Fig 2: Multifactorial functionality of GIT2 in the neurometabolic axis. From our data presented here and from previous research it is becoming clear that GIT2 likely acts as a keystone or hub protein for many physiological or cellular signaling processes involved in the maintenance of homeostatic metabolic function across life span. To illustrate the multidimensional functionality of GIT2 in such a complex system we have selected proteins/transcripts identified in this manuscript to highlight the network-level functional signature of GIT2 activity. The ability of GIT2 to simultaneously control and integrate proteins involved in GPCR signaling/trafficking (Adora2b, adenosine A2b receptor; Kdelr2, Lys–Asp–Glu–Leu ER lumen protein retaining receptor 2; Rgs5, regulator of G-protein signaling 5; Ramp1, receptor activity-modulating protein 1), insulin receptor (InsR) associated signaling (Irs2, insulin receptor substrate 2; Reg3b, regenerating islet-derived 3 beta; Insig 2, Insulin induced gene 2; Sesn1, sestrin 1), metabolite/vesicular transport (Glut2, glucose transporter 2; Rims3, regulating synaptic membrane exocytosis 3; Slc2a13, solute carrier family 2 (facilitated glucose transporter), member 13; Gltp, glycolipid transport protein), DNA damage repair (DDR) (Atm, ataxia-telangiectasia mutated; Wrn, Werner syndrome ATP-dependent helicase; Gadd45a, growth arrest and DNA-damage-inducible protein GADD45 alpha; Parp2, Poly (ADP-ribose) polymerase 2), cytoskeletal remodeling (Vim, vimentin; Flna, filamin A; Spnb3, spectrin beta-3; Als2, Alsin) and energy metabolism (Pebp1, phosphatidylethanolamine binding protein 1; Glo1, glyoxylase 1; Mfn1, mitofusin 1; Gde1, glycerophosphodiester phosphodiesterase 1) clearly demonstrates keystone functionality of GIT2 in neurometabolic aging. Therefore the modulation of the expression of this protein, its post-translational and subcellular distribution are likely to strongly influence the orchestration of these vital functions in both health and pathophysiology.

Fig 3: Pathological manipulation mimicking insulin resistance in murine TC-6 cells affects GIT2 functionality and subcellular disposition. Treatment (1 day) of TC-6 cells with palmitic acid (10 μM) and mimicking an insulin-resistant cellular status potentiates GIT2 and Glut2 expression (A). Long-term palmitic acid treatment (2–4 days) differentially and biphasically regulates GIT2/Glut 2 expression (B). Extracellular signal-regulated kinase 2 (Erk2) was employed as a loading control in (A,B). Attenuation of bolus (5 mM) glucose uptake in TC-6 cells by long-term (4 days) palmitic acid treatment (C). Palmitic acid treatment (4 days) of TC-6 cells affects glucose-driven subcellular redistribution of GIT2 across the cytoplasmic, plasma membrane, and nuclear microdomains (D). Statistical significance is indicated in each figure as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Fig 4: Genomic GIT2 deletion affects pancreatic islet functional structure. (A) DAB- (3,3′-diaminobenzidine) based immunohistochemical identification of GIT2-associated reactivity in discrete islet-like structures in WT murine pancreas. (B) High-magnification inset of DAB-stained, GIT2-immunoreactive pancreatic islet-like structure. (C) GIT2 AF488 immunostaining of WT pancreatic islet. (D) Glucagon AF568 immunostaining of WT pancreatic islet. (E) Merge of panels (C,D) indicating AF488–AF568 colocalization. (F) Insulin AF488 immunostaining of WT pancreatic islet. (G) GIT2 AF568 immunostaining of WT pancreatic islet. (H) Merge of panels (F,G) indicating AF488–AF568 colocalization. MATLAB-based pancreatic islet mathematical automated assessment of WT (black bars) or GIT2KO (white bars) pancreas islet area (I), β-cell percentage (J), α-cell percentage (K), α-cell area (L), β-cell area (M) and islet number/size distribution (N). (O) Insulin AF488, glucagon (P) immunostaining of WT islet [8 month old (m.o.)]. (Q) Merge of panels (O,P). (R) Insulin AF488, glucagon (S) immunostaining of GIT2KO islet (8 m.o.). (T) Merge of panels (R,S). Statistical significance is indicated in a specific panel as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Fig 5: Depletion of GIT2 in murine pancreatic TC-6 cells modulates the expression of multiple proteins involved in stress management and energy regulation. (A) Dose-dependent GIT2 depletion curve for GIT2 siRNA. A random sequence “scramble” siRNA was employed as a negative control. Extracellular signal-regulated kinase 2 (Erk2) was employed as a loading control throughout this figure. GIT2 siRNA-mediated (using 400 nM siRNA concentration) modulation of TC-6 cells expression of GIT2 (B), doublecortin [Dcx: (C)], glyoxylase 1 [Glo1: (D)], NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10 [Ndufb10: (E)], mitochondrial ribosomal protein L12 [Mrpl12: (F)], ataxia telangiectasia mutated [Atm: (G)], regenerating islet-derived 3 beta [Reg3b: (H)] and sestrin 1 [Sesn1: (I)]. Statistical significance is indicated in each figure as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.