. Panels A and B CC by NC-ND [75].

Figure 13

Ependymal cells lining the ventricular cerebrospinal fluid (CSF) system of the aqueduct in female control 20 week old models. Panel A illustrates that not all ependymal cells (EPY) are cuboidal but may lie flattened and be multilayered at the subventricular zone (SVZ) region of the adjacent neuropil. Panel B demonstrates the more classical cuboidal morphologic phenotype of the EPY cells that line the CSF ventricles. Note the close 5 µm distance between the EPY cells and fenestrated capillary within the SVZ neuropil. The adjacent capillary neurovascular units (NVU) in the SVZ do not have a similar coverage by astrocytes as in the cortical regions but pericytes (Pc) are frequently noted and this capillary appears to be fenestrated due the thinning of the ECs. Panel C depicts the ependymal cilia with the classical 9:2 arrangements of cytoskeletal proteins within them in addition to multiple microvilli (Mv) on the apical surface facing the CSF. Panel D demonstrates the overlapping and interdigitations of EPY cells, and also the staining of the very electron dense tight and adherens junction (TJ/AJ) proteins (arrows) and desmosomes that create the barrier functions of the EPY cells in some regions of the ependymal lining of the CSF. While these EPY cells are located in the aqueduct they also are representative of the EPY cells found throughout each of the four ventricles and lining of choroid plexus (CP) that provides the CP with its barrier function. As noted in these images, the EPY cells form the structural and functional barrier of the choroid plexus. CL = capillary lumen.

Figure 14

Choroid plexus (lateral ventricle) with aberrant vacuolization of the basilar infoldings of ependymal cells in obese diabetic female BTBR ob/ob models. Panel A demonstrates the normal ultrastructural morphology of the choroid plexus ependymal cell(s) (CPE) in the control heterozygous model, characterized by compact basilar infoldings (BI) (yellow dashed line), tight and adherens junctions (TJ/AJ) (arrowheads), microvilli (Mv) of the brush border at the apical cerebrospinal fluid (CSF) interface, and the multiple electron dense mitochondria (Mt) in a highly polarized ependymal cell. Panel B depicts aberrant remodeling changes of the (BI) at lower magnification to include multiple CPEs (CPE 1–5) which consist of vesiculation (v)/vacuolization (V) of the BI (yellow dashed lines). Importantly, note the lower fenestrated (f) capillary with positive staining for lanthanum nitrate (LaN) of the endothelial glycocalyx (ecCGx) and the upper capillary (*) that is filled with multiple red blood cells (RBCs) that is highly suggestive of capillary micro-thrombosis and without evidence of LaN staining. Panel C also illustrates the control heterozygote CPEs and this image contains an exploded insert of the BI (outlined in yellow dashed lines). This insert illustrates the compactness of the BIs without v/V. Panel D depicts the v/V of the BI as compared to the control models in Panels A and C. Magnification ×400; scale bar = 5 µm in Panel B and magnification ×1200; scale bar = 2 µm in Panels A–D. n = nucleus.