Fredric B. Kraemer

Hormone-sensitive lipase control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis

Hormone-sensitive lipase (HSL) is an intracellular neutral lipase that is capable of hydrolyzing triacylglycerols, diacylglycerols, monoacylglycerols, and cholesteryl esters, as well as other lipid and water soluble substrates. HSL activity is regulated post-translationally by phosphorylation and also by pretranslational mechanisms. The enzyme is highly expressed in adipose tissue and steroidogenic tissues, with lower amounts expressed in cardiac and skeletal muscle, macrophages, and islets. Studies of the structure of HSL have identified several amino acids and regions of the molecule that are critical for enzymatic activity and regulation of HSL. This has led to important insights into its function, including the interaction of HSL with other intracellular proteins, such as adipocyte lipid binding protein. Accumulating evidence has defined important functions for HSL in normal physiology, affecting adipocyte lipolysis, steroidogenesis, spermatogenesis, and perhaps insulin secretion and insulin action; however, direct links between abnormal expression or genetic variations of HSL and human disorders, such as obesity, insulin resistance, type 2 diabetes, and hyperlipidemia, await further clarification. The published reports examining the regulation, and function of HSL in normal physiology and disease are reviewed in this paper.

The role of lipid droplets in metabolic disease in rodents and humans

Lipid droplets (LDs) are intracellular organelles that store neutral lipids within cells. Over the last two decades there has been a dramatic growth in our understanding of LD biology and, in parallel, our understanding of the role of LDs in health and disease. In its simplest form, the LD regulates the storage and hydrolysis of neutral lipids, including triacylglycerol and/or cholesterol esters. It is becoming increasingly evident that alterations in the regulation of LD physiology and metabolism influence the risk of developing metabolic diseases such as diabetes. In this review we provide an update on the role of LD-associated proteins and LDs in metabolic disease.

Increased plasma inactive renin in diabetes mellitus. A marker of microvascular complications.

Plasma renin exists in an active form or as an inactive zymogen that resembles a prorenin present in homogenates of human kidneys. We examined the relation of diabetes and its microvascular complications with the level of plasma inactive renin activated by dialysis to pH 3.3. Plasma inactive renin was measured in 235 diabetic patients and 90 nondiabetic controls. In the controls, the level of plasma inactive renin increased slightly with age but was never above 50 ng per milliliter per hour. In young diabetic patients studied within three years of the onset of diabetes the concentration of inactive renin was normal, and in some older diabetics without complications it remained within the age-adjusted normal range for many years. However, in patients with retinopathy or albuminuria, plasma inactive renin was above the normal range with few exceptions, reaching levels 50 to 200 per cent above the upper limits of normal in patients with nephropathy. The frequency of neuropathy was also significantly higher among patients with levels above the normal range. In 37 per cent of the diabetics followed during one to three years of conventional treatment, plasma inactive renin increased significantly, but in another group of diabetics under intensive treatment, the level rose in only 7 per cent and fell in 43 per cent. We conclude that there is a close association between a high level of plasma inactive renin and the presence of microvascular complications, and that the level of inactive renin can be modified by intensive treatment of diabetes.

Assessment of Insulin Resistance with the Insulin Suppression Test and the Euglycemic Clamp

Insulin resistance was quantified with two different methods in 30 subjects with varying degrees of glucose tolerance. One method, the insulin suppression test, is performed by continuously infusing epinephrine, propranolol, insulin, and glucose. Epinephrine and propranolol suppress endogenous insulin release, and steady-state plasma levels of exogenous insulin and glucose are reached in all individuals. Because the steady-state insulin level is the same in all subjects, the height of the steady-state plasma glucose level provides a direct estimate of insulin resistance. The other method, the euglycemic clamp technique, produces a steady-state level of exogenous hyperinsulinemia by means of a primed and continuous insulin infusion. Glucose is also infused at a rate sufficient to prevent an insulin-induced fall in glucose concentration, and the amount of glucose required to maintain the basal plasma glucose level provides the estimate of insulin resistance. The results indicated that estimates of insulin resistance generated by the two methods were highly correlated (r = 0.93). Furthermore, both methods of assessing insulin resistance indicated that the greater the degree of glucose intolerance, the more severe the insulin resistance. These results serve to further emphasize the importance of insulin resistance in the pathogenesis of hyperglycemia in type II diabetes.

Do Automated Calls with Nurse Follow-up Improve Self-Care and Glycemic Control among Vulnerable Patients with Diabetes?

PURPOSE We sought to evaluate the effect of automated telephone assessment and self-care education calls with nurse follow-up on the management of diabetes. SUBJECTS AND METHODS We enrolled 280 English- or Spanish-speaking adults with diabetes who were using hypoglycemic medications and who were treated in a county health care system. Patients were randomly assigned to usual care or to receive an intervention that consisted of usual care plus bi-weekly automated assessment and self-care education calls with telephone follow-up by a nurse educator. Outcomes measured at 12 months included survey-reported self-care, perceived glycemic control, and symptoms, as well as glycosylated hemoglobin (Hb A1c) and serum glucose levels. RESULTS We collected follow-up data for 89% of enrollees (248 patients). Compared with usual care patients, intervention patients reported more frequent glucose monitoring, foot inspection, and weight monitoring, and fewer problems with medication adherence (all P -0.03). Follow-up Hb A,, levels were 0.3% lower in the intervention group (P = 0.1), and about twice as many intervention patients had Hb A1c levels within the normal range (P = 0.04). Serum glucose levels were 41 mg/dL lower among intervention patients than usual care patients (P = 0.002). Intervention patients also reported better glycemic control (P = 0.005) and fewer diabetic symptoms (P <0.0001 ), including fewer symptoms of hyperglycemia and hypoglycemia. CONCLUSIONS Automated calls with telephone nurse follow-up may be an effective strategy for improving self-care behavior and glycemic control, and for decreasing symptoms among vulnerable patients with diabetes.

Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes

Phosphorylation of the lipid droplet-associated protein perilipin A (Peri A) mediates the actions of cyclic AMP-dependent protein kinase A (PKA) to stimulate triglyceride hydrolysis (lipolysis) in adipocytes. Studies addressing how Peri A PKA sites regulate adipocyte lipolysis have relied on non-adipocyte cell models, which express neither adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triglyceride catabolism in mice, nor the “downstream” lipase, hormone-sensitive lipase (HSL). ATGL and HSL are robustly expressed by adipocytes that we generated from murine embryonic fibroblasts of perilipin knock-out mice. Adenoviral expression of Peri A PKA site mutants in these cells reveals that mutation of serine 517 alone is sufficient to abrogate 95% of PKA (forskolin)-stimulated fatty acid (FA) and glycerol release. Moreover, a “phosphomimetic” (aspartic acid) substitution at serine 517 enhances PKA-stimulated FA release over levels obtained with wild type Peri A. Studies with ATGL-and HSL-directed small hairpin RNAs demonstrate that 1) ATGL activity is required for all PKA-stimulated FA and glycerol release in murine embryonic fibroblast adipocytes and 2) all PKA-stimulated FA release in the absence of HSL activity requires serine 517 phosphorylation. These results provide the first demonstration that Peri A regulates ATGL-dependent lipolysis and identify serine 517 as the Peri A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphorylated or phosphomimetic serine 517. Thus, serine 517 is a novel “master regulator” of PKA-stimulated adipocyte lipolysis.

Perilipin Promotes Hormone-sensitive Lipase-mediated Adipocyte Lipolysis via Phosphorylation-dependent and -independent Mechanisms

Hormone-sensitive lipase (HSL) is the predominant lipase effector of catecholamine-stimulated lipolysis in adipocytes. HSL-dependent lipolysis in response to catecholamines is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin A (Peri A), an essential lipid droplet (LD)-associated protein. It is believed that perilipin phosphorylation is essential for the translocation of HSL from the cytosol to the LD, a key event in stimulated lipolysis. Using adipocytes retrovirally engineered from murine embryonic fibroblasts of perilipin null mice (Peri–/– MEF), we demonstrate by cell fractionation and confocal microscopy that up to 50% of cellular HSL is LD-associated in the basal state and that PKA-stimulated HSL translocation is fully supported by adenoviral expression of a mutant perilipin lacking all six PKA sites (Peri AΔ1–6). PKA-stimulated HSL translocation was confirmed in differentiated brown adipocytes from perilipin null mice expressing an adipose-specific Peri AΔ1–6 transgene. Thus, PKA-induced HSL translocation was independent of perilipin phosphorylation. However, Peri AΔ1–6 failed to enhance PKA-stimulated lipolysis in either MEF adipocytes or differentiated brown adipocytes. Thus, the lipolytic action(s) of HSL at the LD surface requires PKA-dependent perilipin phosphorylation. In Peri–/– MEF adipocytes, PKA activation significantly enhanced the amount of HSL that could be cross-linked to and co-immunoprecipitated with ectopic Peri A. Notably, this enhanced cross-linking was blunted in Peri–/– MEF adipocytes expressing Peri AΔ1–6. This suggests that PKA-dependent perilipin phosphorylation facilitates (either direct or indirect) perilipin interaction with LD-associated HSL. These results redefine and expand our understanding of how perilipin regulates HSL-mediated lipolysis in adipocytes.

Relationship Between Insulin Resistance, Insulin Secretion, Very Low Density Lipoprotein Kinetics, and Plasma Triglyceride Levels in Normotriglyceridemic Man

We have previously postulated that resistance to insulin-mediated glucose uptake was the basic metabolic abnormality in patients with endogenous hypertriglyceridemia. In this situation, glucose tolerance would tend to deteriorate, and could only be maintained by the increased secretion of insulin. Although the ensuing hyperinsulinemia might prevent the development of glucose intolerance, we suggested that it would also lead to increased hepatic very low density (VLDL) triglyceride (TG) synthesis and secretion. In the current study we have quantified these four metabolic variables in 16 nonobese human subjects with plasma TG concentrations less than 175 mg/dl. The results demonstrate the following degree of correlation: insulin resistance (Formula: see text) insulin response to food (Formula: see text) VLDL-TG secretion rate (Formula: see text) plasma TG concentration. These data indicate that nonobese subjects with normal TG levels have the same relationship between degree of insulin sensitivity, insulin response to food, VLDL-TG secretion, and TG concentration previously described in patients with endogenous hypertriglyceridemia.

IL-17 Regulates Adipogenesis, Glucose Homeostasis, and Obesity

Inflammatory mediators have the potential to impact a surprising range of diseases, including obesity and its associated metabolic syndrome. In this paper, we show that the proinflammatory cytokine IL-17 inhibits adipogenesis, moderates adipose tissue (AT) accumulation, and regulates glucose metabolism in mice. IL-17 deficiency enhances diet-induced obesity in mice and accelerates AT accumulation even in mice fed a low-fat diet. In addition to potential systemic effects, IL-17 is expressed locally in AT by leukocytes, predominantly by γδ T cells. IL-17 suppresses adipocyte differentiation from mouse-derived 3T3-L1 preadipocytes in vitro, and inhibits expression of genes encoding proadipogenic transcription factors, adipokines, and molecules involved in lipid and glucose metabolism. IL-17 also acts on differentiated adipocytes, impairing glucose uptake, and young IL-17–deficient mice show enhanced glucose tolerance and insulin sensitivity. Our findings implicate IL-17 as a negative regulator of adipogenesis and glucose metabolism in mice, and show that it delays the development of obesity.

Lipase-selective functional domains of perilipin A differentially regulate constitutive and protein kinase A-stimulated lipolysis

Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMP-dependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1–300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301–517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1–300 (Ser81, Ser222, and Ser276). PKA-stimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301–517 (Ser433, Ser492, and Ser517). Peri 301–517 promoted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301–517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific “barrier” domains block basal lipolysis by HSL and non-HSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity.