Diabetes is the strongest independent predictor of progression of metabolic dysfunction-associated steatotic liver disease (MASLD) to cirrhosis and hepatocellular carcinoma, yet how glucose injures the liver is poorly understood. In the retina and kidney, hyperglycemia damages the microvascular endothelium through a shared mechanism in which unrestricted glucose entry inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and diverts glycolytic intermediates into the four major pathways of glucotoxicity. The liver sinusoidal endothelial cell (LSEC) is the first cell exposed to portal blood, which carries glucose from the gut at concentrations higher than the systemic circulation reaches, for years before diabetes is diagnosed. LSEC capillarization, the loss of fenestrae and transition to a continuous capillary, precedes steatohepatitis and fibrosis. Here we show that the LSEC is a target of the same glucotoxic mechanism that underlies the classical diabetic complications. Hyperglycemia inhibited GAPDH in primary mouse LSECs and induced capillarization cell-autonomously, and inhibition of GAPDH alone reproduced the defenestration. Of the four glucotoxicity pathways, flux through the hexosamine pathway to protein O-GlcNAcylation was necessary for defenestration under high glucose and sufficient to induce it under low glucose. O-GlcNAcylation of endothelial nitric oxide synthase suppressed nitric oxide production, driving defenestration through cGMP and protein kinase G signaling and Rho kinase-driven actomyosin contraction. Intervening at any step of the cascade reversed the defenestration. Hyaluronan, cleared by the LSEC and normally catabolized through glycolysis, supplied a second, GFAT-independent input to protein O-GlcNAcylation under hyperglycemia. Loss of LSEC nitric oxide was sufficient to increase hepatocyte lipid accumulation in coculture. In mice with established diet-induced steatohepatitis, inhibiting O-GlcNAc transferase restored sinusoidal fenestrae and attenuated fibrosis. The hepatic sinusoid is therefore a site of microvascular complications of hyperglycemia, alongside the retina, kidney and peripheral nerve.
