Jang H. Youn

Anti-obesity effects of α-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase

AMP-activated protein kinase (AMPK) functions as a fuel sensor in the cell and is activated when cellular energy is depleted. Here we report that α-lipoic acid (α-LA), a cofactor of mitochondrial enzymes, decreases hypothalamic AMPK activity and causes profound weight loss in rodents by reducing food intake and enhancing energy expenditure. Activation of hypothalamic AMPK reverses the effects of α-LA on food intake and energy expenditure. Intracerebroventricular (i.c.v.) administration of glucose decreases hypothalamic AMPK activity, whereas inhibition of intracellular glucose utilization through the administration of 2-deoxyglucose increases hypothalamic AMPK activity and food intake. The 2-deoxyglucose-induced hyperphagia is reversed by inhibiting hypothalamic AMPK. Our findings indicate that hypothalamic AMPK is important in the central regulation of food intake and energy expenditure and that α-LA exerts anti-obesity effects by suppressing hypothalamic AMPK activity.NOTE: In the version of this article initially published online, the name of an author was misspelled. The name of the twelfth author should be “Benoit Viollet”. This error has been corrected for the HTML and print versions of the article

Essential Role of Mitochondrial Function in Adiponectin Synthesis in Adipocytes

OBJECTIVE—Adiponectin is an important adipocytokine that improves insulin action and reduces atherosclerotic processes. The plasma adiponectin level is paradoxically reduced in obese individuals, but the underlying mechanism is unknown. This study was undertaken to test the hypothesis that mitochondrial function is linked to adiponectin synthesis in adipocytes. RESEARCH DESIGN AND METHODS—We examined the effects of rosiglitazone and the measures that increase or decrease mitochondrial function on adiponectin synthesis. We also examined the molecular mechanism by which changes in mitochondrial function affect adiponectin synthesis. RESULTS—Adiponectin expression and mitochondrial content in adipose tissue were reduced in obese db/db mice, and these changes were reversed by the administration of rosiglitazone. In cultured adipocytes, induction of increased mitochondrial biogenesis (via adenoviral overexpression of nuclear respiratory factor-1) increased adiponectin synthesis, whereas impairment in mitochondrial function decreased it. Impaired mitochondrial function increased endoplasmic reticulum (ER) stress, and agents causing mitochondrial or ER stress reduced adiponectin transcription via activation of c-Jun NH2-terminal kinase (JNK) and consequent induction of activating transcription factor (ATF)3. Increased mitochondrial biogenesis reversed all of these changes. CONCLUSIONS—Mitochondrial function is linked to adiponectin synthesis in adipocytes, and mitochondrial dysfunction in adipose tissue may explain decreased plasma adiponectin levels in obesity. Impaired mitochondrial function activates a series of mechanisms involving ER stress, JNK, and ATF3 to decrease adiponectin synthesis.

α-Lipoic Acid Prevents Endothelial Dysfunction in Obese Rats via Activation of AMP-Activated Protein Kinase

Objective—Lipid accumulation in vascular endothelial cells may play an important role in the pathogenesis of atherosclerosis in obese subjects. We showed previously that α-lipoic acid (ALA) activates AMP-activated protein kinase (AMPK) and reduces lipid accumulation in skeletal muscle of obese rats. Here, we investigated whether ALA improves endothelial dysfunction in obese rats by activating AMPK in endothelial cells. Methods and Results—Endothelium-dependent vascular relaxation was impaired, and the number of apoptotic endothelial cells was higher in the aorta of obese rats compared with control rats. In addition, triglyceride and lipid peroxide levels were higher, and NO synthesis was lower. Administration of ALA improved all of these abnormalities. AMPK activity was lower in aortic endothelium of obese rats, and ALA normalized it. Incubation of human aortic endothelial cells with ALA activated AMPK and protected cells from linoleic acid–induced apoptosis. Dominant-negative AMPK inhibited the antiapoptotic effects of ALA. Conclusions—Reduced AMPK activation may play an important role in the genesis of endothelial dysfunction in obese rats. ALA improves vascular dysfunction by normalizing lipid metabolism and activating AMPK in endothelial cells.

Plasma Free Fatty Acids Decrease Insulin-Stimulated Skeletal Muscle Glucose Uptake by Suppressing Glycolysis in Conscious Rats

The effects of elevated plasma free fatty acid (FFA) levels on insulin-stimulated whole-body and skeletal muscle glucose transport, glucose uptake, glycolysis, and glycogen synthesis were studied in conscious rats during hyperinsulinemic-euglycemic clamps with (n = 26) or without (n = 23) Intralipid and heparin infusion. Whole-body and skeletal muscle glucose uptake, glycolysis, and glycogen synthesis were estimated using d-[3-3H]glucose and 2-[14C]deoxyglucose (study 1), and glucose transport activity was assessed by analyzing plasma kinetics of l-[14C]glucose and 3-O-[3H]-methylglucose (study 2). Plasma FFA levels decreased during the clamps without intralipid but increased above basal during the clamps with Intralipid infusion (P < 0.01 for both). Elevated plasma FFA levels decreased insulin-stimulated whole-body glucose uptake by ∼ 15% and ∼ 20% during physiological and maximal insulin clamps, respectively (P < 0.01). Similarly, insulin-stimulated glucose uptake was also decreased in individual skeletal muscles with Intralipid infusion (P < 0.05). The most profound effect of elevated plasma FFA levels was a 30–50% suppression of insulin-stimulated glycolysis in whole body and individual skeletal muscles in both clamps. In contrast, physiological insulin-stimulated glycogen synthesis was increased with elevated plasma FFA levels in whole body and individual skeletal muscles (P < 0.05). Glucose-6-phosphate (G-6-P) levels were increased in soleus and extensor digitorum longus (EDL) muscles with Intralipid infusion in both clamps (P < 0.05). Intralipid infusion did not alter the time profiles of plasma l-glucose and 3-O-methylglucose after an intravenous injection during maximal insulin clamps, and compartmental analysis indicated no significant effect of elevated FFA levels on glucose transport activity in insulin-sensitive tissues (P > 0.05). Thus, elevated plasma FFA decreased insulin-stimulated glucose uptake in skeletal muscle by suppressing glycolysis and increasing G-6-P levels. These findings suggest that the classic glucose-fatty acid cycle was the predominant mechanism underlying the inhibitory effect of FFA on skeletal muscle glucose uptake.

Insulin Regulation of Skeletal Muscle PDK4 mRNA Expression Is Impaired in Acute Insulin-Resistant States

We previously showed that insulin has a profound effect to suppress pyruvate dehydrogenase kinase (PDK) 4 expression in rat skeletal muscle. In the present study, we examined whether insulin’s effect on PDK4 expression is impaired in acute insulin-resistant states and, if so, whether this change is accompanied by decreased insulin’s effects to stimulate Akt and forkhead box class O (FOXO) 1 phosphorylation. To induce insulin resistance, conscious overnight-fasted rats received a constant infusion of Intralipid or lactate for 5 h, while a control group received saline infusion. Following the initial infusions, each group received saline or insulin infusion (n = 6 or 7 each) for an additional 5 h, while saline, Intralipid, or lactate infusion was continued. Plasma glucose was clamped at basal levels during the insulin infusion. Compared with the control group, Intralipid and lactate infusions decreased glucose infusion rates required to clamp plasma glucose by ∼60% (P < 0.01), confirming the induction of insulin resistance. Insulin’s ability to suppress PDK4 mRNA level was impaired in skeletal muscle with Intralipid and lactate infusions, resulting in two- to threefold higher PDK4 mRNA levels with insulin (P < 0.05). Insulin stimulation of Akt and FOXO1 phosphorylation was also significantly decreased with Intralipid and lactate infusions. These data suggest that insulin’s effect to suppress PDK4 gene expression in skeletal muscle is impaired in insulin-resistant states, and this may be due to impaired insulin signaling for stimulation of Akt and FOXO1 phosphorylation. Impaired insulin’s effect to suppress PDK4 expression may explain the association between PDK4 overexpression and insulin resistance in skeletal muscle.

Recent Advances in Understanding Integrative Control of Potassium Homeostasis

The potassium homeostatic system is very tightly regulated. Recent studies have shed light on the sensing and molecular mechanisms responsible for this tight control. In addition to classic feedback regulation mediated by a rise in extracellular fluid (ECF) [K(+)], there is evidence for a feedforward mechanism: Dietary K(+) intake is sensed in the gut, and an unidentified gut factor is activated to stimulate renal K(+) excretion. This pathway may explain renal and extrarenal responses to altered K(+) intake that occur independently of changes in ECF [K(+)]. Mechanisms for conserving ECF K(+) during fasting or K(+) deprivation have been described: Kidney NADPH oxidase activation initiates a cascade that provokes the retraction of K(+) channels from the cell membrane, and muscle becomes resistant to insulin stimulation of cellular K(+) uptake. How these mechanisms are triggered by K(+) deprivation remains unclear. Cellular AMP kinase-dependent protein kinase activity provokes the acute transfer of K(+) from the ECF to the ICF, which may be important in exercise or ischemia. These recent advances may shed light on the beneficial effects of a high-K(+) diet for the cardiovascular system.

Metabolic Impairment Precedes Insulin Resistance in Skeletal Muscle During High-Fat Feeding in Rats

To examine whether impairment of intracellular glucose metabolism precedes insulin resistance, we determined the time courses of changes in insulin-stimulated glucose uptake, glycolysis, and glycogen synthesis during high-fat feeding in rats. Animals were fed with a high-fat (66.5%) diet ad libitum for 0, 2, 4, 7, or 14 days (n = 10–11 in each group) after 5 days of a low-fat (12.5%) diet. Submaximal and maximal insulin-stimulated glucose fluxes were estimated in whole body and individual skeletal muscles using the glucose clamp technique combined with D-[3-3H]glucose infusion and 2-[1-14C]deoxyglucose injection. Both submaximal and maximal insulin-stimulated glucose uptake in whole body decreased gradually with high-fat feeding. However, the decreases were minimal and not statistically significant during the initial few days (i.e., 2 and 4 days) of high-fat feeding (P > 0.05). In contrast, insulin-stimulated whole-body glycolysis (both maximal and submaximal) significantly decreased by ∼30% with 2 days of high-fat feeding and remained suppressed thereafter (P < 0.05). Similar patterns of changes in insulin-stimulated glucose uptake and glycolysis were also observed in skeletal muscle. Insulin-stimulated glycogen synthesis and glucose-6-phosphate (G-6-P) concentrations in skeletal muscle increased significantly during the initial few days of high-fat feeding and gradually returned to control levels by day 14, suggesting that increased G-6-P concentrations were responsible for increased glycogen synthesis. Thus, suppression of insulin-stimulated glycolysis and a compensatory increase in glycogen synthesis (presumably arising from the glucose-fatty acid cycle) preceded decreases in insulin-stimulated glucose uptake in skeletal muscle during high-fat feeding. These findings suggest that the insulin resistance may develop as a secondary response to impaired intracellular glucose metabolism.

Fasting does not impair insulin-stimulated glucose uptake but alters intracellular glucose metabolism in conscious rats.

Effects of 24-h and 48-h fasting on maximal insulin-stimulated whole-body and muscle glucose uptake, glycogen synthesis, and glycolysis were studied in conscious rats by combining the glucose clamp technique with tracer methods. Fasting decreased body weight and basal plasma glucose, plasma insulin, hepatic glucose output, and glucose clearance (P < 0.05 for all). However, maximal insulin-stimulated whole-body glucose uptake, normalized to body weight, was almost identical in fed, 24-h fasted, and 48-h fasted rats (191 ± 8, 185 ± 14, and 182 ± 5 μmol · kg−1 · min−1, respectively; P > 0.7). Similarly, rates of insulin-stimulated glucose uptake by four different skeletal muscles, estimated by the 2-deoxyglucose injection technique, were not different among the three groups. In contrast to glucose uptake, insulin-stimulated whole-body glycolysis was decreased significantly after fasting (36% after 48 h fasting; P < 0.05), whereas insulin-stimulated whole-body glycogen synthesis was increased (44% after 48 h fasting; P < 0.05). In fed rats, glycolysis was the major pathway for glucose metabolism during hyperinsulinemia, accounting for 60 ± 5% of glucose uptake. This fraction was decreased significantly by fasting (P < 0.01), so that after a 48-h fast, glycolysis accounted for only 40 ± 3% of insulin-stimulated glucose uptake and glycogen synthesis became predominant pathway, accounting for 60 ± 3% of whole-body glucose utilization. Whole-body patterns of glucose metabolism during hyperinsulinemia were paralleled by glucose metabolism in individual muscles. These data indicate that fasting for up to 48 h in rats had no effect on insulin-stimulated glucose disposal (whole body or muscle) but resulted in a change of the primary metabolic path for insulin-stimulated glucose utilization from glycolysis to glycogen synthesis.

Narrative Review: Evolving Concepts in Potassium Homeostasis and Hypokalemia

Hypokalemia is a common disorder that can result from potassium redistribution between plasma and intracellular fluid (ICF), inadequate potassium intake, or excessive potassium excretion (1). When hypokalemia reflects true potassium depletion, the body activates several mechanisms, especially in the kidney, to conserve potassium (Figure 1). Although short periods of mild potassium depletion are typically well tolerated in healthy individuals, severe potassium depletion can result in glucose intolerance (2) and serious cardiac (3), renal (4), and neurologic (5) dysfunction, including death. Prolonged potassium depletion of even modest proportion can provoke or exacerbate kidney injury or hypertension (6, 7). Indeed, reduced potassium intake correlates directly with higher blood pressure in both normotensive and hypertensive individuals (8). Figure 1. Integrated model of the regulation of body potassium balance. CNS = central nervous system. Left. Classic mechanisms. Right. Additional putative mechanisms. Normal Potassium Balance and Renal Potassium Excretion To set the context, we first summarize key concepts of steady-state potassium handling. Total body potassium is roughly 55 mmol/kg of body weight, with 98% distributed to the ICF (primarily in muscle, the liver, and erythrocytes) and 2% in the extracellular fluid (1). Na,K-ATPase isoforms (see Glossary) actively pump potassium into the cell and maintain and restore the electrochemical gradient between the normal extracellular potassium concentration of 3.5 to 5.0 mmol/L and the intracellular potassium concentration of approximately 150 mmol/L, which is particularly important for normal functioning of excitable cells. -Catecholamines, aldosterone, insulin, pH, and osmolality influence the transcellular potassium distribution and thus how well cells buffer changes in plasma potassium concentration (9). Physicians exploit this fact in the emergency management of severe hyperkalemia when they use insulin or -catecholamines to drive plasma potassium into cells. Normal persons who consume a typical Western diet ingest approximately 70 mmol of potassium per day (10). The intestine absorbs virtually all of the ingested potassium and delivers it to the liver for processing by means of the hepatoportal circulation. Minimal amounts of potassium are excreted in the feces. Renal potassium excretion, the principal defense against chronic potassium imbalances, depends on free filtration at the glomerulus, extensive proximal tubule reabsorption, and a highly regulated secretory process of the distal convoluted tubule and segments of the collecting duct in the cortex and outer medulla (the cortical collecting duct and the outer medullary collecting duct, respectively) (Figure 2). The cortical collecting duct and outer medullary collecting duct consist of at least 2 very different cell types, termed principal cells and intercalated cells (Figure 2). Principal cells, which comprise approximately 70% to 75% of collecting duct cells, mediate sodium reabsorption and potassium secretion and are targets for angiotensin II (11, 12), aldosterone, aldosterone receptor antagonists, and potassium-sparing diuretics (Figure 2). Principal cells exploit the electrochemical gradient established by sodium entry into the cell through a sodium channel at the luminal membrane (the molecular target of amiloride) and the basolateral membrane Na,K-ATPase to drive potassium secretion through 2 classes of luminal membrane potassium channels (13). One of these, the renal outer medullary potassium (known among renal physiologists as ROMK) channels, secrete potassium under normal tubular fluid flow conditions and are inserted into or internalized from the luminal membrane, depending on the demand for potassium secretion. The other class of potassium channels are the big conductance channels (known as BK channels), which are relatively inactive under normal conditions but exhibit increased activity during high tubular flow or high-potassium conditions (13). Factors that regulate principal cell potassium secretion include previous potassium intake; intracellular potassium level; sodium delivery to the cells; urine flow rate; and hormones, such as aldosterone and -catecholamines (14). The other collecting duct cell type, intercalated cells, mediate acidbase transport but upregulate expression of luminal H,K-ATPases (see Glossary) during potassium depletion to enhance potassium reabsorption (1) (Figure 2). Figure 2. Segmental handling of potassium excretion along the nephron and collecting duct cell types under normal conditions or conditions of potassium excess or deficiency. We present a simplified model of potassium handling by collecting duct cell types. Principal cells in the collecting duct are responsible for secretion of excess potassium in the circulation into the tubule lumen and thus into the urine. This secretion is accomplished by luminal membrane potassium channels responding primarily to the electrochemical gradient for potassium generated by the combined actions of the basolateral membrane Na,K-ATPase and a luminal membrane sodium channel (the target of the potassium-sparing diuretic amiloride). In states of potassium depletion, potassium secretion by the principal cells is inhibited and the luminal membrane H,K-ATPase is activated in the intercalated cells to reclaim the potassium that remains in the tubular fluid, thereby limiting urinary potassium wasting. ADP = adenosine diphosphate; ATP = adenosine triphosphate; CCD = cortical collecting duct; DT = distal tubule; Glom = glomerulus; IMCD = inner medullary collecting duct; MTAL = medullary thick ascending limb of Henle loop; OMCD = outer medullary collecting duct; Pi = inorganic phosphate. To summarize, mammalian cells require a steep concentration gradient of potassium between ICF and extracellular fluid to function properly, which requires primary active transport by Na,K-ATPase. The kidney excretes sufficient amounts of potassium to maintain total body homeostasis. Although the proximal nephron reabsorbs the bulk of the potassium filtered at the glomerulus, the collecting duct fine-tunes potassium excretion and is subject to several regulatory influences. Feedback Control of Potassium Balance The use of thermostats to adjust heating or cooling is a common example of feedback control, a control mechanism in a homeostatic system that uses the consequences or outputs of a process to feed back and regulate the process itself. The thermostat detects the error (for example, the room is too hot) and signals for the air conditioner to provide cool air. Once the room reaches the temperature set at the thermostat (the room becomes cool enough), the air conditioner turns off. This example of feedback control also applies to potassium homeostasis. Although feedback control of potassium balance has been recognized for decades, only recently have some of its secrets been discovered. In response to a high-potassium meal that includes glucose, pancreatic insulin secretion activates skeletal muscle and liver Na,K-ATPase, which pumps potassium from the plasma to the ICF of these cells. This mechanism minimizes the postprandial increase in plasma potassium concentration (15). With muscle activity, potassium is released into the plasma and filtered at the glomerulus. To maintain balance, the amount of potassium consumed in the meal (minus the small amount lost in the feces) is secreted into the urine. When potassium consumption increases plasma potassium concentration enough, it triggers aldosterone synthesis and release from the adrenals, which stimulates the activity and synthesis of Na,K-ATPase and luminal potassium channels in collecting duct principal cells to secrete the excess potassium (16) (Figures 1 and 2). Aldosterone also enhances potassium secretion in the distal colon (17), which can be especially important when renal function is compromised. Conversely, if potassium intake is very low or its output is very high, plasma potassium concentration decreases and feedback regulation redistributes potassium from ICF to plasma and minimizes renal potassium excretion. Skeletal muscle becomes insulin-resistant to potassium (but not glucose) uptake even before plasma potassium concentration decreases, which blunts the shift of potassium from plasma into the cell (18). After hypokalemia ensues, the expression of skeletal muscle Na,K-ATPase 2 isoform decreases, which allows a net potassium leak from ICF to the plasma (19). The low plasma potassium concentration suppresses adrenal aldosterone release so that the kidney can reclaim all but about 1% of the filtered potassium (Figure 2). This renal potassium conservation involves downregulation of potassium secretion by means of the ROMK channels in cortical collecting duct principal cells. Chronic potassium depletion activates a renal NADPH oxidase (a relative of the enzyme that produces the respiratory burst in neutrophils) that produces reactive oxygen species to signal for the ROMK channels to be internalized and thus incompetent to conduct membrane transport of potassium (20, 21). In addition, an H,K-ATPase (a relative of the proton pump active in the gastric mucosa) is activated in outer medullary collecting duct intercalated cells to reclaim any remaining filtered potassium into the plasma. Figure 2 summarizes the responses of the different nephron segments to accommodate changes in potassium intake. Feedforward Control of Potassium Balance Feedforward control refers to a pathway in a homeostatic system that responds to a signal in the environment in a predetermined manner, without responding to how the system subsequently reacts (that is, without responding to feedback). The most famous example of feedforward control is the conditioned salivation of Pavlovs dogs in anticipation of food (22). Pavlov implanted small stomach pouches in dogs to measure salivation. He and his assistants would ring bell

Increasing plasma [K+] by intravenous potassium infusion reduces NCC phosphorylation and drives kaliuresis and natriuresis

Dietary potassium loading results in rapid kaliuresis, natriuresis, and diuresis associated with reduced phosphorylation (p) of the distal tubule Na(+)-Cl(-) cotransporter (NCC). Decreased NCC-p inhibits NCC-mediated Na(+) reabsorption and shifts Na(+) downstream for reabsorption by epithelial Na(+) channels (ENaC), which can drive K(+) secretion. Whether the signal is initiated by ingesting potassium or a rise in plasma K(+) concentration ([K(+)]) is not understood. We tested the hypothesis, in male rats, that an increase in plasma [K(+)] is sufficient to reduce NCC-p and drive kaliuresis. After an overnight fast, a single 3-h 2% potassium (2%K) containing meal increased plasma [K(+)] from 4.0 ± 0.1 to 5.2 ± 0.2 mM; increased urinary K(+), Na(+), and volume excretion; decreased NCC-p by 60%; and marginally reduced cortical Na(+)-K(+)-2Cl(-) cotransporter (NKCC) phosphorylation 25% (P = 0.055). When plasma [K(+)] was increased by tail vein infusion of KCl to 5.5 ± 0.1 mM over 3 h, significant kaliuresis and natriuresis ensued, NCC-p decreased by 60%, and STE20/SPS1-related proline alanine-rich kinase (SPAK) phosphorylation was marginally reduced 35% (P = 0.052). The following were unchanged at 3 h by either the potassium-rich meal or KCl infusion: Na(+)/H(+) exchanger 3 (NHE3), NHE3-p, NKCC, ENaC subunits, and renal outer medullary K(+) channel. In summary, raising plasma [K(+)] by intravenous infusion to a level equivalent to that observed after a single potassium-rich meal triggers renal kaliuretic and natriuretic responses, independent of K(+) ingestion, likely driven by decreased NCC-p and activity sufficient to shift sodium reabsorption downstream to where Na(+) reabsorption and flow drive K(+) secretion.