Obese mice on a high-fat alternate-day fasting regimen lose weight and improve glucose tolerance.

Alternate-day fasting (ADF) causes body weight (BW) loss in humans and rodents. However, it is not clear that ADF while maintaining a high-fat (HF) diet results in weight loss and the accompanying improvement in control of circulating glucose. We tested the hypotheses that a high-fat ADF protocol in obese mice would result in (i) BW loss, (ii) improved glucose control, (iii) fluctuating phenotypes on 'fasted' days when compared to 'fed' days and (iv) induction of torpor on 'fasted days'. We evaluated the physiological effects of ADF in diet-induced obese mice for BW, heart rate (HR), body temperature (Tb ), glucose tolerance, insulin responsiveness, blood parameters (leptin, insulin, free fatty acids) and hepatic gene expression. Diet-induced obese male C57BL/6J mice lost one-third of their pre-diet BW while on an ADF diet for 10 weeks consisting of HF food. The ADF protocol improved glucose tolerance and insulin sensitivity, although mice on a fast day were less glucose tolerant than the same mice on a fed day. ADF mice on a fast day had low circulating insulin, but had an enhanced response to an insulin-assisted glucose tolerance test, suggesting the impaired glucose tolerance may be a result of insufficient insulin production. On fed days, ADF mice were the warmest, had a high HR and displayed hepatic gene expression and circulating leptin that closely mimicked that of mice fed an ad lib HF diet. ADF mice never entered torpor as assessed by HR and Tb . However, on fast days, they were the coolest, had the slowest HR, and displayed hepatic gene expression and circulating leptin that closely mimicked that of Chow-Fed mice. Collectively, the ADF regimen with a HF diet in obese mice results in weight loss, improved blood glucose control, and daily fluctuations in selected physiological and biochemical parameters in the mouse.

PGAM-M expression is regulated pretranslationally in hindlimb muscles and under altered loading conditions.

Enzymatic activity from the muscle-specific isoform of phosphoglycerate mutase (PGAM-M) is higher within glycolytic skeletal muscles than in oxidative muscles. The hypothesis that PGAM-M is regulated pretranslationally among muscles of the hindlimb was tested using enzymatic assays, Western blots, and Northern blots. We further investigated the regulatory level(s) at which PGAM-M gene expression is controlled during hindlimb unweighting. PGAM-M mRNA and immunoreactive protein levels were fourfold lower in the rat soleus muscle than in the tibialis anterior (TA), plantaris, and extensor digitorum longus muscles. Four weeks of unweighting induced a 2.5-fold increase in PGAM enzymatic activity within the soleus muscle, a 1.8-fold increase in PGAM-M immunoreactivity, and a 3. 5-fold increase in PGAM-M mRNA. To examine potential transcriptional regulatory mechanisms, the proximal 400 bp of the rat PGAM-M promoter were linked to a firefly luciferase and injected into normal and unweighted TA and soleus muscles. Firefly luciferase activity was elevated two- to threefold in the TA and the unweighted soleus over the normal soleus muscle. These data suggest that PGAM-M expression is pretranslationally regulated among muscle types and within unweighted slow-twitch muscle. Furthermore, the proximal 400 bp of the PGAM-M promoter contains cis-acting sequences to allow muscle-type-specific expression of a reporter gene and responsiveness to soleus muscle unweighting.

Interaction of thyroid hormone and functional overload on skeletal muscle isomyosin expression.

This study examined the interaction of exogenous thyroid hormone 3,5,3'-triiodothyronine (T3) and functional overload on skeletal muscle myosin heavy chain (MHC) expression, studied at both the protein and mRNA level of analysis. Animals were allocated to the following groups: 1) normal control, 2) overload control, 3) hyperthyroid control, and 4) hyperthyroid+overload. Overload of the rat plantaris was accomplished by surgical removal of its synergists (soleus and gastrocnemius), and the animals were made hyperthyroid by injections of T3 (350 micrograms/kg every other day). After overload of 8 wk, muscle enlargement occurred by 53% for both overload groups (P < 0.05). This was accompanied by a 330 and 82% increase in the relative content of type I and IIa MHC, respectively, and a corresponding decrease by 16 and 44% in type IIx and IIb MHC, respectively, in the overload control group (P < 0.05 vs. normal control). Changes in the relative and absolute content of mRNA for these MHCs paralleled the protein response. Exogenous T3 completely reversed the upregulation of type I MHC and the downregulation of type IIx associated with overload at both the protein and mRNA level (P < 0.05). However, T3 was only partially effective in blunting the downregulation of IIb MHC and the upregulation of IIa MHC (protein and mRNA) accompanying the overload.(ABSTRACT TRUNCATED AT 250 WORDS)

Vagal tone dominates autonomic control of mouse heart rate at thermoneutrality.

It is generally accepted that cardiac sympathetic tone dominates the control of heart rate (HR) in mice. However, we have recently challenged this notion given that HR in the mouse is responsive to ambient temperature (T(a)) and that the housing T(a) is typically 21-23 degrees C, well below the thermoneutral zone ( approximately 30 degrees C) of this species. To specifically test the hypothesis that cardiac sympathetic tone is the primary mediator of HR control in the mouse, we first examined the metabolic and cardiovascular responses to rapid changes in T(a) to demonstrate the sensitivity of the mouse cardiovascular system to T(a). We then determined HR in 1) mice deficient in cardiac sympathetic tone ("beta-less" mice), 2) mice deficient in cardiac vagal tone [muscarinic M(2) receptor (M(2)R(-/-)) mice], and 3) littermate controls. At a T(a) of 30 degrees C, the HR of beta-less mice was identical to that of wild-type mice (351 +/- 11 and 363 +/- 10 beats/min, respectively). However, the HR of M(2)R(-/-) mice was significantly greater (416 +/- 7 beats/min), demonstrating that vagal tone predominates over HR control at this T(a). When these mice were calorically restricted to 70% of normal intake, HR fell equally in wild-type, beta-less, and M(2)R(-/-) mice (DeltaHR = 73 +/- 9, 76 +/- 3, and 73 +/- 7 beats/min, respectively), suggesting that the fall in intrinsic HR governs bradycardia of calorically restricted mice. Only when the T(a) was relatively cool, at 23 degrees C, did beta-less mice exhibit a HR (442 +/- 14 beats/min) that was different from that of littermate controls (604 +/- 10 beats/min) and M(2)R(-/-) mice (602 +/- 5 beats/min). These experiments conclusively demonstrate that in the absence of cold stress, regulation of vagal tone and modulation of intrinsic rate are important determinants of HR control in the mouse.

In vivo analysis of the myosin heavy chain IIB promoter region.

The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.

Altered leptin signaling is sufficient, but not required, for hypotension associated with caloric restriction.

Caloric restriction of mammals leads to decreases in blood pressure and heart rate. Although relevant clinically, the mechanisms involved, in terms of hormones and signaling pathways invoked, are currently not known. Circumstantial evidence suggests that leptin signaling may be involved with the bradycardia and hypotension associated with caloric restriction. This hypothesis was specifically tested using leptin-deficient mice (ob/ob) or leptin-receptor rats (Koletsky). Ob/ob mice were hypertensive during the light cycle relative to littermate controls (108 +/- 2 vs. 100 +/- 2 mmHg, respectively). Both ob/ob mice and wild-type mice exhibited hypotension and bradycardia on initiation of a 50% caloric restriction regime, suggesting that the loss of leptin during caloric restriction is not required to explain the cardiovascular effects. Blood pressure in Koletsky rats did not drop in response to caloric restriction during the light cycle, whereas blood pressure in littermate control rats significantly dropped. These data suggest that at least two pathways are involved with cardiovascular effects of caloric restriction: one dependent on leptin signaling and the other independent of the leptin axis.

Interaction of hypertension and caloric restriction on cardiac mass and isomyosin expression.

Previous studies show that elevations in blood pressure induce concomitant increases in both cardiac mass and slow beta-myosin heavy chain (MHC) expression in rodents, whereas caloric restriction of 50% (CR) causes an increase in beta-MHC while modestly lowering blood pressure in normotensive rats. The goals of this study were to 1) determine if beta-MHC expression could be independently regulated by CR and hypertension when these two interventions are combined and 2) determine if CR exerts a lowering of blood pressure in two contrasting models of rodent hypertension. Rodents were assigned to the following groups: 1) normal control (NC); 2) abdominal aortic constriction (Abcon), a model that induces hypertension via renin-angiotensin II; 3) nephrectomy-deoxycorticosterone acetate treatment (DOCA), a model that induces hypertension through increased salt retention; 4) CR; 5) Abcon+CR; 6) DOCA+CR. Results show that both Abcon and DOCA induced significant increases in systemic blood pressures, left ventricular (LV) weight/body weight, and the relative content of beta-MHC compared with NC. When applied in combination with either Abcon or DOCA, CR significantly blunted the changes observed in both systemic blood pressures and LV weight/body weight. In contrast, CR in conjunction with DOCA augmented % beta-MHC expression relative to either DOCA or CR alone. These data suggest 1) caloric restriction exerts a powerful impact on reducing experimentally induced hypertension in rodents and 2) the regulation of beta-MHC expression appears to be regulated by at least two processes, one associated with the stimulus of hypertension and the other involving an independent pathway linked to caloric restriction.

Optimal shortening velocities for in situ power production of rat soleus and plantaris muscles.

Force-velocity (FV) relationships have been used previously to calculate maximal power production and to identify an optimal velocity of shortening (V(opt)-fv) to produce such power in skeletal muscle. The cyclical nature of muscle position during locomotion for muscles such as the soleus and plantaris is such that either constant force or velocity is rarely attained. In the present study, the work loop technique, a technique developed to measure maximal attainable power output from muscles undergoing cyclic length changes, was undertaken to determine whether simulating in vivo function alters the power-velocity relationship of the soleus and plantaris and, in particular, the velocity of shortening that produces maximal power (V(opt)-wl). FV relationships were determined for both soleus (n = 4) and plantaris (n = 4) muscles in situ from adult female Sprague-Dawley rats by measuring shortening velocities during afterloaded isotonic contractions. The velocity that produced maximal power using FV relationships, V(opt)-fv, was 54.6 +/- 0.7 mm/s for the plantaris vs. 20.2 +/- 1.2 mm/s for the soleus. Then, the work loop technique was employed to measure net power from these same muscles at multiple cycling frequencies (1.5 to 4.0 Hz for the soleus; 4.0 to 8.0 Hz for the plantaris). Multiple power-velocity curves were generated (one at each cycle frequency) by varying the strain (1-8 mm). Thus, at each cycle frequency, V(opt)-wl could be identified. For both the plantaris and soleus, V(opt)-wl at each cycle frequency was not different from their respective V(opt)-fv value. Thus both fast and slow skeletal muscles have inherent optimal shortening velocities, identifiable with FV relationships, that dictate their respective maximal attainable mechanical power production using the work loop technique.

bHLH transcription factor MyoD affects myosin heavy chain expression pattern in a muscle-specific fashion.

A strong correlative pattern between MyoD gene expression and myosin heavy chain IIB (MHC IIB) gene expression exists. To test whether this correlative relationship is causative, MHC gene expression in muscles from MyoD(-/-) mice was analyzed. The MHC IIB gene was not detectable in the MyoD(-/-) diaphragm, whereas the MHC IIB protein made up 10.0 +/- 1.7% of the MHC protein pool in the wild-type (WT) mouse diaphragm. Furthermore, the MHC IIA protein was not detectable in the MyoD(-/-) biceps brachii, and the MHC IIB protein was overexpressed in the masseter. To examine whether MyoD is required for the upregulation of the MHC IIB gene within slow muscle after disuse, MyoD(-/-) and WT hindlimb musculature was unweighted. MyoD(-/-) exhibited a diminished response in the upregulation of the MHC IIB mRNA within the soleus muscle as a result of the hindlimb unweighting. Collectively, these data suggest that MyoD plays a role in the MHC profile in a muscle-specific fashion.

Cell Wall Free Space of Cucumis Hypocotyls Contains NAD and a Blue Light-Regulated Peroxidase Activity.

Solutions were obtained from the cell wall free space of red light-grown cucumber (Cucumis sativus L.) hypocotyl sections by a low-speed centrifugation technique. The centrifugate contained NAD and peroxidase but no detectable cytoplasmic contamination, as indicated by the absence of the activity of glucose-6-phosphate dehydrogenase from the cell wall solution. Peroxidase activity centrifuged from the cell wall of red light-grown cucumber hypocotyl section could be resolved into at least three cathodic isoforms and two anodic isoforms by isoelectric focusing. Treatment of red light-grown cucumber seedlings with a 10-minute pulse of high-intensity blue light increased the level of cell wall peroxidase by about 60% and caused a qualitative change in the anodic isoforms of this enzyme. The increase in peroxidase activity was detectable within 25 minutes after the start of the blue light pulse, was maximal at 35 minutes, and declined to control levels by 45 minutes of irradiation. The inhibitory effect of blue light on hypocotyl elongation was more rapid than the effect of blue light on total wall peroxidase activity, leading to the conclusion that growth and peroxidase activity are not causally related.

An E-box within the MHC IIB gene is bound by MyoD and is required for gene expression in fast muscle.

The myosin heavy chain (MHC) IIB gene is selectively expressed in skeletal muscles, imparting fast contractile kinetics. Why the MHC IIB gene product is expressed in muscles like the tibialis anterior (TA) and not expressed in muscles like the soleus is currently unclear. It is shown here that the mutation of an E-box within the MHC IIB promoter decreased reporter gene activity in the fast-twitch TA muscle 90-fold as compared with the wild-type promoter. Reporter gene expression within the TA required this E-box for activation of a heterologous construct containing upstream regulatory regions of the MHC IIB promoter linked to the basal 70-kDa heat shock protein TATA promoter. Electrophoretic mobility shift assays demonstrated that mutation of the E-box prevented the binding of both MyoD and myogenin to this element. In cotransfected C2C12 myotubes and Hep G2 cells, MyoD preferentially activated the MHC IIB promoter in an E-box-dependent manner, whereas myogenin activated the MHC IIB promoter to a lesser extent, and in an E-box-independent manner. A time course analysis of hindlimb suspension demonstrated that the unweighted soleus muscle activated expression of MyoD mRNA before the de novo expression of MHC IIB mRNA. These data suggest a possible causative role for MyoD in the observed upregulation of MHC IIB in the unweighted soleus muscle.

Control of beta-myosin heavy chain expression in systemic hypertension and caloric restriction in the rat heart.

In the rat left ventricle, both pressure overload induced by abdominal aortic constriction (Abcon) and caloric restriction (CR) induce an increase in the steady-state level of the beta-myosin heavy chain (MHC) protein and mRNA. Both models also induce a concomitant decrease in the alpha-MHC protein and mRNA. The goals of this study were to 1) determine if the changes in MHC expression in the models are due to altered transcription and 2) identify the relative levels of some key factors interacting with the regulatory regions of these genes. Female Sprague-Dawley rats were randomly assigned to the following groups: 1) normal control (NC), 2) Abcon, and 3) CR. After 5 wk of experimental manipulations, myocardial nuclei were isolated. These nuclei were used for 1) nuclear run-on assays or 2) nuclear extract, which was prepared and used for gel mobility shift assays (GMSAs). Nuclear run-on assays demonstrated that the increase in beta-MHC mRNA and protein expression in both Abcon and CR can be at least partially attributed to increased transcription. The concomitant decrease in alpha-MHC content can similarly be attributed to a decrease in transcription of this gene. Furthermore, GMSAs demonstrate that nuclear extract from each group interact differently with certain elements known to be important for expression in vitro. CR nuclear extracts have a 25.6 +/- 7.2% decrease (P < 0.05 vs. NC) in interaction with a thyroid-responsive element, a potential repressor of beta-MHC transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunolocalization of rat cardiac beta-MHC protein expression in systemic hypertension and caloric restriction.

Previous studies have shown that both systemic hypertension induced by abdominal aortic constriction (Abcon) and 50% caloric restriction (CR) increase left ventricular (LV) beta-myosin heavy chain (MHC) protein expression in the rat. However, these two physiological states have different effects on hemodynamic load, and information regarding beta-MHC localization across the LV wall in these two models may provide insight into the process of adaptation to chronic stress among myocardial cells. Thus the goal of this study was to determine the pattern of beta-MHC protein expression across the LV wall in Abcon and CR models using a beta-MHC-specific antibody. Adult female Sprague-Dawley rats (approximately 225-250 g) were randomly assigned to one of three groups: 1) normal control (NC), 2) Abcon, and 3) CR. After a treatment period of 5 wk, Abcon LVs hypertrophied 52% relative to NC, accompanying the 42% increase in mean blood pressure. CR rats, however, had a normal LV weight-to-body weight ratio. The relative content of LV beta-MHC protein expression, as assessed by native gel electrophoresis, increased from 3% in NC to 25 and 41% in Abcon and CR rats, respectively. Immunohistochemical analysis of beta-MHC expression demonstrated that the increase in beta-MHC protein in the Abcon group occurred primarily on the endocardial side of the LV. In contrast, the increase in beta-MHC protein in the CR LV occurred equally across the entire LV wall. This suggests that CR has a global effect on MHC isoform expression in LV myocardial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of chronic energy deprivation on cardiac thyroid hormone receptor and myosin isoform expression.

Thyroid hormone (3,5,3'-triiodothyronine; T3) and its receptor (TR) play an important regulatory role for in vivo and in vitro cardiac myosin heavy chain (MHC) isoform gene expression by activating the alpha- and inhibiting the beta-MHC genes. Previous studies have shown that chronic energy deprivation (CED; 50% of normal caloric intake) in the rat impacts cardiac MHC protein expression and hemodynamic parameters in a pattern typically seen with hypothyroidism; yet, unlike hypothyroidism, circulating T3 levels are not depressed. Therefore, the goal of this study was to determine if the altered MHC isoform expression observed in CED is associated with altered TR expression, both at the mRNA and protein levels. Female rats weighing approximately 250 g were allocated into two groups, designated as normal control (NC) and CED. After 5 wk, the relative content of alpha-MHC protein and mRNA levels decreased in CED ventricles by 20% (P < 0.05). In contrast, the relative content of both beta-MHC protein and mRNA levels increased five- to sixfold in CED (P < 0.05). Although there were no changes in TR mRNA levels relative to 18S rRNA in CED, the total number of nuclear TRs decreased 3.5-fold in the CED group (P < 0.05), from a maximum binding capacity of 840 +/- 130 fmol/mg DNA in NC to 241 +/- 118 fmol/mg DNA in CED, with no change in the affinity of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

The calcineurin-NFAT pathway and muscle fiber-type gene expression.

To test for a role of the calcineurin-NFAT (nuclear factor of activated T cells) pathway in the regulation of fiber type-specific gene expression, slow and fast muscle-specific promoters were examined in C2C12 myotubes and in slow and fast muscle in the presence of calcineurin or NFAT2 expression plasmids. Overexpression of active calcineurin in myotubes induced both fast and slow muscle-specific promoters but not non-muscle-specific reporters. Overexpression of NFAT2 in myotubes did not activate muscle-specific promoters, although it strongly activated an NFAT reporter. Thus overexpression of active calcineurin activates transcription of muscle-specific promoters in vitro but likely not via the NFAT2 transcription factor. Slow myosin light chain 2 (MLC2) and fast sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) reporter genes injected into rat soleus (slow) and extensor digitorum longus (EDL) (fast) muscles were not activated by coinjection of activated calcineurin or NFAT2 expression plasmids. However, an NFAT reporter was strongly activated by overexpression of NFAT2 in both muscle types. Calcineurin and NFAT protein expression and binding activity to NFAT oligonucleotides were different in slow vs. fast muscle. Taken together, these results indicate that neither calcineurin nor NFAT appear to have dominant roles in the induction and/or maintenance of slow or fast fiber type in adult skeletal muscle. Furthermore, different pathways may be involved in muscle-specific gene expression in vitro vs. in vivo.

Differential expression of mitochondrial DNA replication factors in mammalian tissues.

Mitochondrial biogenesis and mitochondrial DNA (mtDNA) replication are regulated during development and in response to physiological stresses, but the regulatory events that control the abundance of mtDNA in cells of higher eukaryotes have not been defined at a molecular level. In this study, we observed that expression of the catalytic subunit of DNA polymerase gamma (POLgammaCAT) mRNA varies little among different tissues and is not increased by continuous neural activation of skeletal muscle, a potent stimulus to mitochondrial biogenesis. Increased copy number for the POLgamma locus in a human cell line bearing a partial duplication of chromosome 15 increased the abundance of POLgammaCAT mRNA without up-regulation of mtDNA. In contrast, expression of mitochondrial single-stranded DNA-binding (mtSSB) mRNA is regulated coordinately with variations in the abundance of mtDNA among tissues of mammalian organisms and is up-regulated in association with the enhanced mitochondrial biogenesis that characterizes early postnatal development of the heart and the adaptive response of skeletal myofibers to motor nerve stimulation. In addition, we noted that expression of mtSSB is concentrated within perinuclear mitochondria that constitute active sites of mtDNA replication. We conclude that constitutive expression of the gene encoding the catalytic subunit of mitochondrial DNA polymerase is sufficient to support physiological variations in mtDNA replication among specialized cell types, whereas expression of the mtSSB gene is controlled by molecular mechanisms acting to regulate mtDNA replication or stability in mammalian cells.