Eleazar Shafrir

Diabetes: insulin resistance and derangements in lipid metabolism. Cure through intervention in fat transport and storage

We present multiple findings on derangements in lipid metabolism in type 2 diabetes. The increase in the intracellular deposition of triglycerides (TG) in muscles, liver and pancreas in subjects prone to diabetes is well documented and demonstrated to attenuate glucose metabolism by interfering with insulin signaling and insulin secretion. The obesity often associated with type 2 diabetes is mainly central, resulting in the overload of abdominal adipocytes with TG and reducing fat depot capacity to protect other tissues from utilizing a large proportion of dietary fat. In contrast to subcutaneous adipocytes, the central adipocytes exhibit a high rate of basal lipolysis and are highly sensitive to fat mobilizing hormones, but respond poorly to lipolysis restraining insulin. The enlarged visceral adipocytes are flooding the portal circulation with free fatty acids (FFA) at metabolically inappropriate time, when FFA should be oxidized, thus exposing nonadipose tissues to fat excess. This leads to ectopic TG accumulation in muscles, liver and pancreatic beta‐cells, resulting in insulin resistance and beta‐cell dysfunction. This situation, based on a large number of observations in humans and experimental animals, confirms that peripheral adipose tissue is closely regulated, performing a vital role of buffering fluxes of FFA in the circulation. The central adipose tissues tend to upset this balance by releasing large amounts of FFA. To reduce the excessive fat outflow from the abdominal depots and prevent the ectopic fat deposition it is important to decrease the volume of central fat stores or increase the peripheral fat stores. One possibility is to downregulate the activity of lipoprotein lipase, which is overexpressed in abdominal relatively to subcutaneous fat stores. This can be achieved by gastrointestinal bypass or gastroplasty, which decrease dietary fat absorption, or by direct means that include surgical removal of mesenteric fat. Indirect treatment consists of the compliant application of drastic lifestyle change comprising both diet and exercise and pharmacotherapy that reduces mesenteric fat mass and activity. The first step should be an attempt to effectively induce a lifestyle change. Next comes pharmacotherapy including acarbose, metformin, PPARγ, or PPARγα agonists, statins and orlistat, estrogens in postmenopausal women or testosterone in men. Among surgical procedures, gastric bypass has been proven to produce beneficial results in advance of other surgical techniques, the evidence basis of which still needs strengthening. Copyright

A biochemical and morphologic study of very low density lipoproteins in carbohydrate-induced hypertriglyceridemia

On a high carbohydrate, fat-free diet, control and hypertriglyceridemic subject had a three-fold increase in d < 1.006, very low density lipoprotein (VLDL) triglyceride, and somewhat lesser increases in VLDL cholesterol and protein. Cholesterol and protein in 1.006 < d < 1.21 lipoprotein decreased in a reciprocal fashion, suggesting that these components might have been utilized in VLDL production. Electron microscope studies demonstrated a significant increase in the size of lipoprotein particles of the VLDL class and, in three of four subjects, an apparent increase in particle number. The change in particle size correlated with an increase in the triglyceride/protein ratio of the d < 1.006 lipoprotein. Hypertriglyceridemic individuals differed from the control subjects in that they had greater absolute increases in VLDL triglyceride, cholesterol, and protein, and greater decreases in 1.006 < d < 1.21 cholesterol and protein. In addition, they had larger VLDL particles with a higher triglyceride/protein ratio, both before the study and at the peak of the carbohydrate effect. The data suggest that the increase in plasma triglycerides induced by a high carbohydrate diet is usually due to the appearance in plasma of both greater numbers of VLDL particles and larger particles that are relatively richer in triglyceride content than those isolated during the basal state.

Rat myofibrillar protease: Enzyme properties and adaptive changes in conditions of muscle protein degradation

Abstract Protease activity of rat gastrocnemius myofibrillar fraction, optimally active at the alkaline pH range, was investigated. The activity was inhibited by soybean trypsin inhibitor, tranexamic acid and other inhibitors of proteases, as well as by 2- to 10-m m solutions of FeCl 2 , FeCl 3 , and ZnCl 2 . CaCl 2 , MgCl 2 , and MnCl 2 at the same concentration range did not affect the activity of the enzyme. Treating the myofibrillar fraction with 1 m KCNS or KCl solubilized the enzyme. The protease activity was increased after 4 days of fasting, and on Day 6 was five times higher than in nonfasted rats. This increase in specific activity was significant when expressed in terms of total protein, noncollagen protein, or muscle DNA content. Protease activity also increased in rats after glucocorticoid administration, in rats with alloxan diabetes, in aminonucleoside-nephrotic rats, and in rats with hypoproteinemia induced by plasmapheresis. These findings suggest that the activity of the myofibrillar protease is adaptive, and increases whenever catabolism of muscle proteins and mobilization of amino acids to the liver is required.

Mechanism of fatty acid assimilation in adipose tissue

Abstract The kinetics of fatty acid uptake and esterification by sections of rat epididymal fat tissue was studied. Uptake of [I- 14 C]palmitate in albumin solution followed a first order reaction rate and was markedly enhanced by the addition of glucose. Incorporation into the tissue glyceride esters accounted for almost all the activity removed from the medium during the first 3–6 h of incubation. After this time, the incorporation of radioactivity into the esters ceased, while their specific activity was still much below that of the tissue or medium free acids. Centrifugal fractionation of the tissue proved the existence of an active compartment of tissue esters precipitated with the particles in which the specific activity exceed that of the esters in the floating fat by a factor of IO at least. This active compartment seems to be in equilibrium and interchange with the medium fatty acids. The tissue free fatty acids, on the other hand, do not seem to take a prominent part in assimilation and estrification. This conclusion is based on kinetic considerations and corroborated by experiments in which [I- 14 C]palmitate labeled epididymal tissues were made to release fatty acids with or without the presence of epinephrine. The fatty acids released to the medium exceeded in their specific activity those of the tissue and were therefore assumed to be derived from the active compartment of the tissue glycerides.

Nutritionally induced insulin resistance and receptor defect leading to beta-cell failure in animal models

ABSTRACT: Animals with genetically or nutritionally induced insulin resistance and Type 2 diabetes comprise two groups: those with resilient β‐cells, e.g., ob/ob mice or fa/fa rats, capable of longstanding compensatory insulin hypersecretion and those with labile β‐cells in which the secretion pressure leads to β‐cell degranulation and apoptosis, e.g., db/db mice and Psammomys gerbils (sand rats). Psammomys features low insulin receptor density; on a relatively high energy diet it becomes hyperinsulinemic and hyperglycemic. In hyperinsulinemic clamp the hepatic glucose production is only partially suppressed by insulin, even in the normoglycemic state. The capacity of insulin to activate muscle and liver receptor tyrosine kinase is nearly abolished. GLUT4 content and mRNA are markedly reduced. Hyperinsulinemia was also demonstrated to inhibit insulin signaling and glucose transport in several other species. Among the factors affecting the insulin signaling pathway, phosphorylation of serine/threonine appears to be the prominent cause of receptor malfunction as inferred from the finding of overexpression of PKCɛ isoforms in the muscle and liver of Psammomys. The insulin resistance syndrome progressing in animals with labile β‐cells to overt diabetes and β‐cell failure is a “thrifty gene” characteristic. This is probably also true for human populations emerging from food scarcity into nutritional affluence, inappropriate for their metabolic capacity. Thus, the nutritionally induced hyperinsulinemia, associated with PKCɛ activation may be looked upon from the molecular point of view as “PKCɛ overexpression syndrome.”

Characterization of stages in development of obesity-diabetes syndrome in sand rat (Psammomys obesus).

Sand rats (Psammomys obesus) maintained on a diet providing a free choice between laboratory chow and salt bush (Atriplex halimus) were classified into four groups differing in extent of the diabetic syndrome: A, normoglycemic-normoinsulinemic; B, normoglycemichyperinsulinemic; C, hyperglycemic-hyperinsulinemic; or D, hyperglycemic with reduced insulin levels. The metabolic pattern of these groups was characterized by measuring 1) the uptake of fatty acid-labeled, very-lowdensity lipoprotein-borne triglycerides (VLDL-TG) and [3H]%-2-deoxyglucose (2-DOG) into muscle and adipose tissues; 2) incorporation of [14C]alanine into glycogen in vivo; 3) gluconeogenesis from lactate, pyruvate, and alanine in hepatocytes; 4) the effect of insulin on glycogen synthesis from glucose; 5) the oxidation of albumin-bound [1-14C]palmitate and [14C]glucose in strips of soleus muscle; 6) activities of muscle and adipose tissue lipoprotein lipase; and 7) activities of rate-limiting enzymes of glycolysis, gluconeogenesis, and fatty acid synthesis in liver. In group A, uptake of VLDL-TG and activity of lipoprotein lipase were higher in adipose tissue and lower in muscle than in albino rats. In the liver, gluconeogenesis and the activity of phosphoenolpyruvate carboxykinase, as well as lipid synthesis and the activity of NAOP-malate dehydrogenase,were higher than in albino rats, whereas activity of pyruvate kinase was lower. In group B, uptake of VLDL-TG by adipose tissue and muscle and lipoprotein lipase activity were similar or higher than in group A. Uptake of 2-DOG by muscle and adipose tissue and activity of liver phosphoenolpyruvate carboxykinase were lower than in group A. In groups C and D, uptake of VLDL-TG and lipoprotein lipase activity in muscle were further increased. In adipose tissue a progressive decrease in VLDL-TG uptake and lipoprotein lipase activity was found, and uptake of 2-DOG by muscle and adipose tissue was further reduced. In the liver, gluconeogenesis was increased, and activity of phosphoenolpyruvate carboxykinase reached a maximum in group D. These results suggest that in the hyperinsulinemic stage (group B), uptake of glucose by muscle and adipose tissue is reduced, but insulin suppresses gluconeogenesis and stimulates hepatic synthesis and adipose tissue uptake of TG. Hyperglycemia manifests itself when insulin resistance results in increased gluconeogenesis and a further reduction in peripheral glucose uptake. These characteristics can be regarded as a model for the development of type II diabetes in humans evoked by nutritional affluence.

Effect of longterm sucrose feeding on the activity of some enzymes regulating glycolysis, lipogenesis and gluconeogenesis in rat liver and adipose tissue

Abstract 4- to 6-fold increases in the activity of liver pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), NADP-malate dehydrogenase (EC 1.1.1.40) and acetyl-CoA carboxylase (EC 6.4.1.2), relative to liver protein concentration, were obtained in rats fed for 12 months on a 72% sucrose diet, as compared to rats kept on a standard laboratory chow. Feeding a 72% starch diet resulted in only 2- to 3-fold increases in the activities of these enzymes. The activity of liver 6-phosphogluconate dehydrogenase (EC 1.1.1.44) increased about 2-fold in the sucrose-fed rats and did not rise significantly on a starch diet. In adipose tissue, the activity of pentose shunt enzymes rose significantly only on the sucrose diet, whereas NADP-malate dehydrogenase rose 5-fold in sucrose and 2-fold in starch-fed rats. Fasting for 48 h resulted in a decrease in the activity of enzymes of glucose utilization and lipogenesis in the liver and adipose tissue, which on all diets was of s similar proportion with respect to the activity prior to fasting. The activity of most enzymes was reduced to 50–70% of the prefasting value; that of NADP-malate dehydrogenase decreased more pronouncedly. The activity of liver phosphoenolpyruvate carboxylase (EC 4.1.1 32) was slightly decreased on both starch and sucrose diets, whereas the activity of glucose-6-phosphatase (EC 3.1.3.9) was significantly increased in the sucrose-fed rats, indicating adaptation to gluconeogenesis from the fructose component. Each of the enzymes of gluconeogenesis responded to 48 h fasting with a rise in activity, the extent of which was not apparently related to the previous diet. The more extensive changes in enzyme activities on prolonged feeding of sucrose in comparison to starch are discussed with respect to the large share of the liver in the metabolism of carbohydrate intake on sucrose and to the role of the fructose component of sucrose in producing a pattern of adaptation of liver enzymes conducive to lipogenesis and glucose intolerance.

Characterization of tissue lipolytic and esterolytic activities cleaving full and partial glycerides

Abstract Rat liver, kidney, heart, diaphragm, lung and spleen were found to contain lipolytic activities with increasing rates of cleavage from tri- to monoglycerides of long-chain fatty acids. These activities were assumed to be due to distinct enzymes, as inferred from varying ratios of activity in different tissues, different pH optima and inhibition characteristics. Similarities between tri- and monoglyceride lipase were found in their prevalence in the microsomal fraction of tissue homogenates and in the inhibitory effects on their activity of homogenization in the presence of ATP, cations, especially divalent, and of preincubation with uncouplers of oxidative phosphorylation. Thus, tri- and monoglyceride lipases seem to reside in the same intracellular site and are activated in a similar fashion, but upon homogenization exhibit distinct properties. Cleavage of tri- and monoglycerides of short-chain fatty acids, exemplified by tri- and monobutyrin, did not show the differences encountered between tri- and monoolein. Cleavage of the full and partial butyrate esters was, therefore, attributed to an esterase distinct from the lipases, as seen in the varying ratio of activity between esterolytic and lipolytic activities among the tissues studied, in pH optimum, susceptibility to inhibitors and in the kinetics of cleavage at rising substrate concentration. In analogy to lipases, the esterolytic activity was located in the microsomal fraction and was also somewhat affected by ATP and ions during tissue homogenization, suggesting a common location and similar mode of activation. The implication of lipase content in non-adipose tissues of the rat and the role of ATP and cations in their activation are briefly discussed in conjunction with the lipase activity in adipose tissue.

Cytosolic citrate and malonyl-CoA regulation in rat muscle in vivo

In liver, insulin and glucose acutely increase the concentration of malonyl-CoA by dephosphorylating and activating acetyl-CoA carboxylase (ACC). In contrast, in incubated rat skeletal muscle, they appear to act by increasing the cytosolic concentration of citrate, an allosteric activator of ACC, as reflected by increases in the whole cell concentrations of citrate and malate [Saha, A. K., D. Vavvas, T. G. Kurowski, A. Apazidis, L. A. Witters, E. Shafrir, and N. B. Ruderman. Am. J. Physiol. 272 ( Endocrinol. Metab. 35): E641-E648, 1997]. We report here that sustained increases in plasma insulin and glucose may also increase the concentration of malonyl-CoA in rat skeletal muscle in vivo by this mechanism. Thus 70 and 125% increases in malonyl-CoA induced in skeletal muscle by infusions of glucose for 1 and 4 days, respectively, and a twofold increase in its concentration during a 90-min euglycemic-hyperinsulinemic clamp were all associated with significant increases in the sum of whole cell concentrations of citrate and/or malate. Similar correlations were observed in muscle of the hyperinsulinemic fa/fa rat, in denervated muscle, and in muscle of rats infused with insulin for 5 h. In muscle of 48-h-starved rats 3 and 24 h after refeeding, increases in malonyl-CoA were not accompanied by consistent increases in the concentrations of malate or citrate. However, they were associated with a decrease in the whole cell concentration of long-chain fatty acyl-CoA (LCFA-CoA), an allosteric inhibitor of ACC. The results suggest that increases in the concentration of malonyl-CoA, caused in rat muscle in vivo by sustained increases in plasma insulin and glucose or denervation, may be due to increases in the cytosolic concentration of citrate. In contrast, during refeeding after starvation, the increase in malonyl-CoA in muscle is probably due to another mechanism.In liver, insulin and glucose acutely increase the concentration of malonyl-CoA by dephosphorylating and activating acetyl-CoA carboxylase (ACC). In contrast, in incubated rat skeletal muscle, they appear to act by increasing the cytosolic concentration of citrate, an allosteric activator of ACC, as reflected by increases in the whole cell concentrations of citrate and malate [Saha, A. K., D. Vavvas, T. G. Kurowski, A. Apazidis, L. A. Witters, E. Shafrir, and N. B. Ruderman. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E641-E648, 1997]. We report here that sustained increases in plasma insulin and glucose may also increase the concentration of malonyl-CoA in rat skeletal muscle in vivo by this mechanism. Thus 70 and 125% increases in malonyl-CoA induced in skeletal muscle by infusions of glucose for 1 and 4 days, respectively, and a twofold increase in its concentration during a 90-min euglycemic-hyperinsulinemic clamp were all associated with significant increases in the sum of whole cell concentrations of citrate and/or malate. Similar correlations were observed in muscle of the hyperinsulinemic fa/fa rat, in denervated muscle, and in muscle of rats infused with insulin for 5 h. In muscle of 48-h-starved rats 3 and 24 h after refeeding, increases in malonyl-CoA were not accompanied by consistent increases in the concentrations of malate or citrate. However, they were associated with a decrease in the whole cell concentration of long-chain fatty acyl-CoA (LCFA-CoA), an allosteric inhibitor of ACC. The results suggest that increases in the concentration of malonyl-CoA, caused in rat muscle in vivo by sustained increases in plasma insulin and glucose or denervation, may be due to increases in the cytosolic concentration of citrate. In contrast, during refeeding after starvation, the increase in malonyl-CoA in muscle is probably due to another mechanism.

Displacement of albumin-bound bilirubin by free fatty acids. Implications for neonatal hyperbilirubinemia

The interaction of bilirubin, linoleate and human plasma albumin was investigated in an isolated model system, designed to evaluate the competitive effect of free fatty acids (FFA) on the binding of unconjugated bilirubin by albumin. Increasing amounts of linoleate were added to bilirubin-albumin solutions at a constant molar ratio, and the mixtures passed through Sephadex G-25 columns to absorb the displaced bilirubin, which was then eluted and determined. In solutions with molar ratio bilirubin/albumin of 0.5 or 1.0 no displacement was evident when the molar ratio of linoleate relative to albumin was below 5. Above molar ratio of linoleate/albumin of 6, free bilirubin in the system rose sharply. In solutions with molar ratio bilirubin/albumin of 1.8, bilirubin dissociated from albumin markedly when the molar ratio of linoleate relative to albumin approached 4. Thus, the displacement of bilirubin starts only after most of the FFA binding sites on albumin with high association constants become saturated, and the competition involves mainly multiple binding sites with low association constants. In two groups of infants without or with hyperbilirubinemia requiring exchange transfusion, the molar ratio FFA/albumin in plasma ranged from 1.07 to 2.37. These values are much lower than those initiating the displacement of bilirubin in the in vitro system. The significance of FFA in causing shifts of bilirubin between intra- and extravascular pools is briefly discussed. It is suggested that FFA in neonates, although elevated, do not seem to reach concentrations, relative to albumin, which are effective in bilirubin dissociation.