David Saggerson

Acute effects of insulin on glycerol phosphate acyl transferase activity, ketogenesis and serum free fatty acid concentration in perfused rat liver

In the perfused rat liver insulin inhibits the oxidation of serum free fatty acids [1 ] and endogenous fatty acids [2], increases the secretion of very low density lipoprotein triacylglycerol [1-3] and also opposes the antilipogenic effect of long chain free fatty acids [3]. In isolated adipocytes insulin increases the synthesis of triacylglycerol. Although some of this insulin effect in adipose tissue may be attributed to enhancement of glucose transport and hence provision of triose phosphates for esterification, there is evidence for more direct insulin action upon glyceride synthesis [4]. In vitro adipocyte glycerol phosphate acyltransferase (GPAT) and long-chain fatty acyl CoA synthetase activities have been shown to increase as a result of insulin treatment [5 -7] . It was therefore of interest to examine the acute effects of insulin on activities of these enzymes in the perfused rat liver.

A selective decrease in mitochondrial glycerol phosphate acyltransferase activity in livers from streptozotocin-diabetic rats.

The glycerol phosphate pathway of fatty acid esterification is one of the biosynthetic routes to glycerolipids. Glycerol phosphate acyltransferase (GPAT), is a possible site of control of this process, being the first committed enzyme. Little is known about regulation of glycerolipid synthesis although recent studies in adipose tissue indicate the possibility of acute hormonal regulation of GPAT activity [1,2]. In order to establish a basis for studies of short-term insulin action on hepatic GPAT in the intact animal the effect of mild diabetes on the mitochondrial and microsomal enzymes has been investigated. Effects of insulin on GPAT in the perfused rat liver are presented [3]. A statistically inconclusive study of the effect of starvation on hepatic GPAT has been made [4], but there do not appear to be any previous reports of the effect of diabetes. The present results show an interesting divergence in the responses of the mitochondrial and microsomal activities.

Regulation of mitochondrial carnitine palmitoyl transferases from liver and extrahepatic tissues

Developments in our understanding of the complex CPT enzyme system over the past ten years have been reviewed. Liver CPT1, which is probably distinct from that in several extrahepatic tissues, is subject to up- or down-regulation of its activity and kinetic properties with changing physiological state. Evidence is now accumulating to support the notion that the catalytic and malonyl-CoA-binding entities of CPT1 are separate polypeptides.

Neonatal carnitine palmitoyltransferase-2 deficiency: a case presenting with myopathy

Mitochondria were isolated from liver, heart and skeletal muscle of a 34-day-old female infant who died from a myopathic illness. Muscle biopsy showed lipid accumulation and no obvious pathology in any other organ. Enzymatic analysis of skeletal muscle extracts revealed normal activities of the markers pyruvate dehydrogenase and citrate synthase. Malonyl-CoA-sensitive carnitine palmitoyltransferase (CPT1) was detected but malonyl-CoA-insensitive carnitine palmitoyltransferase (CPT2) appeared to be absent. Quantitative immunoblotting revealed the presence of a normal abundance of CPT2 protein in the patients muscle. It is concluded that enzymically inactive CPT2 protein was present.

Acute regulation of 5′-AMP-activated protein kinase by long-chain fatty acid, glucose and insulin in rat primary adipocytes

Palmitate increased AMPK (5′-AMP-activated protein kinase) activity, glucose utilization and 2-DOG (2-deoxyglucose) transport in rat adipocytes. All three effects were blocked by the AMPK inhibitor Compound C, leading to the conclusion that in response to an increase in long-chain NEFA (non-esterified fatty acid) concentration AMPK mediated an enhancement of adipocyte glucose transport, thereby providing increased glycerol 3-phosphate for FA (fatty acid) esterification to TAG (triacylglycerol). Activation of AMPK in response to palmitate was not due to an increase in the adipocyte AMP:ATP ratio. Glucose decreased AMPK activity and effects of palmitate and glucose on AMPK activity were antagonistic. While insulin had no effect on basal AMPK activity insulin did decrease AMPK activity in the presence of palmitate and also decreased the percentage effectiveness of palmitate to increase the transport of 2-DOG. It is suggested that activation of adipocyte AMPK by NEFA, as well as decreasing the activity of hormone-sensitive lipase, could modulate adipose tissue dynamics by increasing FA esterification and, under certain circumstances, FA synthesis.

Soluble 5′‐Nucleotidase Activities in Rat Brain

Abstract: 5′‐Nucleotidase activity was assayed in 105,000‐g supernatants from rat brain by following conversion of [3H]AMP into adenosine. The effect of ATP on this process was complex and suggested the presence of at least two soluble 5′‐nucleotidase activities: one inhibited by ATP and another activated by ATP. The relative proportions of these activities differed considerably among brain regions. Activity changes induced by hypothyroidism also suggested that these activities may be regulated independently. These findings may have consequences for the regional regulation of adenosine formation in the brain.

Streptozotocin diabetes results in increased responsiveness of adipocyte lipolysis to glucagon

Adipocytes from streptozotocin‐diabetic rats are ∼50‐times more sensitive to the lipolytic action of glucagon. This change is only perceived in the presence of a small quantity of adenosine deaminase which itself has little effect on basal lipolysis. Insulin treatment restores glucagon sensitivity to normal.

Diabetes decreases sensitivity of adipocyte lipolysis to inhibition by G-linked receptor agonists

(1) Streptozotocin-diabetes decreased the responsiveness of noradrenaline- or forskolin-stimulated lipolysis to inhibition by phenylisopropyladenosine (PIA), prostaglandin E1 (PGE1) and nicotinate in rat adipocytes. (2) Diabetes had no effect on high affinity binding of [3H]PIA to adipocyte plasma membranes. (3) Plasma membranes from diabetic animals had increased abundance of alpha-subunits of Gi1 and Gi2. The effect of pertussis toxin in overcoming inhibition of lipolysis by PIA was delayed in adipocytes from diabetic rats. (4) Diabetes decreased the GTP-dependent right-wards shift in the dose-curve for displacement of the antagonist [3H]DPCPX by PIA in adipocyte plasma membranes. (5) It is concluded that, despite increased abundance of Gi in diabetic adipocytes, less of this is functional. This may contribute to reduced sensitivity to PIA, PGE1 and nicotinate and explains some of the loss of control of lipolysis in insulin-dependent diabetes.

Physiological state and the sensitivity of liver mitochondrial outer membrane carnitine palmitoyltransferase to malonyl-coa. correlations with assay temperature, salt concentration and membrane lipid composition

1. Liver mitochondrial outer membranes were pre-exposed to media of low (20 mM phosphate) or high salt concentration (20 mM phosphate + 0.3 M KCl) before assay of carnitine palmitoyltransferase (CPT) at 25 degrees C. 2. With membranes from fed rats, exposure to high salt decreased sensitivity of CPT to malonyl-CoA whereas high salt increased sensitivity of CPT to malonyl-CoA in membranes from 48 hr-fasted rats. These changes were paralleled by alterations in the KD for high affinity binding of [14C]malonyl-CoA to outer membranes. 3. Decreasing the CPT assay temperatures from 25 to 10 degrees C caused qualitatively similar changes to those seen on exposure to high salt. 4. The relative content of sphingomyelin was increased 2-fold and 4-fold in liver mitochondrial outer membranes from fasted and diabetic rats respectively. Fasting had no effect on the content of cholesterol whereas diabetes decreased this by a third.

The role of insulin in fatty acid metabolism

Abstract Insulin is now recognized to have several sites of action in the regulation of adipose and liver fatty acid metabolism.