Stephen Welle

Uptake and release of glucose by the human kidney. Postabsorptive rates and responses to epinephrine.

Despite ample evidence that the kidney can both produce and use appreciable amounts of glucose, the human kidney is generally regarded as playing a minor role in glucose homeostasis. This view is based on measurements of arteriorenal vein glucose concentrations indicating little or no net release of glucose. However, inferences from net balance measurements do not take into consideration the simultaneous release and uptake of glucose by the kidney. Therefore, to assess the contribution of release and uptake of glucose by the human kidney to overall entry and removal of plasma glucose, we used a combination of balance and isotope techniques to measure renal glucose net balance, fractional extraction, uptake and release as well as overall plasma glucose appearance and disposal in 10 normal volunteers under basal postabsorptive conditions and during a 3-h epinephrine infusion. In the basal postabsorptive state, there was small but significant net output of glucose by the kidney (66 +/- 22 mumol.min-1, P = 0.016). However, since renal glucose fractional extraction averaged 2.9 +/- 0.3%, there was considerable renal glucose uptake (2.3 +/- 0.2 mumol.kg-1.min-1) which accounted for 20.2 +/- 1.7% of systemic glucose disposal (11.4 +/- 0.5 mumol.kg-1.min-1). Renal glucose release (3.2 +/- 0.2 mumol.kg-1.min-1) accounted for 27.8 +/- 2.1% of systemic glucose appearance (11.4 +/- 0.5 mumol.kg-1.min-1). Epinephrine infusion, which increased plasma epinephrine to levels observed during hypoglycemia (3722 +/- 453 pmol/liter) increased renal glucose release nearly twofold (5.2 +/- 0.5 vs 2.8 +/- 0.1 mol.kg-1.min-1, P = 0.01) so that at the end of the infusion, renal glucose release accounted for 40.3 +/- 5.5% of systemic glucose appearance and essentially all of the increase in systemic glucose appearance. These observations suggest an important role for the human kidney in glucose homeostasis.

Thermic effect of feeding in man: increased plasma norepinephrine levels following glucose but not protein or fat consumption.

In seven healthy male subjects, intake of 100 g protein hydrolysate produced significantly greater increments in energy expenditure than intake of 100 g glucose, 44 g fat, or a noncaloric control solution during the first 4 hr postcibum. Glucose and fat intake produced similar increments in energy expenditure. In contrast to the effects on thermogenesis, protein and fat intake did not alter sympathetic nervous system (SNS) activity, as estimated by plasma norepinephrine (NE) levels, whereas glucose intake significantly increased NE levels. Plasma levels of immunoreactive insulin were stimulated by glucose intake to a much greater level than by protein intake, and were unaffected by ingestion of the fat and control solutions. Pulse rate significantly increased following ingestion of all nutrients compared to pulse rate changes during the control test. These data support the traditional concept of a greater thermic effect of protein than of carbohydrate or fat, but the possibility of SNS involvement in the thermic effect of protein and fat is not supported.

Skeletal muscle gene expression profiles in 20-29 year old and 65-71 year old women.

Gene expression profiling may provide leads for investigations of the molecular basis of functional declines associated with aging. In this study, high-density oligonucleotide arrays were used to probe the patterns of gene expression in skeletal muscle of seven young women (20-29 years old) and eight healthy older women (65-71 years old). The older subjects had reduced muscle mass, strength, and peak oxygen consumption relative to young women. There were approximately 1000 probe sets that suggested differential gene expression in younger and older muscle according to statistical criteria. The most highly overexpressed genes (>3-fold) in older muscle were p21 (cyclin-dependent kinase inhibitor 1A), which might reflect increased DNA damage, perinatal myosin heavy chain, which might reflect increased muscle fiber regeneration, and tomoregulin, which does not have a defined function in muscle. More than 40 genes encoding proteins that bind to pre-mRNAs or mRNAs were expressed at higher levels in older muscle. More than 100 genes involved in energy metabolism were expressed at lower levels in older muscle. In general, these results support previous observations on the differences in gene expression profiles between younger and older men.

Genetic Deletion of Myostatin From the Heart Prevents Skeletal Muscle Atrophy in Heart Failure

Background— Cardiac cachexia is characterized by an exaggerated loss of skeletal muscle, weakness, and exercise intolerance, although the cause of these effects remains unknown. Here, we hypothesized that the heart functions as an endocrine organ in promoting systemic cachexia by secreting peptide factors such as myostatin. Myostatin is a cytokine of the transforming growth factor-&bgr; superfamily that is known to control muscle wasting. Methods and Results— We used a Cre/loxP system to ablate myostatin (Mstn gene) expression in a cell type–specific manner. As expected, elimination of Mstn selectively in skeletal muscle with a myosin light chain 1f (MLC1f)-cre allele induced robust hypertrophy in all skeletal muscle. However, heart-specific deletion of Mstn with an Nkx2.5-cre allele did not alter baseline heart size or secondarily affect skeletal muscle size, but the characteristic wasting and atrophy of skeletal muscle that typify heart failure were not observed in these heart-specific null mice, indicating that myocardial myostatin expression controls muscle atrophy in heart failure. Indeed, myostatin levels in the plasma were significantly increased in wild-type mice subjected to pressure overload–induced cardiac hypertrophy but not in Mstn heart-specific deleted mice. Moreover, cardiac-specific overexpression of myostatin, which increased circulating levels of myostatin by 3- to 4-fold, caused a reduction in weight of the quadriceps, gastrocnemius, soleus, and even the heart itself. Finally, to investigate myostatin as a potential therapeutic target for the treatment of muscle wasting in heart failure, we infused a myostatin blocking antibody (JA-16), which promoted greater maintenance of muscle mass in heart failure. Conclusions— Myostatin released from cardiomyocytes induces skeletal muscle wasting in heart failure. Targeted inhibition of myostatin in cardiac cachexia might be a therapeutic option in the future.

Deliberate overfeeding in women and men: energy cost and composition of the weight gain.

1. Thirteen adult females and two males were overfed a total of 79-159 MJ (19,000-38,000 kcal) during a 3-week period at the Clinical Research Center, Rochester. The average energy cost of the weight gain was 28 kJ (6.7 kcal)/g, and about half the gain consisted of lean body mass (LBM) as estimated by 40K counting. 2. A survey of the literature disclosed twenty-eight normal males and five females who had been overfed a total of 104-362 MJ (25,000-87,000 kcal) under controlled conditions: twenty-five of these had assays of body composition, and three had complete nitrogen balances. 3. When these values were combined with those from our subjects (total forty-eight), there was a significant correlation between weight gain and total excess energy consumed (r 0.77, P less than 0.01) and between LBM gain and excess energy (r 0.49, P less than 0.01). Based on means the energy cost was 33.7 kJ (8.05 kcal)/g gain and 43.6% of the gain was LBM; from regression analysis these values were 33.7 kJ (8.05 kcal)/g gain and 38.4% of gain as LBM. 4. Individual variations in the response could not be explained on the basis of sex, initial body-weight or fat content, duration of overfeeding, type of food eaten, amount of daily food consumption or, in a subset of subjects, on smoking behaviour. 5. The average energy cost of the weight gain was close to the theoretical value of 33.8 kJ (8.08 kcal)/g derived from the composition of the tissue gained.

The Regulation of Skeletal Muscle Protein Turnover during the Progression of Cancer Cachexia in the ApcMin/+ Mouse

Muscle wasting that occurs with cancer cachexia is caused by an imbalance in the rates of muscle protein synthesis and degradation. The ApcMin/+ mouse is a model of colorectal cancer that develops cachexia that is dependent on circulating IL-6. However, the IL-6 regulation of muscle protein turnover during the initiation and progression of cachexia in the ApcMin/+ mouse is not known. Cachexia progression was studied in ApcMin/+ mice that were either weight stable (WS) or had initial (≤5%), intermediate (6–19%), or extreme (≥20%) body weight loss. The initiation of cachexia reduced %MPS 19% and a further ∼50% with additional weight loss. Muscle IGF-1 mRNA expression and mTOR targets were suppressed with the progression of body weight loss, while muscle AMPK phosphorylation (Thr 172), AMPK activity, and raptor phosphorylation (Ser 792) were not increased with the initiation of weight loss, but were induced as cachexia progressed. ATP dependent protein degradation increased during the initiation and progression of cachexia. However, ATP independent protein degradation was not increased until cachexia had progressed beyond the initial phase. IL-6 receptor antibody administration prevented body weight loss and suppressed muscle protein degradation, without any effect on muscle %MPS or IGF-1 associated signaling. In summary, the %MPS reduction during the initiation of cachexia is associated with IGF-1/mTOR signaling repression, while muscle AMPK activation and activation of ATP independent protein degradation occur later in the progression of cachexia. IL-6 receptor antibody treatment blocked cachexia progression through the suppression of muscle protein degradation, while not rescuing the suppression of muscle protein synthesis. Attenuation of IL-6 signaling was effective in blocking the progression of cachexia, but not sufficient to reverse the process.

Effect of beta-hydroxybutyrate on whole-body leucine kinetics and fractional mixed skeletal muscle protein synthesis in humans.

Because intravenous infusion of beta-hydroxybutyrate (beta-OHB) has been reported to decrease urinary nitrogen excretion, we investigated in vivo metabolism of leucine, an essential amino acid, using L-[1-13C]leucine as a tracer during beta-OHB infusion. Leucine flux during beta-OHB infusion did not differ from leucine flux during normal saline infusion in nine normal subjects, whereas leucine oxidation decreased 18-41% (mean = 30%) from 18.1 +/- 1.1 mumol.kg-1.h-1 (P less than 0.01), and incorporation of leucine into skeletal muscle protein increased 5-17% (mean = 10%) from 0.048 + 0.003%/h (P less than 0.02). Since blood pH during beta-OHB infusion was higher than the pH during saline infusion, we performed separate experiments to study the effect of increased blood pH on leucine kinetics by infusing sodium bicarbonate intravenously. Blood pH during sodium bicarbonate infusion was similar to that observed during the beta-OHB infusion, but bicarbonate infusion had no effect on leucine flux or leucine oxidation. We conclude that beta-OHB decreases leucine oxidation and promotes protein synthesis in human beings.

Transcriptional and post-transcriptional impact of toxic RNA in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is an RNA dominant disease in which mutant transcripts containing an expanded CUG repeat (CUG(exp)) cause muscle dysfunction by interfering with biogenesis of other mRNAs. The toxic effects of mutant RNA are mediated partly through sequestration of splicing regulator Muscleblind-like 1 (Mbnl1), a protein that binds to CUG(exp) RNA. A gene that is prominently affected encodes chloride channel 1 (Clcn1), resulting in hyperexcitability of muscle (myotonia). To identify DM1-affected genes and study mechanisms for dysregulation, we performed global mRNA profiling in transgenic mice that express CUG(exp) RNA, when compared with Mbnl1 knockout and Clcn1 null mice. We found that the majority of changes induced by CUG(exp) RNA in skeletal muscle can be explained by reduced activity of Mbnl1, including many changes that are secondary to myotonia. The pathway most affected comprises genes involved in calcium signaling and homeostasis. Some effects of CUG(exp) RNA on gene expression are caused by abnormal alternative splicing or downregulation of Mbnl1-interacting mRNAs. However, several of the most highly dysregulated genes showed altered transcription, as indicated by parallel changes of the corresponding pre-mRNAs. These results support the idea that trans-dominant effects of CUG(exp) RNA on gene expression in this transgenic model may occur at the level of transcription, RNA processing and mRNA decay, and are mediated mainly but not entirely through sequestration of Mbnl1.

Human kidney and liver gluconeogenesis: evidence for organ substrate selectivity.

To assess the contribution of the human kidney to gluconeogenesis (GN) and its role in conversion of glutamine and alanine to glucose, we used a combination of isotopic and organ balance techniques in nine normal postabsorptive volunteers and measured both overall and renal incorporation of these precursors into glucose before and after infusion of epinephrine. In the postabsorptive basal state, renal incorporation of glutamine (27 ± 2 μmol/min) and alanine (2.1 ± 0.5 μmol/min) into glucose accounted for 72.8 ± 3.3 and 3.9 ± 0.5% of their overall incorporation into glucose (37 ± 2 and 51 ± 6 μmol/min, respectively) and 19.0 ± 3.5 and 1.4 ± 0.2%, respectively, of overall renal glucose release. Infusion of epinephrine, which increased systemic and renal glucose release more than twofold ( P< 0.001), increased overall glutamine and alanine incorporation into glucose (both P < 0.001) and increased renal GN from glutamine ( P< 0.001) but not from alanine ( P = 0.15). Renal glutamine GN now accounted for 90.3 ± 4.0% of overall glutamine GN ( P = 0.01 vs. basal), whereas renal alanine GN still accounted for only 4.8 ± 1.7% of overall alanine GN ( P = 0.36 vs. basal). With the assumption that kidney and liver are the only gluconeogenic organs in humans, these results indicate that glutamine GN occurs primarily in kidney, whereas alanine GN occurs almost exclusively in liver. Isotopic studies of glutamine and alanine incorporation into plasma glucose may provide a selective, noninvasive method to assess hepatic and renal GN.

Expression profile of FSHD supports a link between retinal vasculopathy and muscular dystrophy

Background: Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions within a tandem array of D4Z4 repeats on chromosome 4q35. In addition to muscle degeneration, most patients with FSHD develop abnormalities of the retinal vasculature. Previous work has suggested that muscle degeneration in FSHD results from increased expression of genes proximal to the deletion, including FRG1. Objectives: To reexamine this mechanism and identify pathways that are abnormally regulated early in the disease process. Methods: We prospectively studied gene expression in skeletal muscle in patients with FSHD (n = 19) vs healthy individuals (n = 30) and patients with myotonic dystrophy type 1 (n = 12). We used oligonucleotide microarrays for global analysis of gene expression and reverse transcriptase-PCR (RT-PCR) to assess expression or alternative splicing for particular genes. Results: Expression of FRG1 was not increased in patients with FSHD, either by microarray analysis or quantitative RT-PCR. Among genes on 4q35, only LRP2BP showed upregulation that was specific to FSHD. However, neither LRP2BP nor FRG1 showed imbalance of allelic expression by RT-PCR. After filtering out genes that showed similar dysregulation in other forms of muscular dystrophy, only 44 genes were specifically upregulated early in FSHD. Among these, 34 genes were characterized or partially characterized, of which 11 (32%) had a role in vascular smooth muscle or endothelial cells. Conclusion: Expression of genes on chromosome 4q35 was normally regulated in the early stages of facioscapulohumeral muscular dystrophy. Our results support a possible link between muscular dystrophy and retinal vasculopathy in facioscapulohumeral muscular dystrophy.