Highlights d Spatial proteogenomic single-cell atlas of healthy and obese murine and human liver d Validated flow cytometry and microscopy panels for all hepatic cells d LAMs are differentially located in the lean and obese liver d Evolutionary conserved BMP9/10-ALK1 axis is essential for KC development

Studies in Zucker diabetic fatty rats have led to the concept that chronically elevated free fatty acid (FFA) levels can cause apoptosis of triglyceride-laden pancreatic ␤-cells as a result of the formation of ceramides, which induce nitric oxide (NO)-dependent cell death. This "lipotoxicity" hypothesis could explain development of type 2 diabetes in obesity. The present study examines whether prolonged exposure to FFA affects survival of isolated normal rat ␤-cells and whether the outcome is related to the occurrence of triglyceride accumulation. A dose-dependent cytotoxicity was detected at 5-100 nmol/l of unbound oleate and palmitate, with necrosis occurring within 48 h and an additional apoptosis during the subsequent 6 days of culture. At equimolar concentrations, the cytotoxicity of palmitate was higher than that of oleate but lower than that of its nonmetabolized analog bromopalmitate. FFA cytotoxicity was not suppressed by etomoxir (an inhibitor of mitochondrial carnitine palmitoyltransferase I) or by antioxidants; it was not associated with inducible NO synthase expression or NO formation. An inverse correlation was observed between the percentage of dead ␤-cells on day 8 and their cellular triglyceride content on day 2. For equimolar concentrations of the tested FFA, oleate caused the lowest ␤-cell toxicity and the highest cytoplasmic triglyceride accumulation. On the other hand, oleate exerted the highest toxicity in islet non-␤-cells, where no FFA-induced triglyceride accumulation was detected. In conditions without triglyceride accumulation, the lower FFA concentrations caused primarily apoptosis, both in islet ␤-cells and non-␤-cells. It is concluded that FFAs can cause death of normal rat islet cells through an NO-independent mechanism. The ability of normal ␤-cells to form and accumulate cytoplasmic triglycerides might serve as a cytoprotective mechanism against FFA-induced apoptosis by preventing a cellular rise in toxic free fatty acyl moieties. It is conceivable that this potential is lost or insufficient in cells with a prolonged triglyceride accumulation as may occur in vivo. Diabetes 50:1771-1777, 2001 I t is suspected that chronically elevated concentrations of free fatty acids (FFAs) contribute to the development of type 2 diabetes (1). They have been found to exert negative influences at the level of both insulin action and insulin release. FFAs can induce a state of insulin resistance (2-5) and impair pancreatic ␤-cell function (6 -9). There is also evidence that FFAs may cause ␤-cell death, at least in islets of Zucker diabetic fatty rats. In this animal model of obesity-associated diabetes, the sustained increase in circulating FFAs is held responsible for a triglyceride accumulation in the islet cells and for elevated cellular free fatty acyl levels that are cytotoxic (10). In isolated islets from prediabetic Zucker diabetic fatty rats, FFAs were shown to increase ceramide formation, leading to expression of nitric oxide (NO) synthase and NO-dependent ␤-cell apoptosis (11). These pro...

Summary Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow. Recruited macrophages existed in two subsets with distinct activation states, either closely resembling homeostatic KCs or lipid-associated macrophages (LAMs) from obese adipose tissue. Hepatic LAMs expressed Osteopontin, a biomarker for patients with NASH, linked with the development of fibrosis. Fitting with this, LAMs were found in regions of the liver with reduced numbers of KCs, characterized by increased Desmin expression. Together, our data highlight considerable heterogeneity within the macrophage pool and suggest a need for more specific macrophage targeting strategies in MAFLD.

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This study demonstrates that rat islet ␤ cells constitutively express an apoptotic program which is activated when mRNA or protein synthesis is blocked. Apoptotic ␤ cells were detectable by electron microscopy after treatment with actinomycin D or cycloheximide. With a fluorescence microscopic assay both agents were found to increase the number of apoptotic ␤ cells dose-and time-dependently, up to 70% after 1 wk of culture; virtually no apoptotic ␤ cells occurred in control preparations or in conditions leading to primary necrosis. Thus, survival of ␤ cells seems dependent on synthesis of proteins which suppress an endogenous suicide program. This mechanism explains earlier observed effects of glucose on survival of cultured ␤ cells. Glucose is known to dose-dependently increase the percentage of ␤ cells in active biosynthesis and the percentage that survives during culture. It is now demonstrated that the glucose-induced survival of ␤ cells cultured for 1 wk results from a dose-dependent reduction in the percentage of ␤ cells dying in apoptosis (49% at 3 mM glucose, 40% at 6 mM, 9% at 10 mM). Thus, intercellular differences in glucose sensitivity appear responsible for the heterogeneity in ␤ cell sensitivity to apoptotic conditions. These data indicate that glucose promotes