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The water permeability of biological membranes has been a longstanding problem in physiology, but the proteins responsible for this remained unknown until discovery of the aquaporin 1 (AQP1) water channel protein. AQP1 is selectively permeated by water driven by osmotic gradients. The atomic structure of human AQP1 has recently been defined. Each subunit of the tetramer contains an individual aqueous pore that permits single-file passage of water molecules but interrupts the hydrogen bonding needed for passage of protons. At least 10 mammalian aquaporins have been identified, and these are selectively permeated by water (aquaporins) or water plus glycerol (aquaglyceroporins). The sites of expression coincide closely with the clinical phenotypes -ranging from congenital cataracts to nephrogenic diabetes insipidus. More than 200 members of the aquaporin family have been found in plants, microbials, invertebrates and vertebrates, and their importance to the physiology of these organisms is being uncovered.

(CHIP28), which is the major water-channel protein of erythrocytes (3), present in the renal proximal tubule and descending limb of the loop of Henle (4), and found in several other water-transporting tissues (5). Recently, the nucleotide sequence of a cDNA coding for an additional member of this family of proteins [referred to as the aquaporin family (2)] has been reported by Fushimi et al. (6). This collecting-duct water-channel protein (WCH-CD) is expressed chiefly, if not exclusively, in the renal collecting duct. On the basis of this localization, WCH-CD has been proposed to be the vasopressin-regulated water channel.Vasopressin binds to receptors in the basolateral plasma membrane and, acting through cAMP, increases the water permeability of the apical plasma membrane of principal and IMCD cells. The mechanism whereby the water permeability increases is unknown, although it presumably involves an increase in the number of or unit conductance of water channels in the apical membrane. The most widely accepted theory is the "shuttle" hypothesis (7), which proposes that a reservoir of water channels is contained in the membranes of intracellular vesicles and that vasopressin increases the apical plasma membrane water permeability by triggering exocytosis of these vesicles, delivering the vesicles with their water channels to the apical membrane. Studies using freezefracture electron microscopy in collecting ducts or vasopressin-responsive amphibian-bladder epithelia have supported this model by showing that discrete patches (termed clusters or aggregates) of intramembrane particles appear in the apical membrane with vasopressin exposure and disappear with vasopressin withdrawal (7-9). These clusters appear to be localized to clathrin-coated pits (8). The intramembrane particles have been proposed to contain the water channels. One item of evidence that is lacking, but is crucial for verification ofthe shuttle hypothesis, is direct localization of the water-channel protein at a subcellular level. In the present study, we have prepared polyclonal antibodies to the WCH-CD protein and have used these antibodies to carry out immunohisto-and immunocytochemistry to determine the cellular and subcellular localization of the WCH-CD channel.MATERIALS AND METHODS Preparation of Antibodies. Two peptides were synthesized by standard automated solid-phase techniques (10) based on the predicted amino acid sequence of WCH-CD (6). These were as follows: peptide 1 (P1), CEVRRRQSVELHSPQSL-PRGSKA (amino acids 250-271 of WCH-CD plus an N-terminal cysteine used for conjugation), and peptide 2 (P2), CELHSPQSLPRGSKA (amino acids 258-271 plus an N-terminal cysteine). These peptides were conjugated to keyhole Abbreviations: WCH-CD, collecting-duct water-channel protein;IMCD, inner medullary collecting duct.tTo whom reprint requests should be addressed at: Laboratory of

Elevated FFA concentrations have been shown to reproduce some of the metabolic abnormalities of obesity. It has been hypothesized that visceral adipose tissue lipolysis releases excess FFAs into the portal vein, exposing the liver to higher FFA concentrations. We used isotope dilution/hepatic vein catheterization techniques to examine whether intra-abdominal fat contributes a greater portion of hepatic FFA delivery in visceral obesity. Obese women (n = 24) and men (n = 20) with a range of obesity phenotypes, taken together with healthy, lean women (n = 12) and men (n = 12), were studied. Systemic, splanchnic, and leg FFA kinetics were measured. The results showed that plasma FFA concentrations were approximately 20% greater in obese men and obese women. The contribution of splanchnic lipolysis to hepatic FFA delivery ranged from less than 10% to almost 50% and increased as a function of visceral fat in women (r = 0.49, P = 0.002) and in men (r = 0.52, P = 0.002); the slope of the relationship was greater in women than in men (P < 0.05). Leg and splanchnic tissues contributed a greater portion of systemic FFA release in obese men and women than in lean men and women. We conclude that the contribution of visceral adipose tissue lipolysis to hepatic FFA delivery increases with increasing visceral fat in humans and that this effect is greater in women than in men. IntroductionA predominantly upper-body fat distribution is an important risk factor for the metabolic complications of obesity (1), especially when it is associated with increased intra-abdominal fat (2). Several metabolic abnormalities associated with upper-body obesity can be reproduced by excess FFAs, including insulin resistance with respect to muscle glucose uptake (3) and endogenous glucose production (4) and increased VLDL triglyceride production (5). Increased delivery of FFAs to the liver may be responsible for some of these abnormalities (6, 7). Visceral adipocytes are more lipolytically active than subcutaneous adipocytes in vitro (8, 9), suggesting that the association between greater amounts of visceral fat and the metabolic complications of obesity may reflect excess FFAs originating from visceral adipose tissue lipolysis (6, 7). These FFAs are released directly into the portal vein, exposing the liver to more FFAs than would be predicted from systemic FFA availability data. Thus, enlarged visceral fat stores could increase the proportion of hepatic FFA delivery coming from visceral, as opposed to systemic, sources.The relationship between visceral fat and splanchnic FFA kinetics has not been assessed in humans. We previously reported a slight, but nonsignificant increase in splanchnic FFA (palmitate) release in upper-body obese women compared with lower-body obese and nonobese women (10). Visceral fat was not measured, however, and we did not include women with the full range of obesity-related metabolic abnormalities (10) in whom more significant disturbances of FFA metabolism might be expected. In addition, men were not studied, and obese ...

Motor function in type 2 diabetes is largely unknown. In 36 type 2 diabetic patients and in 36 control subjects matched for sex, age, weight, height, and physical activity, strength of flexors and extensors at elbow, wrist, knee, and ankle was assessed at isokinetic dynamometry. The degree of neuropathy was determined by clinical scores, nerve conduction studies, and quantitative sensory testing. Eventually, all results were summed to obtain a neuropathy rank-sum score (NRSS). The degree of nephropathy and retinal condition were also evaluated. Diabetic patients had a 17 and 14% reduction of strength of ankle flexors (P < 0.02) and ankle extensors (P < 0.03), respectively. At the knee, strength of extensors and flexors was reduced by 7% (NS) and 14% (P < 0.05), respectively. At the elbow and wrist, muscle strength was preserved. The NRSS was related to the strength at the ankle (r ‫؍‬ ؊0.45, P < 0.01) and knee (r ‫؍‬ ؊0.42, P < 0.02). Following multiple regression analysis, the NRSS but not the degree of nephropathy or retinopathy was related to strength at the ankle and knee. In conclusion, type 2 diabetic patients may have muscle weakness at the ankle and knee related to presence and severity of peripheral neuropathy. Diabetes 53:1543-1548, 2004 S ensory symptoms and deficits are frequent in distal diabetic polyneuropathy. Motor symptoms are less dramatic, and motor deficits are more difficult to recognize. Not surprisingly, the literature on this manifestation of diabetic polyneuropathy is sparse. In patients with long-term type 1 diabetes, we have found impaired muscle strength at the ankle and knee closely related to the severity of neuropathy (2). Motor dysfunction is known to occur in type 2 diabetic patients; however, the severity and distribution of the weakness has not been reported (3).In a population-based study from the U.K., a similar frequency of neuropathy was found in type 1 and 2 diabetic patients after age correction (4). Nevertheless, a considerably lower frequency of severe neuropathy, defined as an inability to walk on heels, was observed in type 2 diabetes in a population-based study from Minnesota (3). This observation may indicate less motor dysfunction in type 2 diabetes. However, the relation between inability to walk on heels and muscle strength has not been established, so there is a clear need for quantitative studies of motor function in type 2 diabetes.In the present study, we evaluated muscular performance of lower and upper extremities quantitatively in type 2 diabetic patients, applying isokinetic dynamometry, which has high reliability in the determination of maximal strength in both neuropathic and healthy subjects (5). To study the relationship of muscle strength with the prevalence and severity of diabetic neuropathy, other diabetes complications, and metabolic control, patients were characterized clinically, biochemically, and with electrophysiological and sensory function tests. RESEARCH DESIGN AND METHODSAll patients and control subjects gave informed consent for partici...