there are a number of open issues in the hfd mouse modeling of t2dm : will these mouse models be useful in preclinical investigations?what about the differences identified between wild type mouse strains?why some protocols do not elicit dcm?can the research community agree upon a common hfd feeding protocol ?
comparisons between papers , genetic manipulations , and therapies will be much easier and more informative if a specific diet , length of diet , and analysis protocols can be agreed upon .
why some protocols do not elicit dcm ? can the research community agree upon a common hfd feeding protocol ?
comparisons between papers , genetic manipulations , and therapies will be much easier and more informative if a specific diet , length of diet , and analysis protocols can be agreed upon .  
an updated murine hfd review is urgently required as 223 references for january 2016 were found on pubmed using  high fat diet and mouse  as the search term ( too many to read all ) .
the body of this review will therefore be focused upon a researcher 's point of view .
the goal is that researchers will use this paper to plan their hfd protocols and to create some consistency in future experimental protocols and analysis techniques . due to differences in the literature ,
we will use the following convention : obesity leads to metabolic syndrome which can progress to t2 dm . in humans obesity
is defined by a body mass index ( bmi = weight / height ) of over 30  kg / m .
progression to metabolic syndrome is diagnosed when a patient has 3 of the following 5 pathologies : obesity , hypertension , fasting hyperglycemia , elevated serum triglycerides , and decreased high - density lipoprotein .
after years of the associated hyperinsulinemia the pancreas may falter and the patient will then also suffer from hypoinsulinemia .
the pancreas fails to produce insulin and therefore the patient becomes hyperglycemic , but never hyperinsulinemic . 

examples of knowledge and therapies which have progressed from basic knowledge to preclinical trials in mouse models are numerous ( and some examples are reviewed in [ 35 ] ) . in the obesity and t2 dm fields , mouse models have proven invaluable in the basic science of the diseases by identifying the roles of inflammation , insulin resistance , fat content of the diet , pampk , exercise , and potential treatments . in addition , and very importantly , what has been learned from the mouse models has faithfully been carried over into the human patients .
these physiological similarities between the two species are due to the genetic homology between the two species .
one of the largest problems  or perhaps one of the most advantageous aspects  of mouse models for t2 dm are the different pathologic responses between mouse strains .
multiple publications have identified strain specific differences in hfd susceptibility ( a few recent examples are [ 813 ] ) .
the advantageous aspects of these differences lie in the pursuit of mechanisms which cause a mouse strain to be resistant to hfd pathologies and therefore provide avenues to identify targets for future therapies .
however , researchers must also be aware of these phenotypic differences when making comparisons with earlier publications which have utilized different mouse strains or even different sources for a mouse strain . in the hfd field
many manuscripts have identified dcm after a relatively short diet intervention [ 1417 ] while other experiments did not identify dcm after an 8-month hfd protocol   or 6-month hfd protocol .
some of these disparities are easily identified to lie in specific hfd formulations or specific mouse strains being utilized or even to know which characteristics of dcm are being considered for comparison .
however , some of the disparities are not so easily identified and having standardized hfd protocols will aid in identifying where the differences are arising and if they are worth pursuing for increased t2 dm mechanistic knowledge . for further insights into dcm the reader
is revered to a recent review article   concerning dcm in both t1 dm and t2 dm mouse models . of further interest to researchers is that mice subjected to hfd dependably model other human conditions in addition to t2 dm . among these other diseases
are those usually related to t2 dm but not occurring in all patients or mice .
nonalcoholic fatty liver disease ( nafld ) and the progression of this disease to nonalcoholic steatohepatitis ( nash ) and hepatocellular carcinoma ( hcc ) are common sequela of obesity and t2 dm . these conditions are well modeled with murine long - term hfd strategies ( reviewed in ) .
hfd has also been utilized to model chronic inflammation , which is an important pathogenic mechanism of t2 dm   and many other diseases including aging .
the hfd - mediated chronic inflammation is marked by increased tnf- , il-1 , and il-6 in the circulation . among the additional inflammatory diseases investigated using murine hfd
are wound healing [ 24 , 25 ] , prostate disease , dysbiosis [ 2729 ] , aging , and alzheimer 's .
the hfd mice also provide very good models for investigating exercise benefits   and muscle regeneration .
hfd has also been shown to decrease skeletal muscle regeneration , likely through inflammation and lipotoxicity in the muscle stem cells known as satellite cells ( reviewed in ) .
furthermore , impaired leptin signaling which occurs in hfd fed mice also decreases satellite cell proliferation .
insulin resistance occurs in many tissues , of primary importance in the skeletal muscle , liver , and heart . using acute insulin challenge protocols described below , individual
the  thrifty genome  theory posits that slight insulin resistance may have had some advantage when our ancestors ( and the ancestors of mice in the wild ) underwent times of food scarcity .
this transient insulin resistant after a large meal would cause increased calorie storage for the following times of food scarcity .
however , now during times of constant food overabundance in many cultures , and for the mice , the transient and slight insulin resistance has become chronic and leads to pathologies .
although recent reports do not support the theory [ 3840 ] , there must be an evolutionary reason for the maintenance of loci associated with t2 dm .
this is a very important idea when considering insulin sensitizing agents as therapy , which , if this theory is valid , would cause further cardiac damage as has been seen with rosiglitazone treatment [ 41 , 42 ] . in either case , hfd in mice
a number of recent publications and reviews have addressed the utility of the mouse model hfd protocol .
these reviews have excellent details and i will therefore refer the reader to these details at appropriate sections , instead of being repetitive .
discuss the cardiac disease phenotype of murine hfd feeding and present an important time course of phenotypic progression .
islam and loots very competently present the similarities between the murine hfd model and humans suffering from t2 dm .
another review manuscript describes the use of hfd mice to identify factors involved in obesity resistance or sensitivity .
in addition , a recent chapter has described the utility of mouse models for t2 dm drug discovery .
in addition , a thorough discussion of insulin resistance in multiple tissues was recently published .
additional publications describe genetically engineered t2 dm mouse models , which have recently been reviewed . however , due to the many genes identified which cause t2dm  more than 50 genes having been identified by human gwas studies -none of these murine genetic models can model disease etiology of more than a few patients .
many of these mice faithfully recapitulate some of the t2 dm phenotypes and can therefore be useful for preclinical trials and can be used to investigate specific portions of the phenotype . however , they do not model the disease etiology of the majority of patients .
most t2 dm humans have become sick due to their diets , not their genetics ( cdc , http://www.cdc.gov/diabetes/data/statistics/faqs.html ) .
there are many important considerations for designing a murine hfd protocol ( table 1 ) .
as discussed , which mouse strain to use is also highly important to consider when designing a hfd protocol .
we investigated many manuscripts and identified many different durations for diet intervention ; the most commonly utilized times are listed in table 2 .
clearly this wide array of times ( and diet specifics and age at start ) makes interstudy comparisons very difficult .
such an early adaptation phase was noted in the hfd fed wild type mrl mouse strain after 2 and 3 weeks of hfd .
the mrl mice became slightly hyperglycemic at these early weeks , which they fully recovered from by 7 weeks of diet .
the earliest hfd effects we found in the literature were after 3 days of diet as an increase of pancreatic -cell proliferation .
these authors also followed a time course through 11 weeks of hfd which revealed important differences between early ( 1 week ) and late ( 11 weeks ) results in intraperitoneal gtt and the dynamics of disease progression .
insulin tolerance also progressively worsened over the time course , while hyperinsulinemia was only apparent at 11 weeks of diet and was not seen at 1 or 5 weeks of diet .
this is a great illustration that the length of time of hfd must be carefully considered depending upon which variables one is interested in investigating .
furthermore , the above - mentioned early adaptation phase seen in the mrl mice did not occur in the hfd fed c57bl/6 mice , again stressing the importance of choosing the correct mouse strain depending upon the desired investigations .
others have identified adipocyte baseline differences that is in wild type mice fed a normal diet between male and female mice in glucose metabolism . in a highly comprehensive study morselli et al
. identified that due to hfd changes in the c57bl/6 male hypothalamus the male mice were more susceptible to hfd - mediated chronic inflammation , while the female mice gained as much weight ; they did not suffer from the pathogenic inflammation .
similarly , it has been identified that multiple fat deposits in c57bl/6 t female mice and castrated male mice are more insulin sensitive than those from intact male mice .
it is well known that age - related chronic inflammation correlates strongly with insulin resistance . as t2 dm is still considered a disease of the elderly it is fully appropriate to analyze older mice . however , unless one is contrasting age , comparisons with younger mice must be made with caution .
a great example of the variable responses of different mouse strains to a hfd was recently published .
the authors compared the c57bl/6 mouse strain , containing a typical th1 leaning immune system , to the balb / c mouse strain which is a th2 leaning strain .
the authors identified that the c57bl/6 mice were more susceptible to adiposity , liver inflammation , and liver fibrosis .
furthermore , slight genetic differences due to genetic drift result in significant differences between mouse substrains [ 13 , 65 , 66 ] .
these strains were physically separated in 1951 and kept in different facilities since then . as no selection pressure was applied the current genetic differences are purely due to genetic drift .
the strains are different in the severity of hfd - induced characteristics ; the c57bl/6n strain has a milder phenotype .
another advanced genetic analysis was conducted on some of the bxd mouse strains ( c57bl/6j and dba/2j intercrosses for 20 plus generations , ) .
these authors identified strains that were susceptible and resistant to hippocampal dysfunction elicited by hfd .
the advantage of this strategy is that genome wide association studies and additional genetic analyses will be very informative due to the complex but known sequences .
a further caution must be mentioned for the variations in diet compositions particularly important in light of continued data that the type of the fat is critically important to provide protection or pathology .
the complete review of diets is beyond the scope of this review ; here are a few important points to consider . in the united states
there are a few main suppliers of main suppliers of lab diets : teklad ( http://envigo.com/ ) , labdiet / purina ( http://labdiet.com/ ) , research diets ( http://researchdiets.com/ ) , and bio - serve ( http://bio-serve.com/ ) .
in addition , teklad provides many fat additives to custom make diets with specific lipid types .
not only are the amount and type of fat important but also the grams of carbohydrate in each formulation are highly important to consider . as mentioned before a true
diets high in saturated fatty acids are more obesogenic than mono- and polyunsaturated as the saturated fatty acids are inefficiently used for energy production and are therefore more readily stored .
furthermore , long - chain fatty acids ( c14:0c24:0 ) are more obesogenic than short- and medium - chain because they are transported into the mitochondria less readily and are therefore more likely to be stored . a very interesting study regarding diet composition came from the laboratory of ezaki .
these scientists balanced carbohydrate with different lipid percentages in an isocaloric diet and demonstrated a clear correlation between lipid content and hyperglycemia .
as calories from lipid increased and carbohydrate calories decreased to keep total calories constant , the glucose tolerance of the mice deteriorated . similarly , maiolo et al .
recently published comparisons of a hfd versus high sugar diet versus a diet of high fat and high sugar .
the results demonstrate that the combined high fat and sugar diet presented with most severe symptoms , including hyperglycemia , hypercholesterolemia , and higher levels of inflammatory mediators and lower levels of regulatory t cells . as we are all trying to improve patient lives the diet that best approximates the true western diet ( high fat and high sugar ) is arguably the best diet to use , unless diet specifics are being investigated .
many researchers use a straightforward hfd in their protocols , but some utilize additional stressors to model human t2 dm . an additional stressor is a low dose streptozotocin injection to boost the disease severity and model a later more progressed stage of disease after hyperinsulinemia when hypoinsulinemia has appeared [ 54 , 73 , 74 ] .
the dose of streptozotocin does not cause any phenotypes in chow fed mice and does not always cause dcm in hfd mice . additionally , to compensate for the additional calories provided by the hfd , some scientists have calorie that matched the amount of hfd given to their mouse cohort [ 75 , 76 ] . in this way
the hfd mice are only allowed to consume the same amount of calories as the cd cohort consumed in the previous 24 hours .
these variations provide very useful protocols to investigate different and specific aspects of t2 dm .
although there has been speculation that sucrose with or without fat should also cause t2 dm , this has not been found . in humans
there is retrospective epidemiological evidence that sucrose is not causative to t2 dm . in mice , where the experiment can be conducted with all of the proper controls ,
sucrose did not alter the progression to t2 dm with or without a hfd component .
therefore a straightforward hfd may be the optimum diet to model early t2 dm in mice with an additional streptozotocin injection to model the later stages of disease when hypoinsulinemia is also present .
type 2 diabetes mellitus ( t2 dm ) and the murine hfd model are complex diseases with complex and overlapping pathogenic mechanisms ( figure 1 ) .
the overlapping nature of these mechanisms clearly causes researchers and clinicians anxiety when thinking about prevention and treatment strategies .
it is therefore clear that once t2 dm is present , additional therapeutics beyond glucose control are required .
targets for the additional therapeutics require the consideration of many t2 dm and hfd pathogenic mechanisms .
it appears that endothelial cells are highly sensitive to chronic hyperglycemia most possibly due to their constant proximity to the circulation .
generally it may be that the body 's fuel storage cells ( primarily adipose and liver cells ) adapt to hyperglycemia through decreasing levels of insulin sensitivity .
it may be that what is viewed as the most pathogenic portion of the disease is actually a beneficial adaptation to hyperglycemia .
this view is supported through evolutionary history during which humans were subjected to periods of feast or famine and during times of feast the extra calories needed to be stored in glycogen and fat deposits not utilized immediately for atp production . by temporarily shifting the insulin receptors to resistance the storage pathway would be enhanced .
however , now we ( humans and mice on a chronic hfd ) no longer have the famine times and the resulting chronic hyperglycemia is toxic at many levels . at the onset of a hfd the murine adipose tissue adequately expands and stores the excess calories .
however , during chronic hfd the adipose tissue can no longer store the excess calories and the excess lipids are deposited into other organs .
this can lead to disruption of normal cellular processes and then also to frank lipotoxicity where the excessive lipid molecules damage cellular molecules .
lipotoxicity occurs in multiple tissues post - hfd protocols : heart , skeletal muscle , liver pancreas , and kidneys .
the definition of this pathology can be considered an imbalance in lipid uptake and disposal leading to an accumulation of lipid intermediates in nonadipose cells , which causes impaired cellular function .
excessive lipids appear to cause stress responses often leading to apoptosis and replacement of functioning cells with fibroblasts and extracellular matrix .
it is currently thought that healthy individuals with a varied diet and adequate exercise can adapt to their caloric source , such that eating a few hfd meals in a row will not produce any pathology .
however , after chronic hfd mice and individuals become metabolically inflexible . for example , cardiac tissue often responds to hfd by increasing its carbohydrate utilizing proteins to extract as much energy from glucose as possible [ 16 , 18 ] .
however when the diet then switches to a normal diet the heart draws too many calories from glucose causing a pathologic excessive caloric intake .
inflammation is a central pathogenic mediator in all aspects of t2 dm and murine hfd - induced pathologies including the cardiovascular system [ 8082 ] .
immune response time courses are also required as it has been shown that continued immune responses are pathogenic in many disease models , including t2 dm .
as inflammation is also associated with many age - related diseases and conditions it becomes doubly critical in the older population historically affected by t2 dm .
demonstrated that mice with a th2 leaning immune response ( balb / c ) are spared many of the hfd - induced pathologies , while mice leaning towards a th1 immune response are susceptible to many hfd pathologies .
infiltrating macrophages are known to be involved in pathogenesis . a major contributor to hfd - induced dcm
extensive recent publications describe decreased autophagy [ 89 , 90 ] and in particular mitophagy   as pathogenic in t2 dm and hfd mouse models .
increasing autophagy can alleviate many of the hfd - mediated pathologies [ 92 , 93 ] .
this is clearly an ongoing and interesting research area in the hfd mice . in diabetic humans hyperglycemia
is thought to cause mitochondrial dysfunction and fragmentation , thereby linking hyperglycemia , mitochondrial dysfunction , and temporary insulin resistance characteristics in cardiomyocytes before obvious pathology .
there is significant new data that the insulin signaling cascade directly impacts cardiac mitochondrial fission and fusion ( reviewed in ) .
this data arises from a number of observations in knock - out mouse models and cultured cardiomyocytes in which disrupted mitochondrial morphology and function precede t2 dm . the beneficial effects of metformin on increasing mitochondrial function and volume   are also consistent with mitochondrial pathology being pathogenic for t2 dm .
common human t2 dm and mouse hfd pathologies are as follows : weight gain.hyperglycemia.hyperinsulinemia followed by hypoinsulinemia.insulin resistance.increased fasting leptin levels.decreased fasting adiponectin levels.inflammation with increases in inflammatory cytokines and reactive oxygen species.fatty lipid droplet accumulation in multiple tissues.fibrosis in multiple tissues.increases in blood pressure , but not always seen in the mouse models .
there are many assays to quantify the extent of these pathologies in the hfd mouse model ( table 1 ) .
all of these assays have been described in detail , so i will just highlight some of the important considerations that may be overlooked by busy investigators and direct you to the pertinent references . based upon my own limited experience many of these assays should be done weekly .
we have noticed an adaptation phase in the first few weeks of hfd , which we now wish we had analyzed more closely instead of beginning another cohort of animals through the long and costly procedure .
therefore , all of the nonterminal procedures , weight gain , blood glucose , insulin , adiponectin , echocardiography , dexa , and blood pressure ( noninvasive tail cuff ) , should be conducted weekly .
terminal assays that should be considered at these intervening dates are liver , skeletal muscle , and adipose deposits for weighing , histology , and immunoblot analysis .
interesting results would include identifying changes in molecules used by the tissues in attempts at adaptation .
for example changes in enzymes and molecules controlling glucose and lipid metabolic rates would be of interest to investigate early in the hfd protocol .
a recent chapter from baribault describes the gtt , itt , insulin secretion tests , nonesterified free fatty acid quantification , body composition , and terminal harvesting techniques . as this chapter appears in methods in molecular biology and contains protocol details we will not repeat these descriptions here . although many of these tests appear straightforward some additional considerations are important .
animal mass can be reported as absolute or relative to cd mice or relative to beginning masses .
perhaps the most accurate is comparing the ratio of the animal 's final weight to the animal 's own beginning weight , such that the animal serves as its own control .
however , using this method may not be the optimum method to compare between sexes or mouse strains where an absolute change would be optimal .
another consideration for many of these tests is the time of day performed and if any fasting is required .
as mice are nocturnal many of their diurnal cycling hormones and activities would be opposite of those found in humans .
therefore , as with all biological experiments the time of day must be kept consistent .
multiple publications discuss the appropriate length of fasting to achieve the most accurate data for various tests .
fasting is required to minimize variation and therefore minimize the number of animals needed to uncover differences .
fasting is usually used with the basal blood glucose measurements , gtt , itt , and acute itt .
these authors empirically identified 6 hours of fasting and 2  g / kg of oral glucose as the most informative assay parameters .
the most often used insulin dose is 1  mu / g of humalog ( e. l. lilly , indianapolis ) for itt [ 4 , 12 ] .
these experiments require additional equipment and are not high - throughput . in brief , for a hyperinsulinemic euglycemic clamp , the animals receive a basal insulin delivery ( 3  mu / min / kg ) which is a commonly used dose for rats   and the amount of glucose required to keep the blood sugar in a certain range is quantified .
these studies quantify insulin sensitivity better because they circumvent the counterregulatory responses associated with itt .
clamp techniques are often used in conjunction with labeled glucose so that individual tissues can be analyzed for glucose uptake .
the most high - throughput method is simply weighing some of the various fat masses and comparing this to the animal 's body mass , both measurements should be determined anyway as part of the harvesting protocol .
the next upgrade is to utilize dexa measurements on all or a consistent portion of the mouse .
this technique has the added bonus that it is not a terminal procedure and a time course can be constructed for each animal .
this also benefits from being amenable to establish a time course , although it is the furthest from high - throughput of the three methods and may also be expensive .
human type 2 diabetes mellitus is truly a systemic , multiorgan disease and each of these organs is also negatively affected in mouse models on a hfd .
the mouse organs affected each are being investigated to gain pathologic and therapeutic knowledge into the disease .
the ultimate goal is to identify therapeutics which reduce all systemic and organ pathologies . in this section
additional , tissue - specific analysis methods will also be listed and referenced or discussed .
extensive data identifies that adipose inflammation , including secretion of adipokines and activation of adipose resident macrophages , is pathogenic for all other tissues and organs .
it is indicated that visceral white fat is the most pathogenic type because it secretes large amounts of adipocytokines such as leptin , tnf , il6 , and adiponectin that can disrupt homeostasis when dysregulated due to hfd .
as the visceral fat undergoes hypertrophy and perhaps hyperplasia it secretes more of these potentially unhealthy adipocytokines .
subcutaneous adipose tissue also secretes the adipocytokines but in smaller quantities so this adipose site is not as frequently studied .
the adipocytokines are reliably measured by serum or tissue extract enzyme - linked immunosorbent assays ( elisas ) .
the weights of the various adipose deposits are also very important to obtain at harvest time .
relatively small amounts ( 0.1  mg ) of brown adipose tissue were transplanted into the visceral cavity of hfd recipients .
eight weeks after surgery the recipients had significant improvements in glucose tolerance and at 12 weeks the recipients had significant improvements in insulin tolerance tests . a detailed review of brown adipose tissue physiology assessment has recently been published   and will therefore not be repeated here .
adipocytes usually respond to insulin by dividing , increasing glucose uptake , and inhibiting lipolysis .
when the cells become insulin resistant due to hfd the body is subjected to increased levels of hyperglycemia and lipotoxicity because of the excessive lipolysis . as skeletal muscle
skeletal muscle also provides one of the best methods to cure t2 dm through increasing exercise and disposing of glucose in an insulin independent manner , thus by passing the pathology and reestablishing homeostasis . for these reasons investigating and treating the skeletal muscles of hfd mice are critically important .
skeletal muscle of hfd mice succumbs to many of the above - mentioned pathologies : insulin resistance , lipotoxicity from excessive lipid storage , and inflammation .
insulin resistance can be measured with an acute itt , fifteen minutes after an insulin bolus ( 1  mu / g ) ; skeletal muscle is harvested and immunoblots are used to determine the levels of phosphorylated pakt at t and s .
insulin resistant tissues produce less pakt and this appears to be a linear relationship so that the degrees of insulin resistance can be quantified .
other downstream targets of the insulin receptors can also be monitored for insulin signaling , although specificity of insulin signaling must be considered during data analysis .
the gold standard is perhaps by electron microscopy so that lipid droplet location can also be determined .
lipid droplets in the mitochondria [ 12 , 16 ] versus cytoplasmically located   would reveal specific pathologic mechanisms .
inflammation and specific cells of the invading immune system can be measured by histology or flow cytometry .
the liver of hfd mice is negatively affected in a number of ways [ 21 , 103 ] . as with many hfd - elicited diseases
the pathology usually begins with patchy steatosis / fatty droplet accumulation ( nonalcoholic fatty liver disease ( nafld ) ) , inflammation ( pathogenic cytokines and ros release ) , fibrosis , hepatocyte hypertrophy and hyperplasia , more global steatosis ( nonalcoholic steatohepatitis ( nash ) ) , liver mass gain , cirrhosis , and finally hepatocellular carcinoma ( hcc ) .
the usefulness of a long - term hfd to model the steps of this disease progression is discussed in a review by kanuri and bergheim .
therefore , in an insulin resistant animal or t2 dm patient the liver actually makes and secretes more glucose than normal . in an organism that is
however , due to  selective insulin resistance  this signaling pathway is not inhibited during times of systemic insulin resistance and therefore the liver inappropriately produces lipids in response to the hyperinsulinemia in the later stages of disease . in addition , a large proportion of a health body 's readily available lipid is stored in the liver ; the liver is therefore highly susceptible to lipotoxicity . of interest
 requires a long hfd protocol to be established ; at 60 weeks of hfd still only 54% of the mice displayed hcc .
however , this hfd model is still very useful in liver pathology and treatment investigations .
for example , long - term ( 60 weeks ) hfd feeding establishes nafld , including the hcc , in just over half of the hfd c57bl/6 mice .
the group went on to use this hfd mouse model , at multiple hfd durations , to demonstrate the preclinical efficacy of metformin treatment in reducing hcc if the treatment starts coincident with the diet , but not if metformin treatment is begun after nafld has begun to develop .
these authors also compared various time points of diet duration and treatment to elucidate the mechanisms behind metformin cancer reduction .
histology is very useful to identify the stage of disease progression and to quantify the degree of pathology .
the liver is also well studied by the acute itt to quantify its level of insulin resistance . as many t2 dm patients die of cardiomyopathy without vessel disease or hypertension , investigating diabetic cardiomyopathies
multiple studies have investigated the effects of hfd upon cardiac function , remodeling , and metabolism .
mice fed a hfd for 10 weeks develop myocardial insulin resistance evidenced by a downregulation of insulin receptor activity , downregulation of akt signaling , and increased fatty acid oxidation .
it is exacerbated in chronic ( hfd ) situations and obesity when the body is overwhelmed by free fatty acids ( ffa ) .
even though the heart metabolizes lipids for 6080% of its total basal energy it will store excess ffa and metabolites .
abnormal accumulation of nonoxidative lipid derivatives leads to increased apoptotic signaling , oxidative stress , and broad cellular dysfunction in the heart .
overwhelming evidence indicates that the type of ingested fat is critically important for its place in the pathogenic or protective spectrum ( reviewed in ) .
this is especially evident in the effects of particular fats in the diets on cardiac function .
for example , a diet high in saturated fats is protective in muscular dystrophy mediated cardiomyopathy in hamsters .
rodent hfd models of t2 dm also reproduce the  obesity paradox ,  initially identified in humans .
the situation is labeled a paradox because obese patients are better at overcoming long - term damage after myocardial infarctions . in rats subjected to pressure - overload hypertrophy , those receiving hfd
furthermore , mice on a hfd have less postinfarct myocardial remodeling and dysfunction compared to normal diet controls .
the similarities between the human diseases and the rodent models facilitate investigations into the mechanisms of this paradox and identify if it can be leveraged for human therapeutics .
for example , in a 6-month hfd study the investigators did not find any cardiac pathology by echocardiography . alternatively
for example , in a 12-week hfd study we identified cardiomyocyte hypertrophy , unsuccessful metabolic adaptations , and lipid droplet accumulation .
these results indicate that caution must be used when designing hfd experiments , especially for diabetic cardiomyopathy assessments
. it may be that in these long - term experiments the hearts have compensated fully and have established a new homeostasis and the pathology must be evaluated over a time course .
this is an interesting point requiring further study , because such compensatory mechanisms may reveal therapeutic targets .
these authors compared 8 , 12 , and 16 months of hfd on the c57bl/6 male hearts .
they identified cardiac remodeling at all time points , which became more severe as the diet duration was extended . in the hfd group they identified functional pathology , but only after dobutamine pharmacological stress .
there are a number of possible reasons for the discrepancies regarding hfd - induced dcm .
some of the reasons are easily identified : different diets ( fat type is highly important , w. c. stanley ) , diet duration , different assays , and age of animals .
some of the harder to identify reasons are genetic drift , seasonal variation , and diurnal variation in time of assays .
when designing a hfd protocol these variations each must be considered and minimized as much as possible .
cardiac pathology assessments are critically important to obtain due to the large number of t2 dm patients dying from heart failure .
blood pressure values can be quantified by either terminal ( pressure volume loops ( pv loops ) ) or survival ( tail cuff ) techniques .
the tail cuff procedure benefits from being survival , relatively high - throughput , and inexpensive while the pv loops benefit from being more accurate and accepted by reviewers . the best possible scenario would be to perform tail cuff weekly throughout the hfd protocol and
hypertrophy can occur in two distinct or overlapping characteristics at the organ level and/or cellular level .
the organ level hypertrophy can be assessed with echo and heart weight to tibia length and the cellular hypertrophy can be assessed by histology methods and imagej .
the hyperglycemia caused by the hfd signals the pancreas to secrete more and more insulin . eventually the pancreas falters and hyperinsulinemia is followed by hypoinsulinemia .
these changes in insulin levels are seen in patients and in mice fed a hfd .
of course , because t2 dm is a systemic disease , all cells and tissues are affected by the disease - defining hyperglycemia and lipotoxicity .
principal among these cell types would be endothelial cells , neurons , and beta cells of the pancreas .
the hfd mice have proven invaluable not only for the identification of molecular pathways affected by hfd , but also in preclinical protocols to test potential therapies to reverse the condition and its many associated pathologies .
as the research community identifies the most human - like hfd model the strength of using this model for preclinical protocols can only become better .
recently resveratrol ( rsv , red grape extract ) has come into the news as being antiaging and potentially inhibiting t2 dm symptoms . when given for the 12 weeks of hfd
, rsv produced only a slight reduction of body weight and blood glucose but had a significant reduction on renal fibrosis and renal dysfunction .
other pharmacologies that have been tested in the hfd protocol include metformin , ezetimibe , acarbose , and atorvastatin .
as the research community approaches the ideal hfd protocol , consistent protocols will be undeniably helpful .
as stated previously this would enable ease when comparing study to study , especially important for the preclinical therapeutic trials .
i will now go out on a limb and steel myself for the upcoming onslaught of detractors .
i propose this following standard protocol : using c57bl/6j.feeding from 4 to 20 weeks old.using both male and female mice.using a hfd plus high fructose to mimic the human diet best .
feeding from 4 to 20 weeks old . using both male and female mice . using a hfd plus high fructose to mimic the human diet best .  
it can be also modified to study specific mouse strains , exercise , age , length of diet , and so forth .
for example , it would be of great interest to compare young and old mice in a hfd protocol . although older mice do not have the high levels of chronic inflammation as found in the aged human population
, it would be of interest to establish if the older mice develop t2 dm symptoms at a shorter hfd time period .
 type 2 diabetes mellitus ( t2 dm ) is a worldwide epidemic , which by all predictions will only increase . to help in combating the devastating array of phenotypes associated with t2 dm a highly reproducible and human disease - similar mouse model 
 is required for researchers . 
 the current options are genetic manipulations to cause t2 dm symptoms or diet induced obesity and t2 dm symptoms . 
 these methods to model human t2 dm have their benefits and their detractions . as far as modeling the majority of t2 dm cases 
 , hfd establishes the proper etiological , pathological , and treatment options . 
 a limitation of hfd is that it requires months of feeding to achieve the full spectrum of t2 dm symptoms and no standard protocol has been established . 
 this paper will attempt to rectify the last limitation and argue for a standard group of hfd protocols and standard analysis procedures . 