Insulin stimulates triacylglycerol secretion by perfused livers from fed rats but inhibits it in livers from fasted or insulin-deficient rats implications for the relationship between hyperinsulinaemia and hypertriglyceridaemia.

We determined whether the direction of the acute effect of insulin on hepatic triacylglycerol secretion is dependent on the prior physiological state or on the in vitro experimental system used. The effect of insulin on triacylglycerol secretion was studied using perfused livers isolated from rats under three metabolic conditions: fed normo-insulinaemic, 24-h fasted and fed, streptozotocin-diabetic (insulin-deficient). Insulin acutely activated triacylglycerol secretion (by 43%) in organs from fed, normo-insulinaemic animals, whereas it inhibited triacylglycerol secretion in livers isolated from fasted or insulin-deficient rats (by 30 and 33%, respectively). By contrast, in 24-h-cultured hepatocytes insulin invariably acutely inhibited triacylglycerol secretion irrespective of the metabolic state of the donor animals. It is concluded that the use of perfused livers enables the observation of a switch in the direction of insulin action on hepatic triacylglycerol secretion from stimulatory, in the normo-insulinaemic state, to inhibitory in the fasting or insulin-deficient state. The possible implications of this switch for the relationship between hyperinsulinaemia, increased hepatic very-low-density lipoprotein-triacylglycerol secretion and hypertriglyceridaemia observed in vivo are discussed.     

Alpha-adrenergic suppression of very-low-density-lipoprotein triacylglycerol secretion by isolated rat hepatocytes.

The effect of adrenaline on triacylglycerol synthesis and secretion was examined in isolated rat hepatocytes. Cells were incubated with 0.5 mM-[1-14C]oleate, and the accumulation of triacylglycerol and [14C]triacylglycerol was measured in the incubation medium. Triacylglycerol appearing in the medium was present in a form with properties similar to very-low-density lipoproteins. Triacylglycerol, [14C]triacylglycerol and [14C]phospholipid contents of hepatocytes were also determined. Addition of 10 microM-(-)adrenaline decreased accumulation of glycerolipid in the incubation medium and also decreased cellular [14C]phospholipid content. Prazosin abolished these effects, whereas propranolol did not. The hormone did not affect cellular triacylglycerol content or rates of incorporation of [1-14C]oleate into cell triacylglycerol. The effect of adrenaline on the removal of newly secreted triacylglycerol and the secretion of synthesized glycerolipid was also examined. The catecholamine did not affect rates of removal of newly secreted triacylglycerol. Adrenaline did inhibit the secretion of pre-synthesized lipid by the cells, as assessed by the appearance of radiolabelled triacylglycerol from hepatocytes that had been preincubated with [1,2,3-3H]-glycerol. Adrenaline did not affect rates of fatty acid uptake by hepatocytes, but did stimulate oxidation of [1-14C]oleate, principally to 14CO2.     

Fatty acid metabolism and lipid secretion by perfused livers from rats fed laboratory stock and sucrose-rich diets

To assess the possible role of altered hepatic processing of free fatty acids in dietary sucrose-induced accumulation of triglyceride in the liver and blood plasma, livers from rats fed commercial laboratory stock and high sucrose diets were perfused both with and without oleic acid substrate. Consumption of the sucrose diet exerted a multiplicity of effects on oleic acid metabolism, characterized by decreased conversion to both ketone bodies and carbon dioxide, increased esterification into liver triglyceride, and increased secretion in triglyceride-rich lipoproteins. During the infusion of oleic acid, livers from sucrose-fed rats also exhibited decreased ketogenesis, and increased secretion of triglyceride from endogenous sources. Since oleic acid uptake from the perfusion medium was identical in both groups, the observed effects of sucrose feeding are ascribed to altered rates of intracellular metabolic processes. Mass and radiochemical analyses of perfusate ketone bodies and triglycerides were indicative of greater mobilization of triglycerides from hepatocellular lipid droplets in the livers from sucrose-fed rats. These livers contained more triglyceride and secreted more triglyceride even in the absence of infused oleic acid. In summary, the sucrose-rich diet increased the esterification:oxidation ratio of intracellular free fatty acids derived from both the circulation and endogenous sources within the hepatocyte. In response, secretion of triglyceride-rich lipoproteins by the liver and deposition of triglyceride within the liver were promoted. It is concluded that alterations in the processing of free fatty acids by the liver contribute significantly to the liver and plasma triglyceride accumulation following sucrose consumption.     

Formation of free acetate by isolated perfused livers from normal, starved and diabetic rats

Isolated rat livers perfused in an open system exhibited a continous net release of free acetate. Upon intraportal infusion of hexanoate the net release of total ketone bodies and of free acetate increased significantly in livers from fed and 48 hours starved rats. The ratio ketone body production/acetate production during infusion of hexanoate was similar with livers from fed and starved rats. Livers from diabetic rats, however, did not only exhibit a higher rate of ketone body and acetate production, but also a significant decrease of the ratio ketone body production/acetate production. Intraportal infusion of oleate led also to an enhanced release of free acetate. An examination of the activities of 5 enzymes involved in ketone body and acetate metabolism showed no correlation with the higher rate of acetate production by diabetic livers.     

Failure of insulin to stimulate lipogenesis and triacylglycerol secretion in perfused livers from rats adapted to dietary fish oil

Livers from male rats fed a standard commercial diet supplemented with 8% (w/w) marine fish or safflower oils were perfused for 70 min with undiluted blood in the presence and absence of insulin. Lipogenesis, as measured by the incorporation of ³H₂O into liver and perfusate fatty acids, was inhibited by the feeding of fish oil. Net triacylglycerol secretion was also depressed by this dietary treatment. Infusion of insulin stimulated triacylglycerol secretion and the incorporation of newly synthesised fatty acids into liver and perfusate lipids with dietary safflower oil but not with fish oil. Hepatic cholesterol synthesis was also depressed by feeding fish oil. Net ketogenesis was raised by feeding fish oil and was depressed by insulin with both safflower and fish oil. Blood glucose was raised in the fish oil group but with both dietary oils the hormone exerted a significant hypoglycaemic effect. The data are discussed with respect to the observations that in vivo dietary fish oil (but not safflower oil) opposes the hypertriglyceridaemia arising from the hepatic overproduction of very-low-density lipoproteins.     

Regulation of very-low-density-lipoprotein lipid secretion in hepatocyte cultures derived from diabetic animals.

Hepatocytes were derived from 2-3-day streptozotocin-diabetic rats and maintained in culture for up to 3 days. Compared with similar cultures from normal animals, these hepatocytes secreted less very-low-density-lipoprotein (VLDL) triacylglycerol, but the decrease in the secretion of VLDL non-esterified and esterified cholesterol was not so pronounced. This resulted in the secretion of relatively cholesterol-rich VLDL particles by the diabetic hepatocytes. Addition of insulin for a relatively short period (24 h) further decreased the low rates of VLDL triacylglycerol secretion from the diabetic hepatocytes. The secretion of VLDL esterified and non-esterified cholesterol also declined. These changes occurred irrespective of whether or not exogenous fatty acids were present in the culture medium. Little or no inhibitory effect of insulin was observed after longer-term (24-48 h) exposure to the hormone. Both dexamethasone and a mixture of lipogenic precursors (lactate plus pyruvate) stimulated VLDL triacylglycerol and cholesterol secretion, but not to the levels observed in hepatocytes from normal animals. The low rate of hepatic VLDL secretion in diabetes contrasts with the increase in whole-body VLDL production rate. This suggests that the intestine is a major source of plasma VLDL in insulin-deficient diabetes.     

Stimulation by 3-mercaptopicolinate of net glusose uptake by perfused livers from diabetic rats

Glucose uptake/production was studied as a function of varied glucose loadsin isolated perfused livers from glucagon-treated alloxan-diabetic rats. Uptake of D-[U-¹⁴C]glucose was seen at all levels studied - 9.5–71 mM. In studies with unlabelled D-glucose carried out in the absence of 3-mercaptopicolinate, livers of diabetic rats showed a net production of glucose with perfusate glucose levels less than 22 mM. Above this level, these livers exhibited a time- and concentration-dependent net uptake of glucose for the period of 20–30 min. When 4 mM 3-mercaptopicolinate, which inhibited gluconeogenesis from endogenous substrates, was included in perfusates, a continuous net uptake of unlabelled glucose was observed at all levels above 4 mM. This lowering of the null-point, cross-over glucose concentration was shown to relate mechanistically to the observed reduction in steady hepatic glucose 6-phosphate level produced by mercaptopicolinate. The need for supplemental mechanisms of glucose utilization by high K_ww hepatic enzyme(s) operative in the virtual absence of insulin-dependent glucokinase also is indicated by these observations and by kinetic analysis.     

Secretion and uptake of nascent hepatic very low density lipoprotein by perfused livers from fed and fasted rats

Livers from fed or 24-hr fasted male rats were perfused in a recycling system. VLDL labeled with [1-14C]oleate (95% in triglyceride), produced in separate perfusions of livers from fed rats, was added to the medium as a pulse. Uptake of VLDL 14C-labeled triglyceride by livers from fasted rats was less than that from fed rats regardless of addition of oleate. During the interval in which radioactive triglyceride was taken up, the mass of triglyceride in the medium increased, indicative of the synthesis and net secretion of triglycerides. The rates of secretion of VLDL and uptake of VLDL were both more rapid in livers from fed rats in comparison to those from fasted animals. It was calculated that about 50% of the triglyceride synthesized and secreted by the liver was taken back by livers from fed rats. The VLDL from livers of fasted rats did not contain any apoE detectable by SDS gel electrophoresis or by radioimmunoassay when no fatty acid or 166 mumol of oleic acid was infused. In contrast, apoE comprised 6% of the VLDL apoprotein derived from perfusion of livers from fed animals in the absence of added fatty acid, and 20% when the fed livers were infused with 166 mumol of oleic acid. However, the net output (accumulation) of apoE by fasted liver was only two-thirds that from fed livers. When lipoprotein-free rat plasma containing apoE (4 mg/dl) was used in place of bovine serum albumin, the VLDL secreted by livers from either fed or fasted rats contained apoE and was taken up to a similar extent by such livers. These data suggested that the apoE of the d greater than 1.21 g/ml fraction was transferred to newly secreted VLDL which then stimulated uptake of the VLDL by livers from fasted rats. With further stimulation of secretion of VLDL triglyceride by infusion of 332 mumol of oleic acid/hr, the percent of apoE in the VLDL secreted by livers from fasted rats increased to 20%, which was similar to that of the VLDL produced by livers from fed rats when either 166 or 332 mumol/hr was infused. These data suggest a relationship between rates of hepatic secretion of VLDL (TG) and apoE, and the association of apoE with the secreted VLDL. During fasting, reduced secretion of both VLDL and apoE resulted in a VLDL particle that was considerably diminished in content of apoE and, therefore, that would be taken up by the liver at a reduced rate, in comparison to that observed in the fed animal.     

Differential inhibition of ketogenesis by malonyl-CoA in mitochondria from fed and starved rats.

Rates of ketogenesis in mitochondria from fed or starved rats were identical at optimal substrate concentrations, but responded differently to inhibition by malonyl-CoA. Kinetic data suggest that the K1 for malonyl-CoA is greater in the starved animal. These results indicate that, for the regulation of ketogenesis in the starved state, the lower sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA may be more important than the concentration of malonyl-CoA.     

Glycogen synthase R in livers of starved rats and starved rats given glucose

We reported that when synthase D was converted to synthase I in a rat liver extract, it progressed through a synthase form with activity characteristics which could not be explained by a mixture of synthase D and synthase I (Tan, A. W. H. (1981) Biochem. J. 200, 169-172). In this study we will borrow the "R" nomenclature to describe this "non-D" and "non-I" activity. Using activities measured at five different conditions and simultaneous equations, the amount of the three synthase forms in liver extracts can be estimated. During incubation of the liver extract, the amount of synthase R was found to increase with time and then to decrease as synthase I was generated, a profile typical of an enzyme intermediate. We investigated for the presence of synthase R in rat liver under different in vivo conditions. In contrast to the liver of fed rats which had very little synthase R, the liver of fasted rats was found to have 30% of its synthase in the R form. This synthase R was increased 2-fold when glucose was given and decreased to a very low level when glucagon was given. Synthase I was not detected, even in the livers of starved rats given glucose. Using conditions which were closer to those of the cell, synthase R was found to have relatively high activity, up to 70% that of synthase I. Based on these results, synthase R is proposed to be an active enzyme form responsible for glycogen synthesis in rat liver.     

Ethanol stimulates glycogenolysis in livers from fed rats.

To determine the reason for the lack of a hypoglycemic effect of ethanol in the fed state, the effect of ethanol on glucose turnover, liver glycogenolysis, and glucose metabolites was determined. Chronically catheterized awake and freely moving fed rats received either ethanol (blood ethanol, 37 +/- 10 mmol/liter, n = 11) or saline (n = 13) intravenously for 4 hr. Glucose turnover was determined using a primed continuous infusion of [3-3H]glucose. The liver was freeze clamped at 4 hr for glycogen and metabolite measurements. Plasma glucose (5.8 +/- 0.3 mmol/liter vs 6.3 +/- 0.2 mmol/liter at 4 hr, ethanol versus saline) and the rate of glucose turnover (61 +/- 9 vs 58 +/- 8 moles/kg.min) were similar during the ethanol and saline infusions. Plasma lactate was significantly higher in the ethanol (1.32 +/- 0.05 mmol/liter) than in the saline (0.86 +/- 0.06 mmol/liter, P less than 0.001) study. Concentrations of gluconeogenic intermediates in the liver (glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate, and pyruvate) were all significantly and -30% lower in ethanol-infused than in saline-infused rats. The liver citrate content was similar in ethanol-infused than in saline-infused rats. The liver citrate content was similar in ethanol (0.38 +/- 0.03 mmol/liter) and saline (0.37 +/- 0.04 mmol/liter) studies. Liver glycogen was 75% lower in the ethanol-infused (61 +/- 9 mmol/kg dry wt) than the saline (242 +/- 27 mmol/kg dry wt, P less than 0.001)-infused rats. These data demonstrate that in fed rats given ethanol, glucose turnover is maintained constant by accelerated glycogenolysis. Thus, inhibition of gluconeogenesis by ethanol does not lower hepatic glucose production unless compensatory glycogenolysis can be prevented.     

In vitro formation of glucose-6-phosphate from glyceraldehyde-3-phosphate by liver cytosol from fed and starved rats. Effect of divalent cations on the conversion rate.

Liver cytosol preparations from fed rats are shown to form glucose-6-phosphate from glyceraldehyde-3-phosphate at a rate of 1.6 μmoles·min^?1·g liver wet weight^?1 in presence of 0.4 mM Mg²⁺. This rate is more than doubled by 30 μM EGTA and/or Mg²⁺-concentrations ≥2 mM. 48 hours starvation increases the rate of glucose-6-phosphate formation at 0.4 mM Mg²⁺ to 3.0 μmoles·min^?1·g liver wet weight^?1 and greatly diminishes the effect of EGTA and of higher Mg²⁺-concentrations. Inhibition of glucose-6-phosphate formation by Ca²⁺ and Zn²⁺ is shown to be more pronounced with cytosol from fed than from 48 hours starved rats.     

Abnormal secretion of proteins into bile from colchicine-treated isolated perfused rat livers.

The microtubule poison, colchicine, caused an abnormal output of a variety of proteins into rat bile. After 3 h of exposure to the drug, livers were isolated and perfused with media of defined protein composition. There was no essential change in permeability of the hepatobiliary system to proteins (e.g. bovine serum albumin) entering bile from the perfusion fluid. The rat (serum) albumin and fibrinogen that were secreted into bile from colchicine-treated livers were probably derived from the hepatocytes. Disruption of the microtubular system reduces the secretion of proteins at the sinusoidal face of the hepatocyte and results in an accumulation of secretory vesicles in the cytoplasm. It is suggested that under these conditions some of the vesicles discharge their contents into the bile canaliculus.     

Dietary creatine supplementation lowers hepatic triacylglycerol by increasing lipoprotein secretion in rats fed high-fat diet

Recent studies have shown that dietary creatine supplementation can prevent lipid accumulation in the liver. Creatine is a small molecule that plays a large role in energy metabolism, but since the enzyme creatine kinase is not present in the liver, the classical role in energy metabolism does not hold in this tissue. Fat accumulation in the liver can lead to the development of nonalcoholic fatty liver disease (NAFLD), a progressive disease that is prevalent in humans. We have previously reported that creatine can directly influence lipid metabolism in cell culture to promote lipid secretion and oxidation. Our goal in the current study was to determine whether similar mechanisms that occur in cell culture were present in vivo. We also sought to determine whether dietary creatine supplementation could be effective in reversing steatosis. Sprague–Dawley rats were fed a high-fat diet or a high-fat diet supplemented with creatine for 5 weeks. We found that rats supplemented with creatine had significantly improved rates of lipoprotein secretion and alterations in mitochondrial function that were consistent with greater oxidative capacity. We also find that introducing creatine into a high-fat diet halted hepatic lipid accumulation in rats with fatty liver. Our results support our previous report that liver cells in culture with creatine secrete and oxidize more oleic acid, demonstrating that dietary creatine can effectively change hepatic lipid metabolism by increasing lipoprotein secretion and oxidation in vivo. Our data suggest that creatine might be an effective therapy for NAFLD.     

Insulin secretion reactivation of pancreatic islets from starved rats in vitro.

Starvation of Wistar rats induced a shift of glucose threshold for insulin secretion of isolated islets above 5 mM, which can be restored by pretreatment of the tissue with glucose, mannose, glyceraldehyde, an theophylline, but not with acetylcholine or lactate. The improved insulin secretion is not connected with an enhanced glucose utilization.     

Amino acid control of proteolysis in perfused livers of synchronously fed rats. Mechanism and specificity of alanine co-regulation

The primary control of autophagically mediated proteolysis in perfused rat liver is carried out via two alternate mechanisms in response to specific regulatory amino acids. One (L) elicits direct inhibition at low and high plasma levels, but requires a co-regulatory amino acid to express inhibition at normal concentrations. The second (H) is ineffective at normal levels and below, but active at higher concentrations. Because regulation is subject to unpredictable variability with ad libitum feeding, we have utilized rats synchronously fed 4 h day-1 to stabilize responses. Proteolytic control is seen to evolve in stages: H appears 12 h after the start of feeding; by 18 h L emerges, alternating with H in a statistically predictable way; with omission of the 24-h feeding, H disappears and L remains constant through 42 h. In both 18- and 42-h rats, alanine, glutamate, and aspartate exhibit similar inhibitory activity when added singly to the regulatory group at normal plasma concentrations. However, since alanine, but not glutamate or aspartate, evokes proteolytic acceleration when it is deleted from a full plasma mixture, alanine appears to be the sole co-regulator. Alanine yields co-regulatory effects with normal plasma leucine (0.2 mM) in 18- and 42-h animals and interacts synergistically with 0.8 mM leucine in 42-h but not in 18-h rats where leucine alone inhibits strongly. Because the inactivation of alanine amino-transferase by aminooxyacetate (determined from the conversion of [14C]alanine to glucose) does not alter the co-regulatory and synergistic effects of alanine, regulation by alanine must be mediated from a site of recognition before transamination.