Developmental Alterations of Intestinal SGLT1 and GLUT2 Induced by Early Weaning Coincides with Persistent Low-Grade Metabolic Inflammation in Female Pigs

Early life adversity (ELA) is linked with the increased risk for inflammatory and metabolic diseases in later life but the mechanisms remain poorly understood. Intestinal epithelial glucose transporters SGLT1 and GLUT2 are the major route for intestinal glucose uptake but have also received increased attention as modulators of inflammatory and metabolic diseases. Here we tested the hypothesis that early weaning (EW) in pigs, an established model of ELA, alters the development of epithelial glucose transporters and coincides with elevated markers of metabolic inflammation. Jejunum and ileum of 90 d old pigs previously exposed to EW (16 d wean age), exhibited reduced SGLT1 activity (by ~ 30%, P<0.05), compared with late weaned (LW, 26 d wean age) controls . In contrast, GLUT2-mediated glucose transport was increased (P = 0.003) in EW pigs compared with LW pigs. Reciprocal changes in SGLT1 and GLUT2-mediated transport coincided with transporter protein expression in the intestinal brush border membranes (BBM) that were observed at 90 d and 150 d of age. Ileal SGLT1-mediated glucose transport and BBM expression were Inhibited by the β-adrenergic receptor (βAR) blocker propranolol in EW and LW pigs. In contrast, propranolol enhanced ileal GLUT2-mediated glucose transport (P=0.015) and BBMV abundance (P=0.035) LW pigs, but not EW pigs. Early weaned pigs exhibited chronic elevated blood glucose and C-Reactive Protein (CRP) levels, and adipocyte hypertrophy and upregulated adipogenesis-related gene expression in visceral adipose tissue. Altered development of intestinal glucose transporters by EW could underlie the increased risk for later life inflammatory and metabolic diseases.

Knockout of Syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport which is underpinned by the insulin-stimulated delivery of glucose transporter-4 (GLUT4)- containing vesicles to the plasma membrane where they dock and fuse increasing cell surface GLUT4 levels. Adipocytokines such as adiponectin are secreted via a similar mechanism. We used genome editing to knockout Syntaxin-4 a protein reported to mediate GLUT4-vesicle fusion with the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of HA-GLUT4-GFP showed that Syntaxin-4 knockout cells retain significant GLUT4 translocation capacity demonstrating that Syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that Syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that Syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells.

Diet-induced obesity in mice impairs host defense against Klebsiella pneumonia in vivo and glucose transport and bactericidal functions in neutrophils in vitro

Obesity impairs host defense against Klebsiella pneumoniae but responsible mechanisms are incompletely understood. To determine the impact of diet-induced obesity on pulmonary host defense against K. pneumoniae, we fed 6-week-old male C57BL/6j mice a normal (ND) or high fat diet (HFD) (13% versus 60% fat, respectively) for 16 weeks. Mice were intratracheally infected with Klebsiella, assayed at 24 or 48 h for bacterial colony-forming units, lung cytokines, and leukocytes from alveolar spaces, lung parenchyma, and gonadal adipose tissue were assessed using flow cytometry. Neutrophils from uninfected mice were cultured with and without 2-deoxy-D-glucose (2-DG) and assessed for phagocytosis, killing, reactive oxygen intermediates (ROI), transport of 2-DG, and glucose transporter (GLUT1-4) transcripts, and protein expression of GLUT1 and GLUT3. HFD mice had higher lung and splenic bacterial burdens. In HFD mice, baseline lung homogenate concentrations of IL-1β, IL-6, IL-17, IFN-ɣ, CXCL2, and TNF-ɑ were reduced relative to ND mice, but following infection were greater for IL-6, CCL2, and CXCL2 and IL-1β (24 h only). Despite equivalent lung homogenate leukocytes, HFD mice had fewer intra-alveolar neutrophils. HFD neutrophils exhibited decreased Klebsiella phagocytosis and killing, and reduced ROI to heat-killed Klebsiella in vitro. 2-DG transport was lower in HFD neutrophils, with reduced GLUT1 and GLUT3 transcripts and protein (GLUT3 only). Blocking glycolysis with 2-DG impaired bacterial killing and ROI production in neutrophils from mice fed ND but not HFD. Diet-induced obesity impairs pulmonary Klebsiella clearance and augments blood dissemination by reducing neutrophil killing and ROI due to impaired glucose transport.

Cancer causes dysfunctional insulin signaling and glucose transport in a muscle-type specific manner

Metabolic dysfunction and insulin resistance are emerging as hallmarks of cancer and cachexia, and impair cancer prognosis. Yet, the molecular mechanisms underlying impaired metabolic regulation is not fully understood. To elucidate the mechanisms behind cancer-induced insulin resistance in muscle, we isolated extensor digitorum longus (EDL) and soleus muscles from Lewis Lung Carcinoma tumor-bearing mice. Three weeks after tumor inoculation, muscles were isolated and stimulated with or without a submaximal dose of insulin (1.5 nM). Glucose transport was measured using 2-[3H]Deoxy-Glucose and intramyocellular signaling was investigated using immunoblotting. In soleus muscles from tumor-bearing mice, insulin-stimulated glucose transport was abrogated concomitantly with abolished insulin-induced TBC1D4 and GSK3 phosphorylation. In EDL, glucose transport and TBC1D4 phosphorylation were not impaired in muscles from tumor-bearing mice, while AMPK signaling was elevated. Anabolic insulin signaling via phosphorylation of the mTORC1 targets, p70S6K thr389 and ribosomal-S6 ser235, were decreased by cancer in soleus muscle while increased or unaffected in EDL. In contrast, the mTOR substrate, pULK1 ser757, was reduced in both soleus and EDL by cancer. Hence, cancer causes considerable changes in skeletal muscle insulin signaling that is dependent of muscle-type, which could contribute to metabolic dysregulation in cancer. Thus, skeletal muscle could be a target for managing metabolism in cancer.