Free Fatty Acid Reesterification Research Articles

Role of Dgat2 in Glucose Uptake and Fatty Acid Metabolism in C2C12 Skeletal Myotubes.

Acyl-coenzyme A (CoA):diacylglycerol acyltransferase 2 (DGAT2) catalyzes the last stage of triacylglycerol (TAG) synthesis, a process that forms ester bonds with diacylglycerols (DAG) and fatty acyl-CoA substrates. The enzymatic role of Dgat2 has been studied in various biological species. Still, the full description of how Dgat2 channels fatty acids in skeletal myocytes and the consequence thereof in glucose uptake have yet to be well established. Therefore, this study explored the mediating role of Dgat2 in glucose uptake and fatty acid partitioning under short interfering ribonucleic acid (siRNA)-mediated Dgat2 knockdown conditions. Cells transfected with Dgat2 siRNA downregulated glucose transporter type 4 (Glut4) messenger RNA (mRNA) expression and decreased the cellular uptake of [1-14C]-labeled 2-deoxyglucose up to 24.3% (p < 0.05). Suppression of Dgat2 deteriorated insulininduced Akt phosphorylation. Dgat2 siRNA reduced [1-14C]-labeled oleic acid incorporation into TAG, but increased the level of [1-14C]-labeled free fatty acids at 3 h after initial fatty acid loading. In an experiment of chasing radioisotope-labeled fatty acids, Dgat2 suppression augmented the level of cellular free fatty acids. It decreased the level of re-esterification of free fatty acids to TAG by 67.6% during the chase period, and the remaining pulses of phospholipids and cholesteryl esters were decreased by 34.5% and 61%, respectively. Incorporating labeled fatty acids into beta-oxidation products increased in Dgat2 siRNA transfected cells without gene expression involving fatty acid oxidation. These results indicate that Dgat2 has regulatory function in glucose uptake, possibly through the reaction of TAG with endogenously released or recycled fatty acids.

Exercise increases myocardial free fatty acid oxidation in subjects with metabolic dysfunction-associated fatty liver disease

Background and aimsMetabolic dysfunction-associated fatty liver disease (MAFLD) is associated with dyslipidemia and may promote cardiac lipotoxicity. Myocardial free fatty acids (FFA) oxidation (MOFFA) is normal in pre-diabetes, but reduced in heart failure. We hypothesized that during exercise MOFFA, very low-density lipoprotein triglycerides (VLDL-TG) secretion, hepatic FFA utilization, and lactate production differ among obese subjects with and without MAFLD. MethodsNine obese subjects with MAFLD and 8 matched subjects without MAFLD (Control) without a history of heart failure and cardiovascular disease were compared before and after 90-min exercise at 50% Peak oxygen consumption. Basal and exercise induced cardiac and hepatic FFA oxidation, uptake and re-esterification and VLDL-TG secretion were measured using [11C]palmitate positron-emission tomography and [1–14C]VLDL-TG. ResultsIn the heart, increased MOFFA was observed after exercise in MAFLD, whereas MOFFA decreased in Control (basal vs exercise, MAFLD: 4.1 (0.8) vs 4.8 (0.8) μmol·100 ml−1 min−1; Control: 4.9 (1.8) vs 4.0 (1.1); μmol·100 ml−1 min−1, mean (SD), p < 0.048). Hepatic FFA fluxes were significantly lower in MAFLD than Control and increased ≈ two-fold in both groups. VLDL-TG secretion was 50% greater in MAFLD at rest and similarly suppressed during exercise. Plasma lactate increased significantly less in MAFLD than Control during exercise. ConclusionsUsing robust tracer-techniques we found that obese subjects with MAFLD do not downregulate MOFFA during exercise compared to Control, possibly due to diminished lactate supply. Hepatic FFA fluxes are significantly lower in MAFLD than Control, but increase similarly with exercise. VLDL-TG export remains greater in MAFLD compared to Control. Basal and post-exercise myocardial and hepatic FFA, VLDL-TG and lactate metabolism is abnormal in subjects with MAFLD compared to Control.

RF04 | PSUN333 Divergent Pathways in Liver Triglyceride Storage and Secretion in Partial Lipodystrophy vs. Obesity Associated NAFLD

Abstract Background Partial lipodystrophy (PL) syndromes are characterized by deficiency of subcutaneous fat, increased liver fat and insulin resistance (IR). Nonalcoholic fatty liver disease (NAFLD) is an important cause of morbidity and mortality in both PL and obesity. Hepatic lipid derives from re-esterification of free fatty acids (FFA) from adipose tissue lipolysis and dietary sources, as well as de novo lipogenesis. Hepatic TG can be used for energy by β-oxidation, stored in hepatocytes as lipid droplets or secreted as VLDL. Hepatosteatosis develops when the rate of intrahepatic TG production is greater than the rate of TG disposal (oxidation and secretion). Here we investigate the contributions of insulin resistance and adipose tissue to hepatic steatosis in patients with PL vs non-syndromic, obesity associated NAFLD (NAFLD). Methods Single-center retrospective analysis of subjects with NAFLD (N=73) and PL (N=28). Fibroscan Controlled Attenuated Parameter (CAP) was used to estimate liver fat. A1c, β-hydroxybutyrate, and circulating lipids by NMR were measured simultaneously and within 6 months of CAP measurement. Insulin resistance was estimated by HOMA-IR (fasting glucose x insulin ÷ 405) and LP-IR (a lipoprotein-based measure of IR derived from circulating lipid particles). Results The PL group was 86% female vs 48% in NAFLD (P&amp;lt;0.001) and younger (43±8 vs. 50±14 years, P&amp;lt;0.03). BMI was lower PL vs NAFLD (27±6 vs 33±6 kg/m2, P&amp;lt;0.0001). Fasting insulin was not statistically different between PL and NAFLD (median 26.4 [interquartile range 21.3,51.2]) vs 31.6 mcU/L [15.1-33.4], P=0.052); however, IR was more severe in PL vs NAFLD based on both HOMA-IR (15.5±16.2 vs 7.6±5.2, P=0.003) and LP-IR (65.1±19.3 vs 58.5±16.7, P=0.03). A1c was higher in PL (7.5±2.1 vs 5.9±0.8%, &amp;lt;0.001). CAP was similar in PL and NAFLD (301±57 vs 320±49 dB/m, P=0.1). CAP correlated with HOMA-IR in both PL (r=0.51, p=0.01) and NAFLD (r=0.44, p=0.003), but for any given HOMA-IR liver fat was lower in PL vs NAFLD. FFA levels were comparable between groups (0.52± 0.28 vs 0.50±0.27 mEq/L, P=0.058). TRLP concentration by NMR was higher in PL compared to NAFLD (240±123 vs165±86 nmol/L, p=0.001) as was β-hydroxybutyrate (162±67 vs 115±58 mmol/L, P=0.004). Conclusions Patients with PL have comparable liver fat vs. non-syndromic NAFLD despite worse insulin resistance. To understand the lower liver fat relative to insulin resistance in PL, we examined input, oxidation, and export of lipid from the liver. Surprisingly, despite lower adipose mass in PL vs NAFLD, FFA input to the liver was similar. Hepatic fat oxidation and export, estimated by β-hydroxybutyrate and large VLDL respectively, were both higher in PL. This suggests a shift of fat storage out of the liver into the circulation in PL. Presentation: Saturday, June 11, 2022 1:18 p.m. - 1:23 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Non-adrenergic control of lipolysis and thermogenesis in adipose tissues.

The enormous plasticity of adipose tissues, to rapidly adapt to altered physiological states of energy demand, is under neuronal and endocrine control. In energy balance, lipolysis of triacylglycerols and re-esterification of free fatty acids are opposing processes operating in parallel at identical rates, thus allowing a more dynamic transition from anabolism to catabolism, and vice versa. In response to alterations in the state of energy balance, one of the two processes predominates, enabling the efficient mobilization or storage of energy in a negative or positive energy balance, respectively. The release of noradrenaline from the sympathetic nervous system activates lipolysis in a depot-specific manner by initiating the canonical adrenergic receptor-Gs-protein-adenylyl cyclase-cyclic adenosine monophosphate-protein kinase A pathway, targeting proteins of the lipolytic machinery associated with the interface of the lipid droplets. In brown and brite adipocytes, lipolysis stimulated by this signaling pathway is a prerequisite for the activation of non-shivering thermogenesis. Free fatty acids released by lipolysis are direct activators of uncoupling protein 1-mediated leak respiration. Thus, pro- and anti-lipolytic mediators are bona fide modulators of thermogenesis in brown and brite adipocytes. In this Review, we discuss adrenergic and non-adrenergic mechanisms controlling lipolysis and thermogenesis and provide a comprehensive overview of pro- and anti-lipolytic mediators.

21 Sensitivity of Comparing Serum Triglycerides Levels at “Fasting” and “60 Minutes After Oral Glucose Loading” to Screen and Estimate Insulin Resistance

Insulin function on cellular uptake of plasma triglycerides is affected by insulin potency (proinsulin percentage) and insulin resistances (prereceptor, receptor, and postreceptor insulin resistance types), while its function on plasma free fatty acids re-esterification into triglycerides is not affected by receptor or postreceptor resistance. Glucose loading in oral glucose tolerance test should lead to (1) suppression of cellular “hepatic” utilization of fatty acids, (2) suppression of further stimulation of lipolytic and hyper-glycemic hormones (such as glucagon, adrenaline, ACTH and cortisol, GH, TSH and FT3), and (3) stimulation of insulin secretion, which enhances re-esterification of plasma free fatty acids into triglycerides and induces cellular uptake of plasma triglycerides. Therefore, during oral glucose tolerance test, measuring the “fasting” serum triglycerides and “after 60 minutes” serum triglycerides levels will provide two snapshots of the circulatory triglycerides levels in one hour, and because there is no exogenous intake of lipids and there are almost no endogenous catabolic effects, the comparison between the two triglycerides levels will uncover any lack of concomitance of the rate of triglycerides formation with the rate of its uptake. In metabolic syndrome or diabetes mellitus type 2, where the insulin resistance is significant, the “after 60 minutes” serum triglycerides levels are increased compared to the basal fasting levels, meaning that excess circulatory triglycerides formation exceeds the excess triglycerides uptake by cells (hepatic and adipose tissues). While in normal patients or in diabetes mellitus type 1, where the insulin resistance is not significant, there are no remarkable dissimilarities between the triglycerides levels. Comparison of serum triglycerides levels of “fasting” and “after 60 minutes” in oral glucose tolerance test, is sensitive for screening for insulin resistance (especially receptor or postreceptor resistances); however, disturbances in lipolytic hormones (deficiency or excess) or somatostatin could affect the specificity.

The nuclear retinoid-related orphan receptor-α regulates adipose tissue glyceroneogenesis in addition to hepatic gluconeogenesis.

Circadian rhythms have an essential role in feeding behavior and metabolism. RORα is a nuclear receptor involved in the interface of the circadian system and metabolism. The adipocyte glyceroneogenesis pathway derives free fatty acids (FFA) liberated by lipolysis to reesterification into triglycerides, thus regulating FFA homeostasis and fat mass. Glyceroneogenesis shares with hepatic gluconeogenesis the key enzyme phosphoenolpyruvate carboxykinase c (PEPCKc), whose gene is a RORα target in the liver. RORα-deficient mice (staggerer, ROR(sg/sg)) have been shown to exhibit a lean phenotype and fasting hypoglycemia for unsolved reasons. In the present study, we investigated whether adipocyte glyceroneogenesis might also be a target pathway of RORα, and we further evaluated the role of RORα in hepatocyte gluconeogenesis. In vivo investigations comparing ROR(sg/sg) mice with their wild-type (WT) littermates under fasting conditions demonstrated that, in the absence of RORα, the release of FFA into the bloodstream was altered and the rise in glycemia in response to pyruvate reduced. The functional analysis of each pathway, performed in adipose tissue or liver explants, confirmed the impairment of adipocyte glyceroneogenesis and liver gluconeogenesis in the ROR(sg/sg) mice; these reductions of FFA reesterification or glucose production were associated with decreases in PEPCKc mRNA and protein levels. Treatment of explants with RORα agonist or antagonist enhanced or inhibited these pathways, respectively, in tissues isolated from WT but not ROR(sg/sg) mice. Our results indicated that both adipocyte glyceroneogenesis and hepatocyte gluconeogenesis were regulated by RORα. This study demonstrates the physiological function of RORα in regulating both glucose and FFA homeostasis.

Twelve weeks of endurance training increases FFA mobilization and reesterification in postmenopausal women

We examined the effects of exercise intensity and training on rates of lipolysis, plasma free fatty acid (FFA) appearance (R(a)), disappearance (R(d)), reesterification (R(s)), and oxidation (R(oxP)) in postmenopausal (PM) women. Ten sedentary but healthy women (55 ± 0.6 yr) completed 12 wk of supervised endurance exercise training on a cycle ergometer [5 days/wk, 1 h/day, 65% peak oxygen consumption (Vo(2peak))]. Flux rates were determined by continuous infusion of [1-(13)C]palmitate and [1,1,2,3,3-(2)H(5)]glycerol during 90 min of rest and 60 min of cycle ergometer exercise during one pretraining exercise trial [65% Vo(2peak) (PRE)] and two posttraining exercise trials [at power outputs that elicited 65% pretraining Vo(2peak) (absolute training; ABT) and 65% posttraining Vo(2peak) (relative training; RLT)]. Initial body weights (68.2 ± 4.5 kg) were maintained over the course of study. Training increased Vo(2peak) by 16.3 ± 3.9% (P < 0.05) (Zarins ZA, Wallis GA, Faghihnia N, Johnson ML, Fattor JA, Horning MA and Brooks GA. Metabolism 58: 9: 1338-1346, 2009). Glycerol R(a) and R(d) were elevated in the RLT trial (P < 0.05), but not the ABT trial after training. Rates of plasma FFA R(a), R(d), and R(oxP) were elevated during the ABT compared with PRE trial (P < 0.05). FFA R(s) accounted for most (50-70%) of R(d) during exercise; training reduced FFA R(s) during ABT, but not RLT compared with PRE. We conclude that, despite the large age-related decrease in metabolic scope in PM women, endurance training increases the capacities for FFA mobilization and oxidation during exercises of a given power output. However, after menopause, total lipid oxidation capacity remains low, with reesterification accounting for most of FFA R(d).

Glucose-Dependent Insulinotropic Polypeptide May Enhance Fatty Acid Re-esterification in Subcutaneous Abdominal Adipose Tissue in Lean Humans

OBJECTIVEGlucose-dependent insulinotropic polypeptide (GIP) has been implicated in lipid metabolism in animals. In humans, however, there is no clear evidence of GIP effecting lipid metabolism. The present experiments were performed in order to elucidate the effects of GIP on regional adipose tissue metabolism.RESEARCH DESIGN AND METHODSEight healthy subjects were studied on four different occasions. Abdominal subcutaneous adipose tissue metabolism was assessed by measuring arterio-venous concentration differences and regional adipose tissue blood flow during GIP (1.5 pmol/kg/min) or saline infused intravenously alone or in combination with a hyperinsulinemic-hyperglycemic (HI-HG) clamp.RESULTSDuring GIP and HI-HG clamp, abdominal subcutaneous adipose tissue blood flow, hydrolysis of circulating triacylglycerol (TAG) (P = 0.009), and glucose uptake (P = 0.03) increased significantly while free fatty acid (FFA) output (P = 0.04) and FFA/glycerol release ratio (P = 0.02) decreased compared with saline and HI-HG clamp.CONCLUSIONSIn conclusion, GIP in combination with hyperinsulinemia and slight hyperglycemia increased adipose tissue blood flow, glucose uptake, and FFA re-esterification, thus resulting in increased TAG deposition in abdominal subcutaneous adipose tissue.

Phosphoenolpyruvate carboxykinase (Pck1) helps regulate the triglyceride/fatty acid cycle and development of insulin resistance in mice

The aim of this study was to investigate the role of the cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) in the development of insulin resistance. Previous studies have shown that the roles of Pck1 in white adipose tissue (WAT) in glyceroneogenesis and reesterification of free fatty acids (FFA) to generate triglyceride are vital for the prevention of diabetes. We hypothesized that insulin resistance develops when dysregulation of Pck1 occurs in the triglyceride/fatty acid cycle, which regulates lipid synthesis and transport between adipose tissue and the liver. We examined this by analyzing mice with a deletion of the PPARgamma binding site in the promoter of Pck1 (PPARE(-/-)). This mutation reduced the fasting Pck1 mRNA expression in WAT in brown adipose tissue (BAT). To analyze insulin resistance, we performed hyperinsulinemic-euglycemic glucose clamp analyses. PPARE(-/-) mice were profoundly insulin resistant and had more FFA and glycerol released during the hyperinsulinemic-euglycemic clamp compared with wild-type mice (WT). Finally, we analyzed insulin secretion in isolated islets. We found a 2-fold increase in insulin secretion in the PPARE(-/-) mice at 16.7 mM glucose. Thus, the PPARE site in the Pck1 promoter is essential for maintenance of lipid metabolism and glucose homeostasis and disease prevention.

On the role of glucose-dependent insulintropic polypeptide in postprandial metabolism in humans

We investigated the role of glucose-dependent insulintropic polypeptide (GIP) in the regulation of gastric emptying (GE), appetite, energy intake (EI), energy expenditure (EE), plasma levels of triglycerides (TAG), and free fatty acids (FFA) in humans. First, 20 healthy males received intravenous infusion of GIP (0.8 pmol.kg(-1).min(-1)) or saline for 300 min during and after a fixed meal (protocol 1). GE was measured using paracetamol, appetite sensations using visual analog scales, EE using indirect calorimetry, and EI during a subsequent ad libitum meal (at 300 min). Next, 10 healthy males received intravenous infusions of Intralipid, glucose, or Intralipid plus glucose, with and without GIP (1.5 pmol.kg(-1).min(-1)) for 300 min (protocol 2). In protocol 1, GIP did not have any effect on GE, EI, EE, removal of TAG, or FFA and did not influence the subjective feeling of hunger, satiety, fullness or prospective food consumption compared with saline. In protocol 2, no difference was seen in the plasma TAG on Intralipid + GIP/saline and Intralipid + glucose + GIP/saline days. FFA concentrations were lower on Intralipid + glucose + GIP/saline days (P < 0.05) compared with Intralipid + GIP/saline days and on Intralipid + GIP day (P < 0.004) compared with Intralipid + saline day. Insulin increased on all GIP days compared with saline days (P < 0.05). In conclusion, while confirming its insulinotropic effects, these data suggest that GIP does not affect GE, appetite, energy intake, EE, or the clearance rate of the applied TAG formulation in humans. However, both insulin and GIP lower post-Intralipid FFA concentration, GIP probably via stimulation of insulin secretion, increasing FFA reesterification.

Upregulation of cellular triacylglycerol – free fatty acid cycling by oleate is associated with long-term serum-free survival of human breast cancer cells

We previously showed that exogenous oleate protects human breast cancer cells against palmitate-induced apoptosis in part by increasing esterification of this free fatty acid (FFA) into triacylglycerol (TG). Here, we studied the mechanism whereby oleate protects these cells against apoptosis induced by serum withdrawal. The metabolism of FFA, TG, and glucose, in parallel with long-term cell survival in the absence of serum, was investigated in a panel of human breast cancer cell lines and in nontransformed MCF-10A cells after treatment with exogenous oleate. Short-term (3-24 h) exposure of MDA-MB-231 human breast cancer cells to exogenous oleate resulted in a dose-dependent long-term (10 day) serum-free survival that correlated with the accumulation of TG in lipid droplets and with upregulation of lipolysis. Both effects persisted for several days after oleate removal. Rapid TG lipolysis and FFA re-esterification, supported by high rates of glycolysis that provide the glycerol backbone for TG synthesis, are consistent with the presence of very active TG-FFA cycling in human breast cancer cells. Only the cancer cell lines capable of accumulating TG showed long-term serum-free survival after oleate treatment. The results suggest that upregulation of TG-FFA cycling induced by oleate may be involved in maintenance of human breast cancer cell survival.

Optimized conditions for measuring lipolysis in murine primary adipocytes

The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000-15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells.

Glucose‐Dependent Insulinotropic Polypeptide Modulates Adipocyte Lipolysis and Reesterification

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin released from intestinal K-cells during the postprandial period. Previous studies have suggested that GIP may play an etiologic role in obesity; thus, the GIP receptor may represent a target for anti-obesity drugs. The present studies were conducted to elucidate mechanisms by which GIP might promote obesity by examining the effect of GIP on both glycerol release (indicative of lipolysis) and free fatty acid (FFA) release (indicative of both lipolysis and reesterification), as well as the ability of a GIP-specific receptor antagonist (ANTGIP) to attenuate these effects. Isolated rat adipocytes were perifused on a column with 10 nM GIP alone or in combination with 10 microU/mL insulin, 1 microM isoproterenol, or 1 microM ANTGIP. Samples were collected every minute and assayed for FFA, glycerol, and lactate. GIP significantly increased FFA reesterification (decreased FFA release by 25%), stimulated lipolysis (increased glycerol release by 22%), and attenuated the lipolytic response to isoproterenol by 43%. These properties were similar to those of insulin in vitro, suggesting that GIP possesses insulin-like lipogenic effects on adipocytes. Finally, ANTGIP reversed the effects of GIP on both basal and stimulated adipocyte metabolism. These studies provide further evidence for an important physiological role for GIP in lipid homeostasis and possibly in the pathogenesis of obesity. They also suggest that the GIP receptor may represent an excellent target for the prevention and treatment of obesity and obesity-related type 2 diabetes.

Adipose Overexpression of Phosphoenolpyruvate Carboxykinase Leads to High Susceptibility to Diet-Induced Insulin Resistance and Obesity

Obesity and insulin resistance are associated with increased serum free fatty acids (FFAs). Thus, a reduction in circulating FFAs may increase insulin sensitivity. This could be achieved by increasing FFA reesterification in adipose tissue. Transgenic mice with increased adipose tissue glyceroneogenesis, caused by overexpression of phosphoenolpyruvate carboxykinase (PEPCK), show increased FFA reesterification and develop obesity but are insulin sensitive. Here, we examined whether these transgenic mice were protected from diet-induced insulin resistance. Surprisingly, when fed a high-fat diet for a short period (6 weeks), transgenic mice developed severe obesity and were more hyperinsulinemic, glucose intolerant, and insulin resistant than controls. The high triglyceride accumulation prevented white adipose tissue from buffering the flux of lipids in circulation and led to increased serum triglyceride levels and fat deposition in liver. Furthermore, circulating leptin and FFA concentrations increased to similar levels in transgenic and control mice, while adiponectin levels decreased in transgenic mice compared with controls. In addition, transgenic mice showed fat accumulation in brown adipose tissue, which decreased uncoupling protein-1 expression, suggesting that these mice had impaired diet-induced thermogenesis. These results indicate that increased PEPCK expression in the presence of high-fat feeding may have deleterious effects and lead to severe insulin resistance and type 2 diabetes.

Basal Lipolysis in Epididymal Fat Cells from Streptozotocin-Induced Diabetic Rats

The level of free fatty acid (FFA) in plasma is increased by diabetes. The increase in plasma FFA levels accompanied the stimulation of basal lipolysis (i.e. lipolysis in the absence of lipolytic agents) in fat cells. Injection of streptozotocin with rats resulted in a significant increase in basal FFA production (5.5 fold) in fat cells. However, basal glycerol production in fat cells was increased only 1.5 fold by streptozotocin-induced diabetes, implying that FFA re-esterification in fat cells was decreased by streptozotocin-induced diabetes. The FFA re-esterification in fat cells was also decreased by 1 d of fasting. Although basal lipolysis was increased by streptozotocin-induced diabetes or 1-d fasting, neutral triacylglycerol lipase activity and the immunoreactive HSL protein content in fat cells from streptozotocin-induced diabetic rats or 1-d fasting rats were not significantly changed. Although beta-blockers inhibited lipolysis induced by norepinephrine at a concentration of 10(-4) M, it failed to inhibit the basal lipolysis and FFA re-esterification in fat cells from streptozotocin-induced diabetic rats. Nor did insulin or H-89, another antilipolytic agent, affect basal lipolysis or FFA re-esterification in fat cells from streptozotocin-induced diabetic rats. These results indicate that basal FFA production may be induced by a decrease of re-esterification of FFA in diabetic rats and is not affected by antilipolytic agents such as insulin, beta-blockers or H-89.

Glucose‐dependent Insulin Modulation of Oscillatory Lipolysis in Perifused Rat Adipocytes

We showed glucose-dependent lipolytic oscillations in adipocytes that are modulated by free fatty acids (FFAs). We hypothesized that the oscillations are driven by oscillatory glucose metabolism that leads to oscillatory formation of alpha-glycerophosphate (alpha-GP), oscillatory removal of long-chain coenzyme A (LC-CoA) by alpha-GP to form triglycerides, and oscillatory relief of LC-CoA inhibition of triglyceride lipases. This study examined the effect of insulin on this hypothesis. Samples were collected every minute from perifused rat adipocytes during the basal state followed by insulin (+/-glucose) or isoproterenol (+/-insulin; n = 4 each). Insulin caused a significant increase in glycerol release (18%), with a concomitant significant decrease in FFA release (38%). Without glucose, insulin had no effect on glycerol release while still decreasing FFA release (35%). Insulin (5 microU/mL) attenuated the response of lipolysis to isoproterenol (approximately 3-fold increase with isoproterenol vs. 2-fold increase with insulin + isoproterenol). However, 1 mU/mL insulin amplified the lipolytic response ( approximately 5-fold increase in glycerol release with insulin + isoproterenol), with a concomitant increase in FFA reesterification (no increase in FFA release compared with isoproterenol alone). We interpret these results to be due to insulin's ability to increase glucose uptake and conversion to alpha-GP, thus removing LC-CoA inhibition of triglyceride lipases. While the physiological importance of lipolytic oscillations remains to be determined, we hypothesize that such an oscillation may play an important role in the delivery of FFAs to the liver, beta cells, and other tissues.

Reducing insulin resistance with metformin: the evidence today

Insulin resistance, defined as the inability of insulin to exert a normal biological action at the level of its target tissues, is one of the principal pathogenetic defects of type 2 diabetes. Metformin, the most widely-prescribed insulin-sensitizing agent in current clinical use, improves blood glucose control mainly by improving insulin-mediated suppression of hepatic glucose production, and by enhancing insulin-stimulated glucose disposal in skeletal muscle. Experimental studies show that metformin-mediated improvements in insulin sensitivity may be associated with several mechanisms, including increased insulin receptor tyrosine kinase activity, enhanced glycogen synthesis, and an increase in the recruitment and activity of GLUT4 glucose transporters. In adipose tissue, metformin promotes the re-esterification of free fatty acids and inhibits lipolysis, which may indirectly improve insulin sensitivity through reduced lipotoxicity. The improved glycaemia with metformin is not associated with increased circulating levels of insulin, and the risk of hypoglycaemia with metformin is minimal. The therapeutic profile of metformin supports its use for the control of blood glucose, in diabetic patients and for the prevention of diabetes in subjects with impaired glucose tolerance. Moreover, the improvement by metformin of cardiovascular risk factors associated with the dysmetabolic syndrome may account for the significant improvements in macrovascular outcomes observed in the UK Prospective Diabetes Study.

Effect of thiazolidinediones on glucose and fatty acid metabolism in patients with type 2 diabetes

The current study aimed to compare the effects of treatment (2 months) with thiazolidinediones (TZDs) and placebo on glucose and fat metabolism in patients with type 2 diabetes (T2DM) in a crossover design. Eight patients received placebo (2 months) followed by TZD (2 months). Two-stage (1.5 and 6.0 pmol/kg min) hyperinsulinemic-euglycemic clamps were performed in all 8 patients pre- and post-placebo and post-TZD (post-placebo = pre-TZD). We determined rates of glucose disappearance (GRd), glycolysis (GLS), glycogen synthesis (GS) (all with 3-3H glucose), carbohydrate (CHO) oxidation (indirect calorimetry), endogenous glucose production (EGP), free fatty acid (FFA) release (2H5 glycerol), and oxidation (indirect calorimetry) and re-esterification, as well as plasma adiponectin and leptin concentrations, and fat cell size and number (determined in upper thigh biopsy samples). Placebo treatment had no effects on any of the measured parameters. TZD treatment improved insulin-stimulated glucose uptake (ISGU) from 17.1 to 26.4 μmol/kg min (P < .01) and insulin-stimulated GS from 4.8 to 13.4 μmol/kg min (P < 0.03), potentiated insulin-induced suppression of lipolysis from 4.3 to 2.3 μmol/kg min (P < .03) and FFA re-esterification from 1.9 to 1.0 μmol/kg min (P < .02), increased plasma adiponectin levels from 2.7 to 7.2 μg/mL (P < .05), and decreased plasma leptin levels from 30.8 to 23.4 ng/mL (P < .02). In addition, TZD tended to increase the number of small adipocytes (<50 μm) in subcutaneous adipose tissue. We conclude that TZDs have multiple actions and that many, but perhaps not all, can be accounted for by their action on adipose tissue.

Evidence of insulin resistant lipid metabolism in adipose tissue in familial combined hyperlipidemia, but not type 2 diabetes mellitus

In patients with familial combined hyperlipidemia (FCHL) and type 2 diabetes (DM2) organ-specific differences in insulin resistance may exist. In FCHL and DM2 in vivo insulin mediated muscle glucose uptake and inhibition of lipolysis were studied by euglycemic hyperinsulinemic clamp. Insulin mediated glucose uptake was impaired to the same extent in both FCHL and DM2. Only FCHL subjects showed no reduction in plasma glycerol concentrations during insulin infusion and incomplete suppression of plasma free fatty acid (FFA) concentrations combined. This finding indicated that insulin-induced suppression of lipolysis, or glycerol/FFA utilization, or both, were impaired in FCHL, in contrast to DM2 or control subjects. To analyze these possibilities in more detail, control, FCHL, and DM2 adipocytes were studied in vitro. In contrast to adipocytes from DM2 or control subjects, no reduction in medium FFA concentration was detected with FCHL adipocytes after incubation with insulin. This finding indicated impaired intracellular FFA utilization, most likely impaired FFA re-esterification. Genetic linkage analysis in 18 Dutch families with FCHL revealed no evidence for involvement of LIPE, the hormone sensitive lipase gene, indicating that genetic variation in adipocyte lipolysis by LIPE is not the key defect in FCHL. In conclusion, FCHL as well as DM2 subjects exhibited in vivo insulin resistance to glucose disposal, which occurs mainly in muscle. FCHL subjects showed insulin resistant adipose tissue lipid metabolism, in contrast to DM2 and controls. The different pattern of organ-specific insulin resistance in FCHL versus DM2 advances our understanding of differences and similarities in phenotypes between these disorders.

Increased fatty acid re-esterification by PEPCK overexpression in adipose tissue leads to obesity without insulin resistance.

Adipose tissue glyceroneogenesis generates glycerol 3-phosphate, which could be used for fatty acid esterification during starvation. To determine whether increased glyceroneogenesis leads to increased fat mass and to explore the role of obesity in the development of insulin resistance, we overexpressed PEPCK, a regulatory enzyme of glyceroneogenesis in adipose tissue. Transgenic mice showed a chronic increase in PEPCK activity, which led to increased glyceroneogenesis, re-esterification of free fatty acids (FFAs), increased adipocyte size and fat mass, and higher body weight. In spite of increased fat mass, transgenic mice showed decreased circulating FFAs and normal leptin levels. Moreover, glucose tolerance and whole-body insulin sensitivity were preserved. Skeletal muscle basal and insulin-stimulated glucose uptake and glycogen content were not affected, suggesting that skeletal muscle insulin sensitivity is normal in transgenic obese mice. Our results indicate the key role of PEPCK in the control of FFA re-esterification in adipose tissue and, thus, the contribution of glyceroneogenesis to fat accumulation. Moreover, they suggest that higher fat mass without increased circulating FFAs does not lead to insulin resistance or type 2 diabetes in these mice.