Glucose Phosphorylation Is Essential for the Turnover of Neutral Lipid and the Second Stage Assembly of Triacylglycerol-Rich ApoB-Containing Lipoproteins in Primary Hepatocyte Cultures

Abstract

Abstract-Primary hepatocytes cultured in a medium supplemented with amino acids and lipogenic substrates responded to increased extracellular glucose by increasing the secretion of VLDL apoB. This effect was accompanied by an increased secretion of VLDL triacylglycerol (TAG) derived from endogenous stores. Glucose also stimulated intracellular TAG mobilization via the TAG lipolysis/esterification cycle. All these effects were abolished in the presence of mannoheptulose (MH), an inhibitor of glucose phosphorylation. Glucose also gave rise to a modest (50% to 60%) increase in the incorporation of 35 S methionine into newly synthesized apoB (PϽ0.05) and to a doubling of newly-synthesized apoB secretion as VLDL (PϽ0.05). The magnitude of these effects was similar for apoB-48 and for apoB-100. MH inhibited apoB-48 and apoB-100 synthesis and VLDL secretion at all glucose concentrations. The effects of glucose and MH on the secretion of newly-synthesized apoB-48 or apoB-100 as small dense particles were less pronounced. Glucose had no effects on the posttranslational degradation of newly-synthesized apoB-100 or apoB-48. However, this process was significantly enhanced by MH. The results suggest that glucose stimulates TAG synthesis, turnover, and output as VLDL. These effects are associated with an increased VLDL output of apoB mediated mainly by an increase in the net synthesis of both apoB-48 and apoB-100. All these changes are prevented by interference with glucose phosphorylation. Output of small, dense, apoB-containing particles is relatively unaffected by the glucose and MH-induced changes in TAG synthesis and lipolysis, an observation which suggests that only the bulk lipid addition step of VLDL assembly is affected by changes in glucose metabolism. Key Words: primary hepatocytes Ⅲ apoB Ⅲ glucose Ⅲ lipid recruitment Ⅲ phosphorylation E levated plasma glucose is a characteristic feature of the hypertriglyceridemia that frequently accompanies conditions such as non-insulin-dependent diabetes mellitus (NIDDM) and obesity. [1][2][3][4] The role of glucose in this relationship has been studied for several years and there now seems little doubt that 1 of the direct effects of glucose at the hepatic level is to increase the secretion of VLDL TAG. 5-8 Controversy exists, however, as to whether this effect of glucose is accompanied by a similar increase in the secretion of apoB, and studies with primary cultures of rat hepatocytes 7 and the human hepatoma cell line HepG2 9 showed little effect of glucose in this regard. Recent studies have produced evidence for a glucose-mediated stimulation of apoB output in HepG2, 10,11 and it has been suggested that the precise effect of glucose is dependent on the culture conditions in vitro. 11 We have previously shown that high rates of VLDL output from primary hepatocytes can be maintained only when the culture medium is supplemented with certain amino acids and fatty acid precursors, which provide an environment conducive to vigorous de novo lipogenesis. [12][13][14] It has also ...

Acute Inhibition of Glucose-6-Phosphate Translocator Activity Leads to Increased De Novo Lipogenesis and Development of Hepatic Steatosis Without Affecting VLDL Production in Rats

“…Additionally, insulin is a well-known acute inhibitor of VLDL secretion (52)(53)(54), and insulin resistance is associated with increased VLDLtriglyceride and apoB secretion (9 -11). Furthermore, recent data has shown that phosphorylation of glucose is also of importance in the regulation of assembly and secretion of triglyceride-containing VLDL (19). In our study, surprisingly, we did not find increased VLDL-triglyceride secretion in the S4048-treated rats.…”

“…Overexpression of hepatic glucokinase also leads to hyperlipidemia in fed rats (18). Brown et al (19) showed that the phosphorylation process is important for regulation of assembly and secretion of triglyceride-containing VLDLs by hepatocytes. Little is known about the mechanisms underlying the apparent paradox that hyperlipidemia de-velops in conditions associated with high as well as low G6Pase activity, i.e., diabetes and GSD.…”

Overproduction of large VLDL particles is driven by increased liver fat content in man

“…Therefore, in the setting of increased NEFA flux, hyperglycaemia may enhance TG synthesis. Along these lines, it has been indicated that glucose could influence the bulk lipid addition step of VLDL assembly [43]. Another potential source of liver TG is de novo lipogenesis [29,30].…”

Fatty liver in familial hypobetalipoproteinemia: triglyceride assembly into VLDL particles is affected by the extent of hepatic steatosis

“…These FAs are removed by liver and esterified into TG that may be assembled into VLDL-TG for export. FA may also be derived within liver by de novo synthesis (24)(25)(26). Mature VLDL particles are assembled in two steps (27): complexes form between lipids and apoB in the rough endoplasmic reticulum (ER) to form a primary VLDL particle, and these fuse with a separately formed "apoB-free" lipid particle fashioned in smooth ER (28).…”